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Uncaria tomentosaAdjuvant Treatment for Breast Cancer:
Clinical Trial
1
Department of Chemistry, Federal University of Santa Maria, Avenida Roraima,
Predio18, 97105-900 Santa Maria, Rs, Brazil
2
Santa Maria University Hospital, Federal University of Santa Maria, Avenida
Roraima, Predio18, 97105-900 Santa Maria, Rs, Brazil
3
Department of Biology, Federal University of Santa Maria, Avenida Roraima,
4
Department of Industrial Pharmacy, Federal University of Santa Maria, Avenida
Roraima, Predio18, 97105-900 Santa Maria, Rs, Brazil
5
Department of Morphology, Federal University of Santa Maria, Avenida Roraima,
Received 15 November 2011; Revised 26 April 2012; Accepted 27 April 2012
Academic Editor: Alyson Huntley
Copyright 2012 Maria do Carmo Santos Arajo et al. This is an open access
article distributed under theCreative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original
work is properly cited.
Abstract
Breast cancer is the most frequent neoplasm affecting women worldwide. Some of
the recommended treatments involve chemotherapy whose toxic effects include
leukopenia and neutropenia. This study assessed the effectiveness of Uncaria
tomentosa (Ut) in reducing the adverse effects of chemotherapy through a
randomized clinical trial. Patients with Invasive Ductal CarcinomaStage II, who
underwent a treatment regimen known as FAC (Fluorouracil, Doxorubicin,
Cyclophosphamide), were divided into two groups: the UtCa received chemotherapy
plus 300mg dry Ut extract per day and the Ca group that only received
chemotherapy and served as the control experiment. Blood samples were collected
before each one of the six chemotherapy cycles and blood counts, immunological
parameters, antioxidant enzymes, and oxidative stress were analyzed. Uncaria
tomentosa reduced the neutropenia caused by chemotherapy and was also able to
restore cellular DNA damage. We concluded that Ut is an effective adjuvant
treatment for breast cancer.
1. Introduction
Breast cancer is the most frequent neoplasm affecting women worldwide, both in
terms of incidence and mortality. The disease is more common in developed
countries with its highest incidence being observed in the United Kingdom,
Australia, USA, and Canada. From invasive tumors, ductal carcinoma and its
variants represent 80% of cases [1] and the proportion of women with tumors in
clinical stages I and II increased from 41 to 65% in the last decade [1, 2]. About
70% of breast cancers express Estrogen hormone receptors and/or Progesterone
receptor [3]. These markers along with the HER-2 receptor (c-erbB2) provide
information about the tumor and how it might respond to different treatments [4].
Chemotherapy is among the recommended treatments for breast cancer, which can
be a single or combination therapy with multiple drugs. Chemotherapy drugs have
very narrow therapeutic indexes in terms of nonselective toxic effects on normal
tissues, with neutropenia being the most frequently observed adverse reaction, which
increases the risk of infections [5].
Pharmacological interventions that reduce or prevent adverse effects may have a
substantial impact on cancer treatment. According to World Health Organization
(WHO), 80% of the population use medicinal plants as alternative or complementary
procedures for the treatment of their diseases [6].
Studies have reported the use of herbal medicines in cancer patients to minimize the
effects of chemotherapy.Uncaria tomentosa (Utor Cats Claw) is a medicinal herb
that has been used in the treatment of different diseases including cancer. Patients
who use Cats Claw along with traditional cancer therapies, such as chemotherapy
and radiation, reported fewer adverse effects to those therapies [7]. Uthelps in the
restoration of cellular DNA, preventing mutations and cell damages caused by
chemotherapy drugs [8]. It modulates the activity in the immune system, such as the
proliferation of normal T and B lymphocytes [9], also modulating certain cytokines,
including IL-1 and IL-6, TNF- [10]. In addition, it has antioxidant properties [11].
Its direct myelostimulating effects, through myelopoiesis stimulation and Colony-
Stimulating Factors (G-CSF) [8, 12], seem to be a beneficial option to minimize the
risks associated with neutropenia.
Numerous reports present a theoretical understanding of Ut action mechanisms, but
none of these studies consisted of clinical trials. Thus the objectives of this study are
situated in this context, which consisted of a clinical trial using Uncaria
tomentosa Herbarium tablets, as adjuvant treatment for breast cancer.
2. Methods
2.1. Design and Patients
A randomized interventional study was performed. It was carried out with 40
patients who had undergone complete breast cancer resection, which was
histologically diagnosed as Invasive Ductal CarcinomaStage II [2], and who were
going to begin adjuvant chemotherapy with Doxorubicin-based scheme for six
cycles, at the Santa Maria University Hospital, Brazil.
Patients were randomly divided into two groups: the CaUt group, which was treated
with six cycles chemotherapy + Ut and the cancer group (Ca), which only received
six cycles of chemotherapy, according to the date treatment was started, as follows:
the first patient who agreed to participate in the study was included into the CaUt
group, the second, into the Ca group, and, thus, successively, until the end.
For the control group were invited to participate healthy women, classified by
clinical trial, with similar age of the patients and that did not receive any medication
in the last 30 days or have chronic disease.
Patients were part of the study during 6 chemotherapy cycles, of 21 days each.
Medication dosage in the CaUt group was as follows: FAC (Fluorouracil,
Doxorubicin, and Cyclophosphamide) and 3 tablets of Ut (Unha de Gato
Herbarium), daily, from day 2 to day 21. The dose of Ut was similar to that used in
previous studies, with 250350mg C-MED-100, in aqueous Ut extracts [13].
The calculation to estimate the sample size required for randomized clinical trial was
performed according to Greenberg et al. [14], with constant significance level () of
5%, and statistical power of 90% ( 10%), using as reference the studies of Sheng et
al. [15].
The Human Ethics Committee of the Santa Maria University Hospital, Brazil,
approved the present study and informed consent was obtained from all participants
(protocol number: 0169.0.0242.000-07.). All subjects were invited to participate and
were informed in detail about the design of this study through a Statement of
Consent signed by the researcher and participants. They were informed that they
could be selected randomly for the Ca or UtCa group.
2.2. Materials
Each tablet of Unha de Gato Herbarium contained 100mg of dry Uncaria
tomentosa extract. Biological materials used in the tablets were derived from plants
in their natural habitat. The Uncaria tomentosa extract was prepared by Ultra-turrax
Extraction (Biotron, Kinematica AG) from ground bark (Centroflora) using 70%
ethanol (Dipalcool). The HPLC analysis of the Ut dry extract presents 2.57%
pentacyclic oxindole alkaloids (POAs) content, which was calculated with reference
to external calibration curves of mitraphylline. The extract analysis showed absence
of tetracyclic oxindole alkaloids in the sample, allowing its use for therapeutic and
research purposes in accordance with the U.S. Pharmacopeia.
2.3. Sample Collection
Blood was collected into citrate, EDTA, heparin Vacutainer tubes, without any
anticoagulants, before chemotherapy and after each of the 6 cycles.
2.4. Biochemical Parameters
A COBAS INTEGRA system was used for the quantitative determination of the
blood chemical constituents, and data were acquired through a COBAS INTEGRA
400 Plus apparatus (USA).
2.5. Hemograms
Blood samples were analyzed using a Pentra apparatus (France). The lowest values
were confirmed by observation of slides, using a May Grnwald-Giemsa Stain and
optical microscopy.
2.6. CD3+, CD4+, and CD8+ Cells
Samples were collected in EDTA and analyses were performed using a three-color
fluorescence-activated cell sorter (FACSCalibur, Becton Dickinson Biosciences,
United States) and a Multiset software (Becton Dickinson). FITC-conjugated anti-
CD4, PE-conjugated anti-CD8, and PerCP-conjugated anti-CD3 were used. Immune
subpopulations were measured as a percentage of the total CD3+ cell number.
2.7. Interleukin 6 (IL-6)
ELISA assays of IL-6 were carried out according to a previously published method
[16], at room temperature in Microtiter 96-Well Plates (Nunc-Immuno Plate
MaxiSorp) and optical densities (O.D.) at 490nm, which were determined using a
Microplate Reader (Thermo Scientific Multiskan FC, Vantaa, Finland).
2.8. Single Cell Gel Electrophoresis (Comet Assay)
The alkaline comet assay was performed as described by Singh et al. [17] in
accordance with the general guidelines for use of the comet assay [18, 19].
Lymphocytes were suspended in 0.7% low-melting-point agarose and phosphate-
buffered saline (PBS) at 37C and placed on microscopic slides with a layer of 1%
agarose. The slides were immersed in lysis solution at 4C for 1h and followed by
electrophoresis at 25V, 300mA, for 40min at steady temperature. The slides were
then silver-stained, as described by Nadin et al. [20]. All steps, from sample
collection to electrophoresis, were conducted under yellow light to minimize the
possibility of cellular DNA damage. One hundred cells (50 cells from each of the
two replicate slides) were selected and analyzed. Cells were visually scored
according to tail length and received scores from 0 (no migration) to 4 (maximal
migration). Therefore, the damage index for cells ranged from 0 (all cells with no
migration representing a damage index of 0%) to 400 (all cells with maximal
migration, representing a damage index of 100%). The slides were analyzed under
blind conditions by at least two different individuals [21].
2.9. Carbonylation of Serum Protein
The carbonylation of serum proteins was determined by a modified Levines method
[22]. The absorbance of the supernatant at 370nm was measured using a
spectrophotometer. Carbonyl content was calculated using mM
1
cm
1
as the molar
extinction coefficient, and the results were expressed as nanomoles of carbonyl
groups per milligram protein.
2.10. Determination of Lipid Peroxidation
Lipid peroxidation was estimated by measuring TBARS levels in plasma samples
according to a modified method of Jentzsch et al. [23]. The concentration of
malondialdehyde (MDA) was determined by measuring the absorbance at 532nm
using a spectrophotometer. The results were expressed as nanomoles of MDA per
milliliter of plasma.
2.11. Catalase (CAT) and Superoxide Dismutase (SOD) Activities
CAT activity was determined in accordance with a modified method of Nelson and
Kiesow [24]. The change in absorbance at 240nm was measured for 2min. CAT
activity was calculated using the molar extinction coefficient (0.046mM
1
cm
1
),
and the results were expressed as picomoles of CAT per milligram of protein.
SOD activity was determined based on the inhibition of the radical superoxide
reaction with adrenaline as described by McCord and Fridovich [25]. SOD activity
is determined by measuring the rate of adrenochrome formation, observed at
480nm, in a medium containing glycine-NaOH (50mM, pH 10) and adrenaline
(1mM).
2.12. Statistics
Results are expressed as deviation. The statistical analysis was performed with
Graph-Pad Prism 5.0 (GraphPad Prism 5.0 Software Inc., USA) using the
Students t-test. was considered to represent a significant difference in all tests.
3. Results
All patients (40) included in the trial had Breast Cancer, Invasive Ductal
CarcinomaStages II A or II B, according to the American Joint Committee on
Cancer (AJCC) and the American Cancer Society (ACS) staging systems [2].
The general characteristics of patients and controls who participated in the study are
described in Table 1.

Table 1: Clinical characteristic of patients. It represents age, body mass index
(BMI), total cholesterol levels, estrogen receptor (ER), and progesterone receptors
(PR), as well as the HER-2 status in different groups.
To evaluate the effectiveness of Ut as adjuvant treatment for breast cancer,
haematological parameters were used and analyzed (Table 2). At day zero, the
results of the haematological parameters analyzed in the blood count did not
significantly differ among the Control, the Ca, and the UtCa groups. A greater
reduction in the white blood cell (WBCs) and the neutrophil counts were observed
in the Ca group along the treatment, differently from the UtCa group, which
remained closely the reference values, obtained in the control group (Figure 1).
Considering the lymphocytes number, a significant difference between the control
group and the groups of patients with breast cancer, either treated or not with Ut in
the chemotherapy cycles, was observed. (). Monocytes number in patients with
breast cancer (treated and not treated with Ut) at 5-6 chemotherapy cycles were
higher than control group, but in the UtCa group, this increase was more strong
(Table 2).

Table 2: Leukocytes, neutrophils, lymphocytes, and monocytes levels in breast
cancer patients before treatment and after 6 cycles of chemotherapy without Uncaria
tomentosasupply (Ca group) or receiving 300mg/day of Uncaria tomentosa (UtCa
group).

Figure 1: Values neutrophil granulocytes in patients with breast cancer undergoing
chemotherapy with (UtCa) and without (Ca) supplementation with Uncaria
tomentosaand reference values (control). Data are expressed as deviation.
To evaluate the immune response of patients with breast cancer, CD4
+
T cells,
CD8
+
T cells (absolute count and ratio) and IL-6 levels were analyzed. During the
chemotherapy treatment cycles, no significant difference was observed between
groups. There was no difference between groups for any of the parameters analyzed
(Table 3).

Table 3: Immune status of breast cancer patients before treatment and after 6 cycles
of chemotherapy without Uncaria tomentosa supply (Ca group) or receiving
300mg/day of Uncaria tomentosa (UtCa group).
No correlation between the IL-6, CD4
+
T/CD8
+
T ratio and age, body mass index,
and hormone receptor status was found (data not shown).
Antioxidant defenses were analyzed by the activity of Superoxide Dismutase (SOD)
and Catalase (CAT) compared to treatment cycles zero and six, as well as between
the UtCa and the Ca groups. There were no statistically significant differences
among groups. An increase in SOD enzyme when compared to treatment cycles zero
and six for the group supplemented with Ut was observed, but that difference was
not observed between the groups (UtCa = 11.53U/mg protein, Ca = 11.43U/mg
protein) or at the end of treatment (17.32U/mg protein, 11.74U/mg protein). Lipid
Peroxidation was also estimated by the TBARS scale and the carbonylation of serum
proteins, but there was no difference between groups (UtCa and Ca).
The protective effect of chemotherapy to extract Ut was evaluated by the Comet
Assay. In the start of the treatment (zero cycle), the Ca group and UtCa group
showed no significant difference in the Comet assay index. However, in the sixth
cycle (end of the treatment), it was observed a significant decrease in the index test
in the UtCa group, when compared to the Ca group () Figure 2.

Figure 2: Index test of blood cells in patients with breast cancer treated and not
treated with Ut. *Represents significant difference between all groups ().
**Represents significant difference between the UtCa group (cycle 0) and the UtCa
group (cycle 5-6) (Students t-test).
4. Discussion
Uncaria tomentosa enables the stimulation of the immune system, increasing
resistance to diseases when the body is immunosuppressed due to stress,
malnutrition, or due to the effect of some medication.
Many herbal medicines are used for various purposes, in various combinations
(along with allopathic and homeopathic, medicines, etc.) based on historical or
personal evidences generally not being associated with any adverse effects [26].
Therefore, this study, through a randomized clinical trial, evaluated the efficacy of
Ut as a complementary therapy to chemotherapy.
The cytotoxic effect of chemotherapeutic agents is not selective for neoplastic cells,
being also harmful to other body cells. Hematopoietic suppression is the major
complication limiting dosage of such cytostatic agents; neutropenia and
thrombocytopenia are the most frequent ones [1]. Treatment should be discontinued
when neutrophil count is below 500 cells/mm
3
[27]. Thus, the success of the
treatment process depends on the neutrophils content. Prevention of chemotherapy-
induced neutropenia should be considered a clinical priority [28]. Once it is known
that neutropenia predisposes to serious infections, often resulting in delays in
treatment cycles and dose reductions.
Treatment using a daily dose of 300mg dry Ut extract was effective in reducing the
main chemotherapy effect, which is neutropenia. The effects of chemotherapy on
blood cells tend to become more pronounced during treatment. However, our results
show that in cycle six, which corresponds to the end of chemotherapy, the
differences in the leukocytes and neutrophils counts were even more significant, as
the group that was supplemented with Ut presented values twice as high of
neutrophils when compared to the cancer group (without supplementation). In the
group without supplementation, 67.89% of patients had neutropenia. Similarly, there
was an increase in activated monocytes, as the activated precursors were common to
both strains.
Our findings are corroborated by other studies that had already shown that the Ut
extract has a stimulating effect on growth and differentiates the CFU-GM from mice
bone marrow and spleen, using the model for listeriosis [29]. Increased leukocytes
numbers were also detected using Ut aqueous extract for six consecutive weeks in
volunteers [13]. The recovery of leukocytes was also observed in mice using a
model for chemotherapy-induced leukopenia (Doxorubicin) using Granulocyte
Colony-Stimulating Factor (Neupogen) as a positive control [15].
Our group confirmed these results using a model for ifosfamide-induced neutropenia
in mice, which caused a severe neutropenia. Bioassays showed that treatment with
Ut significantly increased neutrophils counts, and a power of 85.2% was calculated
in relation to Filgrastim (rhG-CSF) at the corresponding doses tested (5 and
15mg/day of Ut, and 3 and 9mcg/day Filgrastim, resp.) [13]. Through in
vitro assays in human hematopoietic stem precursor cells (hHSPCs) obtained from
umbilical cord blood (UCB), we reach the conclusion that this effect happened due
to proliferation of Forming Units-Granulocyte-Macrophage (CFU-GM) [13].
In this study, no differences were observed in lymphocyte counts between groups,
either supplemented or not with Ut, over the chemotherapy cycles; however, its
counts presented decrease due to chemotherapy when compared to the control
group. These differences were not observed in the CD4+ and CD8+ subpopulations.
This paper reports the effects of different Uncaria tomentosa extracts. The aqueous
extract that has the highest concentration of quinic acid and low concentrations of
oxindole alkaloids, being related to immunomodulatory properties is mediated by
cytokines such as TNF- [30]. Clinical studies using 20mg/day of Uncaria
tomentosa extract for 2 to 5 months in patients with HIV, receiving no other therapy,
showed an increase in total peripheral lymphocytes without significant changes in
the proportion of CD4+ and CD8+ [31]. Healthy volunteers receiving 350mg of
aqueous Ut extract for 8 weeks showed leukocytosis, with a tendency to higher
proliferation of lymphocytes [15]. In an animal model, using aqueous extract, which
has the highest concentration of quinic acid and low concentrations of oxindole
alkaloids, an increase in lymphocytes was also observed [15, 32].
Furthermore, alcoholic extracts and/or pentacyclic oxindole alkaloids have higher
myeloproliferative effects [33, 34]. Other studies have shown that the increase in the
lymphocyte counts happens due to increased survival rates rather than proliferation
[32].
Thus, changes observed in lymphocytes are associated with the chemically active
components defined as quinic and bioactive acid esters in vivo, as quinic acid
present in the aqueous extract used by authors. In our study, hydroalcoholic extract
was used.
High levels of circulating IL-6 are associated with worse survival rates for patients
with metastatic breast cancer, being correlated to the extent of the disease [30].
The patients who comprised our sample did not present a negative progression
during the treatment cycles, that is, there was no occurrence of relapses or increased
lesion extent, which could lead to an increase in IL-6.
Different Uncaria tomentosa extracts were tested in vitro in order to determine their
antioxidant activity. Aqueous and alcoholic extracts prevent the production of
reaction products with thiobarbituric acid (TBARS) and, therefore, damage the
cytoplasmic membrane (lipids) and DNA by the nonformation of free radicals
[35, 36] among the evaluated parameters of oxidative stress, such as SOD, CAT,
TBARS, and carbonylated proteins.
Women with breast cancer present an increase in blood concentrations of oxidized
substances, such as products derived from lipids peroxidation, proteins, and DNA
[32, 33].
The only observed differences were in the SOD enzyme between groups, either with
or without supplementation with Ut. These results were also found in an animal
model, where an increase in the activity of this enzyme [10] was perceived. In a
study on women with breast cancer, SOD activity showed a significant increase
regardless of clinical stage and menopausal status [31]. There is evidence that the
state of oxidative stress is higher than the greater degree of the disease stage is
[37, 38].
Similar to results found in IL-6, the fact that all patients in the study had Stage II
cancer may explain the results found.
The ability of doxorubicin to bind itself to the cell membrane lipid can affect a
variety of cellular functions. The reaction of the doxorubicin enzymatic reduction by
a variety of oxidase, reductase, and dehydrogenases genes generates ROS and, thus,
may result in damage to DNA and proteins, triggering apoptosis [39, 40].
The performance of antioxidants in vivo depends on the types of free radicals
formed, where and how these radicals are generated, and what are the doses for
optimal protection. So it is entirely possible for an antioxidant to act as a protector in
any given systems, but it is also possible for it to fail to protect, or even increase
lesions induced in other systems or tissues. Thus, the use of antioxidants in cancer
treatment is controversial.
Ambrosone and colleagues [41] observed that women having breast cancer with
genotypes that result in higher levels of ROS had better survival rates than those
with genotypes associated with lower generations of ROS. Such results indicate that
an increased oxidative stress may increase the effects of chemotherapy and/or
radiotherapy, resulting in improved treatment efficacy and, thus, better survival
rates. The overexpression of SOD is associated with better survival rates for patients
diagnosed with colorectal cancer [42].
Cleveland and Kastan suggest that a promising treatment for some types of cancer
could happen by increasing ROS levels and inhibiting SOD levels [43, 44]. Other
authors report that the overexpression of SOD has presented resistance to
doxorubicin [43, 44], but not 5-fluorouracil in gastric cells [41]. Another study on
breast cancer cells showed an increased resistance to Adriamycin with the
intracellular level of glutathione (GSH) [45].
The protective effect of Ut on DNA was observed during the breast cancer cycles of
treatment, by the Comet test analysis.
Doxorubicin has its own mechanism of action related to its binding to the DNA and
the inhibition of nucleic acid synthesis. Studies have shown that aqueous Ut extracts
present DNA repairing activities [15]. Mammone et al. (2006) showed the ability to
modulate Uncaria tomentosa and repair of DNA in human skin and organ cultures
[46].
In the present study, the results of comet test suggest that Ut had a protective effect
of the DNA during the treatment cycles. However, it is necessary for other studies to
confirm these effects.
5. Conclusions
Uncaria tomentosa, used at dose of 300mg dry extract per day, is effective in the
recovery from neutropenia induced by chemotherapy in women diagnosed with
Invasive Ductal CarcinomaStage II. It is also able to restore cellular DNA. Thus,
it is a safe and effective adjuvant treatment in reducing adverse chemotherapy
effects.
Conflict of Interests
All authors deny any conflict of interests.
Acknowledgments
The authors would like to thank the physicians and patients at the Servio de
Hematologia/Oncologia (Department of Hematology/Oncology Services) of the
Santa Maria University Hospital Brazil and Herbarium Botanic Laboratory. This
work was supported by governmental funds: CNPq and CAPES.
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T
Uncaria tomentosa - tratamiento adyuvante para el cncer de mama: Estudio Clnico
1 Departamento de Qumica de la Universidad Federal de Santa Maria , Avenida
Roraima , Predio18 , 97105-900 Santa Maria , RS, Brasil
Hospital de 2Santa Maria University , la Universidad Federal de Santa Maria ,
Avenida Roraima , Predio18 , 97105-900 Santa Maria , RS, Brasil
3 Departamento de Biologa de la Universidad Federal de Santa Maria , Avenida
4Departamento de Farmacia Industrial, Universidad Federal de Santa Maria ,
Avenida Roraima , Predio18 , 97105-900 Santa Maria , RS, Brasil
5Department de Morfologa de la Universidad Federal de Santa Maria , Avenida
Recibido el 15 de noviembre de 2011; Revisado 26 de abril 2012 ; Aceptado 27 de
abril 2012
Editor Acadmico: Alyson Huntley
Copyright 2012 Maria do Carmo Santos Arajo et al. Este es un artculo de
acceso abierto distribuido bajo theCreative Commons Attribution License , que
permite el uso irrestricto, la distribucin y reproduccin en cualquier medio, siempre
que la obra original es debidamente citados.
abstracto
El cncer de mama es la neoplasia ms frecuente entre las mujeres de todo el
mundo.Algunos de los tratamientos recomendados involucran quimioterapia cuyos
efectos txicos incluyen leucopenia y neutropenia. Este estudio evalu la eficacia de
Uncaria tomentosa( Ut ) para reducir los efectos adversos de la quimioterapia a
travs de un ensayo clnico aleatorizado. Los pacientes con carcinoma ductal
invasivo en etapa II, que se someti a un rgimen de tratamiento conocido como
FAC ( fluorouracilo , doxorrubicina , ciclofosfamida ) , se dividieron en dos grupos:
el utca recibi quimioterapia ms 300 mg de extracto seco Ut por da y el grupo que
slo recibi Ca quimioterapia y servido como el experimento de control . Tomaron
muestras de sangre antes de cada uno de los seis ciclos de quimioterapia y los
recuentos sanguneos, los parmetros inmunolgicos , enzimas antioxidantes, y se
analiz el estrs oxidativo. Uncaria tomentosa redujo la neutropenia causada por la
quimioterapia y tambin fue capaz de restaurar el dao del ADN celular. Llegamos a
la conclusin de que Ut es un tratamiento adyuvante eficaz para el cncer
de mama.
1 . Introduccin
El cncer de mama es la neoplasia ms frecuente entre las mujeres de todo el mundo
, tanto en trminos de incidencia y mortalidad. La enfermedad es ms comn en los
pases desarrollados, con su incidencia ms alta se observa en el Reino Unido,
Australia , EE.UU. y Canad. De los tumores invasivos , el carcinoma ductal y sus
variantes representan el 80 % de los casos [1] y la proporcin de mujeres
contumores en estadios clnicos I y II aumentaron 41-65 % en la ltima dcada [ 1 ,
2 ] . Alrededor del 70 % de los cnceres de mama expresan receptores hormonales
de estrgenos y / o receptores de progesterona [ 3 ] . Estos marcadores junto con el
receptor HER-2 (c- erbB2 ) proporcionan informacin sobre el tumor y cmo podra
responder a diferentes tratamientos [ 4 ] .
La quimioterapia es uno de los tratamientos recomendados para el cncer de mama ,
que puede ser una terapia nica o combinacin con mltiples frmacos . Los
medicamentos de quimioterapia tienen ndices teraputicos muy estrechos en
trminos de efectos txicos no selectivos sobre los tejidos normales , con la
neutropenia siendo la reaccin adversa ms frecuentemente observada , lo que
aumenta el riesgo de infecciones [ 5 ] .
Las intervenciones farmacolgicas que reducen o previenen los efectos adversos
pueden tener un impacto sustancial en el tratamiento del cncer . Segn la
Organizacin Mundial de la Salud ( OMS) , el 80 % de la poblacin utiliza las
plantas medicinales como procedimientos alternativos o complementarios para el
tratamiento de sus enfermedades [ 6 ] .
Los estudios han reportado el uso de hierbas medicinales en los
pacientes de cncer para minimizar los efectos de
chemotherapy.Uncaria tomentosa ( Ua de Gato Utor ) es una
hierba medicinal que se ha utilizado en el tratamiento de
diferentes enfermedades como el cncer . Los pacientes que usan la ua
de gato , junto con las terapias tradicionales contra el cncer , como la quimioterapia
y la radiacin, informaron menos efectos adversos a esas terapias [ 7 ] . Uthelps en
la restauracin de ADN celular , la prevencin de las mutaciones y daos celulares
causados por los medicamentos de quimioterapia [ 8 ] . Se modula la actividad en el
sistema inmune , tales como la proliferacin de linfocitos T y B normales [ 9 ] ,
tambin la modulacin de ciertas citoquinas , incluyendo IL - 1 e IL - 6 , TNF - [ 10
] . Adems , tiene propiedades antioxidantes [ 11 ] . Sus efectos directos
myelostimulating , a travs mielopoyesis estimulacin y factores estimulantes de
colonias (G -CSF) [ 8 , 12 ] , parecen ser una opcin beneficiosa para minimizar los
riesgos asociados con neutropenia.
Numerosos informes presentan una comprensin terica de los mecanismos de
accin Ut , pero ninguno de estos estudios consistieron en ensayos clnicos. As, los
objetivos de este estudio se encuentran en este contexto , que consisti en un ensayo
clnico utilizando tabletas Uncaria tomentosa de herbario , como tratamiento
adyuvante para el cncer de mama.
2 . Mtodos
2.1 . Diseo y pacientes
Se realiz un estudio de intervencin aleatorizado . Se llev a cabo con 40 pacientes
que haban sido sometidos a una reseccin completa del cncer de mama , que se
diagnostic histolgicamente como carcinoma ductal invasivo en etapa II [ 2], y que
se va a comenzar la quimioterapia adyuvante con el esquema basado en
doxorubicina durante seis ciclos, en el Santa hospital Universitario Maria , Brasil.
Los pacientes se dividieron al azar en dos grupos : el grupo Caut , que se trat con
seis ciclos de quimioterapia + Ut y el grupo de cncer ( Ca ) , que slo recibi seis
ciclos de quimioterapia , de acuerdo con la fecha de tratamiento fue comenzado , de
la siguiente manera : la primera pacientes que aceptaron participar en el estudio se
incluy en el grupo Caut , el segundo , en el grupo Ca, y, as , sucesivamente, hasta
el final.
Para fueron invitados el grupo de control a participar las mujeres sanas , clasificados
por ensayo clnico, con la misma edad de los pacientes y que no recibieron ningn
medicamento en los ltimos 30 das o tiene una enfermedad crnica.
Los pacientes fueron parte del estudio durante 6 ciclos de quimioterapia, de 21 das
cada uno. Dosificacin de la medicacin en el grupo Caut fue el siguiente: FAC (
fluorouracilo , doxorrubicina y ciclofosfamida ) y 3 tabletas de Ut ( Unha de Gato
Herbario ) , todos los das , desde el da 2 al da 21 la dosis de Ut fue similar a la
utilizada en . estudios previos , con 250 a 350 mg C -MED- 100 , en Ut acuosa
extractos [ 13 ] .
El clculo para estimar el tamao de la muestra requerida para el ensayo clnico
aleatorizado se llev a cabo de acuerdo con Greenberg et al. [ 14 ] , con nivel
constante de significacin ( ) de 5 % , y el poder estadstico de 90 % ( 10 % ) ,
utilizando como referencia los estudios de Sheng et al . [ 15 ] .
El Comit de tica Humana del Hospital Universitario Santa Mara , Brasil, aprob
el presente estudio y se obtuvo el consentimiento de todos los participantes ( nmero
de protocolo : 0169.0.0242.000-07 . ) . Se invit a todos los sujetos a participar y se
les inform en detalle sobre el diseo de este estudio a travs de una Declaracin de
consentimiento firmado por el investigador y los participantes . Se les inform que
podan ser seleccionados al azar para el grupo Ca o utca .
2.2 . Materiales
Cada comprimido de Unha de Gato Herbario contena 100 mg de extracto de
Uncaria tomentosa seco. Los materiales biolgicos utilizados en las pastillas se
derivaron de las plantas en su hbitat natural. El extracto de Uncaria tomentosa fue
preparado por Ultra- turrax extraccin ( Biotron , Kinematica AG) de la corteza
terrestre ( Centroflora ) utilizando etanol al 70 % ( Dipalcool ) . El anlisis por
HPLC del extracto seco Ut presenta 2,57 % de alcaloides de oxindol pentacclicos (
POAs ) de contenido, que se calculan con referencia a las curvas de calibracin
externos de mitraphylline . El anlisis de extracto mostr ausencia de alcaloides de
oxindol tetracclicos en la muestra , lo que permite su uso para fines teraputicos y
de investigacin de acuerdo con la Farmacopea de EE.UU. .
2.3 . Recogida de muestras
La sangre se recogi en citrato, EDTA, heparina tubos Vacutainer , sin ningn tipo
de anticoagulantes, antes de la quimioterapia y despus de cada uno de los 6 ciclos.
2.4 . Parmetros bioqumicos
Un sistema COBAS INTEGRA se utiliz para la determinacin cuantitativa de los
componentes qumicos de la sangre , y los datos fueron adquiridos a travs de un
aparato 400 Plus COBAS INTEGRA ( EE.UU. ) .
2.5 .hemogramas
Las muestras de sangre se analizaron usando un aparato de Pentra ( Francia ) . Los
valores ms bajos fueron confirmadas por la observacin de diapositivas, utilizando
una mayo - Grnwald Giemsa Stain y microscopa ptica.
2.6 . CD3 + , CD4 + , y CD8 + Clulas
Las muestras se recogieron en EDTA y los anlisis se realizaron usando una de tres
colores de fluorescencia - clasificador de clulas activadas ( FACSCalibur , Becton
Dickinson Biosciences , Estados Unidos ) y un software de Multiset ( Becton
Dickinson ) . Conjugado con FITC anti-CD4 , conjugado con PE anti-CD8 , y PerCP
- conjugado anti - CD3 se utilizaron . Subpoblaciones inmunes se mide como un
porcentaje del nmero total de clulas CD3 + .
2.7 . La interleucina 6 ( IL - 6 )
Ensayos de ELISA de IL - 6 se llevaron a cabo de acuerdo con un mtodo publicado
previamente [ 16 ] , a temperatura ambiente en placas de microtitulacin de 96
pocillos ( placa Nunc - Immuno MaxiSorp ) y las densidades pticas ( DO) a 490
nm, que se determinaron usando un lector de microplacas (Thermo Scientific
Multiskan FC , Vantaa , Finlandia).
2.8 . Single Cell electroforesis en gel ( ensayo cometa )
El ensayo cometa alcalino se llev a cabo como se ha descrito por Singh et al .[ 17 ]
de conformidad con las directrices generales para el uso de la prueba del cometa [ 18
, 19 ] . Los linfocitos se suspendieron en 0,7 % de agarosa de bajo punto de fusin y
solucin salina tamponada con fosfato ( PBS) a 37 C y se colocan en portaobjetos
de microscopio con una capa de 1 % de agarosa . Los portaobjetos se sumergieron
en solucin de lisis a 4 C durante 1 h y seguido por electroforesis a 25 V , 300 mA
, durante 40 min a temperatura constante . Las diapositivas fueron manchados de
plata, segn lo descrito por Nadin et al. [ 20 ] . Todas las etapas , desde la recogida
de muestras a electroforesis , se llevaron a cabo bajo luz amarilla para minimizar la
posibilidad de daos en el ADN celular. Se seleccionaron y analizaron Un centenar
de clulas ( 50 clulas de cada uno de los dos diapositivas repetir ) . Las clulas se
puntuaron visualmente de acuerdo a la longitud de la cola y recibieron puntajes de 0
( sin migracin ) a 4 ( la migracin mxima) . Por lo tanto , el ndice de dao para
las clulas vari de 0 ( todas las clulas sin migracin que representa un ndice de
dao de 0 % ) a 400 ( todas las clulas con la migracin mxima , lo que representa
un ndice de dao de 100 % ) . Los portaobjetos se analizaron bajo condiciones
ciegas por al menos dos individuos diferentes [ 21 ] .
2.9 . Carbonilacin de protenas sricas
La carbonilacin de protenas de suero se determin por el mtodo de una Levine
modificado [ 22 ] . La absorbancia del sobrenadante a 370 nm se midi utilizando un
espectrofotmetro . Se calcul el contenido de carbonilo usando mM - 1 cm - 1
como el coeficiente de extincin molar , y los resultados se expresaron como
nanomoles de grupos carbonilo por miligramo de protena .
2.10 . Determinacin de la peroxidacin lipdica
La peroxidacin lipdica se estim mediante la medicin de los niveles de TBARS
en muestras de plasma de acuerdo con un mtodo modificado de Jentzsch et al .[ 23
] . La concentracin de malondialdehdo ( MDA ) se determin midiendo la
absorbancia a 532 nm usando un espectrofotmetro . Los resultados se expresaron
como nanomoles de MDA por mililitro de plasma .
2.11 . La catalasa (CAT ) y superxido dismutasa (SOD) Actividades
La actividad de CAT se determin de acuerdo con un mtodo modificado de Nelson
y Kiesow [ 24 ] . Se midi el cambio en la absorbancia a 240 nm durante 2 min . La
actividad de CAT se calcul utilizando el coeficiente de extincin molar ( 0,046 mM
- 1 cm - 1 ) , y los resultados se expresaron como picomoles de CAT por miligramo
de protena .
La actividad de SOD se determin sobre la base de la inhibicin de la reaccin de
radicales superxido con adrenalina como se describe por McCord y Fridovich [ 25 ]
. La actividad de SOD se determina mediante la medicin de la tasa de formacin de
adrenocromo , observada a 480 nm , en un medio que contiene glicina-NaOH ( 50
mM, pH 10 ) y adrenalina ( 1 mM ) .
2.12 .estadstica
Los resultados se expresan como desviacin . El anlisis estadstico se realiz con
Graph -Pad Prism 5.0 ( GraphPad Prism 5.0 Software Inc., EE.UU. ) utilizando la
prueba t de Student. se considera que representa una diferencia significativa en todas
las pruebas .
3 . Resultados
Todos los pacientes ( 40 ) incluidos en el estudio tenan cncer de mama , el
carcinoma ductal invasivo - Etapas II A o II B , de acuerdo con el Comit
Americano Conjunto sobre el Cncer ( AJCC) y los sistemas de clasificacin de la
Sociedad Americana del Cncer (ACS ) [ 2 ] .
Las caractersticas generales de los pacientes y los controles que participaron en el
estudio se describen en la Tabla 1.

Tabla 1 : caractersticas clnicas de los pacientes. Se representa la edad , ndice de
masa corporal ( IMC ) , niveles de colesterol total , los receptores de estrgenos ( ER
) , y los receptores de progesterona (PR) , as como el estado de HER- 2 en
diferentes grupos .
Para evaluar la eficacia de Ut como tratamiento adyuvante para el cncer de mama ,
se utilizaron los parmetros hematolgicos y se analizaron (tabla 2 ) . En el da cero,
los resultados de los parmetros hematolgicos analizados en el hemograma no
difirieron significativamente entre el control, el Ca, y los grupos utca . Se observ
una mayor reduccin en los glbulos blancos (GB ) y los recuentos de neutrfilos en
el grupo de Ca a lo largo del tratamiento , a diferencia del grupo tca , que se
mantuvo estrechamente los valores de referencia , obtenida en el grupo control (
Figura 1 ) . Teniendo en cuenta el nmero de linfocitos , una diferencia significativa
entre el grupo control y los grupos de pacientes con cncer de mama , ya sea tratado
o no con Ut en los ciclos de quimioterapia , se observ. ( ) . Nmero de monocitos
en pacientes con cncer de mama ( tratados y no tratados con Ut ) a los 5-6 ciclos de
quimioterapia fueron mayores que las del grupo de control , pero en el grupo tca ,
este aumento fue ms fuerte ( Tabla 2 ) .

Tabla 2 : Los leucocitos , neutrfilos , linfocitos, monocitos y los niveles en
pacientes con cncer de mama antes del tratamiento y despus de 6 ciclos de
quimioterapia sin Uncaria tomentosasupply (grupo Ca) o recibir 300 mg / da de
Uncaria tomentosa (grupo utca ) .

Figura 1 : Valores de los granulocitos neutrfilos en pacientes con cncer de mama
sometidas a quimioterapia con ( utca ) y sin ( Ca) la complementacin con los
valores de referencia (control) Uncaria tomentosaand . Los datos se expresan como
desviacin .
Para evaluar la respuesta inmune de pacientes con cncer de mama , las clulas T
CD4 + , clulas T CD8 + (recuento absoluto y ratio) y se analizaron los niveles de
IL - 6 . Durante los ciclos de tratamiento de quimioterapia , se observ ninguna
diferencia significativa entre los grupos . No hubo diferencias entre los grupos para
ninguno de los parmetros analizados (Tabla 3 ) .

Tabla 3 : estado inmune de los pacientes con cncer de mama antes del tratamiento y
despus de 6 ciclos de quimioterapia sin suministro de Uncaria tomentosa ( grupo
Ca ) o recibiendo 300 mg / da de Uncaria tomentosa ( grupo tca ) .
No existe correlacin entre la IL- 6 , se encontr CD4 + T/CD8 relacin T + y la
edad , ndice de masa corporal y el estado de los receptores hormonales (datos no
presentados).
Defensas antioxidantes se analizaron por la actividad de la superxido dismutasa (
SOD ) y catalasa ( CAT ) en comparacin con los ciclos de tratamiento cero y seis,
as como entre la tca y los grupos Ca . No hubo diferencias estadsticamente
significativas entre los grupos . Se observ un aumento en la enzima SOD en
comparacin con los ciclos de tratamiento cero y seis para el grupo suplementado
con Ut , pero que no se observ diferencia entre los grupos ( tca = 11,53 U / mg
protena , Ca = 11,43 U / mg protena ) o por lo al final del tratamiento ( 17,32
protena U / mg , 11.74 U / mg protena ) . Peroxidacin lipdica tambin se estim
mediante la escala de TBARS y la carbonilacin de protenas de suero , pero no
hubo diferencia entre los grupos ( tca y Ca) .
El efecto protector de la quimioterapia para extraer Ut se evalu por el ensayo
cometa . En el inicio del tratamiento ( ciclo de cero) , el grupo Ca y grupo tca no
mostr ninguna diferencia significativa en el ndice de ensayo de cometa . Sin
embargo , en el sexto ciclo ( final del tratamiento ) , se observ una disminucin
significativa en la prueba del ndice en el grupo tca , en comparacin con el grupo
de Ca ( ) Figura 2 .

Figura 2: Ensayo de ndice de clulas sanguneas en pacientes con cncer de mama
tratados y no tratados con Ut . * Representa una diferencia significativa entre los
grupos ( ) . ** Representa una diferencia significativa entre el grupo utca ( ciclo 0 )
y el grupo utca ( ciclo 5-6) (t de Student ) .
4 . Discusin
Uncaria tomentosa permite la estimulacin del sistema inmune , aumento de la
resistencia a las enfermedades cuando el cuerpo est inmunodeprimido debido al
estrs, malnutricin , o debido al efecto de algunos medicamentos .
Muchas de las medicinas a base de hierbas se utilizan para diversos fines, en
diversas combinaciones (junto con alopticos y homeopticos , medicamentos, etc )
basado en evidencias histricas o personales generalmente no estn asociados con
ningn efecto adverso [26]. Por lo tanto , este estudio , a travs de un ensayo clnico
aleatorizado , evalu la eficacia de Ut como terapia complementaria a la
quimioterapia.
El efecto citotxico de los agentes quimioteraputicos no es selectivo para clulas
neoplsicas , siendo tambin perjudiciales para otras clulas del cuerpo . Supresin
hematopoyticas es la complicacin ms importante que limita la dosis de dichos
agentes citostticos ; neutropenia y la trombocitopenia son las ms frecuentes [ 1 ] .
El tratamiento debe interrumpirse cuando el recuento de neutrfilos es inferior a 500
clulas/mm3 [27]. Por lo tanto , el xito del proceso de tratamiento depende del
contenido de los neutrfilos . La prevencin de la neutropenia inducida por la
quimioterapia debe ser considerada una prioridad clnica [ 28 ] . Una vez que se sabe
que la neutropenia predispone a infecciones graves, a menudo resulta en retrasos en
los ciclos de tratamiento y reduccin de la dosis .
El tratamiento con una dosis diaria de extracto de Ut seco 300 mg fue efectiva en la
reduccin del efecto de la quimioterapia principal, que es la neutropenia . Los
efectos de la quimioterapia en clulas de la sangre tienden a ser ms pronunciada
durante el tratamiento . Sin embargo , nuestros resultados muestran que en el ciclo
de seis , que se corresponde con el final de la quimioterapia , las diferencias en los
leucocitos y neutrfilos recuentos eran an ms significativo , como el grupo que fue
suplementado con Ut present valores dos veces ms altos de los neutrfilos en
comparacin con la grupo de cncer ( sin suplemento ) . En el grupo sin
suplementacin, 67.89 % de los pacientes tenan neutropenia. De manera similar ,
hubo un aumento en los monocitos activados , como los precursores activados eran
comunes a ambas cepas .
Nuestros resultados han sido corroborados por otros estudios que ya haban
demostrado que el extracto de Ut tiene un efecto estimulante sobre el crecimiento y
la diferencia la CFU- GM de mdula sea de ratn y el bazo , utilizando el modelo
de la listeriosis [ 29 ] . El aumento del nmero de leucocitos tambin se detectaron
utilizando extracto acuoso Ut durante seis semanas consecutivas en voluntarios [ 13
] . La recuperacin de los leucocitos tambin se observ en los ratones usando un
modelo para la leucopenia inducida por quimioterapia ( doxorrubicina ) usando
Granulocitos Factor Estimulante de Colonias ( Neupogen ) como control positivo [
15 ] .
Nuestro grupo confirm estos resultados utilizando un modelo para la neutropenia
inducida por la ifosfamida en ratones , lo que caus una neutropenia grave . Los
bioensayos mostraron que el tratamiento con Ut aument significativamente los
recuentos de neutrfilos , y una potencia de 85,2 % se calcul en relacin con
filgrastim ( rhG -CSF ) a las dosis correspondientes a prueba ( 5 y 15 mg / da de Ut
, y 3 y 9 mcg / da filgrastim , resp . ) [ 13 ] . A travs de ensayos in vitro de clulas
precursoras hematopoyticas madre humanas ( hHSPCs ) obtenidos a partir de
sangre de cordn umbilical (SCU ) , llegamos a la conclusin de que este efecto
ocurri debido a la proliferacin de unidades formadoras de granulocitos y
macrfagos ( CFU- GM) [ 13 ] .
En este estudio, no se observaron diferencias en el recuento de linfocitos entre los
grupos , ya sea complementado o no con Ut , durante los ciclos de quimioterapia ;
Sin embargo , sus recuentos presentan disminucin debido a la quimioterapia en
comparacin con el grupo de control . Estas diferencias no se observaron en las
subpoblaciones CD4 + y CD8 + .
Este artculo reporta los efectos de diferentes extractos de Uncaria tomentosa . El
extracto acuoso que tiene la mayor concentracin de cido qunico y bajas
concentraciones de alcaloides de oxindol , por estar relacionados con propiedades
inmunomoduladoras es mediada por citoquinas tales como TNF - [ 30 ] . Los
estudios clnicos utilizando 20 mg / da de extracto de Uncaria tomentosa durante 2 a
5 meses en pacientes con VIH , que no recibi otro tratamiento , mostraron un
aumento en el total de linfocitos perifricos sin cambios significativos en la
proporcin de clulas CD4 + y CD8 + [ 31 ] . Voluntarios sanos que recibieron 350
mg de extracto acuoso Ut durante 8 semanas mostraron leucocitosis , con una
tendencia a una mayor proliferacin de los linfocitos [ 15 ] . En un modelo animal ,
el uso de extracto acuoso , que tiene la mayor concentracin de cido qunico y bajas
concentraciones de alcaloides de oxindol , un aumento en los linfocitos tambin se
observ [ 15 , 32 ] .
Adems, los extractos alcohlicos y / o alcaloides oxindol pentacclicos tienen
mayores efectos mieloproliferativos [ 33 , 34]. Otros estudios han demostrado que el
aumento en los recuentos de linfocitos ocurre debido al aumento de las tasas de
supervivencia en lugar de la proliferacin [ 32 ] .
As, los cambios observados en los linfocitos estn asociados con los componentes
qumicamente activos definidos como steres de cido qunico y bioactivos in vivo ,
presente como cido qunico en el extracto acuoso utilizado por los autores. En
nuestro estudio, se utiliz extracto hidroalcohlico .
Los altos niveles circulantes de IL - 6 se asocian con tasas de supervivencia peor
para los pacientes con cncer de mama metastsico , que se relaciona con el grado
de la enfermedad [ 30 ] .
Los pacientes que formaban nuestra muestra no presentaron una progresin negativa
durante los ciclos de tratamiento , es decir , no hubo aparicin de recadas o aumento
de la medida de la lesin , lo que podra conducir a un aumento de la IL - 6 .
Diferentes extractos de Uncaria tomentosa se ensayaron in vitro con el fin de
determinar su actividad antioxidante . Extractos acuosos y alcohlicos previenen la
produccin de productos de reaccin con cido tiobarbitrico ( TBARS ) y , por lo
tanto , daar la membrana citoplasmtica ( lpidos ) y de ADN por el nonformation
de radicales libres [ 35 , 36 ] entre los parmetros evaluados de estrs oxidativo ,
tales como SOD , CAT, TBARS , y las protenas carboniladas .
Las mujeres con cncer de mama presentan un aumento en las concentraciones en
sangre de sustancias oxidadas , tales como productos derivados de la peroxidacin
de lpidos , protenas , ADN y [ 32 , 33 ] .
Las nicas diferencias observadas fueron en la enzima SOD entre los grupos , ya sea
con o sin suplementacin con Ut . Tambin se encontraron Estos resultados en un
modelo animal , en el que se percibe un aumento en la actividad de esta enzima [ 10
] . En un estudio en mujeres con cncer de mama , la actividad SOD mostr un
aumento significativo , independientemente de la etapa clnica y el estado
menopusico [ 31 ] . Hay pruebas de que el estado de estrs oxidativo es mayor que
el mayor grado de la etapa de la enfermedad est [ 37 , 38 ] .
De manera similar a los resultados encontrados en la IL- 6 , el hecho de que todos
los pacientes en el estudio tenan cncer en estadio II puede explicar los resultados
encontrados .
La capacidad de doxorrubicina de obligar a s mismo a la de lpidos de la membrana
celular puede afectar a una variedad de funciones celulares . La reaccin de la
reduccin enzimtica doxorrubicina por una variedad de oxidasa , reductasa , y
deshidrogenasas genes genera ROS y , por lo tanto , puede resultar en el dao al
ADN y protenas , desencadenando la apoptosis [ 39 , 40 ] .
El rendimiento de los antioxidantes in vivo depende de los tipos de radicales libres
que se forman , dnde y cmo estos radicales son generados , y cules son las dosis
para una proteccin ptima. Por lo tanto, es completamente posible que un
antioxidante para actuar como un protector en cualquier sistema dado , pero tambin
es posible para que no protegen , o incluso aumentar las lesiones inducidas en otros
sistemas o tejidos . Por lo tanto , el uso de antioxidantes en el tratamiento del cncer
es controvertido .
Ambrosone y colegas [ 41 ] observado que las mujeres que tienen cncer de mama
con genotipos que resultan en niveles ms altos de ROS tenan mejores tasas de
supervivencia que aquellos con genotipos asociados con las generaciones inferiores
de ROS . Tales resultados indican que un aumento del estrs oxidativo puede
aumentar los efectos de la quimioterapia y / o radioterapia , lo que resulta en una
mejora de la eficacia del tratamiento y , por lo tanto , mejores tasas de supervivencia
. La sobreexpresin de SOD se asocia con mejores tasas de supervivencia para los
pacientes diagnosticados con cncer colorrectal [ 42 ] .
Cleveland y Kastan sugieren que un tratamiento prometedor para algunos tipos de
cncer puede ocurrir mediante el aumento de los niveles de ROS y la inhibicin de
los niveles de SOD [ 43 , 44 ] . Otros autores informan de que la sobreexpresin de
SOD ha presentado resistencia a la doxorrubicina [ 43 , 44 ] , pero no de 5 -
fluorouracilo en las clulas gstricas [ 41 ] . Otro estudio sobre las clulas de cncer
de mama mostr un aumento de la resistencia a adriamicina con el nivel intracelular
de glutatin ( GSH ) [ 45 ] .
Se observ el efecto protector de UT el ADN durante los ciclos de tratamiento de
cncer de mama , por el anlisis de la prueba del cometa .
La doxorrubicina tiene su propio mecanismo de accin relacionado con su unin al
ADN y la inhibicin de la sntesis de cidos nucleicos . Los estudios han demostrado
que Ut acuosa extrae actividades de reparacin del ADN presentes [ 15 ] .
Mammone et al .( 2006 ) demostraron la capacidad de modular la Uncaria tomentosa
y reparacin de ADN en la piel humana y cultivos de rganos [ 46 ] .
En el presente estudio , los resultados de ensayo del cometa sugerir que Ut tuvo un
efecto protector del ADN durante los ciclos de tratamiento . Sin embargo , es
necesario para otros estudios para confirmar estos efectos .
5 . Conclusiones
Uncaria tomentosa , que se utiliza en dosis de 300 mg de extracto seco por da , es
eficaz en la recuperacin de la inducida por la quimioterapia en mujeres con
diagnstico de carcinoma ductal invasivo - Etapa II neutropenia . Tambin es capaz
de restaurar el ADN celular . Por lo tanto , es un tratamiento adyuvante seguro y
eficaz en la reduccin de los efectos adversos de la quimioterapia .
Conflicto de Intereses
Todos los autores niegan cualquier conflicto de intereses.
Agradecimientos
Los autores desean agradecer a los mdicos y los pacientes en el Servio de
Hematologia / Oncologia ( Departamento de Servicios de Hematologa / Oncologa)
de la Universidad Santa Mara del Hospital Brasil y Laboratorio Botnico Herbario .
Este trabajo fue apoyado por fondos gubernamentales: CNPq y CAPES .

http://www.thefreelibrary.com/Inhibitory+mechanisms+of+two+Uncaria+tomentosa+extracts+aff
ecting+the...-a0265381691
Inhibitory mechanisms of two Uncaria
tomentosa extracts affecting the Wnt-signaling
pathway.
ARTICLE INFO

Keywords: Uncaria tomentosa Cat's claw Una de gato Wnt-signaling pathway cMyc Beta-Catenin
Cancer cells

ABSTRACT

Uncaria tomentosa ("una de gato"; "cat's claw"), a woody vine native to the Amazon rainforest, is
commonly used in South American traditional medicine to treat a broad spectrum of diseases.
Although recent studies have reported anti-inflammatory and anti-proliferative properties of
different alkaloids extracted from this plant, the underlying molecular mechanisms of these effects
have not been elucidated yet. Our study investigates the inhibitory mechanisms of Uncaria
tomentosa extracts on the Wnt-signaling pathway, a central regulator of development and tissue
homoeostasis. A modified cell-based luciferase assay for screening inhibitors of the Wnt-pathway
was used for analysis. Three cancer cell lines displaying different levels of aberrant Wnt-signaling
activity were transfected with Wnt-signaling responsive Tcf-reporter plasmids and treated with
increasing concentrations of two Uncaria tomentosa bark extracts. Wnt-signaling activity was
assessed by luciferase activity and by expression of Wnt-responsive target genes. We show that
both, an aqueous and an alkaloid-enriched extract specifically inhibit Wnt-signaling activity in
HeLa, HCT116 and SW480 cancer cells resulting in reduced expression of the Wnt-target gene: c-
Myc. The alkaloid-enriched extract (B/[S.sub.rt]) was found to be more effective than the aqueous
extract (B/[W.sub.37]). The strongest effect was observed in SW480 cells, displaying the highest
endogenous Wnt-signaling activity. Oownregulation of Wnt-signaling by a dominant negative-TCF-4
variant in non-cancer cells rendered the cells insensitive towards treatment with B/[S.sub.rt].
B/[S.sub.rt] was less toxic in non-cancer cells than in cancer cells. Our data suggest that the broad
spectrum of pharmacological action of Uncaria tomentosa involves inhibition of the Wnt-signaling
pathway, downstream of beta-Catenin activity.

[c] 2010 Elsevier GmbH. All rights reserved.

Introduction

The medicinal vine Uncaria tomentosa (Rubiacea), native to the Amazon rainforest and commonly
known as "una de gato" or "cat's claw" has been traditionally used by indigenous tribes to treat a
broad spectrum of mental and physical disorders (Keplinger et al. 1999). Today, it is commonly
used in tropical American folk medicine to treat viral infections, arthritis, chronic degenerative
diseases, gastric ulcers and cancer. Consequently, it has evoked increasing scientific and
commercial interest and is widely promoted as an alternative treatment for these ailments
(Heitzman et al. 2005).

Despite a growing number of reports describing the clinical and biological effects of U. tomentosa
extracts, its pharmacological effectiveness and molecular targets are largely unknown. The most
common pharmaceutical forms of U. tomentosa are crude water-soluble or ethanol-soluble extracts
derived from its bark or roots for oral consumption as infusions (Keplinger et al. 1999). Active
components identified in these extracts include different oxindolic alkaloids,indole alkaloids,
glycosides (pentacyclic triterpenes with a variety of derivatives such as ursolic acid, quinovic acid
glycosides, sterols and procianidins) and tannins the chemical composition of which can vary
depending on their geographical origin and seasonal harvesting (Heitzman et al. 2005). Thus, the
diverse pharmacological properties of U. tornentosa reported in the literature might be ascribed to
different types and combinations of these compounds.

Most pharmacological in vivo-studies initially investigated how U. tomentosa extracts affect anti-
inflammatory, immunomodulatory and DNA-repair mechanisms (Akesson et al. 2003; Keplinger et
al. 1999; Sheng et al. 2000, 2001; Spelman et al. 2006). In addition, several studies suggest an
anti-tumorigenic activity of both, water-soluble and alkaloid-enriched U. Comentosa extracts:
cytotoxic, antiproliferative and pro-apoptotic effects were reported on different tumor cell lines
(Cheng et al. 2007; Garcia Gimenez et al. 2009; Pilarski et al. 2010; Garcia Prado et al. 2007;
Gonzales and Valerio 2006; Pilarski et al. 2007). Moreover, U. tomentosa preparations with different
oxindole alkaloid compositions and hydroalcoholic extracts, respectively were recently shown to
reduce tumor growth in in vivo solid tumor animal models (Pilarski etal. 2010; Dreifuss etal. 2010).
The mechanism of this antitumor-activity and possible effects on cancer-related signaling pathways
in tumor cells however, have not been yet investigated.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

The broad spectrum of activities claimed for U. tomentosa extracts suggests that it affects a central
regulatory mechanism. One of these major regulators, which are essential for stem cell fate, self-
renewal, development and tissue homoeostasis throughout the animal kingdom, is the Wnt-
signaling pathway (Logan and Nusse 2004; Nusse 2005). Consequently, alterations on this
pathway play a crucial role on human pathophysiology including inflammatory and degenerative
diseases, diabetes, and cancer (Janssens et al. 2006; Jin 2008; Logan and Nusse 2004). The
canonical Wnt-signaling cascade is activated by binding of secreted Wnt glycoproteins to Frizzled
(Fz) receptors via a core set of proteins thereby regulating the ability of beta-Catenin to activate the
transcription of T-cell factor (Tcf)-dependent genes. Full activation of Fz receptors requires
interaction with LRP (low density lipoprotein receptor-related protein) co-receptors. In the absence
of Wnts, most of beta-Catenin is attached to the plasma membrane, where it associates with E-
cadherin at adherence junctions. Newly synthesized beta-Catenin is constitutively targeted for
degradation by a multi-protein complex. Upon activation of this pathway by specific Wnts, beta-
Catenin is released from this complex and transferred to the nucleus where it associates with
transcription factors of the TCF/LEF family to activate target genes that regulate cell proliferation,
differentiation and genes involved in tumorigenesis (see Wnt homepage for list;
http://www.stanford.edu/~rnusse/wntwindow.html). In colorectal epithelium, aberrant activation of
Wnt-signaling by mutations is considered to be the major initiating event of cancer growth. In
addition, this pathway is commonly affected by mutations in an increasing number of other cancer
entities (Behrens and Lustig 2004; Giles et al. 2003; Polakis 2007) as well as in degenerative
diseases (Fuerer et al. 2008; Janssens et al. 2006).

Due to its central regulatory role in human disease, the Wnt-signaling pathway is a major target for
therapeutic intervention and the development of Wnt-specific inhibitors (Barker and Clevers 2006;
Dihlmann and von Knebel 2005; Janssens et al. 2006). This prompted us to investigate whether the
mechanism of action of U. Comentosa extracts involves interference with Wnt-signaling activity.
Since the preparation and composition of extracts affect its biological activities, which is one of the
main problems hindering comparison of different studies, we here used U. tomentosoa extracts
derived from well characterized and standardized preparation procedures (Pilarski et al. 2007,
2010) which result in biologically active composition of the extracts that are characterized by pen-
tacyclic oxindole alkaloids (Wurm et al. 1998).

Materials and methods

Cell lines

The human colorectal cancer cell line HCT116 was obtained from ECACC
(http://www.ecacc.org.uk), the human colorectal cancer cell line SW480, human cervical carcinoma
cell line HeLa and human embryonic kidney epithelial cell line 293T were received from the German
Cancer Research Centre Tumorbank or CLS Cell Lines Services (Heidelberg, Germany). All cell
lines were grown in RPMI 1640 (PAA Laboratories, Germany) supplemented with 10% FCS, 100
U/ml penicillin and l00mg/ml streptomycin using standard conditions.

Uncaria tomentosa extracts

Origin of the plant material, as well as preparation and alkaloid composition of the extracts used for
this study were described earlier (Pilarski et al. 2007, 2010). Briefly, for preparation of the water-
soluble (B/[W.sub.37]) extract, 1 g of bark was extracted in 10 ml of water for 8 h at
37[degrees]C.To obtain the alkaloid-rich bark extract (B/[S.sub.rt]), 10 g of bark was extracted with
50 ml of water (6 h, 37[degrees]C). Next, the sample was centrifuged at 35,000 rpm and an equal
amount of dichloromethane was added to the water supernatant.The organic layer was removed
and evaporated under vacuum at 40'C to dryness. For analysis in cell culture experiments,
B/[W.sub.37] was adjusted in cell culture medium RPMI-1640 to prepare a stock solution of 50
mg/ml, B/Srt was dissolved in DMSO to prepare a stock solution of 50mg/ml.

Transfection and luciferase reporter assays (TOP/FOP assay)

A modified cell-based assay for screening of Wnt-pathway inhibitors was used to determine the
Wnt-signaling activity in cell lines treated with Uncaria tornentosa extracts (Barker and Clevers
2006). Briefly, 2 x 106 cells of each cell line were grown in 6-well plates for 24 h. For reporter
assays, 1 x 106 cells of each cell line were transiently transfected in parallel with either 1.5[micro]g
of a Tcf-reporter construct (TOP-luciferase, which responds to aberrant Wnt-signaling by driving
high levels of luciferase activity) or 1.5[micro]g of a mutated reporter construct (FOP-luciferase,
which is unable to respond to active Wnt-signaling and consequently drives lower levels of
luciferase activity). 0.3[micro]g of a dominant negative pcDNA-dn-hTCF4 was used to biologically
downregulate Wnt-signaling, as indicated in the figures. To normalize the trans-fection efficiency
and non-specific toxicity, 0.5[micro]g of a pRSV-lacZ reporter was included in each sample. All
transfections were performed using Fugene HD transfection reagent (ROCHE Diagnostics,
Mannheim, Germany) according to the manufacturer's instructions. The TCF-responsive reporter
constructs pTOPFLASH and pFOPFLASH were kindly provided by H Clevers; University of Utrecht,
The Netherlands. Control plasmid pRSV-lacZ was obtained fromDKFZ Heidelberg, Germany.
Dominant negative pcDNA-dn-hTCF4 was kindly provided by Bert Vogelstein; The John Hopkins
Oncology Centre, Baltimore, MD, USA. Three hours after transfection, cells were harvested and
plated out into 96-well plates (1 x [10 sup.4] cells/well) in 50[micro]l cell culture medium. Uncaria
tomentosa compounds were added to final concentrations as indicated in the figures. Luciferase
activity was determined 24 h later by adding 50 ulof OneGlo-Luciferase Assay solution (Promega,
Heidelberg, Germany) to each well and incubation for 3min. Lysates were transferred to white
polystyrene plates and luminescence was measured immediately in a 96 well-luminometer
(TECAN-Reader, Genios) for 2 s. For normalization, relative beta-galactosidase activity was
analyzed from corresponding wells in microtiter plates by using an ELISA reader at 570nm
(Dihlmann et al. 2005). Briefly, 15ul of each lysate was diluted in l00ul of phosphate buffer, and 25ul
of a chromogenic substrate (CPRG, ROCHE Diagnostics, Mannheim Germany) was added. Fold
activation was determined by normalizing the light units of the luciferase assays versus values
derived fromabsorbance of the beta-galactosidase assay. Mean values of lysates derived from
FOP-luciferase-transfected, untreated or DMSO-treated cells were set as 1.0.

[FIGURE 3 OMITTED]

WST1-cell viability assay

Cells were plated in microtiter plates at densities of 2x [10.sub.4] cells/well and Uncaria tomentosa
extracts were added at the desired final concentrations (range from 1[micro]g/ml to 300[micro]g
/ml). Cell viability was assessed using 10 u1 of WST-1 cell proliferation reagent (ROCHE
Diagnostics, Mannheim, Germany) after 0, 2, 5, or 23 h of incubation. Cells were grown for 60 min
and absorbance was measured using a microplate (ELISA) reader (absorbance at 450 nm versus
650 nm, according to instructions of the manufacturer).

Immunoblotting

Cells were treated as described in Fig. 2. After 24 h, cells were harvested in lysis buffer
(150[micro]l PBS, 100[micro]M sodium orthovana-date, Proteinase Inhibitor Cocktail (Complete[R],
EDTA-free, ROCHE Diagnostics, Mannheim, Germany)) and lysed by two freeze-thaw cycles.
Protein concentration of lysates was determined by a Protein Assay Kit (BIORAD-Laboratories,
Munchen, Germany), and 20[micro]g of each lysate was applied to SDS-PAGE and blotting using
standard procedures as previously described (Dihlmann et al. 2005). Specific proteins were
detected by incubation with anti-beta-Catenin (1:1000; Transduction Laboratories (BD Biosciences,
Heidelberg, Germany)), anti-cMyc (1:500; clone 2Q.330, Santa Cruz, Heidelberg, Germany) or anti-
Actin (1:5000; clone C4; MP Biomedicals, Heidelberg, Germany) antibodies in blocking buffer (5%
milk/Tris buffered saline/1% Tween20). After incubation of the blots with rabbit-anti-mouse
IgG peroxidase (Dianova, Hamburg, Germany) for 1 h, Western Lightning[R] Plus-ECL, (Perkin
Elmer, Rodgau, Germany) was added as a substrate for visualization by enhanced chemolu-
minescence.

Results

Inhibition of Wnt-signaling activity by water-soluble (B/[W.sub.37]) and alkaloid-enriched (B/Srt)
Uncaria tomentosa bark extracts

We have previously shown that U. tomentosa extracts B/[W.sub.37] and B/Srt reduce proliferation
of various cancer cell lines in different concentrations (Pilarski et al. 2007, 2010). In these studies
however, the growth inhibiting activity of B/[S.sub.rt] and B/[W.sub.37] was very variable. Cancer
cell lines of different origins showed great differences in their sensitivity, and no clear correlation
between U. tomentosa preparation and cancer cell type was observed. This variation may be due to
differently activated cancer signaling pathways. We here applied a cell-based luciferase-reporter
assay to determine the ability of U. tomentosa extracts to inhibit beta-Catenin/Tcf-mediated
transcription, the outcome of canonic Wnt-signaling. This assay is based on the principle that
compounds specifically inhibiting aberrant Wnt-signaling activity will reduce TOP-luciferase activity
without (or to a much lower extent) reducing FOP-luciferase activity (Barker and Clevers 2006).
Thus, specific Wnt-inhibitors will show TOP/FOP-inhibition ratios to be less than 1.0,
whereasunspecific inhibitors will result inTOP/FOP-inhibition ratios greater or equal to 1.0. Fig. 1
shows the effects of a 24-h treatment of HeLa, HCT116 and SW480 cancer cells. In agreement with
previous findings (Dihlmann et al. 2001; Huang et al. 2006), endogenous Wnt-signaling in untreated
controls was moderately activated in HeLa and HCT116 cells (8-10-fold and 12-20-fold,
respectively), whereas it was extensively activated in untreated SW480 colorectal cancer cells (220-
250-fold). Upon treatment with B/W37 extract, Wnt-signaling activity was decreased in a dose-
dependent manner in all cell lines. SW480 and HCT116 cells were more sensitive than HeLa cells,
which is reflected in 50% Wnt-inhibitions (I[C.sub.50(wnt)]) of 190[micro]g/m1, 150[micro]g /m1 and
278 ug/ml, respectively (Fig. 1, left panel). Considering the TOP/FOP ratio, however, the effect was
only specific in HeLa and HCT116 cells at concentrations > 300[micro]g/ml (Table 1). In contrast,
B/Srt displayed a specific inhibitory effect on Wnt-signaling at much lower concentrations (Fig. 1,
right panel). Interestingly, the sensitivity towards B/Srt correlated well with the endogenous Wnt-
signaling activity. SW480 cells displaying the highest Wnt-signaling activity were clearly more
sensitive than HCT116 and HeLa cells (Fig. 1 and Table 1) with an I[C.sub.50(wnt)] of 29[micro]g
/ml compared to 60[micro]g /ml and 38[micro]g /ml, respectively. This finding further argues for a
specific interference of the B/Srt extract with the pathway resulting in down-regulation of beta-
Catenin/Tcf-mediated transcription.
Table 1

TOP/FOP-inhibition ratios of cancer cell lines treated with two
different extracts of Uncaria tomentosa.

Extract Cell 0/DMSO 1 10 50 100 200
line [mu] [mu] [mu] [mu] [mu]
g/ml g/ml g/ml g/ml g/ml

B/[W sub.37] HeLa 1.0 - 0.9 1.0 0.8 1.0
HCT116 1.0 - 1.2 1.1 0.9 0.8
SW480 1.0 - 0.9 1.4 2.1 1.3

B/[S.sub.rt] HeLa 1.0 1.4 1.2 1.2 1.1 0.0
HCT116 1.0 0.8 0.8 1.1 0.4 0.0
SW480 1.0 0.8 2.2 0.2 0.4 0.0
29 3T 1.0 1.3 1.3 0.7 0.3 0.0

Extract 300
[mu]
g/ml

B/[W sub.37] 0.7
0.4
1.1

B/[S.sub.rt] -
-
-
-
Relative inhibition of Wnt-signaling activity in TOP-luciferase
transfected versus FOP-luciferase transfected cells. Ratios,
pointing to a specific effect on Wnt-signaling are presented in bold.

Uncaria tomentosa extracts B/[W.sub.37] and B/Srt down-regulate expression of the Wnt-signaling
target c-Myc without affecting beta-Catenin levels

Activation ofTCF-mediated transcription of Wnt-target genes is modulated by beta-Catenin, the level
of which is tightly regulated by degradation (Polakis 2007). Over-expression of beta-Catenin, due to
mutations that inactivate the beta-Catenin destruction complex or by stabilizing mutations of beta-
Catenin itself, results in activation of Wnt-signaling, as seen in the cancer cell lines used in this
study (Fig. 1, untreated or DMSO treated cells). To determine, whether the signaling-inhibitory
effect of U. tomentosa extracts operates upstream or downstream of beta-Catenin, we investigated
beta-Catenin protein levels in HeLa, HCT116 and SW480 cells in response to B/[W.sub.37] and
B/Srt extracts. As shown by immunoblotting (Fig. 2), neither extract affected significantly beta-
Catenin levels in all cancer cell lines studied. Furthermore, beta-Catenin degradation products in
lysates of U, tomentosa extract treated cells did not increase as compared to the controls (data not
shown). Therefore, it is more likely that B/[W.sub.37] and B/Srt extracts mediate their inhibitory
effect by regulating the transcription of Wnt-target genes, through a mechanism independent of the
beta-Catenin level, rather than by sequestering beta-Catenin. In contrast, expression of the
endogenous Wnt-target cMyc (He et al. 1998) was clearly reduced by both U. tomentosa extracts,
confirming the data obtained by the luciferase reporter assays (Fig. 2). In summary, our data
provide strong evidence that B/[W.sub.37] and B/Srt extracts inhibited Wnt-signaling activity
downstream of beta-Catenin regulation, thereby reducing the expression of Wnt target genes.

Dose-dependent growth inhibition of HeLa, HCT116 and SW480 cells by B/[W.sub.37] and B/Srt
extracts

To establish, whether the here described Wnt-signaling inhibitory effect exhibited by B/[W.sub.37]
and B/Srt extracts was the cause or the effect of reduced cell growth, the proliferation of treated
HeLa, HCT116 and SW480 cells at different time points was determined. As shown in Figs, 3 and 4,
proliferation of all cell lines tested was clearly reduced, in a dose-dependent manner, after 24 h of
treatment with both extracts. Table 2 shows the concentration of the extracts required to inhibit cell
proliferation by 50% (I[C.sub.50]). Similar to the effect on Wnt-signaling, the strongest inhibitory
effect was observed in SW480 cells, resulting in the lowest I[C.sub.50]. In addition, the I[C.sub.50]
for B/Srr after 24 h was lower than that of B/ [W.sub.37], indicating a stronger effect on cell
proliferation (Table 2). Interestingly, growth inhibition started earlier with B/ [W.sub.37] than with
B/Srt indicating that the ability to enter the cells is important. In contrast, growth inhibition by B/Srt
was more intense after 24 h (Fig. 4).
Table 2

Fifty percent growth inhibition (I [C.sub.50]) of two different
Uncaria tomentosa extracts in HeLa, HCT116 and SW480 cells.

Extract Cell I[C.sub.50] I[C.sub.50]
line ([mu] g/ml) ([mu] g/ml)
3h 24 h

B/[W.sub.37] HeLa 280 [much
greater
than] 300

HCT116 200 200

SW480 165 200

B/Srt HeLa [much [much
greater greater
than]200 than] 200

HCT116 [much 140
greater
than]200

SW480 160 95

I[C.sub.50]: concentration of extracts, where cell proliferation
is inhibited by 50%.

[FIGURE 4 OMITTED]

Effect of B/Sn extracts on non-cancer cells

We next investigated the specificity of the alkaloid enriched B/Srt extract in more detail. Since
normal, primary cells of the colon are not available and normal epithelial cells of other entities are
almost impossible to transfect effectively, we used immortalized human embryonic kidney 293T
cells for our analysis. 293T cells are commonly used for studying signaling pathways because they
do not harbor mutations in signaling components and are highly transfectable. Due
toimmortalization by SV40 large T antigen Wnt-signaling is strongly activated in these cells (Fig. 5A,
first column). Accordingly, Wnt-signaling activity was effectively inhibited by B/Srt, displaying an
I[C.sub.50(wnt)] of 43[micro]g/ml and TOP/FOP ratios, indicating a specific effect at concentrations
of not less than 50[micro]g/m1 (Table 1).

To exclude the possibility that the observed down-regulation of Wnt-target gene expression
following exposure to U. tomen-tosa extracts is the result of hitherto unknown growth inhibitory
effects we studied time response of Wnt-signaling inhibition by B/Srt. Analysis of time response to
50[micro]g/mI B/Srt revealed that downregulation of beta-Catenin/Tcf-mediated transcription starts
6h after treatment, reaching 50% of inhibition at 26 h of treatment (Fig. 5B, continuous line). To
investigate whether this effect is indeed dependent on Wnt-signaling activity and not merely a result
of growth inhibition, we transfected 293T cells with a well-known inhibiting variant of the Wnt-
effector TCF4 lacking the N-terminal beta-Catenin binding region (dn-hTCF-4, (Morin et al. 1997)).
Dn-hTCF4-transfected 293T cells, displaying a markedly reduced Wnt-singnaling activity were
insensitive towards 50[micro]g/ml B/Srt (Fig. 5B, dotted line), indicating that the extract specifically
interferes with Wnt-signaling and may thus not affect normal, differentiated cells. Furthermore
proliferation was not inhibited by B/Srt in 293T cells, neither in native cells nor when Wnt-signaling
was downregulated by dn-hTCF-4 (Fig. 5C). In contrast, proliferation was shortly activated within 1
h of B/Sr, treatment and returned to control levels during further treatment. Taken together, our
results indicate that B/Srt is clearly less cytotoxic to non-cancer cells and to cells without activated
Wnt-signaling than to cancer cells with aberrantly activated Wnt-signaling activity.

Discussion

Previous studies on the anti-tumorigenic potential of U. tomentosa extracts focused on inhibition of
neoplastic cell growth without analyzing the underlying mechanisms. We here combined
investigation of growth inhibitory effects of defined U. tomentosa extracts with analysis of Wnt-
signaling, which is a major target for future therapeutic intervention. In general, the antiproliferative
activity of U. tomentosa observed in this study is in good agreement with results obtained
withhematopoietic (Bacher et al. 2006; Pilarski et al. 2007; Sheng et al. 1998), breast (Garcia
Gimenez et al. 2009; Riva et al. 2001), neurologic (Garcia Prado et al. 2007) and other (De Martino
et al. 2006; Pilarski et al. 2010) cancer cell lines, where similar I [C.sub.50] values ranging from
25[micro]g/ml to more than 1000[micro]g/m1 had been determined, depending on the extract
preparation and cell line. Differential activity of B/ [W.sub.37] and B/Srt extracts was observed
previously, without finding a correlation between I [C.sub.50] values and cancer cell types.
Particularly, the B/Srt extract has been shown to have inconsistent activity in various cancer cell
lines which was ascribed to its low solubility in water and/or unknown selectivity for some cell lines
(Pilarski et al. 2010). To avoid problems with solubility, we here used B/Srt extract dissolved in
DMSO, which resulted in good reproducibility of all experiments. Our here presented data strongly
suggest that the selectivity of B/[W.sub.37] and B/Srt extracts on different cancer cell lines may be
ascribed to different endogenous Wnt-signaling activities. Cancer cell lines exhibiting high Wnt-
signaling activity, such as SW480 cells were clearly more sensitive towards B/[W.sub.37] and B/Srt
than cell lines showing a low endogenous Wnt-signaling activity, such as HCT116 and HeLa cells.
Furthermore, inhibition of Wnt-signaling activity by a dominant-negative TCF-4 variant resulted in
insen-sitivity towards B/Srt in non-cancerous cells. Finally, in all cell lines tested, the 50% Wnt-
inhibition (I I[C.sub.50(wnt)]) was much lower than the 50% growth inhibition (I[C.sub.50]), indicating
that the decrease in Wnt-signaling is rather the cause than the result of reduced cell proliferation.
The specificity for Wnt-signaling inhibition is therapeutically important, because inappropriate
regulation and activation of this pathway is associated with several pathological disorders including
cancer, retinopathy, tetra-amelia and arthritis (Janssens et al. 2006). It should be noted, that very
low concentrations of both extracts resulted in slight stimulation of both, growth and Wnt-signaling
activity (Figs. 1, 3 and 4, Table 2). This response has also been observed in colorectal cancer cells
treated with aspirin (Dihlmann et al. 2001), a known inhibitor of colorectal cancer growth (Barker
and Clevers 2006; Dihlmann and von Knebel 2005).

In all assays performed in this study, the B/Srt preparation was more effective than the B/[W.sub.37]
preparation. This strongly suggests that the antiproliferative and Wnt-inhibitory effects of U. tomert-
tosa extracts result from oxindole alkaloids, which account for over 50% of dry mass in B/Srt but
only 0.43% in the B/[W.sub.37] extract (Pilarski et al. 2010). Moreover, as reported earlier (Pilarski
et al. 2010), the alkaloid composition of both extracts is free oftetracyclic oxindole alkaloids and rich
in pentacyclic oxindole alkaloids, which are more biologically active (Wurm et A1 1998). So far it is
unknown, which of the alkaloids contained in the extracts is mainly responsible for the observed
inhibitory effect on Wnt-signaling. Both, the B/[W.sub.37] and B/Sr preparations contain a high
percentage of pteropodine and isomitraphylline (Pilarski et al. 2010). Future testing of single
alkaloids will help to identify the constituents responsible for down-regulation of Wnt-signaling.
Alternatively, the combination of different alkaloids may be important for the specificity.

In addition to testing the specificity of anti-cancer compounds targeting Wnt-signaling, secondary
assays are necessary to ensure their safety and efficiency in vivo. Although comparable studies in
humans have not been performed yet, alkaloid-rich U. Cornentosa extracts and even pure oxindole
alkaloids were reported to be non-toxic and safe to use, since L[D.sub.50] values determined in
mice (Keplinger et al. 1999) point to a lethal poisoning dose for adults of consuming 2 kg of B/Srr
preparation. Moreover, while this paper was in preparation, we and others reported the capacity of
U. tomentosci extracts to reduce solid tumor growth in vivo in different animal models. The
B/[W.sub.37] preparation was shown to significantly inhibit tumor growth in a Lewis lung carcinoma
mouse model compared to control groups when administered for 21 days at doses of 5 and
0.5mg/day (Pilarski et al. 2010). The treatment was well tolerated and non-toxic, as indicated by
blood parameters such as leukocyte number, erythrocytes, platelets, and hemoglobin. The B/Srt
preparation was likewise well tolerated and non-toxic in the Lewis lung carcinoma mouse model,
however, interestingly, it did not reveal any significant anti-tumor activity (Pilarski et al. 2010). A
second in vivo study tested the antitumoral activity of U. tomentosa hydroalcoholic extract in a
Walker-256 cancer model in rats (Dreifuss et al. 2010). These hydroalcoholic extract, containing
high concentrations of pentacyclic oxindole alkaloids similar to B/Srt, markedly reduced the
subcutaneous tumor growth without affecting body weight of the animals. In addition, treatment with
these extracts resulted in altered plasmatic levels of urea and hepatic markers, indicating that the
antioxidant properties of the U. tomentosa extract were responsible for its antitumoral effect
(Dreifuss et al. 2010). The Wnt-signaling activity in Lewis lung carcinoma cells and Walker-256 cells
is unknown. However, considering our findings of a specific Wnt-signaling inhibition by B/Srr, it is
reasonable to speculate that its ineffectiveness in Lewis lung carcinoma cells may result from low or
absent Wnt-signaling activity in these cells. In contrast, the effectiveness of alkaloid-rich U.
tomentosa extracts on Walker-256 cells might result from higher Wnt-signaling activity. Future
analysis will clarify whether different activities of Wnt- and/or other cancer signaling pathways
account for the differential effectiveness of various U. tomentosa extracts on different tumor entities
and which alkaloid-composition is most appropriate.

[FIGURE 5 OMITTED]

In conclusion, our data provide strong evidence that some U. tomentosa extracts target the Wnt-
signaling pathway, explaining in part its broad spectrum of biological activities. In agreement with
these findings, we recently demonstrated that U. tomentosa extracts affected embryogenesis in a
chicken embryo model (Kuras et al. 2009). Bearing in mind that Wnt-signaling plays a central role in
embryonic development, it would be interesting to analyze if the observed in vivo changes likewise
result from the interaction of U. tomentosa extracts with this pathway.

Acknowledgments

We are grateful to S. Garcia for providing helpful comments and suggestions. The work of CM.
Gurrola-Diaz, P.M. Garcia-Lopez and S. Dihlmann was supported by a bilateral travel grant for
German-Mexican collaboration of the Consejo Nacional de Cienciay Tecnologia (CONACYT),
Coordinacion General de Cooperation e Internationalization (CGCI, Universidad de Guadalajara)
Mexico and the Deutsche Forschungsgemeinschaft (DFG), Germany.

0944-7113/$ - see front matter @ 2010 Elsevier GmbH. All rights reserved.
Author: Gurrola-Diaz, Carmen Magdalena; Garcia-Lopez, Pedro Macedonio; Gulewicz, Krzysztof;
Pilarski, Radosl
Publication: Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type: Report
Geographic Code: 4EUGE
Date: Jun 15, 2011
Words: 5753
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Topics: Cancer cells
Physiological aspects
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Plant extracts
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Wnt proteins
Physiological aspects
Research
Mecanismos inhibitorios de dos extractos de Uncaria tomentosa que afectan a la va Wnt -
sealizacin.
ARTCULO INFO

Palabras clave : Clulas garra Ua de Gato va Wnt - sealizacin de Uncaria tomentosa Cat cMyc
Beta - catenina Cncer

RESUMEN

Uncaria tomentosa ( " ua de gato ", " ua de gato "), una trepadora leosa oriunda de la selva
amaznica, se utiliza comnmente en la medicina tradicional de Amrica del Sur para el
tratamiento de un amplio espectro de enfermedades. Aunque los estudios recientes han
informado de propiedades anti - inflamatorias y anti -proliferativos de diferentes alcaloides
extrados de esta planta , los mecanismos moleculares subyacentes de estos efectos no se han
elucidado todava. Nuestro estudio investiga los mecanismos inhibitorios de los extractos de
Uncaria tomentosa en la va Wnt de sealizacin , un regulador central del desarrollo y la
homeostasis del tejido. Un ensayo de luciferasa basado en clulas modificado para el cribado de
inhibidores de la va de Wnt - se utiliz para el anlisis . Tres lneas celulares de cncer que
muestran diferentes niveles de actividad de Wnt - sealizacin aberrante fueron transfectadas con
plsmidos de sealizacin de Wnt - Tcf - informador sensible y se trataron con concentraciones
crecientes de dos extractos de corteza de Uncaria tomentosa . La actividad de Wnt - sealizacin
se evalu mediante la actividad de luciferasa y por la expresin de los genes diana de Wnt -
sensibles . Se demuestra que ambos, una fase acuosa y un extracto alcaloide enriquecido inhiben
especficamente la actividad de Wnt - sealizacin en HeLa , HCT116 y clulas de cncer de SW480
resultantes en la expresin reducida del gen Wnt - objetivo : c-Myc . El extracto alcaloide
enriquecido ( B / [ S.sub.rt ] ) se encontr que era ms eficaz que el extracto acuoso ( B / [
W.sub.37 ] ) . El efecto ms fuerte se observ en las clulas SW480 , que muestra la actividad de
Wnt - sealizacin endgena ms alta . Oownregulation de Wnt- sealizacin por una negativa -
TCF- 4 variante dominante en clulas no cancerosas rendido las clulas insensibles hacia el
tratamiento con B / [ S.sub.rt ] . B / [ S.sub.rt ] fue menos txico en clulas no cancerosas que en
las clulas cancerosas . Nuestros datos sugieren que el amplio espectro de accin farmacolgica de
Uncaria tomentosa implica la inhibicin de la va de Wnt - sealizacin , aguas abajo de la actividad
de la beta-catenina .

[ c] 2010 Elsevier GmbH. Todos los derechos reservados .

introduccin

El medicamento vid Uncaria tomentosa ( rubicea ), nativo de la selva amaznica y comnmente
conocida como " ua de gato " o " ua de gato " se ha utilizado tradicionalmente por las tribus
indgenas para tratar un amplio espectro de los trastornos mentales y fsicas ( Keplinger et al. 1999
) . Hoy en da , se utiliza comnmente en la medicina popular de Amrica tropical para tratar
infecciones virales , artritis, enfermedades degenerativas crnicas , lceras gstricas y cncer. En
consecuencia , se ha evocado un creciente inters cientfico y comercial y es ampliamente
promovido como una alternativa de tratamiento para estas enfermedades ( Heitzman et al. 2005 )
.

A pesar de un nmero creciente de informes que describen los efectos clnicos y biolgicos de los
extractos de U. tomentosa , su eficacia farmacolgica y dianas moleculares son en gran parte
desconocidos . Las formas farmacuticas ms comunes de U. tomentosa son extractos solubles en
agua o solubles en etanol crudo derivados de su corteza o races para el consumo oral como
infusiones ( Keplinger et al . 1999 ) . Componentes activos identificados en estos extractos incluyen
diferentes alcaloides oxindolic , alcaloides de indol , glucsidos triterpenos ( pentacclicos con una
variedad de derivados tales como el cido urslico, glucsidos de cido quinvico , esteroles y
procianidins ) y taninos de la composicin qumica de la cual puede variar en funcin de su origen
geogrfico y la cosecha estacional ( Heitzman et al . 2005 ) . Por lo tanto , las diversas propiedades
farmacolgicas de U. tornentosa reportados en la literatura pueden ser atribuidos a diferentes
tipos y combinaciones de estos compuestos .

La mayora de los estudios in vivo - farmacolgicos inicialmente investigaron cmo extractos de U.
tomentosa afectan antiinflamatoria , inmunomoduladora y mecanismos de reparacin del ADN (
Akesson et al 2003 ; . . Keplinger et al 1999 ; . Sheng et al 2000 , 2001 ; . Spelman et al 2006 ) .
Adems , varios estudios sugieren una actividad anti - tumorignico de ambos, y alcaloide
enriquecido U. Comentosa extrae soluble en agua : citotxicos , antiproliferativos y pro-
apoptticos efectos se informaron en diferentes lneas celulares tumorales ( Cheng et al 2007 ;
Garca Gimenez . et al 2009 ; . . Pilarski et al 2010 ; Garca Prado et al 2007 ; . Gonzales y Valerio
2006 ; . Pilarski et al 2007 ) . Por otra parte , las preparaciones de U. tomentosa con diferentes
composiciones de alcaloides de oxindol y extractos hidroalcohlicos , respectivamente , se mostr
recientemente para reducir el crecimiento del tumor in vivo en modelos animales de tumores
slidos ( Pilarski et al 2010 ; . Dreifuss et al 2010 . ) . El mecanismo de este antitumoral - actividad y
los posibles efectos en las vas de sealizacin relacionadas con el cncer en las clulas tumorales
sin embargo, no han sido an investigado .

[ FIGURA 1 OMITIR]

[ FIGURA 2 OMITIR]

El amplio espectro de actividades reivindicadas para los extractos de U. tomentosa sugiere que
afecta a un mecanismo de regulacin central. Uno de estos importantes reguladores , que son
esenciales para el destino de las clulas madre , la auto-renovacin , el desarrollo y la homeostasis
del tejido en todo el reino animal, es la va Wnt - sealizacin ( Logan y Nusse 2004 ; Nusse 2005 ) .
En consecuencia , las alteraciones en esta va tienen un papel crucial en la fisiopatologa humana,
incluyendo las enfermedades inflamatorias y degenerativas , diabetes y cncer ( Janssens et al
2006 ; . Jin 2008 ; Logan y Nusse 2004 ) . La cascada de sealizacin Wnt - cannica se activa por la
unin de glicoprotenas secretadas de Wnt a Frizzled ( Fz ), los receptores a travs de un conjunto
bsico de protenas regulando de esta manera la capacidad de la beta-catenina para activar la
transcripcin del factor de clulas T (TCF ) que dependen de los genes . La activacin completa de
los receptores Fz requiere la interaccin con la PRL (protena relacionada con el receptor de
lipoprotena de baja densidad) co-receptores . En la ausencia de Wnt , la mayor parte de la beta-
catenina se une a la membrana plasmtica , donde se asocia con E - cadherina en las uniones de
adherencia . Recin sintetizado beta- catenina se dirige de forma constitutiva de la degradacin
por una multi-protena . Tras la activacin de esta va por especfico Wnts , beta-catenina se libera
de este complejo y se transfiere al ncleo donde se asocia con factores de transcripcin de la
familia TCF / LEF para activar genes diana que regulan la proliferacin celular, la diferenciacin y
genes implicados en la tumorignesis ( ver pgina de inicio Wnt para la lista ;
http://www.stanford.edu/ ~ rnusse / wntwindow.html ) . En el epitelio colorrectal , la activacin
aberrante de la sealizacin de Wnt - por mutaciones se considera que es el principal evento de
iniciacin del crecimiento del cncer . Adems , esta va es comnmente afectada por mutaciones
en un nmero creciente de otras entidades de cncer ( Behrens y Lustig 2004 ; Giles et al 2003 ; .
Polakis 2007 ) , as como en las enfermedades degenerativas ( Fuerer et al 2008 ; . Janssens et al
.2006 ) .

Debido a su papel regulador central en la enfermedad humana , la va Wnt - de sealizacin es un
objetivo importante para la intervencin teraputica y el desarrollo de inhibidores de Wnt -
especficos (Barker y Clevers 2006 ; Dihlmann y von Knebel 2005 ; . Janssens et al 2006 ) . Esto nos
llev a investigar si el mecanismo de accin de los extractos de U. Comentosa implica interferencia
con la actividad de Wnt - sealizacin . Desde la preparacin y composicin de los extractos
afectan a sus actividades biolgicas , que es uno de los principales problemas que dificultan la
comparacin de diferentes estudios, los extractos de tomentosoa U. aqu utilizados derivados de
los procedimientos de preparacin bien caracterizados y estandarizados ( Pilarski et al . 2007 ,
2010 ) que resultar en la composicin biolgicamente activa de los extractos que se caracterizan
por los alcaloides de oxindol - pluma tacyclic ( Wurm et al . 1998 ) .

Materiales y mtodos

lneas celulares

La lnea celular de cncer colorrectal humano HCT116 se obtuvo de ECACC (
http://www.ecacc.org.uk ) , la lnea celular de cncer colorrectal humano SW480 , lnea celular de
carcinoma cervical humano HeLa y se recibieron lnea de clulas epiteliales de rin embrionario
humano 293T desde la Investigacin del Cncer del Centro Alemn Tumorbank o CLS lneas
celulares de Servicios (Heidelberg , Alemania). Todas las lneas celulares se cultivaron en medio
RPMI 1640 ( PAA Laboratories , Alemania ) suplementado con 10 % de FCS , 100 U / ml de
penicilina y estreptomicina l00mg/ml utilizando condiciones estndar .

Extractos de Uncaria tomentosa

Origen del material vegetal , as como la preparacin y alcaloide composicin de los extractos
utilizados para este estudio fueron descritas anteriormente ( Pilarski et al . 2007 , 2010 ) . En
resumen, para la preparacin de la soluble en agua ( B / [ W.sub.37 ] ) extraer , se extrajo 1 g de
corteza en 10 ml de agua durante 8 horas a 37 [ grados ] C.To obtener el alcaloide corteza rica en
extraer (B / [ S.sub.rt ] ) , se extrajo 10 g de corteza con 50 ml de agua ( 6 h , 37 [ grados ] C ) . A
continuacin, la muestra se centrifug a 35.000 rpm y una cantidad igual de diclorometano se
aadi a la capa orgnica supernatant.The agua se retir y se evapor a vaco a 40'C a sequedad.
Para el anlisis en experimentos de cultivo de clulas , B / [ W.sub.37 ] se ajust en medio de
cultivo celular RPMI - 1640 para preparar una solucin madre de 50 mg / ml , B / Srt se disolvi en
DMSO para preparar una solucin madre de 50 mg / ml .

Ensayos de transfeccin y luciferasa reportero ( ensayo TOP / FOP )

Un ensayo basado en clula modificada para el cribado de inhibidores de Wnt - va se utiliz para
determinar la actividad de sealizacin de Wnt - en lneas celulares tratadas con extractos de
Uncaria tornentosa (Barker y Clevers 2006 ) . En resumen, 2 x 106 clulas de cada lnea celular se
cultivaron en placas de 6 pocillos durante 24 h . Para los ensayos de reportero , 1 x 106 clulas de
cada lnea celular fueron transfectadas transitoriamente en paralelo , ya sea con 1.5 [ micro ] g de
un constructo de Tcf - reportero ( TOP - luciferasa , que responde a la sealizacin aberrantes de
Wnt - por la conduccin altos niveles de actividad de la luciferasa ) o 1,5 [ micro ] g de un
constructo reportero mutado ( FOP - luciferasa , que es incapaz de responder a la sealizacin de
Wnt - activo y, en consecuencia conduce niveles ms bajos de actividad de la luciferasa ) . Se
utiliz 0,3 [ micro ] g de un dominante negativo pcDNA - DN - hTCF4 a biolgicamente regular por
disminucin la sealizacin de Wnt - , tal como se indica en las figuras. Para normalizar la
eficiencia transfeccin y la toxicidad no especfica , 0.5 [ micro ] g de un reportero pRSV -lacZ se
incluy en cada muestra. Todas las transfecciones se realizaron utilizando Fugene HD reactivo de
transfeccin (Roche Diagnostics, Mannheim, Alemania) de acuerdo con las instrucciones del
fabricante . El reportero TCF- sensible construye pTOPFLASH y pFOPFLASH fueron amablemente
proporcionados por H Clevers ; Universidad de Utrecht , Pases Bajos. Control de plsmido pRSV -
lacZ se obtuvo fromDKFZ Heidelberg, Alemania . Dominante negativo pcDNA- dn- hTCF4 fue
proporcionado amablemente por Bert Vogelstein ; El Centro de Oncologa de John Hopkins ,
Baltimore, MD, EE.UU. . Tres horas despus de la transfeccin , las clulas se cosecharon y se
sembraron en placas de 96 pocillos ( 1 x [ 10 OR4 ] clulas / pocillo) en 50 [ micro ] medio de
cultivo de clulas L . Compuestos de Uncaria tomentosa se aadieron a concentraciones finales
como se indica en las figuras. La actividad de luciferasa se determin 24 h ms tarde mediante la
adicin de 50 ulof solucin OneGlo - de ensayo de luciferasa ( Promega , Heidelberg, Alemania ) a
cada pocillo y la incubacin durante 3 min . Los lisados se transfirieron a placas de poliestireno
blanco y la luminiscencia se midi inmediatamente en un luminmetro bien - 96 ( TECAN - Reader ,
Genios ) durante 2 s . Para la normalizacin , relativa actividad de beta- galactosidasa se analiz a
partir de pocillos correspondientes en placas de microtitulacin usando un lector de ELISA a 570
nm ( Dihlmann et al . 2005 ) . Brevemente , 15ul de cada lisado se diluy en l00ul de tampn de
fosfato , y se aadi 25 ul de un sustrato cromognico ( CPRG , Roche Diagnostics , Mannheim
Alemania ) . Doblar la activacin se determin mediante la normalizacin de las unidades de luz de
los ensayos de luciferasa frente a valores derivados fromabsorbance del ensayo de beta-
galactosidasa . Los valores medios de lisados procedentes de clulas FOP - luciferase transfectadas
, no tratados o tratados con DMSO se fijaron en un 1,0 .

[ FIGURA 3 OMITIR]

Ensayo de viabilidad de clulas WST1

Las clulas se sembraron en placas de microtitulacin a densidades de 2x [ 10.sub.4 ] se aadieron
clulas / pocillo y extractos de Uncaria tomentosa a las concentraciones finales deseadas (rango
de 1 [ micro ] g / ml a 300 [ micro ] g / ml ) . La viabilidad celular se evalu mediante 10 u1 de WST-
1 proliferacin celular reactivo (Roche Diagnostics, Mannheim , Alemania ) despus de 0, 2 , 5, o
23 horas de incubacin . Las clulas se cultivaron durante 60 min se midi la absorbancia y
utilizando una microplaca ( ELISA ) lector ( absorbancia a 450 nm frente a 650 nm , segn las
instrucciones del fabricante ) .

immunoblotting

Las clulas fueron tratadas como se describe en la figura .2 . Despus de 24 h , las clulas se
recogieron en tampn de lisis ( 150 [ micro ] l PBS, 100 [ micro ] M de sodio orthovana fecha,
inhibidor de proteinasa cctel ( Complete [ R] , libre de EDTA , Roche Diagnostics, Mannheim ,
Alemania ) ) y se lisaron mediante dos ciclos de congelacin y descongelacin . La concentracin
de protena de los lisados se determin mediante un kit de ensayo de protenas (BioRad -
Laboratories , Mnchen , Alemania ) , y 20 [ micro ] g de cada lisado se aplic a SDS - PAGE y
transferencia utilizando procedimientos estndar como se describe previamente ( Dihlmann et al .
2005 ) . Las protenas especficas se detectaron por incubacin con anti - beta-catenina ( 1:1000 ;
Transduccin Laboratories ( BD Biosciences , Heidelberg, Alemania ) ) , anti - cMyc ( 1:500 ; clon
2Q.330 , Santa Cruz , Heidelberg, Alemania ) o anti- actina (1:5000 ; clon C4 ; MP Biomedicals ,
Heidelberg, Alemania ) anticuerpos en el amortiguador de bloqueo ( 5 % de leche / solucin salina
amortiguadora Tris / 1 % de Tween 20 ) . Despus de la incubacin de los blots con conejo anti -
IgG de ratn con peroxidasa ( Dianova , Hamburgo , Alemania ) durante 1 h , relmpago occidental
[ R ] - ECL Plus , ( Perkin Elmer , Rodgau , Alemania ) se aadi como un sustrato para la
visualizacin por una mayor chemolu - minescence .

Resultados

La inhibicin de la actividad de Wnt - sealizacin por ( B / [ W.sub.37 ] ) y (B / Srt ) extractos de
corteza de Uncaria tomentosa alcaloides enriquecido soluble en agua

Hemos demostrado previamente que U. tomentosa extrae B / [ W.sub.37 ] y B / Srt reducir la
proliferacin de diversas lneas celulares de cncer en diferentes concentraciones ( Pilarski et al .
2007 , 2010 ) . En estos estudios , sin embargo , la actividad inhibidora de crecimiento de B / [
S.sub.rt ] y B / [ W.sub.37 ] era muy variable. Lneas celulares de cncer de diferentes orgenes
mostraron grandes diferencias en su sensibilidad, y no se observ una correlacin clara entre la
preparacin de U. tomentosa y el tipo de clulas cancerosas. Esta variacin puede deberse a
cncer de las vas de sealizacin activadas de manera diferente . Nosotros aqu aplic un ensayo
de luciferasa - indicador basado en clulas para determinar la capacidad de los extractos de U.
tomentosa para inhibir la transcripcin beta-Catenin/Tcf-mediated , el resultado de la sealizacin
de Wnt - cannica . Este ensayo se basa en el principio de que compuestos que inhiben
especficamente la actividad de Wnt - sealizacin aberrante reducir la actividad de la luciferasa
sin - TOP ( o en un grado mucho menor ) reducir la actividad de la FOP - luciferasa ( Barker y
Clevers 2006 ) . Por lo tanto , Wnt - inhibidores especficos demostrarn / ratios de FOP- inhibicin
TOP ser inferior a 1,0, whereasunspecific inhibidores resultarn / ratios de FOP- inhibicin Intop
mayor o igual a 1.0 . La figura . La figura 1 muestra los efectos de un tratamiento de 24 h de clulas
cancerosas HeLa , HCT116 y SW480 . De acuerdo con los resultados anteriores ( Dihlmann et al
2001 ; . . Huang et al 2006 ) , endgeno Wnt- sealizacin en los controles no tratados se activ
moderadamente en HeLa y clulas HCT116 ( 8-10 veces y 12-20 veces, respectivamente ) ,
mientras que se activa ampliamente en clulas SW480 de cncer colorrectal no tratados (220 -
250- veces) . Tras el tratamiento con extracto de B/W37 , la actividad de Wnt - sealizacin se
redujo de una manera dependiente de la dosis en todas las lneas celulares . Clulas SW480 y
HCT116 fueron ms sensibles que las clulas HeLa , que se refleja en 50 % de Wnt - inhibiciones ( I
[ C50 ( Wnt ) ] ) de 190 [ micro ] g/m1 , 150 [ micro ] g / m1 278 ug / ml , respectivamente (Fig. 1 ,
panel izquierdo) . Teniendo en cuenta la relacin de TOP / FOP , sin embargo , el efecto fue slo
especfico en HeLa y clulas HCT116 en concentraciones > 300 [ micro ] g / ml ( Tabla 1 ) . En
contraste , B / Srt muestra un efecto inhibitorio especfico sobre Wnt de sealizacin a
concentraciones mucho ms bajas (Fig. 1 , panel derecho) . Curiosamente, la sensibilidad hacia B /
Srt una buena correlacin con la actividad de sealizacin Wnt- endgeno. Clulas SW480 que
muestra la actividad ms alta - de sealizacin Wnt eran claramente ms sensibles que las clulas
HCT116 y HeLa (Fig. 1 y Tabla 1 ) con un I [ C50 ( Wnt ) ] de 29 [ micro ] g / ml en comparacin con
60 [ micro ] g / ml y 38 [ micro ] g / ml, respectivamente . Este hallazgo es un argumento ms para
una interferencia especfica del extracto B / Srt con la va que resulta en la baja regulacin de beta-
Catenin/Tcf-mediated transcripcin.
Tabla 1

TOP / ratios de FOP- inhibicin de las lneas celulares de cncer tratados con dos
diferentes extractos de Uncaria tomentosa
I [ C50 ] : concentracin de los extractos , donde la proliferacin celular
se inhibe en un 50 % .

[ FIGURA 4 OMITIR]

Efecto de B / Sn extrae en clulas no cancerosas

A continuacin se investig la especificidad del extracto B / Srt alcaloide enriquecido con ms
detalle . Puesto que las clulas normales , primarios del colon no estn disponibles y clulas
epiteliales normales de otras entidades son casi imposibles de transfectar eficazmente , hemos
utilizado inmortalizados clulas 293T de rin embrionario humano para nuestro anlisis . Clulas
293T se utilizan comnmente para el estudio de las vas de sealizacin , ya que no albergan
mutaciones en los componentes de sealizacin y son altamente transfectables . Debido
toimmortalization por el antgeno T grande de SV40 de Wnt - sealizacin se activa fuertemente
en estas clulas ( Fig. 5A , primera columna ) . Por consiguiente , la actividad de Wnt - sealizacin
se inhibi de manera efectiva por B / SRT, mostrando un I [ C50 ( Wnt ) ] de 43 [ micro ] g / ml y
ratios TOP / FOP , lo que indica un efecto especfico en concentraciones de no menos de 50 [ micro
] g/m1 ( Tabla 1 ) .

Para excluir la posibilidad de que el observado la baja regulacin de la expresin de genes Wnt-
tras una exposicin a U. extractos Tomen - tosa es el resultado de los efectos inhibitorios del
crecimiento hasta ahora desconocidas que estudiamos el tiempo de respuesta de la inhibicin de
la sealizacin de Wnt- B / Srt . Anlisis de la respuesta en el tiempo de 50 [ micro ] g / mI B / Srt
revel que la regulacin negativa de arranques de transcripcin beta-Catenin/Tcf-mediated 6h
despus del tratamiento , alcanzando un 50% de inhibicin a 26 h de tratamiento (Fig. 5B, lnea
continua ) . Para investigar si este efecto es de hecho dependiente de la actividad de Wnt - de
sealizacin y no simplemente una consecuencia de la inhibicin del crecimiento , las clulas 293T
transfectadas con una variante bien conocida de la inhibicin de la TCF4 de Wnt - efector que
carece de la regin de unin a beta-catenina N-terminal ( DN - hTCF - 4 , ( Morin et al . 1997 ) ) .
Clulas 293T transfectadas - Dn- hTCF4 , mostrando una actividad de Wnt - singnaling
marcadamente reducida fueron insensibles hacia 50 [ micro ] g / ml B / Srt (Fig. 5B , lnea de
puntos ) , lo que indica que el extracto interfiere especficamente con Wnt - de sealizacin y
puede por tanto, no afectan a las clulas normales y diferenciadas . Adems proliferacin no fue
inhibida por B / Srt en clulas 293T , ni en clulas nativas ni cuando Wnt - sealizacin se
downregulated por DN - hTCF - 4 (Fig. 5C ) . En contraste , la proliferacin fue poco activa dentro
de 1 h de B / Sr , tratamiento y regres a los niveles de control durante el tratamiento ulterior .
Tomados en conjunto , nuestros resultados indican que B / Srt es claramente menos citotxico
para las clulas no cancerosas y a clulas sin activado Wnt - de sealizacin que a las clulas
cancerosas con actividad de Wnt - de sealizacin activadas aberrantemente .

discusin

Estudios previos sobre el potencial anti- tumorignicos de extractos de U. tomentosa se centraron
en la inhibicin del crecimiento de clulas neoplsicas sin analizar los mecanismos subyacentes .
Estamos aqu, combinamos la investigacin de los efectos inhibitorios del crecimiento de los
extractos de U. tomentosa definidas con el anlisis de la sealizacin Wnt- , que es un objetivo
importante para la futura intervencin teraputica. En general , la actividad antiproliferativa de U.
tomentosa observado en este estudio est en buen acuerdo con los resultados obtenidos
withhematopoietic ( Bacher et al 2006 ; . . Pilarski et al 2007 ; . Sheng et al 1998 ) , de mama (
Garca Gimnez et al 2009 . ; . Riva et al 2001) , neurolgicas (Garca Prado et al 2007 ) y otros ( de
Martino et al 2006 ; . . . Pilarski et al 2010 ) lneas celulares de cncer , donde [ C50 ] valores
similares que van desde 25 [ micro ] g / ml a ms de 1.000 [ micro ] g/m1 se haba determinado ,
en funcin de la preparacin del extracto y lnea celular . Se observ una actividad diferencial de [
W.sub.37 ] y extractos de B / B / Srt anteriormente , sin encontrar una correlacin entre la I [ C50 ]
valores y tipos de clulas cancerosas . En particular , el extracto B / Srt se ha demostrado que
tienen actividad inconsistente en diversas lneas celulares de cncer que se atribuye a su baja
solubilidad en agua y / o selectividad desconocido para algunas lneas celulares ( Pilarski et al .
2010 ) . Para evitar problemas con la solubilidad , que aqu utilizado el extracto de B / Srt disuelto
en DMSO , que dio lugar a una buena reproducibilidad de todos los experimentos . Nuestro aqu
present datos sugieren fuertemente que la selectividad de los extractos y B / [ ] W.sub.37 B / Srt
en diferentes lneas celulares de cncer puede atribuirse a diferentes actividades Wnt de
sealizacin endgenos. Lneas celulares de cncer que presentan alta actividad Wnt- sealizacin
, tales como clulas SW480 eran claramente ms sensible hacia B / [ W.sub.37 ] y B / Srt de lneas
de clulas que muestran una baja actividad de Wnt- sealizacin endgena , como HCT116 y
clulas HeLa . Adems, la inhibicin de la actividad de Wnt - de sealizacin por un TCF - 4 variante
dominante negativo result en - sibilidad insensibilidad hacia B / Srt en las clulas no cancerosas .
Finalmente , en todas las lneas celulares ensayadas , el 50 % de inhibicin de Wnt - ( II [ C50 ( Wnt
) ] ) fue mucho menor que la inhibicin del crecimiento del 50 % (I [ C50 ] ) , lo que indica que el
disminucin de la sealizacin de Wnt - es ms bien la causa que el resultado de la proliferacin
celular reducida . La especificidad para la inhibicin de Wnt - sealizacin es teraputicamente
importante , porque la regulacin inapropiada y activacin de esta va se asocia con varios
trastornos patolgicos incluyendo cncer , retinopata , tetra - Amelia y la artritis ( Janssens et al .
2006 ) . Cabe sealar , que concentraciones muy bajas de ambos extractos dieron como resultado
una ligera estimulacin de ambos, el crecimiento y la actividad de Wnt - sealizacin ( Figs. 1 , 3 y
4 , Tabla 2 ) . Esta respuesta tambin se ha observado en clulas de cncer colorrectal tratados con
aspirina ( Dihlmann et al 2001 . ) , Un conocido inhibidor del crecimiento del cncer colorrectal (
Barker y Clevers 2006 ; Dihlmann y von Knebel 2005 ) .

En todos los ensayos realizados en este estudio , la preparacin B / Srt fue ms eficaz que el / [
W.sub.37 ] la preparacin B . Esto sugiere que los efectos antiproliferativos y Wnt - inhibitorios de
U. Tomert - tosa extractos resultan de alcaloides de oxindol , que representan ms del 50 % de la
masa seca en B / Srt pero slo el 0,43% en el B / [ W.sub.37 ] extraer ( Pilarski et al. 2010 ) . Por
otra parte, como se inform anteriormente ( Pilarski et al. 2010 ), la composicin de alcaloide de
ambos extractos es alcaloides oxindol oftetracyclic y rica en alcaloides oxindol pentacclicos , que
son biolgicamente ms activa ( Wurm et A1 1.998 ) gratis. Hasta el momento no se sabe cul de
los alcaloides contenidos en los extractos es el principal responsable del efecto inhibidor
observado sobre Wnt- sealizacin. Ambos , los [ W.sub.37 ] y B / preparados B / Sr contienen un
alto porcentaje de pteropodine y isomitraphylline ( Pilarski et al. 2010 ) . Pruebas Futuro de
alcaloides individuales le ayudar a identificar los componentes responsables de la baja regulacin
de la sealizacin de Wnt- . Alternativamente , la combinacin de diferentes alcaloides puede ser
importante para la especificidad .

Adems de probar la especificidad de los compuestos contra el cncer dirigidos Wnt- sealizacin ,
ensayos secundarios son necesarios para garantizar su seguridad y la eficacia in vivo. Aunque los
estudios comparables en los seres humanos no se han realizado todava , se inform de alcaloide -
ricos extractos de U. Cornentosa y alcaloides de oxindol incluso puro para ser no txico y seguro
de usar , ya que L [ D50 ] valores determinados en ratones ( Keplinger et al. 1999 ) apuntan a una
dosis letal envenenamiento para los adultos de consumir 2 kg de preparacin B / Srr . Adems, si
bien este documento se estaba preparando , nosotros y otros informaron de la capacidad de U.
tomentosci extrae para reducir el crecimiento de tumores slidos in vivo en diferentes modelos
animales . El / [ W.sub.37 ] la preparacin B ha demostrado inhibir significativamente el
crecimiento del tumor en un modelo de ratn de carcinoma de pulmn de Lewis en comparacin
con grupos de control cuando se administra durante 21 das a dosis de 5 y 0.5mg/day ( Pilarski et
al . 2010 ) . El tratamiento fue bien tolerado y no txico , tal como se indica por los parmetros de
la sangre tales como el nmero de leucocitos , eritrocitos , plaquetas y hemoglobina . La
preparacin B / Srt fue igualmente bien tolerado y no txicos en el modelo de ratn de carcinoma
de pulmn de Lewis , sin embargo , de manera interesante , no revel ninguna actividad anti -
tumoral significativa ( Pilarski et al . 2010 ) . Un segundo estudio in vivo evalu la actividad
antitumoral del extracto hidroalcohlico de U. tomentosa en un modelo de cncer de Walker -256
en ratas ( Dreifuss et al. 2010 ) . Estos extracto hidroalcohlico , que contiene altas
concentraciones de alcaloides de oxindol pentacclico similares a B / SRT, redujo notablemente el
crecimiento del tumor subcutneo sin afectar el peso corporal de los animales . Adems , el
tratamiento con estos extractos dio como resultado niveles plasmticos alterados de urea y
marcadores hepticos , lo que indica que las propiedades antioxidantes del extracto de U.
tomentosa eran responsables de su efecto antitumoral ( Dreifuss et al . 2010 ) . La actividad de
sealizacin Wnt- en clulas de carcinoma de pulmn de Lewis y Walker- 256 clulas se
desconoce. Sin embargo , teniendo en cuenta nuestros hallazgos de una inhibicin de Wnt de
sealizacin especfica por B / Srr , es razonable especular que su ineficacia en clulas de
carcinoma de pulmn de Lewis puede ser el resultado de la actividad baja o ausente de Wnt - de
sealizacin en estas clulas . En contraste , la eficacia de los extractos de U. tomentosa alcaloides
- rica en Walker - 256 clulas podra ser el resultado de una mayor actividad de Wnt - sealizacin .
En futuros anlisis se aclarar si diferentes actividades de Wnt - y / o otras vas de sealizacin del
cncer representan la eficacia diferencial de los diversos extractos de U. tomentosa en diferentes
entidades tumorales y que alcaloide - composicin es el ms apropiado .

[ FIGURA 5 OMITIR]

En conclusin , nuestros datos proporcionan una fuerte evidencia de que algunos extractos de U.
tomentosa se dirigen a la va Wnt - sealizacin , lo que explica en parte su amplio espectro de
actividades biolgicas. De acuerdo con estos resultados, hemos demostrado que los extractos de
U. tomentosa afectados embriognesis en un modelo de embrin de pollo ( Kuras et al. 2009 ) .
Teniendo en cuenta que la sealizacin de Wnt - juega un papel central en el desarrollo
embrionario , sera interesante analizar si el observado en los cambios in vivo del mismo modo
resultar de la interaccin de los extractos de U. tomentosa con esta va .

Agradecimientos

Damos las gracias a S. Garca para proporcionar valiosos comentarios y sugerencias. El trabajo del
CM . Gurrola -Daz , P. M. Garca - Lpez y S. Dihlmann el apoyo de una beca de viaje para la
colaboracin bilateral germano- mexicana del Consejo Nacional de Cienciay Tecnologia (CONACYT
) , Coordinacion General de Cooperacin e Internacionalizacin ( CGCI , Universidad de
Guadalajara ) Mxico y la Deutsche Forschungsgemeinschaft ( DFG ) , Alemania .

0944-7113 / $ - see front matter @ 2010 Elsevier GmbH. Todos los derechos reservados .

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