“IN VITRO CYTOTOXICITY OF NATURAL PRODUCTS

AGAINST HUMAN CANCER CELL LINES”

UNDER THE GUIDANCE OF

Dr.A.K.SAXENA, SCIENTIST F
Department Of Pharmacology
INDIAN INSTITUTE OF INTEGRATIVE MEDICINE (CSIR),

BY
NIKHIL DEEP SINGH
B.Tech (2nd Year)
IIT KANPUR®

1
 #INTRODUCTION

CANCER:
Cancer (medical term: malignant neoplasm) is a class of diseases in which a group
of cells display uncontrolled growth (division beyond the normal limits), invasion
(intrusion on and destruction of adjacent tissues), and sometimes metastasis
(spread to other locations in the body via lymph or blood). These three malignant
properties of cancers differentiate them from benign tumors, which are self-
limited, and do not invade or metastasize. Most cancers arise from stem cells or
early progenitor cells. Stem cells exist at specific locations within a given tissue
(niches) and are permanently resident at this location. They have a proliferative
potential that exceeds an individual’s life time ( Renetan and Potten ., 2001).

CAUSES:
Abnormalities in the genetic material of the transformed cells are the main cause.
(Kinzler, Kenneth W.; Vogelstein, Bert 2002.) This may be due to the effects of
carcinogens, such as tobacco smoke, benzene, radiation, chemicals, or viruses e.g.;
HPV. Other cancer-promoting genetic abnormalities may be randomly acquired
through errors in DNA replication, or are inherited, and thus present in all cells
from birth. The heritability of cancers is usually affected by complex interactions
between carcinogens and the host's genome.

2
GENES RESPONSIBLE

When proto-oncogene are mutated they become oncogene. Often a whole series
of these genes become activated into oncogenes (Dollinger., 1997), giving the cells
new properties, such as hyperactive growth and division, protection against
programmed cell death (Apoptosis), loss of respect for normal tissue boundaries,
and the ability to become established in diverse tissue environments. It’s apparent
that epigenetic abnormalities in the expression of these genes also play an important
role in carcinogenesis (Weinsten., 2000).

Tumor-suppressor genes. (p-53, p-21) are then inactivated in cancer cells,

resulting in the loss of normal functions in those cells, such as accurate DNA
replication, control over the cell cycle, orientation and adhesion within tissues, and
interaction with protective cells of the immune system.

3
TYPES AND TREATMENT:

Cancers are classified by the type of cell that resembles the tumor and, therefore,
the tissue presumed to be the origin of the tumor.Examples of general categories
includes:

 Carcinoma: Malignant tumors derived from epithelial cells. This group

includes the common forms of breast, prostate, lung and colon cancer.

 Sarcoma: Malignant tumors derived from connective tissue, or

mesenchymal cells.

 Lymphoma and leukemia: Malignancies derived from hematopoietic (blood-

forming) cells

 Germ cell tumor: Tumors derived from totipotent cells. In adults most often
found in the testicle and ovary; in fetuses, babies, and young children most
often found on the body midline, particularly at the tip of the tailbone; in
horses most often found at the poll (base of the skull).

The four most deadly cancers are lung, stomach, liver, and colorectal (Parkin et al.,
2001). More than 35% of cancer cases in men are related to the oral cavity, larynx
and pharynx (all tobacco related), and about 40% and 30% of cancer cases in
women are cervical and breast cancer, respectively, in India. (Rao et al., 2002).
Leukemia and lymphoma are malignant tumors of hematopoetic cells of the bone
marrow and account for 9% of cancer. Benign cell is a hyperplasia or non malignant
lesion that is often premalignant (Doll and Pelo., 1981).

4
There are different types of therapies available Surgery e.g.; biopsy, Cryosurgery –
(Extreme cold to kill cancer cells), Radiation Therapy (use of ionizing radiation to
kill cancer cells and shrink tumors ), Cancer Vaccines, Herbal Therapy (A recent
survey lists over 1400 genera of herbs that have a history of use in cancer
treatments. (Hartwell, 1967, 1971), Hormonal Therapy (providing or blocking
certain hormones), Gene Therapy, Immunotherapy (diverse set of therapeutic
strategies designed to induce the patient's own immune system to fight the
tumor), Chemotherapy

5
#REVIEW OF LITERATURE:
Cancer drug is a major transition from the previous pre genomic cytotoxic era to
the new post genomic targeted era ( Workman et al., 2001 ; Guillemard et al.,
2004; Sawyers et al 2004; Segota et al., 2004; Pesgram et al 2005) . Some e.g. of
currently marketed targeted drugs for cancer therapeutics are shown in this table.

Trade Name Company Mechanism

Trastuzumab (Herceptin ) Genentech Humanized monoclonal
antibody against HER 2
Imatinib (Gleevec) Novartis Small molecule Inhibitor
of Bcr-Abl and C-kit
tyrosine kinases
Gefitinib (Iressa) Astrazeneca Small molecule Tyrosine
kinase inhibitor of EGFR
Cetuximab (Erbitux) Imclone/Bristol-Myers Chimeric monoclonal
squibb antibody against EGFR
Bevacizumab (Avastin) Genetech Humanized monoclonal
antibody against vascular
endothelial growth factor
(VEGF)
Bortezomib (Velcade) Millennium co- Small molecule
developed with Proteasome inhibitor
Johnson - and
Johnson

6
DEVELOPMENT OF ANTICANCER DRUGS FROM NATURAL
SOURCES:
• Paclitaxel:

1. A Chemical discovered from the pacific Yew tree Taxus brevifolia in

1971 (Wani et al.,1971)
2. Effective drug for the treatment of breast and ovarian cancers.
3. It binds specifically to the beta-tubulin subunit of microtubules (Schiff
et al., 1979; Horwitz 2004)
• Camptothecin:

1. Alkaloid first found in Camptotheca acuminata.

2. Activity in colorectal, ovarian, and small cell lung cancer (Takimoto and
Arbuck., 2001).
3. Enhances binding of topo-isomerase I to DNA, thus promoting DNA
strand breaks. (Cragg and Newman. 2004)

7
• Mitomycin C:

1. Antibiotic isolated from Streptococcus caespitosus

2. Mitomycin contains an aziridine group and a quinone group in its
structure, as well as a mitosane ring, and each of these participates in the
alkylation reactions with DNA. (Verweij et al... 2001)
• Doxorubicin ( Adiriamycin ):

1. Produced by the fungus Streptococcus peucetius var . caesius

2. Have been used primarily in the acute leukemias , whereas
doxorubicin displays broader activity against human neoplasm,
including a variety of solid tumors

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#IN VITRO STUDIES:

SOME OF THE COMMON CANCER CELL LINES.

CANCER TYPE CELL LINES

BREAST MCF-7
CERVIX HeLa, SiHa
CNS IMR-32, , SK-N-SH, SNB-78,SF-295
COLON Colo-205, HCT-15, HT-29, SW-620, 502713
LEUKEMIA K-562, MOLT-4
LIVER Hep-2
LUNG A-549, HOP-62, H226
PROSTATE DU-145, PC-3
ORAL KB

9
Materials and Methodology

• Chemicals Required:
Growth medium (RPMI), Fetal calf serum, Trypsin , PBS , Tryphan blue,
Ethanol, Penicillin , Streptomycin , Gentamycin, DMSO , Trichloroacetic
acid, Distilled water, Sodium Hydroxide, Tris-EDTA Buffer,m
Sulphorhodamine , Tris buffer, Acetic acid , Sodium bicarbonate,
Mitomycin C ,Palcitaxel (taxol), 5 Fluorouracil , Hydrochloric acid ,
Isopropanol , Tris-Acetate-EDTA Buffer.

• Apparatus:
Tissue culture flasks, Incomplete growth medium (RPMI), 6-well flat
bottomed culture plates Micropipettes 1.5 ml and 0.5 ml eppendorf
centrifuge tubes 15ml centrifuge tubes 50 ml centrifuge tubes, 96-Well cell
culture plates ,Sterile centrifuge tubes, Cryo vials, Glass bottles to store
media etc. Glass pipettes Syringe

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Reagents:

 Prepare RPMI-1640 with 2 mM glutamine medium or MEM in double

distilled water. PH is adjusted to 7.2. Add penicillin (100 units/ ml dissolved
in PBS) and sterilize by filtering through 0.2 µm filters in sterile laminar flow.
Store the media in refrigerator (2-8 o C).

 Complete growth medium: Contains 10 % FCS and 1% Penicillin. The

amount of FCS may vary depending upon the requirements of cell lines
used. Freezing Medium for cryopreservation contains 20 % FCS and 10 %
DMSO in growth medium (RPMI or MEM).

 Phosphate Buffer Saline (PBS): Dissolve 9.6 gm/lt. in distilled water.

PBS is used to prepare solutions of Penicillin and Trypsin EDTA.

 Penicillin Solution: Dissolve 100 units/ml or 625 mg/ ml in PBS. Penicillin

is mixed in RPMI Medium to avoid contamination.

 Gentamycin Solution: Dissolve 50 mg/ ml in PBS. Gentamycin is added in

the medium used in the preparation of dilutions of the test sample to avoid
contamination because of plant extracts.

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 Trypsin EDTA: 0.05% Trypsin and 0.02% EDTA (disodium salt) are dissolved
in PBS. Trypsin EDTA is used to detach the cells while sub-culturing and
splitting the cell line.

 TCA: 50% (w/v) TCA solution is prepared in double distilled water. TCA is
used for fixing the culture cells before washing.

 Acetic acid: Prepare 1% in distilled water. Acetic acid is used to prepare

solution of SRB dye crystals and to remove unbound dye from cells after
staining.

 SRB Dye: Dissolve 0.4% in 1% acetic acid. SRB Dye is used to stain the basic
proteins of cancer cells fixed by 50% TCA.

 Tris buffer: 10 mM (pH 10.5). Tris buffer is used to dissolve protein bound
dye.

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INSTRUMENTS:

Laminar Air Flow Microscope

Elisa Reader

Hemocytometer Cryovial
13
 • Hemocytometer (designed for the counting of blood cells and other
cells),Cryo-containers(for storing cells), Water bath(for incubation),
Filtration assembly, Deep freezer, Mechanical shaker, Centrifuge, Autoclave,
ELISA Reader , Liquid Nitrogen Cylinder, CO2 Gas Cylinder, CO2 Incubator

• Handling of cell lines on arrival:

The cells are first grown in complete RPMI growth medium then incubated
in CO2 incubator (37oc, 5% CO2, 90% R.H) and observed under microscope,
for apparent contamination and proper growth.
After monolayer formation cells are harvested and then stored in Liquid N2
at -196oc.

• Revival and seeding of cells:

The desired cryovial is removed from liquid nitrogen cylinder and thawed.
Nearly 10 ml of complete growth medium is poured into the tissue culture
flask.
The cells are added to tissue culture flask. Mixed properly and incubated at
37oC, 5% CO2 atmosphere and 90% R.H.

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STAGES OF CELL GROWTH DURING CULTURING

 Attachment Stage: Within 24 hours of incubation after seeding or

trypisinization, the cells get attached to the base of tissue culture flask.

 Sub-confluent Stage: It is a stage of rapid growth of cells. In this stage, some

space remains in between the growing cells. The cells are in log phase of their
growth and are used for experimental purposes.

 Confluent Stage: The medium turns turbid as nutrients are utilized & cell debris
and metabolic wastes accumulate in the medium. When the cells are observed
under microscope, they are seen to form a complete monolayer.

Sub-culturing of cell lines:

Sub-culturing of cell lines Is done by trypsinisation to maint
ain the cells in log phase of growth. The cells are detached when they reach sub-
confluent stage and to make single cell suspension for experimental purposes. The
trypsinisation of a cell line should not be done more than 5 times as the cells lose
their potential to grow.

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 Preparation of test material:

 Stock solution (depending upon required conc.) DMSO is used for

dissolving 95 % MeOH / EtOH extracts, 50% aqueous DMSO for 50%
Aqueous MeOH/EtOH extracts and distilled water for hot water extracts.
The working solutions of test samples are prepared in the complete growth
medium. Gentamycin (50 mg/ml) is added to check the microbial
contamination in growth medium.

 Positive control is essential for comparison of the activity of the test

material with the already established drugs against cancer e.g.;
a) 5-fluorouracil: It is a pro-drug undergoing a series of biotransformation
reactions to ribosyl and deoxyriboxyl nucleotide metabolites. One of these
metabolites is 5-fluoro-2-deoxyuridine-5monophosphate (FdUMP) which
gets incorporated in to RNA where it interferes with r-RNA and m-RNA
translation.

b) Mitomycin-C: It is an antibiotic isolated from Streptomyces caespitosus. It

is an alkylating agent that undergoes metabolic activation through an
enzyme-mediated reduction, to generate an alkylating agent that cross-links
DNA.

16
Blank (negative) Control: Blank control is essential to find the OD of medium and
thus that can be reduced from total OD to get the OD of the test sample.
Gentamycin media and test sample solution are added but cell suspension is not
added.

Determination of Cytotoxicity:
In a single 96 well plate, the Cytotoxicity of sample can be determined on 2 cell
lines at a time. The number of plates depends on the number of test samples

*96 Well Plate

17
• Preparation of cell suspension for the assay:
Skehan et al., (1990)
• Grow the desired human cancer cell line in tissue culture flasks at 370 C, in
an atmosphere of 5% CO2 and 90% relative humidity in complete growth
medium to obtain enough number of cells.
• Select the flask with sub-confluent stage of growth.
• Harvest the cells by treating the cells with Trypsin- EDTA.
• Count the number of cells/ ml of suspension with the help of
haemocytometer.
• Adjust the cell density to 10,000 cells /100 ml (or the standard density
depending upon cell line) in the cell suspension.
• Add 100 µl of cell suspension to each well of 96 well plates with the help
of a handy-step.
• Incubate the plates at 370 C, in an atmosphere of 5% CO2 and 90% relative
humidity for 24 hours.
• After 24 hours the 100 µl of working solution of each test material is
added to the wells of 96 well plates.
• The wells 1 to 4 and 9 to 12 of first row in each plate is control growth (CG
i.e. blank) i.e. having the cell suspension and medium. The growth of the
cells in these wells is maximum as no test sample is added.

18
 • The second row of one plate have 100 µ/well positive control (PC) i.e.
having 5-FU or Mitomycin C etc. The growth of these wells is less, as it is
the known inhibitor of cells.

Addition of test material:

19
• If different concentrations of the same test sample are used, they should be
in the successive wells in the same plate.
• 100 ml of working solution of the test material is added to rest of the wells
excluding the first row of each plate and where the negative control
(1%gentamycin medium) has been added.
• Incubate the plates for 48 hours at 37oC, in an atmosphere of 5% CO2 and
90% relative humidity.
• Determine the cell growth after 48 hours of adding sample
• To stop the reaction, gently add 50 µl of chilled 50% TCA (trichloroaceticacid
) to each well of the plate, making final concentration of 10%.

• Incubate the plates at 4C for one hour to fix the cells attached to bottom of
the wells.

• Wash the plates 5-6 times with distilled water.

• Plates are air-dried.

• Add 100 µl of SRB dye(0.4%in1%acetic acid) to each well of the plate and
leave the plates at room temperature for 30 minutes

• Wash the plates with 1% acetic acid after 30 minutes.

• Plates are again air-dried.

20
 • 100 µl of Tris buffer (10.5M) added to each well.

• Shake the plates gently for 10- 15 minutes on a mechanical shaker.

• Record the optical density with ELISA reader at 540nm wavelength and
maintain the data

• Cytotoxicity assay by Sulforhodamine B Dye:

*.figure depicting the absorbance phenomenon of Elisa reader where hѵ

is the energy of the photon coming in and hѵ’ the energy of photon that
reflects back after absorbance by the cells having Sulphorhodamine B
bound to them.

21
• SRB assay is a rapid, sensitive and inexpensive method for measuring the
cytotoxic potential of test substances, based on the cellular protein content
of adhered suspension cultures in 96 well plate. This method is suitable for
ordinary laboratory purposes and for large-scale applications like high
throughput in vitro screening in anticancer drug development.

• Principle of assay:
SRB is a bright pink aminoxanthene dye with 2 sulphonic groups. Under mild
acidic conditions, Sulphorhodamine B binds to the protein’s basic amino acid
residues in TCA fixed cells to provide a sensitive index and cellular protein
content. The anticancer activity is determined by the cytotoxic potential of
the test material using human cancer cell line, which is allowed to grow on
tissue culture plate in the presence of test material

22
 PROCEDURE
Seeded human cancer cell lines
(96 well plates)

Added test samples

Stopped cell growth

Stopped reaction with 50% w/v TCA

Kept at 4*C for 1hr. and washed with distilled water and left
the plates to dry @ room temp.

Added SRB into plates and kept for 30 minutes.

Washed plates with 1% acetic acid, left the plates to dry@
room temp.

Added tris buffer to dissolve bound dye

Read plates on ELISA READER at 540 nm

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 #5.RESULTS AND DISCCUSIONS:
RESULT -1
IN VITRO CYTOTOXICITY AGAINST HUMAN CANCER CELL LINES
CELL LINE TYPE Colon Lung Liver Neuroblastoma
CCL Conc
Inst. Codes 502713 A-549 Hep-2 IMR-32
Codes (μg/ml)
10809 RBH 1762 P13 A001 100 24 35 45 0
10810 RBH 1762 P13 A002 100 20 32 41 15
10811 RBH 1969 P01 A001 100 87 63 84 65
10812 RBH 1982 P01 A001 100 46 30 44 24
10813 RBH 1982 P01 A002 100 23 12 29 1
10814 RBH 2298 P13 A001 100 71 37 41 18
10815 RBH 2307 P04 A001 100 15 0 21 0
10816 RBH 2310 P03 A003 100 4 60 20 23
10817 RBH 2321 P04 A002 100 75 65 54 38
10818 RBH 2325 P14 A002 100 23 41 22 31
10819 RBH 2467 P02 A003 100 28 48 30 19
10820 RBH 2467 P09 A001 100 61 52 57 28
10821 RBH 2476 P01 A002 100 48 60 42 29
10822 RBH 2470 P13 A001 100 26 19 22 15
10823 RBH 2470 P13 A002 100 7 0 13 16
10824 RBH 2470 P13 A003 100 27 13 33 6
10825 RBH 2485 P09 A001 100 37 10 50 27
10826 RBH 2566 P09 A001 100 23 15 33 19
10827 RBH 2575 P13 A001 100 97 43 91 56
10828 RBH 2575 P13 A002 100 94 31 86 41
10829 IHB 1422 P02 A001 100 41 0 28 0
10830 IHB 1422 P02 A002 100 15 20 8 3
10831 IHB 1422 P02 A003 100 51 51 12 24
10832 IHB 1423 P02 A001 100 93 49 36 50
10833 IHB 1423 P02 A002 100 91 40 31 29
10834 IHB 1423 P02 A003 100 93 54 87 99
10835 IHB 1423 P03 A001 100 97 44 63 64
10836 IHB 1423 P03 A002 100 92 26 32 38
10837 IHB 1423 P13 A001 100 73 40 42 22
10838 IHB 1423 P13 A002 100 80 18 40 23
10839 IHB 1423 P13 A003 100 83 49 76 48
10687 RJO 1888 P13 A001 100 83 39 41 80
10688 RJO 1888 P13 A001 100 50 39 35 28
10689 RJO 1888 P13 A001 100 32 1 12 41
5-Fu 2X10-5M 53 - - -
-5
Paclitaxel 1X10 M - 63 - -
Adriamycin 1X10-6M - - - 72
-5
Mitomycin-C 1X10 M - - 69 -

24
DISCUSSION:
Remarks: Stock solution (20 mg/ml) was prepared in DMSO, 50%DMSO and water
and further dilution was carried out with medium. In vitro cytotoxicity against 4
human cancer cell lines was determined using SRB assay.
Criteria of activity: >70% growth inhibition at 100 µg/ml against three cell lines
Discussion: All the samples have been evaluated at 100 µg/ml and one sample
(IHB 1423 P02 A003) shows such growth inhibition.

25
RESULT 2:
Cell line type Colon Lung Liver Prostrate
502713 A-549 Hep-2 PC-3
CCL CODES INST. CODES Conc. GROWTH INHIBITION%
10926 RLB 1241 P05 A001 100ug/ml 59 17 60 60
10927 RLB 2401 P02 A001 100ug/ml 19 2 49 41
10928 RLB 2401 P02 A002 100ug/ml 0 2 25 17
10929 RLB 2401 P02 A003 100ug/ml 0 14 12 13
10930 RLB 2424 P14 A001 100ug/ml 64 42 59 62
10931 RLB 2424 P14 A002 100ug/ml 62 32 56 56
10932 RLB 2424 P14 A003 100ug/ml 0 12 0 0
10933 RLB 2425 P14 A001 100ug/ml 0 9 6 9
10934 RLB 2425 P14 A002 100ug/ml 0 4 5 22
10935 RLB 2425 P14 A003 100ug/ml 0 13 0 16
10943 IHB 1423 P01 A001 100ug/ml 79 17 80 66
10944 IHB 1423 P01 A002 100ug/ml 17 2 25 9
10945 IHB 1423 P01 A003 100ug/ml 90 43 95 93
10946 IHB 1423 P03 A003 100ug/ml 68 33 68 77
10947 IHB 1423 P04 A001 100ug/ml 91 38 94 86
10948 IHB 1423 P04 A002 100ug/ml 83 32 90 73
10949 IHB 1423 P04 A003 100ug/ml 77 19 91 79
10950 IHB 1423 P14 A001 100ug/ml 89 43 95 83
10951 IHB 1423 P14 A002 100ug/ml 82 18 91 75
10952 IHB 1423 P14 A003 100ug/ml 94 14 99 94
10953 IHB 1424 P13 A001 100ug/ml 88 4 70 42
10954 MAP 2601 P13 A001 100ug/ml 61 17 57 34
10955 MAP 2601 P13 A002 100ug/ml 0 16 47 13
10956 MAP 2601 P13 A003 100ug/ml 95 54 98 95
10957 MAP 2603 P02 A001 100ug/ml 60 35 44 49
10958 MAP 2603 P02 A002 100ug/ml 18 27 14 29
10959 MAP 2603 P02 A003 100ug/ml 89 34 40 72
10960 MAP 2603 P03 A001 100ug/ml 13 24 27 35
10961 MAP 2603 P03 A002 100ug/ml 35 26 30 27
10962 MAP 2603 P03 A003 100ug/ml 3 9 10 7
10963 MAP 2609 P13 A001 100ug/ml 17 14 37 12
10964 MAP 2609 P13 A002 100ug/ml 16 20 25 13
10965 MAP 2609 P13 A003 100ug/ml 15 0 0 28
10460 RJO 1932 P13 A001 100ug/ml 47 6 37 36
10461 RJO 1932 P13 A002 100ug/ml 14 2 23 12
10462 RJO 1932 P13 A003 100ug/ml 68 20 50 75
10463 RJO 2351 P13 A003 100ug/ml 0 30 0 14
10464 RJO 2360 P13 A001 100ug/ml 11 21 39 39
5-FU 1X10-6M 44 31 24 33

26
 PACLI 1X10-5M 64 60 88 48
MITO-C 1X10-5M 49 23 69 29

DISCUSSION:
Remarks : Stock solution (20 mg/ml)was prepared in DMSO,50%DMSO and
water and further dilution was carried out with medium. In vitro cytotoxicity against
4 human cancer cell lines was determined using SRB assay.
Criteria of activity: >70% growth inhibition at 100 µg/ml against three cell lines
Discussion : All the samples have been evaluated at 100 µg/ml and eight(IHB 1423
P01 A003, IHB 1423 P14 A002, IHB 1423 P14 A003, IHB 1423 P04 A001, IHB 1423 P04
A003, IHB 1423 P14 A001, IHB 1423 P04 A002, MAP 2601 P13 A003) samples show such
growth inhibition.

27
CALCULATIONS AND DISCUSSIONS:

The viability and growth in the presence of test material is calculated as:

OD Change = Mean of OD of Test sample – Mean of OD of Blank

% Growth in presence of the Control = 100 / OD change in presence of control

% Growth in the presence of Test sample = (% Growth of in presence of control) X

(OD change in presence of test sample)

% Inhibition by the Test sample = 100 - % Growth in the presence of Test sample

Growth in presence of test material

= ------------------------------------------------------- X 100
Growth in absence of test material
T / C value for 50% growth inhibition for each test material is calculated from its
three concentrations

Criteria for activity:

The test material showing >70% activity at 100ug/ml is considered to be active

28
29