Oxadiazoles as potential cytotoxic compounds

Abstract

To better understand the mechanism underlying anti-cancer activity mediated by PBD hybrids,
we performed assays to observe inhibition of cell proliferation, cell cycle distribution, the level
of cell cycle control related protein (p53, p21/WAF1, Chk1, chk2, CDK2) and apoptosis
associated TNFR1, Bax, cytochrome c, caspase-9, caspase-3 and PARP molecules. These factors
have been shown to be strongly associated with the programmed cell death signaling pathway
and are known to be involved in chemosensitivty. This study contributes to the chemoprevention
of melanoma cells (A375) mediated by PBD hybrids with 4i compound (oxadiazoles having two
methoxy groups) being the most effective among all PBD hybrids studied.

Introduction

Anticancer drugs have been shown to modulate the cellular functions in causing cell death in
chemosensitive tumors. Activation of certain genes such as p53 was found to be important in the
regulation of apoptotic pathway induced by various stimuli (Taguchi et al., 2004). Cell death
also called as apoptosis occurs by two pathways. The extrinsic pathway mediated by death
receptors such as Fas and TNF receptors, whereas the intrinsic pathway is mediated by
mitochondria, an important organelle of the cell (Ashkenzai and Dixit, 1998). A number of pro
and antiapoptotic members of Bcl2 protein family regulate the release of cytochrome c from
mitochondria into cytosol. Cytochrome c interacts with procaspase-9 and Apaf-1 to activate
caspase-9 and then switch on caspase-3, 6 and 7 leading to apoptosis (Hengartner, 2000).
Apoptosis is a phenomenon that is characterized by drastic changes in cell morphology,
chromatin condensation, membrane blebbing, nuclear breakdown and cleavage of Poly (ADP-
ribose) Polymerase (PARP), a repair enzyme by caspaase-3 (Berger and Petzold, 1985). Cell
cycle progression is governed by cyclin dependent kinases (CDKs) that are activated by cyclin
binding and inhibited by CDK inhibitor (Sheer and Roberts, 1999). CDK inhibitors are p21 cip1
(CDKNIA) and p27Kip1 (CDKNIB). Drugs which inhibit CDK2 and arrest cell cycle may reduce
the sensitivity of epithelium to many cell cycle active antitumor agents. Members of the Rb
family are important substrates of CDKs and the cell cycle progression at the G1 check point
coincides with phosphorylation and consequent inactivation of Rb protein (obaya and Sedivy,
2002). Induction of p53, a sequence specific transcriptional activator results in expression of a
number of gene products whose function is to either arrest cell growth or promote apoptosis
(Morgan and Kastan, 1997). In response to DNA damage, p53 activity enhances the rate of
transcription of numerous target genes such as p21 and Bax that mediate the plethora of p53-
dependent functions. These functions include the promotion of apoptosis and the induction of G1
cell cycle arrest (el-Deiry, 1998). Melanoma is the malignant types of cancer of melanocyte and
is the most common cancer and rated fifth in man and sixth in woman in United States (Jemal et
al., 2001).

The retinoblastoma gene product (Rb) was first identified as the tumor suppressor
because it is absent or mutated in many human tumors and also has been shown that Rb binds to
E2F and repress the transcription of genes required for the G1 to S phase transition. It is also
known that phosphorylation of Rb can promote cells to proceed to S-phase. When p21, which is
a CDK inhibitor (CDKI) is up regulated then phosphorylation is inhibited and finally leading to
cell cycle arrest at G1 phase (Weingberg, 1995). Upon DNA damage caused by ionizing
radiation (IR), ultraviolet (UV), anticancer chemicals or replication stress the DNA damage
checkpoint blocks the cell cycle at multiple junctions such as G-S and G2-M transitions (Kastan
and Bartek, 2004). This gives an opportunity for the cell to repair the damaged DNA or commit
apoptosis, thus thereby preventing the passage of genetic interactions to the next generation. At
G1 and G2 transitions both Chk1 and Chk2 respectively play a pivotal role. Both Chk1 and
Chk2 activates p53 by phosphorylation which leads to stabilization and accumulation of p53
leading to elevated activation of transcription factor p21, a CDK inhibitor thereby causing G1
cell cycle arrest and apoptosis (Hongtao, 2007). In this paper we have demonstrated the induced
expression of tumor suppressor by these anti-cancer compounds to control cell cycle arrest and
apoptosis.

Results and Discussion

Superior invitrocytotoxicity mediated by PBD hybrids (Oxidiazoles) than DC-81

To investigate the cytotoxic effect mediated by PBD hybrids in A375 cells, WST-1
invitrocytotoxicty was performed and is considered to be more sensitive than already existing
MTT dye for determining invitro cytotoxicity at 420nm. At 4µM concentration these PBD
hybrids have exhibited pronounced cytotoxicity and were found to be more effective than DC-
81, a positive control used.

Cell Cycle effects

The effect of PBD hybrids at 4µM concentration on cell cycle progression was studied in A375
cells using FACS analysis. The control cells showed 56.22% of cells in G0/G1. Dc-81which is a
positive control showed 89% of G0/G1 phase. The compound (4a, 4f, 4g and 4i) treated cells
have shown 80.38%, 91.22%, 87.42% and 95.7% cells arrested in G0/G1 phase of cell cycle.
This data has clearly revealed the cell cycle arrest mediated by PBD hybrids are at G1 phase with
4i showing potent activity on G1 cell cycle arrest and apoptosis as indicated by cells in G0 phase
(subG1 phase).

Enhanced apoptosis in PBD hybrid compound treated tumor cells.

Because hypodiploid DNA content (sub G1 material) is characteristic of apoptosis, treatment of

A375 cells with 4µΜ agents for 24h induced apoptotic effects of 1.59% with control, 15% with
DC-81, 6.15%, 18.37%, 13.29% and 35.71% with 4a, 4f, 4g and 4i compounds. 4i was more
efficient in causing highest subG1 accumulation than DC-81. The cells accumulated in subG1
phase indicated apoptotic cells.

Effect of PBD hybrids on the expression of tumor suppressor proteins

FACS data revealed the G1 cell cycle arrest caused by these PBD hybrid compounds with 4i
being the most potent one. It is known that G1/S check point is regulated by tumor suppressors
such as p53, pRb and Chk2. Moreover studies related to these tumor suppressors may provide
important clues in anticancer approach. The tumor suppressor protein p53 regulates the
transcription p21 which in turn leads to the inhibition of CDK2 and CDK4 and eventually leads
to cell cycle arrest at G1phase (Sampath and Plunkett, 2001). Moreover increased retinoblastoma
protein (pRb) can enforce a G1 block by suppressing the E2F responsive promoters. The
restoration of the function of retinoblastoma protein can lead to the blockade of G1/S phase of
cell cycle (Kaelin, 2009). Chk2 is a tumor suppressor protein and has been implicated in G1
phase of cell cycle and is predicted to act downstream of ATM to stabilize and activate the p53
dependent response by phosphorylating p53 on ser-20 (Shieh et al., 2000). Interestingly
Hongtao, 2007 demostrated that activated chk1 protein control the G1 check point during DNA
damage. A375 cells were treated with PBD hybrids at 4µM concentration for 24h and cell lysates
were subjected to western blot analysis and probed with antibodies against p53, p21, Chk1,Chk2
and Rb. The expression pattern of these tumor suppressor proteins was more in cells treated with
4i compound and revealed the importantance of this compound in controlling cell proliferation
and apoptosis.

Effect of PBD hybrids on the expression of apoptotic proteins

We were further interested to see the proteins that are invoved in the apoptosis caused by these
compounds ( Oxadiazole PBD hybrids). Apoptosis refers to programmed cell death in response
to various intrinsic or extrinsic death signals (Nicholson and Thornberry, 1997). Extrinsic
pathway is mediated by death signals such as Fas, TNF receptors etc. Intrinsic pathway involves
mitochondria that play an important role in regulation of apoptosis (Green and Reed, 1998). To
investigate the possible involvement of extrinsic pathway, we checked the expression of tumor
necrosis factor receptor in the compound treated cells by western blot analysis. To our surprise
DC-81, 4a, 4f, 4g have shown upregulation of TNFR expression but cells treated with 4i
compound have shown negligible upregulation (or almost equal to untreated cells) since 4i was
found to be most effective compound deduced from FACS analysis and western studies of tumor
suppressor protein expression. Previous studies revealed that a number of pro and anti-apoptotic
members of the Bcl2 protein family regulate the release of cytochrome c from mitochondria into
cytosol. Cytochrome c interacts with Procaspase-9 and Apaf-1 to activate caspase-9 and then
switch on caspase-3 leading to apoptosis (Hengartner, 2000). Further we have investigated the
possible involvement of mitochondria in this apoptotic event. As part of intrinsic pathway we
first analysed the release of cytochrome c from mitochondria. An increased level of cytochrome
c protein was observed in all the compound treated cells. Among all the compounds 4i
compound treated cells have shown maximum increase in cytochrome c protein expression.
Further we have also checked other mitochondria mediated apoptotic proteins such as Bax and
PARP. Pronounced upregulation of Bax and PARP was observed particularly in the case of 4i
compound treated cells than control cells. Here DC-81 was used as positive control. The above
data clearly showed the existance of intrinsic pathway in the apoptosis caused by these
compounds

PBD hybrids activate caspase-3 and caspase-9

Caspase-3 and 9 are important executioners of apoptosis (Hengartner, 2000) with caspase-3
being the most effective one. This led us to check the levels of caspase-3 and 9 in the compound
treated cells. Interestingly casapase-9 level was upregulated to 4-fold and 7-fold in case of 4f and
4i compound treated cells than untreated control cells. The caspase-3 levels were upregulated to
2 folds and 3.5 folds in case of 4f and 4i compound treated cells. In both the cases use of
inhibitor has shown drastic reduction in the activity of respective caspase, showing the
specificity of this assay. This assay has clearly shown the pivotal role of mitochondria in the
apoptotic event with 4i being the most effect compound.

Effect of PBD hybrids on the expression of CDK2

We further proceed to observe cyclin-dependent kinase needed for cell proliferation G1 to S

phase transition. The loss of cell cycle control and deregulated cell proliferation is one of the
hallmarks of cancer. The cell cycle progression is regulated by the activities of cyclin dependent
kinases (CDKs) and their subunits known as cyclins (Kong et al., 2003). CDK2 is the crucial
controller of cell cycle progression and associates with cyclin E and cyclin A and controls the
cell cycle progression from G1 to S phase (Akiyama et al., 1992). This led us to check the CDK2
protein levels after compound treatment. To our surprise the 4i compound has shown almost
complete inhibition of the CDK2 levels. This clearly showed the anti-cancer nature of 4i, as it
has shown effective CDK2 inhibition.

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Experimental Section

Cell lines
A375 (Human melanoma cells) was obtained from ATCC, USA. A375 cells were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen), supplemented with 10% fetal calf
serum and 100 U/ml Pencillin and 100mg/ml streptomycin sulfate (Sigma). The cells were
passaged and maintained at 37oC in a humidified atmosphere containing 5% CO2.
Cell viability (MTT Assay)
Cell viability was assessed by the MTT based assay using WST-1 (premix WST-1 cell
proliferation Assay system, Takara), is more sensitive than MTT. Briefly, A375 cells were
seeded in a 96-well plate (TPP) at a cell density of 10,000cells/well. After overnight incubation,
the cells were treated with compounds 4a, 4f, 4g, 4i and DC-81 and incubated for 24 h. The
medium was then discarded and replaced with fresh 100µl media followed by addition of 10µl of
WST-1 dye. Plates were incubated at 37oC for 30 min. Optical density (O.D.) was read at 420
nm using Multimode Varioskan FLASH (Thermoscientifics).
Cell Cycle Analysis
5 X 105 A375 cells were seeded in 60 mm dish and were allowed to grow for
24 h. Compounds 4a, 4f, 4g and 4i and DC-81 at 4µM concentration were added to the culture
media and the cells were incubated for an additional 24 h. Harvesting of cells was done with
Trypsin-EDTA, fixed with ice-cold 70% ethanol at 4oC for 30 min, washed with PBS and
incubated with 1 mg/ml RNaseA solution (Sigma) at 37oC for 30 min. Cells were collected by
centrifugation at 2000 rpm for 5 min and further stained with 250µl of DNA staining solution
[10 mg of Propidium Iodide (PI), 0.1 mg of trisodium citrate, and 0.03 ml of Triton X-100 were
dissolved in 100ml of sterile MilliQ water at room temperature for 30 min in the dark]. The DNA
contents of 20,000 events were measured by flow cytometer (DAKO CYTOMATION, Beckman
Coulter, Brea, CA). Histograms were analyzed using Summit Software.
.
Caspase-3assay
We have used Apoalert caspase -3 fluorescent assay kit (Clonetech, CA) according to the
manufacturer’s recommendations. A375 cells were treated with compounds 4a, 4f, 4g, 4i and
DC-81 at 4µ M concentrations as obtained from FACS analysis. Here the substrate and inhibitor
(I) used are DEVD-AFC and DEVD-CHO respectively. The DEVD-AFC substrate, DEVD-
AFC+ DEVD-CHO is added to the cell lysate and incubation was carried out at 37oC for 1h.
Readings were taken at excitation wavelength 400 nm and emission wavelength 505 nm.
Caspase-9 assay
We have used Apoalert caspase 9/6 fluorescent assay kit (Clonetech, CA) according to the
manufacturer’s recommendations. A375 cells were treated with compounds 4a, 4f, 4g, 4i and
DC-81 at 4µM concentrations as obtained from FACS analysis. Here the substrate and inhibitor
(I) used are LEHD-AMC and LEHD-CHO respectively. The LEHD-AMC substrate, LEHD-
AMC + LEHD-CHO is added to the cell lysate and incubation was carried out at 37oC for 1h.
Readings were taken at excitation wavelength 380 nm and emission wavelength 460 nm.

Protein Extraction and Western Blot Analysis

Total cell lysates were isolated from cultured A375 cells after compound treatments as
mentioned earlier were obtained by lysing the cells in ice-cold RIPA buffer (1XPBS, 1%NP-40,
0.5% sodium deoxycholate and 0.1% SDS containing protease inhibitors). After centrifugation at
12,000 rpm for 10 min, the protein in supernatant was quantified by Bradford method (BIO-
RAD) using Multimode varioscan instrument (Thermo-Fischer Scientifics). Thirty micrograms
of protein per lane was applied in 12% SDS polyacrylamide gel. After electrophoresis, the
protein was transferred to polyvinylidinedifluoride (PVDF) membrane (Amersham Biosciences).
The membrane was blocked at room temperature for 2 h in TBS + 0.1% Tween20 (TBST)
containing 5% blocking powder (Santacruz). The membrane was washed with TBST for 5 min,
and primary antibody was added and incubated at 4oC overnight (O/N). Rabbit polyclonal beta-
actin, Chk1 and Chk2, mouse monoclonal cytochrome c and cleaved PARP were obtained from
Imgenex. Rabbit polyclonal Bax (p-19), TNFR1 (H271), Cdk2 (M2) and mouse monoclonal p53
(pab1801) were from santa cruz and p21 antibody was obtained from upstate. After three TBST
washes, the membrane was incubated with corresponding horseradish peroxidase-labeled
secondary antibody (1:2000) (Santa Cruz) at room temperature for 1h. Membranes were washed
with TBST three times for 15 min and the protein blots were visualized with chemiluminescence
reagent (Thermo Fischer Scientifics Ltd.). The X-ray films were developed with developer and
fixed with fixer solution.
Figure 1: Effect of PP-PBD compounds (4a, 4f, 4g and 4i) on cell viability (in
vitrocytotoxicity). A375 cells were treated with 4µΜ concentration of PBD hybrid compounds as
indicated for 24 h in 96-well plates seeded with 10,000 cells per well. O.D readings were taken at
420nm wavelength to measure the percentage of cell viability after treatment with the respective
compound. DC-81 was used as the positive control. Control: control cells (untreated cells).
Figure 2a. DNA histograms obtained by flowcytometry and the percentages of cells in
sub G0, G1, S, and G2/M cell cycle phase, after the treatment of A375 cells with 4a, 4f, 4g and
4i at 4 µM concentration for 24 h.

Figure 2b. FACS analysis of cell cycle distribution of A375 cells after treatment with PBD
conjugates (4a, 4f.4g and 4i) at 4µΜ concentration for 24 h. DC-81 was used as the positive
control. Con+DMSO is control cells treated with DMSO.
Figure 2c. DNA histograms obtained by flow cytometry and the percentages of cells in
sub G0, indicating the apoptotic cells, after the treatment of A375 cells with 4a, 4f, 4g and
4i at 4 µM concentration for 24 h.

Figure 3. Effect of PBD compounds on the expression of p53, p21, Chk2, pRb and Chk1 protein
levels. A375 cells were treated With PBDs at 4a, 4f, 4g, 4i and DC-81 at 4 µM concentrations
for 24 h. The cell lysates were collected and expression levels p53, p21, Chk2 and pRb were
determined by western blot analysis. β-actin was used as loading control.

Figure 4. Effect of PBD compounds on TNF-R1, cytochrome- c, cleaved PARP and Bax levels
A375 cells were treated with compounds 4a, 4f, 4g, 4i and DC-81 at 4µM concentrations for 24
h. The cell lysates were collected and expression levels of TNFR-1, cytochrome c, cleaved
PARP and BAX were determined by western blot analysis. β-actin was used as loading control.

Figure 5. Effect of PBD compounds on Cdk2 levels. A375 cells were treated with compounds
4a, 4f, 4g, 4i and DC-81 at 4µΜ concentrations for 24 h. The cell lysates were collected and
expression levels of Cdk2 were determined by western blot analysis. β-actin
was used as loading control.
Figure 6a.

Figure 6b

Figure 6a and 6b. Effect of PBD conjugates on caspase-3 and caspase-9 activities in A375 cells.
The increased enzymatic activity of caspse-3 and 9, in apoptosis after the treatment of PBD
hybrids (4a, 4f, 4g and 4i) at 4µΜ concentration was determined by flourimetry. The cleavage of
peptide by caspase-3 releases the fluorophore AFC that was quantified at excitation wavelength
of 400 nm and emission wavelength of 505nm. The cleavage of peptide by caspase-9 releases the
fluorophore AMC that was quantified at excitation wavelength of 380 nm and emission
wavelength of 460 nm. ‘I’ represent the inhibitor used. DEVD-CHO is the inhibitor in case of
caspase-3 and LEHD-CHO is the inhibitor in case of caspase-9. DC-81 was used as the positive
control.
Figure 7. Oxadiazoles activates p53 and Rb and transactivates p21 gene. Activated p21 inhibits
CDK2 protein expression thereby causing G1 cell cycle arrest and finally causing apoptosis
mediated by p53 with the involvement of mitochondria.