pranjal manuscript jan28th dastagiri give 2003

8/9/2019 Pranjal Manuscript Jan28th Dastagiri Give 2003

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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). Cellcycle 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


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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


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MTT dye for determining invitro cytotoxicity at 420nm. At 4M 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 4M 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 4 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


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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 of4fand

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

Dulbeccos modified Eagles 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.


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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 100l media followed by addition of 10l 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 4M 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 250l 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

manufacturers recommendations. A375 cells were treated with compounds 4a, 4f, 4g, 4i and

DC-81 at 4 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

manufacturers recommendations. A375 cells were treated with compounds 4a, 4f, 4g, 4i and

DC-81 at 4M concentrations as obtained from FACS analysis. Here the substrate and inhibitor


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(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.


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Figure 1: Effect of PP-PBD compounds (4a, 4f, 4g and 4i) on cell viability (in

vitrocytotoxicity). A375 cells were treated with 4 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).


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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 M 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 4 concentration for 24 h. DC-81 was used as the positive

control. Con+DMSO is control cells treated with DMSO.


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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 M 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 M concentrations


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for 24 h. The cell lysates were collected and expression levels p53, p21, Chk2 and pRb were

determined by western blot analysis. -actin was used as loading control.

Figure 4. Effect of PBD compounds on TNF-R1, cytochrome- c, cleaved PARP and Bax levelsA375 cells were treated with compounds 4a, 4f, 4g, 4i and DC-81 at 4M 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. -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 4 concentrations for 24 h. The cell lysates were collected and

expression levels of Cdk2 were determined by western blot analysis. -actin

was used as loading control.


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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


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hybrids (4a, 4f, 4g and 4i) at 4 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.


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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.

pranjal manuscript jan28th dastagiri give 2003

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