Inhibition of cancer progression by a novel trans-stilbene derivative through disruption of microtubule dynamics, driving G2/M arrest, and p53-dependent apoptosis

Resveratrol, a trans-stilbene polyphenolic compound and its synthetic analogs are widely used bioactive molecules due to their remarkable chemo-preventive potential. Here, we have identified a novel synthetic trans-stilbene compound, Z-DAN-11 ((Z)-3-(3, 4-dimethoxyphenyl)-2-(3, 4, 5-trimethoxyphenyl) acrylonitrile) which shows remarkable efficacy in blocking tumor growth and progression both in vitro and in vivo. Z-DAN-11 inhibits proliferation of cancer cells in vitro through microtubule depolymerization that induced G2/M arrest and consequently leads to apoptotic cell death. More importantly, Z-DAN-11 shows limited cytotoxicity to normal cells as compared to cancer cells. Quite interestingly, we have found that Z-DAN-11-mediated ROS production helps in dramatic alteration in the mitochondrial redox status which critically contributes to the apoptosis. Mechanistic studies reveal that Z-DAN-11 induces the expression of pro-apoptotic proteins and decreases anti-apoptotic protein expression that decisively helps in the activation of caspase 8, caspase 9, and caspase 3, leading to cleavage of PARP1 and cell death via intrinsic and extrinsic pathways of apoptosis. Moreover, Z-DAN-11-mediated apoptosis of cancer cells is through a partial p53-dependent pathway, since both HCT116 p53−/− cells as well as p53-silenced cells (siRNA) were able to block apoptosis partially but significantly. Importantly, Z-DAN-11 also imparts its anti-tumorigenic effect by inhibiting clonogenic property and anchorage-independent growth potential of cancer cells at concentrations at least 10 times lower than that required for inducing apoptosis. Finally, in vivo study with immuno-competent syngeneic mice shows Z-DAN-11 to be able to impede tumor progression without any adverse side-effects. Hence, we identified a novel, synthetic trans-stilbene derivative with anti-tumorigenic potential which might tremendously help in devising potential therapeutic strategy against cancer.


Introduction
Among the various interventions used therapeutically against cancer, chemotherapy along with radiotherapy has been the most widely used and potential strategies for treating this disease 1,2 . Although, an ideal chemotherapeutic drug should target a cancer cell by acting against a cancer-specific receptor, protein or DNA, in reality, however, chemotherapeutic applications are limited primarily owing to their adverse effects on the normal cells 3,4 . This has increasingly led to studies aimed towards identification of novel compounds, preferably from natural sources having anti-cancer potential with minimal collateral damages to normal cells. Among them, polyphenols, many of which are found to be abundantly available in edible fruits, have shown promise 5,6 ; however, only a few of these went past laboratory studies to being potentially therapeutic against cancer 7 . In recent years, trans-stilbene polyphenolic compounds like Resveratrol have gained tremendous attention due to their diversified biological effects [8][9][10] . Several methoxy and hydroxy derivatives of trans-stilbene have shown to possess anticancer activity in various human cancer cells through growth suppression by inducing cell cycle arrest and apoptosis [11][12][13] . Hence, the need to identify potential leads that can block cancer progression through targeted blocking of cells in particular phase of cell cycle 14 .
In the present study, we synthesized a number of trans-stilbene derivatives in an attempt to identify potential anti-cancer leads which is able to deliver a multi-modal yet targeted anti-cancer therapy, through affecting some of the key cellular processes primarily involved in carcinogenesis. Here, we evaluated the anticancer potential of 22 synthetic trans-stilbene derivatives using several cancer cell lines and identified one lead, Z-DAN-11 ((Z)-3-(3, 4-dimethoxyphenyl)-2-(3, 4, 5-trimethoxyphenyl) acrylonitrile), based on its ability to inhibit proliferation of cancer cells but not normal cells. Z-DAN-11 caused G2/M arrest in cancer cells, primarily by interfering with tubulin polymerization leading to disruption of microtubule formation. Furthermore, Z-DAN-11 was also able to induce genomic instability by DNA damage and consequent activation of p53 leading to activation of the caspase cascade eventually inducing apoptosis. In vivo, Z-DAN-11 demonstrates dose-dependent reduction in tumor burden and tumor progression in Balb/c mice without any significant hepatotoxicity. Hence, trans-stilbene derivative, Z-DAN-11 holds tremendous potential as an ideal lead towards targeted cancer chemotherapy, which either alone or in combination with existing chemotherapy may be used as a potential strategy towards cancer treatment.

Results
Synthesis and comparative cytotoxic efficacies of transstilbene derivatives (Z)-2, 3-diarylacrylonitrile (trans-stilbene) derivatives were synthesized as described earlier 8 . Primarily, the series of compounds (Z-DAN-1 to Z-  shown in Fig. S1 were assessed for their ability to block proliferation of HeLa, MCF-7 (cancer cells), and NKE cells (normal kidney epithelial cell). The anti-proliferative efficacies of the tested compounds in terms of IC 50 were presented in Table S1, which showed three compounds Z-DAN-10, Z-DAN-11, and Z-DAN-12 to possess higher efficacy in inhibiting proliferation of HeLa as well as MCF-7 cells compared to the other derivatives. However, Z-DAN-11 displayed the highest selectivity index (SI) in both MCF-7 (SI = 7.26 ± 0.43) and in HeLa (8.08 ± 0.58) cells over Z-DAN-10 and Z-DAN-12. Thus, based on these results Z-DAN-11 was selected as a potent anti-cancer lead.
Z-DAN-11 interferes with cell cycle progression by inducing G2/M arrest Cycle distribution profile of various cancer cells in response to Z-DAN-11 clearly showed dose-dependent and time-dependent induction of G2/M arrest in both A549 (Fig. 2a,b) and MCF-7 cells (Fig. 2c,d). Interestingly, increase in the proportion of cells in G2/M phase was accompanied by decrease in cell population of G1 phase ( Fig. 2a-d). Similar results were obtained in HeLa cells (Fig. S2a) however; MDA-MB-231 cells (Fig. S2b) showed significant but comparatively lower G2/M arrest. We also observed significant time-dependent increase in phospho-histone H3(Ser10) indicative of mitotic cells with condensed DNA 15 , in presence of 10 µM of Z-DAN-11 (Fig. 2e,f and Fig. S2c). However, MCF-7 and A549 cells showed relatively higher pH3-ser10-positive cells as compared to MDA-MB-231 which correlated with propidium iodide staining. Collectively these results suggest that Z-DAN-11 treatment induce G2/M arrest thereby playing a critical role in blocking cellular proliferation.  . d, e Dose-dependent increase in trypan blue positivity indicating significantly higher cytotoxic ability of the Z-DAN-11 in MCF-7 and A549 cells, as compared to normal cells NKE and WI-38 respectively. f, g Photomicrographs showing dose-dependent (0-10 µM) anti-proliferative effect of Z-DAN-11 in MCF-7 and A549 cells respectively. For all experiments, 0.2% DMSO served as vehicle control. All the data represent mean ± SEM of minimum three independent experiments (*p < 0.05**p < 0.01, ***p < 0.001, ns not significant) Z-DAN-11 induces differential expression of distinct gene sets involved in cell cycle and cell death-related processes In order to observe the systemic impact of Z-DAN-11 treatment in gene expression, we did transcriptome analysis of Z-DAN-11 treated MCF-7 cells at 6 and 12 h, along with untreated and DMSO treated cells using microarray. Comparative analysis revealed that Z-DAN-11 treatment led to time-dependent increase in the were treated with Z-DAN-11 in a dose-dependent (0, 0.5, 1, 2.5, 5, and 10 µM) as well as time-dependent (6, 12, 24 h) manner. Post treatment, cells were harvested and fixed with ethanol followed by PI staining to check cellular distribution in different phases of the cell by flow cytometry. Histograms representing dose-dependent and time-dependent increase in the proportion of A549 cells a, b and MCF-7 cells c, d in G2/M phase along with decrease in G1 phase in response to Z-DAN-11. One-way ANOVA followed by Dunnett's t-test was performed to comparisons of multiple group means (treatments) versus DMSO control. Data are representative of at least three independent experiments and bar graph shows mean ± SEM (*p < 0.05**p < 0.01, ***p < 0.001, ns not significant). Effect of Z-DAN-11 on mitotic DNA condensation marker p-H3 ser10 proteins in e A549 and f MCF-7 cells. Here, cells were incubated with Z-DAN-11 (10 μM) for the indicated time points and fixed with 3.7% formaldehyde. Post fixation cells were permeabilized with triton-X and incubated with anti-p-H3 ser10 primary antibody followed by incubation with Alexa-Fluor 594-conjugated secondary antibody. Counter staining was done using DAPI to visualize the nuclei. Images were acquired in Leica confocal microscope (Magnification: ×40) number of differentially regulated genes (DRGs), however the overall numbers of DRGs were highest at 12 h (Fig. 3a). Hierarchical clustering of the cell samples based on their gene expression, along with a heat map, demonstrate a distinct differential gene expression signature among the Z-DAN-11 treated and control samples Z-DAN-11-mediated disruption of micro-tubular dynamics leads to G2/M cell cycle arrest Microarray data revealed altered expression of several genes involved in cell cycle regulation as well as cell death-related processes in response to Z-DAN-11 in MCF-7 cells, including but not limited to β-tubulin, which were validated by q-PCR ( Fig. S3a-b). Both microarray as well as RT-PCR demonstrated comparable fold changes in the expression of these genes (Fig. S3b). Our western immunoblot and immunofluorescence data showed reduced expression of β-tubulin (Fig. 3e,g) but not αtubulin ( Fig. 3g and Fig. S2d), which play critical role during the M-phase of cell division process 16 . We observed microtubule networks in DMSO as well as paclitaxel (a drug known to stabilize tubulin polymers 17,18 ) treated condition in MCF-7 and A549 cells. On the contrary, there was a time-dependent decrease in the expression of β-tubulin indicative of disrupted microtubule networks in Z-DAN-11 (10 μM) treated cells similar to those treated with the drug, nocodazole 19 (Fig. 3e). Similar results were obtained with MDA-MB-231 cells (Fig. S2e). Further, confirmation of tubulin depolymerization by Z-DAN-11 was obtained from kinetic loop experiment with purified porcine tubulin, which shows that Z-DAN-11-mediated (V max = 13 mOD/ min) tubulin polymerization was comparatively lower than the control condition (V max = 21 mOD/min) while with paclitaxel (V max = 36 mOD/min.), polymerization efficiency was much higher (Fig. 3f). These results indicate that Z-DAN-11 interferes with microtubule polymerization process which leads to G2/M arrest. Interestingly Schneider et al. discussed inhibition of tubulin polymerization for other Resveratrol derivative which is structurally different from Z-DAN-11 but alike Z-DAN-11 it causes G2/M arrest through inhibition of tubulin polymerization 20 . Moreover, western blot analysis of critical cell cycle regulatory proteins (Fig. 3g) show significant time-dependent decrease in the expression level of total Cdc2 along with phospho-cdc2 (Tyr15) in response to Z-DAN-11 treatment in both MCF-7 (Fig. 3g, left panel) and A549 cells (Fig. 3g, right panel). While cyclin B1 expression level initially increased up to 24 h, significant decrease in cyclin B1 levels were observed post 36 h treatment with the compound (Fig. 3g). Phosphocyclin B1 (S126) also followed quite similar expression pattern as cyclin B1 both in MCF-7 and A549 cells. Cdk2 and phospho-cdk2 expression also showed a timedependent decrease in response to Z-DAN-11 treatment, with decrease mostly evident at late time points. Again, while Z-DAN-11 treatment did not affect the expression of cyclin A1 in MCF-7, time-dependent decrease in cyclin A1 was observed in A549 after 36 h (Fig. 3g). Briefly, cells were treated with Z-DAN-11 (10 μM) for the indicated time points and fixed with 3.7% formaldehyde. Post fixation cells were permeabilized with triton-X and incubated with βtubulin antibody. Alexa-Fluor 594-conjugated secondary antibody was used followed by counter staining with DAPI. Finally, images were acquired in Leica confocal microscope (magnification: ×63). f Effect of Z-DAN-11 on in vitro tubulin polymerization. Purified porcine tubulin was incubated with or without Z-DAN-11 or paclitaxel in tubulin polymerization buffer and kinetic loop study was carried out. g Western immunoblot data depicts Z-DAN-11-mediated alternation in the expression of cell cycle-related proteins including α-tubulin and β-tubulin, cyclin A1, cyclin B1, phospho-cyclin B1 (S126), cdc2, phospho-cdc2 (Tyr15), cdk2, and phospho-cdk2 (Tyr15) in MCF-7 (left panel) and A549 cells (right panel) Briefly, cells grown on coverslips were fixed with 3.7% formaldehyde, were washed with 1× PBS and DAPI staining was done. Coverslips with stained cells were mounted on slides and observed in Leica confocal microscope staining also revealed profound DNA damage caused by Z-DAN-11 (Fig. S4c). Moreover, we investigated the effect of Z-DAN-11 on mitochondrial damage by JC-1 staining which showed a drastic alteration of the redox status of cellular mitochondria in response to Z-DAN-11 in a concentration-dependent and time-dependent manner both in MCF-7 and A549 cells ( Fig. 4d and Fig. S5a).The shift of fluorescence from red to green or a decrease in the red/green ratio indicated the increase in the mitochondrial permeability in response to Z-DAN-11. In addition to that we also checked both cellular (  (Fig. 5c,d) like Bax, p21/cip, and cleaved caspase 3, and decrease in a number of anti-apoptotic (survival) proteins (Fig. 5c,d) namely, Survivin, Claspin, c-IAP, and Bcl-2. Although TNFR1 expression did increase significantly at 24 h (Fig. 5c,d), interestingly it decreased to almost normal levels by 72 h. Figure 5d represents mean pixel densities of each protein from two separate proteome profile experiments. A time-dependent induction in phosphorylation of p53 was observed at ser15, ser46, and ser392 (Fig. 5c,d) in Z-DAN-11 treated cells versus the control cells, indicating a possible p53 dependency in Z-DAN-11-mediated apoptosis. Thus, these results suggest induction of an apoptotic cascade involving an imbalance between proapoptotic and anti-apoptotic proteins, which may be p53 dependent.

Z-DAN-11-mediated apoptosis of cancer cells involve both intrinsic and extrinsic pathway
Since, mitochondrial damage ( Fig. 4d and Fig. S5a) play a critical role in apoptosis signaling 22 , the effect of Z-DAN-11 on the levels of key mediators regulating mitochondrial damage were assessed. Western blot data suggest increase in cytosolic AIF (apoptosis-inducing factor) expression with time in both MCF-7 as well as A549 cells time dependently in response to Z-DAN-11 (Fig. 6a), as well as over-expression of the pro-apoptotic proteins Bax, while Bcl-2 expression was significantly lowered (Fig. 6a). Z-DAN-11 also lowered the expression of anti-apoptotic proteins Survivin and Claspin (Fig. 6a). Since, Z-DAN-11induced DNA fragmentation ( Fig. 4c and Fig. S4b-c) expression profile of critical proteins associated with maintenance of genomic integrity was studied in response to treatment with the compound. Z-DAN-11 treatment showed significant decrease in Chek1 expression time dependently (Fig. 6a). More importantly, while level of p-chek1 (ser317) was elevated at early time points, significant decrease in p-chek1 was observed at later time points (Fig. 6a). Marked lowering in expression of Claspin, a DNA-binding protein critical for phosphorylation and activation of the Chek1 protein kinase by ATR in response to DNA damage 23 , was observed in Z-DAN-11-treated conditions versus control (Fig. 6a). These data were corroborated with an elevated expression of the DNA fragmentation marker γ-H2A.X in MCF-7 as well as A549 cells (Fig. 6a). A time-dependent induction in p53 as well as p-p53 ser15 was observed (Fig. 6a) which correlated to the proteome profiler data (Fig. 5c,d).The p21/cip expression increased time dependently while level of p-p21 (Thr145) increased at early time points but significant decrease was observed at later time points. TNFR1 showed profound increase in expression in response to Z-DAN-11 in MCF-7 as well as A549 cells (Fig. 6b), which correlated with time-dependent increase in both FADD and TRADD expression, suggesting formation of the death-inducing signaling complex (DISC). Confirmation of recruitment of the DISC was obtained from Co-IP data (Fig. 6c), where TNFR1 immunoprecipitated with both TRADD as well as FADD in a timesensitive manner, following Z-DAN-11 treatment in MCF-7 cells.
Induction of Z-DAN-11-mediated apoptosis involves activation of the caspase cascade leading to PARP1 cleavage The activation of caspase 3 was demonstrated by a timedependent increase in expression of corresponding cleaved caspase 3 accompanied with decrease in their proform both in MCF-7 and A549 cells (Fig. 6d) following treatment with Z-DAN-11(10 μM) at several time points (6-72 h). However, in both cells the activation of caspase 7 and caspase 8 were evident from decreased expression of their pro-form after 36 h of treatment (Fig. 6d). Further, Z-DAN-11 was found to downregulate pro-caspase 9 levels time dependently, indicating activation of caspase 9 and thereby suggesting mitochondrial involvement in Z-DAN-11-mediated apoptosis. The activity assay also confirmed activation of caspase 3/7 in MCF-7 and A549 cells at later time points (36,48, and 72 h) as shown in Fig. 6e. Additionally, cleavage mediated deactivation of PARP1 was observed in both MCF-7 as well as A549 cells (Fig. 6d). Importantly, MCF-7 and A549 pretreated with 20 µM each of the corresponding inhibitors for caspase 3 (Z-DEVD-FMK), caspase 8 (Z-IETD-FMK), and PAN-caspase (Z-VAD-FMK) conferred significant protection to the cells from Z-DAN-11mediated apoptosis as evidenced from Fig. 6f, confirming that Z-DAN-11-mediated tumor cell apoptosis was caspase dependent. Collectively these results suggest that Z-DAN-11 involves both the extrinsic and intrinsic pathway causing mitochondrial damage, activation of caspases leading to PARP1 cleavage, DNA damage, and eventually tumor cell apoptosis.

Z-DAN-11-mediated apoptosis of tumor cells is p53 dependent
Since, proteome profiler followed by western blot analysis indicated time-dependent phosphorylation of p53 (Fig. 5c,d and Fig. 6a), we wanted to confirm the role of p53 in Z-DAN-11-mediated apoptosis of tumor cells. While in p53 wt HCT116 cells, Z-DAN-11 caused a significant induction of apoptosis (>40% Annexin V+ve cells shown in Fig. 7a,b), in the p53 −/− HCT116 cells it is significantly lower (around 27% Annexin V+ve cells), thereby confirming that p53 plays a definitive role in Z-DAN-11-mediated death of tumor cells. This was further confirmed by silencing p53 levels in both A549 and MCF-7 cell line using specific siRNAs targeted against p53, which conferred significant protection from Z-DAN-11-mediated apoptosis, in A549 (nearly 50% lower than the scrambled, Fig. 7c) and MCF-7 (nearly 35% lower, Fig. 7d) p53-siRNA-transfected cells. Together, these experiments confirmed that Z-DAN-11-mediated tumor cell apoptosis is at least partly but significantly p53-dependent. Taken together, these results suggest Z-DAN-11 depolymerizes microtubules leading to G2/M arrest, and at the same time induce DNA damage, leading to p53-dependent induction of the apoptotic Fig. 7 Z-DAN-11-mediated apoptosis of tumor cells is p53-dependent. a Depicts a representative density plot showing that Z-DAN-11-mediated apoptosis is significantly reduced in HCT116 p53 −/− cells compared to HCT116 p53 wt cells along with b bar graph representing mean ± SEM (*p > 0.05, **p < 0.01, ***p < 0.001, ns not significant) of at least three independent experiments. Bar diagram showing that Z-DAN-11-mediated apoptosis is blocked in p53 siRNA transfected c A549 and d MCF-7 cells compared to the scrambled. Data are represented as mean ± SEM (*p > 0.05, **p < 0.01, ***p < 0.001, ns not significant). e A schematic signaling model proposing how Z-DAN-11-mediated proliferation inhibition is associated with G2/M arrest and apoptosis. Z-DAN-11 binds to tubulin and induces microtubule depolymerization which leads to G2/M arrest. On the other hand Z-DAN-11-induced DNA damage contribute to apoptosis through intrinsic and extrinsic pathways involving chek1, p53, TNFR1, TRADD, FADD etc. and downstream activation of caspase 8, caspase 9, and caspase 3 leading to apoptosis cascade that involves both the intrinsic as well as the extrinsic pathway (Fig. 7e).

Z-DAN-11 caused significant reduction in tumor growth and progression in immuno-competent mice
In vivo data shows a significant dose-dependent inhibition of tumor growth as evidenced from the reduction in tumor volume as shown in Fig. 8a, demonstrating that mice treated with either 10 mg/kg or 20 mg/kg b.w. of Z-DAN-11 have significantly smaller tumor size than the non-treated mice. This is also reflected in the weight of resected tumors from mice treated with Z-DAN-11, which shows dose-dependent decrease in the tumor weight compared to the nontreated mice (Fig. 8b). Z-DAN-11 also caused a dosedependent decrease in tumor progression with time, as shown in Fig. 8c. Microscopic analysis of tumor section with H and E staining showed compact arrangement of cells in control tumor section in contrast to loosely arranged cells in treated conditions. Additionally, more eosin-stained cells were observed in treated condition as compared to control tumor (Fig. 8d). However, there seems to be no significant change in total body weight of mice or the serum levels of alanine transaminase (ALT), aspartate transaminase (AST), creatinine, urea and alkaline phosphatase (ALP), either treated or non-treated, as shown in Fig. S6a. The pharmacokinetics of Z-DAN-11 showed 11.23 ± 0.68% of absolute bioavailability (Fig. S6b).
Z-DAN-11 reduced anchorage-independent growth (AIG) potential and clonogenicity of cancer cells Z-DAN-11 significantly inhibits colony forming ability of the tumor cells at concentrations as low as 250 nM in case of MCF-7 (Fig. 8e) and 500 nM in case of A549 cells (Fig. 8f), with a complete abrogation of colony forming ability of both cells at 1 µM dose of Z-DAN-11 (Fig. 8e,f). Moreover, Z-DAN-11 exhibited inhibition of anchorageindependent growth (AIG) of tumor cells at 500 nM and above (Fig. 8g); indicating Z-DAN-11 treatment results in a loss of ability of the tumor cells to grow anchorage independently. A dose-dependent reduction in the size of individual colonies was evident from the photomicrographs shown in Fig. 8h.

Discussion
One of the major obstacles of cancer chemo-prevention is undesirable toxicity to the normal cells, thereby resulting in adverse side-effects. This study was aimed in identification of a possible candidate with potent chemotherapeutic potential and minimal toxicity. Here, we synthesized a series of trans-stilbene derivatives and identified at least one derivative, named Z-DAN-11 which blocked cellular proliferation selectively in a series of different cancer cell lines in vitro, concentration dependently (Fig.1a-e and Table S1).
Functionally, Z-DAN-11-arrested tumor cells in the G2/ M-phase both time and dose dependently (Fig. 2a-d and  Fig. S2a-b). Z-DAN-11 caused a time-dependent decrease in cdc2, cdk2 and their phosphorylation, which are classical molecular signatures (Fig. 3g) leading to progression through the G2/M-phase, thereby confirming that Z-DAN-11 induces G2/M-phase arrest. Again as microtubule dynamics is essential for cell cycle regulation, perturbation of which triggers cell cycle arrest and apoptosis [24][25][26][27] . Mechanistically, Z-DAN-11 caused a significant downregulation of β-tubulin ( Fig. 3e and Fig. S2e) similar to nocodazole. These results indicate that Z-DAN-11 drives G2/M arrest through microtubule depolymerization which may provide a possible strategy to reverse cell cycle-mediated drug resistance through synchronization of cells 28 .
Moreover, induction of cell cycle arrest was accompanied by a parallel induction of apoptosis, as evidenced from a dose-dependent increase in the number of annexin V/PI and TUNEL positive cells (Fig. 4a-c and Fig. S4a-b). Our experimental evidences (Fig. 4d-f and Fig. S5a-c) confirmed role of Z-DAN-11-mediated ROS generation in dramatic alteration of the mitochondrial redox status that critically contribute to apoptosis, as NAC was able to reduce Z-DAN-11-mediated apoptosis significantly (Fig. 5a,b and Fig. S5d-e). These data clearly suggests that Z-DAN-11-induced ROS production contributes to mitochondrial dysfunction which leads to the cancer cell apoptosis through involvement of both, intrinsic as well as extrinsic pathways. This was confirmed from increase in expression of mediators (Fig. 5c,d and Fig. 6a-d) that leads to mitochondrial damage ( Fig. 4d and Fig. S5a), activation of caspases (Fig. 6d,e) and PARP1 cleavage.
Our findings (Fig. 6a) showed time-dependent decrease in expression of claspin along with a significant decrease in the expression of total chek1 and p-chek1 (ser317) in response to Z-DAN-11 treatment. This was associated with an elevated expression of γ-H2A.X (Fig. 6a), which may provide a possible link between the observed apoptosis and G2/M-phase arrest mediated by Z-DAN-11 treatment. Further, since inhibition of chek1 was known to cause G2 abrogation thereby selectively promoting apoptosis in cancer cells but not in normal cells 29 , Z-DAN-11-mediated chek1 inhibition may also explain the selective cytotoxicity of the compound observed earlier. Thus, chek1 plays a critical role in the observed G2/M arrest, DNA fragmentation and subsequent Z-DAN-11induced apoptosis. As p53 phosphorylation at ser15 (DNA damage response) 30,31 , ser46 (induction of apoptosis) 32,33 and ser392 (growth suppression) 34 play important role in cell proliferation. Interestingly, from proteome profiler and western immunoblot data, we found increased levels of total p53 along with p-p53 ser15, ser46, ser392 as shown in Fig. 5c,d and Fig. 6a, suggesting a critical role of p53 in Z-DAN-11-mediated apoptosis. Significant reduction of Z-DAN-11-mediated apoptosis both in p53 −/− cells (Fig. 7a,b) as well as siRNA-mediated p53-silenced A549 and MCF-7 cells (Fig. 7c,d), confirmed the involvement of p53 in Z-DAN-11-mediated apoptosis. Finally, to establish Z-DAN-11 as an effective anti-cancer lead, we investigated its therapeutic efficacy in a syngeneic mouse tumor model, using 4T1 cells on a Balb/c background. Z-DAN-11-regressed tumor volume as well as tumor weight (Fig. 8a-c) without significant adverse effect on animals (Fig. S6a). Thus, we have identified a novel synthetic trans-stilbene resveratrol derivative with potent anti-tumor activity, the mechanism behind which is its ability to selectively block tumor cell proliferation and induce tumor cell apoptosis, but not normal cells. Hence, Z-DAN-11 may be a promising candidate for targeted and selective therapeutic strategy against cancer.

Synthesis of trans-stilbene compounds
Briefly, to an ice-cold solution of aryl acetonitriles (1-4) (1 mmol) and aldehydes (5-19) (1.1 mmol) in EtOH (10 mL) was added 5% aq. NaOH (0.2 mL) and the reaction mixture was briskly stirred at room temperature for 30 min. Acidification of the reaction mixture using 1 N HCl led to the formation of products as solids. Filtration of the solid products and re-crystallization from EtOH furnished pure products Z-DAN-1−Z-DAN-22.

MTT cell proliferation assay
In order to determine the effect of the synthesized compounds on cell proliferation, MTT assay was performed 35,36 . Briefly, cells were grown in 96 well plate in presence of the synthesized (Z) -2, 3-diarylacrylonitrile (trans-stilbene) derivatives (0-60 µM) for 48 h. 5% FBScontaining phenol red-free DMEM and MTT (200 µL; 0.5 mg/mL) were added to each well and incubated at 37°C for 4 h in a humidified incubator containing 5% CO 2 . The purple-colored formazan crystals formed in the wells were dissolved in DMSO and absorbance was measured at 570 nm with a microplate reader.

Trypan blue exclusion method
Trypan blue exclusion is technique which classifies viable and nonviable cells based on membrane integrity. A viable cell with intact membrane will not take trypan blue whereas a nonviable cell will have trypan blue positive cytoplasm 37 . Post treatment, 1× PBS resuspended cells were mixed with 0.4% trypan blue solution in 1:1 dilution for 4 min and counted with hemocytometer.

Cell cycle profiling assay by propidium iodide staining
For this experiment, double thymidine blocked 38 synchronized human cancer cells including MCF-7, MDA-MB-231, A549, and HeLa were treated with increasing concentrations (0.5 µM, 1 µM, 2.5 µM, 5 µM, and 10 µM) of Z-DAN-11 for 24 h. Nocodazole was used as a reference control for this experiment 39,40 . Post treatment, cells were harvested into single cell suspension and fixed by incubating the cells overnight at −20°C with 75% ethanol. Cells were centrifuged and resuspended in 1× PBS for 2 h followed by RNaseA (20 µm) treatment for 2 h at 37°C. Finally, propidium iodide was added and incubated for 20 min at room temperature. Flow cytometric analysis was immediately performed using a FACS-Verse instrument (BD).

Microarray
Affymetrix PrimeView™ Human Gene Expression Array platform was used to check the differential expression of gene sets in response to Z-DAN-11. Microarray data sets were analyzed using TM4:Microarray Software suite 41 . Gene-level signal estimates were derived from the raw data files. The 'affy' package in R were used to quantile normalize and background adjust the raw CEL files by implementing the Robust Multichip Averaging algorithm. SAM analysis was performed to determine the statistically significant differentially expressed genes between the test and the control groups, using default statistical parameters. Genes with FDR-adjusted p values ≤ 0.05 were further screened to get the final set of DRGs based on fold change ≥2 between test and the control groups. Hierarchical clustering was performed by complete linkage and Pearson's correlation using Cluster 3.0 42 ; results were visualized using Java TreeView (http://jtreeview. sourceforge.net/).

RNA isolation and real-time PCR
Total RNA was extracted using Trizol reagent (Invitrogen). cDNA was prepared from 1 µg of RNA extracted from MCF-7 cells treated with 10 µM dose of Z-DAN-11 for 6, 12, and 24 h along with DMSO-treated control using Verso cDNA synthesis kit (Thermo Scientific). RT-PCR was performed using SYBR green PCR system on 7500 fast (Applied Biosystem). All mRNA quantification data were normalized to β-actin. Real-time primers were listed in Figure S3c.
Immunocytochemistry Immunocytochemistry was performed as described previously 43 . Briefly, 5 × 10 3 cells grown on coverslips were fixed with 3.7% paraformaldehyde, washed with 1× PBS and stained with primary antibody (1:100) overnight at 4°C. Cells were washed with 1× PBS, counterstained with Alexaflour-488/594 secondary antibody (1:500) for 1 h at room temperature. Finally, DAPI staining was done and washed with 1× PBS. Coverslips with stained cells were mounted on slides and observed in Leica confocal microscope.

In vitro microtubule polymerization assay
Purified porcine tubulin (15 μM) was incubated with or without the compounds in tubulin polymerization buffer PEM (80 mM PIPES, 0.5 mM MgCl2, 1 mM EGTA, pH 6.8) with 10% DMSO in ice for 10 min. Subsequently, 1 mM (GTP) was added and kinetic loop study was done using Thermo scientific Multiscan Go Multi Plate reader set at 37°C temperature. The polymerization was monitored over 60 min by measuring the absorbance at 340 nm.

Annexin V-FITC/PI staining
Induction of apoptosis was measured by flow cytomety after annexinV-FITC/PI staining 44 using BD Bioscience kit. Briefly, post treatment 0.5 × 10 6 cells were washed in ice-cold 1× PBS and resuspended in 100 µL of binding buffer and incubated with 5 µL of annexin V-FITC and 5 µL of PI for 15 min at room temperature in a dark place as per manufacturer's guidelines. Flow cytometric analysis was immediately performed using a FACS-Verse instrument (BD).

JC1 staining
Assessment of mitochondrial permeability was measured by JC1 staining as described earlier 45 . MCF-7 cells were treated with test compounds (0, 2.5, 5, and 10 μM) for 6 and 9 h. Cells were washed with 1× PBS buffer and incubated with the JC-1 dye (3 μM final concentration in DMEM media) at 37°C for 30 min in the dark. Cells were again washed twice with 1× PBS buffer and kept back in 1× PBS buffer. Finally images were captured with Leica fluorescent microscope.

Measurement of mitochondrial ROS by using MitoSOX™
MitoSOX™ Red mitochondrial superoxide indicator is a novel fluorogenic dye for highly selective detection of superoxide in the mitochondria of live cells 46 . For this experiment briefly, 5 × 10 4 cells grown on coverslips were fixed with 3.7% formaldehyde, washed with 1× PBS and DAPI staining was done. Coverslips with stained cells were mounted on slides and observed in Leica fluorescence microscope.

Measurement of cellular ROS using DCFDA
Intracellular ROS was measured using DCFDA method 47 . MCF-7 and A549 cells were treated with test compounds (0, 2.5, 5, and 10 μM) for 6 and 9 h. After treatment, the media was discarded and serum-free media was added. Then DCFDA (5 µM final) was added and incubated for 30 min. Post incubation the media was discarded and adherent cells were scrapped out and washed in 1× PBS. Finally, the fluorescent signals from the cells were acquired by FACS-Verse.

Human apoptosis proteome profiler array
Expression pattern of several pro-apoptotic and antiapoptotic proteins were examined in Z-DAN-11 (10 µM) treated and DMSO-treated MCF-7 cells for 24, 48, and 72 h by using Human apoptosis array kit (R&D Biosystem). An aliquot of 300 µg of protein was used for each condition and experiment was as performed as described in our earlier report 48 . Thereafter, cell lysates were subjected to analysis using the Proteome Profiler TM human apoptosis antibody array according to the manufacturer's instructions. Arrays were developed with streptavidin-HRP for 30 min on a rocking platform shaker. Developed signals were densitized using ImageJ software, pixel densities were normalized to untreated sample and expressed as mean pixel density.

TUNEL assay
Visual confirmation of apoptosis was achieved by using Terminal deoxynucleotidyl TUNEL assay 45 . For this experiment, 4 × 10 4 cells were grown to confluence with or without Z-DAN-11 (10 µM) on coverslips, following which cells were washed with 1× PBS and fixed with prewarmed 3.7% formaldehyde. Cells were then permeabilized with 0.1% triton-X at 2-8°C for 5 min. TUNEL reaction mixture was added to the cells and incubated for 1 h in the dark in humidified chamber. DAPI was used as counter stain. Microscopic analysis was done using a confocal microscope.

Western immunoblotting
Briefly, cell lysates were prepared from MCF-7 and A549 cells post treatment with Z-DAN-11 (10 µM) with RIPA buffer method. For immunoblotting, 20-35 µg proteins were resolved on 8-15% SDS-PAGE, transferred onto PVDF membrane (Millipore, USA), blocked with 5% BSA in 1× TBST, and probed with respective primary antibodies followed by corresponding secondary antibodies. Finally blots were developed into X-Ray films by ECL method or by using Chemidoc MP system Bio-Rad.

Caspase 3/7 activation assay
For this experiment cell lysates were prepared from MCF-7 cells after treating with Z-DAN-11 (10 µM) with RIPA buffer method. An aliquot of 150 µg of protein was used for each condition. DTT and DEVD-p-NA substrate were added according to manufactures protocol. Samples were mixed well and incubated at 37°C for 90 min and finally OD was measured at 405 nm in Thermo Multiskan GO microplate reader.

Co-immunoprecipitation
Co-immunoprecipitation was performed as described previously 49 . Cells were lyzed in IP lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, and 5% glycerol). A total of 400 μg of protein lysates were incubated with rabbit monoclonal anti-TNFRI antibody (3 μg/condition) overnight with continuous rotation. Next day, Protein A beads were added and incubated for another 4 h. Then, beads were washed three times using lysis buffer. Finally, beads were resuspended in 40 μL of 2× laemmli buffer, centrifuged at 13,000 rpm. Supernanants were collected and resolved on 12% SDS-PAGE. Finally, western immunoblot was done with FADD, TRADD, and TNFRI antibodies.
si-RNA transfection MCF-7 cells (1.5 × 10 5 ) were grown in 6 well plates in antibiotic-free media for 24 h. Post incubation, p53 si-RNA and scrambled were transfected with RNAi Max (Invitrogen) as per manufacturer's instructions. Cells were grown in opti-MEM for 6 h followed by incubation with complete DMEM for 24 h.

Soft agar colony formation assay and clonogenicity
Clonogenic assay and Soft agar colony formation assay was performed as described earlier 50 . For soft agar assay, 2 × 10 4 cells were resuspended in 1 mL 2× complete DMEM and mixed with 1 mL warmed 0.7% agarose and plated over a solidified 0.75% agarose in 1× DMEM. Following solidification of the top layer, 2 mLof DMEM was added, incubated at 37°C for 30 days with continuous media change (with or without Z-DAN-11) in every 3rd day. Colonies were fixed, stained with 0.05% crystal violet and imaged using Gel Doc XR+ (Bio-Rad). For clonogenicity assay, cells were plated at very low density (200 cells/well in 6 well plate), grown for either 8/10 days, fixed with 3.7% formaldehyde, stained with 0.05% crystal violet, and image was captured using Gel Doc XR+ (Bio-Rad).

4T1 breast tumor model
BALB/c mice 6-8-week-old were obtained from Center for Translational Animal Research (CTAR), Bose Institute, Kolkata, India and were maintained as per the guidelines of the animal ethical committee in accordance with CPCSEA guidelines (IAEC Approval No. IAEC/BI/ 32/2015 Dt. 26/05/2015). For in vivo tumorigenic assay, 4T1 cells (1.5 × 10 6 cells/animal) were subcutaneously injected into the mammary fat pad of Balb/c mice to develop solid tumor. Animals with solid tumor were randomly distributed into three groups each containing six animals. One group was treated with vehicle control (10% DMSO in normal saline) whereas; other two groups were given intraperitoneal injection of Z-DAN-11 (10 mg/ kg and 20 mg/kg) starting after 10 days of tumor development and continued until 31 days (seven doses, 72 h interval). Tumor progression was monitored by measuring the volume of the tumor with vernier calipers on every third day. The tumor volume was calculated by using the formula V = 0.5 × a × b 2 , where "a" and "b" indicate major and minor diameter, respectively 51 . Histopathological examination was performed by haematoxylin and eosin staining as described previously 52 .

Hepatotoxicity and pharmacokinetics studies
Following seven doses Z-DAN-11(10 mg/kg and 20 mg/ kg) each with 3 days interval 100 μL blood sample were taken manually into heparinized capillary tubes by piercing the saphaneous vein with a needle and subsequently into 0.5 mL microcentrifuge tubes. All blood samples were processed for plasma isolation by centrifugation at 1640 rpm for 5 min at 4°C within half an hour of collection. Finally serum profile of ALT, AST, ALP, creatinine, urea nitrogen was checked using respective kit as per manufacture's instruction.
Time course (predose and 0.25, 0.50, 1, 2, 4, 8, 12, 24 h) change in mean plasma concentration of Z-DAN-11 after intravenous (1 mg/kg) and oral (10 mg/kg) administration of Z-DAN-11 was done by isolating plasma samples at each time point post oral and intravenous dose. An aliquot of 100 μL blood sample were taken manually into heparinized capillary tubes by piercing the saphaneous vein with a needle and subsequently into 0.5 mL microcentrifuge tubes. All blood samples were processed for plasma isolation by centrifugation at 1640 rpm for 5 min at 4°C within half an hour of collection. Finally, samples were analyzed using Sciex 5500 Q-Trap LC-MS/MS and agilent 1290 series high performance liquid chromatograph. Kinetex Biphenyl (2.1 30 mm, 5 μm particle) column was used where the injection volume was 20 mL. H 2 O containing 0.1% acetic acid or 90:20 MeCN: H 2 O containing 0.1% acetic acid were used differently as mobile phase.

Statistical analysis
All statistical analysis was performed by one-way ANOVA followed by Dunnett's test or Turkey test for comparing variable groups using GraphPad prism 5 software. In certain case Student's t-test was also performed. Values are expressed as mean ± SEM for control and treated samples. The values were considered statistically significant as per p values (*p < 0.05, **p < 0.01, ***p < 0.001, ns = not significant).