Polyphenols Sensitization Potentiates Susceptibility of MCF-7 and MDA MB-231 Cells to Centchroman

Polyphenols as “sensitizers” together with cytotoxic drugs as “inducers” cooperate to trigger apoptosis in various cancer cells. Hence, their combination having similar mode of mechanism may be a novel approach to enhance the efficacy of inducers. Additionally, this will also enable to achieve the physiological concentrations facilitating significant increase in the activity at concentrations which the compound can individually provide. Here we propose that polyphenols (Resveratrol (RES) and Curcumin (CUR)) pre-treatment may sensitize MCF-7/MDA MB-231 (Human Breast Cancer Cells, HBCCs) to Centchroman (CC, antineoplastic agent). 6 h pre-treated cells with 10 µM RES/CUR and 100 µM RES/30 µM CUR doses, followed by 10 µM CC for 18 h were investigated for Ser-167 ER-phosphorylation, cell cycle arrest, redox homeostasis, stress activated protein kinase (SAPKs: JNK and p38 MAPK) pathways and downstream apoptosis effectors. Low dose RES/CUR enhances the CC action through ROS mediated JNK/p38 as well as mitochondrial pathway in MCF-7 cells. However, RES/CUR sensitization enhanced apoptosis in p53 mutant MDA MB-231 cells without/with involvement of ROS mediated JNK/p38 adjunct to Caspase-9. Contrarily, through high dose sensitization in CC treated cells, the parameters remained unaltered as in polyphenols alone. We conclude that differential sensitization of HBCCs with low dose polyphenol augments apoptotic efficacy of CC. This may offer a novel approach to achieve enhanced action of CC with concomitant reduction of side effects enabling improved management of hormone-dependent breast cancer.


Introduction
Anti-estrogen therapies constitute the mainstay of current treatment for breast cancer [1]. Centchroman (CC), a triphenylethylene Selective Estrogen Receptor Modulator (SERM) has been considered a potential anti-breast cancer drug in ER-positive (MCF-7)/2negative (MDA MB-231) Human Breast Cancer Cells (HBCCs) as well as with all stages of hormone-responsive breast cancer [2][3][4]. Studies have demonstrated that low concentrations of triphenylethylenes in patients cause modest, partial estrogen-like action [5]. Although once a steady level is achieved the drug shows its anti-estrogenic properties whose attainment requires 4-8 weeks [5]. Therefore, to overcome the low dose estrogenic effects of triphenylethylenes, one alternative approach would be to sensitize the cells through supplementing compounds (polyphenols) having similar mode of action. This in turn enables achievement of physiological levels facilitating significant increase in the activity at concentrations which the compound can individually provide. For this reason, polyphenols like Resveratrol (RES) abundant in wine and Curcumin longa derived Curcumin (CUR) a South Asian and Middle Eastern spice were selected as Type I estrogens similar to CC for preconditioning the cells [5]. Moreover, 2-3 times higher retention of polyphenols in local tissue as compared to the plasma levels [nM to low mM (,2 mM ); retention time ,5-6 h] [6,7] may positively influence the cell susceptibility to triphenylethylenes.
Therefore, we pre-treated estrogen-deprived CC treated MCF-7/MDA MB-231 cells with physiological and pharmacological doses of polyphenols to synergize or antagonize the drug action. Both, polyphenols (CUR and RES) and CC bind to ERa and b with lower affinity than E2 [8,9] involving ERdependent and -independent pathways [2,[10][11][12][13]. Hence, sensitization with low dose polyphenols may modulate the receptor levels/redox status overtly improving CC efficacy. Contrarily, high dose polyphenols may disrupt ER and enhance the ER-independent antiproliferative effect in the combination. This may modulate proapoptotic (Bax, Caspase-9), versus antiapoptotic genes (Bcl-2) ratio and transcription factors p53, including c-Jun and phospho-ATF-2 to play a crucial role in sensitizing the caspase-3 deleted and p53 mutant MCF-7 and MDA MB-231 respectively.
Hence, we investigated the mode of action of polyphenolsensitized, CC treated MCF-7/MDA MB-231 cells on cell cycle, redox homeostasis, stress activated protein kinase (SAPKs: JNK and p38 MAPK) and downstream apoptosis effectors. We also employed a series of inhibitors for p53, JNK and p38 pathways critical for time-dependent cytotoxicity of CC and polyphenols. Both RES and CUR at physiological dose potentially sensitize CC-induced apoptosis in MCF-7 cells through modulating the above factors. Pharmacological sensitization significantly arrests the cells (Go/G1 phase; RES and G2/M phase; CUR) rescuing them from CC-induced apoptosis through unaltered factors to the same extent as polyphenols alone. However, on sensitization with low dose RES (JNK/p38-independent)/CUR (JNK/p38dependent), the apoptosis enhanced in CC treated MDA MB-231 cells. Contrarily, with high dose polyphenol sensitization CC in both the combinations showed JNK/p38-dependent apoptosis. This therefore reinforces our hypothesis about the usage of polyphenols to ameliorate Estrogen-dependent/independent breast cancer with enormous translational potential during combination chemotherapy employing Centchroman.

Inhibitor Treatments
ROS inhibition was performed using N-Acetyl-l-Cysteine (L-NAC, 5 mM) 1 h prior to treatment with drugs. For JNK/p38 pathway inhibition, during the 6 h exposure to polyphenols (RES: 10/100 mM and CUR: 10/30 mM), JNK inhibitor (10 mM SP600125)/p38 pathway inhibitor (1 mM SB203580)/p53 pathway inhibitor (15 mM Pif) was added during the last two hours prior to CC exposure. The last regimen with CC was continued until 18 h. The cells were analyzed by FACS (PI 40 mg/mL), western blotting and immunoprecipitation. These concentrations of inhibitors (L-NAC, SP6000125, SB203580 and Pif) were nontoxic to cells under our pre-incubation conditions (data not shown).

Cellular Morphological and Cell-Cycle Analysis
For time-dependent morphological analysis, 0.2610 6 cells were seeded in a 6-welled plate and treated for 3, 6 or 24 h with polyphenols (RES: 10/100 mM and CUR: 10/30 mM) or preconditioned with polyphenols for 6 h and continued with CC (10 mM) for next 18 h. These cells were then photographed by a Nikon Eclipse Ti inverted phase contrast microscope. Cells were also stained with 40 mg/mL PI prior to analysis by a FACS calibur instrument (Becton Dickinson, San Jose, CA, USA) employing the Cell Quest Software [15].

Modulation of Mitochondrial Membrane Potential (Dym) and Intracellular Reactive Oxygen Species (ROS) Generation
Mitochondrial Membrane Potential (Dym) was measured by the uptake of JC-1 which is a dye that stains mitochondria in living cells in a membrane potential dependent fashion. In apoptotic cell, the dye stays in the cytoplasm as a monomer and fluoresces green (527 nm), while in healthy cells, the dye aggregates in the mitochondria and fluoresces red (590 nm). For the experiment, 0.2610 6 cells were plated in a 6-welled plate, treated as above, washed and finally harvested in chilled PBS containing 1 mM JC-1. The samples were incubated at 37uC for 30 min in dark, washed twice with PBS and fluorescence intensities were determined on a Flow Cytometer [15].
Cells were seeded at a concentration of 10610 3 cells per well in black 96-well plates. Cells were treated time-dependently with polyphenols or pre-conditioned with polyphenols for 6 h and continued with CC (10 mM) for next 18 h (total of 24 h). This was followed by incubation with 10 mM 29,79-dichlorofluorescin diacetate (DCFDA) (Molecular Probes, Eugene, OR) for 30 min. Cells were then rinsed with PBS and then placed in a pre-warmed 37uC fluorimeter for time-dependent measurement (DCFDA Ex 480 nm, Em 530 nm). Following this, the supernatant was removed and a protein assay was performed on the cells. The final results have been corrected for variations in the protein concentration between wells and are expressed as a percentage activity in untreated controls being 100%. Flow cytometry analysis of cells stained with DCFDA was performed to confirm results using 5 mM L-NAC. HBCCs were plated in 6-well plates and treated time-dependently with polyphenols or pre-treated with polyphenols accompanied in the presence of DCFDA (10 mM). Fluorescence was measured with a Flow Cytometer (Ex 500 nm, Em 530 nm) [15].

Determination of Total Glutathione (GSH)
Quantification of total GSH was accomplished using a Glutathione Assay Kit from Sigma. According to the manufacturer's instructions, 1610 8 cells were exposed as above and deproteinized with 5% 5-Sulfosalicylic Acid solution after three rounds of alternate freeze-thawing, centrifuged to remove the precipitated protein and the supernatants assayed for GSH. The kit uses a kinetic assay in which catalytic amounts (nM) of GSH cause a continuous reduction of 0.031 mg/mL DTNB (5,59-dithiobis (2nitrobenzoic acid) to TNB (monitored at 412 nm in a SpectraMax M2 Multiwell Plate Reader). The GSSG formed is reduced by glutathione reductase (0.115 units/mL) and 48 mM NADPH to additionally give a positive value. The amount of GSH was calculated from a standard curve generated under similar conditions.

Superoxide Dismutase and Catalase Activities
Superoxide dismutase (SOD) and Catalase (CAT) activities were determined according to McCord and Fridovich, and Beutler [16,17] respectively. Untreated as well as treated 3610 6 MCF-7/ MDA MB-231 cells were collected by scraping, centrifuging and the pellets sonicated in chilled 1.15% KCl. The cell extract was spun at 15006g for 5 min at 4uC. The resulting supernatants were analyzed for enzyme activities. The specific activity of SOD was calculated as follows: Specific Activity = A6dilution fac-tor61000/One unit6volume of enzyme (ml)6mg protein.
''A'' = OD change/min of controlled reaction-OD change/min of experiment, ''One unit'' = (OD change/min of controlled reaction)/2. Here, one unit means 50% reduction in NBT reduction as compared to control. The specific activity of CAT was calculated by the following equation: Specific Activity = OD change/min6dilution factor61000/0.003946volume of enzyme (ml)6mg protein = mmol H 2 O 2 reduced/min/mg protein. Each data point was performed in triplicate and the results reported as Mean Absorption 6 SD. supplemented with protease and phosphatase inhibitor cocktail as per manufacturer's protocol. The supernatant was assayed for protein content [18]. 50 mg of protein per lane were processed for western blotting by SDS-PAGE and electro-transferred to nitrocellulose membranes. Membranes were probed with the appropriate primary antibodies against MnSOD, Catalase, Total p53, phospho p53 (Ser-15), JNK 1/2, non-activated p38, Bcl-2, Bax, active Caspase-9 and Ser-167 phosphorylation of AF-1 region of ERa. All primary antibodies were used at recommended dilution in 1% bovine serum albumin in Tris buffered saline with 0.1% Tween TM 20. Reactions were visualized with biotin conjugated secondary antibody and subsequently with avidin linked Alkaline-Phosphatase enzyme conjugate according to the manufacturer's protocol. Immuno-detection was accomplished using insoluble colored substrate BCIP/NBT.

Assay for JNK and p38 Activity
KinaseStar TM JNK Activity Assay Kit (BioVision) was used to assess JNK activity according to the manufacturer's instructions. Briefly, activated JNK was immunoprecipitated by incubating cell lysates for 45 min with an immobilized JNK specific antibody at room temp. The in vitro kinase assay was then performed using c-Jun protein/ATP mixture as substrate. Levels of phosphorylated c-Jun were then detected by western blotting using an anti phospho-c-Jun (Ser 73) antibody.
p38 activity was assessed using a non-radioactive p38 MAP Kinase Assay Kit (Cell Signalling) according to the manufacturer's instructions. Briefly, phosphorylated p38 was immunoprecipitated by incubating cell lysates overnight with an immobilized phospho-p38 antibody at 4uC. An in vitro kinase assay was then performed using an ATF-2 fusion protein as substrate. Levels of phosphorylated ATF-2 were then detected by western blotting using a phospho-ATF-2 (Thr71) antibody.

Statistical Analysis
The results are expressed as Mean 6 SD from one of three similar experiments each performed in triplicate. Student's 't-test' was used to determine the level of significance and the following values assigned * P,0.05; y P,0.01; # P,0.001, calculated compared to control unless stated otherwise. At 24 h, low dose polyphenol sensitizes CC treated cells showing higher degree of distortion compared to either drug alone. On sensitizing with high doses of polyphenols, CC depicts adverse morphology as compared to CC and polyphenols alone in both the cells ( Fig. 1A and B).

Morphological and Cell-Cycle Changes in Polyphenols
To determine whether polyphenols pre-treatment augments apoptosis in CC treated MCF-7/MDA MB-231 cells, a sub-G0/ G1 DNA peak, suggestive of apoptotic DNA was tested. However, the number of cells in this population on treatment with 10 mM RES increased significantly with 6 h which extended up to 24 h. Even though the cells were undergoing apoptosis, they passed from G1 to S and G2 phase. The transition of cells from S to G2/ M phase was blocked within 6 h and the inhibition persisted up to 24 h, the time point at which most of the cells were arrested in S phase. With higher dose of RES, the cells were arrested in G0/G1 phase as early as 3 h and continued up till 24 h. This goes on along with unaltered S phase and time-dependent decrease in G2/ M phase ( Fig. 2A). In MDA MB -231 cells, 10 mM RES showed increased cell death by 24 h accompanied with decreased G2/M phase. However, it failed to evoke any S-phase arrest compared to MCF-7 cells. Conversely, 100 mM RES showed equivalent sub-G0/G1 compared to 10 mM RES, significant accumulation of G0/G1 and decline in G2/M cells, initiated at 3 h and continued up till 24 h (Fig. 2B).
CC when added to the culture separately, the transition of cells from G1 to S phase was blocked within 3 h but with longer treatment, the inhibition was released and the cells started cycling again accompanied with apoptosis in MCF-7 cells. Timedependent increase in sub-G0/G1 cells was observed in MDA MB-231 cells (Fig. 2C). The cell number in sub-G0/G1 population increased with time up to 24 h with 10 mM CUR treatment with concomitant decrease in G0/G1 population in MCF-7 cells (Fig. 2D), while remaining unaltered G0/G1 in MDA MB-231 cells (Fig. 2E). With 30 mM CUR, the cells undergoing apoptosis passed from G1 to S and G2/M phases MCF-7 cells pre-treatment with 10 mM RES for 6 h, CC significantly up-regulates sub-G0/G1 peak, S phase arrest and down-regulates G0/G1, G2/M phase than either drug alone (Fig. 2F). For MDA MB-231 cells under similar conditions, higher sub-G0/G1 and decreased G2/M was observed (Fig. 2G). In 100 mM RES sensitized CC treated cells G0/G1, S and G2/M phases mirrored with 100 mM RES apart from significant decline in sub-G0/G1 population in MCF-7 cells and unaltered in MDA MB-231 cells ( Fig. 2F and G). Upon comparing with CC alone, the G0/G1 arrest improved whereas sub-G0/G1, G2/M phases showed remarkable decline in MCF-7 cells and remain unchanged for MDA MB-231 cells ( Fig. 2F and G). With 10 mM CUR sensitization, CC significantly augments sub-G0/G1 peak leaving the rest unaltered as compared to individual drugs per se in MCF-7 (Fig. 2F). Interestingly, 10 mM CUR sensitized CC treated MDA MB-231 cells displayed G2/M phase arrest with enhanced sub-G0/G1 peak as compared to drugs alone (Fig. 2G). In 30 mM CUR sensitized CC treated MCF-7 cells, major decline in sub-G0/G1 phase vis-à-vis CUR alone was observed whereas reverse was true for MDA MB-231 cells. On the other hand, G0/G1 and G2/M under similar situations remain unaltered. On comparison with CC, G0/G1 population decreases whereas G2/M phase shows remarkable up-regulation in MCF-7 cells. Significant increase in sub-G0/G1 peak was observed in MDA MB-231 cells ( Fig. 2F and G).  (Fig. 3A and B). Unlike the low dose polyphenols in MCF-7 cells, 100 mM RES induces ROS at 3 h, with significant reduction at 24 h. Vis-à-vis, 30 mM CUR shows drastic generation of ROS as early as 3 h sustained until 24 h (Fig. 3A). The polyphenols associated dichotomy in cell-cycle owes itself to ROS generation significantly pronounced at the higher dose is a unique observation. As ROS generation is cell-cycle dependent phenomenon [19,20], RES (100 mM) arrests the cells in G0/G1 phase (3 h) resultantly letting the cells evade the remainder events of the cycle accompanied with minimal ROS generation. Conversely, 30 mM CUR at 3-24 h permits the transition of cells from G1 to S phase to final arrest in G2/M phase owing to persistent ROS generation. Time-dependent gradual decrease in 100 mM RES and persistent ROS generation leads to disruption of DYm in MCF-7 cells [21] ( Fig. 3A and B).
However, low dose polyphenols sensitized CC treated cells display increase in ROS and concomitant disruption of DYm than CC alone both in MCF-7 and MDA MB-231 cells (Fig. 3C and  D). Conversely, decreased ROS was observed with 100 mM RES sensitized CC treated cells vis-à-vis the ligands per se in MCF-7 cells remaining unaltered in MDA MB-231 (Fig. 3C). On addition of CC to 30 mM CUR sensitized cells, ROS generation declined versus CUR but significantly enhanced vis-à-vis CC (Fig. 3C) in both the cell types. The high dose polyphenol combination severely disrupts DYm compared to CC alone in MCF-7 cells only. As compared to high dose polyphenols alone, DYm remained unaltered in both the cell types (Fig. 3D). Further, cytometry reveals that 5 mM L-NAC, inhibits ROS generation by low dose of polyphenols separately or with sensitized cells in MCF-7 and MDA MB-231 cells (Fig. 3E). In MCF-7 cells, L-L-NAC failed to respond with 100 mM RES either or in CC treated whereas it inhibited 30 mM CUR without or with CC generated ROS. Additionally, 100 mM RES/30 mM CUR alone or in CC treated MDA MB-231 cells induced ROS, was also inhabitable by L-L-NAC (Fig. 3E).

Differential Anti-oxidant Apparatus Expression in Polyphenol Sensitized, CC Treated MCF-7/MDA MB-231 Cells
A sustained flux of ROS results in an imbalance of the intracellular redox state regulated by Glutathione (GSH), Superoxide Dismutases (SOD) and Catalases (CAT) [22,23]. RES and CUR both strongly inuenced total GSH dose-dependently where the former down-regulated while the converse was true for the latter in MCF-7 cells (Fig. 4A). However, in MDA MB-231 cells total GSH remained unaltered through dose-dependent RES/ CUR (Fig. 4A). The disparity between the relative regulations of GSH in MCF-7 cells may be attributed to structural dissimilarities, extent of conjugation, half-life, stability etc. of the polyphenols in question [24,25]. This coupled with the other sequel of events may finally determine the fate of the cells. Sensitization with low dose polyphenols in CC treated cells further reduced the total GSH than with ligands alone in MCF-7 cells. Sensitization with 10 mM RES in CC treated MDA MB-231 cells displays unaltered or further reduced total GSH with RES and CUR respectively compared to drugs alone. 100 mM RES sensitized CC treated cells reduced the total GSH compared to CC alone in MCF-7. However, this remains unaltered compared to RES alone in both cell types (Fig. 4A). 30 mM CUR sensitized CC treated cells reduced the total GSH compared to CUR to increase with CC both in MCF-7 and MDA MB231 cells (Fig. 4A). Moreover, compared to control unlike impervious MnSOD expression with low dose polyphenols in both MCF-7/MDA MB-231 cells, the SOD activity enhanced to decline at their respective higher dose. However, in MDA MB-231 cells, polyphenols at both the doses showed equivalent decline (Fig. 4B, 5 and 6). CC alone did not alter the expression although the activity rose in MCF-7 and decreased in MDA MB-231 cells. With 10 mM RES/CUR sensitization, the level and activity has inverse relationship as compared to CC alone in MCF-7 cells. With 10 mM RES/CUR sensitization in CC treated MDA MB-231 cells, MnSOD level remain unaltered while activity declined in both the cases compared to CC per se. With high dose polyphenols sensitization, compared to CC or polyphenols alone, reduction (MCF-7) and no alteration (MDA MB-231) was observed (Fig. 4B, 5 and 6). Interestingly, CAT expression remained unaltered throughout the low dose exposure either separately or together versus untreated control cells. However, activity remain unaltered at low doses with RES alone or in sensitized cells, while CUR sensitization in CC treated cells displayed decline in activity as compared to the CUR alone in MCF-7 cells (Fig. 4B and 5). In MDA MB-231 cells, low dose polyphenols down-regulated the activity of CAT which further remained unaltered in sensitized cells as compared to polyphenols alone and decreased vis-à-vis CC (Fig. 4B and 6). Further, CAT expression remains unaltered with high dose polyphenols alone in MCF-7 and decline in MDA MB-231 cells.
With high dose polyphenols sensitization, compared to CC or polyphenols alone, reduction and no alteration in activity was observed (Fig. 4B, 5 and 6).
For MTT analysis, 3-24 h exposure with CC/low dose polyphenols slightly inhibited JNK, p38 and p53 pathway by 24 h. However with 100 mM RES and 30 mM CUR, the significant decline of these parameters starts at 6 h until 24 h (Fig. 7A). We therefore propose that at 6 h in MCF-7 cells, the activation of these pathways correlates well with mitochondrial potential supported by elevated ROS. In MDA MB-231 cells, 10 and 100 mM RES displayed no involvement of JNK and p38 pathways, while 10 and 30 mM CUR displayed their involvement at 24 h/6 h until 24 h respectively (Fig. 7B).
Based on the above, for the flow studies on cells during 6 h sensitization with the polyphenols, the inhibitors were individually added during the last 2 h. Subsequently, CC treatment until 18 h was undertaken. Significantly reduced apoptosis through CC and polyphenols exposure per se correlating well with MTT studies was observed. More, in case of inhibitors (Z-VAD-FMK, SP 600125/ SB 203580/Pif) low dose polyphenols sensitized CC treated cells registered decline in apoptosis to an extent greater than with ligands per se in MCF-7 cells. 100 mM RES by itself significantly decreased apoptosis with inhibitors. Notably, with 30 mM CUR plus Z-VAD-FMK/SP 600125/Pif the magnitude of cell death was lower unlike SB 203580 indicating the divergence of pathways at higher molarity. Additionally, the inhibitors fail to exert any effect at high dose of polyphenols sensitized CC treated MCF-7 cells (Fig. 7C). However, p53 null MDA MB-231 cells were expectedly insensitive to p53 inhibitor Pif. In these cells, 10 and 100 mM RES alone as well as in sensitized cells displayed no involvement of JNK and p38 pathways, however caspasemediated cell death was observed. With 10 and 30 mM CUR alone and in sensitized cells, involvement of Caspases, JNK and p38 pathways was noticed. In case of CC with slight inhibition of apoptosis, other three pathways were unaffected (Fig. 7D).
In MCF-7 cells, stress activated protein kinases (SAPKs) expression indicates pan-unaltered total JNK1/2 and p38. Concomitantly, phosphorylation of c-Jun and ATF-2 was upregulated at low dose polyphenol sensitized CC treated cells versus CC and polyphenols alone. This is supported by the data through respective inhibitors (Fig. 5). With 100 mM RES sensitization phospho c-jun enhanced as compared to drugs alone, while with 30 mM CUR sensitization the expression remains unaltered. The  (Fig. 5). SAPKs down-regulate Ser-167 phosphorylation of ER, a site that influences AF-1 ER-dependent transcriptional activity (non-genomic events) [8,28]. With low dose polyphenol sensitization, the Ser-167 phosphorylation decreased as compared to polyphenols while reverse was true vis-à-vis CC exposure. However, on sensitization with high dose polyphenols the combination displayed decline in Ser-167 phosphorylation visà-vis both the drugs (Fig. 8). The knowledge of stress induced Ser-15 phosphorylation of p53-mediated apoptosis through JNK and p38 SAPKs [2,15] made us examine the role of SP 600125 and SB 203580 respectively. Compared to untreated control, phosphor-ylation of p53 was up-regulated dose-dependently with polyphenols and CC alone suppressible by the inhibitors. At low and high dose polyphenols sensitization in CC treated cells, phosphorylation rises and falls respectively compared to either drug. The phosphorylation of p53 in low dose polyphenols sensitized CC treated cells was inhibited both by SP 600125 and SB 203580 which at higher dose remained unaltered or decreased respectively. Total p53 showed constitutive expression (Fig. 5). Phosphorylation of p53 promotes apoptosis whose downstream effectors e.g. Bax/Bcl-2 ratio and caspase-9 [29] examined through western analysis. CC alone and polyphenols dosedependently up-regulate their activation compared to untreated control. With low dose polyphenols sensitization in CC treated cells, the markers increased to subsequently decrease at higher dose than either drug alone (Fig. 5). In p53 mutant MDA MB-231 cells [2] the gene regulates Bax expression where total p53, Ser-15 phosphorylation of p53 and Bax was found to be unaltered in CC/RES/CUR exposed cells with or without inhibitors (Fig. 6, A-D). RES dose-dependently did not display any significant increase in the apoptosis (unaltered Bcl-2 and Caspase-9) accompanied with no phosphorylation of cjun/ATF-2 (Fig. 6, A and B). On sensitization with 10 mM RES in CC treated cells, these factors further remained unaffected with no involvement of JNK/p38 pathway. Conversely, involvement of JNK/p38 pathway through phosphorylation of c-Jun and ATF-2 was evidenced in100 mM RES sensitized CC treated cells versus CC and polyphenols alone (Fig. 6, A and B). However, progressive phosphorylation of c-jun/ATF-2, down-regulation of Bcl-2 and unaltered Caspase-9 was observed dose-dependently in CUR treated cells. Further, on sensitization with low dose CUR in CC treated cells the phosphorylation of c-jun/ATF-2 further rose and was subsequently inhibited both by SP 6000125 and SB 203580 respectively. With 30 mM CUR sensitization, phosphorylation of c-jun/ATF-2 and Caspase-9 were down-regulated compared to CUR alone or remained unaltered vis-à-vis CC respectively. Total JNK/p38 showed constitutive expression. These events were further supported by the data through respective inhibitors. Further, MnSOD and Catalase were also evaluated in presence of SP 6000125 and SB 203580 whose expression remained unaltered compared to respective treatments (Fig. 6, C and D). Finally, the SAPKs regulated Ser-167 phosphorylation of ER alpha in MDA MB-231 cells also displayed no change in expression in CC/RES/ CUR alone or in combination treated cells (Fig. 8).

Discussion
Despite decades of research, the use of dietary antioxidant supplementation during conventional chemo-and radiation therapy remains controversial vis-à-vis the efficacy and safety of complementary treatment [30,31]. Several in vitro studies have demonstrated that the concurrent administration of natural products viz. RES [14,15,22,31], CUR [22,[31][32] with chemoor radiation-therapy reduces treatment-related side effects without/with improving the efficacy of chemotherapy. Here, we determine the role of Centchroman (CC)/Polyphenols alone plus polyphenols sensitized CC treated cells during stress signaling and intracellular redox status in vitro using the established Estrogen receptor (ER) positive (+ve) MCF-7 and negative (-ve) MDA MB-231 Human Breast Cancer Cells (HBCCs). Polyphenols beyond pharmacological concentrations ($10 mM) elicit ER-independent non-genomic effects [33]. Conversely, sub-pharmacological concentration (#10 mM) induces sub-lethal oxidative stress through ER-dependent non-genomic phenomena [20]. Hence, sensitizing HBCCs with such doses may synergize/antagonize the action of CC. On an overall basis, this may be through the increased generation of Reactive Oxygen Species (ROS) affecting MAPK and similar stress-signaling cascades [33][34][35].
The number of cells in sub-G0/G1 population on treatment with low dose RES/CUR increased significantly by 6 h extending up to 24 h in MCF-7/MDA MB-231 cells ( Fig. 2A, B, D and E). However, with RES treated MCF-7 cells, the majority was arrested in S phase (24 h) [36] (Fig. 2A) while in CUR, the cells continued cycling accompanied with significant apoptosis [37] (Fig. 2D). For MDA MB-231 cells, no arrest was observed in both the ligands (Fig. 2B and E). Unlike MDA MB-231 cells, similar phosphorylation of Ser-167 of AF-1 region of ER alpha containing MCF-7 cells through low dose of RES/CUR supports comparable ER-dependent non-genomic transcriptional events compared to control [20] (Fig. 8). Polyphenols induce ROS via SAPK (JNK and p38 MAPK) capable of phosphorylating p53 under cell-cycle arrest or apoptosis is well known for MCF-7 cells [21]. p53 deficient MDA MB-231 cells proceed differently which may explain the dichotomy of apoptosis in the two cell types [2]. In relation to this we found, low dose polyphenol at 3 and 6 h neither showed significant ROS generation nor involvement of JNK, p38, p53 pathways and correlates well with unaltered morphology in MCF-7/MDA MB-231 cells ( Fig. 3A and B, 5 and 6, 7A-D, 1A and B). However, at 24 h significant increase of ROS was observed both in MCF-7/MDA-MB-231 cells. RES decreased GSH in MCF-7 or remained unaltered in MDA MB-231 cells. Conversely, CUR enhanced total GSH as compared to control in both cell types as reported previously [25] (Fig. 4A). These events were accompanied with increased MnSOD activity with unchanged CAT in MCF-7 and decreased MnSOD, CAT in MDA MB-231 leads to generation of ROS whose levels do not affect the JNK/p38 signaling as well as mitochondria mediated apoptotic events (Fig. 4, 5 and 6). We therefore, infer that polyphenols preincubation sensitizes MCF-7 cells for improved CC action.
The enormity of apoptosis increased significantly in low dose RES/CUR sensitized CC treated MCF-7/MDA MB-231 cells with treatment time. Enhanced S phase arrest (RES sensitized)/ apoptosis (CUR sensitized) increases phosphorylation of p53 in MCF-7 cells (Fig. 2 F and G). Simultaneously, increased JNK/p38 and decline of DYm activates downstream events such as Bax/Bcl-2 ratio and caspase-9 (Fig. 3D, 4 and 5). ROS burst with concomitant increase of SOD activity/unaltered CAT indicates altered redox homeostasis in low dose polyphenol sensitized MCF-7 cells (Fig. 3C, 4B). Increased apoptosis (RES sensitized)/G2/M phase arrest (CUR sensitized) in the absence of phosphorylation of p53 in MDA MB-231 cells shows the differential mode of action of the two polyphenols. Unlike RES, CUR showed JNK/p38 mediated apoptosis in MDA MB-231 cells (Fig. 6). The foregoing favorably contributes to the enhanced pro-apoptotic action in CUR sensitized CC treated MDA MB-231 cells as compared to either drug alone. We therefore, deduce that RES/CUR enhances the CC action through ROS mediated JNK/p38 as well as mitochondrial pathway in MCF-7 cells. However, RES/CUR sensitization enhanced apoptosis in MDA MB-231 cells without and with involvement of ROS mediated JNK/p38. Thus, despite being similar, RES and CUR have subtle differences in their molecular mode of action. The review by Howells [31] and references therein have shown evidences for synergistic actions of RES and CUR with various drugs in different cell lines.
Nonetheless, sensitization with high dose polyphenols renders the cells under arrest in Go/G1 (RES)/G2/M phase (CUR) as early as 3 h in MCF-7 versus 24 h in MDA MB-231cells preventing CC action and resultant apoptosis ( Fig. 2F and G). This thereby leaves the factors unaltered in CC treated polyphenols sensitized cells than with polyphenols per se in both cell types. Interestingly, CC plus RES displayed the involvement of JNK/p38 pathways in MDA MB-231 cells (Fig. 6). This also implies that high dose polyphenols by themselves invoke cell death. This may be however, unachievable physiologically owing to poor bioavailability etc. hence is more of academic value. Efforts therefore should be directed towards modifying the structure of polyphenols to overcome this paradox. Therefore, low dose polyphenols sensitization with demonstrated safety vis-à-vis normal cells can augment apoptotic efficacy of hormonal therapeutics like CC in ER-positive/negative MCF-7/ MDA MB-231 cells. Hence, sensitization through polyphenol under in vivo situation may improve the efficacy of hormonaltherapy with reduced treatment-related morbidity.