Redox Mechanisms in Centchroman Mediated Antineoplasticity in Human Breast Cancer Cell Lines

Abstract

The mere mention of the word ‘Cancer’ itself quivers our mind in general context. It is the form of cellular proliferation which escapes the body’s innate defence mechanism. It is considered as one of the major cause of mortality second to cardiac related diseases around the world. In India, womenfolk in villages are thought much healthier than their urban counterparts due to their natural way of living. Alarmingly, the global statistics focussing on Breast Cancer (BC) has forced to think again as facts speak otherwise. According to a very recent report (July, 2009) of Cancer Detection Centre for Women at Tej Bahadur Sapru (Beli) Hospital, Allahabad, U.P, India, 38 rural women were out of total 42 confirmed cases of cervix (32 cases) and breast cancer (8 cases) identified among the urban and rural population of Allahabad and the neighbouring regions. In India, BC accounts for approximately 20% of total cancer with an increasing incidence amongst urban women. Globally, 18.4% of women are on the verge of its fatality. Even the male population has been a rare victim of BC, (approx. 1%), disregarding the metaphor being a feminine disease. Intense basic and clinical investigations conclusively have brought forth the etiological role of estrogens (E2) in the basis of increased BC risk. E2 regulated actions are mediated via Estrogen Receptor (ER) isoforms- α and -β belonging to the family of nuclear, ligand-activated transcription factors. BC recurrence and death has been treated for the last two decades using adjuvant chemotherapy involving a combination of cytotoxic anticancer drugs and Hormonal Replacement Therapy (HRT) depriving cancer cells of E2. Partial successful procedures have led into search for potent antagonist to E2 biosynthesis or its action like that of antiestrogens (AE) or Selective Estrogen Receptor Modulators (SERM).Tamoxifen (TAM) being one such drug blocks the E2 action by binding to ER expressing differential action according to the concentration. Despite TAM being in the frontline chemotherapy for BC, it is associated with an elevated risk of developing endometrial carcinoma and chronic treatment causes drug resistance. TAM and other similar AE acts as cytoproliferative/- static or -toxic agents. Thus, search for potentially safer therapeutic drug for long-term is desired. An in-house developed non-steroidal SERM, Centchroman (CC), possesses potent antiestrogenic activity. It was primarily developed as an oral contraceptive already being marketed in India. As a candidate antibreast cancer agent, it is undergoing Phase III Multicentric Clinical Trials in Stage III/IV Breast Cancer. Earlier we have reported that apoptosis in conjunction to oxidative stress accomplice in CC induced cytotoxicity in MCF-7 and MDA MB-231 Human Breast Cancer Cells (HBCCs) portraying its antineoplastic potential. The molecular basis of the druginduced cell death provides an exemplar to predict the sensitivity and enables a rational approach to drug design and therapy by utilizing knowledge of signalling pathways as targets. This led us to investigate the molecular events involved in antiproliferative and cytotoxic action of CC on HBCCs, which can be mediated through apoptosis or other pathways of cell death including oxidative stress. Studies with AE like TAM depict their binding to ER, thereby modulating gene expression, leads to the inhibition of proliferation and augmentation of apoptosis in BC cells. Besides these ER-mediated genomic response, there are reports regarding TAM used beyond pharmacological concentrations (>5μM) that display ERindependent non-genomic response in various cell types. TAM and its metabolites have been shown to be an antioxidant. TAM induced apoptotic cell-death involving oxidative stress mechanisms initiated at the cell membrane has already been reported. This may possibly be either through autoxidation of the phenolic moiety, producing superoxides and hydrogen peroxide through redox cycling, or by virtue of its high lipophilicity and partitioning into the cell membrane determining a transmembrane signal transduction cascade like ERK or activation of JNK1 and caspases. Apart from that, E2 mediated non-genomic signalling is mediated by membrane associated ER, possibly inferring that TAM and may be other AEs including CC are also capable of activating various receptors and/or other signalling pathways that are convergent. The intracellular redox status has been concerned to play an important role in the cell survival and death. Oxidative stress occurs when the critical balance of intracellular redox status is disrupted because of excess ROS, biochemical antioxidants depletion, or both. Oxidative stress evokes many intracellular events, such as gene activation, cell-cycle arrest and modulates apoptosis when the cells are under acute stress. In contrast, under non-stressed conditions ROS induces cell proliferation while enhanced generation in the cells under acute stress agents, including anticancer drugs, promote apoptosis by stimulating pro-apoptotic signalling molecules such as ASK1, JNK, p38 and p53 etc. For the study undertaken here, it was necessitated to evaluate the human breast cancer cell line model in vitro for the potential drug targets and for the probable therapeutic purpose. MCF-7 and MDA MB-231 cells derived by pleural effusion from post-menopausal patient proved to be an in vitro model of choice due to the presence and absence of ER respectively. These have been used to deduce estrogen receptor dependent and independent mode of drug action. Moreover, it further strengthens the cause of minimization of in vivo experiments urgently needed due to the primary reason of animal ethics. Effect of CC on normal (HEK, hPGF and MDCK) versus other cancerous cell types were also explored. Morphological and growth-kinetics investigations were made using Phase Contrast microscopy to optimize the culturing of cells. The cytotoxicity of the drug was ascertained by Sulforhodamine-B assay. Cytotoxicity studies reveal that CC was comparatively unsusceptible towards the normal cells. Loss of Membrane integrity study reveals the high lipophilic nature of the ligands corroborating to the cytotoxicity profile. Cell-cycle was studied in a dose-dependent manner using Flow-Cytometry which confirms its disparate effects on cell-cycle of MCF-7 and MDA MB-231 cells at different concentrations of the ligands. To explore the mitochondrial events involved, Reactive Oxygen Species (ROS) generation was determined quantitatively and qualitatively by Flow Cytometry and Fluorescence microscopy respectively. Next, to ascertain the pro- or anti-oxidant nature of the ligands, their free radical scavenging activity was evaluated using the DPPH assay. Biochemical enzymatic assays were performed to determine the basic antioxidant enzyme Catalase (CAT) and Total Glutathione (GSH) status in a cell upon drug exposure. This was performed basically to ascertain the oxidative stress involved in the mechanistic approach of cellular death. To explore the molecular events of oxidative stress involved, expression of various antioxidant enzymes were delineated through Western Blotting. Expectedly, CC enabled modulation of enzymes like Superoxide dismutase (Cu,Zn-SOD, MnSOD), Catalase (CAT), Glutathione Transferase (GST) as well as Cytochrome P450 (CYP 450) evaluated for their expression. Next important factor determined was to check for the DNA damage induced by ROS generation. Although it was found that Abasic (AP) site damage was not induced but internucleosomal fragmentation was observed which has already been reported by us. The present studies however, succeeded in exploring the oxidative stress signalling mechanisms involved in CC induced cytotoxicity in MCF-7 and MDA MB- 231 HBCCs. However, this study put forward a notion about the involvement of Oxidative stress in conjunction to apoptosis. These mechanisms could possibly be exploited to improve the approach in the design and modification of this Selective Estrogen Receptor Modulator (SERM) as well as therapeutic response to the treatment of patient. The following conclusions were drawn from the studies reported in the present work: 1. CC induces dose-dependent toxicity in both the breast cancer cell lines with a disparate degree of response. The MCF-7 cells were found to be more susceptible than MDA MB-231 cells. 2. In normal cell types CC elicited cytotoxicity at comparatively higher doses (>25μM for HEK 293, Hep G2 and MDCK whereas ≈25μM in hGF) suggesting inactivity of CC towards the normal cells. 3. Flow Cytometry studies indicate that CC induces cell-cycle arrest, possibly due to the overexpression of Cyclin dependent kinase inhibitors (CDKIs) p21Waf1/Cip1 and p27Kip1 as reported. 4. At higher doses, the cells are pushed into sub-G0/G1 phase i.e. apoptotic phase failing to undo the drug induced damage. 5. CC initiates apoptosis even at the low dose of 1μM. 6. CC induces mitochondrial events through the generation of ROS at lower doses and subsequently it decreased due to severity of the drug concentration confirmed qualitatively and quantitatively. 7. CC was examined for the Free Radical Scavenging property in a cell-free system. It was performed to determine the need of the drug metabolization as any xenobiotics administered is metabolized in a cell. As the drug was in its pro form, so it had null effect on the scavenging of free radicals at any of the concentration tested. 8. Catalase (CAT) enzyme specific activity was almost equivalent or significantly downregulated which suggests of high level of oxidative stress environment in the cells upon CC treatment to both the cell types. 9. Total Glutathione (GSH) downregulation also confirms a high level of oxidative stress even in the ER-ve cells as compared to ER+ve cells. 10. CC disturbs the intracytosolic redox milieu and thereby modulates expression of various oxidative stress factors involved. 11. Cu,Zn SOD upregulation signify the cellular innate defence mechanism upon elevated ROS generation. 12. There was a downregulation of MnSOD in ER-ve MDA MB-231 cells in comparison to ER+ve MCF-7 cells signifying a high level of oxidative stress even in the ER-ve cells. 13. CAT expression was almost equivalent at all the concentration signifying the generation of stress like environment by creating a load of H2O2 in the cells thereby eliciting downstream signalling for the cellular death. 14. CC induces CYP 450 family of drug metabolizing enzymes in ER+ve cells while was downregulated in ER-ve cells suggestive of the role of ER in the drug metabolism and subsequent signalling mechanism. 15. Although there was no AP site DNA damage observed but we have already reported of internucleosomal DNA fragmentation, a typical hallmark of apoptosis. CC is an in-house developed molecule with known antiestrogenic profile. Cumulatively, the results suggest that CC causes cytotoxicity in HBCCs and membrane deformation due to high lipophilic nature. Apart from the role of ER in the drug’s action, CC was found to accomplish its non-genomic mode of action in causing cell death, at least in part, along with apoptosis. However, non-genomic signalling mechanisms are rapidly activated to adjust the dynamic changes of the cell environment. The facts evidenced in this study may provide an insight into the possible substitute for drug resistant malignant breast carcinomas which needs further elaboration. In contrast to the studies reported earlier, preliminary role of CC mediated similar response was elucidated determining the plausible mechanisms of oxidative stress induced cell death. Conclusively, it is a fact that there are multiple pathways involved in the cytotoxic effect of any anti-cancerous drug involving receptor -dependent or -independent induction of oxidative stress.