Studies on Selective Estrogen Receptor Modulator Induced Molecular Events in Cancer Cells

Abstract

Estrogen is an important steroid hormone involved in the regulation of cellular differentiation and proliferation (1). However, aberrant regulation of estrogen induced effects leads to severe hormonal dysfunctions resulting in cellular transformation followed by initiation and progression of several hormone dependent cancers (e.g breast, endometrium, ovary, prostate and colon etc.). As the growth stimulatory effect of estrogen is mediated through the estrogen receptors (ERs), hormonal therapies targeted towards the blockage of estrogen receptors (ERs) or inhibition of estrogen synthesis have been exploited as therapeutic strategies for the treatment of these cancers (2-3). The pharmacophores that target the ER fall into two major classes; Selective Estrogen Receptor Modulators (SERMs) and the pure antiestrogens. The pure antiestrogens suppress the growth of tumors but they also abolish the beneficial effects of estrogen involved in the regulation of the normal physiological processes resulting in severe side effects and hormonal imbalances in the body (2). Therefore the tissue selective effect of SERMs is considered to be a better therapeutic option for treating the estrogen sensitive cancers. Based on this concept Tamoxifen, a SERM, has been clinically approved and used successfully as an anticancer drug in breast cancer treatment. However, the efficacy of Tamoxifen in many patients with breast cancer is limited by a number of factors. Only 60–70% of the breast tumors are ER-positive, and potent anti-tumor activity of Tamoxifen is observed exclusively in tumors that express ER. Almost all patients with advanced breast cancer who initially respond to Tamoxifen therapy, eventually develop chemo resistance. Even though the development of acquired resistance to Tamoxifen does not exclude response to other types of treatments including other endocrine agents (4) the knowledge of the molecular mechanisms responsible for chemo resistance to SERMs and assessment of novel pharmacophores/drugs with similar effects is extremely important in the development of novel strategies for treatment of these cancers. In this context, synthetic SERMs have been one of the most vigorously studied pharmacophores, especially in case of estrogen sensitive cancers. And even after two decades of research, the interest in these groups of compounds still shows no signs of subsidence. Apart from the well known SERM Tamoxifen, two additional SERMs have been approved by the FDA for clinical use in the USA: i.e Toremifen for the treatment of advanced breast cancer and Raloxifene for the prevention and treatment of postmenopausal osteoporosis and ER-positive breast cancers (3-4). However it is a matter of debate whether Raloxifene would have advantages over Tamoxifen as a chemopreventive anti-breast cancer agent. Like Tamoxifen, Toremifene is a derivative of triphenylethylene and thus has very similar efficacy and side effects to that of Tamoxifen, and exhibits cross-resistance with Tamoxifen (5-6). On similar lines based on the Tamoxifen structure a synthetic SERM, Centchroman (67/20; INN: Ormeloxifene) has been developed by the Central Drug Research Institute (CDRI), Lucknow. It is successfully marketed as a non steroidal birth control pill in the Indian market (7-8).CC has a very good pharmacokinetic profile, is devoid of cytotoxicity and has an excellent therapeutics index (7).Preliminary reports are also available for its tumor suppressive and apoptotic effects in breast cancer cells (9). However, its anticancer properties in other cancer types has not been studied in detail. Furthermore there are some promising estrogen receptor modulators from the natural source with established anticancer properties (10-11). These molecules are advantageous because of their non steroidal nature and lack of side effects ( 10-11 ). These plant derived molecules that have been traditionally used for chemotherapeutic agents (10-11). Therefore, it is imperative to assess the effect of such molecules in estrogen dependent and independent cancers. Overwhelming publications on this area (10-11) suggest that one naturally derived SERM, Shikonin can be a potent chemotherapeutic agent in cancer cells, however it is imperative to study its SERM like / unlike effects in cancers of diverse origin to assess its potency as an anticancer agent. Gaps in Knowledge The clinical limitations of the first generation SERMs like Tamoxifen, has fueled an intense search for newer and better SERMs. The ultimate goal of the SERM research is to find a molecule that 1) has all the benefits of estrogen 2) minimal side effects of the conventional SERMs and 3) is effective against hormonal dependent/ independent cancers. Therefore it is important to develop a better understanding of the effects of novel SERMs/SERM like pharmacophores in various cancer types. Based on this backdrop we have selected one indigenous synthetic SERM centchroman (CC) and a naturally derived SERM, Shikonin for the present study. Centchroman (DL-Centchroman: 67/20; INN: Ormeloxifene; CC) is a non-steroidal SERM developed by Central Drug Research Institute, Lucknow, with specific ERα inhibitory effect (8). CC has favorable pharmacokinetic, toxicity and bioavailability profiles, excellent therapeutic index and has reported anti-tumor activity in breast cancer cells. However, these reports are inconclusive and nothing known in other cancers. Studies on cancer patients suggested that that CC can be a useful agent for the treatment of advanced breast cancer in both males and females (7). However, the details of its effects on cancer cell signaling pathways is not clearly defined. Although the natural SERM Shikonin has multiple pharmacological effects like anti-inflammatory, anti-gonadotropic, osteogenic, HIV-1 protease inhibiting and anti-tumorogenic activities with, multiple mechanisms of action (12-13). However, its estrogen dependent/independent effects in cancer cells have not been studied in detail. Therefore the present study was planned with the following objectives: 1. To investigate the pharmacodynamics of the synthetic SERM, Centchroman/Ormeloxifene in breast and prostate cancer cells in vitro. 2. To investigate the effects of Centchroman/Ormeloxifene on the cell cycle dynamics in breast and prostate cancer cells in vitro. 3. To investigate the effect of Centchroman/Ormeloxifene the on the cellular signaling pathways in breast, prostate and ovarian cancer cells in vitro. 4. To study the effect of Centchroman/Ormeloxifene on radio sensitivity of prostate cancer cells. 5. To study the effect of the naturally derived SERM, Shikonin induced cell death signaling in prostate / cervical cancer cells.