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In developed countries, endometrial cancer is the fourthmost common malignant tumor in women, after breast, colon, and lung cancers7. Majority of patients are in the post-menopausal age group with 91% of cases occur in women aged 50 and older7. Although the incidence has remained stable, the death rate has increased over 100% in the last two decades8. Tumor cells have some features: (1) they are self-sufficient in mitogenic/growth signals; (2) they have intensive anti-growth signals; (3) they have limitless replication performance; (4) they undergo sustained angiogenesis and (5) they achieve tissue invasion and metastasis9. These features allow tumor cells to evade apoptosis, a cell suicide program to eliminate damaged and unwanted cells in order to maintain homeostasis10.Induction of apoptosis is not only an important defense against cancer, but also has recently attracted attention as a novel target for cancer chemoprevention11.
Apoptosis induced by CC is associated with oxidative stresses derived from reactive oxygen species (ROS), and mitochondrial dysfunction6. ROS including hydrogen peroxide(H2O2), superoxide anion (O2•-), and hydroxyl radical (•OH)have been implicated in the regulation of many important cellular events such as transcription factor activation, cell proliferation,and differentiation12. Particularly, excessive production of ROS gives rise to the activation of events leading to death in many cell types13.
Up to date, many studies have shown that CC inhibits cell proliferation in many human cancer cell lines, including head and neck cancer cells and breast cancer cells14, 15. However, there is no study have examined the effect of CC in endometrial cancer cells. Moreover, the precise mechanism for the effect of CC on the cell cycle and apoptotic pathway in endometrial cancer cells is not completely understood.
Previous year study examined the effects of CC on the modulation of cell death, cell cycle arrest and apoptosis in human endometrial carcinoma cells. The results showed that CC significantly inhibited Ishikawa cell growth at micro molar concentrations and the antiproliferative effect of CC is mediated through cell cycle arrest at the G1 phase.
7. Report of the work done since its inception with particular emphasis on the Following:
a) Aims and objectives:
To delineate the Apoptotic events in Centchroman (CC) induced apoptosis in Ishikawa Endometrial Cancer Cells.
b) Material and methods:
The plan has been executed in vitro using HECCs Ishikawa Cells.
Ishikawa cells procured from The European Collection of Cell Cultures, Wiltshire (UK) were cultured in DMEM medium containing Penicillin (100U/ml), Streptomycin (200μg/ml), Gentamycin (50μg/ml) supplemented with 10% Fetal Bovine Serum (FBS) and incubated at 37 °C in a humidified, 5% C02 incubator.
For experimental purposes, as we previously published  Ishikawa cells were grown in estrogen-free medium i.e. phenol red-free DMEM containing Penicillin (100U/ml), Streptomycin (200μg/ml), Gentamycin (50μg/ml) supplemented with Charcoal stripped Fetal Bovine Serum (DCC/FBS) 16. The cells were cultured for a total of 4 days in phenol red-free DMEM contain 10% DCC/FBS Including with the CC during the last 2 days.
Morphological assessment of apoptotic cells by Acridine Orange/Propidium Iodide (AO/PI) staining:
Ishikawa cells were seeded in 12-well plate and incubated at 37 - C in 5% CO2. After 48h, the medium in each well was removed and replaced with the CC at 1-25 µM for 48h. After incubation, treated and control cells were harvested, washed with PBS, incubated with 5μL of acridine orange (10μg/mL) and propidium iodide (10μg/mL) at a ratio of 1:1 and Freshly stained cell suspension was dropped into a glass slide and covered by a cover slip. Slides were observed under UV-fluorescence microscope (Nikon Eclipse Ti, Japan) within 30 min before the fluorescence fade. AO and PI are intercalating nucleic acid-specific fluorochromes which emit green and orange fluorescence, respectively, when they are bound to DNA and only AO can cross the plasma membrane of viable and early apoptotic cells. The criteria for identification are as follows: (i) viable cells appear to have green nucleus with intact structure; (ii) Apoptosis exhibit a both Green and Orange nucleus through compromised cell membranes and were often less round because of apoptotic blebbing; and (iii) Orange intact nucleus depicting secondary necrosis17. This assay provides a useful quantitative evaluation of apoptosis.
The level of apoptosis induced by CC was determined by Annexin V/PI double staining assay by flow cytometry. For evaluating apoptosis, 0.2-106 cells were plated into 6 well culture plates and treated with 1-25μM CC. Annexin V-FITC and PI staining according to manufacturer's instructions (Sigma).
Apoptisis inhibition/ROS inhibition was performed using Z-VAD FMK (30µM), and N-Acetyl-l-Cysteine (L-NAC, 5 mM) respectively, 1 h prior to drugs treatment. The cells were analyzed by Annixin V/ PI. These concentrations of inhibitors (Z-VAD FMK, L-NAC) were nontoxic to cells under our pre-incubation conditions.
In vitro measurement of Caspase-3 activity assay.
To analyze the role of Caspase-3, the cells were cultured at a density of 3x106cells and treated with 1-20μM CC for 48 h and 20µM CC for 12-48 h in T-75 flasks. Activities of caspase-3 were determined using a colorimetric assay based on the protocol we followed previously5.
Briefly, the cells were lysed in 500μl of lysis buffer (Sigma Chemical Co, St. Louis, MO, USA) at 4 °C by Sonication. 200μg of cell lysate protein was mixed in assay buffer in a final volume of 100μl, followed by the addition of 10μl of 2 mM of substrates specific for Caspase-3 Z-DEVD-pNA was respectively employed. Following incubation of the substrate with cell lysate at 37 °C for 30 min, the liberated p-nitroaniline (pNA) was read at 405 nm on SpectraMAX M2e Microplate Reader. The activity of caspase-3 was calculated according to Manufacturer instruction.
Superoxide Dismutase and Catalase Gene Product Expression (Specific Activity) in HECCs
Superoxide dismutase (SOD) and Catalase (CAT) activities were determined according to McCord and Fridovich, and Beutler 15, 18 respectively. After drug treatments Ishikawa cells were collected by scraping, centrifuging and the pellets sonicated in chilled 1.15% KCl. The cell extracts was spun at 1500xg for 5 min at 4°C. The resulting supernatants were analyzed for enzyme activities. The specific activity of SOD was calculated as follows:
Specific Activity = A x dilution factor x 1000/One unit x volume of enzyme (ml) x mg 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/min x dilution factor x 1000/0.00394 x volume of enzyme
(ml) x mg protein =mmol H2O2 reduced/min/mg protein.
V. Statistical Analysis:
All the experiments were carried out in triplicate, and the results were expressed as Mean, ± S. E. Student's 't' test were performed to determine the level of significance.
Induction of apoptosis in Ishikawa cells as a result:
Treatment of cultured Ishikawa cells with Centchroman (CC) for 48 h induced apoptosis. Acridine orange and propidium iodide staining methods were used to determine the apoptosis and necrosis rates on Ishikawa cells after 48 h incubation with CC. (Fig. ). An increase in the percentage of apoptotic cells and small population of necrotic cells were observed with increasing concentrations of CC from 1-25µM (Fig. 2).
Centchroman induces ROS through suppression of catalase activity.
For detoxification, catalase converts H2O2 to H2O and O2. We examined if CC inhibits catalase activity, resulting in elevated H2O2 levels in the cells. As shown in Figure, IC50 dose of CC caused significant suppression of catalase activity in Ishikawa cells at 48h but starting as early as 12h catalase activity was elevated. There were no detectable changes in the expression level of catalase in CC-treated cells (Fig. 4b). A similar inhibitory effect on catalase activity by CC was also detected in MCF-7 and MDA MB-231 cells6, 15.
d) Conclusions and major results achieved:
Ishikawa cells have been discovered in 1985 19, it was several years before the Ishikawa Endometrial Cancer Cell line was characterized in detail in 2002 20. In agreement with the reported studies, we found that the cells do have a polygonal shape, follow a typical growth profile and also respond to the SERMs, TAM/CC. To determine efficacy of these ligands we have employed various techniques such as: 1) Morphology, 2) Viable cell counting, 3) Protein content, 4) SRB, 5) MTT assays and 6) FACS analysis to ascertain that the antineoplastic action of CC can be elicited in these cells. Normal growth profile was also quantified and it was observed that The Growth Rate (Growth Rate = number of doublings that occur per unit of time) was 0.0159 and doubling time was 43.55 hrs.
CC, a CDRI developed prototype drug for breast cancer treatment and sold as a contraceptive by Hindustan Latex Limited, has been found to be an estrogen antagonist like TAM in the breast 4, 5. In this study, we investigated the action of CC on HECCs. For this purpose, we used the ER +ve HECCs, Ishikawa Cell line. Result exhibited upto 10 µM there was no significant cytotoxic effect, however, dose-dependent cytotoxicity was noticed beyond 10 µM within 48 hrs and IC50was observed at 20 µM. Concluded with the quantitative data through FACS for the percentage of apoptotic nuclei (sub G0/G1 population) was found much greater with CC than TAM at various doses (Fig.3.2) which shows apoptosis induced by CC in Ishikawa cells more than the TAM dose-dependently upto 20 µM. Exposure to 20μM CC in Ishikawa cells causes 10 fold increase in percentapoptotic nuclei whereas the corresponding rise by TAM. This illustrates higher susceptibility of Ishikawa cells with CC. However, further analyses of the intrinsic and extrinsic pathways of apoptosis and their regulation with CC need exploration for its future employment