Elucidate the mechanism of anticancer activity of Piper nigrum extract on colon cancer cell line Akila Prashant, MVSST Subbarao, Devananda D, Prashant Vishwanath, Suma MN Centre of Excellence in Molecular Biology and Regenerative Medicine, Department of Biochemistry, JSS Medical College, JSS University, Mysore, Karnataka ABSTRACT RESULTS INTRODUCTION RESULTS Cont… Cyclin D1 BMI 1 P53 β-Actin Introduction: The present study was taken up considering the rising trend of colorectal cancer and death caused due it in India and our previous studies with ethanolic extract of Piper nigrum (EEPN) showing effective anticancer activity Aims and objectives: Here we have revealed the mechanism of the anticancer activity of EEPN on colon cancer cell line Materials and methods: The EEPN in concentrations of 3µg/ml (PN1) and 6µg/ml (PN2) were used to treat the colon cancer cell line HCT116. The cell death in the treated flasks was compared to that of untreated flasks (control) as well as that treated with 1% dimethyl sulfoxide (vehicle control). The apoptotic cells was examined under fluorescent microscope using dual fluorescent staining solution (1 µl), containing 100 µg/ml of acridine orange and 100 µg/ml of ethidium bromide. The relative change in expression of cyclin D1 and P53 in the treated and untreated flasks were studied by western blot and quantitative real time PCR. Results and observations: No significant apoptosis was detected in the control group and in vehicle control group. Both early and late apoptotic cells were seen in cells treated with 3µg/ml of EEPN. Late-stage apoptotic cells, with concentrated and asymmetrically localized orange nuclear ethidium bromide staining, were detected in cells treated with 6µg/ml of EEPN. Flow cytometric analysis of cell cycle parameters showed an arrest in the G2/M phase. The western blot analysis showed down regulation of cyclin D1 and BMI1 along with upregulation of P53 protein. Conclusion: Treatment of HCT116 colon cancer cell lines with the EEPN confirmed apoptosis showing dose dependent arrest of the cells in G2/M phase. The differential expressions of cyclin D1, p53 and BMI1 in HCT116 colon cancer cells after EEPN treatment may play critical roles in the G2/M cell cycle arrest that blocks cell proliferation and induces apoptosis. Dietary phytochemicals which are currently being investigated for their possible clinical application in cancer prevention or treatment have shown to have little or no toxicity. Piper nigrum considered as “The King of Spices” is a valuable medicinal plant, which is known to possess many interesting pharmacological actions, anticancer activity being one of them. Our earlier studies have shown significant cell death in colon cancer cell lines when treated with ethanolic extract of Piper nigrum (EEPN). In this study, we would like elucidate the mechanism of cell death caused by this extract on human colon carcinoma cells Goal : : Here we have revealed the mechanism of the anticancer activity of EEPN on colon cancer cell line A B C D Figure 1: (A) Untreated group with no apoptotic cells. (B) Vehicle control group with one apoptotic cell. (C) Cells treated with 3µg/ml of EEPN showing both early and late apoptotic cells. (D) Cells treated with 6µg/ml of EEPN showing late apoptotic cells. Figure 3: Effect of ethanolic extract of Piper nigrum on Cyclin D1, BMI1 and p53 activities by Western blotting assay, HCT 116 cells were treated with Piper nigrum extract (6 and 3µg/ml) for 48 h and were compared with cell control. A) Untreated B) DMSO Treated DISCUSSION Downregulation of BMI1 leads to de-repression of Ink4a, which encodes tumor suppressors p16Ink and p19Arf. This p16 and p19 proteins are subjected to inhibit the binding of Cyclin D to CDK4/6 which results in the suppression of retinoblastoma (pRb) and causes induction of cell cycle arrest p19 induces p53 and causes cell cycle arrest. This may be the possible mechanism of cell death seen in colon cancer cell line HCT116 on treatment with Piper nigrum extract. METHODS AND MATERIALS ETHIDIUM BROMIDE (EB) AND ACRIDINE ORANGE (AO) STAINING TO DETECT APOPTOSIS The cultured cells were treated with appropriate doses of EEPN and incubated for 48 hrs. 25 µl of cell suspension from each of the sample groups were transferred to the glass slide as a droplet. Dual fluorescent staining solution (1 µl), containing 100 µg/ml of AO and 100 µg/ml of EB was added to each suspension and then covered with a coverslip. The morphology of apoptotic cells was examined using a fluorescent microscope. CELL CYCLE ANALYSIS BY PI STAINING Propidium Iodide (PI) staining method was used for cell cycle analysis in which fluorescent nucleic acid dye i.e. PI (Propidium iodide) was used to identify the proportion of cells that are in one of the three interphase stages of the cell cycle. WESTERN BLOT ANALYSIS The cultured cells were treated with appropriate concentration of EEPN and incubated for 48 hrs. The collected cell lysates were subjected to western blotting using antibodies against cyclin D1 and P53. The level of expression of protein of interest was compared with housekeeping gene. Relative quantification was done using imageJ software. C) Oxaloplatin Treated D) 3µg/ml of EEPN CONCLUSIONS Treatment of HCT116 colon cancer cell lines with the EEPN confirmed apoptosis showing dose dependent arrest of the cells in G2/M phase. The differential expressions of cyclin D1, p53 and BMI1 in HCT116 colon cancer cells after EEPN treatment may play critical roles in the G2/M cell cycle arrest that blocks cell proliferation and induces apoptosis. F) Percentage of cells E) 6µg/ml of EEPN CONTACT Figure 2. Flow cytometric analysis of cell cycle parameters. HCT116 colon cancer cells were incubated for 48 h in the presence of 3µg/ml of EEPN (D), or 6µg/ml of EEPN (E), or Oxaloplatin (C) or vehicle DMSO (B), or without additive (untreated) (A). Cells were then harvested by trypsinization, fixed, and stained with propidium iodide for analysis by flow cytometry. Each histogram indicates the percent of cells in sub G1 (blue fraction), G0/G1 (red fraction), S (green fraction) and G2/M (purple fraction) phases of the cell cycle (F). Dr. Akila Prashant CEMR Laboratory, JSS Medical College, JSS University, Mysore Email: akilaprashant@jssuni.edu.in Phone: 9008097970 Website: http://jssuni.edu.in/cemr/home REFERENCES Baldin V1, Lukas J, Marcote MJ, Pagano M, Draetta G. Cyclin D1 is a nuclear protein required for cell cycle progression in G1. Genes Dev. 1993;7(5):812-21. Nancy Y Assad, Kandil Mona A, Mokhtar Nadia M. Prognostic value of Cyclin D1 and p53 protein in colorectal carcinoma. Journal of Egyptian Nat.Can.Inst. 2000; 12(4):283-292. Acknowledgements: We would like to acknowledge Vision Group on Science and Technology, Government of Karnataka for funding this project under their Establishment of Centres of Excellence in Science, Engineering and Medicine programme in the year 2012-13