ISOLATION OF CANCER STEM CELLS EXPRESSING THE SELECTED BIOMARKERS FROM COLORECTAL CANCER AND EVALUATING THE THERAPEUTIC EFFICACY OF PIPER NIGRUM AGAINST THESE CANCER STEM CELLS

Abstract

newlineABSTRACT newline newlineOBJECTIVE newlineTo study the In vitro anticancer activity of ethanolic extract of Piper Nigrum(EEPN) against colon cancer cell lines. To investigate the mechanism of cell death after treating with EEPN in colon cancer cell line. Isolation of CRC stem cells expressing the biomarkers CD133, CD44 and CD66c from human colon cancer specimens. To study the effect of EEPN on the expression of stem cell related genes and proteins in colon cancer cell line. newlineMETHODS newlineColorectal carcinoma cell lines (HCT 116, HT29 and HCT15) were procured and cultured in complete DMEM media. EEPN was prepared by graded ethanol fractionation method. Cytotoxic effect of EEPN was determined by measuring the cell number using sulforhodamine B assay. IC50 values were calculated using Prism software. The EEPN in concentrations of 3and#956;g/ml and 6and#956;g/ml were used to treat HCT116 for further studies. The apoptotic cells were examined under fluorescent microscope using dual fluorescent staining solution, containing 100 and#956;g/ml of acridine orange and 100 and#956;g/ml of ethidium bromide. Propidium Iodide (PI) staining was used for cell cycle analysis in which fluorescent nucleic acid dye was used to identify the proportion of cells that are in one of the three interphase stages of the cell cycle. The relative change in expression of cyclinD1, P53 and BMI-1 in the treated and untreated flasks was studied by western blot and quantitative real time PCR. Cells expressing CD133, CD44 and CD66 were isolated using magnetic assorted cell sorting, grown in serum free media and observed for the formation of tumor spheres. The relative change in expression of these markers upon treatment with EEPN was studied by western blot, real time PCR and flow cytometry. newline newlineCONCLUSION newlineEEPN treatment blocks the cell proliferation and induces apoptosis by arresting the cells in the G2/M phase of the cell cycle and decreases the population of stem cells expressing CD133+/CD44+. newline