Asian Research Thesis Index

Abstract

Breast cancer is the most prevalent disease in women all-around the world. It is the leading cause of death in women. About 0.5 million deaths of breast cancer are reported worldwide. Options available for breast cancer treatment are surgery, radiation therapy and chemotherapeutics, e.g., doxorubicin, paclitaxel and cyclophosphamide. To improve therapeutics in breast cancer there is a need to develop better compounds with good efficacy and fewer side effects. The prime objective of present study is to analyze the activity of acetamide derivative, N-(2-hydroxyphenyl) acetamide (NA-2) and benzimidazoles derivative, 2-Amino-1 Me Benzimidazole (3Ve) on human breast cancer cell line MCF-7. Cytotoxic effects of NA-2 and 3Ve was determined by MTT assay. Doxorubicin was used as a standard drug in this study. Both NA-2 and 3Ve have shown dose dependent growth inhibition of MCF-7 cell line following 48h treatment. To explore the effects of these compounds on the mechanisms of apoptosis, cell cycle, angiogenesis and proliferation the cells were treated with IC50 doses of these drugs. The process of wound healing was assessed by scratch assay. Both the compounds delayed the wound healing process significantly. The effect of these compounds on cell cycle stages was monitored by flowcytometry. Both NA-2 and 3Ve arrested the cell cycle at G0/G1 phase and DOX has shown G2/M arrest. The apoptosis was assessed though ANNEXIN-V FITC assay. The morphology was seen by using fluorescent microscope and quantification was done by flowcytometry. The cells after treatment with the test compounds and standard have demonstrated the morphology of apoptotic cells. NA-2 and DOX produced late appearance of apoptotic events and 3Ve resulted in early apoptosis.  The effects of NA-2, 3Ve and DOX on BAX (proapoptotic) and BCL2 (antiapoptotic) were also studied. Following 48 hours treatment, NA-2, 3Ve and DOX have enhanced BAX expression and attenuated BCL2 expression as compared to untreated control group. The results were also significant when we studied the effects of the compounds on BAX/ Bcl 2 on protein expression level by immunocytochemistry. Angiogenesis was assessed by using the markers VEGF and AKT. To observe antiangiogenic effect of the compounds RT-PCR was performed but none of these was effective to decrease the gene expression of  XIV   aforementioned markers.The anti proliferative effect of the compounds was further assessed by cellular markers of signal transduction pathway involved in breast cancer progression. These are JAK1, JAK3, STAT2 and STAT5A, which are highly expressed in breast cancer. The effects of the compounds were assessed on both gene and protein expression level. The results of both the compounds were found more significant on JAK 1 and STAT 2 expression levels. The same pattern was seen with Doxorubicin. While in case of JAK3, NA-2 did not diminish either gene or protein expression while 3Ve and DOX decreased the expression at genetic as well as protein level. The gene and protein expression of STAT5A following 3Ve treatment decreased after 48 h significantly. NA-2 and DOX were only effective to diminish the protein expression of STAT5A.  We therefore draw a conclusion that NA-2 and 3Ve can inhibit proliferation of breast cancer cell line MCF-7 by induction of apoptosis and arrest of cell cycle at different stages. NA-2 and 3Ve have also attenuated different markers of JAK/STAT pathway.

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