Characterization On Anti-Proliferation Properties Of Nepenthes Gracilis (Pitcher Pot Plant) Fluid In Breast Cancer Cell Lines
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Date
2017-08
Authors
Sahgal, Geethaa
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Breast cancer remains the second highly notorious killer among women and in the coming years, the age of patients inclining to be younger. Due to the adverse effect of estrogen receptor alpha-36 (ERα-36), there is emerging resistance in chemotherapy treatment for breast cancers. Thus, ER-α36 may be a potential membrane target for drug design against resistance in breast cancer chemotherapy via plant based substance. N. gracilis is a carnivorous plant and well-known for their digestive enzymes. The pitcher fluid of N. gracilis was collected and subjected to pH and protease inhibitory assays. The crude fluids were fractionated (G1 and G2 fractions) and tested to determine the properties on cell detachment, cell proliferation, migration, invasion, estrogen receptor (ER) activity, the expression level of PKCα and phosphorylation state of ERK 1/2, p38 MAPK and JNK in MDA-MB-231 cells. It has been found that the optimum pH for proteolytic activity of N. gracilis pitcher fluid was at pH 3.0 and contained the high content of aspartic proteinases. However, the effect of cell detaching by the crude and fractionated N. gracilis pitcher fluids was found to be significantly lower compared to trypsin-EDTA. MTT assay results have indicated that proliferation of MDA-MB-231 was significantly reduced by fractionated N. gracilis pitcher fluid at 48 hr post-treatment. The IC50 values of G1 and G2 fractions have been found to be at 1.89 ± 0.38 μg/mL; and 6.98 ± 0.17 μg/mL respectively. The growth of non-cancer origin MCF-10A was affected by crude N. gracilis pitcher fluid but not by the G1 and G2 fractions of N. gracilis pitcher fluid. At IC50 value the migration and invasion activities of MDA-MB-231 were decreased by the fractions at 24 hr post-treatment. To evaluate the involvement of ER-α36 in MDA-MB231 cells, the cells were treated with ICI 182 780 (EGFR inhibitor) to block the EGFR which is involved in cell proliferation activity. The estrogen receptor inhibitory assay proved that, ER-α36 was certainly involved in MDA-MB-231 cells proliferation activity. The N. gracilis pitcher fluid fractions treated MDA-MB-231 cells were used to determine the PKCα level and it is showed that, anti-proliferative activity of MDA-MB-231 cells were functioning through EGFR-ER-α36 associated signaling pathway and not from membrane-bound ER-α36 signaling mechanism. G1 fraction didn’t significantly reduce the expression of ERK 1/2 but it’s did reduce significantly the expression of p38 MAPK and JNK. G2 fraction did reduce the expression of ERK 1/2, p38 MAPK and JNK. Hence, the fractions demonstrate that, its involvement in the cellular mechanism of the MDA-MB-231 cell proliferation, migration, and invasion. The protein expression via western blot accentuates that, there is unsuccessful dissociation of the EGFR-ER-α36 complex by G2 N. gracilis pitcher fluid fraction contribute to the interruption of the MAPK/ERK signaling pathway and results in anti-proliferation of MDA-MB-231 cells. In consequence, N. gracilis pitcher fractions create a new area for the development of EGFR-ER-α36 complex target treatment on invasive MDA-MB-231 mammary carcinoma cells.
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Keywords
Characterization on anti-proliferation properties , of nepenthes gracilis fluid in breast cancer cell lines