The Mechanism Of Rapamycin And Andrographolide Action On The Growth Of Breast Cancer By In Vivo And In Vitro Study
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Date
2016-03
Authors
Mohd Khairi, Wirdatul-Nur
Journal Title
Journal ISSN
Volume Title
Publisher
Universiti Sains Malaysia
Abstract
Breast cancer is a major disease that kills women worldwide. Recent advancement in breast cancer treatments such as surgery, chemotherapy, radiation therapy and hormone dependent targeted therapy have improved patient survival rate. However, the results of chemotherapy to date remain unsatisfactory, prompting a need to identify new potential therapeutic agents that could target cancer cells efficiently. Rapamycin, a macrolide isolated from S. hygroscopicus and Andrographolide which isolated from A. paniculata (Hempedu Bumi) have been reported to exert impressive anti-cancer properties against various human malignant diseases. Therefore, this study aims to investigate the effect of Rapamycin and Andrographolide together with their combination on apoptosis signaling pathway and cell cycle arrest in in vivo NMU-induced rat mammary carcinoma model as well as in vitro breast cancer model. In the in vivo study, the histopathology of breast tumour samples were evaluated by Haematoxylin and Eosin staining, while the gene and protein expression of apoptosis related markers such as p53, Fas-Ligand, Bcl-2, Bax, Caspase 3, Caspase 8 and Caspase 9 were analysed using immunohistochemistry staining and Real-Time PCR assay. For in vitro study, the minimal inhibition concentration (IC50) values of Rapamycin and Andrographolide as mono or dual therapy were determined using MTS assay. Cell morphological changes was examined by microscopy. Meanwhile, apoptosis and cell cycle distribution and
apoptosis proteins expressions were determined by flow cytometry and western blot, respectively. The in vivo results showed that Rapamycin (0.2 μg/μl), Andrographolide (100 mg/kg) and combination (0.2 μg/μl + 100 mg/kg) treatments significantly inhibited breast tumour growth in NMU-induced rat model. In addition, Rapamycin, Andrographolide and combination treatments effectively inhibited proliferation of MCF-7 and MDA-MB-231 breast cancer cells in a time and dose dependent manner with IC50 of 0.5 μM, 25 μM and 0.1 μM + 20 μM (MCF-7) and 0.8 μM, 35 μM and 0.1 μM + 30 μM (MDA-MB-231), respectively. The anti-cancer activity of Rapamycin and Andrographolide in both in vivo and in vitro model were associated with up regulation of p53, Fas-Ligand, Bax, Caspase 3, Caspase 8, Caspase 9 and down regulation of Bcl-2 protein expression as well as induction of G0/G1, S and G2/M phase cell cycle arrest. Unfortunately, Rapamycin in combination with Andrographolide do not synergistically inhibited the growth of NMU induced breast tumour, MCF-7 and MDA-MB-231 breast cancer cells. Indeed, combination treatment do not synergistically increased the expression of p53, Fas-Ligand, Bax, Caspase 3, Caspase 8, Caspase 9 as well as down regulation of Bcl-2 protein. In conclusion, Rapamycin and Andrographolide as a single agent represent a potential effective anti-cancer drugs for treatments in both breast cancer models but their combination is likely to be antagonistic to reduce breast cancer cell growth.
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Keywords
Effect of Rapamycin and Andrographolide , on apoptosis signaling pathway.