Integrative analysis of the epigenetic modification in a breast cancer cell line treated with a bioactive extract of bidens pilosa

Abstract

Breast cancer is the leading cause of female deaths in the world. Varying types of therapy options are available, yet these conventional treatments for the malignancy are known to have numerous side effects. Similar to other diseases, herbal remedies are being explored as alternative treatment options as well as starting points for development of new drugs to treat breast cancer. Bidens pilosa is a weed distributed throughout the world with known medicinal properties. Its anti-cancer activity has been established in various cancers. This study aimed to investigate the epigenetic patterns affected by a bioactive extract of B. pilosa in breast cancer. A crude methanol extract of B. pilosa was fractionated with n-hexane, chloroform, ethyl acetate, n-butanol, 65% methanol and water. Healing properties of plants are often an attribute of the presence of phenolic compounds within the plant and the sub-fractions of the methanol extract of B. pilosa were, therefore, assayed for these compounds. The water sub-fraction showed the highest content of total phenolic compounds, however, when the sub-fractions were analysed for the presence of two classes of specific phenolic compounds, the butanol sub-fraction boasted the highest concentration of flavonoids and tannins, affording it superior antioxidant activity in a quantitative DPPH assay. Distribution of the antioxidant compounds in TLC-DPPH analysis also supported this finding. Despite its high antioxidant compound content, cytotoxicity of the butanol sub-fraction in MCF-7 breast cancer cells was not impressive in the MTT viability assay. Treatment with varying concentrations of the chloroform sub-fraction resulted in a better dose- and time-dependent decrease in cell viability of MCF-7 cells than all the other sub-fractions as well as the crude methanol extract. Analysis of breast cancer genes affected by the chloroform sub-fraction on the Human Breast Cancer RT2 Profiler PCR array showed repression in BRCA1 and BRCA2, genes classified as tumour suppressors. Bisulfite pyrosequencing showed no significant modification in methylation of selected CpG islands within the promoter regions of both genes. Results of the array also showed decreased expression of CDH1 which is associated with invasiveness and aggression of tumours. Its investigated CpG island was also shown not to be differentially methylated by treatment of the cells with the chloroform sub-fraction of the extract. As a well-appreciated biomarker for breast cancer risk, BRCA1 protein expression was further investigated. Western blot analysis showed parallel findings to those of the PCR array, with down-regulation of BRCA1 within 24 hours of treatment of MCF-7 cells with the sub-fraction. Repression of the BRCA genes is strongly linked to arrest of cells at the G2/M phase of the cell division cycle, and this was therefore also assessed. Treatment of MCF-7 cells with the chloroform sub-fraction effected a dose-dependent accumulation of cells at the G2/M phase of the cell cycle as determined by flow cytometry. Results of global DNA methylation analysis showed an increase in chromosomal instability by a significantly reduced level of methylation of the genome. This hypomethylation also supports arrest of the cells at the G2/M phase of the cell cycle, as cells accumulate at this checkpoint, awaiting repair to prevent segregation of broken chromosomes during mitosis. However, the lack of BRCA1 suggests that repair proteins were not recruited to the sites of repair and the cells were consequently directed to apoptosis. Analysis of the effect of the chloroform sub-fraction of the methanol extract of B. pilosa in the Mitopotential assay showed an increase in the number of dead cells with depolarised mitochondrial membranes, alluding to the intrinsic mode of apoptotic cell death in MCF-7 cells treated with the sub-fraction. Down-regulation of BRCA1 is further associated with telomerase inactivation in cancer cells. Treatment of MCF-7 cells with the chloroform sub-fraction reduced telomerase activity within 24 hours of treatment, with an absence of activity following treatment with 100 and 125 μg/ml of the sub-fraction. This lack of telomerase activity resulted in shortened telomeres which limit proliferative ability of the cells. Characterisation of the six sub-fractions of the methanol extract of B. pilosa with GC-MS showed an abundance of fatty acids in the chloroform sub-fraction, specifically α-linolenic acid, palmitic acid and linoleic acid. Palmitic acid is alleged to play a role in down-regulation of BRCA1 and its abundance in this sub-fraction leads to the conclusion that palmitic acid may be responsible for the decreased expression of BRCA1 in MCF-7 breast cancer cells. The down-regulation results in hypomethylation of the genome leading to cell cycle arrest at the G2/M checkpoint and subsequent apoptosis as a result of this repression of BRCA1. Repression of BRCA1 also leads to inactivation of telomerase, inhibiting cell proliferation. Taken together, the observed antioxidant activity and pro-apoptotic potential attributed to epigenetic modifications validate B. pilosa as an anticancer agent. Our findings merit the plant for use in development of potential breast cancer drugs.