Investigation of Effect of Metformin and Hesperidin in Combination with Doxorubicin in Experimentally Induces Breast Cancer

Abstract

Breast cancer is the leading cause of cancer worldwide affecting women. There is an increase in the breast cancer incidence and mortality among women. For high-risk women patients, tamoxifen and raloxifene are being used as a chemopreventive approach. Metformin, widely used as anti-diabetic, has gained momentum for its preventive potential and delaying various cancers including breast cancer owing to its high safety profile and better tolerability. Hesperidin, a bioflavonoid, is commonly found in citrus fruits like lemon and orange, which is an integral part of daily diet and consumed worldwide. Hesperidin possess potent antioxidant, anti-inflammatory, anticancer and chemopreventive activity in various cancers. Even though, doxorubicin (Dox) is routinely used for breast cancer treatment but toxicity and drug resistance limit its use. Hence, the aim of the current study was to evaluate the protective effect of metformin and hesperidin alone and in combination with doxorubicin in experimentally-induced breast cancer. In vitro assays such as cell viability assay, scratch assay, annexin V FITC assay and cell cycle analysis were carried out using MDA-MB-468 and MDA-MB-231 human breast cancer cell lines. IC50 concentration of doxorubicin showed potentiation of antiproliferative effect of doxorubicin when combined with lower concentration of metformin or hesperidin or both. The assays revealed that metformin and hesperidin in combination with doxorubicin showed decrease in migration rate of cells, increase in the cells undergoing apoptosis and shift of cells from G0/G1 phase of cells to S phase of cell cycle, ultimately leading to cell cycle arrest in S phase. Whereas, in in vivo study, breast cancer (BC) was induced by administration of 7,12-dimethylbenz(a)anthracene (DMBA) through subcutaneous injection into the 3rd right mammary gland of female Wistar rats. The experimental groups comprised treatment groups and preventive groups with metformin and hesperidin receiving as pretreatment 2 weeks prior to DMBA induction. Metformin and hesperidin pretreated groups showed reduced tumor incidence, tumor volume and increased survival rate as compared to DMBA-induced group of animals. Organ-to-body weight ratios and histopathology of heart, liver and lungs of metformin and hesperidin pretreated groups treated with doxorubicin revealed lesser toxicity than doxorubicin alone treated DMBA-induced groups. There was a noteworthy reduction in the levels of lipid peroxidation marker (MDA) and substantial improvement in the levels of reduced glutathione (GSH) of metformin and hesperidin pretreated groups as compared to DMBA-induced group. In metformin and hesperidin pretreated groups, a significant declin was noticed in the levels of inflammatory markers like IL-6, IL-1β, TNF-α and NF-κB as compared to DMBA-induced group. Histopathology and Ki67 expression of breast tumors depicted better control of tumor development with metformin and hesperidin pretreated groups as compared to DMBA-induced group. In conclusion, metformin and hesperidin possesses protective effect against experimentally induced breast cancer in female rats that appears to be related to attenuation of Ki67 expression. Also, metformin and hesperidin in combination with doxorubicin revealed potentiation of antiproliferative effect of doxorubicin and decrease in toxicity of doxorubicin when given alone.e