Effects of curcumin in combination with cyclophosphamide on canine mammary tumour cell lines

In recent years, significant emphasis has been placed on combination chemotherapy in cancer using cytotoxic agents and plant derived-bioactive substances that are capable of selectively arresting cell growth and inducing apoptosis in tumour cells. The present study was undertaken to evaluate the possibility that the combination of curcumin and cyclophosphamide could show synergistic anti-proliferative effects towards CMT-U27 and CMT-U309 canine mammary cancer cells and, if so, to clarify the mechanism involved. The anti-proliferative activities of curcumin, cyclophosphamide and a combined treatment on CMT cells were determined using the MTT and LDH assays. The concentration of drug required for 50% inhibition of cell viability (IC50) and combination index (CI) values were calculated from log dose-response curves of fixed-combinations of curcumin and cyclophosphamide generated from MTT assays. Apoptosis was detected using a DNA fragmentation assay and Annexin-V/propidium iodide staining followed by flow cytometry. Cell cycle analyses were also performed using flow cytometry. The expression of the apoptosis-related proteins Bax and Bcl-2 was determined by immunocytochemical staining. MTT and LDH assays showed that curcumin and cyclophosphamide induced a dose- and a time-dependent decrease in cell viability. Isobole analysis revealed that the substances exhibited a synergistic interaction when IC50 and 1/2 IC50 concentrations of curcumin and cyclophosphamide were added concurrently to the cultures. This synergy was characterised by a significant increase in the percentage of early and late apoptotic CMT-U27 and CMT-U309 cells. However, internucleosomal fragmentation of DNA was not observed in the DNA fragmentation assay. Cells treated with curcumin and cyclophosphamide arrested at the G2/M and S phases of the cell cycle, respectively. In combined treatments cells were arrested in both phases of the cell cycle. Furthermore, immunocytochemical stainings demonstrated that the curcumin induced apoptosis in CMT cells by the modulation of Bcl-2/Bax protein expression, as the expression of Bcl-2 was decreased and that of Bax increased. This effect was more pronounced in combination treatments. In conclusion, our study shows that a combination of curcumin and cyclophosphamide shows synergistic growth inhibitory activity on CMT cells via induction of apoptosis and cell cycle arrest accompanied by modulation of Bcl-2/Bax protein expression. This finding provides a molecular basis for the development of natural compounds as novel anticancer agents and will allow lowering the dose of cytotoxic agents, which will in turn lead to more specific and less toxic therapies for mammary cancer in dogs.