Methotrexate induced cell death mechanisms in MCF-7 adenocarcinoma breast cancer cells: Enhanced cytotoxicity following dff45-siRNA pre-treatment

Abstract

There are many efforts to diminish risks associated with the use of cancer chemotherapeutic agents through design and utilization of new strategies including gene therapy. We previously showed knockdown of dff45 or overexpression of dff40, the important apoptosis regulators, resulted in enhanced sensitization of cancer cells to chemotherapeutic agents. Here we show that pre-treatment with dff45-siRNA additively increased the overall cytotoxic effects of methotrexate (MTX), at its partially safe concentration (30 μM with almost 37% of killing potency), up to 85%. This is approximately 51% greater than MTX treatment, and about 20% greater than siRNA treatment alone. The main mechanism of cell death by MTX treatment was through AIF-induced caspase-independent apoptosis, as well as the less common Atg5 and DRAM-induced apoptosis. Investigation of apoptosis with dff45-siRNA treatment showed the canonical caspase-dependent apoptosis as the main mechanism of cell death. This was also true for MTX+siRNA treatment. We did not observe a sharp increase in expression of autophagy genes in cells incubated with dff45-siRNA. However, treatment with MTX+siRNA may trigger autophagy via an Atg5-independent pathway.