The bovine dialyzable leukocyte extract, immunepotent CRP, synergically enhances cyclophosphamide-induced breast cancer cell death, through a caspase-independent mechanism

Abstract

Breast cancer (BC) is one of the leading causes of cancer death worldwide. Cyclophosphamide (CTX) remains a mainstay in cancer therapy despite harmful adverse effects and cell death-resistances. To face this, combinational therapy of chemotherapies and immunotherapies has been proposed. IMMUNEPOTENT CRP (ICRP) is an immunotherapy that has cytotoxic effects in several cancer cells without affecting peripheral blood mononuclear cells (PBMC) and CD3+ cells. The aim of this study was to evaluate cytotoxicity, the type of cytotoxic effect, and several features involved in cell death induced by the combination of CTX with ICRP (ICRP+CTX) in breast cancer cells as well as their effect on healthy cells. For this purpose, human and murine breast cancer cells, MCF-7, MDA-MB-231 and 4T1, or PBMC were treated for 24 hours with ICRP, CTX or ICRP+CTX in different combination ratios for the assessment of cell death. Flow cytometry and microscopy were used to determine biochemical and morphological characteristics of cell death. Assays showed that ICRP in combination with CTX induce potentiated cell death manifested with morphological changes, loss of mitochondrial membrane potential, reactive oxygen species (ROS) production, and caspase activation. In addition, it was determined that ICRP+CTX-cell death is caspase-independent in all the breast cancer cells assessed. On the other hand, ICRP did not affect CTX-cytotoxicity in PBMC. For all the above, we can propose that the combination of ICRP with CTX an effective combination therapy, promoting their use even in tumoral cells with defects on proteins implicated in the apoptotic pathway.