Induction of Mitochondrion-mediated Apoptosis by Semecarpus anacardium in the BCR–ABL+ 12B1 Leukemia Cell Line: Possible Mechanism of Therapeutic Action In Vivo

Abstract

Background/Purpose Chronic myeloid leukemia (CML) is a clonal disorder leading to massive proliferation of hematopoietic cells. It is characterized by the BCR–ABL gene, resulting in production of a 210-kD chimeric tyrosine kinase that plays a major role in its etiopathogenesis. There is an increasing interest in identifying potent preventive and therapeutic agents for leukemia. Semecarpus anacardium Linn. nut milk extract (SA) has antileukemic activity in murine BCR–ABL+ leukemia in vivo. The present study aimed to elucidate the mechanism of action of SA by assessing its antiproliferative and apoptotic effects in the BCR–ABL+ 12B1 murine leukemic cell line. Methods Cell viability and apoptosis assay using MTT analysis, propidium iodide staining, DNA fragmentation, intracellular reactive oxygen species/calcium, mitochondrial membrane potential, reverse transcriptase polymerase chain reaction (RT-PCR) and western blotting were determined in 12B1 cells. Results Cell viability of SA-treated 12B1 cells was dose and time dependent. SA-induced cell death was considered to be apoptotic by observing the typical apoptotic morphological changes seen on propidium iodide staining and DNA ladder formation on electrophoresis. A reduction in the BCR–ABL + mRNA levels, induced by SA, was observed by RT-PCR. This may have been the result of a reduction in the transcription of the BCR–ABL fusion gene, with concomitant activation of apoptosis. SA induced a rapid increase in intracellular Ca2+ ions, which led to increased levels of reactive oxygen species. Concomitantly, we observed a significant loss of mitochondrial membrane potential and release of cytochrome c into the cytosol in 12B1 cells. These effects of SA are accompanied by downregulation of Bcl-2, upregulation of Bax, and activation of the caspase-3 and caspase-9 genes. Conclusion These results provide unprecedented evidence that SA triggers mitochondrial receptor pathways to induce apoptotic signals in 12B1 cells. This study highlights the beneficial effect of SA in inducing apoptosis in cancer cells and thereby revealing a possible mechanism of therapeutic action in vivo.