Flex-Hets differentially induce apoptosis in cancer over normal cells by directly targeting mitochondria

Abstract

Flex-Het drugs induce apoptosis in multiple types of cancer cells, with little effect on normal cells. This apoptosis occurs through the intrinsic mitochondrial pathway accompanied by generation of reactive oxygen species (ROS). The objective of this study was to determine if direct or indirect targeting of mitochondria is responsible for the differential sensitivities of cancer and normal cells to Flex-Hets. Mitochondrial effects and apoptosis were measured using JC-1 and Annexin V-FITC dyes with flow cytometry. Bcl-2, Bcl-x L , and Bax were measured by Western blot. Flex-Hets induced mitochondrial swelling and apoptosis in ovarian cancer cell lines but had minimal to no effects in a variety of normal cell cultures, including human ovarian surface epithelium. Effects on inner mitochondrial membrane (IMM) potential were variable and did not occur in normal cells. Two different antioxidants, administered at concentrations shown to quench intracellular and mitochondrial ROS, did not alter Flex-Het -induced mitochondrial swelling, loss of IMM potential, or apoptosis. Inhibition of protein synthesis with cycloheximide also did not prevent FlexHet mitochondrial or apoptosis effects. Bcl-2 and Bcl-x L levels were decreased in an ovarian cancer cell line but increased in a normal culture, whereas Bax expression was unaffected by Flex-Hets treatment. In conclusion, ROS seems to be a consequence rather than a cause of mitochondrial swelling. The differential induction of apoptosis in cancer versus normal cells by Flex-Hets involves direct targeting of mitochondria associated with alterations in the balance of Bcl-2 proteins. This mechanism does not require IMM potential, ROS generation, or protein synthesis.

Synthesis and cytotoxicity studies of 1-propenyl-1,3-dihydro-benzimidazol-2-one

“…In order to study mitochondrial membrane potential, JC-1 dye was widely used in fluorescent microscopy for a variety of cell types, including myocytes and neurons, as well as in intact tissues and isolated mitochondria. Cells with good mitochondrial health show punctuated red staining along with green scattered staining, whereas the cells with mitochondrial dysfunction show only scattered green staining without any red punctuation [30]. To check the efficacy of this molecule, cells were treated with compound 4.…”

Examining impacts of ceranib-2 on the proliferation, morphology and ultrastructure of human breast cancer cells

“…Ceramidase enzyme metabolize ceramide to sphingosine-1-phosphate that is an antiproliferative metabolite in the ceramide pathway (Beckham et al 2013). Ceramidase inhibitors cause augmented ceramide levels in the cells and in turn lead to cell cycle arrest and apoptosis by inhibition of exogenous ceramides (Liu et al 2007a, b).…”

“…MCF7 cells were washed twice with assay buffer (19) at room temperature and pellets were vortexed in 0.5 mL of assay buffer and then analyzed by flow cytometry. A Mitochondrial Membrane Potential Detection Kit (BD Pharmingen, Franklin Lakes, NJ, USA) was used (Liu et al 2007a, b).…”

The Development of Pro-Apoptotic Cancer Therapeutics

“…Whilst nuclear retinoic acid receptor activation accounts for most of the antitumour properties of heteroarotinoids, Flex-Hets were found to have a distinct mechanism of action which entails the activation of the intrinsic mitochondrial pathway as well as the generation of reactive oxygen species (ROS) [66], as a consequence of mitochondrial swelling [68]. In addition, it was also demonstrated that the observed lines where the expression of these proteins increases; pro-apoptotic Bax proteins levels remained unaltered in both cell types [68].…”