Autophagy inhibitors regulate TRAIL sensitivity in human malignant cells by targeting the mitochondrial network and calcium dynamics.

In a variety of cancer cell types, the pharmacological and genetic blockade of autophagy increases apoptosis induced by various anticancer drugs. These observations suggest that autophagy counteracts drug­induced apoptosis. We previously reported that in human melanoma and osteosarcoma cells, autophagy inhibitors, such as 3­methyladenine and chloroquine increased the sensitivity to apoptosis induced by tumor necrosis factor­related apoptosis­inducing ligand (TRAIL). In the present study, we report that different autophagy inhibitors regulate the mitochondrial network and calcium (Ca2+) dynamics in these cells. We found that compared to tumor cells, normal fibroblasts were more resistant to the cytotoxicity of TRAIL and autophagy inhibitors used either alone or in combination. Notably, TRAIL increased the autophagic flux in the tumor cells, but not in the fibroblasts. Live­cell imaging revealed that in tumor cells, TRAIL evoked modest mitochondrial fragmentation, while subtoxic concentrations of the autophagy inhibitors led to mitochondrial fusion. Co­treatment with TRAIL and subtoxic concentrations of the autophagy inhibitors resulted in severe mitochondrial fragmentation, swelling and clustering, similar to what was observed with autophagy inhibitors at toxic concentrations. The enhanced aberration of the mitochondrial network was preceded by a reduction in mitochondrial Ca2+ loading and store­operated Ca2+ entry. On the whole, the findings of this study indicate that co­treatment with TRAIL and autophagy inhibitors leads to increased mitochondrial Ca2+ and network dysfunction in a tumor­selective manner. Therefore, the co­administration of TRAIL and autophagy inhibitors may prove to be a promising tumor­targeting approach for the treatment of TRAIL­resistant cancer cells.

Plasma-stimulated medium kills TRAIL-resistant human malignant cells by promoting caspase-independent cell death via membrane potential and calcium dynamics modulation.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cold plasma-stimulated medium (PSM) have been shown to exhibit tumor-selective cytotoxicity and have emerged as promising new tools for cancer treatment. However, to date, at least to the best of our knowledge, no data are available as to which substance is more potent in killing cancer cells. Thus, in this study, we systematically compared their abilities to kill human malignant cells from different origins. We found that PSM dose-dependently killed TRAIL-resistant melanoma, osteosarcoma and neuroblastoma cells. Moreover, PSM had little cytotoxicity toward osteoblasts. PSM was more potent than TRAIL in inducing caspase-3/7 activation, mitochondrial network aberration and caspase-independent cell death. We also found that PSM was more potent in inducing plasma membrane depolarization (PMD) and disrupting endoplasmic-mitochondrial Ca2+ homeostasis. Moreover, persistent PMD was caused by different membrane-depolarizing agents; the use of the anti-type II diabetes drug, glibenclamide, alone caused mitochondrial fragmentation and enhanced TRAIL-induced Ca2+ modulation, mitochondrial network abnormalities and caspase-independent cell killing. These results demonstrate that PSM has a therapeutic advantage over TRAIL owing to its greater capacity to evoke caspase-independent cell death via mitochondrial network aberration by disrupting membrane potential and Ca2+ homeostasis. These findings may provide a strong rationale for developing PSM as a novel approach for the treatment of TRAIL-resistant malignant cells.