Ca2+-induced changes in energy metabolism and viability of melanoma cells.

Cancer cells are characterized by a high rate of glycolysis, which is their primary energy source. We show here that a rise in intracellular-free calcium ion (Ca2+), induced by Ca2+-ionophore A23187, exerted a deleterious effect on glycolysis and viability of B16 melanoma cells. Ca2+-ionophore caused a dose-dependent detachment of phosphofructokinase (EC 2.7.1.11), one of the key enzymes of glycolysis, from cytoskeleton. It also induced a decrease in the levels of glucose 1,6-bisphosphate and fructose 1,6-bisphosphate, the two stimulatory signal molecules of glycolysis. All these changes occurred at lower concentrations of the drug than those required to induce a reduction in viability of melanoma cells. We also found that low concentrations of Ca2+-ionophore induced an increase in adenosine 5'-triphosphate (ATP), which most probably resulted from the increase in mitochondrial-bound hexokinase, which reflects a defence mechanism. This mechanism can no longer operate at high concentrations of the Ca2+-ionophore, which causes a decrease in mitochondrial and cytosolic hexokinase, leading to a drastic fall in ATP and melanoma cell death. The present results suggest that drugs which are capable of inducing accumulation of intracellular-free Ca2+ in melanoma cells would cause a reduction in energy-producing systems, leading to melanoma cell death.     

Diosgenin induces cell cycle arrest and apoptosis in human leukemia K562 cells with the disruption of Ca2+ homeostasis

Purpose Diosgenin is a steroidal sapogenin with estrogenic and antitumor properties. In order to elucidate the mechanism of its antiproliferative activity, we investigated its effects on the cell cycle and apoptosis in human chronic myelogenous leukemia K562 cells.Methods Cell viability was assessed via an MTT assay. Apoptosis was investigated in terms of nuclear morphology, DNA fragmentation, and phosphatidylserine externalization. Cell cycle analysis was performed via PI staining and flow cytometry (FCM). Western blotting and immunofluorescence methods were used to determine the levels of p53, cell cycle-related proteins and Bcl-2 family members. FCM was also used to estimate the changes in mitochondrial membrane potential (MMP), intracellular Ca²⁺ concentration and reactive oxygen species (ROS) generation.Results Cell cycle analysis showed that diosgenin caused G₂/M arrest independently of p53. The levels of cyclin B1 and p21^Cip1/Waf1 were decreased, whereas cdc2 levels were increased. Subsequent apoptosis was demonstrated with the dramatic activation of caspase-3. A dramatic decline in intracellular Ca²⁺ concentration was observed as an initiating event in the process of cell cycle arrest and apoptosis, which was followed by the hyperpolarization and depolarization of MMP. Generation of ROS was observed in the progression of apoptosis. The antiapoptotic Bcl-2 and Bcl-x_L proteins were downregulated, whereas the proapoptotic Bax was upregulated.Conclusions Diosgenin inhibits K562 cell proliferation via cell cycle G₂/M arrest and apoptosis, with disruption of Ca²⁺ homeostasis and mitochondrial dysfunction playing vital roles.     

Release of Ca2+ from the endoplasmic reticulum and its subsequent influx into mitochondria trigger celastrol-induced paraptosis in cancer cells

Celastrol, a triterpene extracted from the Chinese “Thunder of God Vine”, is known to have anticancer activity, but its underlying mechanism is not completely understood. In this study, we show that celastrol kills several breast and colon cancer cell lines by induction of paraptosis, a cell death mode characterized by extensive vacuolization that arises via dilation of the endoplasmic reticulum (ER) and mitochondria. Celastrol treatment markedly increased mitochondrial Ca²⁺ levels and induced ER stress via proteasome inhibition in these cells. Both MCU (mitochondrial Ca²⁺ uniporter) knockdown and pretreatment with ruthenium red, an inhibitor of MCU, inhibited celastrol-induced mitochondrial Ca²⁺ uptake, dilation of mitochondria/ER, accumulation of poly-ubiquitinated proteins, and cell death in MDA-MB 435S cells. Inhibition of the IP₃ receptor (IP₃R) with 2-aminoethoxydiphenyl borate (2-APB) also effectively blocked celastrol-induced mitochondrial Ca²⁺ accumulation and subsequent paraptotic events. Collectively, our results show that the IP₃R-mediated release of Ca²⁺ from the ER and its subsequent MCU-mediated influx into mitochondria critically contribute to celastrol-induced paraptosis in cancer cells.