We have shown that hydroxycobalamin (vitamin B_12b) increases the toxicity of diethyldithiocarbamate (DDC) to tumor cells by catalyzing the formation of disulfiram (DSF) oxi-derivatives. The purpose of this study was to elucidate the mechanism of tumor cell death induced by the combination DDC + B_12b. It was found that cell death induced by DDC + B_12b differed from apoptosis, autophagy, and necrosis. During the initiation of cell death, numerous vacuoles formed from ER cisterns in the cytoplasm, and cell death was partially suppressed by the inhibitors of protein synthesis and folding, the IP3 receptor inhibitor as well as by thiols. At this time, a short-term rise in the expression of ER-stress markers BiP and PERK with a steady increase in the expression of CHOP were detected. After the vacuolization of the cytoplasm, functional disorders of mitochondria and an increase in the generation of superoxide anion in them occurred. Taken together, the results obtained indicate that DDC and B_12b used in combination exert a synergistic toxic effect on tumor cells by causing severe ER stress, extensive ER vacuolization, and inhibition of apoptosis, which ultimately leads to the induction of paraptosis-like cell death.

The opening of the permeability transition pore (mPTP) in mitochondria initiates cell death in numerous diseases. The regulation of mPTP by NAD(H) in the mitochondrial matrix is well established; however, the role of extramitochondrial (cytosolic) NAD(H) is still unclear. We studied the effect of added NADH and NAD⁺ on: (1) the Ca²⁺-retention capacity (CRC) of isolated rat liver, heart, and brain mitochondria; (2) the Ca²⁺-dependent mitochondrial swelling in media whose particles can (KCl) or cannot (sucrose) be extruded from the matrix by mitochondrial carriers; (3) the Ca²⁺-dependent mitochondrial depolarization and the release of entrapped calcein from mitochondria of permeabilized hepatocytes; and (4) the Ca²⁺-dependent mitochondrial depolarization and subsequent repolarization. NADH and NAD⁺ increased the CRC of liver, heart, and brain mitochondria 1.5–2.5 times, insignificantly affecting the rate of Ca²⁺-uptake and the free Ca²⁺ concentration in the medium. NAD(H) suppressed the Ca²⁺-dependent mitochondrial swelling both in KCl- and sucrose-based media but did not induce the contraction and repolarization of swollen mitochondria. By contrast, EGTA caused mitochondrial repolarization in both media and the contraction in KCl-based medium only. NAD(H) delayed the Ca²⁺-dependent depolarization and the release of calcein from individual mitochondria in hepatocytes. These data unambiguously demonstrate the existence of an external NAD(H)-dependent site of mPTP regulation.

Pioglitazone (PIO) is an insulin-sensitizing antidiabetic drug, which normalizes glucose and lipid metabolism but may provoke heart and liver failure and chronic kidney diseases. Both therapeutic and adverse effects of PIO can be accomplished through mitochondrial targets. Here, we explored the capability of PIO to modulate the mitochondrial membrane potential (ΔΨ_m) and the permeability transition pore (mPTP) opening in different models in vitro. ΔΨ_m was measured using tetraphenylphosphonium and the fluorescent dye rhodamine 123. The coupling of oxidative phosphorylation was estimated polarographically. The transport of ions and solutes across membranes was registered by potentiometric and spectral techniques. We found that PIO decreased ΔΨ_m in isolated mitochondria and intact thymocytes and the efficiency of ADP phosphorylation, particularly after the addition of Ca²⁺. The presence of the cytosolic fraction mitigated mitochondrial depolarization but made it sustained. Carboxyatractyloside diminished the PIO-dependent depolarization. PIO activated proton transport in deenergized mitochondria but not in artificial phospholipid vesicles. PIO had no effect on K⁺ and Ca²⁺ inward transport but drastically decreased the mitochondrial Ca²⁺-retention capacity and protective effects of adenine nucleotides against mPTP opening. Thus, PIO is a mild, partly ATP/ADP-translocase-dependent, uncoupler and a modulator of ATP production and mPTP sensitivity to Ca²⁺ and adenine nucleotides. These properties contribute to both therapeutic and adverse effects of PIO.

Fusaricidins and related LI-F compounds are effective bactericides and fungicides. Recently, we have found that they are highly toxic to mammalian cells. Here, we studied the effect of fusaricidin-type compounds (FTCs) on the membranes of mammalian cells. Ethanol extracts from Paenibacillus polymyxa strains, RS10 and I/Sim, were fractionated and analyzed by HPLC and mass spectrometry. The effects of FTCs on mitochondrial functions and integrity were studied by standard methods: measurements of swelling, membrane potential (ΔΨ_m), respiration rate, cytochrome c release, and pore sizes. Superoxide flashes were registered by 3,7-dihydro-2-methyl-6-(4-methoxyphenyl)imidazol[1,2-a]pyrazine-3-one (MCLA). Plasma membrane permeability was assessed by propidium iodide (PI) staining and ATP release. FTCs caused the permeabilization of the inner mitochondria membrane (IMM) to ions and low-molecular-weight (~750 Da) solutes. The permeabilization did not depend on the permeability transition pore (mPTP) but was strongly dependent on ΔΨ_m. Fusaricidins A plus B, LI-F05a, and LI-F05b–LI-F07b permeabilized IMM with comparable efficiency. They created pores and affected mitochondrial functions and integrity similarly to mPTP opening. They permeabilized the sperm cell plasma membrane to ATP and PI. Thus, the formation of pores in polarized membranes underlays the toxicity of FTCs to mammals. Besides, FTCs appeared to be superior reference compounds for mPTP studies.