Ekhson L. Holmuhamedov

Recent works identified ClpXP, mitochondrial caseinolytic protease, as the only target of imipridones, a new class of antitumor agents. Our study of the mechanism of imipridone derivative TR-57 action in SUM159 human breast cancer cells demonstrated mitochondrial fragmentation, degradation of mitochondrial mtDNA and mitochondrial dysfunction due to inhibition of Complex I and Complex II activity. Complete inhibition of oxidative phosphorylation accompanied 90, 94, 88 and 87% decreases in the content of Complex I, II, III and IV proteins, respectively. The content of the F_OF₁-ATPase subunits decreased sharply by approximately 35% after 24 h and remained unchanged up to 72 h of incubation with TR-57. At the same time, a disappearance of the ATPIF1, the natural inhibitor of mitochondrial F_OF₁-ATPase, was observed after 24 h exposure to TR-57. ATPase inhibitor oligomycin did not affect the mitochondrial membrane potential in intact SUM159, whereas it caused a 65% decrease in TR-57-treated cells. SUM159 cells incubated with TR57 up to 72 h retained the level of proteins facilitating the ATP transfer across the mitochondrial membranes: VDAC1 expression was not affected, while expression of ANT-1/2 and APC2 increased by 20% and 40%, respectively. Thus, our results suggest that although TR-57 treatment leads to complete inhibition of respiratory chain activity of SUM159 cells, hydrolysis of cytoplasmic ATP by reversal activity of F_OF₁-ATPase supports mitochondrial polarization.

Recent works identified ClpXP, mitochondrial caseinolytic protease, as the only target of imipridones - new class of antitumor agents. Our study of the mechanism of imipridone derivative TR-57 action in SUM159 human breast cancer cells demonstrated mitochondrial fragmentation, degradation of mitochondrial mtDNA and mitochondrial dysfunction, due to inhibition of Complex I and Complex II activity. Complete inhibition of oxidative phosphorylation accom-panied with 90, 94, 88 and 87% decrease in the content of Complex I, II, III and IV proteins, respectively. The content of the FOF1-ATPase subunits decreased sharply by approximately 35% after 24 hours and remained unchanged up to 72 hours of incubation with TR-57. At the same time, a disappearance of the ATPIF1, natural inhibitor of mitochondrial FOF1-ATPase observed after 24 h exposure to TR-57. ATPase inhibitor oligomycin had no effect on the mitochondrial membrane potential in intact SUM159, whereas it caused a 65% decrease in TR-57-treated cells. SUM159 cells incubated with TR57 up to 72 hours retained the level of proteins facilitating the ATP transfer across the mitochondrial membranes: VDAC1 expression was not affected, while expression of ANT-1/2 and APC2 increased by 20% and 40%, respectively. Thus, our results suggest that alt-hough TR-57 treatment lead to complete inhibition of respiratory chain activity of SUM159 cells, hydrolysis of cytoplasmic ATP by reversal activity of FOF1-ATPase supports mitochondrial polarization.

ONC201, the anticancer drug, targets and activates mitochondrial ATP-dependent caseinolytic peptidase P (ClpP), a serine protease located in the mitochondrial matrix. Given the promise of ONC201 in cancer treatment, we evaluated its effects on the breast ductal carcinoma cell line (BT474). We showed that the transient single-dose treatment of BT474 cells by 10 µM ONC201 for a period of less than 48 h induced a reversible growth arrest and a transient activation of an integrated stress response indicated by an increased expression of CHOP, ATF4, and GDF-15, and a reduced number of mtDNA nucleoids. A prolonged exposure to the drug (>48 h), however, initiated an irreversible loss of mtDNA, persistent activation of integrated stress response proteins, as well as cell cycle arrest, inhibition of proliferation, and suppression of the intrinsic apoptosis pathway. Since Natural Killer (NK) cells are quickly gaining momentum in cellular anti-cancer therapies, we evaluated the effect of ONC201 on the activity of the peripheral blood derived NK cells. We showed that following the ONC 201 exposure BT474 cells demonstrated enhanced sensitivity toward human NK cells that mediated killing. Together our data revealed that the effects of a single dose of ONC201 are dependent on the duration of exposure, specifically, while short-term exposure led to reversible changes; long-term exposure resulted in irreversible transformation of cells associated with the senescent phenotype. Our data further demonstrated that when used in combination with NK cells, ONC201 created a synergistic anti-cancer effect, thus suggesting its possible benefit in NK-cell based cellular immunotherapies for cancer treatment.

Purpose to study the tumor-forming activity of wild-type MC F-7 cells carrying a full set of porins (VDAC 1, VDAC 2, VDAC 3), as well as their genetically modified cells, from which one of the isoforms was removed (MC F-7 VDAC 1 KO, MC F-7 VDAC 2 KO, MC F -7 VDAC 3 KO).Material and Methods. The study was aimed at establishing of an animal model of orthotopic tumors in the mammary gland of immunodeficient BAL B/c nude mice by implanting a suspension of human breast cancer cells (MC F-7) and derivatives of these cells generated by targeted knockout of one of the selected mitochondrial porin isoforms (VDAC 1, VDAC 2 or VDAC 3). Suspensions of either wild-type MC F-7 cell lines containing all three porin isoforms (VDAC 1, VDAC 2 and VDAC 3) or their VDAC -deficient derivatives (MC F-7 VDAC 1 KO, MC F-7 VDAC 2 KO and MC F-7 VDAC 3 KO) were injected into mammary fat pads of BAL B/c nude mice at a dose of 4x106 cells per injection. A pathomorphological analysis of the place of implantation of tumor cells, the tumor itself, as well as the organs of the abdominal and thoracic cavity was carried out.Results. The study shows the feasibility of successful creation of orthotopic tumors in the adipose tissue of immunodeficient BAL B/c nude mice with MC F-7 human breast cancer epithelial cells containing a complete set of mitochondrial porin isoforms and their VDAC -deficient derivatives. The tumor-forming activity of the implanted cells was shown to correlate with their cytotoxic effect on the internal organs of animals. Pathological analysis showed that all implanted cell cultures, such as MC F-7 WT, MC F-7 VDAC 2 KO and MC F-7 VDAC 3 KO, except for MC F-7 VDAC 1 KO cells, which did not form tumors, caused pathological changes in the lungs, liver and spleen, as well as the presence of other tumor-like lesions.Conclusion. The data obtained will be used to optimize the injection volume and cell number, as well as to refine the dynamics of tumor growth, suitable for studying the effect of anticancer drugs on tumors formed by human breast cancer cells (MC F-7) and its genetically modified VDAC -deficient derivatives.

The effect of the cytostatic antitumor drug ONC201 on the number of mitochondrial nucleoids and the average size of mitochondria in the BT474 human breast

Excessive cardiac fibrosis, characterized by increased collagen-rich extracellular matrix (ECM) deposition, is a major predisposing factor for mechanical and electrical dysfunction in heart failure (HF). The human ventricular fibroblast (hVF) remodeling mechanisms that cause excessive collagen deposition in HF are unclear, although reports suggest a role for [Ca2+]i in fibrosis. Therefore, we determined the association of differences in cellular Ca2+ dynamics and collagen secretion/deposition between hVFs from failing and normal (control) hearts. Histology of left ventricle sections (Masson trichrome) confirmed excessive fibrosis in HF vs normal. In vitro, hVFs from HF showed increased secretion/deposition of soluble collagen in 48 hours of culture compared with control [85.9±7.4 μg/106 vs 58.5±8.8 μg/106 cells, P<0.05; (Sircol™ assay)]. However, collagen gene expressions (COL1A1 and COL1A2; rt-PCR) were not different. Ca2+ imaging (fluo-3) of isolated hVFs showed no difference in the thapsigargin-induced intracellular Ca2+ release capacity (control 16±1.4% vs HF 17±1.1%); however, Ca2+ influx via store-operated Ca2+ entry [SOCE /CRAC (Ca2+ release-activated)] channels was significantly (P≤0.05) greater in HF-hVFs (47±3%) compared with non-failing (35±5%). Immunoblotting for ICRAC channel components showed increased ORAI1 expression in HF-hVFs compared with normal without any difference in STIM1 expression. The Pearson's correlation coefficient for co-localization of STIM1/ORAI1 was significantly (P<0.01) greater in HF (0.5±0.01) than control (0.4±0.01) hVFs. The increase in collagen secretion of HF vs control hVFs was eliminated by incubation of hVFs with YM58483 (10 μM), a selective ICRAC inhibitor for 48 hours (66.78±5.87μg/106 cells vs 55.81±7.09 μg/106 cells, P=0.27). In conclusion, hVFs from HF have increased collagen secretion capacity vs non-failing hearts and this is related to increase in Ca2+ entry via SOCE and enhanced expression of ORAI, the pore-forming subunit. Therapeutic inhibition of SOCE may reduce the progression of cardiac fibrosis/HF.

Fibroblasts, the most abundant cells in the heart, contribute to cardiac fibrosis, the substrate for the development of arrythmogenesis, and therefore are potential targets for preventing arrhythmic cardiac remodeling. A chamber-specific difference in the responsiveness of fibroblasts from the atria and ventricles toward cytokine and growth factors has been described in animal models, but it is unclear whether similar differences exist in human cardiac fibroblasts (HCFs) and whether drugs affect their proliferation differentially. Using cardiac fibroblasts from humans, differences between atrial and ventricular fibroblasts in serum-induced proliferation, DNA synthesis, cell cycle progression, cyclin gene expression, and their inhibition by simvastatin were determined. The serum-induced proliferation rate of human atrial fibroblasts was more than threefold greater than ventricular fibroblasts with faster DNA synthesis and higher mRNA levels of cyclin genes. Simvastatin predominantly decreased the rate of proliferation of atrial fibroblasts, with inhibition of cell cycle progression and an increase in the G0/G1 phase in atrial fibroblasts with a higher sensitivity toward inhibition compared with ventricular fibroblasts. The DNA synthesis and mRNA levels of cyclin A, D, and E were significantly reduced by simvastatin in atrial but not in ventricular fibroblasts. The inhibitory effect of simvastatin on atrial fibroblasts was abrogated by mevalonic acid (500 μM) that bypasses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibition. Chamber-specific differences exist in the human heart because atrial fibroblasts have a higher proliferative capacity and are more sensitive to simvastatin-mediated inhibition through HMG-CoA reductase pathway. This mechanism may be useful in selectively preventing excessive atrial fibrosis without inhibiting adaptive ventricular remodeling during cardiac injury.