TR-57 Treatment of SUM159 Cells Induces Mitochondrial Dysfunction without Affecting Membrane Potential.

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 FOF1-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 FOF1-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 FOF1-ATPase supports mitochondrial polarization.

ONC201-Induced Mitochondrial Dysfunction, Senescence-like Phenotype, and Sensitization of Cultured BT474 Human Breast Cancer Cells to TRAIL.

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.