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 cancer cells was studied. It was shown that incubation of BT474 cells with ONC201 (10 μM) for 24 h reduces the number
Recent demonstration that mechanism of action of small molecule imipridone ONC201 could be mediated through mitochondrial targeting (Graves et al., 2019; Greer et al., Ishizawa et al., 2019), was confirmed by identification of mitochondrial caseinolythic protease (ClpP), as the only target of ONC201 and its chemical analog TR57 (Graves et al., 2019). We further studied direct effect of these drugs on mitochondrial functions and mtDNA distribution in human breast cancer cells. Using respirometry and confocal microscopy, we demonstrated dose- and time dependent suppression of mitochondria oxygen consumption and degradation of mtDNA in cultured human BT-474 cell lines, treated with ONC201 and TR57. Both drugs, used in this study, induced significant and reversible decrease in the rate of proliferation as well as the content of mtDNA within the mitochondria (> by 50%), which was released into the cytosol. Semi-quantitative analysis of confocal images of mitochondrial matrix (MTDR, red fluorescence) and mtDNA (SYBR Green-1, green fluorescence) demonstrated 30-35% release of mtDNA from mitochondria/mitochondrial fragments. Exposure to ONC201 (10µM) or TR57 (50nM) for 48 h decreased mtDNA content by 60% and 85%, respectively. Effect of these drugs was reversible and washout of drugs restored the rate of proliferation as well as mtDNA content in time-dependent manner. Within 24h of washout, the total cellular mtDNA reached almost 90% of initial level, demonstrating fast activation of mtDNA synthesis upon removal of toxins. RT-PCR approach to monitor mtDNA in the cytosol and mitochondria confirmed release of mtDNA into the cytosol and associated decrease of mtDNA within mitochondria/mitochondrial fragments, following ONC201 or TR57 treatment of BT-474 cells. Taken together, our data demonstrate that these drugs reversibly modulate and regulate intracellular mtDNA content, through ONC201 and TR57 dependent regulation of mitochondrial biogenesis in human breast cancer cells. In conclusion, ONC201 & TR57 dependent reversible inhibition of cell proliferation and decrease in mtDNA content allow suggesting drug-dependent regulation of mitochondrial biogenesis in these BCC. Our observation warrants new direction in the elucidation of the mechanism of action of these drugs and search for novel efficient anti-cancer drugs. Support of Funding Information: Russian Science Foundation № 19-75-20145 Citation Format: Margarita I. Kobyakova, Serazhutdin Abdullaev, Yana V. Evstratova, Artem Mishukov, Irina Odinokova, Lee M. Graves, Ekhson Holmuhamedov. ONC201 & TR57 reversibly depletes mtDNA content & regulates mitochondrial biogenesis in BT-474, human breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 991.
The cytotoxicity and cytostatic effects of imipridone family drugs ONC201 and its analog TR57 (Madera, USA) in variety of cultured breast cancer cell line (estrogen dependent BT474) was manifested through induction of ISR (Integrated Stress Response), UPRER and UPRMT. The major consequences of drug treatment are inhibition of proliferation and mitochondrial structural and functional damage(s). Our preliminary data demonstrated that 24 h exposure of BT474 leads to dysregulation and suppression in mitochondrial Ca2+ uptake and Ca2+ capacity (Odinokova et al., 2021). In the present work, we extend our study to include the effect of these drugs on the rate of migration intact and treated cells in culture. Treatment of BT474 cells in culture for 24h resulted in decreased motility of cells from 17 ± 2 µm/h in the absence of the drugs to 5 ± 1 µm/h and to 3±1 µm/h in cells treated with 10µM of ONC201 or 50nM of TR57 (n=5). This suppression was fully reversible and removal of drugs for 120h resulted in time dependent restoration of the initial motility of BT474 cells, and TR57 caused larger inhibitory effect on motility as compared with ONC201. Buffering of extracellular Ca2+ with 5 mM EGTA or intracellular Ca2+ with 10 µM BAPTA, 30 min suppressed motility of cells (19 ± 2 µm/h vs 6 ± 1 µm/h). Gd3+, selective blocker of store operated Ca2+ entrance (SOCE) pathway at concentration of 10 µM suppressed the motility of BT474 cells, and rate of migration changed for from 16 ± 2 µm/h in control to 7± 2 µm/h, in Gd3+ treated cells. In line with our preliminary data, taken together these data indicate that ONC201 and/or TR57 induced dysfunction in mitochondrial Ca2+ homeostasis, which translates into the suppression of the migration of human breast cancer cells in culture. Support of Funding Information: Russian Science Foundation № 19-75-20145 Citation Format: Yana Evstratova, Margarita Kobyakova, Irina Odinokova, Alexander Stolyarov, Artyom Mishukov, Lee M. Graves, Ekhson Holmuhamedov. Reversible and dose-dependent suppression of motility of human breast cancer cells with ONC201 and/or TR57 in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2888.
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