Low expression of ANT1 confers oncogenic properties to rhabdomyosarcoma tumor cells via modulating metabolism and death pathways

ABSTRACTRhabdomyosarcoma (RMS) is the most frequent form of pediatric soft-tissue sarcoma. It is divided into 2 main subtypes: ERMS (embryonal) and ARMS (alveolar). Current treatments are based on chemotherapy, surgery and radiotherapy. 5-year survival rate remains of 70% since 2000, despite several clinical trials.RMS cells are thought to derive from muscle lineage precursors. During development, myogenesis is characterized by primary expansion of myoblasts, elimination of those in excess by cell death and the differentiation of the remaining ones into myotubes and myofibers. The idea that these processes could be hijacked by tumor cells to sustain their oncogenic transformation has emerged, while RMS is being considered as the Mister Hyde’s side of myogenesis. Thus, focusing on myogenic developmental programs could help understanding RMS molecular aetiology.Following this idea, we decided to concentrate on ANT1, which is involved in myogenesis and is the underlying cause of genetic disorders associated with muscle degeneration. ANT1 is a mitochondrial protein, which has a functional duality, as it is involved both in metabolism via regulation of ATP/ADP release from mitochondria, but also in apoptosis as part as the mitochondria Permeability Transition Pore (mPTP). By bioinformatic analysis of transcriptomic datasets, we observed that ANT1 is expressed at low levels in RMS. Using CRISPR-Cas9 technology, we showed that decreased ANT1 expression confers selective advantages to RMS cells in terms of proliferation and resistance to stress-induced death. These effects result notably from a metabolic switch. Restoration of ANT1 expression using a Tet-On system is sufficient to prime tumor cells to death and to increase their sensitivity to chemotherapies. Thus, modulation of ANT1 activity could appear as an appealing therapeutic approach in RMS management.

Synergistic Effects of Calcium-Protein Energy Malnutrition and Methanolic Extract of Plumbago zeylanica Root on Mitochondria Permeability Transition Pore: An In Vitro Study

The purpose of this study was to investigate the synergistic effects of calcium ion-protein energy malnutrition (Ca2+-PEM) and methanolic extract of Plumbago zeylanica (Pz) root on mitochondria permeability transition pore (MPTP). Twenty-four male Wistar rats were studied. The Wistar rats were divided into two groups (experimental and control) of 12 each. The experimental rats were fed with protein-deficient diet, and the control rats were fed with normal rat chow and water ad libitum for 42 days. To monitor MPTP induction and inhibition in both experimental and control Wistar rats, 3 mM Ca2+, 1 mM Mg2+, 120, 160, and 200 μg/ml of Pz extract were used. The rats were sacrificed, and mitochondria were isolated from the livers to monitor MPTP. Our study showed that Ca2+ and Mg2+ induced and inhibited MPTP, respectively. However, PEM drastically increased Ca2+ and Mg2+ MPTP induction and inhibition, respectively, when compared to control. At varying dose and timing, Pz extracts steadily induce MPTP in both experimental and control Wistar rats. Taken together, the results suggest that Ca2+-PEM increased the MPTP induction, while PEM decreased the MPTP induction of Pz extract in dose- and time-dependent pattern when compared to the control that plausibly suggests that PEM may increase Ca2+ induction of MPTP as well mitigate therapeutic effects of Pz extract in diseases related to mitochondria targeting.

A novel estrogen receptor GPER inhibits mitochondria permeability transition pore opening and protects the heart against ischemia-reperfusion injury

Several studies have recently demonstrated that G protein-coupled receptor 30 (GPER) can directly bind to estrogen and mediate its action. We investigated the role and the mechanism of estrogen-induced cardioprotection after ischemia-reperfusion using a specific GPER agonist G1. Isolated hearts from male mice were perfused using Langendorff technique with oxygenated (95% O2 and 5% CO2) Krebs Henseleit buffer (control), with G1 (1 μM), and G1 (1 μM) together with extracellular signal-regulated kinase (Erk) inhibitor PD-98059 (5μM). After 20 min of perfusion, hearts were subjected to 20 min global normothermic (37°C) ischemia followed by 40 min reperfusion. Cardiac function was measured, and myocardial necrosis was evaluated by triphenyltetrazolium chloride staining at the end of the reperfusion. Mitochondria were isolated after 10 min of reperfusion to assess the Ca2+ load required to induce mitochondria permeability transition pore (mPTP) opening. G1-treated hearts developed better functional recovery with higher rate pressure product (RPP, 6140 ± 264 vs. 2,640 ± 334 beats·mmHg−1·min−1, P < 0.05). The infarct size decreased significantly in G1-treated hearts (21 ± 2 vs. 46 ± 3%, P < 0.001), and the Ca2+ load required to induce mPTP opening increased (2.4 ± 0.06 vs. 1.6 ± 0.11 μM/mg mitochondrial protein, P < 0.05) compared with the controls. The protective effect of G1 was abolished in the presence of PD-98059 [RPP: 4,120 ± 46 beats·mmHg−1·min−1, infarct size: 53 ± 2%, and Ca2+ retention capacity: 1.4 ± 0.11 μM/mg mitochondrial protein ( P < 0.05)]. These results suggest that GPER activation provides a cardioprotective effect after ischemia-reperfusion by inhibiting the mPTP opening, and this effect is mediated by the Erk pathway.

Abstract P25: Hypothermia Treatment Inhibits Cardiomyocytes Calcium Overload and Mitochondria Transition Permeability after Hypoxia- Reoxygenation Injuries

Introduction: Hypothermia treatment can provide cardiac protection against ischemia reperfusion injuries, but underlying mechanisms remain unclear. Hypothesis: Hypothermia-related cardiomyocyte protection is through the mitochondrial dependent pathways. Methods : H9c2 rat cardiomyocytes were cultured in 37°C, 5% CO 2 incubators. After initiation of hypoxia-reoxygenation (H-R) treatment, the H9c2 cells were moved to hypothermia (31°C) or kept in normothermia (37°C) environments until cells harvested. Cell damage, intracellular and mitochondria calcium loads were studies. Mitochondria permeability transition and transmembrane potentials were studies by flowcytometric studies. Results: Hypothermia treatment ameliorates H9c2 cardiomyocytes survival after H-R injuries (68.1±11.8% vs. 85.0±12.7 %, P=0.025). Intracellular and mitochondria calcium overloading after H-R injuries was improved under 31°C environment (153.5±16.4 % vs. 957.1±311.7 %, P<0.01 for intracellular calcium; 101.8±28.5% vs. 159.4±32.5%, P=0.014 for mitochondria calcium). Mitochondria reductase activity were more preserved under hypothermia treatment after H-R injuries (55.7±10.9% vs. 8.5±1.2%, P<0.01). Hypothermia treatment decreased the continuous opening of mitochondria permeability transition pore after H-R damage by less reduction of mitochondria calcein fluorescence (15.6±13.7 % vs. 52.8±28.1 %, P=0.003). Release of cytochrome c into the cytoplasm from mitochondria after H-R injuries was more evident in normothermia condition by confocal microscopy study. Activation of caspase-9, which is down-streaming to cytochrome c, was down-regulated under hypothermia (62.1±21.9% vs. 87.5±7.3%, P=0.019). Loss of mitochondria integrity with decreasing of mitochondria transmembrane potential was less evident in 31°C than 37°C environments (55.9±23.3% vs. 102±20.2%, P=0.016). Conclusion: Hypothermia treatment at 31°C provides cardiomyocyte protection against hypoxia-reoxygenation injuries. The mechanisms are related to the decreasing intracellular and mitochondria calcium overloading, preserving the integrity of mitochondria by reduction of mitochondria permeability transition pore opening and cytochrome c release.