The role of reactive oxygen species (ROS) in the metabolic reprogramming of cells adapted to hypoxia and the interplay between ROS and hypoxia in malignancy is under debate. Here, we examined how ROS levels are modulated by hypoxia in human cancer compared to untransformed cells. Short time exposure (20 min) of either fibroblasts or 143B osteosarcoma cells to low oxygen tension down to 0.5% induced a significant decrease of the cellular ROS level, as detected by the CellROX fluorescent probe (−70%). Prolonging the cells’ exposure to hypoxia for 24 h, ROS decreased further, reaching nearly 20% of the normoxic value. In this regard, due to the debated role of the endogenous inhibitor protein (IF₁) of the ATP synthase complex in cancer cell bioenergetics, we investigated whether IF₁ is involved in the control of ROS generation under severe hypoxic conditions. A significant ROS content decrease was observed in hypoxia in both IF₁-expressing and IF₁- silenced cells compared to normoxia. However, IF₁-silenced cells showed higher ROS levels compared to IF1-containing cells. In addition, the MitoSOX Red-measured superoxide level of all the hypoxic cells was significantly lower compared to normoxia; however, the decrease was milder than the marked drop of ROS content. Accordingly, the difference between IF₁-expressing and IF₁-silenced cells was smaller but significant in both normoxia and hypoxia. In conclusion, the interplay between ROS and hypoxia and its modulation by IF₁ have to be taken into account to develop therapeutic strategies against cancer.

Cancer cells overexpress IF₁, the endogenous protein that inhibits the hydrolytic activity of ATP synthase when mitochondrial membrane potential (Δμ_H⁺) falls, as in ischemia. Other roles have been ascribed to IF₁, but the associated molecular mechanisms are still under debate. We investigated the ability of IF₁ to promote survival and proliferation in osteosarcoma and colon carcinoma cells exposed to conditions mimicking ischemia and reperfusion, as occurs in vivo, particularly in solid tumors. IF₁-silenced and parental cells were exposed to the FCCP uncoupler to collapse Δμ_H⁺ and the bioenergetics of cell models were validated. All the uncoupled cells preserved mitochondrial mass, but the implemented mechanisms differed in IF₁-expressing and IF₁-silenced cells. Indeed, the membrane potential collapse and the energy charge preservation allowed an increase in both mitophagy and mitochondrial biogenesis in IF₁-expressing cells only. Interestingly, the presence of IF₁ also conferred a proliferative advantage to cells highly dependent on oxidative phosphorylation when the uncoupler was washed out, mimicking cell re-oxygenation. Overall, our results indicate that IF₁, by allowing energy preservation and promoting mitochondrial renewal, can favor proliferation of anoxic cells and tumor growth. Therefore, hindering the action of IF₁ may be promising for the therapy of tumors that rely on oxidative phosphorylation for energy production.

Purpose: Gonadotropins and anti-Müllerian hormone (AMH) regulate reproductive development and ovarian function. While AMH plays a well-established role in early folliculogenesis by counteracting gonadotropins, luteinizing hormone (LH) becomes more active later, during the antral phase, when granulosa and theca cells express their respective receptors. There are hints suggesting the existence of an interplay between these hormones regulating granulosa cell functions at late stages of the folliculogenesis., but the mechanisms remain unclear. In this context, we explored whether gonadotropin LH can modulate AMH action in cells of ovarian origin. Methods: Primary human granulosa lutein cells isolated from in vitro fertilization (IVF) patients were pretreated with recombinant LH, followed by stimulation with recombinant AMH. AMH receptor type II (AMHR2) expression, AMH signaling activation, and the expression of AMH-responsive genes were assessed through RT-PCR, ELISA, and Western blotting. The involvement of the LH receptor was confirmed using siRNA-mediated knockdown. Results: Our findings showed that LH treatment downregulates AMHR2 transcripts and protein, impairing AMH-induced phosphorylation of small mothers against decapentaplegic (SMAD) 1, 5, and preventing osterix ( OSX ) and matrix metalloprotease 2 ( MMP2 ) gene expression. Conclusions: These data are consistent with the possible interplay between gonadotropins and AMH in cells from antral/luteal stages and provide the molecular basis for further studies evaluating the impact of LH in modulating AMH signaling and follicular responsiveness during the antral stage.