Acquisition of Chemoresistance in Gliomas Is Associated with Increased Mitochondrial Coupling and Decreased ROS Production

Temozolomide (TMZ) is an alkylating agent used for treating gliomas. Chemoresistance is a severe limitation to TMZ therapy; there is a critical need to understand the underlying mechanisms that determine tumor response to TMZ. We recently reported that chemoresistance to TMZ is related to a remodeling of the entire electron transport chain, with significant increases in the activity of complexes II/III and cytochrome c oxidase (CcO). Moreover, pharmacologic and genetic manipulation of CcO reverses chemoresistance. Therefore, to test the hypothesis that TMZ-resistance arises from tighter mitochondrial coupling and decreased production of reactive oxygen species (ROS), we have assessed mitochondrial function in TMZ-sensitive and -resistant glioma cells, and in TMZ-resistant glioblastoma multiform (GBM) xenograft lines (xenolines). Maximum ADP-stimulated (state 3) rates of mitochondrial oxygen consumption were greater in TMZ-resistant cells and xenolines, and basal respiration (state 2), proton leak (state 4), and mitochondrial ROS production were significantly lower in TMZ-resistant cells. Furthermore, TMZ-resistant cells consumed less glucose and produced less lactic acid. Chemoresistant cells were insensitive to the oxidative stress induced by TMZ and hydrogen peroxide challenges, but treatment with the oxidant L-buthionine-S,R-sulfoximine increased TMZ-dependent ROS generation and reversed chemoresistance. Importantly, treatment with the antioxidant N-acetyl-cysteine inhibited TMZ-dependent ROS generation in chemosensitive cells, preventing TMZ toxicity. Finally, we found that mitochondrial DNA-depleted cells (ρ°) were resistant to TMZ and had lower intracellular ROS levels after TMZ exposure compared with parental cells. Repopulation of ρ° cells with mitochondria restored ROS production and sensitivity to TMZ. Taken together, our results indicate that chemoresistance to TMZ is linked to tighter mitochondrial coupling and low ROS production, and suggest a novel mitochondrial ROS-dependent mechanism underlying TMZ-chemoresistance in glioma. Thus, perturbation of mitochondrial functions and changes in redox status might constitute a novel strategy for sensitizing glioma cells to therapeutic approaches

Sphingolipid accumulation causes mitochondrial dysregulation and cell death

Sphingolipids are structural components of cell membranes that have signaling roles to regulate many activities, including mitochondrial function and cell death. Sphingolipid metabolism is integrated with numerous metabolic networks, and dysregulated sphingolipid metabolism is associated with disease. Here, we describe a monogenic yeast model for sphingolipid accumulation. A csg2Δ mutant cannot readily metabolize and accumulates the complex sphingolipid inositol phosphorylceramide (IPC). In these cells, aberrant activation of Ras GTPase is IPC-dependent, and accompanied by increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial mass. Survival or death of csg2Δ cells depends on nutritional status. Abnormal Ras activation in csg2Δ cells is associated with impaired Snf1/AMPK protein kinase, a key regulator of energy homeostasis. csg2Δ cells are rescued from ROS production and death by overexpression of mitochondrial catalase Cta1, abrogation of Ras hyperactivity or genetic activation of Snf1/AMPK. These results suggest that sphingolipid dysregulation compromises metabolic integrity via Ras and Snf1/AMPK pathways

Impacts of post‐radiotherapy lymphocyte count on progression‐free and overall survival in patients with stage III

Background: We evaluated the impact of thoracic radiation in patients withnon-small cell lung cancer (NSCLC), considering the depletion of total lympho-cytes, use or not of chemotherapy, and radiation doses in healthy lung tissue.Methods: Patients with stage III NSCLC, ECOG 0 to 2, receiving radiothera pywith or without chemotherapy were prospectively evaluated. All patients shouldbe treated with three-dimensional radiotherapy and received biologically effectivedoses (BED10α/β 10) of 48 to 80 Gy. Peripheral blood lymphocyte total countswere measured at the start of radiotherapy and at 2, 6 and 12 months after radio-therapy. Along with lymphocytes, PTV and doses of 5 Gy and 20 Gy in healthylung tissue were also evaluated as potential factors inuencing overall survival(OS) and progression-free survival (PFS).Results: A total of 46 patients were prospectively evaluated from April 2016 toAugust 2019, with a median follow-up of 13 months (interquartile range, 1–39months). The median of OS of all cohort was 22,8 months (IC 95% 17,6–28,1)and the median PFS was 19,5 months (IC 95%: 14,7–24,2). Most patientsreceived concurrent or neoadjuvant chemotherapy (43; 93.4%). No patientreceived adjuvant immunotherapy. The lower the lymphocyte loss at 6 monthsafter radiotherapy (every 100 lymphocytes/mcL), the greater the chance of PFS(HR, 0.44; 95%CI, 0.25–0.77; P = 0.004) and OS (HR, 0.83; 95%CI, 0.70–0.98;P = 0.025; P = 0.025). BED was a protective factor for both PFS (HR, 0.52; 95%CI 0.33 –0.83; P = 0.0006) and OS (HR, 0.73; 95%CI 0.54–0.97; P = 0.029).Conclusions: Our results suggest that lymphocyte depletion after radiotherapyreduces tumor control and survival in patients with stage III lung cancer. Radia-tion doses equal or higher than 60 Gy (BED1072 Gy) improve PFS and OS, butthey negatively affect lymphocyte counts for months, which reduces survival andthe potential of immunotherapy