Mechanistic insights into reactive oxygen species (ROS) homeostasis in ovarian cancer chemoresistance

Background : Ovarian cancer remains one of the most lethal gynecologic malignancies, with disease recurrence and platinum resistance representing major therapeutic challenges. Reactive oxygen species (ROS) homeostasis and cancer stem cells (CSC) contribute critically to chemoresistance; however, the molecular mechanisms that sustain CSC expansion under oxidative stress remain insufficiently defined. This study investigated how ROS regulation in ovarian cancer spheroids engages NOTCH1/HES1 and IL11/STAT5-mediated signaling to drive stemness and resistance to cisplatin. Methods : Ovarian cancer cell-derived spheroids and/or patinum-resistant patient-derived organoids were used to evaluate ROS homeostasis, antioxidant enzyme expression, and CSC phenotypes. ROS levels and MnSOD/HO-1 expression were assessed by fluorescence detection and immunoblotting. Expression, promoter activity and/or activation of P38/NRF2, NOTCH1/HES1, and IL11/STAT5 signaling were examined by qPCR, Western blotting, and reporter assays. Gene silencing of MnSOD, HO-1, HES1 or NRF2 was achieved using siRNA. Pharmacologic inhibition was performed using GSI-MK0752 (NOTCH1/HES1) and CYT387 (JAK2/STAT5). CSC activity was quantified by ALDH assays, CD44 expression, and spheroid formation capacity. An orthotopic ovarian cancer xenograft model was used to evaluate therapeutic efficacy of cisplatin given alone or in combination with pathway inhibitors. Results : Ovarian cancer spheroids maintained a low-ROS environment through induction of key antioxidants MnSOD and HO-1. Cisplatin activated spheroids P38/NRF2 signaling, which consequently upregulated NOTCH1/HES1 signaling. Silencing of MnSOD, HO-1, or NRF2 impaired spheroids NOTCH1/HES1 activation. Pharmacologic inhibition of either NOTCH1/HES1 or IL11/STAT5 signaling confirmed the NOTCH1/HES1 and IL11/STAT5 signaling axis and decreased spheroids CSC markers and ALDH⁺/CD44⁺ CSC subpopulations, while dual inhibition produced synergistic effects. Moreover, CSC subpopulations enhanced cisplatin responsiveness. Cisplatin induced organoids NOTCH1 activation and promoted STAT5 signaling and expanded ALDH⁺/CD44⁺ CSC subpopulations. In vivo, combined pathway blockade with cisplatin significantly reduced tumor burden and ascites formation compared with monotherapies. Conclusion : A ROS-responsive NOTCH1/HES1 and IL11/STAT5 signaling axis plays a central role in maintaining CSC populations and promoting platinum resistance in ovarian cancer. Dual targeting of these signaling pathways disrupts the redox-regulated circuit and enhances cisplatin efficacy, supporting further investigation of this combinatorial strategy as a potential therapeutic approach for platinum-resistant ovarian cancer. Acknowledgement: HMRF (18191641).