Disruption of mitochondrial homeostasis with artemisinin unravels anti-angiogenesis effects via auto-paracrine mechanisms.

Rationale: Tumor angiogenesis promotes tumor development, progression, growth, and metastasis. Metronomic chemotherapy involves the frequent administration of low-dose chemotherapeutic agents to block angiogenic activity and reduce side effects. Methods: MDA-MB-231 cells were treated with various concentrations of artemisinin (ART) and vinorelbine (NVB) and the cytotoxic effects of ART/NVB were determined using the CCK-8 assay. Mitochondrial reactive oxygen species (ROS) levels, mitochondrial membrane potential (∆Ψm) and mass were assessed using MitoSOX, TMRE and MitoTracker green staining. Western blot analysis was used to quantify the expression of autophagy-related proteins. Herein, by using bioinformatics analysis and experimental verification, we identified CREB as a master in MDA-MB-231 cells. Results: We found that artemisinin (ART), which exhibits anti-angiogenic and anti-cancer effects via mitochondrial regulation, synergized with vinorelbine (NVB) to inhibit MDA-MB-231 cell proliferation. ART and NVB cooperated to regulate mitochondrial biogenesis. CREB acted as a crucial regulator of PGC1α and VEGF, which played critical roles in NVB-dependent growth factor depletion. Moreover, CREB suppression significantly reversed mitochondrial dysfunction following ART/NVB co-treatment. In addition, combination treatment with ART and NVB significantly suppressed tumor growth in a nude mouse xenograft model, with downregulated CREB and PGC1α expression levels observed in tumor biopsies, in agreement with our in vitro and ex vivo data. Conclusions: These findings support the hypothesis that ART affects cancer and endothelial cells by targeting the auto-paracrine effects of VEGF to suppress mitochondrial biogenesis, angiogenesis, and migration between cancer cells and endothelial cells.