Toosendanin, a late-stage autophagy inhibitor, sensitizes triple-negative breast cancer to irinotecan chemotherapy

Background: Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer that develops resistance to chemotherapy frequently. Autophagy has been reported as a pro-survival response to chemotherapeutic drugs in TNBC, and suppression of autophagy can be a strategy to overcome drug resistance. 

Methods: The efficacy of toosendanin (TSN) in blocking autophagy flux was measured by western blot analysis of autophagy markers, and the fluorescent imaging of RFP-GFP-LC3 probe. The co-localization of autophagosomes and lysosomes was analyzed by fluorescent imaging. Then, lysosome function was determined by measuring the lysosomal pH value and the activity of lysosomal hydrolytic proteases. For in vitro study, human triple-negative breast cancer MDA-MB-231 and MDA-MB-436 cell lines were used for evaluating the anti-proliferative effect. For in vivo study, the RFP-GFP-LC3 MDA-MB-231 xenograft nude mice received intraperitoneal injection of irinotecan (10 mg/kg), TSN (0.5 mg/kg) or a combination, and the autophagy activity and cell apoptosis were determined in tumor tissue. The degree of pathological injury of tissue was evaluated by liver index. 

Results: The natural autophagy inhibitor TSN, a triterpenoid extracted from Melia toosenda Sieb. et Zucc, potently inhibited late-stage autophagy in TNBC cells. This effect was achieved via elevating lysosome pH rather than blocking the fusion of autophagosomes and lysosomes. We further investigated the effects of TSN on the in vitro and in vivo TNBC models, in combination with chemotherapeutic drug irinotecan (or its active metabolite 7-ethyl-10-hydroxycamptothecin), a topoisomerase I inhibitor showing therapeutic potential for TNBC. The data showed that TSN blocked 7-ethyl-10-hydroxycamptothecin (SN-38)/irinotecan-induced protective autophagy, and significantly induced apoptosis in TNBC cells and tumor xenograft models when compared to SN-38/irinotecan alone group.