Jadomycin B, produced by the soil bacterium Streptomyces venezuelae ISP5230, induces cytotoxicity in human breast cancer cells in vitro and has antitumoral effects in animal models. In models of multidrugresistant, triple-negative breast cancer, jadomycin B has shown promise as it is not a substrate of ABCB1 and ABCG2 drug efflux transporters. The generation of reactive oxygen species and inhibition of topoisomerases are potential mechanisms of jadomycin B-mediated DNA damage and apoptosis. However, the mechanisms of jadomycin B's anticancer activity have not been fully elucidated. By gradually exposing MDA-MB-231 triple-negative human breast cancer cells to jadomycin B, we hypothesized that resistance could be selected to further understand jadomycin B's pharmacological mechanisms. A 3-fold increase in the jadomycin B IC50 was observed in MDA-MB-231 cells exposed to increasing jadomycin B concentrations (0-3 mu M) over 7 months, herein 231-JB cells. The 231-JB cells were cross-resistant to jadomycin F and S but not to the comparator drugs mitoxantrone, doxorubicin, and SN-38. The 231-JB cells did not have increased mRNA expression of topoisomerase-2 nor ABCB1 and ABCG2. Cyclooxygenase-2 (COX-2) increased by 25-fold, but expression of prostaglandin E2 receptor 4 did not significantly change. Cotreatment with celecoxib (15-45 mu M), a COX-2 inhibitor, resensitized the 231-JB cells to jadomycin B (IC50 1/4 1.41 +/- 0.24 to 0.75 +/- 0.31 mu M vs 2.28 +/- 0.54 with 0 mu M celecoxib). To our knowledge, this work represents the first report of the involvement of COX-2 in jadomycin B activity in vitro, proving to be an exciting new target for the exploration of jadomycin B anticancer activity. Significance Statement: Cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin production, is associated with procancer signaling. COX-2, ABCB1, and ABCG2 overexpression are typically correlated in cancer, contributing to chemotherapy resistance. We observed increased COX-2, but not ABCG2 or

Breast cancer is one of the most common and deadly cancers in women worldwide. Current treatments for breast cancer have limitations, such as toxicity, resistance, and side effects. Therefore, there is a need to develop new and effective anti-cancer agents from natural sources. Spinosyn A (SPA) is a natural product derived from soil bacteria. SPA has been reported to have anti-parasitic, insecticidal, and anti-bacterial activities. However, its anti-cancer effects and mechanisms are not well understood. In this study, we investigated the effects of SPA on T47-D, estrogen receptor-positive breast cancer cells. We found that SPA inhibited cell proliferation and migration and induced apoptosis and cell cycle arrest. Flow cytometry and holographic imaging microscopy revealed that SPA activated MAPK and PI3K signaling pathways and altered cellular morphology. Finally, RNASeq analysis revealed that SPA treatment altered the expression of 1380 genes in T47-D cells, which were enriched in various biological processes and signaling pathways related to cell proliferation, cholesterol metabolism, growth factor activity, amino acid transport activity, extracellular matrix, PI3K-Akt signaling pathway, neuroactive ligand-receptor interaction, and PPAR signaling pathway. Our results suggest that SPA exerts multiple anti-cancer effects on T47-D cells by modulating multiple pathways and cellular processes involved in cell growth, survival, and motility. Our findings provide new insights into the molecular mechanisms of SPA action on breast cancer cells and its potential applications as a novel anti-cancer agent.