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.

Background: Gut microbiome dysbiosis is related to the pathogenesis of nonalcoholic fatty liver disease (NAFLD), and the role of toll-like receptor 2 (TLR2) in its molecular mechanism is controversial. Here, we investigated the effects and mechanisms of Escherichia coli-derived lipopolysaccharide (LPS) on lipid accumulation and lipotoxicity in palmitic acid (PA)-treated L02 cell as an NAFLD cell model, and the role of TLR2 in this process. Methods: Oil red O staining assay and free fatty acid (FFA) content test were performed to determine the effects of LPS on lipid accumulation in a PA-induced NAFLD cell model with or without TLR2 inhibition. The levels of IL-6 and TNF-alpha were measured to investigate inflammation conditions. Hoechst 33342 staining assay and Caspase-3 activity assay were used to test cell apoptosis, and the expression levels of proteins in the IRS1/PI3K/AKT signaling pathway, TLR2/MyD88/IKK alpha/NF-kappa B signaling pathway, and mitochondrion-dependent apoptotic signaling pathway were detected using Western blot. Results: Lipid accumulation, pro-inflammatory cytokine release, and cell apoptosis with high levels were observed in the PA-induced NAFLD cell model, and LPS aggravated these processes. Whereas TLR2 inhibition could significantly ameliorate PA-induced and LPS-amplified lipid accumulation, inflammatory, and cell apoptosis, it had no significant effect on L02 cells treated with LPS alone. Conclusions: These results were confirmed by activation or inhibition of the IRS1/PI3K/AKT signaling pathway, TLR2/MyD88/IKK alpha/NF-kappa B signaling pathway, and mitochondrion-dependent apoptotic signaling pathway, and were reflected by changes on their proteins expression. TLR2 is involved in PA-induced lipid accumulation and lipotoxicity in L02 cells, which could be aggravated by LPS, although LPS-induced amplification might not be through direct interaction with TLR2.

Background: MicroRNA (miRNA) plays an important role in hepatic stellate cell (HSCs) activation and liver fibrosis. The purpose of this study is to explore the effect of hypoxia on the differential expression of mRNAs and miRNAs in rat HSCs. Methods: HSC-T6 cells were treated with cobalt chloride (CoCl2), and the activity of HSC-T6 cells was measured by the CCK-8 assay. The mRNA expression levels of hypoxia inducible factor-1 alpha (HIF-1 alpha), collagen type I, transforming growth factor-131 (TGF-131), and Smad7 were measured by RT-qPCR. The protein expression levels of HIF-1 alpha, Bax, Bcl-2, and caspase-3 were assayed by western blot. We used basal medium and 400 mu mol/L CoCl2 medium to treat HSC-T6 cells for 48 h. Cells were harvested after 48 h to extract RNA. Transcriptome sequencing was performed to investigate differentially expressed miRNAs and mRNAs (fold change >2; P<0.05). Bioinformatics analysis was performed to predict the functions of differentially expressed miRNAs and mRNAs. Further, we used RT-qPCR to detect the expression of mRNAs and miRNAs to confirm the accuracy of sequencing. Results: With the increase of CoCl2 concentration, the activity of HSC-T6 cells decreased (P<0.05). The mRNA expression levels of HIF-1 alpha, collagen I, TGF-131, and Smad7, and the protein expressions levels of HIF-1 alpha, Bax, caspase-3, and the Bcl-2/Bax ratio were increased compared with the control group (P<0.05), while the expression of Bcl-2 decreased. A total of 54 miRNAs (20 upregulated and 34 downregulated) and 1,423 mRNAs (685 upregulated and 738 downregulated) were differentially expressed in the 400 mu mol/L CoCl2 medium group compared to the control basal medium group. Further bioinformatics analysis demonstrated that the differentially expressed mRNAs and miRNAs were mainly enriched in the synthesis of extracellular matrix. In addition, we used RT-qPCR to detect the expression of mRNAs and miRNAs to confirm the accuracy of sequencing. Conclusions: Our results presented the profiles of mRNAs and miRNAs in hypoxia-induced HSC-T6 cells in rats, the signaling pathways, and co-expression networks. These findings may suggest novel insights for the early diagnosis and treatment of HSC activation and liver fibrosis.