Stanniocalcin 2 Regulates Autophagy and Ferroptosis in Mammary Epithelial Cells of Dairy Cows Through the Mechanistic Target of Rapamycin Complex 1 Pathway.

BACKGROUND: Stanniocalcin 2 (STC2), a glycoprotein hormone, is extensively expressed in various organs and tissues, particularly in the mammary gland. STC2 plays a crucial role in enabling cells to adapt to stress conditions and avert apoptosis. The efficiency of milk production is closely linked to both the quantity and quality of mammary cells. Yet, there remains a dearth of research on the impact of STC2 on mammary cells' activity in dairy cows. OBJECTIVES: The objective of this study was to investigate the effects of STC2 on the viability of mammary epithelial cells in dairy cows and to elucidate the underlying mechanisms. METHODS: First, the Gene Expression Profiling and Interactive Analysis database was employed to perform survival analysis on STC2 expression in relation to prognosis using The Cancer Genome Atlas and GETx data. Subsequently, the basic physical and chemical properties, gene expression, and potential signaling pathways involved in the growth of dairy cow mammary epithelial cells were determined using STC2 knockdown. RESULTS: STC2 knockdown significantly suppressed autophagy in mammary epithelial cells of dairy cows. Moreover, STC2 knockdown upregulated glutathione peroxidase 4 protein expression, elicited an elevation in lipid ROS concentrations, and inhibited the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway, consequently repressing downstream genes involved in lipid synthesis regulated by mTORC1 and ultimately inducing ferroptosis. CONCLUSIONS: The findings of our study suggest that STC2 suppresses autophagy and ferroptosis through the activation of mTORC1. Mechanically, STC2 exerts an inhibitory effect on ferroptosis by activating antioxidative stress-related proteins, such as glutathione peroxidase 4, to suppress lipid ROS production and stimulating the mTORC1 signaling pathway to enhance the expression of genes associated with lipid synthesis.

Dimerized sesquiterpenoid [4 + 2] adducts with ferroptosis-promoting activity from Inula britannica Linn.

Inubritanolides C and D (1 and 2), two exo sesquiterpenoid [4 + 2] adducts with unprecedented interconverting conformations of twist-chair and chair, together with two previously undescribed endo [4 + 2] dimers (3 and 4) were discovered from Inula britannica flowers. Dimers 1 and 2 have an undescribed carbon skeleton comprising of eudesmanolide and guaianolide units with the linkage mode of C-11/C-1' and C-13/C-3' via a Diels-Alder cycloaddition reaction. Their structures were elucidated using 1D/2D NMR, X-ray diffraction, ECD, and variable-temperature NMR experiments. Dimer 2 displayed a strong inhibitory effect on breast cancer cells by promoting lipid ROS production, showing its potential as ferroptosis inducer.

Role of the p38/AKT Pathway in the Promotion of Cell Proliferation by Serum Heat Inactivation.

Serum is a common biomaterial in cell culture that provides nutrients and essential growth factors for cell growth. Serum heat inactivation is a common treatment method whose main purpose is to remove complement factors and viruses. As serum contains many heat-labile factors, heat inactivation may affect cell proliferation, migration, differentiation, and other functions. However, the specific mechanism of its effect on cell function has not been studied. Thus, we investigate the exact effects of heat-inactivated FBS on the viability of various cells and explore the possible molecular mechanisms. We treated HCT116, HT-29, and HepG2 cell lines with heat-inactivated (56 °C for 30 min) medium, DMEM, or fetal bovine serum (FBS) for different times (0, 10, 15, 30, 60, or 90 min); we found that heat-inactivated FBS significantly promoted the viability of these cells, whereas DMEM did not have this effect. Moreover, heat-inactivated FBS stimulated cells to produce a small amount of ROS and activated intracellular signaling pathways, mainly the p38/AKT signaling pathway. These results indicate that heat-inactivated FBS may regulate the p38/AKT signaling pathway by promoting the production of appropriate amounts of ROS, thereby regulating cell viability.

Resveratrol Promotes Gluconeogenesis by Inhibiting SESN2-mTORC2-AKT Pathway in Calf Hepatocytes.

BACKGROUND: The glucose requirement of dairy cows is mainly met by increasing the rate of hepatic gluconeogenesis. However, due to negative energy balance, the liver of periparturient cows is under oxidative stress induced by lipid over-mobilization, and hepatic gluconeogenesis is reduced. Studies have demonstrated that resveratrol, which is widely known for its antioxidant properties, can alter hepatic gluconeogenesis. However, it is not clear whether resveratrol could regulate hepatic gluconeogenesis by its antioxidant properties. OBJECTIVES: This study aims to investigate the precise effect of resveratrol in hepatic gluconeogenesis, the role of resveratrol on hydrogen peroxide (H2O2)-induced oxidative stress in hepatocytes and the potential mechanism using primary hepatocytes. METHODS: Primary hepatocytes were isolated from 5 healthy Holstein calves (1 d old, 30 to 40 kg, fasted) and treated with different concentrations of resveratrol (0, 5, 10, 25, or 50 µM) combined with or without H2O2 (0, 100, or 200 µM) induction for 12 h. RESULTS: Resveratrol enhanced the expression of gluconeogenic genes of calf hepatocytes in a dose-dependent manner (P < 0.05). Conversely, H2O2 suppressed the expression of gluconeogenic genes and induced oxidative stress (P < 0.05), which was improved by resveratrol in calf hepatocytes (P < 0.001). Furthermore, the mechanistic target of rapamycin complex 2 (mTORC2)-AKT pathway was found to negatively regulate gluconeogenesis. An AKT inhibitor was used to assess the role of the mTORC2-AKT pathway in the effects of resveratrol. The results showed resveratrol promoted hepatic gluconeogenesis by inhibiting the mTORC2-AKT pathway. Moreover, sestrin 2 (SESN2) upregulated the activity of mTORC2. We further found that resveratrol decreased SESN2 levels (P < 0.001). CONCLUSIONS: This study indicated that resveratrol enhances the gluconeogenic capacity of calf hepatocytes by improving H2O2-induced oxidative stress and modulating the activity of the SESN2-mTORC2-AKT pathway, implying that resveratrol may be a promising target for ameliorating liver oxidative stress in transition cows.

Butyrate dictates ferroptosis sensitivity through FFAR2-mTOR signaling.

Evidence shows that short-chain fatty acids (SCFAs) play an important role in health maintenance and disease development. In particular, butyrate is known to induce apoptosis and autophagy. However, it remains largely unclear whether butyrate can regulate cell ferroptosis, and the mechanism by which has not been studied. In this study, we found that RAS-selective lethal compound 3 (RSL3)- and erastin-induced cell ferroptosis were enhanced by sodium butyrate (NaB). With regard to the underlying mechanism, our results showed that NaB promoted ferroptosis by inducing lipid ROS production via downregulating the expression of solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4). Moreover, the FFAR2-AKT-NRF2 axis and FFAR2-mTORC1 axis accounts for the NaB-mediated downregulation of SLC7A11 and GPX4, respectively, in a cAMP-PKA-dependent manner. Functionally, we found that NaB can inhibit tumor growth and the inhibitory effect could be eliminated by administrating MHY1485 (mTORC1 activator) and Ferr-1 (ferroptosis inhibitor). Altogether, in vivo results suggest that NaB treatment is correlated to the mTOR-dependent ferroptosis and consequent tumor growth through xenografts and colitis-associated colorectal tumorigenesis, implicating the potential clinical applications of NaB for future colorectal cancer treatments. Based on all these findings, we have proposed a regulatory mechanism via which butyrate inhibits the mTOR pathway to control ferroptosis and consequent tumorigenesis.

Role of FLCN Phosphorylation in Insulin-Mediated mTORC1 Activation and Tumorigenesis.

The amino acid-stimulated Rag GTPase pathway is one of the main pathways that regulate mechanistic target of rapamycin complex 1 (mTORC1) activation and function, but little is known about the effects of growth factors on Rag GTPase-mediated mTORC1 activation. Here, a highly conserved insulin-responsive phosphorylation site on folliculin (FLCN), Ser62, that is phosphorylates by AKT1 is identified and characterized. mTORC2-AKT1 is localized on lysosomes, and RagD-specific recruitment of mTORC2-AKT1 on lysosomes is identified as an essential step in insulin-AKT1-mediated FLCN phosphorylation. Additionally, FLCN phosphorylation inhibits the activity of RagC GTPase and is essential for insulin-induced mTORC1 activation. Functionally, phosphorylated FLCN promotes cell viability and induces autophagy, and also regulates in vivo tumor growth in an mTORC1-dependent manner. Its expression is also positively correlated with mTORC1 activity in colon cancer, clear cell renal cell carcinoma, and chordoma. These results indicate that FLCN is an important intermediate for cross-talk between the amino acid and growth factor pathways. Further, FLCN phosphorylation may be a promising therapeutic target for diseases characterized by mTORC1 dysregulation.

Pharmacological vitamin C inhibits mTOR signaling and tumor growth by degrading Rictor and inducing HMOX1 expression.

Pharmacological vitamin C (VC) is a potential natural compound for cancer treatment. However, the mechanism underlying its antitumor effects remains unclear. In this study, we found that pharmacological VC significantly inhibits the mTOR (including mTORC1 and mTORC2) pathway activation and promotes GSK3-FBXW7-mediated Rictor ubiquitination and degradation by increasing the cellular ROS. Moreover, we identified that HMOX1 is a checkpoint for pharmacological-VC-mediated mTOR inactivation, and the deletion of FBXW7 or HMOX1 suppresses the regulation of pharmacological VC on mTOR activation, cell size, cell viability, and autophagy. More importantly, it was observed that the inhibition of mTOR by pharmacological VC supplementation in vivo produces positive therapeutic responses in tumor growth, while HMOX1 deficiency rescues the inhibitory effect of pharmacological VC on tumor growth. These results demonstrate that VC influences cellular activities and tumor growth by inhibiting the mTOR pathway through Rictor and HMOX1, which may have therapeutic potential for cancer treatment.

Promotion of intestinal epithelial cell apoptosis by enterotoxigenic Escherichia coli via PKA-mediated inhibition of mTORC1 activation.

Enterotoxigenic Escherichia coli (ETEC) is the main causative pathogen of diarrhea. It causes acute watery diarrhea that leads to rapid dehydration and prostration within hours. ETEC is still an important cause of neonatal and post-weaning diarrhea in pigs. However, the mechanism underlying ETEC-induced diarrhea is not yet clear. In this study, we investigated these mechanisms and found that the mTORC1 pathway plays a role in the host response to ETEC F4 infection. Specifically, we found that ETEC F4 treatment significantly repressed mTORC1 activity as well as cell proliferation, promoted apoptosis and regulated the expression of diarrhea-related genes via the promotion of PKA-mediated phosphorylation of SIN1, which plays a critical role in the assembly of mTORC2. These findings indicate that PKA is a checkpoint for ETEC-induced diarrhea. In terms of potential therapeutic strategies, we found that ZnSO4 dramatically rescued ETEC F4-induced the inhibition of mTORC1 activity and cell viability and the induction of apoptosis and alterations in the expression of diarrhea-related genes. Thus, the present findings demonstrate that ETEC F4 influences mTORC1 activation by inhibiting the assembly of mTORC2 through PKA-mediated phosphorylation of SIN1. Further, supplementation with ZnSO4 is an effective strategy for blocking the effect of ETEC F4 on mTORC1 activation, and it may have potential clinical applications in the treatment of ETEC F4-induced diarrhea.

Trans-gnetin H isolated from the seeds of Paeonia species induces autophagy via inhibiting mTORC1 signalling through AMPK activation.

Paeonia is a well-known species of ornamental plants, traditional Chinese medicines, and emerging oilseed crops. Apart from nutritional unsaturated fatty acids, the seeds of peonies are rich in stilbenes characterized by their wide-ranging health-promoting properties. Although the typical stilbene resveratrol has been widely reported for its multiple bioactivities, it remains uncertain whether the trimer of resveratrol trans-gnetin H has properties that regulate cancer cell viability, let alone the underlying mechanism. Autophagy regulated by trans-gnetin H was detected by western blotting, immunofluorescence, and quantitative real-time PCR. The effects of trans-gnetin H on apoptosis and proliferation were examined by flow cytometry, colony formation and Cell Counting Kit-8 assays. Trans-gnetin H significantly inhibits cancer cell viability through autophagy by suppressing the phosphorylation of TFEB and promoting its nuclear transport. Mechanistically, trans-gnetin H inhibits the activation and lysosome translocation of mTORC1 by inhibiting the activation of AMPK, indicating that AMPK is a checkpoint for mTORC1 inactivation induced by trans-gnetin H. Moreover, the binding of TSC2 to Rheb was markedly increased in response to trans-gnetin H stimulation. Similarly, trans-gnetin H inhibited the interaction between Raptor and RagC in an AMPK-dependent manner. More importantly, trans-gnetin H-mediated autophagy highly depends on the AMPK-mTORC1 axis. We propose a regulatory mechanism by which trans-gnetin H inhibits the activation of the mTORC1 pathway to control cell autophagy.

Suffruticosol C-Mediated Autophagy and Cell Cycle Arrest via Inhibition of mTORC1 Signaling.

Paeonia species are well-known ornamental plants that are used in traditional Chinese medicines. The seeds of these species are rich in stilbenes, which have wide-ranging health-promoting effects. In particular, resveratrol, which is a common stilbene, is widely known for its anticancer properties. Suffruticosol C, which is a trimer of resveratrol, is the most dominant stilbene found in peony seeds. However, it is not clear whether suffruticosol C has cancer regulating properties. Therefore, in the present study, we aimed to determine the effect of suffruticosol C against various cancer cell lines. Our findings showed that suffruticosol C induces autophagy and cell cycle arrest instead of cell apoptosis and ferroptosis. Mechanistically, suffruticosol C regulates autophagy and cell cycle via inhibiting the mechanistic target of rapamycin complex 1 (mTORC1) signaling. Thus, our findings imply that suffruticosol C regulates cancer cell viability by inducing autophagy and cell cycle arrest via the inhibition of mTORC1 signaling.