Résumé
Purpose To investigate the effect of autophagy intervention on ferroptosis and drug resistance of colorectal canc-er cells and its molecular mechanism.Methods The human colorectal cancer cell lines HCT-8,COLO205,HCT-116,SW620,and SW480 were cultured.HCT-116 cells with moder-ate expression of LC3 were screened,and the expression differ-ences of LC3,p62,Keap1,Nrf2,GPX4 proteins,Fe2+,GSH,and MDA between them and OXA-resistant HCT-116/OXA cell lines were detected.The expression levels of LC3,p62,Keap1,Nrf2,GPX4,Fe2+,GSH and MDA were assessed in HCT-116/OXA cells through the intervention of autophagy and ferroptosis intervention agent combined with oxaliplatin.The proliferative activity and sensitivity to oxaliplatin in each group were detected by CCK-8 assay.Cell growth and invasion ability of each group were detected by plate cloning and Trans well assay.Results LC3,p62 and GPX4 expression levels of HCT-116 cells in the 5 groups were moderate.Compared with HCT-116 cells,HCT-116/OXA was less sensitive to oxaliplatin,and the proteins of p62,Nrf2 and GPX4 were highly expressed,LC3 and Keap1 were lowly expressed,and the expression of Fe2+,GSH and MDA were increased(P＜0.05).The levels of LC3,Keap1 protein,Fe2+and MDA in Rapa and Rapa+Fer-1 groups were higher than those in Fer-1 and control groups,while p62,Nrf2,GPX4 and GSH levels were lower.The expressions of GPX4 pro-tein and GSH in Rapa+Fer-1 group were lower than those in Rapa group(P＜0.05).In the autophagy inhibitor group,LC3,p62,Nrf2,GPX4 and GSH were highly expressed in the CQ and CQ+Erastin groups compared with the control and Eras-tin groups,while Keap1 protein,Fe2+and MDA were low.The levels of GPX4 protein and GSH in Erastin group were lower than those in the other three groups,and the levels of Fe2+and MDA were higher than those in the other three groups(P＜0.05).The combination of autophagy activator OXA showed that Rapa intervention group had higher chemical sensitivity to OXA,less number of migrating cells and lower cell proliferation activity than the other three groups.The sensitivity of Rapa+Fer-1 group to oxaliplatin was lower than that of Rapa group,but higher than that of Fer-1 group and control group(P＜0.05).There was no significant difference between Fer-1 group and con-trol group(P＜0.05).Compared with the control group,the cell activity,migration capacity and clonogenesis capacity of Erastin,CQ+Erastin and CQ groups were decreased when auto-phagy inhibitor was combined with OXA,and the Erastin group was the lowest,while the CQ+Erastin group was higher than the Erastin group,and lower than the CQ group(P＜0.05).Con-clusion In colorectal cancer,autophagy is involved in the regu-lation of ferroptosis,and intervention in autophagy can regulate ferroptosis in colorectal cancer cells through the p62-Keap1/Nrf2-GPX4 pathway,thereby reversing oxaliplatin resistance.
Résumé
Endometriosis is a common chronic gynecological disease with endometrial cell implantation outside the uterus.Angiogenesis is a major pathophysiology in endometriosis.Our previous studies have demon-strated that the prodrug of epigallocatechin gallate(ProEGCG)exhibits superior anti-endometriotic and anti-angiogenic effects compared to epigallocatechin gallate(EGCG).However,their direct binding targets and underlying mechanisms for the differential effects remain unknown.In this study,we demonstrated that oral ProEGCG can be effective in preventing and treating endometriosis.Additionally,1D and 2D Proteome Integral Solubility Alteration assay-based chemical proteomics identified metadherin(MTDH)and PX domain containing serine/threonine kinase-like(PXK)as novel binding targets of EGCG and ProEGCG,respectively.Computational simulation and BioLayer interferometry were used to confirm their binding affinity.Our results showed that MTDH-EGCG inhibited protein kinase B(Akt)-mediated angiogenesis,while PXK-ProEGCG inhibited epidermal growth factor(EGF)-mediated angiogenesis via the EGF/hypoxia-inducible factor(HIF-1a)/vascular endothelial growth factor(VEGF)pathway.In vitro and in vivo knockdown assays and microvascular network imaging further confirmed the involvement of these signaling pathways.Moreover,our study demonstrated that ProEGCG has superior therapeutic effects than EGCG by targeting distinct signal transduction pathways and may act as a novel anti-angiogenic therapy for endometriosis.
Résumé
Objective:To investigate the expression characteristics of phosphorylated enhancer of Zeste homolog 2(EZH2)in head and neck squamous cell carcinoma(HNSCC)and their effect on chemosensitivity.Methods:Fifty-three patients with HNSCC treated between January 2018 and March 2021 in Tianjin Medical University Cancer Institute&Hospital were selected.Expression levels of p-EZH2S21,p-STAT3Y705,HIF-1α,and Ki-67 in HNSCC tissues were analyzed by immunohistochemical staining.Western blot was used to detect p-EZH2S21 expression in HNSCC tissues and cell lines.HNSCC cell lines stably transfected with wild type(EZH2-WT)or S21 mutant EZH2(EZH2-S21A)were construc-ted.CCK8 and colony formation assays were performed to detect the effect of EZH2 and S21 phosphorylation on HNSCC cell proliferation and their sensitivity to cisplatin(DDP)and 5-fluorouracil(5-FU).Results:Elevated levels of p-EZH2S21 were observed in HNSCC tissues and positively correlated with p-STAT3Y705(P<0.05)and HIF-1α(P<0.01)levels.p-EZH2S21 level correlated positively with lymph node metastasis(P<0.000 5),T(P<0.05),N(P<0.000 1)and AJCC stages(P<0.05).In vitro experiments confirmed that EZH2 expression promoted cell prolifer-ation and attenuated chemosensitivity of HNSCC cells.Inhibition of p-EZH2S21 restored HNSCC cell sensitivity to DDP and 5-FU.Conclusions:p-EZH2S21 plays an important role in tumor progression in HNSCC,and phosphorylation at S21 is an important way for EZH2 to affect HNSCC cell proliferation and chemotherapy sensitivity.