| According to the latest statistics,19.29 million new cancer cases and 9.96 million deaths occurred worldwide in 2020,while 4.57 million new cancer patients and 3million cancer deaths occurrred in China,ranking China first in the world in terms of new cases and deaths.Among many cancers,breast cancer ranked first in the world and fourth in China.After traditional surgery,chemotherapy and radiotherapy,patients with breast cancer are found to have poor effects,and tumors are prone to recurrence and metastasis,and their bodies are seriously affected by side effects.Therefore,it is urgent to find new therapeutic targets and more effective drugs for breast cancer treatment.The study of natural plant compounds is a feasible way to develop anti-tumor drugs and improve the survival rate of cancer patients.Celastrol is a pentacyclic triterpenoid derived from Chinese herbal Tripterygium wilfordii Hook.F,which has a wide range of biological and pharmacological functions,and a number of studies have shown that it also has obvious antitumor effects.It has been reported that TMEM100 protein is closely related to the epithelial-mesenchymal transformation,angiogenesis,metastasis,invasion and apoptosis of many tumor cells,and its expression level in cancer cells is significantly lower than the normal level.In this study,human breast cancer cell lines MCF-7 and MDA-MB-231 were used as experimental subjects to investigate the effects of natural compound Celastrol(Cel)on apoptosis,autophagy and metastasis,and to explore its possible mechanism.The following experiments were carried out: MCF-7 and MDA-MB-231 cells were incubated with Celastrol for a certain period of time.The cell viability was measured by MTT assay,the normal morphology of the cells and the morphological changes of the cells treated with Celastrol were observed under the microscope,the colony formation of the cells was detected by crystal violet staining,and the migration ability was detected by scratch healing test and Transwell test.The morphological changes of apoptosis were observed by AO/PI staining and Hoechst staining,autophagy was observed by MDC staining,cell cycle and apoptosis were detected by Flow cytometry staining,and transcription of TMEM100 and TGFβ signaling pathway-related genes was detected by qRT-PCR.Western blot was used to detect the expression levels of TMEM100,cyclin,EMT-related proteins,apoptotic signature proteins,autophagy signature proteins and TGFβ signaling pathway related proteins.The apoptosis rate and TMEM100 protein expression were detected after treatment of ALK5 inhibitor SD208 alone and combined with Celstrol.The results of this study are as follows:(1)Celastrol inhibited the activity of MCF-7 and MDA-MB-231 cells in a concentration-dependent and time-dependent manner,and the inhibitory effect on the activity of MDA-MB-231 cells was stronger.Short-term treatment with low concentration of Celastrol is non-toxic to normal breast epithelial cells MCF-10A;Celastrol inhibited breast cancer cell activity more effectively than chemotherapeutic drug 5-Fu.(2)Celastrol inhibited cell colony formation in a dose-dependent manner.(3)Celastrol blocked MCF-7 cell cycle in S phase and MDA-MB-231 cell cycle in G1 phase by downregulating cyclin D1.(4)Celastrol reversed epithelial-mesenchymal transformation by up-regulating E-cadherin and down-regulating N-cadherin protein,thus inhibiting cell migration,and the inhibition effect is positively correlated with time and dose.(5)After treatment with Celastrol,the number of apoptotic bodies increased,and the expression levels of apoptotic proteins Cleaved capase-12,CHOP were up-regulated.Celastrol induced apoptosis through endoplasmic reticulum pathway,and effect was stronger at higher and had a better apoptosis-promoting effect than 5-Fu.(6)The autophages increased in the cells treated with Celastrol,autophagosomal marker protein LC3 II was up-regulated,and p62 protein was down-regulated.The effect was more obvious with the increase of concentration.(7)Celastrol significantly upregulated TMEM100 protein at both transcriptional and translational levels.(8)The expression of TGFβ,ALK5 and Smad2 genes related to the TGFβsignaling pathway were significantly decreased with the increase of Celastrol concentration,and the expression of Smad2 protein and p-Smad2 protein were also significantly decreased,Celastrol inhibited the TGFβ signaling pathway.(9)Compared with ALK5 inhibitor SD208 alone,combined treatment with Celastrol,the apoptosis-promoting ability of MCF-7 and MDA-MB-231 cells was enhanced,and the upregulation ability of TMEM100 protein was enhanced.To sum up,Celastrol inhibits TGFβ/ALK5/Smad2 signaling pathway by attenuating Smad2 protein phosphorylation,thereby up-regulating the expression of TMEM100 protein,blocking cell cycle,reversing epithelial mesenchymal transition and inhibiting cell metastasis,inducing endoplasmic reticulum pathway-mediated apoptosis,and promoting autophagy.It also enhanced the apoptotic effect of ALK5 inhibitor SD208 on cells.This study preliminarily investigated the possible mechanism of action of Celastrol against breast cancer,and provided theoretical basis and experimental basis for the clinical application of tripterine against breast cancer. |
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