| Introduction:vulvar cancer is one of the four gynecological malignant tumors,which commonly occurs in middle-aged and elderly women.Vulvar squamous cell carcinoma?VSCC?is the most common type of vulvar cancer,accounting for more than 90%of vulvar malignant tumors[1,2].The 5-year survival rate of early stage patients is 80-90%,and that of late stage patients is only about 15-60%[3].Due to the relatively low incidence,researches on the pathogenesis and treatment of vulvar squamous cell carcinoma have been lagging behind those of other tumors,and there is a lack of effective alternative treatment strategies in the treatment of VSCC.Clinically,surgical resection is the main treatment method for VSCC patients.Postoperative radiotherapy and chemotherapy have also been shown to be effective in preventing recurrence and improving the prognosis of patients with VSCC.However,tumor recurrence and lymph node involvement often lead to poor prognosis in patients with advanced VSCC.Although the patients are currently treated with adjuvant chemotherapy and radiotherapy,the recurrence rate of VSCC is still as high as 40%,which is mainly attributed to the insensitivity of VSCC to conventional chemotherapy and the adverse side effects associated with radiotherapy,indicating that the current adjuvant therapy is not ideal[46].Therefore,it is urgent to find new therapeutic strategies and drugs to effectively control the malignant progression and metastasis of vulvar squamous cell carcinoma,which is of great clinical significance.In recent years,with the continuous development of immunotherapy,traditional Chinese medicine and targeted alternative therapy has greatly improved the 5-year survival rate of cancer patients.Among them,traditional Chinese medicine has become a promising therapeutic strategy,and its clinical application and research have attracted more and more attention from domestic and foreign scholars[7].Combined with thousands of years of Chinese history,traditional Chinese medicine has showed a wide range of pharmacological effects,not only to treat a variety of common diseases,but also has a markedly inhibitory effect on tumors.A large number of studies have reported that traditional Chinese medicine have multi-target,multi-effect and multi-link anti-tumor characteristics,and combined chemotherapy drugs can play a synergistic and detoxification role in the treatment of tumors[810].In China,two-thirds of cancer patients are treated with traditional Chinese medicine,and its efficacy has been recognized internationally.Astragalus membranaceus is one of the most commonly used traditional Chinese medicine,can nourishe Qi and blood with non-toxicity.For thousands of years,it has been used in the treatment of various diseases,and has accumulated rich experience in clinical medication[11,12].Astragaloside ??AS-??is one of the characteristic components of Astragalus membranaceus,and has high physiological activity and medicinal value,and has a broad prospects for clinical application[13,14].Studies in the past few decades have found that AS-? is highly effective in a variety of diseases,including fibrosis,inflammation,autoimmunity and cardiovascular diseases[1517].In recent years,it has been reported that AS-? plays an outstanding anti-cancer activity in prostate cancer[18],breast cancer[19],gastric cancer[20],lung cancer[21],glioma[22]and other different tumors,and it can inhibit cell proliferation,invasion and metastasis.However,the role of the AS-? in VSCC has not yet been reported.Malignant progression and metastasis of tumors are the main causes of cancer-related death.Transforming growth factor TGF-?is a multifunctional cytokine,which is secreted by various cells and it is essential for the cell survival,growth and differentiation.During tumorigenesis and development,tumor cells and their surrounding stromal cells can secrete and activate TGF-?in large quantities,that leads to malignant proliferation and metastasis of tumors[23,24].It was reported,not only TGF-?regulates the classical TGF-?/Smad signal to determine tumor fate,but also participates in the transduction of non-Smad signals,such as PI3K/AKT pathway,FAK pathway,ERK pathway and MAPK pathway,etc.,they are closely related to the proliferation,differentiation,migration and invasion of tumor cells[25,26].It has been reported,the expression of TGF-?was increased in VSCC[27].In the present study,we aimed to explore the effects of AS-? on the cell proliferation,apoptosis,cycle,invasion and metastasis of VSCC in vitro and its potential association with TGF-?signaling pathway.The first part:Methods:In our study,SW962 cells of the vulvar squamous cell line was studied in vitro.MTT and plate colony formation assay examined the cell proliferation.Flow cytometry analyzed cell cycle and apoptosis.The morphological changes of SW962cells were observed photographed under an inverted fluorescence microscope after acridine orange?AO?staining.Western blot assay detected the expression of some relevant proteins in cell proliferation,apoptosis and autophagy,as well as TGF-?/Smad signaling pathway.Results:AS-??0-800?g/ml?inhibited the proliferation of SW962 cells in a concentration-and time-dependent manner,reduced the rate of cell colony formation,and up-regulated cyclin P53 and P21 to induce cell cycle arrest at G0/G1 phase.Moreover,as-iv can up-regulate the expression of pro-apoptotic protein Bax,cleaved caspase-3,down-regulate the anti-apoptotic protein Bcl-2,and significantly increase the early and late apoptosis rate of SW962 cells.We also found that as-iv up-regulated the expression levels of Beclin-1 and LC3B,down-regulated the autophagy substrate protein P62,and increased the autophagy activity of SW962 cells.Beclin-1 and LC3B protein expression decreased and P62 expression increased after 3-MA?3-methyladenine?was given to inhibit autophagy activity.Moreover,the effect of AS-? on promoting apoptosis was weakened,indicating that the effect of AS-? on enhancing the apoptosis of epithelial squamous cell carcinoma cells was related to the induction of autophagy.Combined with the results of previous immunohistochemistry,the expression of TGF-?RII and Smad4 in vulvar squamous cell carcinoma decreased.In our study,AS-? not only up-regulated the expression of TGF-?RII and Smad4,but also reversed the down-regulation of TGF-?RII and Smad4 induced by TGF-?1.It can also up-regulate Beclin-1,LC3B,Cleaved caspase-3,down-regulate Bcl-2,inhibit TGF-?1-mediated cell proliferation and anti-apoptosis.Conclusions:1.AS-? inhibits VSCC cells proliferation and growth.2.AS-? induces VSCC cells apoptosis by up-regulating autophagy activity.3.AS-? inhibits the cell proliferation and promotes apoptosis throughTGF-?/Smad signaling pathway.The second part:Methods:MTT,plate colony-formation assay and cellular morphological changes were used to assessed the cytotoxicity of AS-? to SW962 cells in vitro.Transwell and scratch wound-healing assays analyzed the cell migration and invasion.SW962 cell morphological changes undergoing epithelial-mesenchymal transformation?EMT?was observed photographed under an inverted fluorescence microscope after acridine orange?AO?staining.Western blot assay detected the expression of some relevant proteins in terms of EMT,invasion and metastasis,as well as the TGF-?/FAK/AKT signaling pathway.Results:AS-??0-800?g/ml?inhibited the activity of SW962 cells in a dose-dependent manner.At the dose of AS-? was 200?g/ml,SW962 cells were abnormally atrophied,became smaller,the number of living cells decreased,and the colony formation rate decreased significantly.When the dose was 100?g/ml,the cell activity and colony forming ability remain unchanged.However,it has a significant inhibitory effect on the migration and invasion ability of cells,and is accompanied by a decrease in the expression of invasion markers?MMP-2 and MMP-9?.Cultured SW962 cells of TGF-?1?10 ng/ml?for 24 h,it was found that SW962 cells with epithelioid phenotype gradually changed from oval,clustered distribution to elongated spindle fibroblast-like morphology,with reduced adhesion between cells,reduced expression of epithelial marker protein E-cadherin,and increased expression of mesenchymal marker protein N-cadherin and Vimentin.In addition,the phosphorylation levels of FAK and AKT,as well as the protein levels of MMP-2 and MMP-9 are also up-regulated by TGF-?1,and the ability of cells to migrate and invade is enhanced.Interestingly,the expression levels of N-cadherin,Vimentin,p-FAK,p-AKT,MMP-2 and MMP-9 were down-regulated,while the expression of E-cadherin was up-regulated and the ability of cell migration and invasion was weakened.Conclusions:1.AS-? significantly inhibits VSCC cells migration and invasion.2.AS-? reverses TGF-?1-mediated VSCC cells EMT.3.AS-? inhibits the cell migration and invasion through blocking the TGF-?/FAK/AKT signaling pathway. |
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