The Molecular Mechanism Of Oxymatrine And Tanshinone ?A Of Traditional Chinese Medicine Monomer Against Vulvar Squamous Cell Carcinoma

Objective:Vulvar cancer is the fourth most common malignancy of the female reproductive tract.The histological type is mainly squamous cell carcinoma?SCC?,accounting for 90%of all cases.In the past decade,the incidence and mortality of new vulvar cancers have been increasing year by year.At present,the main treatment is surgery,chemoradiotherapy as a supplement.The 5-year survival rate during the early stage is 80–90%,decreasing to 25–50%in the late stage.The leading causes of death and poor clinical outcomes for patients with VSCC are recurrence and metastasis.The limitations of local radiotherapy of the vulva and the resistance of the tumor to radiotherapy and chemotherapy.In addition,molecular targeting and immunotherapy are rarely used in vulvar squamous cell carcinoma?VSCC?.Therefore,it is urgent to find new treatments.In cancer treatment,Chinese herbs are often the main source of anticancer drugs.Studies have shown that they are relatively less toxic than traditional chemotherapy,and therefore have fewer side effects on patients.In most cancers,multiple genes and pathways are deregulated,which promotes excessive proliferation of cancer cells and their survival.Therefore,multi-target therapies that can modulate these pathways are needed.Because Chinese herbs may target multiple signaling pathways simultaneously,they have an advantage in the treatment of cancer.As we all know,Sophora flavescens and Salvia miltiorrhiza are traditional Chinese medicines with a history of thousands of years.Oxymatrine?OMT?is a quinazine alkaloid extracted from Sophora flavescens and it is a hydrophilic monomer.Tanshinone ?A?Tan ?A?is a fat-soluble diterpene component extracted from the root of Salvia miltiorrhiza.Recent studies have shown that OMT and Tan ?A not only have anti-inflammatory,antiallergic and antiarrhythmic pharmacological effects,but also have anti-tumor effects.It has been reported that they have anticancer activity in gastric,colorectal,breast,lung,liver and other tumors.However,their research on VSCC has not been reported.Transmembrane tyrosine kinases are closely related to the proliferation and metastasis of a variety of human cancers.The RAS/RAF/MEK/ERK and FAK/PI3K/AKT signaling pathways are two of the most common kinase cascade disorders in human cancer.The reason is that the key genes of these pathways often undergo molecular changes,resulting in abnormal activation or dysregulation of the signal,resulting in uncontrolled cell growth and survival,and ultimately leading to the transformation and progression of cancer.In our previous studies,it was found that the expression of RAS gene in VSCC was higher than in normal skin,which was associated with poor prognosis.Abnormal mutations of RAS and BRAF can be detected in VSCC,¹⁴ which are transmitted to proteins through transcription or translation,leading to sustained phosphorylation and activation of ERK.In addition,mutations in PIK3CA can be detected in about 9%of VSCC and these mutations can increase the activity of downstream PI3Ks kinase,thereby activating the PI3K/AKT pathway and promoting tumor proliferation and migration.Therefore,we speculated that abnormal activation of the RAS/RAF/MEK/ERK and FAK/PI3K/AKT signaling pathways may promote proliferation and invasion of VSCC cells.This study aimed to exporle whether OMT and Tan ?A inhibited proliferation and invasion of VSCC cells by inhibiting the RAS/RAF/MEK/ERK and FAK/PI3K/AKT signaling pathways and lay an experimental foundation for the selection of effective Chinese medicine prescriptions for the treatment of anti-vulvar cancer in the future.Methods:We selected SW962 and A431 VSCC cell lines.Cell proliferation was examined using MTT assay.Cell cycle and apoptosis were detected using flow cytometry.Migration and invasion were evaluated using transwell and wound-healing assays.The relevant protein expression and signaling pathways were analyzed using western blotting.Results:Oxymatrine inhibited the proliferation of SW962 and A431 VSCC cells in a time-and dose-dependent manner.Oxymatrine induced cell cycle arrest in the G2/M phase by increasing the protein expression of P21 and decreasing levels of cyclin B1 and CDC2.Oxymatrine upregulated the expression of cleaved-caspase 3 and BAX and downregulated the expression of BCL2,which led to an increase in apoptosis.Oxymatrine also suppressed the migration and invasion of SW962 and A431 cells by reducing levels of MMP2 and MMP9.After treatment with oxymatrine or a RAS inhibitor?salirasib?,expression levels of RAS,p-RAF,p-MEK,p-ERK,C-MYC,and MMP2 were reduced.When TGF-?1 was used to stimulate SW962 and A431 cells,the expression of the above proteins increased;this increase was reversed by using oxymatrine or salirasib again.Subsequently,our in vivo studies showed that administration of oxymatrine induced a significant decrease in tumor growth.In addition,we discovered that Tanshinone ?A significantly inhibited the proliferation of SW962 and A431cells in a time-and dose-dependent manner.In addition,Tanshinone ?A arrested the cell cycle in the G0/G1 phase by increasing the protein expression of P21 and decreasing the levels of Cyclin D1 and C-MYC.Tanshinone ?A upregulated the expression of cleaved-caspase3,cleaved-caspase9 and BAX,and downregulated the expression of the BCL2,which resulted in an increase in apoptosis.Furthermore,Tanshinone ?A suppressed the migration and invasion of SW962 and A431 cells by reducing the expression of MMP2 and MMP9.After treatment with Tanshinone ?A or FAK inhibitor?PF-562271?,not only the protein expression of p-FAK,p-PI3K and p-AKT were downregulated,but also that of the C-MYC,BCL2 and MMP2expression decreased.When VEGF was used to stimulate SW962 and A431 cells,the expression of the above proteins increased;this increase was reversed by using Tanshinone ?A or the FAK inhibitor again.Conclusion:1.The inhibition of cells proliferation,migration,invasion and the induction of apoptosis in response to oxymatrine in VSCC cells,may function through the suppression of RAS/RAF/MEK/ERK pathway,which was considered as the vital signaling pathway in regulating tumorigenesis.2.The inhibition of cells proliferation,migration,invasion and the induction of apoptosis in response to Tanshinone ?A in VSCC cells,may function through the suppression of FAK/PI3K/AKT pathway,which was considered as the vital signaling pathway in regulating tumorigenesis.