Inhibition Of Teramethylpyrazine On Proliferation,Invasion And Metastasis Of A498 Cell By EMT Mechanism

BackgroundsIn recent decades,the incidence of renal cell carcinoma(RCC)as is a form of kidney cancer,is increasing at an alarming rate,which second only to bladder cancer in urogenital system tumors.Currently,Patients with early,localized RCC have a good prognosis,but those with advanced disease do not respond to most traditional treatment such as radiotherapy,chemotherapy,surgical and pHarmaceutical interventions.However,a growing understanding of the molecular mechanisms underlying RCC has led to the development of novel targeted agents.Therefore,the development of novel targeted agents was one of the research hotspots of RCC in molecular immunity.Tumor invasion and metastasis are complex and multifarioust,in which the molecular mechanism provides targets for inhibiting the invasion and metastasis.Epithelial-mesenchymal transformation(EMT)has become widely accepted as a mechanism by which injured renal tubular cells transform into mesenchymal cells that contribute to tumor invasion and metastasis.The degree of EMT in tumors directly determines the malignant degree of tumors.The most prominent feature of EMT is the down-regulated expression of epithelial marker E-cadherin,while up-regulated the expression of N-cadherin interstitial cell marker protein and related transcription factors.EMT can be activated by different signaling pathways,such as Notch and Wnt,those are regulated by intracellular transcription factors Snail and Slug to induce EMT.Therefore,we predict that by regulating transcription factors involved in the process of EMT can prevent or inhibit the cancer cells invasion and metastasis.Ligusticum chuanxiong is the dried rhizome of umbelliferae,it has many functions,such as promoting blood circulation and promoting qi,dispelling wind and relieving pain,and promoting qi and depression.Tetramethyipyrazine(TMP)is one of the pHarmacological active ingredients isolated from the rhizomes of Ligusticum chuanxiong.It has been widely used in the prevention and treatment of ischemic stroke and cardiovascular diseases for a long time.In addition,TMP also has the pHarmacological effects of anti-free radical and improving microcirculation.Increasing studies have confirmed that TMP has certain therapeutic effect on lung cancer,breast cancer and other malignant tumors.In 2013,Huang Qi et al.found that TMP can effectively improve renal interstitial fibrosis by inhibiting the up-regulation of p-Smad2 protein and alpHa-SMA protein induced by TGF-beta 1,thereby inhibiting the activation of Smad2 and blocking the transduction of epithelial mesenchymal(EMT).Other studies have confirmed that TMP can inhibit the lung cancer cells invasion and metastasis by up-regulating the expression of E-cadherin,down-regulating the expression of N-cadherin and reversing the process of EMT.Therefore,we attempt to explore the molecular mechanism of TMP inhibiting the RCC invasion and metastasis,and provide valuable new targets for clinical research of Anti-renal cell cancer drugs.ObjectivesTo research the influence of TMP on the proliferation,metastasis and invasion of renal carcinoma A498 cell,and primarily explore the molecular mechanism of the function of TMP.MethodsThis project is divided into three partsThe first part,the influence of TMP on the proliferation,metastasis and invasion of A498 cell.The influence of TMP on the proliferation of A498 cell was detected by MTT method.Flow cytometry was used to observe the effect of different concentrations of TMP on the cell cycle of A498.The effect of TMP on the cloning ability of A498 cell was detected by plate cloning experiment.The effect of TMP on the metastasis ability of A498 cell was observed by Trans-well cell metastasis experiment.The effect of TMP on the invasion ability of A498 cell was observed by Trans-well cell invasion experimentThe second part,the effect of TMP on the proliferation,metastasis and invasion of A498 cell in nude mice.Firstly,we inoculated A498 cell into the skin of nude mice to construct a subcutaneous tumor-bearing model in nude mice.The effect of TMP on the proliferation of subcutaneous xenograft in nude mice was observed by comparison with the control group.Secondly,we used the method of injecting A498 cell into the tail vein of nude mice to establish an animal model of invasion and metastasis of A498 cell in the lung.The effect of TMP on the invasion and metastasis of A498 cell was detected by comparison with the control group.The third part,the molecular mechanism of the inhibition of TMP on A498 cell was studied.EMT plays an important role in the process of tumor invasion and metastasis.In order to further explore the molecular mechanism of TMP inhibiting renal carcinoma A498 cell,we examined the expression effect of three marker proteins,N-cadherin,Snail and Slug in the function process of TMP over EMT mechanism of renal carcinoma by Western blot method.ResultsIn the experiments of the first part,MTT method,flow cytometry,and plate cloning experiment show that TMP can inhibit A498 cell in a time and dose dependent manner.Trans-well cell metastasis and cell invasion experiments show that TMP can inhibit the metastasis and invasion of A498 cell in a time and dose dependent manner.The second part,the result of subcutaneous tumor-bearing experiment in nude mice shows that the subcutaneous tumor growth rate of the nude mice in the TMP treatment group is slower than that of the control group,and the tumor volume is smaller than that of the control group,indicating that TMP can inhibit the tumor growth rate of A498 cell in nude mice.In the experiment of metastasis tumor formation in nude mice,we found that 100%of the nude mice in the tail vein injection control group formed lung metastases,and only 37.5%of the mice in the TMP treatment group formed lung metastases,and the tumor metastasis lesion number is significantly lower than that of the control group,indicating that TMP can inhibit the invasion and metastasis of A498 cell in nude mice.The third part,the result of Western blot shows that compared with the control group,the expressions of N-cadherin,Snail and Slug proteins in the experimental group are inhibited,and the inhibition effect becomes more obvious with the increase of medicine concentration and time,which leads to the conclusion:TMP can inhibit EMT mechanism of A498 cell,thus inhibiting the invasion and metastasis abilities of A498 cell by inhibiting the expressions of N-cadherin,Snail and Slug proteins.ConclusionsIn vitro cell experiments in this study show that TMP can inhibit the cell proliferation,metastasis and invasion of renal clear cell carcinoma A498 cell,indicating that TMP can inhibit the metastasis of renal clear cell carcinoma and the ability to form distant lesions.In addition,in vivo animal experiments in nude mice by subcutaneous implantation of tumor models and tail vein injection in vivo tumor formation model,we found that mice with the treatment of TMP can significantly inhibit the subcutaneous tumor formation and metastasis in nude mice,which is consistent with the cell experiments.Western blot result shows that with the increase of concentration and time of administration,TMP can inhibit the expressions of N-cadherin,Snail and Slug,and the occurrence of EMT,thus inhibiting the proliferation,metastasis and formation of distant lesions of renal clear cell carcinoma.