Baicalin Inhibits Metastasis Of Melanoma By Targeting TRPV4

Bcakground and PurposeTumor metastasis is the leading cause of tumor death.At present,the most commonly used tumor treatment strategies in clinical practice are surgical resection,radiotherapy and chemotherapy,molecular targeted inhibitors,and biopharmaceutical therapy.Although these treatments can achieve the purpose of inhibiting tumor growth or even eliminating in situ tumors,a large amount of data indicates that Chemotherapy-based tumor therapy does not significantly improve the survival of cancer patients.The cause of this phenomenon,on the one hand,is due to the low immunity of cancer patients caused by radiotherapy and chemotherapy;on the other hand,tumor therapy stimulation promotes the occurrence of tumor metastasis.Tumor metastasis refers to tumor cells in situ tumors or tumors that are dormant after surgical resection into blood vessels and lymphatic vessels through blood transfer and lymphatic metastasis,reaching metastases and forming secondary tumors.Inhibition of tumor metastasis may enable tumor-bearing survival in tumor patients and promote long-term survival of cancer patients.However,there is currently no drμg targeting tumor metastasis.Transient receptor potential vanilloid receptor 4(TRPV4)is a kind of cation channel that can be activated by various physiological stimuli.Previous studies have shown that TRPV4 can promote tumor metastasis in some tumors.The role,and some natural small molecule compounds can inhibit tumor metastasis by acting on the TRPV4 channel.The focus of this study was to screen for small molecule compounds that act on the TRPV4 channel and to explore the mechanisms by which the TRPV4 channel is targeted to inhibit tumor metastasis.Research Contents and ResultsThis study first demonstrated the association of TRPV4 with tumor metastasis and determined the type of cancer studied by using a network database search.The study found that gastric cancer,lung adenocarcinoma,breast cancer,ovarian cancer,melanoma and other tumor patients had a significant negative correlation between survival and TRPV4 expression.In the Cancer Cell Line Encyclopedia database,the melanoma cell line TRPV4 was the most highly expressed,and the TCGA-derived data showed the highest expression of TRPV4 in melanoma and esophageal cancer.In addition,the HPA database included immunohistochemical staining by professional personnel.In the figure,12 patients with TRPV4 and 5 normal subjects were found to have consistently high expression of TRPV4 in melanoma cells at the metastatic site.The high expression of TRPV4 in melanoma was confirmed by Western Blot,Real-time PCR and immunofluorescence.In addition,the expression level of TRPV4 was also closely related to the survival of melanoma patients.Then explore the role of TRPV4 in the process of tumor metastasis.Firstly,the expression level of TRPV4 in A375 metastatic tumors was significantly higher than that in subcutaneous xenografts by immunohistochemistry.TRPV4 knockout cell lines were constructed using CRISPR/Cas9 technology,and in vitro and in vivo experiments demonstrated that TRPV4 gene knockdown inhibits melanoma metastasis.Since the expression of TRPV4 is related to the metastatic ability of melanoma,and TRPV4 is a type of non-selective cation channel,the biological effect is based on open channel,and calcium imaging experiments demonstrate the expression of TRPV4 and calcium influx caused by channel opening.The effect is proportional.Therefore,for melanoma with a high TRPV4 expression rate,it may be possible to inhibit melanoma metastasis by inhibiting TRPV4 channel opening.Next,it is experimentally explored whether inhibition of TRPV4 channel opening can inhibit tumor metastasis.First,TRPV4 positive agonist GSK1016790A and antagonist HC067047 were used,and concentration screening was performed by a proliferation inhibition experiment.Calcium imaging experiments were performed using concentrations that did not inhibit cell proliferation.The results of the experiment demonstrated that GSK1016790A can activate the opening of TRPV4 channels,while HC067047 can antagonize the agonistic effects of GSK1016790A.Scratch experiments and Transwell experiments demonstrated that antagonism of TRPV4 channel opening can inhibit tumor metastasis.In order to investigate whether the traditional Chinese medicine component acts to inhibit tumor metastasis by acting on the TRPV4 channel,component screening was carried out.The screening of common anti-cancer Chinese medicines was screened by virtual screening experiments of structural similarity fitting of compounds.The calcium imaging experiments confirmed that the selected compound baicalin can antagonize the opening of TRPV4 channels.It was confirmed by cell thermal conversion analysis and drug affinity analysis experiments that baicalin can bind to TRPV4 protein.Transwell cell migration experiments,inverted invasion,scratches and other experiments have demonstrated that baicalin has a pharmacological effect of inhibiting tumor migration.It was also concluded that baicalin can inhibit the metastasis of melanoma by nude mice and C56BL6/N mouse tumor metastasis model experiments.In order to further explore the specific mechanism of baicalin and HC067047 antagonizing TRPV4 channel to inhibit tumor metastasis,baicalin was firstly excluded from A375 cell apoptosis and cell cycle arrest.Experiments by ATP assay showed that TRPV4 may promote metastasis by promoting intracellular energy levels,promoting cell viability,and improving tumor migration ability.Therefore,we suspect that the main link in the role of TRPV4 is tumor migration and invasion.In the process of invasion and exudation of tumor metastasis,Cortactin is essential for the generation of pseudopods.Immunohistochemistry results show that the phosphorylation level of Cortactin in A375 metastases is higher than that in in situ tumors,also shown in the TRIP database.TRPV4 interacts with the Cortactin upstream protein SRC kinase.The results showed that the phosphorylation level of Cortactin was significantly decreased in TRPV4-knockout A375 cells,and the use of baicalin and TRPV4 antagonists significantly reduced the phosphorylation of Cortactin in A375 cells.Compared with A375 cells,A375M cells with higher metastatic ability have higher FAK expression levels and higher SRC kinase phosphorylation levels,while high levels of SRC kinase phosphorylation,phosphorylation of Cortactin and FAK expression can be affected by baicalin,HC067047 was reversed and also exhibited low levels in A375 TRPV4 KO cells.CDC42 is also regulated by SRC and associated with cytoskeletal assembly.The results show that baicalin and HC067047 can significantly reduce the expression of RHOA,CDC42 and other proteins.Cofilin is a functional protein capable of depolymerizing the backbone network regulated by SRC kinase and intracellular calcium ions.When it is phosphorylated,its protein function is inhibited.The results show that TRPV4 agonists can inhibit the phosphorylation of Cofilin,while TRPV4 antagonizes The agent can promote the phosphorylation of Cofilin and inhibit the production of pseudopodia.The expression level of Cofilin at the edge of cell movement is an indicator of the ability of cell motility.It has been found that the use of baicalin and HC06747 can inhibit the recruitment of Cofilin to the cell edge of cells,thereby inhibiting cell movement.Conclusion and SignificanceBased on the above results,we first elucidated that antagonism of TRPV4 can inhibit tumor metastasis.Moreover,through the method of virtual screening and screening of active experiments,it was determined that baicalin can act on the TRPV4 channel to inhibit tumor metastasis,and further investigation revealed that TRPV4 antagonist may inhibit Src-Cortactin and CDC42-The Cofilin pathway inhibits microfilament microtubule assembly to inhibit tumor metastasis.