Explore The Mechanism Of Hypaconitine On Melanoma Metastasis Through Targeting TRPV4

Background and PurposeSyndrome differentiation of cold and heat is the basic rule of law for TCM to recognize and treat diseases.Clarifying the modern scientific connotation of TCM theory is the key to promoting the modernization and internationalization of TCM.Our previous investigation found that the syndrome of cold and heat still has important guiding significance for the prescription of modern Chinese medicine for clinical tumor treatment.What is the relationship between "cold and hot"herbs and "cold and hot" tumor syndromes?What is its scientific connotation behind?It drives us to pay close attention to the Transient Receptor Potential(TRP)channels super family.Among them,transient receptor potential(TRP)vanillic acid subtype 4(TRPV4)has been reported to play an important role in tumor metastasis and abnormally expressed in melanoma,which is also closely related to patient prognosis.After comparing the cold and hot Chinese medicine active components with TRP agonists/inhibitors,we found that the structure of main active ingredient in Aconitum carmichaelii Debx,hypoconitine had a higher fitting result with TRPV4 agonist GSK1016790A.Further investigations found that "hot" traditional Chinese medicines such as aconite,are mainly used in the treatment of "cold syndrome" tumors such as liver cancer and stomach cancer,but not used in the treatment of skin cancer.What is the effect of hypaconitine on the metastasis of melanoma with high expression of TRPV4,and whether aconite should be used with caution in the clinical treatment of TRPV4 high expression melanoma?This study aims to clarify the effects of hypoconitine on melanoma metastasis,and to explore its possible mechanism through TRPV4,trying to provide new ideas and strategies for clinical use of aconite for tumor treatment in Chinese medicine.Research Contents and ResultsIn this study,the TCGA,Cancer Cell Line Encyclopedia,and The Human Protein Atlas databases were used to identify and select melanoma that overexpress TRPV4 as the main research objects.It is found that the expression of TRPV4 in melanoma cell lines was higher than that of other tumor cells,and the results of immunohistochemistry showed that the expression of TRPV4 was higher in melanoma than that of normal tissues,and the prognosis of patients was negatively correlated with the expression of TRPV4.Western Blot and immunofluorescence were used to verify that human melanoma cell lines(A375,Sk-Mel-24,and Malme-3)had higher TRPV4 expression than human epithelial cells(HaCaT),and it is confirmed that A375 had the highest TRPV4 expression level.A375TRPV4-/-cells were constructed to confirm that aconitine can stimulate TRPV4 and cause calcium influx of A3 75.In vivo and in vitro experiments found that TRPV4 agonists GSK1016790A and hypoconitine can promote the metastasis of melanoma A3 75 through activating TRPV4.Immunofluorescence was used to detect the polarity of A375 cells in a single-cell suspension state after administration of hypaconitine,and to construct Ezrin-EGFP A375 cells for the observe of the tumor cell retention in the liver and lung blood vessels after tail vein injection.At the same time,nude mice were divided into a model group,a hypoconitine low-dose group,and a hypoconitine high-dose group for in vivo experiments.A375 cells that were pre-treated with hypoconitine were injected to observe the polarization caused by hypoconitine.It was found that hypaconitine can promote melanoma A375 metastasis by promoting A375 polarization(single cell polarity).Using immunofluorescence and Western Blot to test the effects of hypaconitine on Ezrin phosphorylation at different concentrations and different intervention times,and immunohistochemical detection of p-Ezrin levels in metastatic sites,we found that the effect of hypaconitine on A375 cell polarization may regulated through promoting Ezrin phosphorylation.The effect of hypoconitine on amoeboid movement of A375 cells was observed by a long-term live cell observation system,and the effects of hypoconitine on Rho/ROCK axis and MLC phosphorylation were detected by immunohistochemistry,immunofluorescence and Western Blot.It is found that hypoconitine can promote melanoma cells to invade surrounding tissues through amoeboid movement through the Rho/ROCK-MLC signal pathway.Subcutaneous tumor-bearing experiments with B16F10 and A375 further verified the effect of Rho/ROCK/MLC signal axis activation on the invasiveness of melanoma cells.Fluorescence-labeled dextran leakage experiments,immunofluorescence,and Western Blot tests found that hypaconitine can cause changes in the secretion characteristics of A375 cells through the Rho/ROCK/MLC signal axis,and ultimately destroy the tight junction of HUVEC in the extravasate process.Conclusion and SignificanceBased on the above experimental results,it was found that the activation of the ROCK/MLC signal axis led to melanoma cells acquiring amoeboid motion characteristics(resulting in increased self-invasiveness)and special secretory properties(resulting in the cytoskeleton remodeling of the endothelial cells and their penetration,so that after it reaches the metastatic site from in situ,it has the advantage of further forming metastases in the distal tissue.It is demonstrated that the activation of the Rho/ROCK pathway promote tumor cells invade to the surroundings,and the adhesion of circulating tumor cells to the vascular endothelium,destroys the tight connection of the vascular endothelium,and then extravasates finally lead the occurrence of distal metastases of melanoma.The results of this study indicate that there is a certain scientific connotation in the use of large doses of radix aconiti carmichaeli in the treatment of "cold tumor".Combining with our previous research results that baicalin,the main active ingredient of the cold herb Scutellariae,can inhibit the metastasis of "hot tumor" melanoma,it is seems that the syndrome of "cold-hot" has scientific connotation.