Comparative Study On The Effect Of Cool And Hot Compounds On Biological Behavior Of Tumor Based On Tumor Metabolism

PurposeTo study the difference between the effect of compounds characteristic of cool and hot on biological behaviors of tumor and its potential mechanisms.Research Contents and MethodsⅠ To study the effect of compounds characteristic of cool and hot on biological behaviors of malignant tumor.In vivo experiment1)Mice experimental lung metastasis model was used to study the effect of capsaicin and menthol on B16F10 melanoma lung metastasis;2)Nude mice subcutaneous transplantation tumor model was used to study the effect of capsaicin and menthol on tumor growth and lung metastasis of A3 75 melanoma cells.In vitro experiment1)MTT was used to study the effect of capsaicin and menthol on proliferation of B16,B16F10 and A375 melanoma cells in vitro.2)Wound healing was used to study the effect of capsaicin and menthol on migration of B16,B16F10 and A375 melanoma cells in vitro.3)Transwell experiment was used to study the effect of capsaicin and menthol on invasion of B16,B16F10 and A375 melanoma cells in vitro.Ⅱ Mechanism study on the effect of compounds characteristic of cool and hot on biological behaviors of malignant tumor.1)Study on the metabolic types of B16F10 and A375melanoma cells.Glycolysis inhibitor 3-bromopyruvate and oxidative phosphorylation inhibitors oligomycin combined with MTT were used to study the metabolic types of B16F10 and A375melanoma cells.2)Study on the effect of capsaicin and menthol on the basic process of glycolysis of melanoma cells.Glucose consumption detection kit,lactic acid determination kit and ATP detection kit were used to study the effect of capsaicin and menthol on glucose consumption,generation of lactic acid and ATP.3)Study on the effect of capsaicin and menthol on the enzyme activities of three key kinases in glycolysis.Hexokinase(HK)enzyme activity assay kit,Fructose-6-phosphate kinase(PFK)enzyme activity test kits and Pyruvate kinase(PK)enzyme activity determination kit were used to study the effect of capsaicin and menthol on such above three key kinases.4)Study on the protein and mRNA expressions of TRPV1 and TRPM8.Western blot and Q-PCR methods were used to detect the protein and mRNA levels of TRPV1 and TRPM8 receptors in B16,B16F10 and A375 melanoma cells.Another two normal skin cell lines HACAT and JB6 were also examined.5)To determine whether the effect of capsaicin and menthol on the enzyme activities of three key kinases in glycolysis were receptors-dependent.TRPM8 inhibitor clotrimazole and TRPV1 inhibitor ruthenium red were used combined with HK and PK enzyme activity detection kits,to study whether the regulation of enzyme activity of HK by menthol was TRPM8 dependent and whether the regulation of enzyme activity of PK by capsaicin was TRPV1 dependent?ResultsIn vivo experiment results:1)B16F10 melanoma experimental lung metastasis model showed that capsaicin promoted the lung metastasis of B16F10 while menthol inhited it.Nude mice subcutaneously transplanted tumor model showed that both capsaicin and menthol inhibited the growth of A3 75 melanoma.Both capsaicin and menthol had no obvious effect on body weight of nude mice during the experiment,which indicated that long-term use of capsaicin and menthol had no system toxicity.HE results also showed that capsaicin promoted the lung metastasis of A3 75 melanoma while menthol inhibited it.In vitro experiment results:2)The experimental results showed that both capsaicin and menthol inhibited the proliferation of melanoma cells with a dose-dependent manner in certain concentrations;Scratch experiments showed that capsaicin promoted lateral migration of A3 75 and B16F10 melanoma cells while menthol palyed an opposite effect on them.Transwell experiments showed that capsaicin promoted the invasion of both A375 and B16F10 melanoma cells while menthol exerted an opposite effect.Both capsaicin and menthol showed no significant effect on migration and invasion of B16 melanoma cells.3)By using the oxidative phosphorylation inhibitors oligomycin combined with MTT,we found that the proliferation of both A3 75 and B16F10 were slight suppressed after inbition of oxidative phosphorylation.However,using of glycolysis inhibitor 3-bromine pyruvic acid combined with MTT,we found that the proliferation of both A375 and B16F10 were significant inhibited after suppresion of glycolysis.4)Capsaicin promoted the glucose consumption of melanoma cells while menthol inhibited it;capsaicin promoted the generation of lactic acid while menthol inhibited it;capsaicin promoted the generation of ATP while menthol inhibited it.5)Capsaicin promoted the enzyme activity of PK but had no effect on PFK and HK while menthol inhibited the enzyme activity of HK but had no obvious effect on PFK and PK.6)Considering TRPV1 and TRPM8 as the direct receptors of capsaicin and menthol,we then examined the protein expression of such two proteins in B16F10,A375 and B16 melanoma cells,we also examine another two normal skin cell lines,i.e,JB6 and HACAT cells.The results showed that the expression of both TRPV1 and TRPM8 were higher compared to that of the normalcells.Also,the results of Q-PCR showed that the mRNA level of TRPV1 and TRPM8 in melanoma cells were higher than in normal cells.Then TRPV1 inhibitor ruthenium red and TRPM8 inhibitor clotrimazole were used combined with scratch experiment,and the results showed that the migration of melanoma cells was significant inhibited after inhibition of TRPV1 while the migration of melanoma cells was significant promoted after inhibition of TRPM8.7)TRPM8 inhibitor clotrimazole and TRPV1 inhibitor ruthenium red were used combined with HK and PK enzyme activity detection kits,to study whether the regulation of enzyme activity of HK by menthol was TRPM8 dependent and whether the regulation of enzyme activity of PK by capsaicin was TRPV1 dependent?The results showed that the inhibition effect on enzyme activity of HK by menthol disappeared when inhibited TRPM8.Also,the promotion effect on enzyme activity of PK by capsaicin disappeared when inhibited TRPV1.Collectively,the above results indicated that the regulation of enzyme activity by capsaicin and menthol were both receptor-dependent.Conclusions and SignificanceThis project focused on the effect of cold-heat nature of compounds on biology behavior of melanoma.Meanwhile,the potential mechanisms underlying capsaicin and menthol in regulation of melanoma metastasis based on tumor metabolism.The conclusions are as following:(1)Capsaicin promotes migration and invasion of melanoma cells in vitro and promotes melanoma metastasis in vivo.Menthol inhibits migration and invasion of melanoma cells in vitro and inhibits melanoma metastasis in vivo.(2)Energy metabolism type of both B16F10 and A375 are glycolysis.Capsaicin promotes the process of glycolysis while menthol inhibited such process.(3)Of all three key kinases mediating glycolysis,capsaicin promotes the enzyme activity of PK while menthol inhibits the enzyme activity of HK.(4)The regulation of enzyme activity of HK by menthol is TRPM8-dependent while the regulation of enzyme activity of PK by capsaicin is TRPV1-dependent.This project selected menthol and capsaicin as cold-heat nature of compounds,respectively.We try to clarify the difference between cold and hot foods or even cold and hot TCM on disease progression of tumor patients,which would be helpful in clarifying the effect of cold and hot foods on tumor progression,also it will be help to promote the in-depth study on TCM as well as promote the standardization of clinical Chinese medicine prescription.