6-Shogaol Downregulate PKM2 Expression To Inhibit The Proliferation And Metastasis Of Melanoma Cells

[Background and Purpose]Tumor cells under anoxic and aerobic conditions, are primarily through the glycolytic pathway of metabolism, this is known as aerobic glycolysis, the famous " Warburg Effect". Modern studies have shown that aerobic glycolysis is one of the characteristic phenotype of tumor cells, and the study found that the highly activated glycolysis could promote the proliferation and metastasis of tumor cells.Numerous studies have confirmed that the Warburg Effect of tumor cells and key enzymes in glycolysis pathway is closely related to the abnormal activation of the pyruvate kinase (PK) is the most prone to abnormal glycolytic pathway of one of the key enzymes. Modern research proves the main expression in tumor cells of type 2 pyruvate kinase (PKM2), the excessive activation of promote glycolysis of tumor and tumor proliferation and metastasis. Existing research shows that melanoma is highly active glycolytic activity, and high expression of PKM2 closely associated with melanoma metastasis and poor prognosis, prompt PKM2 is probably inhibit melanoma metastasis and effective potential targets for the treatment of melanoma.6-Shogaol (6-ShogaoL 6-SHO) is one of the traditional Chinese medicine rhizoma zingiberis alkyl phenolic compound, is one of the main active component in ginger, the early stage of the laboratory study,6-Shogaol can inhibit melanoma cell proliferation, and melanoma cells glycolysis has certain inhibition effect, therefore, we will delve into this phenomenon, whether 6-Shogaol inhibit the proliferation and metastasis of melanoma by inhibiting the glycolysis of melanoma, to further reveal the 6-Shogaol the molecular mechanisms of resistance to melanoma.[Experimental Approach]The first part of study from the level of cells in vitro, determined by MTT, scratch experiment, transwell little room experiment, inspected the 6-Shogaol to melanoma cell proliferation, migration and invasion. The second part firstly determined by MTT experiment, glucose uptake, lactic acid generated examines the melanoma cells and normal skin sensitivity of glycolysis inhibitors and oxidative phosphorylation inhibitor and glycolysis level, preliminary judgment melanoma metabolic phenotype; Then by glucose uptake, lactic acid, producing experiment examined 6-Shogaol on melanoma cell glycolysis level. The third part probes the mechanism of pyruvate kinase by western blot and immunofluorescence looked at 6-Shogaol of PKM2 protein levels and the influence of nuclear transfer, at the same time, using western blot is investigated within 6-Shogaol of PKM2 nuclear regulatory protein STAT3 protein expression and the influence of nuclear transfer, and the downstream target protein STAT3:Cyclin Dl, the Bcl-xL, MMP-2 and MMP-9, N-cadherin, E-cadherin expression is verified. Metastases to the fourth part from the body, subcutaneous transplantation tumor two models to verify the 6-Shogaol’s influence on the growth and metastasis of melanoma, and through immunohistochemical study 6-Shogaol PKM2 in tumor metastases and transplantation, the influence of p-STAT3 expression, further validation of 6-Shogaol effect of proliferation and metastasis of melanoma.[Research results]The first part of the determined by MTT results showed that 6-Shogaol can dose dependent and time dependent inhibit melanoma cells (B16F10, A375) proliferation. Scratch the experimental results show that 6-Shogaol can to a certain extent the level of inhibition of melanoma cell migration; Transwell experimental results show that 6-Shogaol can inhibit melanoma cells invasion. The second part, first of all, examines the melanoma and normal skin cell metabolism phenotype, according to the results of melanoma cells more sensitive to glycolysis suppression, is not sensitive to oxidative phosphorylation inhibitors, and glycolysis were significantly higher than that of normal skin cells, preliminary judgment melanoma cells is biased towards glycolysis metabolic phenotype. Application 6-Shogaol after the intervention, found that 6-Shogaol to a certain extent, inhibit melanoma cells glycolysis level of glucose uptake, lactic acid and enzyme activity at the same time it was found that 6-Shogaol in glycolytic pathway of pyruvate kinase has certain inhibition effect, pyruvic acid and pyruvate kinase downstream products, ATP has inhibition effect, further verified the 6-Shogaol on pyruvate kinase inhibitory effect. The third part, according to the results of PKM2 expression is significantly higher than normal skin melanoma cells, the height of the tip PKM2 in melanoma cells activation. After application of 6-Shogaol, to a certain degree of inhibition of PKM2 protein expression; Also found that 6-Shogaol to reverse PKM2 agonist to melanoma cell migration and invasion of promotion effect; And within the PKM2 nucleus expression has inhibitory effect. Further experiments found that 6-Shogaol on the STAT3 phosphorylation of is regulated by the PKM2 inhibition effect, and inhibit the STAT3 phosphorylation level, also have inhibition effect to downstream target protein STAT3. Metastases to the fourth part of the body, subcutaneous transplantation tumor experimental results show that 6-Shogaol on the growth and metastasis of melanoma has certain inhibition effect, and can inhibit the metastatic tumor and subcutaneous transplantation tumor of PKM2 and p-STAT3, from the body to further validate the 6-Shogaol inhibition effect on the proliferation and metastasis of melanoma.[Conclusion and significance]This topic of the experimental results show that the main active substance in the 6-Shogaol can inhibit tumor glycolytic pathway of PKM2, reduce the glycolysis process of melanoma cells, inhibit melanoma proliferation; At the same time, 6-Shogaol inhibit STAT3 activation, and inhibit the nuclear PKM2 transposition, then inhibit nuclear activation of STAT3 in further, thus inhibiting the melanoma cell migration and invasion, exert its effect of anti proliferation and metastasis of melanoma.This study first revealed the 6-Shogaol resistance to the effect of the proliferation and metastasis of melanoma, and preliminary from the interference melanoma glycolysis level expounds 6-Shogaol resistance to the mechanism of the proliferation and metastasis of melanoma. Also to study traditional Chinese medicine, especially drug edible kind traditional Chinese medicine (TCM) by interfering with the glycolysis process develop its antitumor effect of tumor provides a new research idea and starting point.