The Effects And Mechanisms Of CD147 On Glycolysis In Malignant Melanoma

BackgroundGlycolysis is increased in tumor cells and the anhanced glycolysis is the essential characteristic of energy metabolism in various tumors, including skin tumors and so on. Lactate, the end product and main metabolite of glycolysis, is excreted from cells through the monocarboxylate transporter (MCT), leading to a decrease in the extracellular pH of tumors and then the enhanced proliferation, invasiveness, metastasis, and angiogenesis of tumor cells. CD147/asigin (CD 147) is an integral plasma membrane protein of the immunoglobulin superfamily, and its expression is increased in various tumor cells. CD 147 and low extrancellular pH have almost exactly the same role on tumorigenesis and tumor development. Previous studies have shown that CD 147 is colocalized with the members of the MCT family and mediates their correct expression on plasma membrane, and might involve in the transport of lactate.Malignant melanoma (MM) is highly malignant because of its rapid growth, advanced stage, and metastatis properties. At 6.4-7.3, the pH of human MM lesions was lower than in normal skin (normal range 7.3-7.6).Human MM cells cultured at acidic pH exhibited enhanced invasiveness and the metastatic potential of experimental pulmonary tumors in an athymic nude mice was increased at acidic pH. The aim of the present study was to determine that whether CD 147, by its close association with MCTs, plays a pivotal role in the glycolysis and provide new insights into possible prevention or progression of cancers such as MM.Objective1. To investigate whether CD 147 is involved in glycolysis to transport lactate.2. To investigate the mechanism of CD 147 involving in glycolysis.3. To investigate whether CD 147 involving in glycolysis contributes to the progression of A375 melanoma cells.Methods1. Western Blotting was used to determine the expression of CD 147, MCT1, and 4 on the Normal Human Melanocyte and A375 melanoma cells.2. The colocalization of MCT1 and 4 with CD 147 in A375 cells was detected by Confocal immunofluorescence microscopy.3. Specific siRNA targeting CD147mRNA was used to knock down CD 147 expression on A375 cells.4. Western Blotting was used to determine the expression of CD 147, MCT1 and 4 on A375 melanoma cells after CD 147 siRNA transfection.5. The colocalization of MCT1 and 4 with CD 147 in A375 cells with CD 147 siRNA transfection was detected by Confocal immunofluorescence microscopy. 6. To determine the lactate concentration, the A375 cells with or without CD 147 siRNA transfection were preincubated with oligomycin. After replacing the culture medium with medium supplemented with mitochondrial H+-ATP synthase inhibitor. The samples were used for the enzymatic determination of lactate.7. The extracelluar pH value of A375 cells with or without CD 147 siRNA transfection was measured with an electronicpH meter.8. ATP levels were measured of A375 cells with or without CD 147 siRNA transfection by the luciferin-luciferase method using an ATP determination Kit.9. CD 147 siRNA transfected A375 cells with or without exogenous lactate were used for the following experiments:MTT assay was used to determine cell proliferation; the in vitro invasion assay was performed using a Boyden chamber with a Matrigel-coated membrane; quantitative evaluation of VEGF in cell culture supernatants was with ELISA; reverse transcription-PCR analysis was used to detect the VEGF mRNA level.Results1. NHMC did not express CD 147. Its expression was remarkably enhanced in A375. Expression of MCT1 and 4 was also up-regulated in A375 compared with NHMC.2. Both MCT1 and MCT4 co-localized with CD 147 on the plasma membrane of A375 cells.3. Two new A375 clones C1 and C2 were established that effectively and stably inhibited the CD 147 expression using pSUPER/CD147siRNA. pSUPER-vector was used as control.4. MCT1 and MCT4 protein expression was significantly decreased both in whole celllysates and in the membrane fraction of C1 and C2, compared with A375 cells.5. The extracellular lactate concentration was significantly increased in A375 cells compared with NHMC in which lactate was barely detectable. Compared to A375 cells, in C1 and C2 it was remarkably reduced to 27.2%and 22.4%(p＜0.001), respectively, by CD 147 silencing. A375-vector showed no significant difference in lactate concentration compared with A375 cells,6. Extracellular pH was lower in A375 (7.18) than in NHMC and restored by CD147 silencing in C1 (7.36) and C2 (7.34).7. Compared to A375 cells, ATP levels were drastically reduced in C1 and C2 to 60.8% and 51.9%(p<0.001), respectively, by CD 147 silencing.8. Compared with A375 cells, the proliferation and invasiveness were significantly inhibited and the VEGF and mRNA level were reduced in C1 and C2. The proliferation, invasiveness, and VEGF were recovered by incubation with 20 mM lactate in C1 and C2. Conclusions1. CD 147 enhanced the glycolytic activity of malignant melanoma.2. CD 147, by its close association with MCT1 and MCT4, plays a pivotal role in the glycolysis reflected by the transmembrane transport of lactate.3. CD 147 maybe enhanced the proliferation, invasiveness, and angiogenesis of tumors cells via lowering the environmental pH.