Cancer Letters

Cancer Letters

Volume 273, Issue 1, 8 January 2009, Pages 140-147
Cancer Letters

A CD147-targeting siRNA inhibits the proliferation, invasiveness, and VEGF production of human malignant melanoma cells by down-regulating glycolysis

https://doi.org/10.1016/j.canlet.2008.07.034Get rights and content

Abstract

Cancer cells require glycolysis for energy; this results in excessive lactate production and secretion. Lactate, the end product of glycolysis, reduces the extracellular pH and contributes to the proliferation, invasiveness, metastasis, and angiogenesis of tumor cells. Our previous results revealed that the over-expressed CD147/basigin plays a critical role in malignant melanoma (MM) invasiveness, metastasis and angiogenesis; CD147 has also been implicated in a specific and strong interaction with monocarboxylate transporters (MCT) 1 and 4 that mediate the transport of lactate. In the present study, we investigated whether CD147/basigin is involved, via its association with MCT1 and 4 to transport lactate, in glycolysis and then contributes to the progression of A375 melanoma cells. A375 cells expressed remarkably higher CD147, MCT1 and 4 and showed increased glycolysis rate compared with normal human melanocytes (NHMC). CD147/basigin co-localized with MCT1 and 4 in the A375 cell membrane. Furthermore, silencing of CD147/basigin in A375 cells by a siRNA clearly abrogated the expression of MCT1 and 4 and their co-localization with CD147/basigin and dramatically decreased the glycolysis rate, extracellular pH, and the production of ATP. Thus, cell proliferation, invasiveness, and VEGF production were significantly decreased by siRNA. These results strongly suggest that highly-expressed CD147 interacts with MCT1 and 4 to promote tumor cell glycolysis, resulting in the progression of MM.

Introduction

Glycolysis, promoted by hypoxic stimuli or injury to the mitochondrial respiration, is the result of an anaerobic enzymatic conversion of glucose to pyruvate or lactate to produce energy stored in the form of adenosine triphosphate (ATP) [1], [2]. It is enhanced in tumor cells and the extracellular pH is lower in tumors than in normal tissue [3]. This is considered to be the consequence of an excessive efflux of lactate, the end product of glycolysis. In 1950’s, Warburg [4] first reported that cancer cells depend on glycolysis for energy and that this results in excessive levels of lactate even in the presence of oxygen. Meienhofer et al. [5] suggested that tumor cell growth and survival depend on the switch from anaerobic to aerobic glycolysis. Tumor cells become addicted to aerobic glycolysis for several reasons including the increased demand for rapid energy production and for the metabolites of glycolysis to support malignant behavior. Lactate is excreted from cells through an H+/lactate co-transporter, the monocarboxylate transporter (MCT), leading to a decrease in the extracellular pH of tumors [6]. Tumor acidity produces a more aggressive phenotype of cancer cells with a number of altered behaviors. Turner [7] first proposed the role of acidic pH in tumor metastasis. Low pH treatments clearly induced experimental metastasis [8], [9] and the invasive behavior of tumor cells [10], [11], [12]. In addition, low pH contributed to the in vivo induction of VEGF expression in brain tumors [13] and human pancreatic adenocarcinoma [14].

CD147/basigin (CD147), an integral plasma membrane protein of the immunoglobulin superfamily, is expressed widely on many cell types and its expression is increased in various tumor cells [15], [16]. Elevated CD147 stimulates matrix metalloproteinase (MMP)-production in stromal fibroblasts and endothelial cells, leading to extracellular matrix degradation, tumor growth promotion, and metastasis [17], [18], [19]. CD147 also stimulates the expression of vascular endothelial growth factor (VEGF) [19], [20]. Interestingly, CD147 associates closely with two members of the proton-coupled MCT family, MCT1 and 4 [21] and acts as an essential chaperone for their correct plasma membrane expression and catalytic activity [21], [22]. MCT1 and 4 catalyze the proton-linked transport of monocarboxylates, of which lactate is metabolically the most important. The rapid transport of lactate by MCTs is of critical importance for almost all cells, especially tumor cells with elevated glycolysis, resulting in a decrease in extracellular pH.

Malignant melanoma (MM) is highly malignant because of its rapid growth, advanced stage, and metastatic properties [23]. We previously showed that CD147 expression was enhanced in MM cells and that CD147 played an important role in tumor proliferation, the production of MMPs, angiogenesis, invasiveness, and metastatic activity in vitro and in vivo [18], [19]. Because the role of CD147 on tumor progression is similar to that of low extracellular Ph and CD147 is closely associated with MCT1 and 4, we hypothesized that it is involved in MM glycolysis and in the transport of lactate across the plasma membrane as well as the changes in the behavior of MM cells. Therefore, after knocking down the expression of CD147 in an MM cell line with siRNA we examined the co-localization of CD147 with MCT1 or 4 on the MM cell membrane by confocal microscopy and assayed the glycolysis rate, extracellular pH, ATP synthesis, and the tumor cellular activities. We found that the association between CD147 and MCT1 or 4 on the plasma membrane of MM cells was remarkably weakened and that the level of lactate and ATP were decreased. The environmental pH and cellular activities were restored after inhibition of CD147 in A375 cells. Our findings suggest that its ability to lower the pH of the microenvironment, resulting in the enhanced proliferation, invasiveness, metastasis, and angiogenesis of tumor cells, may be the fundamental property of CD147 that affects tumorigenesis and tumor development.

Section snippets

Cell culture

The human MM cell line A375, normal human fibroblasts (NHFBs), and normal human melanocytes (NHMCs) were preserved in our laboratory (Kagoshima University). A375 cells and NHFBs were grown in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution (Invitrogen) and incubated at 37 °C in a humidified atmosphere containing 5% CO2. NHMCs were maintained in Ham’s F-10 (Sigma), supplemented with 5% FBS, 20 μg/mL bovine pituitary extract (Sigma), 50 ng/mL TPA

Enhanced expression of CD147 and MCT1 and -4 in human MM cell line A375

As shown in Fig. 1, Western blotting demonstrated that CD147 expression, not detected in NHMCs, was enhanced in A375 cells. The expression of MCT1 and 4 was also up-regulated in A375 cells compared with NHMC.

siRNA-mediated silencing of CD147 expression

Specific siRNA targeting CD147 mRNA was used to knock down CD147 expression. We established two new clones C1 and C2 that effectively and stably inhibited the CD147 expression using pSUPER/CD147 siRNA1, but not pSUPER/CD147 siRNA2 that were accordant to our previous report (data not shown)

Discussion

This study showed that the expression of CD147, MCT1, and 4 was enhanced and that CD147 and MCT1 or 4 co-localized on the plasma membrane of A375 MM cells. Silencing of CD147 by means of siRNA technology down-regulated the expression of MCT1 and 4 and abrogated the co-localization of CD147 with these MCTs. It also demonstrated that glycolysis was activated and extracellular pH was decreased in A375 cells compared with NHMC and that the glycolysis rate, extracellular pH and the production of ATP

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China; Program for New Century Excellent Talents at University; The Hunan Science Fund for Distinguished Young Scholars. J. Su was the recipient of a grant from Jiaikai Foundation, Kagoshima, Japan.

References (50)

  • R.J. Gillies et al.

    MR imaging of the tumor microenvironment

    J. Magn. Reson. Imag.

    (2002)
  • O. Warburg

    On respiratory impairment in cancer cells

    Science

    (1956)
  • M.C. Meienhofer et al.

    Regulation of genes for glycolytic enzymes in cultured rat hepatoma cell lines

    Eur. J. Biochem.

    (1978)
  • R.A. Cardone et al.

    The role of disturbed pH dynamics and the Na+/H+ exchanger in metastasis

    Nat. Rev. Cancer

    (2005)
  • G.A. Turner

    Increased release of tumour cells by collagenase at acidic pH: a possible mechanism for metastasis

    Experientia

    (1979)
  • O.K. Schlappack et al.

    Glucose starvation and acidosis: effect on experimental metastasic potential, DNA content and MTX resistance of murine tumour cells

    Br. J. Cancer

    (1991)
  • D.C. Bennett et al.

    Experimental metastasis and differentiation of murine melanoma cells: actions and interactions of factors affecting different intracellular signalling pathways

    Clin. Exp. Metast.

    (1994)
  • R. Martinez-Zaguilan et al.

    Acidic pH enhances the invasive behavior of human melanoma cells

    Clin. Exp. Metast.

    (1996)
  • J. Rozhin et al.

    Pericellular pH affects distribution and secretion of cathepsin B in malignant cells

    Cancer Res.

    (1994)
  • C. Cuvier et al.

    Exposure to hypoxia, glucose starvation and acidosis: Effect on the invasive capacity of murine tumor cells and correlation with cathepsin (L+B)

    Clin. Exp. Metast.

    (1997)
  • D. Fukumura et al.

    Hypoxia and acidosis independently up-regulate vascular endothelial growth factor transcription in brain tumors in vivo

    Cancer Res.

    (2001)
  • Q. Shi et al.

    Regulation of vascular endothelial growth factor expression by acidosis in human cancer cells

    Oncogene

    (2001)
  • T. Miyauchi et al.

    Basigin, a new, broadly distributed member of the immunoglobulin superfamily, has strong homology with both the immunoglobulin V domain and the H-chain of major histocompatibility complex class II antigen

    J. Biochem.

    (1990)
  • T. Kanekura et al.

    Basigin, a new member of the immunoglobulin superfamily: genes in different mammalian species, glycosylation changes in the molecule from adult organs and possible variation in the N-terminal sequences

    Cell Struct. Funct.

    (1991)
  • C. Biswas et al.

    The human tumor cell-derived collagenase stimulatory factor (renamed EMMPRIN) is a member of the immunoglobulin superfamily

    Cancer Res.

    (1995)
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