In the GLUT family glucose transporter GLUT is
In the GLUT family, glucose transporter 1(GLUT1) is primarily responsible for basal glucose uptake and maintenance of glucose basal metabolism in cells (Olson and Pessin, 1996). High GLUT1 expression occurred in various types of human cancers, like brain tumor, lung cancer and esophageal cancer (Nishioka et al., 1992; Sasaki et al., 2012; Mu et al., 2007). The level of GLUT1 expression is associated with malignant characteristics of cancer cells. GLUT1 plays an important role in tumor cell growth and proliferation (Yan et al., 2015). In malignancy of brain tumors, the main metabolic substrate of brain cells is indeed glucose. Previous studies demonstrated that GLUT1 blockage or inhibition weakened the malignant phenotypes of tumor cells and enhanced drug sensitivity of tumor cells (Matsushita et al., 2012; Liu et al., 2014; Chan et al., 2011; Zhou et al., 2009). For example, when NB cell was treated with 3-bromopyruvate M344 synthesis (3-BrPA), GLUT1 expression was downregulated and the proliferation of NB cell was also obviously reduced (Matsushita et al., 2012). Whereas GLUT1 overexpression in colon cancer cells was associated with tumor cell resistance to 5-Fu and WZB117, a selective GLUT1 inhibitor, could reverse the 5-Fu resistance by inhibition of GLUT1 expression (Liu et al., 2014). Moreover, after reducing GLUT1 expression by STF-31 or RNA interference, renal cell carcinoma cells underwent necrosis (Chan et al., 2011). Laryngeal carcinoma cells showed inhibition of glucose uptake and proliferation through reducing GLUT1 expression (Zhou et al., 2009).
Materials and methods
Discussion In the ischemia anoxic environment, NB just like other malignant tumors, imported a large amount of extracellular glucose into cells by increasing in expression of glucose transporters. Therefore, to enhance glycolysis in response to hypoxic, low sugar and ischemia conditions (Abdul Muneer et al., 2011). Indeed, cancer cells appear to be “addicted” to and heavily rely on glucose and glycolysis for their growth and survival, in which tumor cells could obtain both energy and building materials to facilitate cell growth needs (Heiden et al., 2009). ATP produced by glycolysis is a major source of energy for the survival of cancer cells and in this regard, cancer cells are so sensitive than normal cells to changes in glucose concentration. Thus, it could help us to cure cancers by controlling glucose supply and glycolysis (Pelicano et al., 2015). In this study, we assessed the effects of a selective GLUT1 inhibitor WZB117 on regulation of NB cell line SH-SY5Y viability, cell cycle and glycolysis in vitro. GLUTs are the main carriers of glucose uptake into cells. In the GLUT family, GLUT1 is responsible for primary glucose uptake and maintenance of basal glucose metabolism in cells (Takata et al., 1990). Previous studies showed that GLUT1 activity inhibition by WZB117 effectively reduced glycolysis and cell proliferation, induced cell-cycle arrest in laryngeal carcinoma and lung cancer (Zhou et al., 2009; Liu et al., 2012). In the current study, we extended to assess the effects of WZB117 on NB cells. WZB117 treatment suppressed level of GLUT1 protein, although GLUT1 mRNA level was induced. This could be explained as a compensatory effect for cells to meet the energy needs for growth in the context of reduced glucose supply. In other words, WZB117 inhibition of GLUT1 decreased glucose supply in cancer cells, resulting in an urgent need to bring glucose level to “normal state”. Thus, level of GLUT1 mRNA upregulated. However, Level of GLUT1 protein did not increase, which is because of the limited supply of glucose required for the processing of glycosylated membrane-bound proteins, including GLUT1 (Liu et al., 2012). However, future study of the underlying gene regulations will provide precise details about GLUT1 regulation in cells. Furthermore, the proliferation was significantly inhibited after NB cells was treated with WZB117 for 24 h and further inhibited more at 48 h and 72 h. The cause is that inhibiting the glucose transporters cut off the energy input in tumor cells. Therefore, growth of NB cells was reduced. Moreover, WZB117 treatment arrested approximately 19% cells at the G0–G1 phase of the cell cycle at 24 h, while 36% at 48 h and 46% at 72 h. In order to explore the mechanism of the cell cycle arrest, we analyzed expression of CDK2, cyclin E2, pRb and p53 proteins in NB cells after WZB117 treatment and found the decline of CDK2, cyclin E2 and pRb expression, but p53 protein upregulated. P53 is the first confirmed tumor suppressor gene involved in cell growth, differentiation and death. P53 induces apoptosis by activating transcription of certain genes, such as Bax, Fas. It also can regulate the G1 phase of the cell cycle by regulating the transcription of its downstream effector gene, such as CIP1/WAF1 (Vogelstein et al., 2000; Gottlieb and Oren, 1998). Cyclin and cyclin-dependent kinase (CDK) are key macromolecules in cell cycle regulation. Cyclin E2 binds with CDK2 to form cyclinE-CDK2 complex, which is the key kinase complex from G1 to S phase (Krude et al., 1997; Dulic et al., 1992). Several reports have shown that pRb is a major G1 checkpoint, blocking S-phase entry and cell growth, which is regulated by cyclin E2-CDK2 complexes (Giacinti and Giordano, 2006; Navins, 2001). Thus, the possible reason of cell cycle arrest at the G1 phase in this study is that increase of p53 and reduction of CDK2, cyclin E2 and pRb. Similar experimental results have been reported in other studies (Zhou et al., 2009; Rastogi et al., 2007; Oh et al., 2017). For example, knockdown of GLUT1 by GLUT1 shRNA in triple-negative breast cancer cells resulted in cell cycle arrest at the G1 phase and decrease in cell proliferation. Moreover, the migration and invasion were inhibited by modulation of the EGFR/MAPK and integrin β1/Src/FAK signaling pathways (Oh et al., 2017).