It is well established that tumor cells remodel their metabolism and energy production through aerobic glycolysis ( Warburg effect )[14]. Recent studies have found, in addition to glucose, that glutamine is also an important nutrient for tumor cells. Some tumor cells are more dependent on glutamine to maintain the energy requirements for their growth, exhibiting cell death follows Gln-deprivation, such phenomenon is defined as " Gln addiction "[15–17]. Gln addiction has long been determined a characteristic of tumor cells. It has been shown that malignant cells such as glioma, lung cancer and kidney cancer were addicted to glutamine. In the present study, we confirmed that liver cancer cells strongly relied on glutamine uptake, which was in line with results of other researches [18, 19]. The inhibitory effect of Gln-deprivation on HCC cells growth is consistent with the results caused by glucose-deprivation. Being an important component of glutamine metabolism, glutaminolysis also plays a critical role in cancer cell metabolism, cell signaling, and cell growth in many cancers, which suggests a critical role of glutamine related downstream metabolites in tumor cells. Our experimental data further demonstrated the necessity of NEAAs and α-KG, two of the main metabolites of glutamine, on HCC cells growth. When HCC cells were supplemented with 0.1 mM exogenous NEAAs or 4 mM α-KG, respectively, the number of dead cells were markedly less than those in condition of Gln-deprivation. Moreover, the cell growth inhibition was essentially completely reversed when these two metabolites were present simultaneously.
Previous studies have suggested that glucose and glutamine are the two main energy sources required for the rapid proliferation of tumor cells [20, 21]. Notably, some cancers seem to prefer aerobic glycolysis [22.23], others rely more on glutaminolysis pathway [24] Additionally, there are also some cancers tend to a combination of such two metabolic pathways[25]. Given the important role of glucose and glutamine in cell proliferation and survival, it is of great significance to clarify the relationship between glucose metabolism and glutaminolysis in HCC cells. In our study, HCC cells SK-Hep-1 and PLC/PRC/5 underwent glucose and glutamine deprivation could hardly survive, implying the synergistic effect of glutamine and glucose on HCC cells growth and survival. Besides, it is well known that glutaminolysis is a highly coordinated process catalyzed by numerous enzymes. For example, GDH catalyzes glutaminolysis to form α-KG, which enters the TCA cycle. By contrast, transaminases including GOT, GPT and PSAT promote the generation of NEAAs to maintain cellular events. Therefore, regulation of such key metabolic enzymes and pathways appears particularly important in glutaminolysis. However, the interrelations between these glutaminolysis-associated metabolic enzymes and glucose metabolism in cancers has not yet been exactly elucidated. In this study, our data showed that glutaminolysis was still warranted to maintain cell growth when glucose is sufficient, no to mention glucose starvation. Interestingly, our study validated that under different glucose concentrations, the glutaminolysis pathways that played a leading role in cell survival were different. Specific manifestation was as follows: GOT1 mediated pathway played a dominant role in regulating HCC cells growth under high concentration of glucose conditions, yet was not activated when glucose was limited. However, GDH1 mediated enzymatic reaction was activated under glucose deprivation (Fig. 5D). Surprisingly, we also reported that there was a potential negative correlation of GDH1expression with GOT1expression in low-glucose HCC tissues, but our research didn't reveal any obviously regulatory effect of other aminotransferases, including GOT2, GPT1, GPT2 and PSAT1, on cell growth of HCC. These results highlight the prominent places of GDH1 and GOT1 in the glutaminolysis process of HCC cells, suggesting that novel therapeutic approaches based on such two enzymes may be more beneficial to HCC treatment. Above all, the underlying opposing relationship between GDH1 and GOT1 supports the point that cancer cells adapt to nutrient-deprived tumor microenvironment during progression via adjusting the level and function of metabolic enzymes, that is, liver cancer cells maintain proliferation and survival under different nutritional conditions through the metabolic flexibility of their glutamine-related enzymes.
It has been uncovered that glucose supplement and extra-cellular glucose concentration in tumor tissues are much lower than surrounding normal tissues[13], which indicated the irreplaceable role of GDH1 in the growth and survival of cancer cells, especially under low-glucose conditions. GDH1 is a key enzyme for glutaminolysis. Several studies have reported that GDH1 provides metabolic advantages for cancer cell proliferation and tumor metastasis via regulating the production of α-KG [26,27 ]. Nevertheless, the pathological relationship between changes in GDH1 content and occurrence and development of HCC remains to be clarified in detail. Results of an earlier study by Jin et al. revealed that GDH1 predominantly controlled intracellular α-KG and subsequent fumarate levels, and contributed to redox homeostasis by activating GPx1, thereby promoting the cancer cell multiplying and tumor growth [28]. Another published research showed that phosphorylated ELK1 activated by EGFR/MEK/ERK signaling pathway enriched in the promoter of GDH1 to stimulate the transcription of GDH1, then promoted glutamine metabolism [29]. In the current study, we found an increased glutamine consumption in low-glucose cultured HCC cells, further demonstrated the necessity of glutaminolysis in HCC cells survival. It was reported that pyruvate carboxylase was highly expressed in glutamine-independent cancer cells, contributing to maintain anaplerosis under glutamine-deprivation conditions. In contrast, glutamine-dependent cell lines consume glutamine as the preferred anaplerotic substrate to drive TCA cycle[30]. Results of our study found that NH4+, a specific metabolite produced in a reaction catalyzed by GDH1, was markedly elevated under glucose-limiting conditions, providing evidences that up-regulated GDH1 drives increased entry of glutamine-derived carbon into the TCA cycle in response to glucose starvation. Our finding concerning the driving role of GDH1 on TCA cycle under limited glucose status agreed well with the previously reported results, which further demonstrates a critical role for GDH1 in HCC cell proliferation and tumor growth.