Inhibition Of ASCT2 Suppresses Mitochondrial Metabolism And Progression Of Hepatocellular Carcinoma

Backgrounds:Hepatocellular carcinoma(HCC)is a cancer with high incidence in the world and the sixth most common cancer in the world and an important cause of cancer-related deaths.Since the early clinical symptoms of HCC are not obvious,most patients are diagnosed at an advanced stage.Although the current treatment methods include surgical resection of liver cancer tissue,liver transplantation with good function,radiofrequency ablation,radiotherapy and chemotherapy,etc.,which have been widely used in clinical practice,but many patients are still prone to recurrence and metastasis after receiving treatment,which indicates that the current treatment methods have certain deficiencies.Metabolic changes are one of the main markers of tumorigenesis.Cancer cells are highly dependent on their specific nutrients and have changes in various metabolism-related genes,which may become unique targets for cancer treatment.To be precise,most of the life activities of tumor cells need a large amount of energy,namely ATP,to support their normal operation.So what are the main sources of ATP production that cancer cells need to synthesize biological macromolecules? There are several theories about the energy metabolism inside cancer cells.The first is the classic Warburg effect,which explains how cancer cells metabolize through glycolysis,fermenting glucose as a source of ATP production.Warburg believed that glucose was metabolized to form the energy source of cancer cell growth through fermentation rather than oxidative respiration.Until now,it has been widely believed that glucose is the main source of ATP metabolism in cancer cells.However,this occurs only when glucose is the only nutrient substrate in anoxic conditions.The second theory is the theory of cancer cell metabolism,it argues that the tumor cells by complex symbiotic relationship survival: a first cancer cells use glucose to produce lactic acid and ATP(Warburg effect),adjacent to the cancer cells around it after,using it in the process of metabolism of lactic acid,through the Krebs cycle(TCA)and metabolism of oxidative phosphorylation to generate ATP.Some cancer cells use lactic acid as substrate of TCA intermediate,and then participate in ATP generation through monocarboxylic acid transporter.Through the action of lactate dehydrogenase(LDH)and citrate lyase,lactate can also participate in the synthesis of fatty acids.Therefore,the symbiosis theory of cancer cells may play a more important role in the synthesis of biomacromolecules in cancer cells than ATP production.A third theory is that glutamine is metabolized to glutamate,which is then metabolized by glutamate dehydrogenase to form α-ketoglutarate(α-kg).Then α-kg participates in ATP synthesis through TCA cycle.This means that glutamine can also be used as a raw material for ATP production.And in recent years,the role of glutamine in metabolic research is taken seriously increasingly,the research results show that the glutamine is a special kind of amino acid,which belongs to a set of conditional necessary amino acid,especially in the stress environment need to quickly a large number of catabolism,such as sepsis after surgery,injury,or the several cases,kidney,gastrointestinal tract,etc of glutamine consumption will rise sharply.Intestinal mucosal cells are particularly dependent on glutamine,and will quickly die after glutamine depletion.Glutamine is also consumed and utilized in large quantities in rapidly dividing cells,especially cancer cells,for energy production and as a source of carbon and nitrogen.In the absence of glutamine,death occurs in some cancer cells and progresses rapidly.These results reflect that glutamine plays a very important role in the proliferation and survival of cancer cells.Glutamine itself can promote nucleotide synthesis and the synthesis of Uridine Diphosphate N-acetylglucosamine to support protein folding and transport,and can also be converted to glutamic acid through the action of glutaminase.Glutamate promotes the synthesis of glutathione,a tripeptide composed of cysteine,glutamate,and glycine,which plays an important role in maintaining REDOX homeostasis in cells.Targeting cancer cells’ REDOX metabolism can lead to elevated levels of intracellular reactive oxygen species(ROS),which have historically been considered deadly metabolic byproducts of cellular respiration and protein folding.Low level of ROS can regulate cell proliferation and cell adaptation to metabolic stress.However,with the increase of ROS level,cell death signaling pathway is activated,causing damage to cells,causing structural damage to mitochondrial membrane,oxidative damage to cells,affecting mitochondrial metabolism,and even cell death.Glutamine is required to enter mammalian cells via transporters such as alanine-serine-cysteine transporter 2(ASCT2).Cell metabolism in the tumor microenvironment is influenced by the nutritional conditions and the active signal exchange and metabolite exchange between tumor cells and stromal cells.Similarly,compared with nutrients,nutrient transporters also play an important role in promoting dynamic metabolism in tumor cells.In rapidly proliferating cells,ASCT2 expression is up-regulated to meet the increased demand for glutamine consumption.Interestingly,ASCT2 expression is also elevated in many cancers due to the same amplification energy requirements.Previous research results of our group showed that compared with normal liver cells HL-02,the expression level of ASCT2 in Hep G2 Hep3 B SMMC-7721 Bel-7404 QGY-7701 was significantly increased.These results raise an interesting speculation as to whether ASCT2 could be a potential target for HCC therapy?Research contents:1.SMMC-7721 and BEL-7404 cells with high ASCT2 expression were selected,by lentivirus transfection on knockdown of ASCT2 expression in cells,the fluorescence were observed expression of its transfection efficiency,and then use puromycin for drug screening for the cells after transfection,to establish a stable cell line.Then Western blotting was used to verify ASCT2 knockdown effect after lentivirus transfection.2.ROS kit was used to detect whether ASCT2 knockdown had an effect on REDOX homeostasis in HCC.3.The tumorigenesis experiment in nude mice was conducted to detect whether ASCT2 knockdown had an effect on the tumorigenesis ability of HCC cells in vivo.4.Seahorse was used to detect whether ASCT2 knockdown had an effect on mitochondrial metabolism of HCC.5.ATP kit was used to test whether ASCT2 knockdown had an effect on the energy production in HCC cells. 6.Western blot was used to detect whether ASCT2 knockdown had an effect on the phosphorylation of S6.Research results:1.The SMMC-7721 cells and BEL-7404 cells were successfully transfected and carried fluorescence.ASCT2 protein expression in SMMC-7721 sh ASCT2 group(knockdown group)and BEL-7404 sh ASCT2 group(knockdown group)was lower than that in NC group(negative control group)by Western Bolt detection.2.ROS test results showed that ROS content in HCC cells increased after ASCT2 knockdown.3.The tumor formation test results of nude mice indicated that ASCT2 knockdown could inhibit the tumor formation ability of HCC cells in vivo.4.In terms of metabolism,seahorse test results showed that ASCT2 knockdown could inhibit oxidative phosphorylation in mitochondria of HCC.5.ATP production level detection results show that ASCT2 knockdown can inhibit ATP production of HCC cells.6.In terms of mechanism,Western blot results showed that the phosphorylation level of S6 was decreased after ASCT2 knockdown.Conclusion:1.ASCT2 knockdown leads to increased ROS accumulation in HCC cells. 2.Knockdown of ASCT2 inhibits tumorigenesis of HCC cells in nude mice.3.Knockdown of ASCT2 inhibited oxidative phosphorylation and ATP production in mitochondria of HCC cells.4.Knockdown of ASCT2 inhibits the phosphorylation level of S6.