| Background and objectivesLiver cancer is a common malignant tumor of the digestive system,with a high case fatality rate,which greatly threatens the life and health of humans.China has the largest number of carriers of hepatitis B in the world,with the largest number of primary liver cancer patients.The incidence of liver cancer in China accounts for about 50%of new liver cancer every year,ranking the third mortality rate in the world.The incidence of liver cancer in China accounts for about 50%of new liver cancer every year,ranking the third mortality rate in the world.And the treatment of HCC mainly focuses on radiotherapy,chemotherapy,intervention,minimally invasive therapy,and targeted drugs,among which the targeted treatment drug sorafenib seriously affects the efficacy of rapid drug resistance.The efficacy of first-line chemotherapy drugs is also seriously affected by the production of multidrug resistance.5-fluorouracil(5-FU)is one of the commonly used clinical chemotherapy drugs for HCC,which can effectively kill or inhibit the growth of liver cancer cells.However,its long-term use can induce drug resistance in cancer cells,leading to poor therapeutic efficacy.Compared with normal tissues,cancer cells have a large number of metabolic abnormalities.And there is metabolic heterogeneity between different tumors and different development stages of cancers.In particular,new metabolic phenotype dependence will appear due to treatment resistance in the later stage of cancer progression.Therefore,we will study the correlation between metabolic reprogramming and the resistance of hepatoma cells to 5-FU.As the most abundant amino acid in the body,glutamine participates in multiple metabolic pathways,including the tricarboxylic acid cycle,de novo nucleotide biosynthesis pathway,non-essential amino acid synthesis pathway,maintenance of redox balance,and so on.Compared to normal tissue,cancer cells consume glutamine beyond the rate of glutamine biosynthesis.Therefore,the metabolic reprogramming of glutamine can play an essential role in the malignant progression of tumors.As the rate-limiting enzyme in the glutamine metabolic pathway,Glutaminase(GLS)can convert glutamine to glutamate which can convert into α-ketoglutarate involved in the regulation of other metabolic pathways.According to the TCGA database,the expression of GLS1 was higher in tumor tissues compared to normal tissues,such as gastric adenocarcinoma(STAD),hepatocellular carcinoma(LIHC),and colon adenocarcinoma(COAD).Based on cancer signature analysis tools,the function of GLS 1 in cancer is associated with cellular energetics,redox homeostasis,maintenance of proliferative signaling,induction of apoptosis,driving tumor progression,invasion and metastasis,and poor prognosis.To solve the problem of poor therapeutic effect of patients with liver cancer cells due to drug resistance,this study will analyze the metabolic characteristics of drug-resistant HCC cells,solve the resistance of the conventional chemotherapeutic drug(5-FU)in clinical liver cancer by tumor metabolism reprogramming,and improve the chemotherapy effect of anti-tumor drugs,provide the experimental basis and theoretical support for promoting rational clinical drug-using.Methods and results1.The drug resistance multiple of Bel7402/5FU cells was detected by MTT.After 48 h or 72 h treatment with 5-FU,the drug multiples of cells at 48 h or 72 h were 98.14 or 66.08 times,respectively.2.The glutamine-dependent differences between the two cells were detected by MTT and clonogenic assay.We found that glutamine metabolism reprogramming occurred after cell resistance to 5-FU.3.The GSH level in Bel7402 and Bel7402/5FU cells was detected by GSH detection kit.The results showed that the GSH level in Bel7402/5FU cells was significantly higher compared with Bel7402 cells,and the GSH level in Bel7402/5FU cells was decreased under glutamine deficiency.4.The ROS level in Bel7402 cells and Bel7402/5FU cells was detected by DCFH-DA staining,and it was found that the ROS level of 5-FU-resistant cells was increased.5.The factors explored that support the change of redox level of Bel7402/5FU cells after drug resistance.The experimental results showed that the G6PD protein expression level of Bel7402/5FU cells decreased,resulting in the reduction of NADPH produced by the pentose phosphate pathway,which lead to the change of redox level after drug resistance.6.The function of GLS in cancer and the correlation with prognosis was analyzed through the TCGA database.It was found that the high expression of GLS was associated with poor prognosis.7.The GLS activity in Bel7402 cells and Bel7402/5FU cells was detected with Gln detection kit.The results showed that the GLS activity of Bel7402/5FU cells was significantly higher than that of Bel7402 cells.8.The inhibitory effect of CB-839(glutaminase inhibitor)on the proliferation of Bel7402 cells and Bel7402/5FU cells was detected by MTT assay.It was found that CB-839 had a stronger anti-proliferation effect on Bel7402/5FU cells.9.The change in intracellular glutamate level after using CB-839 was detected with a Gln detection kit.The results showed that the glutamate level of Bel7402/5FU cells decreased significantly after using CB-839,indicating that activity was inhibited by CB-839.10.The anti-proliferation effect of CB-839 combined with 5-FU on Bel7402/5FU cells was detected by MTT assay.The results showed that there was a synergistic anti-proliferation effect of CB-839 combined with 5-FU.11.The effect of the combination of CB-839 and 5-FU on the invasion and migration ability of Bel7402/5FU cells was detected by cell scratch wound-healing and the Tranwell test.The results showed that the combination of CB-839 and 5-FU inhibited the invasion and migration ability of Bel7402/5FU cells,and the protein expression levels of E-cadherin,N-cadherin,and Vimentin were affected.12.The effect of CB-839 and 5-FU on the cell cycle distribution of Bel7402/5FU was detected by flow cytometry;The results showed that the combination of the two drugs arrested Bel7402/5FU cell cycle at S and G2 phases.Western blot results showed that the protein levels of CDK1,Cyclin B1(G2/M phase-related protein),and Cyclin A2(S phase-related protein)decreased.13.The effect of CB-839 and 5-FU combination on the PI3K/Akt/mTOR signal pathway was detected by Western blot.The results showed that the expression of PI3K/Akt signal pathway protein was inhibited after using the combination of CB-839 and 5-FU.14.The effects of CB-839 and 5-FU on the apoptosis and autophagy of Bel7402/5FU cells were detected by Acridine orange staining assay,annexin V-FITC/PI double staining assay and Hoechst 33342 staining assay.The results showed that the combination of the two drugs had no significant effect on the apoptosis and autophagy of Bel7402/5FU cells.15.The GSH level of Bel7402/5FU cells after using 5-FU and CB-839 was detected by GSH kit.The results showed that the use of 5-FU increased the intracellular GSH level in response to the increase of reactive oxygen species level caused by 5-FU.The use of CB-839 reduced the GSH level due to the inhibition of glutamine metabolism.The combination of the two drugs finally inhibited the increase of GSH level,thus promoting the oxidative stress of 5-FU on cells.16.The effect of the combination of CB-839 and 5-FU on the ROS level in Bel7402/5FU cells was detected by DCFH-DA staining.The results showed that the ROS level in cells increased respectively after the use of 5-FU or CB-839,and the ROS level increased significantly after the combination of the two drugs.17.The effects of the combined application of CB-839 and 5-FU on iron ion and MDA levels in Bel7402/5FU cells and the changes in Nrf2-HO-1-GPX4 pathway protein levels were detected by Western blot.The results showed that the combined application of CB-839 and 5-FU significantly increased the iron ion and MDA levels in Bel7402/5FU cells.In addition,the protein level of the Nrf2-HO-1-GPX4 pathway decreased,suggesting that the death mode was related to iron death.18.The growth inhibition of CB-839 combined with 5-FU on Bel7402/5FU transplanted tumor in nude mice was detected.The experimental results showed that CB-839 and 5-FU combination significantly inhibited the growth of xenograft tumors.In addition,except for the combined group,the other three groups of nude mice showed different degrees of liver metastasis.This suggests that the combined treatment of CB-839 and 5-FU can inhibit the metastasis of liver cancer to a certain extent.Western blot assay was used to detect the expression changes of GPX4 and HO-1,and immunohistochemistry was used to detect the expression of GPX4.The results showed that the expression of GPX4 and HO-1 decreased significantly in the co-treatment group.The above results indicated that the combined treatment of CB-839 and 5-FU effectively inhibited the occurrence and development of tumors by promoting the iron death of cancer cells.ConclusionIn this study,it was found that the metabolic dependence of Bel7402 cells was changed after drug resistance to 5-FU,and the dependence on glutamine was enhanced.The glutaminase in Bel7402/5FU cells was over-activated,glutathione synthesis increased,and the level of intracellular reactive oxygen species also increased.The combination of glutaminase(GLS)inhibitor CB-839 and 5-FU can synergically inhibit cell proliferation,invasion,and migration and cause cycle arrest,but do not increase apoptosis and autophagy.In addition,the combination of CB-839 and 5-FU can destroy the redox homeostasis of Bel7402/5 FU cells,reduced the intracellular glutathione level and increased the ROS level,causing intracellular oxidative stress,and increase of Fe2+and MDA levels in Bel7402/5FU cells,which induced iron death and inhibited the malignant progression of cancer.SignificanceIn this paper,we investigated the changes in glutamine metabolism reprogramming and reactive oxygen species level in Bel7402/5FU cells.Therefore,GLS inhibitor has strong cytotoxicity and targeting on Bel7402/5FU cells,and CB-839 combination with 5-FU has obvious synergistic inhibitory effects on the proliferation,invasion and migration,and promotion of cell ferroptosis.The purpose of this study is to provide better treatment plans and new treatment ideas employing the metabolic characteristics of drug-resistant cells for clinical drug-resistant patients. |
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