Roles And Mechanisms Of GRP75 Regulating Anti-oxidation/Apoptosis And Metabolic Reprogramming In Drug Resistance Of Gastric Cancer And Hepatocellular Carcinoma

Gastric cancer(GC)and hepatocellular carcinoma(HCC)were the most common malignant tumors in China.GC was the second leading mortality and HCC was the first leading mortality of digestive system cancers.Cisplatin was one of the first-line agents for advanced GC chemotherapy.TACE and Sorafenib were the main treatment methods for patients with advanced HCC.However,due to the heterogeneity of tumor,acquired resistance to drugs always occurred after multiple cycles of treatment,leading to the overall treatment effect and the benefit time of patients were less than satisfactory.Glucose-regulated protein 75(GRP75)was the stress-inducible molecular chaperones that belong to the heat shock protein 70 family.Overexpression of GRP75was closely associated with drug resistance and poor prognosis in various human cancers.The mechanism may be related to the inhibition of p53.However,the p53 was one of the most frequently mutated genes in GC and HCC of China.It was suggested that there might be a p53 independent way for GRP75 to induce and maintain drug resistance in GC and HCC,and the specific mechanism was rarely reported,which needed further study.Under physiological conditions,endogenous 27HC(27-hydroxycholesterol,27HC)was enriched in the liver and was metabolized out of the body.However,under tumor pathological conditions,liver metabolic dysfunction/loss resulted in high concentration of 27HC in local HCC tissues.In order to protect cells from 27HC toxicity,some hepatocellular carcinoma cells have established a special hydroxysterol metabolism system.It is suggested that long-term exposure of 27HC in the process of hepatocellular carcinoma can induce the adaptive mechanism of cells,and its functional role also changes from hepatotoxicity to cancer promoting.Therefore,an in-depth study of the potential mechanisms of drug resistance in GC and HCC,and the exploration of accurate methods to reverse drug resistance,could effectively improve the overall efficacy and prolong the survival time of patients.Our study was aimed to investigate the mechanism and clinical significance of GRP75 in inducing/maintaining drug resistance in GC and HCC.Methods1.Establishment of cisplatin resistance cells in GC and 27HC long-term exposure induced multi-drug resistance in HCCGC cisplatin resistance cell line(SGC7901^CR)was obtained from Key GENE Biotechnology Co.Ltd.For maintenance of the cisplatin resistance phenotype,SGC7901^CR cells were cultured in RPMI-1640 medium containing 1?g/mL cisplatin.After 24 h of conventional culture,BEL-7402 cells were cultured in RPMI-1640medium containing 1.25?M 27HC for 48-72 h,and then were cultured in RPMI-1640medium containing 2.5?M 27HC for 1 week.After adaption,cells were continued cultured for 1 week with a concentration of 27HC increased to 5?M.Finally,cells were cultured with 10?M 27HC for high-dose shock.After being exposed to 27HC with increasing concentration for 50 generations(about 5 months),cells could grow stably in the medium containing 2.5?M 27HC,and Bel^27HC cells were successfully established.Bel-7402 cells in control culture were recorded as Bel^PC.2.Quantitative PCR(qPCR)After total RNA extraction and concentration determination,cDNA reverse transcription and primer amplification,qPCR-mix reagents were added to detect and calculate the fold changes of gene expression by formula 2^-(??Ct).3.Western blotAfter extraction of total/nuclear proteins,measurement of their concentrations,and SDS-PAGE followed by transferring the protein to PVDF membranes,immune complexes were detected by enhanced chemiluminescence.4.Cell viabilities and calculation of IC50The cells were treated with cisplatin or 27HC of different concentrations for 24 h.Cell viabilities were determined via using a Cell Counting Kit-8.The IC₅₀s were calculated via a graph-pad 8.0 software.5.Bioinformatics AnalysisThe microarray raw data were downloaded from the GEO database.The limma package was used to identify the differentially expressed genes(DEG)between cisplatin-resistant and cisplatin-sensitive cell lines/tissues.KEGG-pathway and GO-terms were conducted to reveal functional and biological characteristics attributes of the DEGs based on DAVID online database.Network Analyst was used to analyze the protein-protein interaction of GRP75 according to the KEGG database.6.Mitochondrial membrane potential(MMP)measurementThe cells treated with cisplatin or 27HC for 24h were incubated with 1 ml JC-1staining working fluid at 37? for 20 min.Fluorescent intensity of the JC-1 monomers and aggregates was detected on a multi-well plate reader.7.Intracellular reactive oxygen species(ROS)determinationTreated cells were incubated with DCFH-DA and the fluorescent signal was observed via a fluorescence microscope,the DCFH fluorescence intensity was measured via a multi-well plate reader at Ex(?)488 nm and Em(?)525 nm.Cells were incubated with superoxide detection working medium and treated with27HC for different times.The absorbance value was measured at the wavelength of450 nm of the enzyme scale.Bel-7402 cells treated with 27HC for different times were mixed with hydrogen peroxide detection lysate.Cell samples and hydrogen peroxide detection reagents were mixed for 30 min.Determine A₅₆₀ and calculate the concentration of hydrogen peroxide in the sample according to the standard curve.8.Apoptosis assayCells were collected,washed twice with cold PBS and re-suspended in binding buffer containing fluorescein isothiocyanate(FITC)-Annexin V and PI.The samples were assessed using a FACS Calibur flow cytometer.9.Glucose uptake assayCells were treated with cisplatin or 27HC for 6 h,respectively.The cells were gently incubated with 100?M 2-NBDG at 37? for 30 min.Fluorescent intensity was detected on a microplate reader(Ex(?)465 nm;Em(?)540 nm).10.Cell growth assayFor the determination of growth kinetics,1×105 cells were seeded in six-well plates,and cultured for 24 h with or without treatment.Cells were then collected and counted in triplicate using a hemocytometer under a microscope.11.Xenografts and treatmentsFor xenograft study,2×10⁷ SGC7901^CR cells in 100?l matrigel were injected subcutaneously into the flanks of the mice for 5 weeks.To determine the effects of GRP75 on the cisplatin-resistance of GC,we performed the intratumoral and intraperitoneal injection assay.Tumors were measured every week.After 5 weeks,the mice were sacrificed,and tumor tissues were removed for further investigation.12.Immunohistochemistry(IHC)Sections were incubated in 10%normal bovine serum albumin for 5 min,followed by incubation with primary antibody at 4? overnight.The slides were then incubated with a horseradish peroxidase-conjugated secondary antibody at room temperature for another 30 min.Samples were then visualized,dehydrated,cleared,mounted,and photographed under a panoramic-scan digital slice scanning system.The graphs were analyzed using Image-Pro-Plus 6.0 software.13.Statistical analysisData was presented as the mean?±?SEM.The statistical significance of results was calculated via graph-pad 8.0 software.Overall survival analysis was performed using the Kaplan-Meier method and log-rank test.Clinicopathological features were analyzed by a?2 test.A Cox proportional hazards regression model was used to identify independent prognostic factors associated with overall survival.The p value<?0.05 was defined as statistically significant.Results1.Effects of GRP75 on cisplatin-resistance in GC and multi-drug resistance in HCCWe found markedly increased GRP75 expressions in SGC7901^CR cells and Bel^27HCcompared with their parental cells.SGC7901^CR or Bel^27HC and their parental cells were treated with different concentrations of cisplatin or 27HC.We found that the IC₅₀s of drugs increased significantly in drug resistant cells.Knockdown of GRP75 in SGC7901^CR or Bel^27HC cells,the results showed that GRP75 knockown significantly inhibited the IC₅₀s compared with control groups.On the contrary,GRP75 was overexpressed in SGC7901 or BEL-7402 cells had the the opposite effect.2.Potential mechanisms underlying GRP75 caused cisplatin-resistance in GC and multi-drug resistance in HCCWe downloaded microarray datasets GSE122130,GSE14209,GSE129071,GSE73571 from GEO.The results showed that oxidation-reduction,apoptotic process,response to drug/hypoxia,metabolic pathway and PI3K/AKT signaling pathway were the key processes or pathways leading to the difference of drug resistance.KEGG pathway analysis of the PPI network with GRP75 was carried out by network analyst.It was found that metabolic pathway also played an important role in the PPI network of GRP75 and its interacting proteins.3.Effects of GRP75 on anti-oxidation/apoptosis caused cisplatin resistance in GC and 27HC induced multi-drug resistance in HCCHere,the intracellular ROS levels were elevated in SGC7901^CR and Bel^27HC cells in comparison with its parental counterparts.Knockdown of GRP75 abolished the maintenance of MMP,decreased the level of NRF2 and its downstream target genes(HO-1 and NQO1),further enhanced the intracellular ROS generations and apoptosis induced by cisplatin or 27HC in SGC7901^CR and Bel^27HC cells.In contrast,overexpression of GRP75 in SGC7901 and BEL-7402 cells showed the opposite effect.4.Effects of GRP75 on metabolic reprogramming caused cisplatin resistance in GC and 27HC induced multi-drug resistance in HCCWe observed raised p-AKT,HIF-1?and c-myc level in SGC7901^CR and Bel^27HC cells compared with their parental cells.Moreover,in SGC7901^CR and Bel^27HC cells,knockdown of GRP75 decreased the p-AKT,HIF-1?,c-myc level and their downstream targets related to glycolysis(HK2,PDK1 and LDHA).On the contrary,overexpression of GRP75 in SGC7901 and BEL-7402 cells showed the opposite effect.5.Confirmation the in vitro data in a xenograft modelThe xenograft data indicated that treatment with cisplatin alone or knockdown of GRP75 alone could inhibit the tumor growth;however,cisplatin treatment combining with GRP75 knockdown significantly facilitated the cisplatin induced inhibition of tumor growth.Moreover,IHC and qPCR assays showed that cisplatin plus GRP75si RNA significantly decreased the expressions of Ki-67,GRP75,NRF2,p-AKT and downstream targets compared with cisplatin treatment alone,but increased the apoptosis.6.Potential mechanism of 27HC regulating GRP75 in HCCThe results showed that the levels of ROS,superoxide and hydrogen peroxide increased first and then decreased at the end of the treatment,and the levels were significantly higher than that at the beginning of the treatment.After treatment with superoxide dismutase inhibitor,the expression of GRP75 mRNA and protein were significantly higher than control.It is suggested that 27HC treatment could induce the increase of intracellular oxidative stress signal level,and induce the increase of GRP75.7.Identification of GRP75 as a characteristic cancer-promoting factor and the clinical significance of GRP75The levels of GRP75 of HCC tissues in TCGA database and GC tissues were significantly higher than adjacent tissues.A stronger staining for GRP75 was also observed with increasing TNM Classification of malignant tumors stage.The level of GRP75 was positively correlated with the transverse diameters.Kaplan-Meier survival analysis also showed that GRP75 was related to poor prognosis.Multivariate analysis identified that,GRP75 was an independent predictor for overall survival.Conclusions1.GRP75 promotes the cisplatin resistance of GC and 27HC induced multi-drug resistance of HCC via regulating anti-oxidation/apoptosis and metabolic reprogramming.2.The expression of GRP75 is higher in GC and HCC tissues or drug-resistant cells,and high level of GRP75 is related to the poor prognosis of gastric cancer.3.GRP75 could be a valuable additive prognostic predictor in guiding clinical management of GC patients.Targeting GRP75 could be a potential therapeutic approach,which restored the drug response in cisplatin resistance cells.4.GRP75 is not only a key regulatory molecule for the role of 27HC in HCC,but also a potential therapeutic target for reversing the multi-drug resistance induced by long-term exposure of 27HC.