| Prostate cancer is one of the malignant tumors in elderly men and remains the third leading cause of cancer-related deathb,behind only digestive system and respiratory system cancers.Prostate cancer has its specific dependence on androgen and castration therapy(testicle resection or drug blockage)is the best choice for primary prostate cancer patients.Though in the early stage androgen deprivation therapy shows effectively tumor-suppressor ability,about 1 year after the castration therapy,most of them progresses to castration-resistant prostate cancer(CRPC)with distal metastasis.Docetaxel is the only chemotherapy agent with the proven survival benefit for treating metastatic castration-resistant prostate cancer(mCRPC),however,the same with other chemotherapy drugs,the multidrug resistance(MDR)caused by docetaxel chemotherapy is still inevitable.Chemotherapy resistance means a very poor prognosis and a high mortality.The mechanism of prostatic resistance has been well studied,including cell membrane,cytoplasm,nucleus and tumor microenvironment mediated resistance mechanism,such as ATP binding channel transport protein activation,?-tubulin mutation or subtype alteration,continued activation of PI3K/AKT/mTOR signaling pathway,epigenetic changes,metabolic changes and so on.Reversed drugs were designed according to drug resistance mechanism,such as Cabazitaxel with the low affinity to ABC trasporter.However,the mechanism of drug resistance in prostate cancer is complex,with few specific targets and few effective reversal schemes.Therefore,exploring new drug-resistant mechanisms of prostate cancer,and finding new effective drug targets and reversal agents have been important topics for solving clinical needs.Part ? Analysis of changes in gene expression in multidrug-resistant cells of prostate cancer by microarray data and screening the reversal agents1.Construction of low dose docetaxel induced multidrug-resistant prostate cancer cell lineProstate cancer DU 145 and PC3 cells are hormone independent tumors,with a strong anti-apoptotic ability and high malignancy degree.According to the pattern of clinical medication,we treated PC3 and DU 145 cells with six cycles of Docetaxel(Doc).After screening,we obtained the PC3/Doc and DU145/Doc tolerated from chemotherapy.The resistance index analysis showed that the IC50 for docetaxel in both of the drug-resistant cells were 3.8 and 2.4 times higher than their parental sensitive cells.They also show resistance to doxorubicin(topoisomerase ? targeted drug)and vincristine(microtubule targeting drugs),without obvious cisplatin resistance.PC3/Doc and DU145/Doc cell acquired multidrug resistance.2.Analysis of differential expression profiles between drug resistant cells and parental cells1)Drug-resistant cells have significant changes in the metabolic related genes:By clustering analysis of the differential expression chips,the most varied genes were concentrated in the metabolic pathway.Analyzing key metabolic genes,the anaerobic glycolysis and oxidation decarboxylation of three carboxylic acid cyclic did not increase significantly in PC3/Doc cells,while the reduction metabolism of TCA cycle,glutamine metabolism and fatty acid synthesis related genes increased significantly,indicating that the cellular metabolism especially mitochondria metabolism in drug-resistant cells is changed.2)Drug-resistant cells were sensitive to glutaminolysis inhibitors and HDAC inhibitors:Screening the reversal agents with 8 metabolic inhibitors,compared with parental cells,PC3/Doc cells were more sensitive to the glutamine metabolic inhibitor BPTES(bis-2-(5-phenylacetamido-1,3,4-thiadizaol-2-yl)ethyl sulfide),the IC50 was 3.12?M,and PC3 was 7.50?M,but no significant response to glycolytic inhibitor 2-DG(IC50:5.87mM vs PC3 was 2.96mM).It was also found that drug-resistant cells were most sensitive to histone deacetylase(HDAC)inhibitor TSA and the sensitivity increased nearly 10 times(IC50 was 0.33?M vs PC3 cells 2.87?M).Therefore,inhibition of glutamine metabolism can significantly inhibit the proliferation of drug-resistant cells,indicating that glutamine metabolism plays an important role in the drug resistance of prostate cancer,and the drug-resistant cells displayed a significant response to HDAC inhibitors,indicating that acetylation of protein also plays an important role in drug resistance,and the mechanism needs further analysis.Part ? Aberant glutamine metabolism contributes to the multidrug-resistance in prostate cancerCancer cells mainly utilize glucose to produce lactic acid through Warburg effect,which conferred continual loss of pyruvate for the mitochondrial TCA cycle.Another energy source glutamine replenish TCA intermediates and produce ATP.Glutamine uptake in the cytoplasm was mediated by transporter,such as Na+ dependent Solute carrier family 1(neutral amino acid transporter)member 5(SLC1A5,also ASCT2),L-type amino acid transporter 1(LAT1),etc.In the mitochondria,glutamine can be converted into glutamate by glutaminase,then glutamate converted into a-ketoglutarate(a-KG)that is shuttled to the Krebs cycle to produce ATP.Therefore,enhanced glutamine metabolism plays a key role in the tumor malignant transformation by providing carbon sources and nitrogen sources.When lack of glucose,the intermediate metabolites of the TCA cycle,such as oxaloacetic acid,enter the glycolysis pathway,accompanied by citric acid shuttling from mitochondria to the cytoplasm.The Acetyl-CoA produced by ATP citric acid lyase(ACLY)is the main substrate for lipid synthesis,acetylation of proteins,etc.At the same time,glutamate is also used to synthesize glutathione(GSH)to remove the intracellular ROS.It has been confirmed that prostate cancer is different from other tumors and more dependent on glutamine metabolism and lipid metabolism.Using bioinformatics to analyze the differential expression profiles between multidrug-resistant PCa induced by docetaxel and its parental cells,we found that top pathway is the metabolic pathway,in which the gene related to glutamine metabolism is significantly increased.Drug-resistant cells displayed increased sensitivity to the GLS inhibitor BPTES.Therefore,we first analyzed the role of glutamine in the drug resistance of prostate cancer,and achieved the following research results:1.Glutamine metabolism promotes the malignant progression of prostate cancer 1)The proliferation of drug-resistant PCa cells is more dependent on glutamine Tumor recurrence,metastasis and drug resistance are all manifestations of malignant transformation.Our results showed that the glutamine transporter ASCT2 and LAT1 increased in varying degrees,and the expression and activity of glutaminase increased significantly.After the removal of glutamine,the survival of the parental cells were not affected,only with a slowing proliferation,but the resistance cells did not proliferate,and the survival rates decreased gradually.Therefore,drug-resistant cells is glutamine"addictive".2)High expression of glutaminase was negatively correlated with the recurrence of prostate cancer after treatmentIn order to further confirm the effect of glutamine on the malignant transformation of prostate cancer,we used the TCGA database to analyze the recurrence of 285 patients with chemotherapy or surgical resection of prostate cancer,and the association with the levels of ASCT2,LAT1,or GLS.The results showed that the prostate cancer patients with high GLS expression had early recurrence,and their disease-free survival time was significantly shorter than those with low GLS expression,but the expression of ASCT2 and LAT1 had no significant correlation with PCa recurrence.Therefore GLS,a key enzyme regulating glutamine metabolism,plays an important role in the progression of prostate cancer.2.Inhibition of glutaminase,reversed drug resistance,and promote drug-resistant cell apoptosis1)Inhibition of glutaminase significantly increased the sensitivity of drug-resistant cells to docetaxel.In view of the significant difference in glutaminase between the drug resistant PCa cells and their parental cells,we used glutaminase inhibitor BPTES to detect the response in drug resistant cells.MTT results showed that drug-resistant cells were more sensitive to BPTES compared with their parental cells,and low concentration of BPTES could significantly increase the sensitivity of drug-resistant cells to docetaxel,suggesting a strong synergistic effect between BPTES and Doc.Therefore,glutaminase plays an important role in drug resistance of prostate cancer.2)Inhibiting the activity of glutaminase increased the ROS level.We previously found that the level of ROS in the drug resistant cells was lower than that of the parental cells,and had the stem cell like characteristics.BPTES decreased the GSH/GSSH ratio and increased ROS level in drug-resistant cells.3.Inhibition of glutaminase promoted the apoptosis of drug-resistant cellsBPTES significantly induced DNA damage in drug-resistant cells,with increasing the expression of gamma-H2AX,and promoted apoptosis as evidenced by increasing cleaved PARP.Part ? Increased glutaminolysis facilitates protein hyper-acetylationthat contributes to the sensitivity of chemo-resistant prostateto HDAC inhibitorsWhen screening the reversal agents in drug-resistant cells,we found that drug-resistant cells were more sensitive to HDAC inhibitors than the parental cells,as evidenced by the IC50 value decreasing by nearly 10 times.Therefore,the inhibition mechanism of HDAC inhibitors was studied in detail.As prostate cancer is more dependent on theglutamine metabolism,glutamine metabolism replenishing TCA cycle can promote citric acid shuttling to the cytoplasm to produce Acetyl-CoA,which is used to synthesize fatty acids.At the same time,Acetyl-CoA is an essential group for histone and non-histone acetylation through acetyl transferase mediated protein acetylation,and HDAC inhibitors increase protein acetylation and increase the demands for Acetyl-CoA.Therefore,we speculated that the response of drug-resistant cells to HDAC inhibitors may be related to Acetyl-CoA,protein acetylation,and the epigenetic regulation of chromosomes.1.The protein acetylation level in drug-resistant PCa cells significantly increased 1)The acetylation level of total protein increased in the drug-resistant PCa cells.We first analyzed the difference in the protein acetylation level between the parental and drug-resistant cells by western blot.The results showed that the acetylation of H3,H4,and the acetylation of non-histone were significantly increased.We also detected another paired Prostate cancer cells,DU 145 cells and drug-resistant DU145/Doc,the western blotting results showed that the acetylation level of total protein and histone in DU145/Doc was also significantly increased.2)The changes of the acetylation level in drug-resistant cells may be related to the chemotherapy drugs.To determine whether the changes of the acetylation level in drug-resistant cells are related to the tumor type or the drug type.We used lung cancer H460 cells and taxol induced H460/RT cells,oral epithelial cell carcinoma KB cells and drug-resistant KB/VCR cells induced by vincristine,esophageal cancer EC 109 cells and cisplatin resistant EC109/CDDP cells for further analyzed.The results of western blotting showed that the protein acetylation increased in the drug-resistant KB/VCR cells,and there was no significant difference in the protein acetylation level of H460/RT and EC109/CDDP cells from their parental cells.It indicated that the change of acetylation state is not related to the tumor type,but related to drugs,which need further confirmation.2.Glutamine metabolism improves nucleocytoplasm Acetyl-CoA in drug resistant cells1)The cytoplasm of drug-resistant PCa contains a high level of Acetyl-CoA.As the acetylation level of histone and non-histone significantly increased in drug-resistant cells,we used overspeed centrifugation to isolate the mitochondria and nucleus components of PC3/Doc cells and analyzed the Acetyl-CoA level in these organelles.The results showed that the Acetyl-CoA in the nucleocytoplasm of drug-resistant cells are far higher than those of drug-sensitive cells.After TSA treatment,the protein acetylation level increased significantly,leading to significantly increasing demands for Acetyl-CoA,and the Acetyl-CoA in nucleocytoplasm decreased significantly.2)The increased activity of citrate lyase in drug-resistant cells:The Acetyl-CoA in nucleocytoplasm needs ATP dependent citrate lyase(ACLY)in the cytoplasm.The results showed that ACLY in drug-resistant cells was significant higher than that in its parent cells,at the same time,the expression of acetyl-coenzyme A carboxylase(ACC)was also significantly increased in drug resistant cells,indicating that lipid metabolism was enhanced.Therefore,the glutamine metabolism in drug-resistant cells provided sufficient Acetyl-CoA for the replenish of TCA cycle,in addition to promoting lipid metabolism,it also increased the level of histone acetylation and then promotes the transcription of chromosomes.3.The acetylation level of drug-resistant cells affects its sensitivity to HDAC inhibitors1)High acetylation status in resistant cells displayed more sensitivity to HDAC inhibitors:We used MTT to analyze the response of different drug-resistant cells to the HDAC inhibitor TSA.The results showed that DU 145/Doc and KB/VCR with high acetylation levels of the total protein were more sensitive to TSA,while compared to their parental cells,the response to TSA was similar in the drug-resistant H460/RT and EC109/CDDP cells,whose acetylated levels were not significantly different from their parental cells.Similarly,another HDAC inhibitor SAHA showed similar phenomenon in drug-resistant PCa cells.2)TSA further enhanced protein acetylation of drug-resistant cells:Western blotting results showed that TSA treatment for 30min significantly increased the acetylation of total protein and histone protein in the drug-resistant PCa cells,and increased acetylation with the prolongation of TSA treatment.In addition,after the TSA treatment for 1h,the acetylation of H3K9 and H4K16 significantly increased in drug-resistant cell and kept at a high acetylation state as the treatment prolonged.Also,the level of acetylation of H3K9 and H4K16 in the tumor tissues of TSA treatment group significantly increased,and that of docetaxel treatment group slightly increased.These results indicated that HD AC inhibitors can simultaneously promote histone and histone acetylation4.Increased HDAC and MOF in drug-resistant prostate cancer cells1)Increased expression of HD ACS in drug-resistant cells:Due to the sensitivity of drug-resistant cells to HDAC inhibitors,we further analyzed the expression level of HDAC in drug-resistant cells.Wertern blotting results showed that the expression of HDAC5 in drug-resistant cells was increased compared with the parental PC3 cells,while HDAC1,HDAC2,HDAC4,HDAC6 were not significantly changed.MTT showed that the sensitivity of drug-resistant cells to docetaxel was unchanged after knockdown HDAC5.Therefore,HDAC5 had no significant effect on drug-resistant cells.2)TSA inhibited the activity of HDACs in drug-resistant cells:the expression of HDAC was not down regulated after TSA treatment.We further analyzed the activity of the enzyme.The results showed that the total HDAC activity in the drug-resistant cells increased significantly.After TSA treatment,the activity of HDACs enzyme was significantly inhibited,indicating that TSA inhibited the HDACs activity of drug resistant cells and had no significant effect on its expression.However,the increase of HDAC activity leads to a decrease in acetylation level of histone,suggesting that there may be other mechanisms for high acetylation level in drug-resistant cells.3)The expression of acetyltransferase in drug resistant cells increased:the acetylation of protein was double regulated by HDAC and acetyltransferase.As the level of acetylation of H3K9 and H4K16 increased,we also analyzed the expression level of histone acetylation related to acetyltransferase p300 and MOF.The results showed that the expression of MOF in the drug resistant cells was high,but the expression of P300 was not significantly increased,indicating that acetyl-transfer was not significantly increased.Acetyltransferase MOF may involved in enhancing the acetylation level of drug-resistant cells,and its mechanism needs further study.5.HDAC inhibitors promoted drug-resistant cells apoptosis by enhancing endoplasmic reticulum stress1)TSA significantly induced the apoptosis of drug-resistant cells:EdU incorporation experiments showed that 0.1 ?M TSA significantly inhibited the proliferation of PC3/Doc cells,but the inhibition on PC3 cells was not obvious;0.2?M TSA could induce caspase-3 activation and cleaved PARP,while even 1?M TSA could not cause the cleaved PARP and apoptosis in PC3 cells.2)TSA significantly induced endoplasmic reticulum stress:Endoplasmic reticulum stress induced protein GRP78 appeared at TSA treatment for 1 hours and increased after 9h.p-PERK began to increase at 3 hours.In addition,apoptosis related proteins of the endoplasmic reticulum ATF3 and CHOP/GADD153 increased significantly at 18h.The above results indicated that endoplasmic reticulum stress induced by TSA increased the expression of apoptosis related proteins in the later stage.3)Inhibitory gene expression significantly reversed the apoptosis induced by TSA:Protein synthesis inhibitor CHX or transcriptional inhibitor Act D preprocessed for 2h,can significantly reversed TSA induced cell death,without caspase-3 activation and cleaved PARP.4)TSA significantly increased the transcription of stress genes:ChIP experiments detected by Ac-H4K16 antibodies and PCR,showed that Ac-H4K16 on HSP5 and ATF4 promoters(HSP5 was a gene encoding GRP78)increased.5)TSA activated PI3K/Akt survival pathway and promoted endoplasmic reticulum stress:In short time observation showed that the activation of AKT/mTOR was earlier than GRP78 change.Inhibition of AKT/mTOR pathway can reduced endoplasmic reticulum stress.6)Low dose of TSA significantly inhibited tumor growth in drug-resistant PCa bearing mice:Tumor volume in TSA treatment group decreased significantly(538.57±349:56 mm3),inhibition rate was 61.3%,and TSA significantly inhibited the proliferation of drug-resistant cells(TSA group 10.79±3.22%vs control group 35.43±5.40%).Part ? Conclusions and Innovations1.Conclusions1)Glutamine metabolism and fatty acid synthesis are vigorous,and mitochondria metabolism are abundant in drug-resistant PCa cells.Glutaminase plays an important role in the process of drug resistance.2)The survival of drug-resistant cells was highly dependent on glutamine,while the glutaminase inhibitor BPTES significantly inhibited the proliferation of drug-resistant cells,and the low concentration of BPTES significantly increased the sensitivity of drug-resistant cells to docetaxel.3)Glutamine inhibitor BPTES induces drug-resistant cell apoptosis by inducing ROS and DNA damage.4)The exuberant glutamine metabolism of drug-resistant cells increased the replenish on the TCA cycle.Compared with the parent cells,the activity of citrate lyase in the drug-resistant cells increased and the amount of Acetyl-CoA in the nucleocytoplasm significantly increased.5)High levels of Acetyl-CoA in nucleocytoplasm promote lipid metabolism and increase histone and histone acetylation levels in drug resistant cells.6)The increased acetyltransferase,such as MOF,increased the acetylation of histone and total protein in drug resistant cells,while the higher HDAC activity of drug resistant cells may have no significant effect on the protein acetylation.7)Highly acetylated drug-resistant cells are more sensitive to HDAC inhibitors.The acetylation level of drug-resistant cells may be related to drug types.8)HDAC inhibitors activated PI3K/Akt pathway in a short time,increased protein synthesis by promoting the acetylation of histone H3K9 and H4K16,and the transcription of stress genes,eventually aggravated endoplasmic reticulum stress,leading to the apoptosis of drug-resistant cells.2.Innovation1)This study first reported glutaminase took part in drug resistance in prostate cancer,and a low dose of BPTES was found to significantly increased the sensitivity of drug-resistant cells to docetaxel,and the two had synergistic effects.2)This study first reported total protein acetylation level was responsible for the sensitivity to HDAC inhibtors in drug-resistant prostate cancer cells.3)This study first reported HDAC inhibitors promoted the apoptosis of prostate resistant cells by activating the AKT pathway and enhancing ER stress.3.Defects1)The mechanism of metabolism reprogramming in drug-resistant cells mediated by docetaxel needs further investigation.2)The mechanism of docetaxel regulates glutaminase should be further analyzed.Eff-ect of glutaminase on acetyl-coenzyme A in drug-resistant should be further analyzed.3)The role of glutaminase in drug-resistant PCa and the resensitization effect to docetaxel still need animal pharmacodynamic experiments.4)The mechanism of increased acetylation level in drug resistant cells needs careful analysis. |
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