| Prostate cancer(PCa)is one of the most common malignancies in men,and the risk increases with age.The over-all 5 years survival of men diagnosed with late metastases disease is only 30%.Docetaxel is a taxane antimitotic agent currently used as a standard treatment for advanced metastatic PCa.Nevertheless,in the course of treatment,the majority of patients ineluctably develop resistance to pharmaceuticals.Current researches suggested that the docetaxel resistant mechanisms were related to beta tubulin subtype mutation,activation of drug efflux proteins,androgen receptors,tumor microenvironment and autophagy.Nevertheless,mechanisms of docetaxel resistant need further exploration.As an important component of tumor microenvironment,nerve plays an important role in tumor development.Sympathetic signals are transmitted by neurotransmitters mainly acting on β2 adrenergic receptors(β2-AR).Propranolol,a β-AR blocker,synergistic with doxorubicin has been reported to increase the sensitivity to docetaxel in sarcoma cells,suggesting that β2AR may have relationship with docetaxel resistance.At present,the study on the effect of β2-AR signal on docetaxel resistance in PCa has not been reported.Currently,there still lack of effective treatment methods for docetaxel resistance patients in clinical practice.It is an urgent problem to study the molecular mechanism of docetaxel resistance and find new effective targets and treatment methods.Objective1.To investigate the expression of β2-AR in benign hyperplasia tissues and PCa tissues with different pathological features and the tissues with preand post-docetaxel chemotherapy.2.To explore the effect of β2-AR on docetaxel sensitivity and drug resistance.3.To study the mechanism of β2-AR affecting the resistance to docetaxel in PCa cells.4.To explore the effect of the combination of β-AR antagonist and other agents on docetaxel sensitivity in vivo by constructing subcutaneous tumorigenesis model.Methods1.Bioinformatics analysis and cell detection analysis.By downloading the GEO database in NCBI,the expression of β2-AR in benign hyperplasia tissues and PCa tissues with different pathological characteristics and the tissues before and after docetaxel chemotherapy was analyzed.The expression of β2-AR in PCa cells treated with docetaxel was detected by Western blot and qPCR.The expression of β-AR subtypes in PCa cells was detected by Western blot and qPCR.2.Cell experiments.The effect of propranolol,a β-AR inhibitor,on docetaxel resistance in castration-resistant PCa cells was detected by CCK8.The effect of β2-AR on docetaxel resistance though autophagy and apoptosis in castration-resistant PCa cells was also detected.The molecular mechanism of β2-AR affecting docetaxel sensitivity through apoptosis was investigated by flow cytometry,mitochondrial membrane potential and Western blot.The effect of propranolol on the autophagosomes was detected by TEM.LC3B-labeled stable cells were constructed by lentivirus infection,and the effects of propranolol and docetaxel on autophagy flux were observed with confocal microscope.The effect of glucose serum starvation on the expression of β2-AR,the effect of β2-AR agonist isoproterenol and antagonist propranolol on autophagy,and the expression of autophagy related signaling pathways were detected by Western blot assay.The molecular mechanism of autophagy induced by propranolol and docetaxel was examined though constructing shAtg5 knockdown stable cells by infection with lentvirus.Docetaxel-resistant PCa cell lines were constructed by the method of dose-escalation.Cell resistance was detected by CCK8 assay,plate colony formation assay and flow cytometry.The sensitivity of parental and resistant cells to propranolol was measured by CCK8.We detected the expression of β2-AR in parental and drug-resistant cells by Western blot and qPCR.Western blot and TEM were used to detect the changes of autophagy in the parental and drug-resistant cells.We further examined the effects of propranolol on docetaxel sensitivity and drug resistance through autophagy by CCK8.3.Cellular mechanism studies.We detected the interaction between the key autophagy factor BECN1 with Vps34,BECN1with the anti-apoptotic factor Bcl-2 after the treatment of propranolol by performing immunoprecipitation assay.Detection of the proteins expression of p38 MAPK and JNK signaling pathways,which are important for the balance between autophagy and apoptosis were detected by performing Western blot assay.The changes of propranolol and docetaxel induced the expression of autophagy and apoptotic proteins with the addition of p38 MAPK and JNK inhibitors were detected by Western blot assay.The effects of propranolol on FOXO3a protein synthesis and decomposition were detected by Western blot.The expression and distribution of FOXO3a protein in cells treated with propranolol were detected by immunofluorescence.4.In vivo experiments.We established a subcutaneous tumor-forming model and intraperitoneally injected the drug into nude mice.The effects of propranolol and chloroquine on docetaxel sensitivity in cells growth in vivo were detected.The expression of p-p38 MAPK,p-c-Jun,FOXO3a and Ki67 in tumor were detected by immunohistochemical staining.Results1.GEO database download data and analysis showed that compared with benign hyperplasia tissues,the expression of β2-AR in primary PCa and advanced PCa was increased.Compared with primary PCa,β2-AR expression was decreased in castration-resistant PCa.In addition,β2-AR expression was also reduced in PCa tissues of patients treated with docetaxel chemotherapy.Protein levels of β2-AR decreased and mRNA levels increased in cells treated with docetaxel.By detecting the expression of β1AR,β2-AR,β3-AR in different PCa cell lines PC-3,22Rv1,DU145,LNCaP,β2-AR was found to be the main β-AR subtype in those cells.The expression of β2-AR in LNCaP cells was lower than other cells.2.The effect of β2-AR inhibition on docetaxel sensitivity was examined by CCK8 assay.Propranolol,a β-AR antagonist,was found to increase the cell sensitivity to docetaxel by autophagy and apoptosis.Propranolol further increases docetaxel-induced apoptosis by reducing mitochondrial membrane potential and activating apoptotic cascade signals.3.Propranolol promoted the progress of autophagy and autophagic flux in PCa cells.The expression of β2-AR decreased in docetaxel and starvation treated cells in which autophagy was induced.The β2-AR agonist isoproterenol inhibited the expression of autophagy associated proteins,and propranolol promoted the expression of autophagy associated proteins.Isoproterenol inhibited autophagy by reducing autophagogenesis,and propranolol promoted autophagy by increasing autophagogenesi s.Propranolol further increased docetaxel-induced autophagy through Atg5/AMPK/mTOR signaling pathway.4.The IC50 of docetaxel-resistant cells was much higher than parental cells,and the growth of the drug-resistant cells was not significantly affected with continuous docetaxel treatment,indicating the successful construction of drug-resistant cells.The resistant cells were more sensitive to propranolol than parental cells.The protein and mRNA expression of β2-AR in drugresistant cells was higher than that in parental cells.Autophagy decreased in PC-3 resistant cells and increased in 22Rv1 resistant cells compared with parental cells.In the parental cells,cell activity was significantly decreased in the propranolol+docetaxel+autophagy inhibitor group compared with the propranolol+docetaxel group.In drug resistant cells,cell activity in the propranolol+docetaxel+autophagy inhibitor group was also significantly decreased compared with the propranolol+docetaxel group.5.Propranolol reduced the interaction between BECN1 and Bcl-2,and increased the interaction between BECN1 and Vps34 to promote autophagy.The expression of p38 MAPK,p-p38 MAPK,JNK,c-Jun,p-c-Jun S63,p-cJun S73 and FOXO3a decreased after propranolol treatment.Compared with the propranolol group or the propranolol+docetaxel group,the addition of p38 MAPK inhibitor further increased the expression of autophagic protein LC3BⅡ and anti-apoptotic protein Bcl-2,and further decreased the expression of apoptotic protein BAX.Compared with the propranolol group or the propranolol+docetaxel group,the addition of JNK inhibitor further increased the expression of anti-apoptotic protein Bcl-2,and decreased the expression of autophagy protein LC3BⅡ and apoptotic protein BAX.Propranolol downregulated FOXO3a expression.6.Propranolol combined with chloroquine further inhibit tumor growth in vivo.In addition,the combination of propranolol or chloroquine with docetaxel further enhanced the inhibitory effect of docetaxel on cell growth in vivo.The expression of p-p38,p-c-Jun,FOXO3a and Ki-67 in the tumor tissues treated with propranolol,chloroquine,docetaxel and combination group all decreased.Conclusions1.β2-AR may play a critical role in the progression of PCa and the anticancer effect of docetaxel on PCa.2.Inhibition of β2-AR activity promoted autophagy and apoptosis,resulting in increased cell sensitivity and reduced drug resistance to docetaxel.3.The expression of β2-AR in the resistant cells was up-regulated compared with the parental cells,and β2-AR affected the resistance to docetaxel.4.β-AR antagonist promoted autophagy by decreasing the interaction between BECN1 and Bcl-2 and increasing the interaction between BECN1 and Vps34.5.β-AR antagonist promoted autophagy and apoptosis through p38 MAPK and JNK signaling pathway,thus affecting drug sensitivity to docetaxel.6.β-AR antagonist affected the balance between autophagy and apoptosis by inhibiting FOXO3a expression,a key factor regulating in autophagy and apoptosis homeostasis.7.Propranolol in combination with chloroquine or separately with docetaxel further inhibited cell growth in vivo. |
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