| BACKGROUNDProstate cancer is one of the leading causes of death in male worldwide.Although it is curable at early stage,the median survival in man with advanced metastatic castration-resistant prostate cancer is less than 2 years.The first choice for early stage prostate cancer is radical surgery.Patients with locally advanced and metastatic prostate cancer underwent endocrine therapy and transformed to CRPC have to receive chemotherapy.Paclitaxel(PTX),a first-line antitumor drug,which is widely approved chemotherapy that is effective in extending the overall survival of prostate cancer patients.However,the side effects are obvious and most patients develop resistance and experience relapse.In fact,tumors easily acquire resistance to PTX and its derivatives,such as docetaxel,even with the assistance of various adjuvants and the growth and invasion speed will be increased.The medical institutions then will be forced to replace PTX for other drugs such as abiraterone.But it is difficult to widely used on account of its high cost of treatment.Thus,it's very important to develop new therapies to enhance the effect of PTX and reduce the dosage of paclitaxel for prostate cancer patients.Morin is a flavonoid,which can be used against a numerous types of cancer including breast cancer,ovarian cancer,lung cancer and leukaemia.Studies have reported that morin could regulate cell survival and proliferation-related genes,promote apoptosis and chemo-sensitivity in multiple cancer cell lines as well as some other flavonoids.It has been reported that significant changes in the level of apoptosis in LNcap cells could be observed after treatment with morin.In a number of studies,researchers have found that the combination of a variety of flavonoids,including morin,combined with PTX can enhance the anticancer effect of PTX.Oral administration of morin and PTX can significantly improve the pharmacodynamics of PTX and enhance the antitumor effect of paclitaxel.Apigenin,naringenin,baicalein,quercetin and myricetin could enhance the oral bioavailability of docetaxel.Nagaprashantha et al reported VCN-2,a new flavonoid,could inhibit the vitality of LNcap cells,DU 145 cells and PC-3 cells as well as the apoptosis of these cells were promoted.They also found that the effect of VCN-2 when combined with docetaxel was more significant than either of the single agents in androgen-independent prostate cancer.OBJECTIVEIn this research,we aimed to research whether morin can enhance the chemotherapeutic activity of PTX in prostate cancer..If positive results could be observed,we will try to research the mechanism.METHODSProstate cancer cell lines DU145 and PC-3 were selected as the research objects.We studied the effect of morin on paclitaxel in the treatment of prostate cancer and its molecular mechanism in vitro and in vivo.1.Evaluate the influence of morin on PTX in treating prostate cancer cells in vitro and in vivo(1)The cell viabilities and apoptosis rates of DU145 and PC-3 cells cultured in the environment of morin(0,10 ?M,20 ?M,50 ?M)and PTX(50 nM)were evaluated using CCK-8 assay and flow cytometry.The same amount of DMSO was treated in control groups.Then a suitable effective concentration of morin was determined.(2)The cell viabilities and apoptosis rates of DU 145 and PC-3 cells cultured in the environment of morin(50 ?M)and PTX(0-100 nM)were evaluated.(3)We investigated the inhibitory function of morin as an adjuvant to paclitaxel in treating DU 145 derived prostate cancer in nude mice models,in which nude mice were divided into 4 groups:?DMSO;?PTX;?morin;?PTX +morin.PTX and morin were injected by tail vein injection for 25 days.The tumors were excised and weighed after 25 days since the day of injection.2.Research the mechanism of morin enhancing the chemotherapeutic activity of PTX in prostate cancer in vitro and in vivo2.1 Determine the target miRNA of morin? MicroRNA(miRNA)microarray was used to detect the changes of miRNA profile in morin treated DU 145 cells.? The results from microarrays were further certified by quantitative real-time reverse transcription-PCR(qRT-PCR)in prostate cancer cell lines DU145 and PC-3.MiR-155 was determined as the research miRNA.? MiR-155 silenced DU145,PC-3 cell lines and miR-155 overexpressed DU145 and PC-3 cell lines were constructed for research.2.2 Research the effects of miR-155 on the proliferation and apoptosis of tumor cells in prostate cancer? The miR-155 in DU 145 and PC-3 cells was silenced or overexpressed,and the cells were treated with PTX(0-100nM).Then the cell viabilities and apoptosis rates were detected as described.? DU145 or PC-3 cells were transfected with miR-155 mimics,or control miR-NC,and were treated with morin and PTX.Then the cell viabilities and apoptosis rates were detected.2.3 Research the downstream effector of miR-155? We performed in silico analyses to determine the underlying targets of miR-155 through Targetscan and miRWalk,then qRT-PCR was performed to analyze the transcription of these proteins in miR-155 mimic transfected cells.? GATA3,the underlying targets of miR-155 was then verified using luciferase assays.? Western blot assays were performed to evaluate the effect of miR-155 on the expression of GATA3 protein.2.4 Research the function of GATA3 expression in prostate cancer?The expression level of GATA3 mRNA in prostate cancer normal adjacent tissues and tumor tissues were detected.? The cell viabilities and apoptosis rates were detected in GATA3 silenced or overexpressed DU 145 and PC-3 cell lines.2.5 Evaluate the regulatory effects of morin on the expression of GATA3 through miR-155? Western blot assays were used to detect the effect of morin treatment on the expression of GATA3 in DU145 and PC-3 cells in vitro.? The expression level of GATA3 in nude mice tumors was then detected.3.Immunohistochemistry analysisKi67,cleaved caspase-3 and GATA3 were immunohistochemistry analysed in DU 145 derived tumors in nude mice to evaluate the effect of morin on the proliferation and apoptosis of prostate cancer cells and expression of target gene in tumor cells.4.Data analysisAll data was analyzed using SPSS version 13.0(IBM,Armonk,NY,USA).The results were presented in the formation of mean ± standard deviation(SD).One or two-way ANOVA analyses was employed for analyzing statistical differences between multiple groups.Student T tests were used to verify the significance of differences between two groups.P<0.05 was considered as statistical significance.RESULTS1.Morin enhances chemotherapy effects of PTX in prostate cancer in vitro(1)In the reseach of the effects of morin((0,10? M,20 ?M,50 ?M)acting on PC-3 and DU 145 cells combined with PTX,50 ?M morin provided most significant effects,then 50?M was determined as the concentration of morin in following research.(2)When PTX was treated at the same time,the viabilities of morin-treated groups in both of the cell lines were significantly lower than control groups.Similar to the results in viability assay,morin led to significant increase in cell apoptosis compared with control groups in both of the cell lines.2.Morin enhanced the chemotherapy effects of PTX on prostate cancer in vivoBoth the data of tumor size and weight indicated that morin significantly promoted the anti-tumor function of PTX during the process of prostate cancer development in vivo.3.Morin inhibits miR-155 expression in prostate cancer cell lines(1)A total of 39 microRNAs were found to be altered in morin treated cells,with the most significant one being miR-155.(2)We assessed the effect of morin on the intracellular concentrations of miR-155 in DU 145 and PC-3 cell by qRT-PCR.The results revealed that the transcription of miR-155 was both reduced in morin-treated cells compared with control cells.4.MiR-155 silencing could enhance the chemotherapy effects of PTX while overexpression of miR-155 reduces chemotherapy effects of PTX and reverses the effects of morin on chemotherapy activity of PTX in tumor cells(1)In both cell lines,the viabilities of miR-155 silenced cells were lower than the control groups after PTX treatment.Apoptosis assays showed that miR-155 silenced cells exhibited elevated percentages of apoptosis cells compared with control cells.(2)The viabilities of miR-155 overexpressed cells were higher than the control groups after PTX treatment and apoptosis assays showed that miR-155 overexpressed cells exhibited reduced percentages of apoptosis cells.(3)The cell viabilities of negative transfected DU145 and PC-3 cells which were treated with morin combined with paclitaxel,were significantly lower than that of the single paclitaxel groups,and the apoptosis rates were significantly higher than that of the single paclitaxel groups.The cell viabilities of cells overexpressing miR-155 were significantly higher than that of negative transfected DU145 and PC-3 cells,and the level of apoptosis was significantly lower than that of negative transfected cells.5.GATA3 is a downstream effector of miR-155(1)Targetscan and miRWalk showed 3 genes among the 5348 genes evaluated maybe the downstream effectors of miR-155:PAK2,XRN1 and GATA3.(2)The transcription of these proteins in miR-155 mimics transfected DU 145 cells and control cells were then analyzed.The expressions of all of the three proteins were suppressed in different degree.However,the suppression effect of miR-155 mimic to PAK2 and XRN1 were limited comparing to its effects to GATA3.(3)Luciferase reporter assays and Western blotting both showed that the expression level of GATA3 in both cell lines were significantly suppressed by overexpression of miR-155 in comparison to the control group.6.The expression of GATA3 was significantly decreased in prostate cancer.Morin enhanced the antitumor activity of PTX by up-regulating GATA3 expression(1)The expression of GATA3 was significantly decreased in prostate cancer tissues(n=33)compared with prostate cancer normal adjacent tissues(n=15),which was conformity with the conclusions reported by the predecessors.(2)The overexpression of GATA3 increased the chemosensitivity of DU 145 and PC-3 cells to PTX.Silencing GATA3 reduced the chemosensitivity of DU145 and PC-3 cells to PTX.(3)We then evaluated the influences of morin and PTX on the expressions of GATA3 in DU 145 and PC-3 cell lines.The treatment of PTX or morin alone elevated the expression of GATA3,and morin enhanced the effects of PTX.The elevated expression of GATA3 under the treatment of morin was inhibited by miR-155 mimics.(4)In nude mice tumors,morin significantly enhanced the expression of GATA3,and the expression of GATA3 in tumors of the mice which were treated with PTX and morin group were highest.7.Immunohistochemistry analysis revealed morin could inhibit the proliferation and promote the apoptosis of tumor cells in prostate cancerMorin combined with PTX reduces expression of Ki67 and enhances expression of cleaved caspase-3.No positive staining of GATA3 was observed in all 4 groups of tumors.Our study revealed that the expression of GATA3 can be up-regulated by morin treatment via down-regulating the expression of miR-155.The overexpression of GATA3 could significantly increase the chemosensitivity of DU145 and PC-3 cells to PTX.Morin acted as an adjuvant could enhance the antitumor activity of PTX.The mechanism maybe that morin could inhibit miR-155 and then increase the activity of GATA3 gene and promote its expression,so as to enhance the effect of PTX on prostate cancer cell lines.CONCLUSIONSMorin may enhance the chemotherapy effects of PTX in prostate cancer cells DU145 and PC-3,significantly reducing the proliferation of these two cells and significantly increasing the apoptosis rates of DU 145 and PC-3 cells.Morin combined with paclitaxel significantly slows down the growth of tumor in nude mice.Morin enhanced the antitumor effect of paclitaxel in vivo.Morin treatment could significantly inhibit the expression of miR-155 in the cells and up-regulate the expression of GATA3.GATA3 is the direct target gene of miR-155.Morin can enhance the antitumor activity of PTX in prostate cancer cells.The main molecular mechanism maybe morin could inhibit miR-155 and then increase the activity of GATA3 gene and promote its expression. |
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