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Objective@#To observe the effect of Yishen Tonglong Decoction (YTD) on the epithelial-mesenchymal transition (EMT) and Ras/ERK signaling pathway in human PCa DU-145 cells and explore its action mechanism.@*METHODS@#We treated human PCa DU-145 cells with normal plasma (the blank control) or plasma containing 5% (low-dose), 10% (medium-dose) and 15% (high-dose) YTD. After intervention, we examined the proliferation of the DU-145 cells in different groups with CCK-8 and their apoptosis by Annexin V/PI double staining. We detected the cell cycle by PI assay, the invasion and migration of the cells using the Transwell chamber and scratch test, and the expressions of the proteins and genes related to the EMT and Ras/ERK signaling pathways in the cells by Western blot and RT-PCR.@*RESULTS@#Compared with the blank control group, high-, medium- and low-dose YTD significantly inhibited the proliferation of the PCa DU-145 cells, decreased their adherence and growth (P < 0.05, P < 0.01), promoted their apoptosis (P < 0.01), regulated their cell cycles (P < 0.05, P < 0.01), and reduced their in vitro invasion and migration abilities (P < 0.05), all in a dose-dependent manner. The results of Western blot and RT-PCR revealed down-regulated protein and mRNA expressions of N-cadherin, zinc finger transcription factor (Snail), Ras, p-ERK1/2 and ERK1/2, but up-regulated protein and mRNA expressions of E-cadherin in the PCa DU-145 cells treated with YTD (P < 0.05, P < 0.01).@*CONCLUSIONS@#Yishen Tonglong Decoction can effectively inhibit the proliferation, promote the apoptosis, regulate the cell cycle and suppress the invasion and migration abilities and EMT process of human PCa DU-145 cells. The mechanism of Yishen Tonglong Decoction acting on PCa may be associated with its inhibitory effect on the EMT process and expression of the Ras/ERK signaling pathway in PCa cells./.
Subject(s)
Humans , Male , Drugs, Chinese Herbal , Epithelial-Mesenchymal Transition , Prostatic Neoplasms , Signal TransductionABSTRACT
@#[Abstract] Objective: To study the effect of microRNA-141(miR-141) expression regulation on cell proliferation, cell cycle, apoptosis, invasion and migration of human prostate cancer cell line DU145 and its mechanism. Methods: MiR-141 mimics (miR-141 up group) and miR-141 inhibitors (miR-141 down group) were transfected into human prostate cancer DU145 cells by using liposome lipofectamine 2000, and the un-transfection group (Control group) and non-sense miRNA sequence transfection group (NC group) were set. The expression of miRNA-141 in DU145 cells in each group before and after transfection was detected by qPCR. MTT assay was used to detect the proliferation viability and sensitivity to cisplatin (DDP) in DU145 cells of each group. Cell cycle and apoptosis rate of DU145 cells under DDP treatment were detected by Flow cytometry; the changes in cell invasion and migration ability were detected by Transwell method. The protein expressions of VEGF and EGFR in DU145 cells of each group were detected by Western blotting. Results: Compared with the Control and NC group, the level of miRNA-141 expression in the miR-141-down group decreased to (0.18±0.08), the cell proliferation viability decreased significantly while its sensitivity to DDP increased significantly, the cell cycle was blocked in the G0+G1 phase, and the apoptosis rate significantly increased to (46.67±5.86)% while cell invasion rate and migration rate significantly decreased to (44.34±8.32)%, (57.73±6.19)%, and the relative expression levels of VEGF and EGFR decreased to (0.47±0.06), (0.36±0.06), (P<0.05 or P<0.01). But in the miR-141-up group, the level of miRNA-141 expression increased to (4.23±0.53), the cell proliferation viability significantly increased while its sensitivity to DDP decreased significantly, and the cell cycle was promoted into S and G2 phase, the apoptosis rate significantly decreased to (18.77±4.24)% while cell invasion and migration rate significantly increased to (89.94±6.34)%, (94.44±5.84)%, and the relative expression levels of VEGF and EGFR were up to (0.89±0.07), (0.73±0.06),(P<0.05 or P<0.01). Conclusion: miR-141 can act as a growth promoting factor in prostate cancer DU145 cells. miR-141 down-regula‐tion can significantly inhibit the proliferation viability, cell cycle, migration and invasion of DU145 cells, and promote cell apoptosis and DDP-sensitivity, and the mechanism of which may be related with inhibition of VEGF and EGFR protein expressions.
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Objective: To investigate the effects and mechanism of gochnatiolide A from Ainsliaea in anti-prostate cancer. Methods: The activities of gochnatiolide A on the proliferation of DU145 cells were tested by CCK-8 and colony formation experiments. Apoptosis morphological changes of cells were observed by DAPI staining. The apoptosis and cell cycle were evaluated by flow cytometry. The expressions of cleaved Poly ADP ribose polymerase (cleaved-PARP), cleaved cystein-containing aspartate specific protease 9 (cleaved Caspase-9), tumor suppressor protein (p53), b cell lymphoma-2 (Bcl-2), transcription factors-κB p65 (NF-κB p65) and IκB kinase α, β (IKKα, IKKβ) proteins in DU145 cells were investigated by Western blotting. Results: The natural product gochnatiolide A significantly inhibited the proliferation of prostate cancer DU145 cells, with IC50 values of 4.15, 2.80 and 1.74 μmol/L at 12 h, 24 h, and 48 h, respectively. Meanwhile, gochnatiolide A promoted DU145 cells apoptosis and induced cell cycle arrest at G2 and S phases in a concentration dependent manner. In addition, gochnatiolide A also up-regulated apoptosis proteins cleaved-PARP, cleaved Caspase-9 and p53 proteins, and down-regulated Bcl-2 protein. Comparing the control group, the expression of NF-κB p65, IKKα and IKKβ proteins in the NF-κB pathway were decreased after treatment with gochnatiolide A. Conclusion: The natural product gochnatiolide A remarkably inhibited the proliferation and promoted apoptosis in DU145 cells, and the possible mechanism was related to the inhibition of NF-κB pathway.
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Background and purpose:miR-17-92 gene cluster overexpression has been observed in various cancers, such as lung cancer, liver cancer, gastric cancer and prostate cancer. In this study, we established the stable cell line overexpressingmiR-17-92 to explore the inlfuence ofmiR-17-92 on the migration, invasion abilities and cisplatin resistance of the prostate cancer DU145 cells.Methods:miR-17-92 overexpression vectors were constructed. DU145 cells were infected with the viral supernatants produced by Phoenix A packaging system. Real-time lfuorescent quanti-tative polymerase chain reaction (RTFQ-PCR) was conducted to detect the expression level of miR-17-92 in the cells. The migration and invasion abilities were measured by a real-time xCELLigence system. The scratch healing assay was carried out to investigate the migration abilities. The expression of integrin β1 was detected by Western blot, and the activities of matrix metalloprotein-2 (MMP-2) and matrix metalloprotein-9 (MMP-9) were measured by gelatin zymography experiment. The cell growth of the two cell lines after the treatment of cisplatin was detected by a real-time xCELLigence system. The mRNA expression ofERCC1 was measured by RTFQ-PCR. Western blot was conducted to investigate the protein expressions of ERCC1, ERK1/2 and pERK1/2.Results:DU145-miR-17-92 cells migrated faster than DU145-control cells during the 24 h continuous monitoring (P<0.01). The scratch healing assay indicated that DU145-miR-17-92 cells migrated from the edge towards the scratch center faster than DU145-control cells. DU145-miR-17-92 cells invaded through matrigel markedly faster than DU145-control cells (P<0.01). The protein expression level of integrin β1 and the MMP-9 activities in DU145-miR-17-92 cells were increased than those in DU145-control cells. After the treatment of cisplatin, DU145-miR-17-92 cells grew faster than DU145-control cells, presenting cisplatin resistance (P<0.01). The phosphorylation of ERK1/2 in DU145-miR-17-92 cells was constantly at a high level regard-less of the treatment of cisplatin. Compared with DU145-control cells, the expression of drug resistance-related gene ERCC1 was dramatically increased in DU145-miR-17-92 cells after the treatment of cisplatin.Conclusion:miR-17-92 overexpression increases the migration and invasion abilities of the prostate cancer DU145 cells, which is associated with the upregulated expression of integrin β1 and the increased activity of MMP-9. Besides,miR-17-92 overexpression enhances the cisplatin resistance of DU145, which is correlated with the increased phosphorylation level of ERK and the upregulated expression of ERCC1 at both the mRNA and protein levels.
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Objective@#To investigate the anti-prostate cancer (PCa) effect of roemerine in vitro and in vivo in the mouse model of PCa.@*METHODS@#We detected the effects of roemerine on the proliferation, apoptosis and migration of PCa cells DU145, LNCaP, PC-3 and 22RV1, screened out the sensitive cell line and constructed a tumor-bearing model in mice for verification of the antitumor efficacy of roemerine in vivo.@*RESULTS@#Roemerine inhibited the proliferation and migration of the DU145, LNCaP, PC-3 and 22RV1 cells and induced their apoptosis in different degrees, particularly those of the LNCaP cells. The average tumor weight was less in the roemerine intervention group (［1.99±0.95］ g) than in the control (［2.95±1.04］ g), the least in the high-dose roemerine (30 mg/kg) plus paclitaxel intervention group (［0.90±0.16］ g). The mean heart, liver, and kidney indexes were markedly lower in the roemerine (0.58±0.06, 6.20±0.42 and 1.49±0.33) than in the paclitaxel group (0.66±0.04, 6.99±0.72 and 1.95±0.34), while the mean spleen and thymus indexes were remarkably higher in the former (0.54±0.11 and 0.06±0.01) than in the latter (0.41±0.09 and 0.05±0.01). Pathological staining showed a lower degree of malignancy and metastasis in both the roemerine and the roemerine + paclitaxel intervention group than in the control, as well as a lower degree of visceral injury in the roemerine and roemerine + paclitaxel groups than in the paclitaxel group.@*CONCLUSIONS@#Roemerine has some anti-PCa effect and alleviates adverse reactions in paclitaxel combination administration.
Subject(s)
Animals , Male , Mice , Alkaloids , Therapeutic Uses , Antineoplastic Agents, Phytogenic , Therapeutic Uses , Apoptosis , Cell Line, Tumor , Cell Movement , Cell Proliferation , Disease Models, Animal , Drug Therapy, Combination , Methods , Drugs, Chinese Herbal , Therapeutic Uses , Mice, Nude , Paclitaxel , Therapeutic Uses , Prostatic Neoplasms , Drug TherapyABSTRACT
Objective@#To study the expression of long noncoding RNA (lncRNA) H19 in human prostate cancer tissue and its effect on the glycometabolism and growth of human prostate cancer cells.@*METHODS@#Realtime quantitative RTPCR (qRTPCR) was employed to detect the expression of lncRNA H19 in human prostate tissues from 20 patients with prostate cancer (10 cases of highGleason score prostate cancer ［HGPC］ and 10 cases of lowGleason score prostate cancer ［LGPC］) and another 5 with benign prostatic hyperplasia (BPH). After transfection of H19 siRNA into the DU145 and PC3 prostate cancer cells, the growth of the cells and the H19 expression in the cells were determined by MTT and qRTPCR respectively, and the changes in the glycometabolism of the prostate cancer cells were analyzed by measuring the contents of glucose and lactate in the culture medium. Nontransfected and transfected negative vectors were used as blank and negative controls respectively.@*RESULTS@#The relative expression of H19 was significantly increased in both the HGPC and LGPC tissues (0.725±0.385 and 2.086±0.542) as compared with that in the BPH tissue (0.210±0.068) (P< 0.01), even higher in the HGPC than in the LGPC tissue (P< 0.01). After transfection of H19 siRNA, the expressions of H19 were remarkably decreased in the DU145 and PC3 prostate cancer cells in comparison with those in the blank control and negative control groups (P< 0.01), and so were the proliferation of and the glucose and lactate levels in the DU145 and PC3 cells (P< 0.01).
Subject(s)
Humans , Male , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Glucose , Metabolism , Lactic Acid , Metabolism , Prostate , Metabolism , Prostatic Hyperplasia , Metabolism , Prostatic Neoplasms , Metabolism , Pathology , RNA, Long Noncoding , Genetics , Metabolism , RNA, Small Interfering , TransfectionABSTRACT
BACKGROUND: Bile acids have anti-cancer properties in a certain types of cancers. We determined anticancer activity and its underlying molecular mechanism of ursodeoxycholic acid (UDCA) in human DU145 prostate cancer cells. METHODS: Cell viability was measured with an MTT assay. UDCA-induced apoptosis was determined with flow cytometric analysis. The expression levels of apoptosis-related signaling proteins were examined with Western blotting. RESULTS: UDCA treatment significantly inhibited cell growth of DU145 in a dose-dependent manner. It induced cellular shrinkage and cytoplasmic blebs and accumulated the cells with sub-G1 DNA contents. Moreover, UDCA activated caspase 8, suggesting that UDCA-induced apoptosis is associated with extrinsic pathway. Consistent to this finding, UDCA increased the expressions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor, death receptor 4 (DR4) and death receptor 5 (DR5), and TRAIL augmented the UDCA-induced cell death in DU145 cells. In addition, UDCA also increased the expressions of Bax and cytochrome c and decreased the expression of Bcl-xL in DU145 cells. This finding suggests that UDCA-induced apoptosis may be involved in intrinsic pathway. CONCLUSIONS: UDCA induces apoptosis via extrinsic pathway as well as intrinsic pathway in DU145 prostate cancer cells. UDCA may be a promising anti-cancer agent against prostate cancer.
Subject(s)
Humans , Apoptosis , Bile Acids and Salts , Blister , Blotting, Western , Caspase 8 , Cell Death , Cell Survival , Cytochromes c , Cytoplasm , DNA , Necrosis , Prostate , Prostatic Neoplasms , Receptors, TNF-Related Apoptosis-Inducing Ligand , Ursodeoxycholic AcidABSTRACT
Objective:To investigate the anti-tumor effect of Hyperoside.Methods: Human γδT cells were amplified by isopentenyl pyrophosphate from peripheral blood cells.The proliferation capacity of γδT cells was measured with CCK-8 assay after treated with different concentrations of Hyperoside.Cytotoxicity of γδT cells was detected with LDH assay , and the expression of granzyme,perforin CD107a and IFN-γonγδT cells were measured by flow cytometry before and after treatment.Results: Hyperoside could significantly stimulate the proliferation of γδT cells at the concentration of 3.13-12.5 μg/ml.Cytotoxicity and expression of granzyme,perforin and IFN-γofγδT cells were increased after treatment.Conclusion:Hyperoside could enhance cytotoxicity of humanγδT cells through up-regulation of granzyme ,perforin CD107 a and IFN-γexpression.
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<p><b>Objective</b>To investigate the potential role of the RhoA/Rock signaling pathway in the formation of prostate cancer and the effects of the Rock inhibitor fasudil on the invasion, migration and apoptosis of human prostate cancer cells.</p><p><b>METHODS</b>Human prostate cancer cell lines PC3 and DU145 were treated with fasudil at the concentrations of 5, 10, 20, 40, 80, and 160 μmol/L, respectively, and those as negative controls cultured in the Ham's-F12 medium, all for 24 hours. Then, MTT assay was used to measure the cell inhibition rate and half maximal inhibitory concentration (IC50) value of fasudil, with 1/4 of IC50 as the medication dose for further investigation. The expressions of RhoA, RockⅠ, and RockⅡ proteins in the PC3 and DU145 cells were detected by Western blot and immunohistochemistry, and the invasion, migration and apoptosis of the cells were determined using the Transwell chamber, scratch wound healing assay and flow cytometry.</p><p><b>RESULTS</b>Fasudil inhibited the proliferation of the PC3 cells from (9.29±1.23)% at 5 μmol/L to (81.37±3.97)% at 160 μmol/L and that of DU145 from (7.59±1.54)% to (76.53±2.67)%, both in a dose-dependent manner (P<0.05 ). Significantly fewer PC3 and DU145 cells migrated into the lower compartment in the experimental group (39.2±8.4 and 34.2±6.7) than in the negative control (116.8±9.3 and 112.5±10.8) (P<0.05 ). The wound healing rates of the PC3 and DU145 cells were remarkably lower in the former (［37.26±1.17］% and ［32.38±2.73］%) than in the latter (［78.12±4.16］% and ［69.47±6.71］%) (P<0.05 ). Annexin V-FITC/PI double staining showed markedly increased apoptosis rates of PC3 and DU145 cells treated with fasudil (［31.88±2.49］% and ［28.65±2.99］%) as compared with the negative controls (［7.51±2.28］% and ［7.13±1.61］%) (P<0.05 ). The expressions of RockⅠ and RockⅡ were significantly reduced in the fasudil-treated cells in comparison with those of the control group (P<0.05 ) while that of RhoA showed no significant difference between the two groups (P>0.05 ).</p><p><b>CONCLUSIONS</b>The RhoA/Rock signaling pathway may play an important role in the formation of prostate cancer. Fasudil can significantly inhibit the proliferation, migration, and invasion and promote the apoptosis of human prostate cancer PC3 and DU145 cells by reducing RhoA/Rho kinase activity.</p>
Subject(s)
Humans , Male , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine , Pharmacology , Apoptosis , Cell Line, Tumor , Cell Movement , Cell Proliferation , Prostatic Neoplasms , Drug Therapy , Pathology , Signal Transduction , rho-Associated KinasesABSTRACT
Objective To observe the influence of sirtinol,a silent information regulator 1(SIRT1)inhibitor,in the cell proliferation, cell cycle progression and the expression levels of positive regulator proteins of the cell cycle including Cyclin D1,CDK4 and pRb in prostate cancer DU145 cells,and to explore the possible mechanism of SIRT1 in occurrence of prostagte cancer.Methods The DU145 cells at logarithmic growth phase were cultured in vitro and divided into control group(DMSO)and different doses (10,25,50μmol·L-1 )of sirtinol groups.The inhibitory rate of growth of DU145 cells was detected with MTT method,the SIRT1 mRNA and protein expression levels were determined by RT-PCR and Western blotting method, and the cell cycle was measured by flow cytometry.The Cyclin D1,CDK4 and pRb protein expression levels were examined by Western blotting method. Results Compared with control group, the inhibitory rates of growth of the DU145 cells in different doses of sirtinol groups were increased markedly in a dose-dependent manner(P0.05).Conclusion SIRT1 inhibition by sirtinol can inhibit the cell growth of prostate cancer DU145 cells in a dose-dependent manner and arrest the cell cycle progression,and its mechanism may be related to decreasing the CyclinD1 and pRb protein expressions.
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Objective:To construct a specific small hairpin RNA (shRNA) expressing vectors against human receptor for advanced glycation end product (RAGE) gene and study its inhibitory effect on the proliferation of androgen-independent prostate cancer cells DU145. Methods:Four RAGE specific oligonucleotides were designed and synthesized. These oligonucleotides were annealed to forill double strand DNA fragments and this fragment was cloned into psi-U6 plasmid. The recombinants were transfected into RAGE-overexpressing sub DU145-2C1 cells. Cellular morphology and transfection efficiency were observed under fluorescence microscope. The inhibitory effect of RAGE shRNA construct on RAGE mRNA and protein expression was examined with semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The cellular proliferation was detected with cell counting kit-8 (CCK-8). Scratch test was used to observe the migration of DU145 cells.Results:RAGE shRNA expression plasmids were successfully constructed and transfected into sub DU145-2C1 cells. It can effectively inhibit the expression of RAGE mRNA (P<0.05). The inhibitory effects of shRNA RAGE-1 (R1) was the most stronger. The RAGE mRNA expression was inhibited by 84% and RAGE protein expression was inhibited by 27%. Compared with negative control, the proliferation potential was significantly decreased in shRNA RAGE-transfected cells. The cell migration capability had no significant changes. Conclusion:RAGE shRNA effectively inhibited the expression of RAGE mRNA and protein and suppressed the proliferation of DU145 cells in vitro.
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The differences in intracellular and extracellular protein expressions between human prostate cancer fines LNCap and DU145 were examined. The proteins of the two cell lines were extracted and condensed by using protein extraction kits. And the intracellular and extracellular proteins were quantitatively detected on a micro-plate reader by using bicinchoninie acid (BCA) method. The proteins in cell culture fluid were qualitatively assayed by SELDI-TOF-MS. The results showed that the intracellular protein contents of LNCap cells were extremely higher than those of DU145 cells. After serum-free culture, both intracellular and extracellular protein contents of LNCap and DU145 were decreased to some extent. And the intracellular proteins were decreased by 5% in LNCap and by 36% in DU145 respectively, while the extracellular proteins were decreased by 89% in LNCap and 96% in DU145 respectively. SELDI assay revealed that there were 5 marker proteins in LNCap and 6 in DU145. Although both LNCap and DU145 cell lines originated from human prostate cancer, they had some differences in protein expression.
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0.05). CAT expression of pCAT 1.28 and pCAT 0.65 are 2 and 1.6 times as high as the control group's. CAT expression of pCAT 1.28, pCAT 0.65 and pCAT 0.54 were 2.5, 2.2 and 1.3 times as high as the control group's. Aspirin inhibited the promoter activities of pCAT 1.28, pCAT 0.65. And the inhibition of Aspirin was reversed by PMA. CONGCLUSION:Aspirin can inhibit the expression of human MMP-9 gene in the transcriptional level and can inhibit the formation and development of prostatic carcinoma.
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Prostatic cancer is the typical hormone dependant cancer and several kinds of hormones are used for the treatment of prostate cancer. Since Harris had proposed the hypothalamo-pituitary gonadal axis, the hypothalamus is believed the exclusive organ producing GnRH. According to the recent researches, several organs were proved to be the extrahypothalamic GnRH sources in human and animal. In this research, the expression of the GnRH and GnRH receptor mRNA, detection of the GnRH which prostate cancer cell produced and effect of the GnRH on the prostate cancer cell proliferation using three human prostate cancer cell lines, ALVA 41, ALVA 101 and DU-145 were studied. In Situ Hybridization method was used for the detection of the expression of the GnRH and GnRH receptor mRNA. The charcoal stripped serum and high performance liquid chromatography were used for the detection of the GnRH produced from prostate cancer cells. Thymidine incorporation assay was used for the evaluation of the effect of the GnRH on the prostate cancer cell proliferation. The GnRH mRNA were detected in 96.7% of ALVA 41, 91.5% of ALVA 101 and 95.3% of DU-145 and GnRH receptor mRNA expression signals were detected in almost all of the examined prostate cancer cells, more than 95%, in three cell lines. The number of signals of the GnRH receptor mRNA were more than GnRH mRNA. The GnRH produced from the rostate cancer cell was detected at culture medium with retention time 19.40 minutes. The cancer cells cultured with peptide hormone deficient medium using charcoal stripped serum showed more than 20% growth retardation to the cancer cells grown at the medium used normal serum. The treatment of the GnRH on the cancer cells growing at the peptide hormone deficient medium showed statistically insignificant dose dependant growth retardation. The RESULTS of our research showed that the human prostate cancer cells, including two hormone refractory prostate cancer cell lines, produce the GnRH and the GnRH receptor in the same cell which could be suggest that the role of GnRH produced from the prostate cancer cell would be autocrine action. And the prostate cancer cell growth was down regulated by unknown complex of various peptide hormones and the GnRH does not has the significant effect on the proliferations of the prostate cancer cells. With those RESULTS we obtained in this research and other's data, it seems that there is a system that contains production of GnRH and GnRH receptor and metabolic mechinary within prostate cancer cell. And there should be the some changes in the hypothalamo-pituitary gonadal axis and the mechanism using GnRH analogues for the treatment of prostate cancer aside from central mechanism.