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Objective: To investigate whether ethanol extracts of Hizikia fusiforme could induce apoptosis in human prostate cancer PC3 cells.Methods: Cell viability was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. Apoptosis and mitochondrial membrane potential (MMP) were measured using flow cytometry in PC3 cells. DNA damage was assessed by nuclear staining and DNA fragmentation assay. Expressions of apoptosis-associated proteins were determined by Western blotting assays. Activities of caspase-3, -8, and -9 were determined by colorimetric assay. Moreover, intracellular reactive oxygen species (ROS) generation was detected using a flow cytometer and fluorescence microscope. Results: Treatment of PC3 cells with ethanol extracts of Hizikia fusiforme inhibited proliferation, which was associated with induction of apoptosis, and accompanied by increased expression of Fas, Fas-ligand (FasL), Bax and tBid, and decreased expression of Bcl-2. In addition, ethanol extracts of Hizikia fusiforme reduced c-Flip expression and activated caspase-8, -9 and -3, resulting in an increase in poly (ADP-ribose) polymerase (PARP)cleavage. However, in the presence of a pan-caspase inhibitor, ethanol extracts of Hizikia fusiforme-mediated growth inhibition and apoptosis were significantly attenuated. Ethanol extracts of Hizikia fusiforme also destroyed the integrity of mitochondria due to the loss of MMP, leading to cytosolic release of cytochrome c. Moreover, the levels of ROS were markedly increased by treatment with ethanol extracts of Hizikia fusiforme, which was significantly suppressed by the ROS scavenger N-acetyl-L-cysteine. Further investigation of whether ethanol extracts of Hizikia fusiforme-induced apoptosis was related to the generation of ROS was conducted and the results showed that N-acetyl-L-cysteine fully blocked ethanol extracts of Hizikia fusiforme-induced apoptotic events including loss of MMP, activation of caspase-3, the cytosolic release of cytochrome c and cytotoxicity.Conclusions: Ethanol extracts of Hizikia fusiforme have chemopreventive potential via induction of ROS-dependent apoptosis. Therefore, ethanol extracts of Hizikia fusiforme may be useful for developing effective and selective natural sources to inhibit cancer cell proliferation.
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Objective: To investigate whether ethanol extracts of Hizikia fusiforme could induce apoptosis in human prostate cancer PC3 cells. Methods: Cell viability was evaluated using 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide. Apoptosis and mitochondrial membrane potential (MMP) were measured using flow cytometry in PC3 cells. DNA damage was assessed by nuclear staining and DNA fragmentation assay. Expressions of apoptosis-associated proteins were determined by Western blotting assays. Activities of caspase-3, -8, and -9 were determined by colorimetric assay. Moreover, intracellular reactive oxygen species (ROS) generation was detected using a flow cytometer and fluorescence microscope. Results: Treatment of PC3 cells with ethanol extracts of Hizikia fusiforme inhibited proliferation, which was associated with induction of apoptosis, and accompanied by increased expression of Fas, Fas-ligand (FasL), Bax and tBid, and decreased expression of Bcl-2. In addition, ethanol extracts of Hizikia fusiforme reduced c-Flip expression and activated caspase-8, -9 and -3, resulting in an increase in poly (ADP-ribose) polymerase (PARP)cleavage. However, in the presence of a pan-caspase inhibitor, ethanol extracts of Hizikia fusiforme-mediated growth inhibition and apoptosis were significantly attenuated. Ethanol extracts of Hizikia fusiforme also destroyed the integrity of mitochondria due to the loss of MMP, leading to cytosolic release of cytochrome c. Moreover, the levels of ROS were markedly increased by treatment with ethanol extracts of Hizikia fusiforme, which was significantly suppressed by the ROS scavenger N-acetyl-L-cysteine. Further investigation of whether ethanol extracts of Hizikia fusiforme-induced apoptosis was related to the generation of ROS was conducted and the results showed that N-acetyl-L-cysteine fully blocked ethanol extracts of Hizikia fusiforme-induced apoptotic events including loss of MMP, activation of caspase-3, the cytosolic release of cytochrome c and cytotoxicity. Conclusions: Ethanol extracts of Hizikia fusiforme have chemopreventive potential via induction of ROS-dependent apoptosis. Therefore, ethanol extracts of Hizikia fusiforme may be useful for developing effective and selective natural sources to inhibit cancer cell proliferation.
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Objective To investigate the effect of interfering with E2F3 gene expression on the invasion and migration of prostate cancer cells and its mechanism. Methods The expression of E2F3 gene in human prostate cancer Du145 cells was knocked down by siRNA. The cells were divided into three groups: control group, Du145 NC group (siRNA-NC)and Du145-siRNA group(siRNA-E2F3). Cell migration and invasion were detected by Transwell invasion and wound healing assay. The expressions of E2F3, E-cadherin and MMP-9 proteins were detected by western blotting. Results After transfection, the expression of E2F3 protein in the Du145-siRNA group was significantly lower than the control group(P< 0.01). The number of invasive cells and wound healing rate of Du145 cells in the Du145-siRNA group were significantly lower than the control group(P < 0.01). Furthermore, the protein expression of E-cadherin was significantly increased(P< 0.01)while MMP-9 decreased(P< 0.01)in the Du145-siRNA group. Conclusions E2F3 silencing can inhibit the invasion and migration ability of prostate cancer Du145 cells, and this might be accomplished by regulating E-cadherin and MMP-9 protein.
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Objective To prepare a granulocyte targeting-mediated magnetic-fluorescent nanoprobe for detecting prostate cancer PC3 cells in vitro. Methods The nanometer materials with magnetic and red fluorescence, which were prepared using Fe3O4 as the core, and SiO2 and rhodamine isothiocyanate as the shell, were mixed with normal human peripheral blood granulocytes in different proportions, and co incubated for different periods to examine the toxicity of nanometer materials to granulocytes. The best proportion was selected to combine the nanometer materials and granulocytes in vitro. Finally we obtained the granulocyte targeting-mediated magnetic-fluorescent nanoprobes. We mixed PC3 cells and normal human whole blood cells in different proportions, added the nanoprobes, and then observed the targeting situation of the nanoprobes under a fluorescence microscope. Results The nanoprobe had no obvious influence on the survival rate of granulocytes at different concentrations and action times set in this study. The nanoprobes were enriched around the PC3 cells with a “petal-like” structure, but the peripheral blood cells were not enriched by probes. Conclusion The magnetic-fluorescent nanometer materials prepared in this study have no toxicity to granulocytes, and it can effectively detect tumor cells by the biological targeting effect of granulocytes on tumor cells.
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Objective: To study the chemical constituents of Myrrha and their antitumor activities. Methods: The constituents were isolated and purified by recrystallization, and open silica gel, Sephadex LH-20, ODS column chromatography, as well as preparative HPLC. The structures were elucidated based on the chemical and spectroscopic methods. Furthermore, the cytotoxicities of these chemical components against PC-3 cell lines were measured by MTT method. Results: Eleven compounds were obtained from the chloroform extract of myrrh, and were established as (4α,11α)-2-oxo-8,11-dihydroxycadina-1(6),7,9-trien-12-oic acid γ-lactone (1), (4α,11β)-2-oxo-8,11-dihydroxycadina-1(6),7,9-trien-12-oic acid γ-lactone (2), dihydropyrocurzerenone (3), orientalol E (4), guaianediol (5), cryptomeridiol (6), cycloartane-1α,2α,3β,25-tetrol (7), cycloartan-24-ene-1α,2α,3β-triol (8), cycloartan-24-ene-1α,3β-diol (9), 29-norlanost-8,24-dien-1α,2α,3β-triol (10), and octadecane-1,2S,3S,4R-tetrol-1-O-α-L-rhamnopyranoside (11). Conclusion: Compounds 1 and 2 are two new compounds named as (+)-myrrhalactone A and (-)-myrrhalactone A, respectively. Compounds 4 and 6 are isolated from the genus Commiphora Engl. for the first time. Compounds 8, 10, and 11 show moderate cyctotoxic activity against PC-3 cell lines.
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CONTEXT: Trichosanthin produced in the root tube of Trichosanthes kirilowii shows anti-tumor activity on a series of cancer cells including Hela, MCF-7, HL-60. But there is little information about its effect on the carcinogenesis of prostate cancer. OBJECTIVE: This work was designed to study the role of trichosanthin on prostate cancer cells PC3. MATERIALS AND METHODS: Trichosanthin was expressed in BL21 strain and purified by affinity chromatography. MTT assay was designed to determine the effect of trichosanthin on growth of PC3 cells at doses of 10, 20, 40, 60, 80, and 120 µg/ml.Then the effect of 50 µg/ml rTCS alone or combined with 2 µM IL-2 on PC3 cell proliferation was analyzed. And the mechanism of rTCS was studied by western blot. After that the in vivo effect of rTCS combined with IL-2 was explored in mice bearing PC3 xenograft tumor. RESULTS: Trichosanthin was successfully expressed in BL21 and purified by 100 mM imidazole. It was shown to inhibit proliferation of PC3 cells in a dose-dependent manner with IC50 50.6 µg/ml. When combined with cytokine IL-2, a significant synergic effect was obtained. The inhibition rate on PC3 was around 50 % in combination group while only 35.5 % in single rTCS group at 50 µg/ml. Further, the expression of full length caspase-8 and Bcl-2 decreased significantly while cleaved caspase-8 and Bax were up-regulated, which suggest that caspase-8-mediated apoptosis pathway may be activated by rTCS in PC3 cells. Moreover, our data demonstrated that tumor volume and tumor weight were significantly reduced in rTCS-treated or rTCS/IL-2-treated nude mice bearing PC3 xenograft tumor compared with control. And significant difference was also found between rTCS and rTCS/IL-2 group. CONCLUSIONS: This study demonstrates that rTCS is a potential agent with high in vitro and in vivo anti-tumor activity on PC3 cells. And rTCS combined with IL-2 is a promising strategy in treating patients with prostate cancer in future.
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Animals , Male , Female , Mice , Prostatic Neoplasms/drug therapy , Trichosanthin/pharmacology , Antineoplastic Agents, Phytogenic/pharmacology , Prostatic Neoplasms/pathology , Tetrazolium Salts , Time Factors , Recombinant Proteins/pharmacology , Blotting, Western , Reproducibility of Results , Apoptosis/drug effects , Cell Line, Tumor , Tumor Burden , Cell Proliferation/drug effects , FormazansABSTRACT
Objective To study the radiosensitizing effect of 2'-hydroxyflavanone (2'-HF) on prostate cancer cells,and to preliminarily investigate its mechanism.Methods Colony formation assay,tert-butylhydroperoxide (TBHP) oxidative stress assay,Hoechst staining,and apoptosis flow cytometry using Annexin V-FITC and propidium iodide (PI) were performed to measure the impact of 2'-HF on the radiosensitivity of VCaP prostate cancer cells.Western blot was used to determine the effects of 2'-HF on expression of AKT,phosphorylated AKT (p-AKT),and aldo-keto reductase 1 C3 (AKR1 C3) in VCaP cells and preliminarily investigate the mechanism.Data were analyzed by t test and factorial analysis of variance.Results The results of colony formation assay indicated that after exposure to radiation,VCaP cells treated with 2'-HF had a significantly lower proliferation level than cells in the control group (P=0.010),yielding a sensitization enhancement ratio of 1.19.The resuhs of TBHP oxidative stress assay suggested that VCaP cells treated with 2'-HF had significantly weaker anti-oxidative capacity than cells in the control group (P=0.015).Hoechst staining and apoptosis flow cytometry with Annexin V-FITC and PI indicated that 2'-HF treatment plus irradiation significantly enhanced apoptosis in VCaP cells (P=0.001.The results of Western blot suggested that 2'-HF treatment significantly inhibited the protein expression of p-AKT and AKR1C3 in VCaP cells (P=0.013 and P=0.016).Conclusions 2'-HF can enhance the radiosensitivity of prostate cancer cells,which is probably associated with its inhibitory effects on AKT pathway and AKR1C3 expression in prostate cancer cells.
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Objective To study the effects of IL-4 in prostate cancer cells glycometabolism and proliferation. Methods We used IL-4 to treat PC3 cells, then tested the changes of LDH-A expression by RT-qPCR, Western Blot, CCK-8 and lactate production assay. Results Our data showed that IL-4 induced LDH-A up-expression in PC3 cells at mRNA and protein levels. Also, IL-4 promoted the proliferation activation and increased lactate production in PC3 cells. Conclusion IL-4 can strengthen the proliferation activation in PC3 cells by up-regulating LDH-A expression.
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Objective To identify the role of miR-873, which may regulate the expression of survivin,in human pros-tate cancer PC3 cells invasion.Methods PC3 cells were cultured in vitro, and changes of cellular morphology were ob-served by phase contrast microscope.miR-873,which might regulate the expression of survivin,was predicted by bioinforma-tics and identified using dual luciferase report system.Expressions of miR-873 and survivin were determined using real-time quantitative PCR( qRT-PCR) and Western blotting after transfection of miR-873 mimics.The invasion of PC3 cells was de-tected in vitro by Transwell chamber.Results The expression of survivin was positive by immunofluorescence cytochemis-try.Using dual luciferase reporter system, miR-873 could inhibit the expression of survivin by binding to its mRNA 3′UTR.Results of qRT-PCR and Western blotting showed that overexpression of miR-873 down-regulated the expression of survivin.The invasion of PC3 cells was suppressed by over-expression of miR-873.Conclusion MiR-873 may negatively regulate the expression of survivin in human prostate cancer PC3 cells and inhibit cell invasion.
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Objective To explore the preliminary mechanism of mesenchymal stem cells (MSCs) in promoting prostate cancer proliferation in tumor inflammatory microenvironment.Methods From April 2013 to October 2013,MSCs pretreated with inflammatory cytokine IL-1α (MSCs (IL-1α)) and its culture supernatants mixed with RM-1 cells,which origined from C57BL/6 mice,were subcutaneously administered in the armpit area of C57BL/6 or BALB/c mice to establish homologous or heterologous transplant animal mode and to detect the tumor growth.Meanwhile the influence of MSCs on the proliferation of spleen cells was detected in vitro.Results In homologous transplant model,the relative tumor weight of prostate cancer cells prtreated with MSCs and MSCs (IL-1α) and their culture supernatant were (3.4 ± 0.2),(3.3 ±0.2),(4.9±0.5),and (5.2±0.6) g.The results were statistically significant (P＜0.05) compared with the control group (2.4±0.2) g.In heterologous model,the ratio of tumor formation of the pretreated groups were 50％,50％,80％ and 80％,respectively,compared with the control group of 0％.The results were statistically significant (P＜0.05).In proliferation experiments of spleen cells,the number of spleen cell pretreated with IL-1α were significantly lower than that in control group and unpretreated group (P ＜ 0.05).Conclusions MSCs pretreated with IL-1α could effectively promote the growth of prostate cancer cell in vivo.The reason may be due to inflammatory cytokines induce immune suppression of MSCs and then lead to immune escape of cancer cells.
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Objective To investigate the effect of platelet-derived growth factor-D (PDGF-D) on the prostate cancer cells migration and its possible mechanism. Methods The expressions of PDGF-D in LNCaP and PC-3 cells were detected with western blot.PDGF-D siRNA was synthesized according to mRNA sequence of PDGF-D gene and was transfected into PC-3 cell.The cells were treated with PDGF-D and PDGF-D siRNA,the cell migration was examined by Boyden chamber migration assay.The expression changes of VEGF and MMP-9 mRNA were detected by RT-PCR. Results The results of western blot indicated that the PDGF-D protein expression level was lower in LNCaP cells (29.47 ± 1.68) than that in PC-3 cells (63.43 ±2.10),(P ＜ 0.05).PDGF-D siRNA could down-regulate the PDGF-D protein expression in the transfected group (35.19 ± 1.51).The exogenous PDGF-D could promote migration of LNCaP and PC-3cells,and up-regulate the expression of VEGF,MMP-9 mRNA in PC-3 cells (P ＜ 0.05,compared with control group).PDGF-D siRNA inhibited PC-3 cells' migration and decreased the level of VEGF,MMP-9mRNA expression (0.72 ± 0.09 vs 0.43 ± 0.18,0.65 ±0.07 vs 0.22 ± 0.08) (P ＜ 0.05). Conclusion PDGF-D is involved in the promotion of prostate cancer invasion and angiogenesis.
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OBJECTIVE: 6-Gingerol, one component of ginger (Zingiber officinale) compound, has been known to possess anti-inflammatory, analgesic, anti-emetic, and anti-cancer effects. In this study, the apoptotic ability of 6-gingerol was investigated in human prostate cancer cells. METHODS: 3-(4,5-Dimethylthiazol-2-yl)- 2,5-diphenyl-tetrazolium bromide (MTT) assay, flow cytometry, and western blot analysis were done in LNCaP human prostate cancer cell lines treated with the various doses of 6-gingerol for the different durations of drug exposure. RESULTS: 6-Gingerol in doses ranging from 100 to 300 microM induced dose- and time-dependent inhibition of cell viability in prostate cancer cells by using MTT assay. Maximal inhibition of cell viability was observed at 300 microM of 6-gingerol for 48 hours treatment in LNCaP cells. 6-Gingerol at the dose of 100 microM did not produce any significant change in apoptotic cells in flow cytometry analysis. However, significant increase in sub-G0/G1 phase was observed in cells treated with 200 and 300 microM of 6-gingerol. Any significant cell cycle arrest was not induced by 6-gingerol. In western blotting analysis, expression of caspase-3 was not evident in cells treated with 6-gingerol for 24 hours. However, 48 hours treatment with 6-gingerol altered the expression of caspase-3 in LNCaP cells. Expression of cleaved poly showed the dose-dependent fashion in both 24 hours and 48 hours treatment of 6-gingerol. CONCLUSION: These observations suggest that 6-gingerol may induce apoptosis in LNCaP human prostate cancer cells.
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Humans , Apoptosis , Blotting, Western , Caspase 3 , Catechols , Cell Cycle Checkpoints , Cell Line , Cell Survival , Fatty Alcohols , Flow Cytometry , Zingiber officinale , Poly(ADP-ribose) Polymerases , Prostate , Prostatic NeoplasmsABSTRACT
PURPOSE: Apoptosis is a form of cell death characterized by specific morphological changes in the dying cell including contraction of cytoplasm, chromatin condensation, and cellular fragmentation into membrane-bound bodies. A common biological marker of apoptosis is the degradation of nuclear DNA resulting in a ladder of nucleosome-sized DNA fragments when resolved by electrophoresis. The potential therapeutic implications of simultaneous activation of apoptosis in androgen-dependent and androgen-independent prostatic cells are clearly very important in the development of cancer treatment modalities for advanced prostate cancer. The efficacy of chemotherapeutic agents correlates with their ability to induce apoptosis, Therefore, quantification of experimentally induced apoptosis in cancer cell lines is likely to be a predictor of the outcome of treatment. The main objective of this study was to examine the induction of apoptosis as a new strategy for cancer therapy by cis-diamminedichloroplatinum (CDDP) or 12-0-tetradecanoyl phorbol 13-acetate (TPA) in human prostate (androgen-dependent LNCaP and androgen-independent DU-145), and breast cancer cells (MCF-7). MATERIALS AND METHODS: DNA gel electrophoresis, flow cytometry and transmission electron microscopy for morphological analysis were used to further characterize drug response in human prostate and breast cancer cells. RESULTS: Treatment of the LNCaP and DU-145 cells with CDDP or TPA resulted in dose-dependent growth inhibition and accumulation of cells in Ao (apoptotic region), and caused significant degradation of the genomic DNA into intemucleosomal-sized DNA fragments, indicating apoptosis. In contrast, MCF-7 cells showed little or no DNA fragmentation. CONCLUSION: These studies suggest that a differential susceptibility to apoptosis and chemosensitivity may be related to the efficacy of chemotherapeutic .agents. CDDP and TPA may have clinical implication in the treatment of prostate cancer. In particular, cytotoxic effects of TPA may well lead to new possibilities for improved strategy.