Evaluation of the effect of hyperthermia and electron radiation on prostate cancer stem cells

The aim of this study was to investigate the effect of hyperthermia, 6 MeV electron radiation and combination of these treatments on cancer cell line DU145 in both monolayer culture and spheroids enriched for prostate cancer stem cells (CSCs). Flowcytometric analysis of the expression of molecular markers CD133⁺/CD44⁺ was carried out to determine the prostate CSCs in cell line DU145 grown as spheroids in serum-free medium. Following monolayer and spheroid culture, DU145 cells were treated with different doses of hyperthermia, electron beam and combination of them. The survival and self-renewing of the cells were evaluated by colony formation assay (CFA) and spheroid formation assay (SFA). Flowcytometry results indicated that the percentage of CD133⁺/CD44⁺ cells in spheroid culture was 13.9-fold higher than in the monolayer culture. The SFA showed significant difference between monolayer and spheroid culture for radiation treatment (6 Gy) and hyperthermia (60 and 90 min). The CFA showed significantly enhanced radiosensitivity in DU145 cells grown as monolayer as compared to spheroids, but no effect of hyperthermia. In contrast, for the combination of radiation and hyperthermia the results of CFA and SFA showed a reduced survival fraction in both cultures, with larger effects in monolayer than in spheroid culture. Thus, hyperthermia may be a promising approach in prostate cancer treatment that enhances the cytotoxic effect of electron radiation. Furthermore, determination and characterization of radioresistance and thermoresistance of CSCs in the prostate tumor is the key to develop more efficient therapeutic strategies.     

Activated eosinophils upregulate the metastasis suppressor molecule E-cadherin on prostate tumor cells.

Cell adhesion molecules (CAMs) play an important role in cancer metastasis by facilitating attachment to vascular endothelia, invasion and spread into secondary tissue sites. We have shown that activated eosinophils (EosA) inhibited the growth of prostate cancer (Pca) cells in vitro. In the present study, we examined the ability of EosA 24 hr conditioned supernatants (EosAcs) to modulate the expression of ICAM-1, VCAM-1, ELAM-1, E-cadherin and N-cadherin expression on human Pca cell lines, Du-145 and PC-3 by flow cytometry. TNF-alpha, IL-10 and IL-12 were also evaluated. ICAM-1, expressed on PC-3 and DU 145 cells, was enhanced by TNF-alpha and IL-10. ELAM-1 was present on DU 145 cells but absent on PC-3. TNF-alpha and IL-10 enhanced ELAM-1 on DU 145, but EosA 24 hr supematants failed to do so. All three cytokines, namely IL-10, IL-12 and TNF-alpha-induced ELAM-1 on PC-3 tumor cells. Although VCAM-1 was absent on DU 145 and PC-3 cells, it was expressed on DU-145 cells after exposure to EosA: tumor cell co-cultures, and was expressed on PC-3 following exposure to IL-10 and IL-12. N-cadherin and E-cadherin were both expressed on DU-145. While N-cadherin was expressed on PC-3 cells, E-cadherin was not. N-cadherin was enhanced on DU-145 and PC-3 cells following exposure to EosA co-culture and upregulated on PC-3 by IL-10 and EosA 24 hr supernatants, but decreased by IL-12. E-cadherin was up-regulated on DU 145 cells following co-culture with EosA and was induced on PC-3 by IL-10 and IL-12, but not by EosA co-culture and 24 hr supematants. In conclusion, inflammatory and non-inflammatory cytokines modulate CAM expression on Pca cells; EosA and EosA 24 hr supernatants also exerted modulatory activity of CAM expression. Most significantly, the metastasis suppressor molecule, E-cadherin was enhanced on DU 145 cells by EosA and induced on PC-3 by IL-10 and IL-12 both of which are produced by EosA. This suggests potential use of these cytokines in immunotherapeutic strategies for prostate cancer and its metastasis.     

Salinomycin-induced apoptosis of human prostate cancer cells due to accumulated reactive oxygen species and mitochondrial membrane depolarization

The anticancer activity of salinomycin has evoked excitement due to its recent identification as a selective inhibitor of breast cancer stem cells (CSCs) and its ability to reduce tumor growth and metastasis in vivo. In prostate cancer, similar to other cancer types, CSCs and/or progenitor cancer cells are believed to drive tumor recurrence and tumor growth. Thus salinomycin can potentially interfere with the end-stage progression of hormone-indifferent and chemotherapy-resistant prostate cancer. Androgen-responsive (LNCaP) and androgen-refractive (PC-3, DU-145) human prostate cancer cells showed dose- and time-dependent reduced viability upon salinomycin treatment; non-malignant RWPE-1 prostate cells were relatively less sensitive to drug-induced lethality. Salinomycin triggered apoptosis of PC-3 cells by elevating the intracellular ROS level, which was accompanied by decreased mitochondrial membrane potential, translocation of Bax protein to mitochondria, cytochrome c release to the cytoplasm, activation of the caspase-3 and cleavage of PARP-1, a caspase-3 substrate. Expression of the survival protein Bcl-2 declined. Pretreatment of PC-3 cells with the antioxidant N-acetylcysteine prevented escalation of oxidative stress, dissipation of the membrane polarity of mitochondria and changes in downstream molecular events. These results are the first to link elevated oxidative stress and mitochondrial membrane depolarization to salinomycin-mediated apoptosis of prostate cancer cells.     

Anti-proliferative effects of paeonol on human prostate cancer cell lines DU145 and PC-3

Paeonol (Pae) is the main active ingredient from the root bark of Paeonia moutan and the grass of Radix Cynanchi Paniculati. Numerous reports indicate that Pae effectively inhibits several types of cancer lines. In this study, we report that Pae hinders prostate cancer growth both in vivo and in vitro. Human prostate cancer lines DU145 and PC-3 were cultured in the presence of Pae. The xenograft tumor in mice was established by subcutaneous injection of DU145 cells. Cell growth was measured by MTT, and the apoptosis was detected by the flow cytometry. Expression of Bcl-2, Bax, Akt, and mTOR were tested by western blotting assay. DU145 and PC-3 showed remarkable sensitivity to Pae, and exposure to Pae induced dose-and time-dependent growth inhibitory responses. Moreover, treatment of Pae promoted apoptosis and enhanced activities of caspase-3, caspase-8, and caspase-9 in DU145. Further work demonstrated Pae reduced expression of Bcl-2 and increased expression of Bax in DU145. Interestingly, we observed that Pae significantly decreased phosphorylated status of Akt and mTOR, and inhibitory effects of Pae and PI3K/Akt inhibitor on DU145 proliferation were synergistic. Finally, we confirmed that oral administration of Pae to the DU145 tumor-bearing mice significantly lowered tumor cell proliferation and led to tumor regression. Pae possesses inhibitory effects on prostate cancer cell growth both in vitro and in vivo, and the anti-proliferative effect may be closely related to its activation of extrinsic and intrinsic apoptotic pathway and inhibition of the PI3K/Akt pathway.     

DU-145 and PC-3 human prostate cancer cell lines express androgen receptor: implications for the androgen receptor functions and regulation

The majority of human prostate cancer cell lines, including the two "classical" cell lines DU-145 and PC-3, are reported to be androgen receptor (AR)-negative. However, other studies have provided evidence that the DU-145 and PC-3 cell lines express AR mRNA. These contradictory observations prompted us to investigate whether DU-145 and PC-3 cell lines express the androgen receptor. Using antipeptide antibodies directed against three distinct regions of the human AR protein and an improved method to detect AR protein in immunoblotting, we report that DU-145 and PC-3 cell lines express AR protein. We found that the relative levels of the AR mRNA and protein that were detected in DU-145 and PC-3 cell lines were lower than the LNCaP, an AR-positive cell line. Moreover, the antibody directed against the non-variant region (amino acids 299-315), but not the variant N- or C-terminal region (amino acids 1-20 and 900-919, respectively) of the human AR protein, detected the expression of AR in all prostate cancer cell lines. Notably, treatment of these cell lines with dihydrotestosterone (DHT) resulted in measurable increases in the AR protein levels and considerable nuclear accumulation. Although, treatment of DU-145 and PC-3 cells with DHT did not result in stimulation of the activity of an AR-responsive reporter, knockdown of AR expression in PC-3 cells resulted in decreases in p21(CIP1) protein levels, and a measurable decrease in the activity of the p21-luc-reporter. Our observations demonstrate the expression of AR protein in DU-145 and PC-3 prostate cancer cell lines.     

Cholestane-3β, 5α, 6β-triol Suppresses Proliferation,Migration, and Invasion of Human Prostate Cancer Cells

Oxysterols are oxidation products of cholesterol. Cholestane-3β, 5α, 6β-triol (abbreviated as triol) is one of the most abundant and active oxysterols. Here, we report that triol exhibits anti-cancer activity against human prostate cancer cells. Treatment of cells with triol dose-dependently suppressed proliferation of LNCaP CDXR-3, DU-145, and PC-3 human prostate cancer cells and reduced colony formation in soft agar. Oral administration of triol at 20 mg/kg daily for three weeks significantly retarded the growth of PC-3 xenografts in nude mice. Flow cytometric analysis revealed that triol treatment at 10–40 µM caused G1 cell cycle arrest while the TUNEL assay indicated that triol treatment at 20–40 µM induced apoptosis in all three cell lines. Micro-Western Arrays and traditional Western blotting methods indicated that triol treatment resulted in reduced expression of Akt1, phospho-Akt Ser473, phospho-Akt Thr308, PDK1, c-Myc, and Skp2 protein levels as well as accumulation of the cell cycle inhibitor p27^Kip. Triol treatment also resulted in reduced Akt1 protein expression in PC-3 xenografts. Overexpression of Skp2 in PC-3 cells partially rescued the growth inhibition caused by triol. Triol treatment suppressed migration and invasion of DU-145, PC-3, and CDXR-3 cells. The expression levels of proteins associated with epithelial-mesenchymal transition as well as focal adhesion kinase were affected by triol treatment in these cells. Triol treatment caused increased expression of E-cadherin protein levels but decreased expression of N-cadherin, vimentin, Slug, FAK, phospho-FAK Ser722, and phospho-FAK Tyr861 protein levels. Confocal laser microscopy revealed redistribution of β-actin and α-tubulin at the periphery of the CDXR-3 and DU-145 cells. Our observations suggest that triol may represent a promising therapeutic agent for advanced metastatic prostate cancer.     

miR-155 通过p53/p21 调控前列腺癌细胞周期

目的:探讨miR-155对前列腺癌细胞周期的影响及其分子机制。方法:通过转染anti-miR-155抑制前列腺癌DU145和PC-3细胞中miR-155水平后,采用流式细胞术观察细胞周期的变化,western blot和RT-PCR观察p53和p21蛋白及CDK2和cyclin蛋白和m RNA表达的变化。结果:与对照组相比,DU145和PC-3细胞转染anti-miR-155后,G2/M期细胞阻滞,S期细胞数比例显著增加(P0.05),p53和p21蛋白和m RNA表达水平显著增加(P0.01),CDK2和cyclin E蛋白和m RNA表达均显著降低(P0.01)。结论:miR-155可影响人前列腺癌细胞的周期,可能与其调节p53、p21及其下游的CDK2和cyclin E的表达相关。     

Prostate tumor CXC-chemokine profile correlates with cell adhesion to endothelium and extracellular matrix

Though chemokines of the CXC family are thought to play key roles in neoplastic transformation and tumor invasion, information about CXC chemokines in prostate cancer is sparse. To evaluate the involvement of CXC chemokines in prostate cancer, we analyzed the CXC coding mRNA of both chemokine ligands (CXCL) and chemokine receptors (CXCR), using the prostate carcinoma cell lines PC-3, DU-145 and LNCaP. CXCR proteins were further evaluated by Western blot, CXCR surface expression by flow cytometry and confocal microscopy. The expression pattern was correlated to adherence of the tumor cells to an endothelial cell monolayer or to extracellular matrix components. Based on growth and adhesion capacity, PC-3 and DU-145 were identified to be highly aggressive tumor cells (PC-3>DU-145), whereas LNCaP belonged to the low aggressive phenotype. CXCL1, CXCL3, CXCL5 and CXCL6 mRNA, chemokines with pro-angiogenic activity, were strongly expressed in DU-145 and PC-3, but not in LNCaP. CXCR3 and CXCR4 surface level differed in the following order: LNCaP>DU-145>PC-3. The differentiation factor, fatty acid valproic acid, induced intracellular CXCR accumulation. Therefore, prostate tumor malignancy might be accompanied by enhanced synthesis of angiogenesis stimulating CXC chemokines. Further, shifting CXCR3 and CXCR4 from the cell surface to the cytoplasm might activate pro-tumoral signalling events and indicate progression from a low to a highly aggressive phenotype.     

Docetaxel/zoledronic acid combination triggers apoptosis synergistically through downregulating antiapoptotic Bcl-2 protein level in hormone-refractory prostate cancer cells

Docetaxel, a semi-synthetic taxane analogue, is used effectively in the treatment of metastatic prostate cancer. Zoledronic acid, the most potent member of bisphosphonates, has shown pleiotropic anti-tumoral effects on prostate cancer cells. We have explored the possible additive/synergistic effects and the apoptotic pathways induced by combination treatment of docetaxel and zoledronic acid in hormone and drug refractory, PC-3 and DU-145 prostate cancer cells. Combination of docetaxel and zoledronic acid synergistically inhibits cell growth in PC-3 and DU-145 cells. Moreover, this effect was due to downregulation of antiapoptotic protein Bcl-2 in PC-3 and DU-145 cells. In conclusion, docetaxel/zoledronic acid combination is potentially a novel and effective approach for the treatment of prostate cancer.     

Inhibitory effects of the HDAC inhibitor valproic acid on prostate cancer growth are enhanced by simultaneous application of the mTOR inhibitor RAD001

AimsTo analyze the combined impact of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell growth.Main methodsPC-3, DU-145 and LNCaP cells were treated with RAD001, VPA or with an RAD001–VPA combination for 3 or 5 days. Tumor cell growth, cell cycle progression and cell cycle regulating proteins were then investigated by MTT assay, flow cytometry and Western blotting, respectively. Effects of drug treatment on cell signaling pathways were determined.Key findingsSeparate application of RAD001 or VPA distinctly reduced tumor cell growth and impaired cell cycle progression. Significant additive effects were evoked when both drugs were used in concert. Particularly, the cell cycle regulating proteins cdk1, cdk2, cdk4 and cyclin B were reduced, whereas p21 and p27 were enhanced by the RAD001–VPA combination. Signaling analysis revealed deactivation of EGFr, ERK1/2 and p70S6k. Phosphorylation of Akt was diminished in DU-145 but elevated in PC-3 and LNCaP cells.SignificanceThe RAD001–VPA combination exerted profound antitumor properties on a panel of prostate cancer cell lines. Therefore, simultaneous blockage of HDAC and mTOR related pathways should be considered when designing novel therapeutic strategies for treating prostate carcinoma.     

Inhibition of cell survival signal protein kinase B/Akt by curcumin in human prostate cancer cells

Although curcumin has been shown to inhibit prostate tumor growth in animal models, its mechanism of action is not clear. To better understand the anti-cancer effects of curcumin, we investigated the effects of curcumin on cell survival factor Akt in human prostate cancer cell lines, LNCaP, PC-3, and DU-145. Our results demonstrated differential activation of Akt. Akt was constitutively activated in LNCaP and PC-3 cells. Curcumin inhibited completely Akt activation in both LNCaP and PC-3 cells. The presence of 10% serum decreased the inhibitory effect of curcumin in PC-3 cells whereas complete inhibition was observed in 0.5% serum. Very little or no activation of Akt was observed in serum starved DU-145 cells (0.5% serum). The presence of 10% serum activated Akt in DU-145 cells and was not inhibited by curcumin. Results suggest that one of the mechanisms of curcumin inhibition of prostate cancer may be via inhibition of Akt. To our knowledge this is the first report on the curcumin inhibition of Akt activation in LNCaP and PC-3 but not in DU-145 cells.     

Adrenomedullin inhibits prostate cancer cell proliferation through a cAMP-independent autocrine mechanism

Adrenomedullin (AM) is a multifunctional peptide expressed in the normal and malignant prostate, and in prostate cancer cells. To elucidate the potential role of AM in prostate cancer, we have transfected the human AM gene into PC-3, DU 145, and LNCaP prostate cancer cells. Northern blot, Western blot, and radioimmunoassay techniques confirmed an increase in the synthesis and secretion of the 6kDa mature peptide, in the AM-transfected clones. Proliferation and cell cycle assays demonstrated that AM overexpression inhibited cell proliferation in PC-3 and LNCaP cells through a G0/G1 cell cycle arrest, but not in DU 145 cells. In vivo growth assays also confirmed that, at least in PC-3, AM produced a very significant reduction of tumor volume. In addition, the three cell lines expressed the CL/RCP/RAMP-2 receptor complex by RT-PCR, which suggests that AM peptide acts through an autocrine loop in prostate cancer cells. Although cAMP elevation is the most common pathway involved in AM signalling, stimulation of PC-3, DU 145, and LNCaP with synthetic AM did not increase intracellular cAMP. However, short-term stimulation of PC-3 cells with synthetic AM increased ERK1/2 activation. On the contrary, long-term stimulation, or AM overexpression, caused a reduction in the basal activation of ERK1/2. In summary, our results demonstrate that AM (either overexpressed or exogenously added) causes an inhibition of prostate cancer cell growth. This inhibition does not depend on changes in intracellular cAMP levels, but may be related to ERK1/2 activation.     

Natural antibodies against nerve growth factor inhibit in vitro prostate cancer cell metastasis

Prostate cancer is a major cause of death in older men, and bone metastasis is the primary cause of morbidity and mortality in prostate cancer. Prostate is an abundant source of nerve growth factor (NGF) that is secreted by malignant epithelial cells and utilized as an important autocrine factor for growth and metastasis. We previously showed that intravenous gammaglobulin (IVIg) contains natural antibodies against NGF, which inhibit growth and differentiation of the NGF-dependent cell line PC-12. In the present study, we examined the effects of these natural antibodies on in vitro migration or metastasis of two prostate cancer cell lines namely DU-145 and PC-3. Cancer cell migration was assessed using these cell lines in the upper chambers of Matrigel invasion chambers. The effects of IVIg and affinity-purified anti-NGF antibodies on cell migration through membrane into the lower chamber were assessed in dose/response experiments by a colorimetric method. Affinity-purified natural IgG anti-NGF antibody inhibited DU-145 migration by 38% (p = 0.01) and PC-3 migration by 25% (p = 0.02); whereas, a monoclonal anti-NGF antibody inhibited DU-145 migration by 40% (p = 0.01) and PC-3 migration by 37% (p = 0.02), at the same concentration. When IVIg was depleted of NGF-specific IgG by affinity chromatography, there was no significant inhibition of migration of the DU-145 and PC-3 cells at a concentration of 1 mg/well. Removal of the NGF-specific antibody from the IVIg was also demonstrated by a lack of effect on PC-12 cell differentiation. Therefore, IVIg is able to inhibit the migration of prostate cancer cell lines, through Matrigel chambers in vitro, only when the natural NGF-specific antibodies actively are present in IVIg.     

SRJ23, a new semisynthetic andrographolide derivative: in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis in prostate cancer cells

Purpose

3,19-(3-Chloro-4-fluorobenzylidene)andrographolide (SRJ23), a new semisynthetic derivative of andrographolide (AGP), exhibited selectivity against prostate cancer cells in the US National Cancer Institute (NCI) in vitro anti-cancer screen. Herein, we report the in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis induced by SRJ23.

Methods

3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used in assessing in vitro growth inhibition of compounds against prostate cancer (PC-3, DU-145 and LNCaP) and mouse macrophage (RAW 264.7) cell lines. Flow cytometry was utilised to analyse cell cycle distribution, whereas fluorescence microscopy was performed to determine morphological cell death. DNA fragmentation and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry were done to confirm apoptosis induced by SRJ23. Quantitation of cell cycle and apoptotic regulatory proteins were determined by immunoblotting.

Results

AGP and SRJ23 selectively inhibited the growth of prostate cancer cells compared with RAW 264.7 cells at low micromolar concentrations; however, SRJ23 was more potent. Mechanistically, SRJ23-treated PC-3 cells displayed down-regulation of cyclin-dependent kinase (CDK) 1 without affecting levels of CDK4 and cyclin D1. However, SRJ23 induced down-regulation of CDK4 and cyclin D1 but without affecting CDK1 in DU145 and LNCaP cell lines. DNA histogram analysis revealed that the SRJ23 induced G2/M in PC-3 cells but G1 arrest in DU-145 and LNCaP cells. Morphologically, both compounds induced predominantly apoptosis, which was further confirmed by DNA fragmentation and annexin V-FITC staining. The DNA fragmentation was inhibited in the presence of caspase 8 inhibitor (Z-IETD-FMK). Apoptosis was associated with an increase in caspase 8 expression and activation. This thought to have induced cleavage of Bid into t-Bid. Additionally, increased expression and activation of caspase 9 and Bax proteins were apparent, with a concomitant down-regulation of Bcl-2 protein. Similar apoptosis cascade of events was observed in SRJ23-treated DU145 and LNCaP cell lines.

Conclusion

SRJ23 inhibited the growth of prostate cancer cells by inducing G₂/M and G1 arrest via down-regulation of CDK1, and CDK4 and cyclin, respectively, and initiated caspase-8-mediated mitochondrial apoptosis. Taken together, these data support the potential of this compound as a new anti-prostate cancer agent.     

Adrenomedullin prevents apoptosis in prostate cancer cells

The 52-aminoacid peptide adrenomedullin (AM) is expressed in the normal and malignant prostate. We have previously shown that prostate cancer cells produce and secrete AM, which acts as an autocrine growth inhibitory factor. We have evaluated in the present study the role of AM in prostate cancer cell apoptosis, induced either by serum deprivation or treatment with the chemotherapeutic agent etoposide (which acts as an inhibitor of topoisomerase II). For this purpose we over-expressed AM in PC-3, DU 145 and LNCaP cells, which were transfected with an expression vector carrying AM. We also treated the parental cell lines with synthetic AM in normal culture conditions and in conditions of induced-apoptosis. After serum removal, AM prevented apoptosis in DU 145 and PC-3 cells, but not in LNCaP cells. When treated with etoposide, AM prevented apoptosis in PC-3 and LNCaP cells, but not in DU 145 cells. Cell cycle analysis demonstrated a significant decrease in the percentage of AM-overexpressing PC-3 cells in the subG0/G1 phase after treatment with etoposide, as compared to the percentage of mock-transfected PC-3 treated cells. Western blot showed that protein levels of phosphorylated ERK1/2 increased in parental PC-3 cells after treatment with etoposide. In PC-3 cells overexpressing AM, phosphorylated ERK1/2 basal levels were lower than basal levels of parental PC-3 cells, and treatment with etoposide did not result in such an increase. Etoposide produced a significant increase in cleaved PARP in parental PC-3 cells. However, PC-3 clones overexpressing AM that were treated with etoposide only showed a mild increase in fragmented PARP. The ratio Bcl-2/Bax was reduced in parental or mock-transfected PC-3 cells after treatment with etoposide. On the contrary, this ratio was not reduced in PC-3 clones with AM overexpression that were treated with etoposide. All these data demonstrate that AM plays a protective role against induced apoptosis in prostate cancer cells. These results may have important implications in prostate cancer resistance to chemotherapeutic agents.     

miR-182在前列腺癌组织中的表达及其对前列腺癌细胞增殖和凋亡的影响

目的:观察前列腺癌组织及不同前列腺癌细胞系中miR-182的表达,并探讨下调其表达对前列腺癌细胞增殖和凋亡的影响及机制。方法:采用实时荧光定量PCR(q RT-PCR)检测30例前列腺癌组织和30例相应的癌旁组织以及前列腺正常上皮RWPE-1细胞、前列腺癌PC-3、LNCa P和DU145细胞中miR-182的表达,进一步采用Lipfectamine 2000脂质体转染miRNA-182 inhibitor和阴性对照miRNA于PC-3细胞后,通过噻唑蓝(MTT)比色法检测细胞增殖情况,流式细胞术检测细胞凋亡率,免疫印迹(Western blot)法检测转录因子FOXO1、血管内皮生长因子(VEGF)和抑癌基因p53蛋白的表达。结果:miR-182在前列腺癌组织中的表达明显高于癌旁组织(P0.05);miR-182在前列腺癌细胞系PC-3、LNCa P和DU145中的表达均高于前列腺正常上皮细胞RWPE-1(P0.05),其中PC-3细胞中miR-182表达水平最高。转染miRNA-182 inhibitor至PC-3细胞成功下调miR-182表达后,细胞的增殖能力明显受到抑制,细胞凋亡能力明显增强,FOXO1表达水平显著升高,VEGF和p53的表达明显降低,差异均具有统计学意义(P0.05)。结论:miR-182在前列腺癌组织及细胞中呈高表达,下调miR-182的表达可能通过增加FOXO1的表达并减少VEGF和p53的表达,抑制前列腺癌细胞增殖并诱导细胞凋亡。     

A novel synthetic protoapigenone analogue, WYC02-9, induces DNA damage and apoptosis in DU145 prostate cancer cells through generation of reactive oxygen species

The protoapigenone analogue WYC02-9, a novel synthetic flavonoid, has been shown to act against a variety of experimental tumors. However, its effects on prostate cancer and its mechanism of action are unknown. Thus, WYC02-9 was investigated for its cytotoxicity against DU145 prostate cancer cells, as was the underlying mechanisms by which WYC02-9 might induce DNA damage and apoptotic cell death through reactive oxygen species (ROS). WYC02-9 inhibited the cell growth of three prostate cancer cell lines, especially DU145 cells. In DU145 cells, WYC02-9 increased the generation of intracellular ROS, followed by induction of DNA damage and activation of the ATM-p53-H2A.X pathway and checkpoint-related signals Chk1/Chk2, which led to increased numbers of cells in the S and G2/M phases of the cell cycle. Furthermore, WYC02-9 induced apoptotic cell death through mitochondrial membrane potential decrease and activation of caspase-9, caspase-3, and PARP. The above effects were all prevented by the ROS scavenger N-acetylcysteine. Administration of WYC02-9 in a nude mouse DU145 xenograft model further identified the anti-cancer activity of WYC02-9. These findings therefore suggest that WYC02-9-induced DNA damage and mitochondria-dependent cell apoptosis in DU145 cells are mediated via ROS generation.