Zuowen Liang

Plasmid-based Stat3 siRNA delivered by hydroxyapatite nanoparticles suppresses mouse prostate tumour growth in vivo

DNA vector-based Stat3-specific RNA interference (si-Stat3) blocks Stat3 signalling and inhibits prostate tumour growth. However, the antitumour activity depends on the efficient delivery of si-Stat3. The effects on the growth of mouse prostate cancer cells of si-Stat3 delivered by hydroxyapatite were determined in this study. RM-1 tumour blocks were transplanted into C57BL/6 mice. CaCl₂-modified hydroxyapatite carrying si-Stat3 plasmids were injected into tumours, and tumour growth and histology were determined. The expression levels of Stat3, pTyr-Stat3, Bcl-2, Bax, Caspase3, VEGF and cyclin D1 were measured by western blot analysis. Amounts of apoptosis in cancer cells were analysed with immunohistochemistry and the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay. The results showed that hydroxyapatite-delivered si-Stat3 significantly suppressed tumour growth up to 74% (P < 0.01). Stat3 expression was dramatically downregulated in the tumours. The immunohistochemistry and TUNEL results showed that si-Stat3-induced apoptosis (up to 42%, P < 0.01). The Stat3 downstream genes Bcl-2, VEGF and cyclin D1 were also strongly downregulated in the tumour tissues that also displayed significant increases in Bax expression and Caspase3 activity. These results suggest that hydroxyapatite can be used for the in vivo delivery of plasmid-based siRNAs into tumours.

Effects of a human plasma membrane-associated sialidase siRNA on prostate cancer invasion

Human plasma membrane-associated sialidase (Neu3) is one of several sialidases that hydrolyze sialic acids in the terminal position of the carbohydrate groups of glycolipids and glycoproteins. Neu3 is mainly localized in plasma membranes and plays crucial roles in the regulation of cell surface functions. In this study, we investigated the effects and molecular mechanisms of Neu3 on cell invasion and migration in vivo and in vitro. Initially, we found that the levels of Neu3 expression were higher in prostate cancer tissues and cell lines than in normal prostate tissues based on RT-PCR and Western blotting analyses. We then applied a Neu3 siRNA approach to block Neu3 signaling using PC-3M cells as model cells. Transwell invasion assays and wound assays showed significantly decreased invasion and migration potential in the Neu3 siRNA-transfected cells. RT-PCR and Western blotting analyses revealed that Neu3 knockdown decreased the expressions of the matrix metalloproteinases MMP-2 and MMP-9. In vivo, mice injected with PC-3M cell tumors were evaluated by SPECT/CT to determine the presence of bone metastases. Mice treated with attenuated Salmonella carrying the Neu3 siRNA developed fewer bone metastases than mice treated with attenuated Salmonella carrying a control Scramble siRNA, attenuated Salmonella alone or PBS. The results for bone metastasis detection by pathology were consistent with the data obtained by SPECT/CT. Tumor blocks were evaluated by histochemical, RT-PCR and Western blotting analyses. The results revealed decreased expressions of MMP-2 and MMP-9 at the mRNA and protein levels. Taken together, the present findings suggest that Neu3 is a promising molecular target for the prevention of prostate cancer metastasis.

Flavonoids intake and risk of prostate cancer: a meta-analysis of observational studies.

The aim of the study was to assess the association between total flavonoids/flavonoid subclasses intake and prostate cancer risk. Several databases were searched to select eligible studies with predefined criteria. Risk ratios (RRs) with 95% confidence intervals (CIs) were used as the effect size. Publication bias and sensitivity analysis were performed. A total of five studies including four prospective cohort studies and one case–control study were included in the meta‐analysis. The pooled result demonstrated a significantly increased risk of prostate cancer with higher intake of total flavonoids (RR = 1.12, 95% CI: 1.02–1.23, P = 0.013). However, sensitivity analysis indicated that there lacked a significant association after removing the study of Wang et al. (RR = 1.17, 95% CI: 0.94–1.46). Subgroup analysis stratified by flavonoids subclasses found that higher intake of anthocyanidins and flavan‐3‐ols were significantly associated with increased prostate cancer risk (RR = 1.12, 95% CI: 1.03–1.21, P = 0.011; RR = 1.21, 95% CI: 1.10–1.32, P < 0.001). Sensitivity analysis also indicated that after removing Wangs study, no significant association between anthocyanidins intake and prostate cancer risk was detected (RR = 1.22, 95% CI: 0.97–1.54). In conclusion, higher intake of flavonoids may not be associated with prostate cancer risk.

Atrazine promotes RM1 prostate cancer cell proliferation by activating STAT3 signaling

Atrazine, a widely used pesticide, is frequently detected in soil and surface water, which alarms epidemiologists and medical professionals because of its potential deleterious effects on health. Indeed, atrazine is a potent endocrine disruptor that increases aromatase expression in some human cancer cell lines. Both animal and human studies have suggested that atrazine is possibly carcinogenic, although discrepant results have been reported. In this study, RM1 cells were used to explore the atrazine effects on prostate cancer. Proliferation, migration and invasion of RM1 cells were assessed by colony formation, wound-healing and invasion assays, respectively, after in vitro exposure to atrazine. In addition, an RM1 cell xenograft model was generated to evaluate the effects of atrazine in vivo. To explore the molecular mechanisms, qRT‑PCR, immunohistochemistry, and western blot analyses were employed to detect mRNA and protein levels of STAT3 signaling and cell cycle related proteins, including p53, p21, cyclin B1 and cyclin D1. Interestingly, RM1 cell proliferation was increased after treatment with atrazine, concomitantly with STAT3 signaling activation. These results suggest that atrazine promotes RM1 cell growth in vitro and in vivo by activating STAT3 signaling.

Relationship between the expression of CD133, HIF-1α, VEGF and the proliferation and apoptosis in hypoxic human prostate cancer cells

This study measured the levels of expression of CD133, hypoxia-inducible factor (HIF)-1α and vascular endothelial growth factor (VEGF) in human prostate cancer cells grown under hypoxic and non-hypoxic conditions to compare the values to resulting amounts of proliferation and apoptosis in the cells. Human prostate cancer cell line LNCaP cells were routinely thawed, cultured and passaged. Actively growing cells were divided into batches. Cells in the control group were grown under 5% CO2 + 20% O2, and those in the hypoxia group were grown under 5% CO2 + 1% O2. The experiments were performed after 12, 24 and 72 h under each growth condition. The percentages of CD13+ cells were detected by flow cytometry, the expression of HIF-1α and VEGF was detected by western blot analysis, the cell proliferation rate was detected by the MTT assay, and the apoptotic rate was detected by flow cytometry. The results showed that the percentage of CD133+ cells, and the expressions of HIF-1α and VEGF for the cells in the hypoxia group increased gradually from 12 to 24, to 72 h, while there were no equivalent changes in the control group. Cell proliferation in the two groups increased gradually from 12 to 24, to 72 h, but was significantly higher at all time-points in the hypoxia group (p<0.05). There was no significant difference in terms of the amount of apoptotic cells at any of the three different time-points in either group, but the apoptotic cells in the hypoxia group were significantly less than those in the control group at each time-point, and the difference was statistically significant (p<0.05). We conclude that the expression of CD133+, HIF-1α and VEGF in human prostate cancer cells is related to conditions of hypoxia, which ultimately promotes the proliferation and reduces apoptosis in these cells.

Comparison of miRNA and gene expression profiles between metastatic and primary prostate cancer

The present study aimed to identify the regulatory mechanisms associated with the metastasis of prostate cancer (PC). The microRNA (miRNA/miR) microarray dataset GSE21036 and gene transcript dataset GSE21034 were downloaded from the Gene Expression Omnibus database. Following pre-processing, differentially expressed miRNAs (DEMs) and differentially expressed genes (DEGs) between samples from patients with primary prostate cancer (PPC) and metastatic prostate cancer (MPC) with |log2 fold change (FC)| >1 and a false discovery rate <0.05 were selected using the Linear Models for Microarray and RNA-seq Data 4 package of R. Next, a DEM-DEG regulatory network was constructed by downloading miRNA-DEG pairs from the miRNA.org database. Finally, functional annotation of each DEM-DEG module was performed using the Database for Annotation, Visualization and Integrated Discovery based on the Gene Ontology database. The upregulated miRNAs, including miR-144, miR-494 and miR-181a, exhibited a higher degree of connections compared with other nodes, including in the DEM-DEG regulatory network, and regulated a number of downregulated DEGs. According to the functional annotation of the DEM-DEG modules, miR-144 and its targeted DEGs enriched the highest number of biological process terms (36 terms), followed by miR-494 (24 terms), miR-30d (18 terms), miR-181a (15 terms), hsa-miR-196a (8 terms), miR-708 (7 terms) and miR-486-5p (2 terms). Therefore, these miRNAs may serve roles in the metastasis of PC cells via downregulation of their corresponding target DEGs.

Effects of Hydroxyapatite Nanoparticles on Apoptosis of Prostate Cancer

The incidence rate of prostate cancer has increased rapidly in China, and the disease will recur in about one third of patients and there is no cure for metastatic prostate cancer. The aim of this study was to investigate the effects of hydroxyapatite nanoparticles on prostate cancer cells. Our results showed that hydroxyapatite nanoparticles significantly reduced cell proliferation, invasion and induced apoptosis in RM-1 cells. This study suggests that hydroxyapatite nanoparticles may be a potential therapeutic agent with possible prostate cancer clinical applications.

Ablation of Ggnbp2 impairs meiotic DNA double-strand break repair during spermatogenesis in mice

Gametogenetin (GGN) binding protein 2 (GGNBP2) is a zinc finger protein expressed abundantly in spermatocytes and spermatids. We previously discovered that Ggnbp2 resection caused metamorphotic defects during spermatid differentiation and resulted in an absence of mature spermatozoa in mice. However, whether GGNBP2 affects meiotic progression of spermatocytes remains to be established. In this study, flow cytometric analyses showed a decrease in haploid, while an increase in tetraploid spermatogenic cells in both 30‐ and 60‐day‐old Ggnbp2 knockout testes. In spread spermatocyte nuclei, Ggnbp2 loss increased DNA double‐strand breaks (DSB), compromised DSB repair and reduced crossovers. Further investigations demonstrated that GGNBP2 co‐immunoprecipitated with a testis‐enriched protein GGN1. Immunofluorescent staining revealed that both GGNBP2 and GGN1 had the same subcellular localizations in spermatocyte, spermatid and spermatozoa. Ggnbp2 loss suppressed Ggn expression and nuclear accumulation. Furthermore, deletion of either Ggnbp2 or Ggn in GC‐2spd cells inhibited their differentiation into haploid cells in vitro. Overexpression of Ggnbp2 in Ggnbp2 null but not in Ggn null GC‐2spd cells partially rescued the defect coinciding with a restoration of Ggn expression. Together, these data suggest that GGNBP2, likely mediated by its interaction with GGN1, plays a role in DSB repair during meiotic progression of spermatocytes.