br The effect of green tea polysaccharide on the growth
3.6. The effect of green tea polysaccharide on the growth of PC-3 Bay 11-7821BAY 11-7082 and apoptotic protein expression
To study the effect of cytotoxic activity of GTP on cell viability, PC-3 cells were treated with increasing concentrations of GTP (25, 50 and 100 μg/ml). After 48 h, growth of PC-3 cells was significantly suppressed
by GTP in a dose-dependent manner (Fig. 6A), indicating that growth inhibitory effect of GTP on the PC-3 cells in vitro. More importantly, GTP hardly affects proliferation of a normal prostate epithelial cell line (PrEC), suggesting its safety (data not shown).
In order to explore whether GTP displayed cytotoxicity to PC-3 cells through inducing apoptosis, the percentage of apoptotic cells were fur-ther quantified using annexin V-FITC/PI Apoptosis Assay Kit by flow cy-tometry (Fig. 6B). In the absence of GTP, a small percentage of the total cell population (2.41 ± 0.36%) was apoptotic. In contrast, 24.3% ± 2.8% and 73.2% ±2.9% of the cell population were apoptotic in a concentration-dependent manner after treatment with 25, 50 and 100 μg/ml of GTP, respectively, which are all significantly different from the untreated cells (P b 0.05 or P b 0.01). Thus the annexin V/PI double staining assay confirmed GTP induces cell death via apoptosis in PC-3 cells.
Apoptosis is a type of programmed cell death and is controlled by a series of apoptosis related proteins such as Bcl-2 gene family, caspase family, cytochrome c, poly (ADP-ribose) polymerase (PARP), extracellu-lar signal-regulated kinase-1/2 (ERK1/2) and so on [31,32]. In our
Fig. 4. (A) Tumorigenesis of prostate cancer PC-3 cells in nude mice after miR-93 mimic or miR-NC injection on day 28; (B) gross growth observation of PC-3 xenograft tumor masses after miR-93 mimic or miR-NC injection on day 8, 18 and 28; (C) the PC-3 xenograft tumor growth curve after miR-93 mimic or miR-NC injection in nude mice. PC-3 cells stably expressing miR-93 or vector were injected to nude mice and xenograft tumor growth was assessed. These experiments were repeated three times and the data are represented as the mean ± SD. P b 0.05, P b 0.01, P b 0.001 vs. the miR-NC group.
current work, miR-93 plays a pivotal role in regulation of apoptosis and proliferation of PC-3 cells. We quantified the level of miR-93 in PC-3 cells following GTP treatment (25, 50 and 100 μg/ml) using qRT-PCR technique. As anticipated in Fig. 6C, miR-93 level decreased significantly with increasing GTP concentration in comparison with that in untreated PC-13 cells (P b 0.05 or P b 0.01), suggesting contribution of suppression of the miR-93 to GTP-induced apoptosis. As a continuing research, here we only detect several important apoptosis related protein (bax, bcl-2, caspase-3) in PC-3 cells after exposure of increasing concentrations of GTP (25, 50 and 100 μg/ml) by means of Western blot analysis. The levels of β-actin were monitored to ensure equal loading of protein samples. Treatment with GTP increased the expression levels of bax, and caspase-3, but decreased the levels of bcl-2 in the cell lines studied (Fig. 6D). All these findings confirmed that the possibility of miR-93 in-volvement in GTP-induced apoptosis in PC-13 cells.
4. Discussion and conclusions
In recent years, a growing number of PC patients develop clinical re-currence, and their survival period is limited and extremely variable . The challenge lies in the identification of those patients most at
risk for relapse. Accumulated evidence suggests that miRNAs are crucial regulators in the progression of PC [34,35] and have potential as diag-nostic and prognostic biomarkers in PC [36,37]. Indeed, miRNAs targeting either oncogenes or tumor suppressive genes are also promis-ing therapeutic tools for PC . Therefore, it is of great importance to identify PC associated miRNAs for use as biomarkers for diagnosis and treatment.
In present study, we used qRT-PCR and in situ hybridization to ana-lyze miR-93 levels in PC tissues and cell lines. miR-93 was significantly upregulated in PC cells and tissues when compared with human benign prostatic hyperplasia tissues. Overall survival rate analysis based on 60 PC patients by the Kaplan–Meier method indicated that miR-93 overex-pression was correlated with the poor survival of PC patients, confirming that miR-93 may serve as a potential prognostic biomarker for PC. To gain more insight into the role of miR-93 in PC growth, gain and loss-of-function studies were carried out in MTT, colony formation, cell migration and invasion analysis. The results showed that overex-pression of miR-93 by using mimics significantly promoted PC-3 cell proliferation, migration and invasion, while depletion of miR-93 by using its inhibitor significantly restrained this effect in DU145 cells. In addition, we explored the effect of overexpression of miR-93 on