Exogenous P27 Expression Induces Anti-tumor Action And Its Correlation To EGFR/PI3K/Akt Signaling Pathway In PC3 Cells

Prostate cancer is one of the leading causes of cancer death in the developed countries and the incidence is increasing around the world. Although most cases initially respond to androgen deprivation, eventually this therapy fails and the patients die of recurrent hormone-refractory prostate cancer (HRPC). Currently, there is no effective therapeutic option for this form of disease. The precise molecular mechanisms of HRPC development are poorly understood, however, to elucidate such mechanisms is the frist step toward improving treatment outcomes of HRPC.Progression toward hormone-refractory correlates with up-regulation of autocrine and paracrine growth factor loops, including overexpression of epidermal growth factor recepror (EGFR). EGFR plays a critical role in tumor growth, and the prostate tissue becomes more susceptible to the growth-promoting action of EGF family growth factors during androgen withdrawal The general inhibition of tyrosine kinase signaling pathways provides therapeutic advantage against prostate cancer metastasis .Therefore, inhibiting the activation of growth factor receptors, especially EGFR may be a promising strategy for the treatment of prostate cancer.Expression of the cyclin-dependent kinase inhibitor (CKI) p27 is lost in a large fraction of human prostate cancer, and reduced or absent expression of p27 correlates with poor clinical outcome . Exogenous p27 overexpression results in cell cycle regulation and an increase in cell apoptosis in the human prostate carcinoma cell lines In addition, some studies demonstrate that EGF-induced stimulation of growth in prostate cancer cells is associated with down-regulation of p27 . Based on those theories, we hypothesis that the anti-tumor action of (CKI) p27 may be a mechanism by which EGF signaling pathway be involved in prostate cancer cells.The goal of this study was to determine whether restoring of p27 expression in human hormone-independent prostate cancer PC3 cells had major effect on proliferation, apoptosis and EGFR signaling pathway altering of PC3 cells. PC3 and LNCaP, derived from human prostate cancer, were obtained from the American Type Culture Collection (Rockville, MD, USA). These cells were maintained at 37℃ in a humidified atmosphere of 5% CO₂ /95% air and serially passaged in RPMI-1640 (Hyclone), supplemented with 10% fetal bovine serum.Total RNA was isolated from normal prostate tissue using Trizol^TM (Invitrogen, Inc) according to the manufacturer's instructions. Two micrograms of total RNA was used for cDNA synthesis (Superscript? II Reverse Transcriptase; Invitrogen) and one-tenth of the obtained cDNA was used for PCR to amplify p27 and p27-HA-tag. The conditions of PCR for each individual gene were optimized to analyzed amplified product in the linear range of amplification by adjusting amplification cycles for each set of primer. The nucleotide sequences of the primers used to amplify the p27 and p27-HA-tag genes were as follows: p27, sense primer 5'-CGC GAA TTC GCG ATG TCA AAC GTG CGA GTG TCT-3' and antisense primer 5'-CCG GGA TCC GGC GTT TGA CGT CTT CTG AGG CC-3', p27-HA-tag, sense primer 5'-CGC GAA TTC GCG ATG TCA AAC GTG CGA GTG TCT-3' and antisense primer 5'-GCC GGA TCC TTA AGC GTA ATC CGG AAC ATC GTA TGG GTA CGT TTG ACG TCT TCT GAG GCC-3', respectively.The amplified p27-HA-tag and p27 DNA were cloned into a pGEM^?-T Easy vector (Promega), following the protocol provided by the manufacturer. The DNA was digested with EcoR I and BamH I restriction enzymes and the fragment representing p27-HA-tag and p27 cDNA were excised from a 1% agarose gel. The DNA was purified using a QIA quick Gel extraction Kit (Qiagen). The resulting p27-HA-tag fragment was directionally cloned into the EcoR I and BamH I restriction sites of plasmid pcDNA3.1n (-) (Invitrogen). p27 was cloned into pEGFP-N1(Clontech) at BamH I and EcoR I sites. The successful cloning was confirmed by sequencing the plasmid.Cell viability was evaluated by MTT assay. The viable cell number was monitored with absorbance at 570nm. At the same time, we used a trypan blue dye exclusion assay to determine cell viability after the treatments. After the cells were collected by trypsinization, they were stained with trypan blue, and the total number cells and the number of viable cells in each well were counted.We used flow cytometry to analysis the cell cycle and apoptosis. The cells transfected with various plasmids DNA for 24 h or 48 h were assayed for cell cycle progression by the propidium iodide (PI) staining method. However, Annexin V (Annexin V-FITC) and PI double staining were used to determine apoptosis.To prodive further confirmation that anti-tumor effect of p27 induces apoptosis in PC3 cells, we also analyzed apoptotic-related proteins using western blot method.To investigate the effects of EGFR on PC3 cells, we analyzed signaling molecules related to the EGFR pathway. The EGFR, PI3K (p85), Akt and p-Akt (S473) were detected by western blotting with antibodies.PC3 cells were transfected with pEGFP-Nl-p27 24 h and 48 h after transfection, reporter gene expression was monitored by fluorescence microscopy and the fluorescence signal was quantified by flow cytometry. Many brightly fluorescence cells were found in transfected PC3 cells. The GFP fusion protein was readily detectable by flow cytometry, transfection efficiency about 50%⁵5% at 24 h and about 45%-47% at 48 h.Proliferation status was assessed by MTT assay. Transfection of PC3 cells with p27 gene produced a significantly lower absorbance compared with the control group (p<0.05) at the 48 h time point after transfection, but failed to produce a measurable difference at the 24 h time point after transfection.Consistent with our cell proliferation studies, transfected of PC3 cells with p27 gene induced increase in cell accumulation at G0/G1 phase and a corresponding decrease in the number of cells in S and G2/M phases. The results indicate that antiproliferative effect by p27 was associated with cell cycle arrest in the G0/G1 phase.The results from an annexin-V FITC binding assay showed that the higher proportion of annexin V positive cells were observed in p27 gene transfected group at the 24 and 48 h time point after transfection, with values of 14.59% and 16.60%. These values were greater than in control-plasmid transfected group.The effect of p27 on cellular apoptosis corresponds to the decrease in caspase-3 protein expression (caspase-3 activation is presented by the loss of its pro-form), which is concurrent with decreasing Bcl-2 protein and increasing Bax and Bad proteins expression in transfected cells. Consistent with the caspase-3 activation results, the p27 markedly increased the level of cleaved PARP The results suggesting that cellular apoptosis induced by the p27 was triggered by activation of caspase-3 and regulated by Bcl-2 protein. EGFR signaling might be one of the most critical signaling mechanisms for cancer cells, including prostate cancer cells To investigate the effects of EGFR on PC3 cells, we analyzed signaling molecules related to the EGFR pathway. The EGFR, PI3K (p85), Akt and p-Akt (S473) were detected by western blotting with antibodies. As shown in, the expression of EGFR decrease after transfection of p27 gene, densitometry analysis of EGFR protein levels was inhibited by 47% and 62% at the 24 h and 48 h time point after transfection.To analyze the downstream signaling events of the EGFR pathway, we next examined phosphorylation of PI3K (p8S), which also showed significant inhibition by the exogenous expression of p27. In addition, when we examined the downstream signaling molecules Akt and p-Akt (S473), levels of p-Akt (S473) was significantly inhibited by exogenous p27 in PC3 cell. Densitometry analysis shows that p-Akt (S473) protein levels are decreased by 43% and 50% at 24 h and 48 h time point after transfection. These data suggest that the exogenous expression of p27 can inhibit EGFR-mediated cell survival signals such as PI3K and Akt.In summary, we demonstrated that plasmid transfection of p27 gene into PC3 cells that do not express mis gene inhibited cell growth, resulted in accumulation of cells in the G1 phase of the cell cyle, and induced apoptosis. This results, at least in part, by direct inhibition EGFR/PI3K/Akt signaling pathway. Therefore, our results not only define a molecular mechanism that provides an explanation for the exogenous p27 expression induces anti-tumor action but also suggest that the p27 gene and its regulated pathway are targets for treatment of HRPC.