Epigenetic upregulation of urokinase plasminogen activator promotes the tropism of mesenchymal stem cells for tumor cells.

A major obstacle for the effective treatment of cancer is the invasive capacity of the tumor cells. Previous studies have shown the capability of mesenchymal stem cells (MSC) to target these disseminated tumor cells and to serve as therapeutic delivery vehicles. However, the molecular mechanisms that would enhance the migration of MSCs toward tumor areas are not well understood. In particular, very little is known about the role that epigenetic mechanisms play in cell migration and tropism of MSCs. In this study, we investigated whether histone deacetylation was involved in the repression of urokinase plasminogen activator (uPA) expression in MSCs derived from umbilical cord blood (CB) and bone marrow (BM). Induction of uPA expression by histone deacetylase inhibitors trichostatin A and sodium butyrate was observed in CB- and BM-derived MSCs examined. In vitro migration assays showed that induction of uPA expression by histone deacetylase inhibitors in CB- and BM-derived MSCs significantly enhanced tumor tropism of these cells. Furthermore, overexpression of uPA in CB-MSCs induced migration capacity toward human cancer cells in vitro. In addition, our results showed that uPA-uPAR knockdown in PC3 prostate cancer cells significantly inhibited tumor-specific migration of uPA-overexpressing MSCs. These results have significant implications for the development of MSC-mediated, tumor-selective gene therapies.

Small interfering RNA-directed knockdown of uracil DNA glycosylase induces apoptosis and sensitizes human prostate cancer cells to genotoxic stress.

Uracil DNA glycosylase (UNG) is the primary enzyme responsible for removing uracil residues from DNA. Although a substantial body of evidence suggests that DNA damage plays a role in cancer cell apoptosis, the underlying mechanisms are poorly understood. In particular, very little is known about the role of base excision repair of misincorporated uracil in cell survival. To test the hypothesis that the repair of DNA damage associated with uracil misincorporation is critical for cancer cell survival, we used small interfering RNA (siRNA) to target the human UNG gene. In a dose-dependent and time-dependent manner, siRNA specifically inhibited UNG expression and modified the expression of several genes at both mRNA and protein levels. In LNCaP cells, p53, p21, and Bax protein levels increased, whereas Bcl2 levels decreased. In DU145 cells, p21 levels were elevated, although mutant p53 and Bax levels remained unchanged. In PC3 cells, UNG inhibition resulted in elevated p21 and Bax levels. In all three cell lines, UNG inhibition reduced cell proliferation, induced apoptosis, and increased cellular sensitivity to genotoxic stress. Furthermore, an in vitro cleavage experiment using uracil-containing double-stranded DNA as a template has shown that siRNA-mediated knockdown of UNG expression significantly reduced the uracil-excising activity of UNG in human prostate cancer cells, which was associated with DNA damage analyzed by comet assay. Taken together, these findings indicate that RNA interference-directed targeting of UNG is a convenient, novel tool for studying the biological role of UNG and raises the potential of its application for prostate cancer therapy.

Matrix metalloproteinase-1 promotes prostate tumor growth and metastasis.

Cell migration and invasion are critical events during the progression to metastasis. Matrix metalloproteinase-1 (MMP-1) is involved in the progression of human malignancies, but the precise role of MMP-1 in tumor invasion and metastasis remains unclear. In the present study, we investigated the role of MMP-1 in tumor cell invasion and metastasis by overexpressing MMP-1 in prostate cancer cells. Overexpression of MMP-1 in prostate cancer cells increases cell invasion and migration as measured by modified transwell assays. Furthermore, the results from a bioluminescence tumor/metastasis model showed that the overexpression of MMP-1 significantly induces prostate tumor growth and the incidence of lung metastasis. We observed that this increase in tumor growth correlates with an increase in tumor angiogenesis. In addition, we assessed the importance of MMP-1 expression in cell invasion and migration by inhibiting MMP-1 activity with specific inhibitor and antibodies. Blockade of MMP-1 activity inhibited prostate cancer cell migration and invasion in vitro. Treatment of mice with an MMP-1 specific inhibitor significantly decreased prostate tumor growth and incidence of lung metastasis in vivo. Collectively, our findings suggest that MMP-1 plays an important role in prostate cancer progression during the invasive and metastatic stages of the disease.

Inhibition of histone deacetylase activity promotes invasion of human cancer cells through activation of urokinase plasminogen activator.

Histone acetylation plays an important role in chromatin remodeling and gene expression. The molecular mechanisms involved in differential regulation of urokinase plasminogen activator (uPA) gene expression are not fully understood. In this study, we investigated whether histone deacetylation was involved in repression of uPA expression in human cancer cells. Induction of uPA expression by histone deacetylase (HDAC) inhibitors trichostatin A (TSA), sodium butyrate, and scriptaid was observed in all three different types of human cancer cells examined. Chromatin immunoprecipitation assays showed that the induction of uPA expression by TSA was accompanied by a remarkable increase of acetylation of histones H3 and H4, which are associated with the uPA promoter region in human cancer cells. These results were further substantiated by the findings of a restriction enzyme accessibility assay and TSA-stimulated uPA promoter activity through the inhibition of HDAC activity. In vitro Matrigel invasion assays showed that induction of uPA expression by HDAC inhibitors in human cancer cells resulted in a significant increase of cancer cell invasion. Furthermore, HDAC1 knockdown by small interference RNA stimulated uPA expression and cancer cell invasion. In conclusion, this study demonstrates the important role of histone modifications in regulating uPA gene expression and raises a possibility that the use of HDAC inhibitors in patients as cancer therapy may paradoxically establish metastasis through up-regulation or reactivation of uPA.

Small interfering RNA directed reversal of urokinase plasminogen activator demethylation inhibits prostate tumor growth and metastasis.

Recent studies have shown that small interfering RNA (siRNA) silences genes at the transcriptional level in human cells. However, the therapeutic potential of siRNA-mediated transcriptional gene silencing remains unclear. Here, we show that siRNA targeted to the urokinase plasminogen activator (uPA) promoter induced epigenetic transcriptional silencing in human prostate cancer cells. This silencing resulted in a dramatic reduction of tumor cell invasion and angiogenesis in vitro. Furthermore, the results from a bioluminescence tumor/metastasis model showed that the silencing of uPA significantly inhibits prostate tumor growth and the incidence of lung metastasis. Our findings represent a potentially powerful new approach to not only epigenetic silencing of metastasis or growth-promoting genes as a cancer therapy, but also as a means to shed light on how aberrant de novo methylation during cancer progression might be targeted to specific sequences.

Demethylation-linked activation of urokinase plasminogen activator is involved in progression of prostate cancer.

Increased expression of urokinase plasminogen activator (uPA) has been reported in various malignancies including prostate cancer. However, the mechanism by which uPA is abnormally expressed in prostate cancer remains elusive. Here, we show that uPA is aberrantly expressed in a high percentage of human prostate cancer tissues but rarely expressed either in tumor-matched nonneoplastic adjacent tissues or benign prostatic hyperplasia samples. This aberrant expression is associated with cancer-linked demethylation of the uPA promoter. Furthermore, treatment with demethylation inhibitor S-adenosylmethionine or stable expression of uPA short hairpin RNA significantly inhibits uPA expression and tumor cell invasion in vitro and tumor growth and incidence of lung metastasis in vivo. Collectively, these findings strongly suggest that DNA demethylation is a common mechanism underlying the abnormal expression of uPA and is a critical contributing factor to the malignant progression of human prostate tumors.

Activation of p53/p21Waf1/Cip1 pathway by 5-aza-2'-deoxycytidine inhibits cell proliferation, induces pro-apoptotic genes and mitogen-activated protein kinases in human prostate cancer cells.

The tumor suppressor gene p53 plays an essential role in cell proliferation and apoptosis. Due to its relevance to cancer therapy, most studies have focused on the cellular consequences of p53 activation in relation to cytotoxic drugs. 5-aza-2'-deoxycytidine (5-aza-CdR) is widely used as an anti-cancer drug for the treatment of leukemia and solid tumors. However, the mechanism by which 5-aza-CdR exerts its anti-neoplastic activity remains unclear. Here, we address the role of p53 in regulating cellular responses to 5-aza-CdR treatment in human prostate cancer cells. We found that 5-aza-CdR induces p53 and p21Waf1/Cip1 expression associated with inhibition of cell proliferation in LNCaP cells (p53 wild-type), but not in DU145 cells (p53 mutant). By using pifithrin-alpha, a chemical inhibitor of p53, we confirmed that the increase in p21Waf1/Cip1 expression and inhibition of cell proliferation in LNCaP cells by 5-aza-CdR is p53-dependent. Also, the activation of p53 and p21Waf1/Cip1 pathway by 5-aza-CdR modified multiple gene expressions including apoptotic target genes and MAP kinases in LNCaP cells. 5-aza-CdR-induced apoptosis in LNCaP cells is assessed by DNA fragmentation analysis. Furthermore, knockdown of p53 by pU6-p53 siRNA vector suggests the involvement of MAP kinases in the process of 5-aza-CdR-mediated activation of p53 pathway to inhibit cell proliferation and induce apoptosis. Finally, the comet or SCGE assay and methylation-sensitive restriction analysis demonstrated that 5-aza-CdR induced p53 and p21Waf1/Cip1 expression as a consequence of DNA damage and independent of DNA demethylation. Our findings suggest that 5-aza-CdR induces anti-neoplastic activity primarily through the activation of p53 pathway in response to DNA damage and subsequently leads to inhibition of cell proliferation as well as induction of apoptosis. Therefore, our data indicate that p53 status in tumor cells may be critical for the clinical efficacy and toxicity of 5-aza-CdR.