Phenylboronic acid is a more potent inhibitor than boric acid of key signaling networks involved in cancer cell migration

Previous studies from our lab have shown that both boric (BA) and phenylboronic acid (PBA) inhibit the migration of prostate cancer cell lines, as well as non-tumorigenic prostate cells. Our results indicate that PBA is more potent than BA in targeting metastatic and proliferative properties of cancer cells. Here we focus on the impact of BA and PBA on Rho family of GTP-binding proteins and their downstream targets. Treatment with 1 mM PBA and BA decreases activities of RhoA, Rac1 and Cdc42 in DU-145 metastatic prostate cancer cells, but not in normal RWPE-1 prostate cells. Furthermore, ROCKII activity and phosphorylation of myosin light chain kinase decrease as a result of either PBA or BA treatment in DU-145 cells, suggesting these compounds target actomyosin-based contractility.Key words: boric acid, phenylboronic acid, migration, prostate cancer, natural compounds, DU-145     

Inhibition of macrophage migration inhibitory factor or its receptor (CD74) attenuates growth and invasion of DU-145 prostate cancer cells

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is overexpressed in prostate cancer, but the mechanism by which MIF exerts effects on tumor cells remains undetermined. MIF interacts with its identified membrane receptor, CD74, in association with CD44, resulting in ERK 1/2 activation. Therefore, we hypothesized that increased expression or surface localization of CD74 and MIF overexpression by prostate cancer cells regulated tumor cell viability. Prostate cancer cell lines (LNCaP and DU-145) had increased MIF gene expression and protein levels compared with normal human prostate or benign prostate epithelial cells (p < 0.01). Although MIF, CD74, and CD44 variant 9 expression were increased in both androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells, cell surface of CD74 was only detected in androgen-independent (DU-145) prostate cancer cells. Therefore, treatments aimed at blocking CD74 and/or MIF (e.g., inhibition of MIF or CD74 expression by RNA interference or treatment with anti-MIF- or anti-CD74- neutralizing Abs or MIF-specific inhibitor, ISO-1) were only effective in androgen-independent prostate cancer cells (DU-145), resulting in decreased cell proliferation, MIF protein secretion, and invasion. In DU-145 xenografts, ISO-1 significantly decreased tumor volume and tumor angiogenesis. Our results showed greater cell surface CD74 in DU-145 prostate cancer cells that bind to MIF and, thus, mediate MIF-activated signal transduction. DU-145 prostate cancer cell growth and invasion required MIF activated signal transduction pathways that were not necessary for growth or viability of androgen-dependent prostate cells. Thus, blocking MIF either at the ligand (MIF) or receptor (CD74) may provide new, targeted specific therapies for androgen-independent prostate cancer.     

Phenylboronic acid selectively inhibits human prostate and breast cancer cell migration and decreases viability

We compared the in vitro effect of boric acid (BA) versus phenylboronic acid (PBA) on the migration of prostate and breast cancer cell lines and non-tumorigenic cells from the same tissues. Treatment at 24 hours with BA (≤ 500 µM) did not inhibit chemotaxis on fibronectin in any cell line. However, treatment over the same time course with concentrations of PBA as low as 1 µM significantly inhibited cancer cell migration without effecting non-tumorigenic cell lines. The compounds did not affect cell adhesion or viability at 24 hours but did alter morphology; both decreased cancer cell viability at 8 days. These results suggest that PBA is more potent than BA in targeting the metastatic and proliferative properties of cancer cells.     

Selective apoptosis induction by the cancer chemopreventive agent N-(4-hydroxyphenyl)retinamide is achieved by modulating mitochondrial bioenergetics in premalignant and malignant human prostate epithelial cells

Prostate tumorigenesis is coupled with an early metabolic switch in transformed prostate epithelial cells that effectively increases their mitochondrial bioenergetic capacity. The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) inhibits prostate cancer development in vivo, and triggers reactive oxygen species (ROS)-dependent prostate cancer cell apoptosis in vitro. The possibility that 4HPR-induced ROS production is associated with mitochondrial bioenergetics and required for apoptosis induction in transformed prostate epithelial cells in vitro would advocate a prospective mechanistic basis for 4HPR-mediated prostate cancer chemoprevention in vivo. We investigated this tenet by comparing and contrasting 4HPR’s effects on premalignant PWR-1E and malignant DU-145 human prostate epithelial cells. 4HPR promoted a dose- and/or time-dependent apoptosis induction in PWR-1E and DU-145 cells, which was preceded by and dependent on an increase in mitochondrial ROS production. In this regard, the PWR-1E cells were more sensitive than the DU-145 cells, and they consumed roughly twice as much oxygen as the DU-145 cells suggesting oxidative phosphorylation was higher in the premalignant cells. Interestingly, increasing the [Ca²⁺] in the culture medium of the PWR-1E cells attenuated their proliferation as well as their mitochondrial bioenergetic capacity and 4HPR’s cytotoxic effects. Correspondingly, the respiration-deficient derivatives (i.e., ρ⁰ cells lacking mitochondrial DNA) of DU-145 cells were markedly resistant to 4HPR-induced ROS production and apoptosis. Together, these observations implied that the reduction of mitochondrial bioenergetics protected PWR-1E and DU-145 cells against the cytotoxic effects of 4HPR, and support the concept that oxidative phosphorylation is an essential determinant in 4HPR’s apoptogenic signaling in transformed human prostate epithelial cells.     

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.     

Role of protein kinase C-iota in transformed non-malignant RWPE-1 cells and androgen-independent prostate carcinoma DU-145 cells

Prostate cancer is one of the leading causes of death among men in the USA.
Objective:  In this study, we investigated the role of atypical protein kinase C-iota (PKC-ι) in androgen-independent prostate DU-145 carcinoma cells compared to transformed non-malignant prostate RWPE-1 cells.
Materials and methods:  Western blotting and immunoprecipitations demonstrated that PKC-ι is associated with cyclin-dependent kinase activating kinase (CAK/Cdk7) in RWPE-1 cells, but not in DU-145 cells.
Results:  Treatment of prostate RWPE-1 cells with PKC-ι silencing RNA (siRNA) decreased cell viability, cell-cycle accumulation at G₂/M phase, and phosphorylation of Cdk7 and Cdk2. In addition, PKC-ι siRNA treatment caused less phosphorylation of Bad at ser-155, ser-136, and greater Bad/Bcl-x_L heterodimerization, leading to apoptosis. In DU-145 cells, PKC-ι was anti-apoptotic and was required for cell survival. Treatment with PKC-ι siRNA blocked increase in cell number, and inhibited G₁/S transition by accumulation of cells in G₀/G₁ phase. In addition to cell-cycle arrest, both RWPE-1 and DU-145 cells underwent apoptosis due to mitochondrial dysfunction and apoptosis cascades, such as release of cytochrome c, activation of caspase-7, and poly (ADP-ribose) polymerase (PARP) cleavage.
Conclusion:  Our results suggest that PKC-ι is required for cell survival in both transformed non-malignant prostate RWPE-1 cells and androgen-independent malignant prostate DU-145 cells, whereas suppressing PKC-ι lead to apoptosis in DU-145 prostate cells.     

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.     

MicroRNA-Dependent Regulation of <Emphasis Type="Italic">IGF1R</Emphasis> Gene Expression in Hormone-Sensitive and Hormone-Resistant Prostate Cancer Cells

Using multiple parallel sequencing on Illumina platform, we identified eight microRNAs that showed significant opposite changes of gene expression in cells of the hormone-sensitive LNCaP prostate cancer cell line and in cells of the hormone-resistant DU-145 cell line, in comparison to the microRNA expression in the normal prostate tissue cells. We found that the insulin-like growth factor 1 receptor (IGF1R) gene is a target of five microRNAs whose expression is increased in LNCaP cells and reduced in DU-145 cells.     

Rapid molecular and morphological responses of prostate cell lines to cell-cell contact

Cell-cell adhesion in 2-D PZ-HPV-7 prostate epithelial and DU-145 prostate cancer cell aggregates (monolayers), synchronously and rapidly (within 30 s) formed in suspension in an ultrasound trap has been examined over 60 min. The intracellular distributions of the cadherin/catenin complex components for both cell lines were time-dependent and were clearly identifiable as early as 150 s following cell-cell contact in the trap, while equilibrium positions were reached within 60 min following cell-cell contact. The accumulation of E-cadherin at the cell-cell interface was greater for PZ-HPV-7 than for DU-145 cells over 60 min in the trap, with the apparent formation of adherens junctions over that time scale in PZ-HPV-7 but not in DU-145 cells. The amounts of F-actin, alpha-, beta-, and gamma-catenins recruited to the cell-cell interface of PZ-HPV-7 cells were on average 2.4 times higher than those of DU-145 cells. The ability of different cell types to spread along neighboring cells was 1.5-fold greater for the PZ-HPV-7 than for the DU-145 cells. These results, discussed also in the context of earlier studies of cell adhesion in an ultrasound trap, characterize a reduced adhesiveness of DU-145 cells compared to PZ-HPV-7 cells.     

The effect of chelerythrine on cell growth, apoptosis, and cell cycle in human normal and cancer cells in comparison with sanguinarine

We compared the effects of chelerythrine (CHE) and sanguinarine (SA) on human prostate cancer cell lines (LNCaP and DU-145) and primary culture of human gingival fibroblasts. CHE and SA treatment of cell lines for 24 h resulted in (1) inhibition of cell viability in a dose-dependent manner in all tested cells (as evaluated by MTT test and bromodeoxyuridine incorporation assay); (2) dose-dependent increase in DNA damage in all tested cells (as evaluated by DNA comet assay); (3) changes in apoptosis (assessed by western blot analysis and TUNEL assay); and (4) significant induction of cyclin kinase inhibitors p21^Waf1/Cip1 and p27^Kip1 in prostate cancer cells (identified by western blot analysis). Our study demonstrates that CHE had significant cytotoxic effect, independent of p53 and androgen status, on human prostate cancer cell lines. Normal gingival fibroblasts and DU-145 cells were more sensitive to the treatment with both alkaloids than were LNCaP cells. CHE and SA may be prospective natural molecules for use in the treatment of prostate cancer owing to their involvement in apoptosis and cell cycle regulation.     

DU-145 prostate carcinoma cells that selectively transmigrate narrow obstacles express elevated levels of Cx43

The formation of aqueous intercellular channels mediating gap junctional intercellular coupling (GJIC) is a canonical function of connexins (Cx). In contrast, mechanisms of GJIC-independent involvement of connexins in cancer formation and metastasis remain a matter of debate. Because of the role of Cx43 in the determination of carcinoma cell invasive potential, we addressed the problem of the possible Cx43 involvement in early prostate cancer invasion. For this purpose, we analysed Cx43-positive DU-145 cell subsets established from the progenies of the cells most readily transmigrating microporous membranes. These progenies displayed motile activity similar to the control DU-145 cells but were characterized by elevated Cx43 expression levels and GJIC intensity. Thus, apparent links exist between Cx43 expression and transmigration potential of DU-145 cells. Moreover, Cx43 expression profiles in the analysed DU-145 subsets were not affected by intercellular contacts and chemical inhibition of GJIC during the transmigration. Our observations indicate that neither cell motility nor GJIC determines the transmigration efficiency of DU-145 cells. However, we postulate that selective transmigration of prostate cancer cells expressing elevated levels of Cx43 expression may be crucial for the “leading front” formation during cancer invasion.     

Prostate-specific kallikreins-2 and -4 enhance the proliferation of DU-145 prostate cancer cells through protease-activated receptors-1 and -2

A major characteristic of prostate cancer is the elevation of serum levels of prostate-specific antigen (hK3) and hK2, which are tumor markers that correlate with advancing stages of disease. Including hK4, these three kallikrein serine proteases are almost exclusively produced by the prostate. Prostate cancer cells have been recently shown to overexpress protease-activated receptors (PAR), which can be potentially activated by kallikreins and can regulate tumor growth. Here, we show that recombinant hK2 and hK4 activate ERK1/2 signaling of DU-145, PC-3, and LNCaP prostate cancer cells, which express both PAR1 and PAR2. These kallikreins also stimulate the proliferation of DU-145 cells. Pretreatment of hK2 and hK4 with the serine protease inhibitor, aprotinin, blocks the responses in DU-145 cells, and small interfering RNA against PAR1 and PAR2 also inhibits ERK1/2 signaling. To determine which PAR is activated by hK2 and hK4, a cell line that expresses a single PAR, a PAR1 knockout mouse lung fibroblast cell line transfected with PAR1 (KOLF-PAR1) or PAR2 (KOLF-PAR2) was used. hK4 activates both PAR1 and PAR2, whereas hK2 activates PAR2. hK4 generates more phosphorylated ERK1/2 than hK2. These data indicate that prostatic kallikreins (hK2 and hK4) directly stimulate prostate cancer cell proliferation through PAR1 and/or PAR2 and may be potentially important targets for future drug therapy for prostate cancer.     

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.     

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.     

Quantitative analysis of the effect of cancer invasiveness and collagen concentration on 3D matrix remodeling

Extracellular matrix (ECM) remodeling is a key component of cell migration and tumor metastasis, and has been associated with cancer progression. Despite the importance of matrix remodeling, systematic and quantitative studies on the process have largely been lacking. Furthermore, it remains unclear if the disrupted tensional homeostasis characteristic of malignancy is due to initially altered ECM and tissue properties, or to the alteration of the tissue by tumor cells. To explore these questions, we studied matrix remodeling by two different prostate cancer cell lines in a three-dimensional collagen system. Over one week, we monitored structural changes in gels of varying collagen content using confocal reflection microscopy and quantitative image analysis, tracking metrics of fibril fraction, pore size, and fiber length and diameter. Gels that were seeded with no cells (control), LNCaP cells, and DU-145 cells were quantitatively compared. Gels with higher collagen content initially had smaller pore sizes and higher fibril fractions, as expected. However, over time, LNCaP- and DU-145-populated matrices showed different structural properties compared both to each other and to the control gels, with LNCaP cells appearing to favor microenvironments with lower collagen fiber fractions and larger pores than DU-145 cells. We posit that the DU-145 cells' preference for denser matrices is due to their higher invasiveness and proteolytic capabilities. Inhibition of matrix proteases resulted in reduced fibril fractions for high concentration gels seeded with either cell type, supporting our hypothesis. Our novel quantitative results probe the dynamics of gel remodeling in three dimensions and suggest that prostate cancer cells remodel their ECM in a synergistic manner that is dependent on both initial matrix properties as well as their invasiveness.