Sweaty Koul

Oxidative stress is inherent in prostate cancer cells and is required for aggressive phenotype

Reactive oxygen species (ROS) and the coupled oxidative stress have been associated with tumor formation. Several studies suggested that ROS can act as secondary messengers and control various signaling cascades. In the present studies, we characterized the oxidative stress status in three different prostate cancer cells (PC3, DU145, and LNCaP) exhibiting various degree of aggressiveness and normal prostate cells in culture (WPMY1, RWPE1, and primary cultures of normal epithelial cells). We observed increased ROS generation in cancer cells compared with normal cells, and that extramitochondrial source of ROS generator, NAD(P)H oxidase (Nox) systems, are associated with the ROS generation and are critical for the malignant phenotype of prostate cancer cells. Moreover, diphenyliodonium, a specific Nox inhibitor, blocked proliferation, modulated the activity of growth signaling cascades extracellular signal-regulated kinase (ERK)1/ERK2 and p38 mitogen-activated protein kinase as well as AKT protein kinase B, and caused cyclin B-dependent G(2)-M cell cycle arrest. We also observed higher degrees of ROS generation in the PC3 cells than DU145 and LNCaP, and that ROS generation is critical for migratory/invasiveness phenotypes. Furthermore, blocking of the ROS production rather than ROS neutralization resulted in decreased matrix metalloproteinase 9 activity as well as loss of mitochondrial potential, plausible reasons for decreased cell invasion and increased cell death. Taken together, these studies show, for the first time, the essential role of ROS production by extramitochondrial source in prostate cancer and suggest that therapies aimed at reducing ROS production might offer effective means of combating prostate cancer in particular, and perhaps other malignancies in general.

Oxidative stress in prostate cancer.

As prostate cancer and aberrant changes in reactive oxygen species (ROS) become more common with aging, ROS signaling may play an important role in the development and progression of this malignancy. Increased ROS, otherwise known as oxidative stress, is a result of either increased ROS generation or a loss of antioxidant defense mechanisms. Oxidative stress is associated with several pathological conditions including inflammation and infection. ROS are products of normal cellular metabolism and play vital roles in stimulation of signaling pathways in response to changing intra- and extracellular environmental conditions. Chronic increases in ROS over time are known to induce somatic mutations and neoplastic transformation. In this review we summarize the causes for increased ROS generation and its potential role in etiology and progression of prostate cancer.

p38 Mitogen-Activated Protein Kinase–Driven MAPKAPK2 Regulates Invasion of Bladder Cancer by Modulation of MMP-2 and MMP-9 Activity

In transitional cell carcinoma, the most common form of bladder cancer, overexpression of the matrix metalloproteinases MMP-2 and MMP-9 offers prognostic value as markers of disease-specific survival. These molecules have been implicated in metastasis of bladder cancer, but the underlying mechanisms through which they are controlled are poorly defined. In this study, we investigated a role of p38 mitogen-activated protein kinase (MAPK) in this process, using bladder cancer cell lines HTB9 and HTB5 that were derived from different tumor stages. p38 MAPK modulated MMP-2/9 mRNA levels at the levels of transcript stability and MMP-2/9 activity along with invasive capacity. We defined a downstream effector of p38 MAPK, MAPK-activated protein kinase 2 (MAPKAPK2), that was associated with MMP-2/9 activation. Ectopic expression of wild-type or constitutively active forms of MAPKAPK2 increased MMP-2/9 activities and invasive capacity. Conversely, p38 MAPK inhibition blocked the MAPKAPK2-mediated increase in MMP-2/9 activities and the invasive capacity of the cancer cells. Our findings implicate p38 MAPK and MAPKAPK2 in mediating bladder cancer invasion via regulation of MMP-2 and MMP-9 at the level of mRNA stability.

Mitogen Activated Protein kinase signal transduction pathways in the prostate

The biochemistry of the mitogen activated protein kinases ERK, JNK, and p38 have been studied in prostate physiology in an attempt to elucidate novel mechanisms and pathways for the treatment of prostatic disease. We reviewed articles examining mitogen-activated protein kinases using prostate tissue or cell lines. As with other tissue types, these signaling modules are links/transmitters for important pathways in prostate cells that can result in cellular survival or apoptosis. While the activation of the ERK pathway appears to primarily result in survival, the roles of JNK and p38 are less clear. Manipulation of these pathways could have important implications for the treatment of prostate cancer and benign prostatic hypertrophy.

Hypoxia-associated p38 mitogen-activated protein kinase-mediated androgen receptor activation and increased HIF-1α levels contribute to emergence of an aggressive phenotype in prostate cancer

Androgen receptor (AR) signaling is involved in the development and progression of prostate cancer. Tumor microvasculature contributes to continual exposure of prostate cancer cells to hypoxia–reoxygenation, however, the role of hypoxia–reoxygenation in prostate cancer progression and modulation of AR signaling is not understood. In this study, we evaluated the effects of hypoxia–reoxygenation in LNCaP cells, a line of hormone responsive human prostate cancer cells. Our results demonstrate that hypoxia–reoxygenation resulted in increased survival, higher clonogenicity and enhanced invasiveness of these cells. Moreover, hypoxia–reoxygenation was associated with an increased AR activity independent of androgens as well as increased hypoxia inducible factor (HIF-1α) levels and activity. We also observed that the activation of p38 mitogen-activated protein (MAP) kinase pathway was an early response to hypoxia, and inhibition of p38 MAP kinase pathway by variety of approaches abolished hypoxia–reoxygenation induced increased AR activity as well as increased survival, clonogenicity and invasiveness. These results demonstrate a critical role for hypoxia-induced p38 MAP kinase pathway in androgen-independent AR activation in prostate cancer cells, and suggest that hypoxia–reoxygenation may select for aggressive androgen-independent prostate cancer phenotype.

Loss of PDEF, a prostate-derived Ets factor is associated with aggressive phenotype of prostate cancer: Regulation of MMP 9 by PDEF

BackgroundProstate-derived Ets factor (PDEF) is expressed in tissues of high epithelial content including prostate, although its precise function has not been fully established. Conventional therapies produce a high rate of cure for patients with localized prostate cancer, but there is, at present, no effective treatment for intervention in metastatic prostate cancer. These facts underline the need to develop new approaches for early diagnosis of aggressive prostate cancer patients, and mechanism based anti-metastasis therapies that will improve the outlook for hormone-refractory prostate cancer. In this study we evaluated role of prostate-derived Ets factor (PDEF) in prostate cancer.ResultsWe observed decreased PDEF expression in prostate cancer cell lines correlated with increased aggressive phenotype, and complete loss of PDEF protein in metastatic prostate cancer cell lines. Loss of PDEF expression was confirmed in high Gleason Grade prostate cancer samples by immuno-histochemical methods. Reintroduction of PDEF profoundly affected cell behavior leading to less invasive phenotypes in three dimensional cultures. In addition, PDEF expressing cells had altered cell morphology, decreased FAK phosphorylation and decreased colony formation, cell migration, and cellular invasiveness. In contrast PDEF knockdown resulted in increased migration and invasion as well as clonogenic activity. Our results also demonstrated that PDEF downregulated MMP9 promoter activity, suppressed MMP9 mRNA expression, and resulted in loss of MMP9 activity in prostate cancer cells. These results suggested that loss of PDEF might be associated with increased MMP9 expression and activity in aggressive prostate cancer. To confirm results we investigated MMP9 expression in clinical samples of prostate cancer. Results of these studies show increased MMP9 expression correlated with advanced Gleason grade. Taken together our results demonstrate decreased PDEF expression and increased MMP9 expression during the transition to aggressive prostate cancer.ConclusionsThese studies demonstrate for the first time negative regulation of MMP9 expression by PDEF, and that PDEF expression was lost in aggressive prostate cancer and was inversely associated with MMP9 expression in clinical samples of prostate cancer. Based on these exciting results, we propose that loss of PDEF along with increased MMP9 expression should serve as novel markers for early detection of aggressive prostate cancer.

Vascular endothelial growth factor (VEGF) gene therapy using a nonviral gene delivery system improves erectile function in a diabetic rat model

Erectile dysfunction (ED) is a cause of decreased quality of life in more than 70% of diabetic men. Vascular endothelial growth factor (VEGF) has shown to improve overall endothelial and smooth muscle cell dysfunction in models of ED. We describe a novel technique for nonviral, in vivo gene transfection of VEGF in the rat corpus cavernosum. Diabetic rats were transfected with DNA encoding a fusion VEGF/green fluorescent protein (GFP) complex and fluorescence microscopy was used to monitor the expression of VEGF–GFP fusion protein. Western blot and PCR analyses confirmed the expression of the GFP–VEGF fusion protein and mRNA. Functional studies using cavernous nerve stimulation revealed maximal intracavernous pressures (ICPs) of 63.1 mm Hg, and 30.7 mm Hg in the normal and diabetic control groups, respectively, and 47.4 mm Hg in VEGF–GFP-transfected diabetic group. Immunohistochemical analysis of the cavernosal tissue from transfected rats showed increased smooth muscle content compared with the diabetic control group. We show for the first time in our animal model that expression of the transfected VEGF in cavernosal tissue leads to an overall improvement of maximal ICP and smooth muscle content. On the basis of these results, it is tempting to speculate that our nonviral vector system offers an excellent system for gene delivery into cavernosal tissue, and that VEGF gene therapy using this system could be useful in improving erectile function in diabetic men.

Focal adhesion kinase controls aggressive phenotype of androgen-independent prostate cancer.

Overexpression of focal adhesion kinase (FAK) has been well correlated with tumor development and/or the maintenance of tumor phenotype. In addition, inappropriate activation of the extracellular regulated kinase (ERK) signaling pathway is common to many human cancers. In the present study, we investigated the interplay between FAK and ERK in androgen-independent prostate cancer cells (PC3 and DU145 cells). We observed that suppression of FAK expression using small interfering RNA–mediated knockdown decreased the clonogenic activity, whereas overexpression of FAK increased it. We also observed that detachment of PC3 and DU145 cells from their substrate induced tyrosine phosphorylation of FAK. ERK knockdown diminished FAK protein levels and tyrosine phosphorylation of FAK as well as FAK promoter-reporter activity. We also tested the effect of MEK inhibitors and small interfering RNA–mediated knockdown of ERK1 and/or ERK2 on cell proliferation, invasiveness, and growth in soft agar of PC3 and DU145 cells. Inhibition of ERK signaling grossly impaired clonogenicity as well as invasion through Matrigel. However, inhibition of ERK signaling resulted in only a modest inhibition of 3H-thymidine incorporation and no effect on overall viability of the cells or increased sensitivity to anoikis. Taken together, these data show, for the first time, a requirement for FAK in aggressive phenotype of prostate cancer cells; reveal interdependence of FAK and ERK1/2 for clonogenic and invasive activity of androgen-independent prostate cancer cells; suggest a role for ERK regulation of FAK in substrate-dependent survival; and show for the first time, in any cell type, the regulation of FAK expression by ERK signaling pathway. (Mol Cancer Res 2008;6(10):1639–48)

Kidney injury molecule-1 is up-regulated in renal epithelial cells in response to oxalate in vitro and in renal tissues in response to hyperoxaluria in vivo.

Oxalate is a metabolic end product excreted by the kidney. Mild increases in urinary oxalate are most commonly associated with Nephrolithiasis. Chronically high levels of urinary oxalate, as seen in patients with primary hyperoxaluria, are driving factor for recurrent renal stones, and ultimately lead to renal failure, calcification of soft tissue and premature death. In previous studies others and we have demonstrated that high levels of oxalate promote injury of renal epithelial cells. However, methods to monitor oxalate induced renal injury are limited. In the present study we evaluated changes in expression of Kidney Injury Molecule-1 (KIM-1) in response to oxalate in human renal cells (HK2 cells) in culture and in renal tissue and urine samples in hyperoxaluric animals which mimic in vitro and in vivo models of hyper-oxaluria. Results presented, herein demonstrate that oxalate exposure resulted in increased expression of KIM-1 m RNA as well as protein in HK2 cells. These effects were rapid and concentration dependent. Using in vivo models of hyperoxaluria we observed elevated expression of KIM-1 in renal tissues of hyperoxaluric rats as compared to normal controls. The increase in KIM-1 was both at protein and mRNA level, suggesting transcriptional activation of KIM-1 in response to oxalate exposure. Interestingly, in addition to increased KIM-1 expression, we observed increased levels of the ectodomain of KIM-1 in urine collected from hyperoxaluric rats. To the best of our knowledge our studies are the first direct demonstration of regulation of KIM-1 in response to oxalate exposure in renal epithelial cells in vitro and in vivo. Our results suggest that detection of KIM-1 over-expression and measurement of the ectodomain of KIM-1 in urine may hold promise as a marker to monitor oxalate nephrotoxicity in hyperoxaluria.

Differential effects of valproic acid on growth, proliferation and metastasis in HTB5 and HTB9 bladder cancer cell lines

High grade invasive bladder cancer is a leading cause of cancer deaths and treatment options are limited for this type of cancer. Recent studies have reported anticancer effects of valproic acid in many cancers and also in superficial bladder cancer. Acute valproic acid administration suppressed cell proliferation in a time- and dose-dependent manner in two muscle-invasive human bladder cancer cell lines (HTB5 and HTB9), with accompanying G1 phase cell cycle arrest. A significant decrease in colony formation ability and invasiveness was seen with valproic acid treatment though the effectiveness varied with cell type. Our results suggest a role for valproic acid in inhibiting growth and invasion of muscle-invasive bladder cancer.