Hari Koul

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.

Oxalate toxicity in LLC-PK1 cells, a line of renal epithelial cells

PURPOSE The present studies assessed the possibility that high concentrations of oxalate may be toxic to renal epithelial cells. MATERIALS AND METHODS Subconfluent cultures of LLC-PK1 cells were exposed to oxalate, and the effects on cell morphology, membrane permeability to vital dyes, DNA integrity and cell density were assessed. RESULTS Oxalate exposure produced time- and concentration-dependent changes in the light microscopic appearance of LLC-PK1 cells with higher concentrations ( > 140 microM.) inducing marked cytosolic vacuolization and nuclear pyknosis. Exposure to oxalate also increased membrane permeability to vital dyes, promoted DNA fragmentation and, at high concentrations (350 microM. free oxalate), induced a net loss of LLC-PK1 cells. CONCLUSIONS Since high concentrations of oxalate can be toxic to renal epithelial cells, hyperoxaluria may contribute to several forms of renal disease including both calcium stone disease and end-stage renal disease.

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.

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)

Oxalate Transport in LLC-PK1 Cells: Evidence for Oxalate Transport by Anion Exchange

Previous studies of renal oxalate handling have demonstrated that oxalate can be transported by a number of anion transport systems within the kidney including the SO4 2-/oxalate exchanger on basolateral (abluminal) and apical (luminal) membranes1, 2 and the Cl/oxalate exchanger on apical membranes3, 4. These studies demonstrated that pathway(s) exist for transcellular oxalate flux, but technical limitations with studies on membrane vesicles (limited timecourse for transport due to the small intravesicular volumes) and with micropuncture studies (limited control of the composition of the extracellular space) have made it difficult to predict the magnitude or direction of this flux under normal and pathological conditions. Thus the present studies have examined LLC-PK1 cells, a renal epithelial cell line with many characteristics of proximal tubular cells5 as a possible model system for assessing renal oxalate handling.

Oxalate-Induced Changes in Intracellular Calcium Levels in Renal Papillary Cells

Alterations in renal tubular and cellular function have been reported in clinical and experimental nephrolithiasis1-3, and it has been suggested that the presence of calcium oxalate stones within the kidney elicits these changes as a result of mechanical injury to the tubular cells. Evidence in support of this possibility was provided by studies in experimental animals where the induction of calcium oxalate crystal formation led to renal tubular damage (enzymuria, proteinuria, etc,4, 5). The present studies assessed the possibility that oxalate produces alterations in cell function in the absence of overt crystal formation. Specifically we determined whether or not exposure to physiological levels of oxalate would elicit changes in intracellular calcium levels in isolated papillary cells from normal rat kidneys.

Abstract 253: Tetrandrine promotes prostate cancer cell apoptosis in part by up-regulation of death receptors

Introduction: Tumor necrosis factor-related apoptosis-inducing-ligand (TRAIL) is a member of the tumor necrosis factor superfamily. TRAIL, a valuable agent for cancer therapy, induces apoptosis in the majority of cancer cells, but development of resistance limits its clinical use in many malignancies including prostate cancer. We have demonstrated that Tetrandrine derivatives (TET), promote apoptosis in PCa cells in vitro and in vivo. In the present study, we investigated the ability of TET to sensitize tumor cells to TRAIL. We also investigated the role of two-death receptors- DR4 and DR5 in overcoming TRAIL resistance in prostate cancer cells. Methods: Prostate cancer cell lines LNCaP and LNCaP derived C4-2b were used for this study. Cell lines were treated with TET and TRAIL alone or in various combinations of these two for different time points. mRNA levels were quantitated via Real Time-PCR and changes in protein expression were analyzed by Western Blot. Cytotoxicity was evaluated using the Crystal violet and MTT survival assay. Cells were transfected with Lentiviral-shRNA to generate stable DR4 and DR5 single and double knockdown cells. Apoptosis assay was done using BD Pharmingen FITC Annexin-V Apoptosis Detection kit I. Results: LNCaP and C4-2b cells did not show a significant response following exposure to TRAIL. Exposure of LNCaP and C4-2b cells to TET resulted in increased mRNA and protein levels of death receptors DR4, DR5. Pretreatment of both cell lines with TET for 12h, followed by TRAIL treatment for another 24h increased the apoptosis inducing potential of TRAIL in these cells. Exposure of TET treated cells in vitro to soluble h-TRAIL in combination with TET resulted in synergistic apoptosis response. shRNA targeting DR4 and DR5 significantly reduced the levels of DR4 and DR5 protein. There was a reduced expression of DR4 and DR5 in the knock down cells in response to TET in time-dependent and concentration-dependent manner. In comparison to scrambled cells, DR4, DR5 single knockdown and DR4/DR5 double-knock down cells showed reduced sensitivity to TRAIL and TET. These cells were less apoptotic as compared to scrambled LNCaP cells as revealed by our apoptosis assay data. Taken together these data suggest that TET induced sensitization of PCa cells to TRAIL is mediated in part by DR4/DR5. Conclusion: Our results suggest that TET induces apoptosis in part by inducing the expression of DR4 and DR5, and provide the proof-of-principle for a novel therapeutic strategy to sensitize cancer cells to apoptosis. Acknowledgement: Financial support from following Sources is gratefully acknowledged: (VA Merit Award 1BX001258; NCI RO1-CA161880); Carroll W. Feist Endowed Chair Funds, FWCC support and Chair commitment LSUHSC-Shreveport). Citation Format: Gauri Shishodia, Sweaty Koul, Qin Dong, Sergey Slepenkov, Hari K. Koul. Tetrandrine promotes prostate cancer cell apoptosis in part by up-regulation of death receptors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 253.

Gene therapy for erectile dysfunction

Erectile dysfunction is a disease that affects half of American men aged over 50 years. Many men respond to oral phosphodiesterase inhibitors but many do not. For this reason, many researchers are focusing their efforts on developing novel gene therapies for the treatment of erectile dysfunction. Aided by the meticulous characterization of the molecular cascades involved in the physiology of erection, several groups around the world are studying gene therapies in animal models, and one in a human clinical trial. Here we provide a review of the pathophysiology of erectile dysfunction and how it relates to the molecular targets of novel gene therapeutics. The field of gene therapy for the treatment of erectile dysfunction is continually growing, and this decade will likely see exciting results as the expansion from animal models to human clinical trials continues.

Polarized Distribution of Oxalate Transport Systems in A Line of Renal Epithelial Cells (LLC-PK1)

Evidence from clinical1 and experimental2 studies demonstrated a significant increase in cellular oxalate handling associated with urolithiasis. These findings led to the speculation that altered oxalate handling within the kidney may contribute to stone formation. However, our current understanding of renal oxalate handling has been limited by a variety of technical difficulties. Studies on intact kidneys are compromised by limited access to various regions of the nephron, difficulty in regulating the ionic composition in these different regions, regional heterogeneity, etc. Biochemical studies on isolated membrane fractions from the kidney have other inherent difficulties (contamination of membrane fractions, small intravesicular volumes and hence limited timecourse for transport studies, etc). Thus our laboratory has explored the possibility of using a simpler model system, LLC-PK1 cells, for studies on renal oxalate handling.

MP12-06 OXALATE AND COM-CRYSTALS ACTIVATE TOLL-LIKE RECEPTOR 4 (TLR4)-MEDIATED NF-?B SIGNALING PATHWAY AND PROINFLAMMATORY RESPONSE IN HUMAN RENAL EPITHELIAL CELLS

RESULTS: The urinary oxalate in C and D group was significantly higher than that in A group and B group (P<0.05). Von Kossa staining showed that crystal deposition of A, B, C, D group were 0, 0, 81.25% and 93.75%, respectively. The crystal density of group C was significantly higher than that in group D (P<0.05). OPN, CD44, IL-1, caspase-1 and NLRP3’s mRNA and protein expression of C, D group was significantly higher than the other two groups (P<0.05), the C group was significantly higher than the D group (P<0.05). CONCLUSIONS: Metformin inhibits the production of NLRP3 in rats with high oxalate, which can reduce the activation of the inflammatory bodies and thus reduce the production of kidney stones.