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Box-plot analysis of cDNA microarrays

Table S5. ChIP PCR primer set. Table S6. Patient characteristics of microdissected prostate tumors. Table S7. In silico analysis of hnRNPH1 3'UTR

Additional detailed methods than can be incorporated in the body of the article

Figure S5. Growth kinetics of hnRNPH1 siRNA-silenced PC-3 cells; Figure S6, Effect of hnRNPH1 overexpression on growth of LNCaP cells in nude mice.

Purpose: The causes of disproportionate incidence and mortality of prostate cancer among African Americans (AA) remain elusive. The purpose of this study was to investigate the mechanistic role and assess clinical utility of the splicing factor heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1) in prostate cancer progression among AA men.Experimental Design: We employed an unbiased functional genomics approach coupled with suppressive subtractive hybridization (SSH) and custom cDNA microarrays to identify differentially expressed genes in microdissected tumors procured from age- and tumor grade–matched AA and Caucasian American (CA) men. Validation analysis was performed in independent cohorts and tissue microarrays. The underlying mechanisms of hnRNPH1 regulation and its impact on androgen receptor (AR) expression and tumor progression were explored.Results: Aberrant coexpression of AR and hnRNPH1 and downregulation of miR-212 were detected in prostate tumors and correlate with disease progression in AA men compared with CA men. Ectopic expression of miR-212 mimics downregulated hnRNPH1 transcripts, which in turn reduced expression of AR and its splice variant AR-V7 (or AR3) in prostate cancer cells. hnRNPH1 physically interacts with AR and steroid receptor coactivator-3 (SRC-3) and primes activation of androgen-regulated genes in a ligand-dependent and independent manner. siRNA silencing of hnRNPH1 sensitized prostate cancer cells to bicalutamide and inhibited prostate tumorigenesis in vivo.Conclusions: Our findings define novel roles for hnRNPH1 as a putative oncogene, splicing factor, and an auxiliary AR coregulator. Targeted disruption of the hnRNPH1-AR axis may have therapeutic implications to improve clinical outcomes in patients with advanced prostate cancer, especially among AA men. Clin Cancer Res; 22(7); 1744–56. ©2015 AACR.

Table S1. TMA-4 patient characteristics. Table S2. qPCR primers. Table S3. miRNA primers. Table S4. EMSA oligonucleotides

PDF file - 3MB, Percentages of PIN and invasive adenocarcinoma in different prostatic lobes.

PDF file - 63K, PCR Primer Sequences

PDF file - 7.6MB, A. Thickness of stroma surrounding each gland in different mouse postatic lobes. B. MMP7 staining in the dorsal prostatic lobes.

PDF file - 6.6MB, Immunohistochemical staining of laminin in whole prostate sections.

Renal Cell Carcinoma (RCC) is the most common form of kidney cancer, with clear cell RCC (ccRCC) representing about 85% of all RCC tumors. There are limited curable treatments available for metastatic ccRCC because this disease is unresponsive to conventional targeted systemic pharmacotherapy. Exosomes (Exo) are small extracellular vesicles (EVs) secreted from cancer cells with marked roles in tumoral signaling and pharmacological resistance. Ketoconazole (KTZ) is an FDA approved anti-fungal medication which has been shown to suppress exosome biogenesis and secretion, yet its role in ccRCC has not been identified. A time-course, dose-dependent analysis revealed that KTZ selectively decreased secreted Exo in tumoral cell lines. Augmented Exo secretion was further evident by decreased expression of Exo biogenesis (Alix and nSMase) and secretion (Rab27a) markers. Interestingly, KTZ-mediated inhibition of Exo biogenesis was coupled with inhibition of ERK1/2 activation. Next, selective inhibitors were employed and showed ERK signaling had a direct role in mediating KTZ’s inhibition of exosomes. In sunitinib resistant 786-O cells lines, the addition of KTZ potentiates the efficacy of sunitinib by causing Exo inhibition, decreased tumor proliferation, and diminished clonogenic ability of RCC cells. Our findings suggest that KTZ should be explored as an adjunct to current RCC therapies.

Background Testosterone (T) deficiency is associated with erectile dysfunction (ED). The relaxant response of T on the corporal smooth muscle through a non-genomic pathway has been reported; however, the in vitro modulating effects of T on human corpus cavernosum (HCC) have not been studied. Objectives To compare the effects of various concentrations of T on nitric oxide (NO)-dependent and nitric oxide-independent relaxation in organ bath studies and elucidate its mode of action, specifically targeting the cavernous NO/cyclic guanosine monophosphate (cGMP) pathway. Materials and methods Human corpus cavernosum (HCC) samples were obtained from men undergoing penile prosthesis implantation (n = 9). After phenylephrine (Phe) precontraction, the effects of various relaxant drugs of HCC strips were performed using organ bath at low (150 ng/dL), eugonadal (400 ng/dL), and hypergonadal (600 ng/dL) T concentrations. The penile tissue measurements of endothelial nitric oxide synthase (eNOS), neuronal (n)NOS, and phosphodiesterase type 5 (PDE5) were evaluated via immunostaining, Western blot, cGMP and nitrite/nitrate (NOx) assays. Results Relaxation responses to ACh and EFS in isolated HCC strips were significantly increased at all T levels compared with untreated tissues. The sildenafil-induced relaxant response was significantly increased at both eugonadal and hypergonadal T levels. Normal and high levels of T are accompanied by increased eNOS, nNOS, cGMP, and NOx levels, along with reduced PDE5 protein expression. Conclusion This study reveals an important role of short-term and modulatory effects of different concentrations of T in HCC. T positively regulates functional activities, inhibition of PDE5 expression, and formation of cGMP and NOx in HCC. These results demonstrate that T indirectly contributes to HCC relaxation via downstream effects on nNOS, eNOS, and cGMP and by inhibiting PDE5. This action provides a rationale for normalizing T levels in hypogonadal men with ED, especially when PDE5 inhibitors are ineffective. T replacement therapy may improve erectile function by modulating endothelial function in hypogonadal men.

Background Aging is the most important risk factor for prostate cancer (PCa), but how age contributes to PCa is poorly understood. Aging is characterized by low-grade systemic inflammation (i.e., inflammaging) that is often attributed to the progressive activation of immune cells over time, which may play an important role in prostate carcinogenesis. Th17 response is elevated in aging humans and mice, but it remains unknown whether it is increased in prostate tissue or contributes to prostate carcinogenesis during aging. In this study, we aimed to determine the role of age-related Th17 response in PCa cell growth, migration, and invasion. Methods C57BL/6J (B6) mouse was used as an aging animal model and the prostate histopathology during aging was analyzed. Splenic CD4+ T cells were isolated from young (16-20 weeks old) and aged (96-104 weeks old) mice, and cultured in the presence of plate-bound anti-CD3/anti-CD28, with or without Th17 differentiation conditions. The cells were collected and used for subsequent flow cytometry or quantitative reverse transcription polymerase chain reaction. The supernatant was collected and used to treat PCa cell lines. The treated PCa cells were analyzed for cell viability, migration, invasion, and nuclear factor kappa B (NF-κB) signaling. Results Aged mice had enlarged prostate glands and increased morphological alterations, with not only increased inflammatory cell infiltration but also increased Th17 cytokines in prostate tissue, compared to young mice. Naive CD4+ T cells from aged mice differentiated increased interleukin (IL)-17-expressing cells. CD4+ T cells from aged mice spleen had increased Th17 cells, Th17 cytokines and Th17/Treg ratio compared to young mice. Factors secreted from aged CD4+ T cells, especially from ex vivo differentiated Th17 cells, not only promoted PCa cell viability, migration, and invasion but also activated the NF-κB signaling in PCa cells compared to young mice. Conclusions These results indicate that age-related CD4+ T cells, especially Th17 cells-secreted factors have the potential to contribute to prostate carcinogenesis. Our work could prompt further research using autochthonous PCa mouse models at different ages to elucidate the functional role of Th17 response in prostate carcinogenesis during aging.

Although well-differentiated papillary thyroid cancer may remain indolent, lymph node metastases and the recurrence rates are approximately 50% and 20%, respectively. No current biomarkers are able to predict metastatic lymphadenopathy and recurrence in early stage papillary thyroid cancer. Hence, identifying prognostic biomarkers predicting cervical lymph-node metastases would prove very helpful in determining treatment.The database of the Cancer Genome Atlas included 495 papillary thyroid cancer samples. Using this database, we developed a machine learning model to define a gene signature that could predict lymph-node metastasis (N0 or N1). Kruskal-Wallis tests, univariate and multivariate logistic and Cox regression models, and Kaplan-Meier analyses were performed to correlate the gene signature with clinical outcomes.We identified a panel of 25 genes and constructed a risk score that can differentiate N0 and N1 papillary thyroid cancer samples (P.001) with a sensitivity of 86%, a specificity of 62%, a positive predictive value of 93%, and a negative predictive value of 42%. This panel represents an independent biomarker to predict metastatic lymphadenopathy (OR = 8.06, P.001) specifically in patients with T1 lesions (OR = 7.65, P = .002) and disease-free survival (HR = 2.64, P = .043).This novel 25-gene panel may be used as a potential prognostic marker for accurately predicting lymph-node metastasis and disease-free survival in patients with early-stage papillary thyroid cancer.

Background: Tyrosine kinase inhibitors (TKI) were initially demonstrated as an efficacious treatment for renal cell carcinoma (RCC). However, after a median treatment length of 14 months, a vast majority of patients develop resistance. This study analyzed a combination therapy of tipifarnib (Tipi) + sunitinib that targeted exosome-conferred drug resistance. Methods: 786-O, 786-O-SR (sunitinib resistant), A498, A498-SR, Caki-2, Caki-2-SR, and 293T cells were cultured. Exosomes were collected using differential ultracentrifugation. Cell proliferation, Jurkat T cell immune assay, and immunoblot analysis were used for downstream analysis. Results: SR exosomes treatment displayed a cytotoxic effect on immune cells. This cytotoxic effect was associated with increased expression of PD-L1 on SR exosomes when compared to sunitinib-sensitive (SS) exosomes. Additionally, Tipi treatment downregulated PD-L1 expression on exosomes derived from SR cell lines. Tipi’s ability to downregulate PD-L1 in exosomes has a significant application within patients. Exosomes collected from patients with RCC showed increased PD-L1 expression over subjects without RCC. Next, exosome concentrations were then compared after Tipi treatment, with all SS cell lines displaying an even greater reduction. On immunoblot assay, 293T cells showed a dose-dependent increase in Alix with no change in either nSMase or Rab27a. Conversely, all the SS and SR cell lines displayed a decrease in all three markers. After a cell proliferation employed a 48-h treatment on all SS and SR cell lines, the drug combination displayed synergistic ability to decrease tumor growth. Conclusions: Tipifarnib attenuates both the exosome endosomal sorting complex required for endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways, thereby blocking exosome biogenesis and secretion as well as downregulating PD-L1 on SS and SR cells.

Extracellular vesicles (EVs) of various types are released or shed from all cells. EVs carry proteins and contain additional protein and nucleic acid cargo that relates to their biogenesis and cell of origin. EV cargo in liquid biopsies of widespread interest due to its ability to provide a retrospective snapshot of cell state at the time of EV release. For the purposes of EV cargo analysis and repertoire profiling, multiplex assays are an essential tool in multiparametric analyte studies but are still being developed for high-parameter EV protein detection. Although bead-based EV multiplex analyses offer EV profiling capabilities with conventional flow cytometers, the utilization of EV multiplex assays has been limited by the lack of software analysis tools for such assays. Therefore, to facilitate robust EV repertoire studies, we have developed a free, open-source software (MPAPASS) for stitched multiplex analysis, EV database-compatible reporting, and visualization of EV repertoires.

INTRODUCTION AND OBJECTIVE:Renal cell carcinoma remains an aggressive malignancy with known substantial cross talk between cells. Exosomes (exo) are small extracellular vesicles (EVs) secreted from...

BACKGROUND. Aging is the most important risk factor for prostate cancer (PCa), but how age contributes to PCa is poorly understood. Aging is characterized by low-grade systemic inflammation (i.e., inflammaging) that is often attributed to the progressive activation of immune cells over time, which may play an important role in prostate carcinogenesis. Th17 response is elevated in aging humans and mice, but it remains unknown whether it is increased in prostate tissue or contributes to prostate carcinogenesis during aging. In this study, we aimed to determine the role of age-related Th17 response in PCa cell growth, migration, and invasion. METHODS. C57BL/6J (B6) mouse was used as an aging animal model and the prostate histopathology during aging was analyzed. Splenic CD4(+) T cells were isolated from young (16–20 weeks old) and aged (96–104 weeks old) mice, and cultured in the presence of plate-bound anti-CD3/anti-CD28, with or without Th17 differentiation conditions. The cells were collected and used for subsequent flow cytometry or qRT-PCR. The supernatant was collected and used to treat PCa cell lines. The treated PCa cells were analyzed for cell viability, migration, invasion, and NF-κB signaling. RESULTS. Aged mice had enlarged prostate glands and increased morphological alterations, with not only increased inflammatory cell infiltration but also increased Th17 cytokines in prostate tissue, compared to young mice. Naïve CD4(+) T cells from aged mice differentiated increased interleukin (IL)-17-expressing cells. CD4(+) T cells from aged mice spleen had increased Th17 cells, Th17 cytokines and Th17/Treg ratio compared to young mice. Factors secreted from aged CD4(+) T cells, especially from ex vivo differentiated Th17 cells, not only promoted PCa cell viability, migration, and invasion, but also activated the NF-κB signaling in PCa cells compared to young mice. CONCLUSIONS. These results indicate that age-related CD4(+) T cells, especially Th17 cells-secreted factors have the potential to contribute to prostate carcinogenesis. Our work could prompt further research using autochthonous PCa mouse models at different ages to elucidate the functional role of Th17 response in prostate carcinogenesis during aging.

Androgen receptor (AR) signaling is fundamental to prostate cancer (PC) progression, and hence, androgen deprivation therapy (ADT) remains a mainstay of treatment. However, augmented AR signaling via both full length AR (AR-FL) and constitutively active AR splice variants, especially AR-V7, is associated with the recurrence of castration resistant prostate cancer (CRPC). Oxidative stress also plays a crucial role in anti-androgen resistance and CRPC outgrowth. We examined whether a triterpenoid antioxidant drug, Bardoxolone-methyl, known as CDDO-Me or RTA 402, can decrease AR-FL and AR-V7 expression in PC cells. Nanomolar (nM) concentrations of CDDO-Me rapidly downregulated AR-FL in LNCaP and C4-2B cells, and both AR-FL and AR-V7 in CWR22Rv1 (22Rv1) cells. The AR-suppressive effect of CDDO-Me was evident at both the mRNA and protein levels. Mechanistically, acute exposure (2 h) to CDDO-Me increased and long-term exposure (24 h) decreased reactive oxygen species (ROS) levels in cells. This was concomitant with an increase in the anti-oxidant transcription factor, Nrf2. The anti-oxidant N-acetyl cysteine (NAC) could overcome this AR-suppressive effect of CDDO-Me. Co-exposure of PC cells to CDDO-Me enhanced the efficacy of a clinically approved anti-androgen, enzalutamide (ENZ), as evident by decreased cell-viability along with migration and colony forming ability of PC cells. Thus, CDDO-Me which is in several late-stage clinical trials, may be used as an adjunct to ADT in PC patients.

To determine if the insulin-like growth factor-1 (IGF-1) pathway is involved in the improvement in erectile function recovery in rats after nerve crush injury treated with pioglitazone (Pio). Sprague-Dawley rats were divided into four groups. The first group received sham operation (n = 5). The second group underwent bilateral cavernous nerve injury (BCNI, n = 7). The third group received BCNI and Pio treatment (BCNI + Pio, n = 7), whereas the fourth group underwent BCNI with Pio treatment and IGF-1 inhibition (BCNI + Pio + JB-1, n = 7). The IGF-1 receptor (IGF-1R) was inhibited by JB-1, a small molecular antagonist of the receptor. After 14 days of treatment, erectile function was measured via intracorporal pressure normalized to mean arterial pressure (ICP/MAP) and the major pelvic ganglion and cavernous nerve harvested for western blot and immunohistochemistry (IHC) of phosphorylated-IGF-1Rβ (p-IGF-1Rβ), phosphorylated-ERK1/2 (p-ERK1/2), and neuronal NOS (nNOS). BCNI + Pio animals exhibited improvements in ICP/MAP, similar to Sham animals, and BCNI + Pio + JB-1 rats demonstrated a reduced ICP/MAP similar to BCNI-only rats at all measured voltages. Western blot results showed upregulation of p-IGF-1Rβ was observed in the BCNI + Pio group. Low levels of p-ERK1/2 were seen in the JB-1-treated animals. The immunoblot results were supported by IHC findings. Intense IHC staining of nNOS was detected in the BCNI + Pio group. The group treated with JB-1 showed minimal protein expression of p-ERK1/2, nNOS, and p-IGF-1Rβ. Pio improves erectile function in rats undergoing BCNI via an IGF-1-mediated pathway.

MicroRNAs (miRNAs) are a small functional non-coding RNAs that post-transcriptionally regulate gene expression through mRNA degradation or translational repression. miRNAs are key regulatory components of various cellular networks. Current evidence support that multiple mammalian genome-encoded miRNAs impact the cellular biology, including proliferation, apoptosis, differentiation, and tumorigenesis, by targeting specific subsets of mRNAs. This minireview is focused on the current themes underlying the interactions between miRNAs and their mRNA targets and pathways in prostate tumorigenesis and progression, and their potential clinical utility as biomarkers for prostate cancer. Impact statement The primary goal of this article was to review recent literature on miRNA biogenesis and further elaborate on the identity of newly discovered miRNAs and their potential functional significance in the complex biological network associated with prostate tumorigenesis and disease progression and as biomarkers for prostate cancer.

The mitochondrial Bit1 protein exerts tumor-suppressive function in NSCLC through induction of anoikis and inhibition of EMT. Having this dual tumor suppressive effect, its downregulation in the established human lung adenocarcinoma A549 cell line resulted in potentiation of tumorigenicity and metastasis in vivo. However, the exact role of Bit1 in regulating malignant growth and transformation of human lung epithelial cells, which are origin of most forms of human lung cancers, has not been examined. To this end, we have downregulated the endogenous Bit1 expression in the immortalized non-tumorigenic human bronchial epithelial BEAS-2B cells. Knockdown of Bit1 enhanced the growth and anoikis insensitivity of BEAS-2B cells. In line with their acquired anoikis resistance, the Bit1 knockdown BEAS-2B cells exhibited enhanced anchorage-independent growth in vitro but failed to form tumors in vivo. The loss of Bit1-induced transformed phenotypes was in part attributable to the repression of E-cadherin expression since forced exogenous E-cadherin expression attenuated the malignant phenotypes of the Bit1 knockdown cells. Importantly, we show that the loss of Bit1 expression in BEAS-2B cells resulted in increased Erk activation, which functions upstream to promote TLE1-mediated transcriptional repression of E-cadherin. These collective findings indicate that loss of Bit1 expression contributes to the acquisition of malignant phenotype of human lung epithelial cells via Erk activation-induced suppression of E-cadherin expression.

The increased expression of phosphatase of regenerating liver-3 (PRL‑3) has been shown to be associated with the aggressive and metastatic phenotype of different solid tumors. However, it is not known whether PRL‑3 plays a similar role in the progression of prostate cancer (PCa). In this study, immunoblot analysis of androgen receptor (AR)-positive PCa lines (LNCaP and LNCaP‑SF) revealed the constitutive cytoplasmic expression of PRL‑3, and stimulation with R1881 (AR agonist) rapidly increased the nuclear translocation of PRL‑3. The AR-negative cell lines exhibited negligible PRL‑3 expression, and the ectopic overexpression of PRL‑3 increased both the proliferative and invasive potential of PC3 and DU145 cells. In addition, we measured PRL‑3 protein expression in human prostate tumor sections. A high-density prostate tumor microarray (TMA) was immunostained to assess whether PRL‑3 expression and its subcellular localization (cytoplasmic and nuclear levels) is associated with the Gleason score (GS), Gleason grade (GG) and tumor stage (T-stage). Digital image analysis (DIA) revealed that PRL‑3 expression was significantly higher in the malignant cores, as compared to the non‑malignant areas. Increases in both total and nuclear PRL‑3 levels were also associated with a higher GS and GG. Metastatic tumors (T4‑stage) had lower cytoplasmic, but higher nuclear PRL‑3 levels. Furthermore, the nuclear/cytoplasmic ratio for PRL‑3 in the tumors graded as GS7 could effectively distinguish between indolent (3+4) and aggressive (4+3) disease. Thus, our experiments using PCa lines suggested that PRL‑3 is an AR-regulated gene and its androgen-induced nuclear localization may increase the aggressive behavior of PCa cells. Furthermore, the digital analysis of immunostained tumor sections suggested that PRL‑3 may be an effective biomarker of high-grade PCa, and its nuclear/cytoplasmic ratio may be used to distinguish between indolent vs. aggressive tumors.

Prostate cancer (PCa) cells expressing full-length androgen receptor (AR-FL) are susceptible to androgen deprivation therapy (ADT). However, outgrowth of castration-resistant prostate cancer (CRPC) can occur due to the expression of constitutively active (ligand-independent) AR splice variants, particularly AR-V7. We previously demonstrated that sulforaphane (SFN), an isothiocyanate phytochemical, can decrease AR-FL levels in the PCa cell lines, LNCaP and C4-2B. Here, we examined the efficacy of SFN in targeting both AR-FL and AR-V7 in the CRPC cell line, CWR22Rv1 (22Rv1). MTT cell viability, wound-heal assay, and colony forming unit (CFU) measurements revealed that 22Rv1 cells are resistant to the anti-androgen, enzalutamide (ENZ). However, co-exposure to SFN sensitized these cells to the potent anticancer effects of ENZ (P

// Xin Yao 1 , Tri Pham 1 , Brandi Temple 1 , Selena Gray 1 , Cornita Cannon 1 , Camry Hardy 1 , Kamari Fletcher 1 , Shubha Kale Ireland 1 , Ahamed Hossain 1 , Renwei Chen 2 , Asim B. Abdel-Mageed 3 and Hector Biliran 1 1 Department of Biological and Public Health Sciences, Xavier University of Louisiana, New Orleans, LA 70125, USA 2 Center for Bioengineering, University of California, Santa Barbara, CA 93106, USA 3 Tulane Cancer Center, Tulane University School of Medicine, New Orleans, LA 70112, USA Correspondence to: Hector Biliran, email: hbiliran@xula.edu Keywords: TLE1, anoikis, E-cadherin, tumorigenecity, ZEB1 Received: April 27, 2017 Accepted: June 26, 2017 Published: July 31, 2017 ABSTRACT The Transducin-like enhancer of split 1 (TLE1) corepressor protein is overexpressed in human lung tumors and is a putative lung-specific oncogene. However, the molecular mechanism underlying its oncogenic function remains to be delineated. Here, we report an important role of TLE1 in promoting lung tumorigenesis by a mechanism involving induction of anoikis resistance. Using the human lung adenocarcinoma A549 and immortalized bronchial epithelial BEAS-2B cell lines, we observed that TLE1 inhibits anoikis through transcriptional repression of E-cadherin gene. In support of E-cadherin as a downstream target of TLE1 to block anoikis, forced expression of E-cadherin attenuated TLE1-induced anoikis resistance while E-cadherin downregulation decreased the anoikis sensitivity of TLE1 knockdown cells. Furthermore, we determined that E-cadherin expression is transcriptionally induced upon loss of cell attachment and functions as an effector of anoikis. Loss of E-cadherin via the siRNA strategy or exogenous TLE1 expression was sufficient to attenuate anoikis in A549 and BEAS-2B cells. Importantly, we demonstrated that the ZEB1 transcriptional factor is required for TLE1-mediated E-cadherin repression and anoikis resistance. ZEB1 interacted with and recruited the TLE1 to the E-cadherin promoter to impose histone deacetylation and gene silencing. In vivo , TLE1 strongly promoted tumorigenicity of A549 cells in a ZEB1-dependent manner. Underscoring its role in anoikis insensitivity of lung cancer cells, the TLE1-mediated E-cadherin repression was negatively regulated by the tumor suppressor Bcl-2 inhibitor of transcription 1 (Bit1) to effect anoikis. These findings identify the ZEB1/TLE1/E-cadherin transcriptional mechanism as a novel pathway that promotes anoikis resistance and oncogenicity of lung cancer cells.

Prostate cancer (PCa) is associated with advanced age. To better understand how age impacts PCa, it is critical to use PCa animal models generated at different ages (aged vs. non-aged). The PB-Cre4 driven phosphatase and tensin homolog (Pten) conditional knockout mouse model, which closely imitates human PCa initiation and progression. However, the Pten deletion is triggered in a 2-week-old prostate, when comparing the extent of PCa between aged and non-aged mice, it is difficult to distinguish the extent to which the onset and progression of PCa are due to the acceleration of the normal aging process or due to the manifestation of PCa pathologies over time. We present here a protocol to inject Cre-expressing adenovirus with luciferin tag intraductally into the prostate anterior lobes of Pten floxed mice, thus, the Pten-loss will be triggered at different ages post-Cre expression. The in vivo imaging of luciferin signals following viral infection was conducted to confirm the Cre expression and activity. Immunohistochemical staining was performed to confirm the Cre expression, Pten loss, and p-Akt and p-S6 activation. Prostate weight and histopathology were compared between aged and non-aged mice. The results showed that the virus infection was limited in the prostate glands and aged mice had significantly increased PCa onset and progression compared to young mice. Although technical skill is required to carry out this procedure and the success rate of viral infection is about 80%, this model of PCa is of great use to all investigators in the aging and cancer research field.

Oxidative stress and inflammation are key factors in the development of salt sensitive hypertension (SSH). Enhanced sodium reabsorption in the distal nephron, particularly the collecting duct (CD) plays a critical role in SSH development. Although the decrease in nitric oxide (NO) production (oxidative stress) and the increase in tumor necrosis factor-alpha (TNFα; pro-inflammatory cytokine) are common factors involved in SSH, the interactive role of these factors in modulating sodium reabsorption in CD cell is not yet clearly understood. Activation of TNF-α receptor type 1 (TNFR1) induces natriuresis by inhibiting ENaC, which is abundant in renal CD cells. The present study examined the effect of NO inhibition on TNFR1 protein expression in cultured M-1 cells (ATCC® CRL-2038™) that maintain the characteristics of renal cortical collecting duct cells. These cells were grown in DMEM-F12 growth medium containing 10% FBS and 1% Penstrep at 37 0 C, 5% CO2, 99% relative humidity up to 90% confluence. Then these cells were trypsinized and seeded into 100mm petri-dishes and treated with NO inhibitor, Nitro-L-arginine methyl ester (L-NAME; 0.5 mM) alone and in combination with a NO donor compound (DETA NONOate; 0.5 mM) along with a vehicle (PBS) treated group for 24 hours followed by harvesting and lysed with protease inhibitor-containing RIPA buffer. Extracted Protein concentration was determined by BCA assay kit, and a predetermined protein concentration was loaded in each well for SDS-PAGE. Immunoblots were detected for TNFR1 using primary antibodies for TNFR1 (19139; Abcam) and GAPDH. It was observed that TNFR1 expression significantly increases to 68±9.9 % (n=6) in L-NAME treated cells compared to vehicle treated control cells (n=4). This increase was prevented in cells co-treated with DETA NONOate (n=6). Immunofluorescence staining for TNFR1 protein in these treated cells also showed qualitatively similar findings as in western blot analysis. As TNFR1 mediates TNFα induced natriuretic response in the kidney, these data suggest that enhanced TNFR1 activity during NO inhibition in CD cells helps to minimize excessive sodium retention, and thus plays a counter-regulatory role in the development of SSH induced in oxidative stress conditions.

© 2019 Elsevier Inc. The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data. The Extracellular RNA Communication Consortium (ERCC) presents progress toward understanding the biology of extracellular RNAs and their use as biomarkers and therapeutics.

INTRODUCTION AND OBJECTIVES:Collagenase Clostridium histolyticum (CCH) (Xiaflex®; Endo, Chesterbrook, PA) is the first licensed drug for the treatment of Peyronie's disease (PD). Although PD is str...

PubMedID: 31741421 Objective: To evaluate the effect of the If channel inhibitor, ivabradine on human corpus cavernosum (HCC) smooth muscle tone. Methods: HCC samples were obtained from erectile dysfunction(ED) patients (n = 12) undergoing penile prosthesis surgery. Concentration–response curves for ivabradine were exposed to various inhibitory and stimulatory agents. The relaxant and contractile responses to electrical field stimulation (EFS, 10 Hz and 80 Hz) were examined in the presence or absence of ivabradine (10 µM). HCN3 and HCN4 channel expression and localization were determined by Western blot and immunohistochemical analyses of HCC tissues. Results: Increasing ivabradine concentrations dependently reduced the maximal contractile responses of isolated HCC strips induced by KCl (59.5 ± 2.5%) and phenylephrine (84.0 ± 9.8%), which was not affected by nitric oxide synthase and soluble guanylyl cyclase inhibitors after phenylephrine-induced contraction. Nifedipine and tetraethylammonium inhibited the maximum relaxation to ivabradine by 75% and 39.3%, respectively. Fasudil and sildenafil increased the relaxation response to ivabradine without altering the maximum response. Pre-incubation with ivabradine significantly increased relaxant responses to EFS (p < 0.01) and reduced the contractile tension evoked by EFS (72.3%) (p < 0.001). Ivabradine incubation did not affect the expression and localization of HCN3 and HCN4 channels in the HCC smooth muscle cells. Conclusions: Ivabradine exhibits a relaxant effect on HCC tissues, which is likely to be attributed to the blocking of L-type Ca2+ channels and the opening of K+ channels, independent of changes in the activation of the nitric oxide/cyclic guanosine monophosphate system. Inhibition of HCN channels localized in cavernosal smooth muscle cells may offer pharmacological benefits for patients with cardiovascular risk factors. © 2019, © 2019 Informa UK Limited, trading as Taylor & Francis Group.