Effect of Testosterone on Insulin Stimulated IRS1 Ser Phosphorylation in Primary Rat Myotubes—A Potential Model for PCOS-Related Insulin Resistance

Polycystic ovary syndrome (PCOS) is characterized by a hyperandrogenic state and frequently develops skeletal muscle insulin resistance. We determined whether testosterone adversely affects insulin action by increasing serine phosphorylation of IRS-1(636/639) in differentiated rat skeletal muscle myotubes. The phosphorylation of Akt, mTOR, and S6K, downstream targets of the PI3-kinase-IRS-1 complex were also studied.Primary differentiated rat skeletal muscle myotubes were subjected to insulin for 30 min after 16-hour pre-exposure to either low (20 ng/ml) or high (200 ng/ml) doses of testosterone. Protein phosphorylation of IRS-1 Ser(636/639), Akt Ser(473), mTOR-Ser(2448), and S6K-Thr(389) were measured by Western blot with signal intensity measured by immunofluorescence.Cells exposed to 100 nM of insulin had increased IRS-1 Ser(636/639) and Akt Ser(473) phosphorylation. Cells pre-exposed to low-dose testosterone had significantly increased insulin-induced mTOR-Ser(2448) and S6K-Thr(389) phosphorylation (p<0.05), and further increased insulin-induced IRS-1 Ser(636/639) phosphorylation (p = 0.042) compared to control cells. High-dose testosterone pre-exposure attenuated the insulin-induced mTOR-Ser(2448) and S6K-Thr(389) phosphorylation.The data demonstrated an interaction between testosterone and insulin on phosphorylation of intracellular signaling proteins, and suggests a link between a hyperandrogenic, hyperinsulinemic environment and the development of insulin resistance involving serine phosphorylation of IRS-1 Ser(636/639). These results may guide further investigations of potential mechanisms of PCOS-related insulin resistance

Androgen Excess Produces Systemic Oxidative Stress and Predisposes to β-Cell Failure in Female Mice

In women, excess production of the male hormone, testosterone (T), is accompanied by insulin resistance. However, hyperandrogenemia is also associated with β-cell dysfunction and type 2 diabetes raising the possibility that androgen receptor (AR) activation predisposes to β-cell failure. Here, we tested the hypothesis that excess AR activation produces systemic oxidative stress thereby contributing to β-cell failure. We used normal female mice (CF) and mice with androgen resistance by testicular feminization (Tfm). These mice were exposed to androgen excess and a β-cell stress induced by streptozotocin (STZ). We find that following exposure to T, or the selective AR-agonist dehydrotestosterone (DHT), CF mice challenged with STZ, which are normally protected, are prone to β-cell failure and insulin-deficient diabetes. Conversely, T-induced predisposition to β-cell failure is abolished in Tfm mice. We do not observe any proapoptotic effect of DHT alone or in the presence of H2O2 in cultured mouse and human islets. However, we observe that exposure of CF mice to T or DHT provokes systemic oxidative stress, which is eliminated in Tfm mice. This work has significance for hyperandrogenic women; excess activation of AR by testosterone may provoke systemic oxidative stress. In the presence of a prior β-cell stress, this may predispose to β-cell failure

Increased frequency and nocturia in a middle aged male may not always be due to Benign Prostatic Hypertrophy (BPH): a case report

Primary signet ring cell carcinoma of urinary bladder is a rare type of bladder tumor and carries a very high mortality rate. It may have a clinical presentation similar to common diseases like Benign Prostatic Hypertrophy (BPH) and the management options are extremely limited. We report a case of 58 year old Caucasian male who presented with a 5 month history of increased frequency of urination, nocturia and weight loss without any fever or hematuria. He was found to have an increased creatinine of 2.8 mg/dl and a prostate specific antigen level of 0.18 ng/ml. His azotemia was thought to be secondary to BPH. A foley catheter was initially placed with a plan for outpatient follow up. On removal of the catheter his problems persisted and he returned to the hospital. Diagnostic work up including abdominal ultrasonography, computed tomography (CT) scan, retrograde pyelogram, cystography and cystoscopic biopsies revealed the diagnosis of primary signet ring cell carcinoma of urinary bladder. Although cystectomy was planned, our patient passed away before this could be done

Diagnostic algorithm for papillary urothelial tumors in the urinary bladder

Papillary urothelial neoplasms with deceptively bland cytology cannot be easily classified. We aimed to design a new algorithm that could differentiate between these neoplasms based on a scoring system. We proposed a new scoring system that enables to reproducibly diagnose non-invasive papillary urothelial tumors. In this system, each lesion was given individual scores from 0 to 3 for mitosis and cellular thickness, from 0 to 2 for cellular atypia, and an additional score for papillary fusion. These scores were combined to form a summed score allowing the tumors to be ranked as follows: 0–1 = UP, 2–4 = low malignant potential (LMP), 5–7 = low-grade transitional cell carcinoma (TCC), and 8–9 = high-grade TCC. In addition to the scoring system, ancillary studies of MIB and p53 indexes with CK20 expression pattern analyses were compared together with clinical parameters. The MIB index was strongly correlated with disease progression. Four of the 22 LMP patients (18.2%) had late recurrences, two of these four (9.1%) had progression to low-grade carcinoma. The MIB index for LMP patients was strongly associated with recurrence (recurrence vs. non-recurrence, 16.5 vs. 8.1, p < 0.001). The proposed scoring system could enhance the reproducibility to distinguish papillary urothelial neoplasms

Co-expression of RON and MET is a prognostic indicator for patients with transitional-cell carcinoma of the bladder

Recepteur d'Origine Nantais (RON) is a distinct receptor tyrosine kinase in the c-met proto-oncogene family. We examined the mutational and expression patterns of RON in eight human uroepithelial cell lines. Biological effects of RON overexpression on cancer cells were investigated in vitro, and the prognostic significance of RON and/or c-met protein (MET) expression was analysed in a bladder cancer cohort (n=183). There was no evidence of mutation in the kinase domain of RON. Overexpression of RON using an inducible Tet-off system induced increased cell proliferation, motility, and antiapoptosis. Immunohistochemical analysis showed that RON was overexpressed in 60 cases (32.8%) of primary tumours, with 14 (23.3%) showing a high level of expression. Recepteur d'Origine Nantais expression was positively associated with histological grading, larger size, nonpapillary contour, and tumour stage (all P<0.01). In addition, MET was overexpressed in 82 cases (44.8%). Co-expressed RON and MET was significantly associated with decreased overall survival (P=0.005) or metastasis-free survival (P=0.01) in 35 cases (19.1%). Recepteur d'Origine Nantais-associated signalling may play an important role in the progression of human bladder cancer. Evaluation of RON and MET expression status may identify a subset of bladder-cancer patients who require more intensive treatment

Low frequency of defective mismatch repair in a population-based series of upper urothelial carcinoma

BACKGROUND: Upper urothelial cancer (UUC), i.e. transitional cell carcinomas of the renal pelvis and the ureter, occur at an increased frequency in patients with hereditary nonpolyposis colorectal cancer (HNPCC). Defective mismatch repair (MMR) specifically characterizes HNPCC-associated tumors, but also occurs in subsets of some sporadic tumors, e.g. in gastrointestinal cancer and endometrial cancer. METHODS: We assessed the contribution of defective MMR to the development of UUC in a population-based series from the southern Swedish Cancer Registry, through microsatellite instability (MSI) analysis and immunohistochemical evaluation of expression of the MMR proteins MLH1, PMS2, MSH2, and MSH6. RESULTS: A MSI-high phenotype was identified in 9/216 (4%) successfully analyzed patients and a MSI-low phenotype in 5/216 (2%). Loss of MMR protein immunostaining was found in 11/216 (5%) tumors, and affected most commonly MSH2 and MSH6. CONCLUSION: This population-based series indicates that somatic MMR inactivation is a minor pathway in the development of UUC, but tumors that display defective MMR are, based on the immunohistochemical expression pattern, likely to be associated with HNPCC

Identification of S100A8-correlated genes for prediction of disease progression in non-muscle invasive bladder cancer

<p>Abstract</p> <p>Background</p> <p><it>S100 calcium binding protein A8 </it>(<it>S100A8</it>) has been implicated as a prognostic indicator in several types of cancer. However, previous studies are limited in their ability to predict the clinical behavior of the cancer. Here, we sought to identify a molecular signature based on <it>S100A8 </it>expression and to assess its usefulness as a prognostic indicator of disease progression in non-muscle invasive bladder cancer (NMIBC).</p> <p>Methods</p> <p>We used 103 primary NMIBC specimens for microarray gene expression profiling. The median follow-up period for all patients was 57.6 months (range: 3.2 to 137.0 months). Various statistical methods, including the leave-one-out cross validation method, were applied to identify a gene expression signature able to predict the likelihood of progression. The prognostic value of the gene expression signature was validated in an independent cohort (n = 302).</p> <p>Results</p> <p>Kaplan-Meier estimates revealed significant differences in disease progression associated with the expression signature of <it>S100A8</it>-correlated genes (log-rank test, <it>P </it>< 0.001). Multivariate Cox regression analysis revealed that the expression signature of <it>S100A8</it>-correlated genes was a strong predictor of disease progression (hazard ratio = 15.225, 95% confidence interval = 1.746 to 133.52, <it>P </it>= 0.014). We validated our results in an independent cohort and confirmed that this signature produced consistent prediction patterns. Finally, gene network analyses of the signature revealed that <it>S100A8</it>, <it>IL1B</it>, and <it>S100A9 </it>could be important mediators of the progression of NMIBC.</p> <p>Conclusions</p> <p>The prognostic molecular signature defined by <it>S100A8</it>-correlated genes represents a promising diagnostic tool for the identification of NMIBC patients that have a high risk of progression to muscle invasive bladder cancer.</p

Small molecule FGF receptor inhibitors block FGFR-dependent urothelial carcinoma growth in vitro and in vivo

BACKGROUND: Activating mutations of FGFR3 are frequently identified in superficial urothelial carcinoma (UC) and increased expression of FGFR1 and FGFR3 are common in both superficial and invasive UC. METHODS: The effects of inhibition of receptor activity by three small molecule inhibitors (PD173074, TKI-258 and SU5402) were investigated in a panel of bladder tumour cell lines with known FGFR expression levels and FGFR3 mutation status. RESULTS: All inhibitors prevented activation of FGFR3, and inhibited downstream MAPK pathway signalling. Response was related to FGFR3 and/or FGFR1 expression levels. Cell lines with the highest levels of FGFR expression showed the greatest response and little or no effect was measured in normal human urothelial cells or in UC cell lines with activating RAS gene mutations. In sensitive cell lines, the drugs induced cell cycle arrest and/or apoptosis. IC(50) values for PD173074 and TKI-258 were in the nanomolar concentration range compared with micromolar concentrations for SU5402. PD173074 showed the greatest effects in vitro and in vivo significantly delayed the growth of subcutaneous bladder tumour xenografts. CONCLUSION: These results indicate that inhibition of FGFR1 and wild-type or mutant FGFR3 may represent a useful therapeutic approach in patients with both non-muscle invasive and muscle invasive UC

Bladder and upper urinary tract cancers as first and second primary cancers

Background Previous population-based studies on second primary cancers (SPCs) in urothelial cancers have focused on known risk factors in bladder cancer patients without data on other urothelial sites of the renal pelvis or ureter. Aims To estimate sex-specific risks for any SPCs after urothelial cancers, and in reverse order, for urothelial cancers as SPCs after any cancer. Such two-way analysis may help interpret the results. Methods We employed standardized incidence ratios (SIRs) to estimate bidirectional relative risks of subsequent cancer associated with urothelial cancers. Patient data were obtained from the Swedish Cancer Registry from years 1990 through 2015. Results We identified 46 234 urinary bladder cancers (75% male), 940 ureteral cancers (60% male), and 2410 renal pelvic cancers (57% male). After male bladder cancer, SIRs significantly increased for 9 SPCs, most for ureteral (SIR 41.9) and renal pelvic (17.2) cancers. In the reversed order (bladder cancer as SPC), 10 individual FPCs were associated with an increased risk; highest associations were noted after renal pelvic (21.0) and ureteral (20.9) cancers. After female bladder cancer, SIRs of four SPCs were significantly increased, most for ureteral (87.8) and pelvic (35.7) cancers. Female bladder, ureteral, and pelvic cancers associated are with endometrial cancer. Conclusions The risks of recurrent urothelial cancers were very high, and, at most sites, female risks were twice over the male risks. Risks persisted often to follow-up periods of >5 years, motivating an extended patient follow-up. Lynch syndrome-related cancers were associated with particularly female urothelial cancers, calling for clinical vigilance.Peer reviewe

The pharmacokinetics of the interstitial space in humans

BACKGROUND: The pharmacokinetics of extracellular solutes is determined by the blood-tissue exchange kinetics and the volume of distribution in the interstitial space in the different organs. This information can be used to develop a general physiologically based pharmacokinetic (PBPK) model applicable to most extracellular solutes. METHODS: The human pharmacokinetic literature was surveyed to tabulate the steady state and equilibrium volume of distribution of the solutes mannitol, EDTA, morphine-6-glucuronide, morphine-3-glucuronide, inulin and β-lactam antibiotics with a range of protein binding (amoxicillin, piperacillin, cefatrizine, ceforanide, flucloxacillin, dicloxacillin). A PBPK data set was developed for extracellular solutes based on the literature for interstitial organ volumes. The program PKQuest was used to generate the PBPK model predictions. The pharmacokinetics of the protein (albumin) bound β-lactam antibiotics were characterized by two parameters: 1) the free fraction of the solute in plasma; 2) the interstitial albumin concentration. A new approach to estimating the capillary permeability is described, based on the pharmacokinetics of the highly protein bound antibiotics. RESULTS: About 42% of the total body water is extracellular. There is a large variation in the organ distribution of this water – varying from about 13% of total tissue water for skeletal muscle, up to 70% for skin and connective tissue. The weakly bound antibiotics have flow limited capillary-tissue exchange kinetics. The highly protein bound antibiotics have a significant capillary permeability limitation. The experimental pharmacokinetics of the 11 solutes is well described using the new PBPK data set and PKQuest. CONCLUSIONS: Only one adjustable parameter (systemic clearance) is required to completely characterize the PBPK for these extracellular solutes. Knowledge of just this systemic clearance allows one to predict the complete time course of the absolute drug concentrations in the major organs. PKQuest is freely available