Altered expression of genes identified in rats with prostatic chronic inflammation in a prostate spheroid model treated by estradiol/testosterone.

Rats are the standard model for male reproductive toxicity testing. Rat prostates are physiologically and anatomically different from those of humans. Drug and chemical toxicity testing would benefit from an in vitro model of human prostate cells. Recently, spheroids derived by three-dimensional culture of human cell lines have been used for assessing drug and chemical toxicity in vitro as they mimic in vivo environments more closely than two-dimensional culture. However, forming consistently sized, uniform spheroids is technically challenging for toxicity testing. The purpose of this study was to identify potential genetic markers for assessing prostatic toxicity in spheroids. We formed prostate spheroids using agarose-coated plates seeded with human primary prostate epithelial cells. Prostate spheroids were treated with either 17ß-estradiol (E2) or testosterone (T) on days 2-7 of culture. Samples were harvested on culture day 7. qPCR was used to examine gene expression levels previously identified in rats with chronic inflammation exposed to estradiol benzoate, E2 and/or T. Changes in some gene expression levels were observed in the spheroids treated with E2 or T. We found that treatment with 1 nM E2 and/or 10 µM T significantly altered spheroid proliferation and viability, as well as the expression levels of genes including Nanog homeobox (NANOG), C-C motif chemokine ligand 2 (CCL2) and bone morphogenetic protein receptor type 2 (BMPR2). Further studies using biologically active molecules with prostatic toxicity are needed to verify the results and to determine whether gene expression changes in the spheroid are specific to E2 or T treatment.

Identification of Altered Proteins in the Plasma of Rats With Chronic Prostatic Inflammation Induced by Estradiol Benzoate and Sex Hormones.

The cause of nonbacterial chronic prostatitis is unknown, yet its prevalence accounts for more than 90% of all prostatitis cases. Whole blood, plasma, and serum have been used to identify prostate cancer biomarkers; however, few studies have performed protein profiling to identify prostatitis biomarkers. The purpose of this study was to identify protein biomarkers altered by chronic prostatitis. To perform the study, we chemically induced chronic prostate inflammation in Sprague Dawley rats using estradiol benzoate (EB), testosterone (T), and estradiol (E) and then examined protein levels in their plasma. Plasma was collected on postnatal days (PNDs) 90, 100, 145, and 200; plasma proteins were profiled using liquid chromatography-tandem mass spectrometry. Chronic inflammation was observed in the rat prostate induced with EB on PNDs 1, 3, and 5. Rats then were dosed with T+E during PNDs 90-200 via subcutaneous implants. We identified time-specific expression for several proteins (i.e., CFB, MYH9, AZGP1). Some altered proteins that were expressed in the prostate (i.e., SERPINF1, CTR9) also were identified in the rat plasma in the EB+T+E group on PNDs 145 and 200. These findings suggest that the identified proteins could be used as biomarkers of chronic prostatitis. Further studies are needed to verify the results in human samples.

Comparison of germ cell differentiation of rat testis fragments cultured in knockout serum replacement versus Albumax™ I.

BACKGROUND: Spermatogenesis complexity makes reliable in vitro testis model development challenging. Previously, we evaluated an in vitro mouse testis organ culture system for assessing testicular toxicity. However, rat models are commonly used for drug/chemical toxicity testing; therefore, we assessed the effects of media on germ cell differentiation in cultured rat testis fragments. METHODS: Testes from postnatal day 5 Sprague-Dawley (Hsd:SD) rats were cultured in knockout serum replacement (KSR) or Albumax™ I (Albumax) medium. For testis morphology and germ cell differentiation, rat testis fragments were collected on days 20, 27, 35, 42, 49, and 63 of culture for histology/immunohistochemistry using antibodies to spermatogenesis-specific markers. The fragments collected on days 20, 27, 42, 49, and 63 were used for qPCR. RESULTS: Pachytene spermatocyte (PS) differentiation was observed in rat testis fragments cultured in KSR and Albumax. However, there were more seminiferous tubules (STs) with PS in rat testis fragments cultured in Albumax than in KSR. Over 60% of STs with germ cell differentiation were observed in rat testis fragments when cultured in Albumax on days 20, 27, and 35, whereas this figure showed only on day 20 when cultured in KSR. CONCLUSIONS: This study found only PS differentiation in rat testis fragments. Compared to KSR, Albumax appears to contribute to increased PS production. This in vitro rat testis organ culture system may be useful for assessing testicular toxicity. However, PS differentiation per ST is lower in rat testis fragments; further studies are required to improve this rat testis organ culture system.

Gene expression profiling in dorsolateral prostates of prepubertal and adult Sprague-Dawley rats dosed with estradiol benzoate, estradiol, and testosterone.

The imbalance of testosterone to estradiol ratio has been related to the development of prostate diseases. Although rat models of prostate diseases induced by endocrine-disrupting chemicals (EDCs) and/or hormone exposure are commonly used to analyze gene expression profiles in the prostate, most studies utilize a single endpoint. In this study, microarray analysis was used for gene expression profiling in rat prostate tissue after exposure to EDCs and sex hormones over multiple time points (prepubertal through adulthood). We used dorsolateral prostate tissues from Sprague-Dawley rats (male offspring) and postnatally administered estradiol benzoate (EB) on postnatal days (PNDs) 1, 3, and 5, followed by treatment with additional hormones [estradiol (E) and testosterone (T)] on PNDs 90-200, as described by Ho et al. Microarray analysis was performed for gene expression profiling in the dorsolateral prostate, and the results were validated via qRT-PCR. The genes in cytokine-cytokine receptor interaction, cell adhesion molecules, and chemokines were upregulated in the EB+T+E group on PNDs 145 and 200. Moreover, early-stage downregulation of anti-inflammatory gene: bone morphogenetic protein 7 gene was observed. These findings suggest that exposure to EB, T, and E activates multiple pathways and simultaneously downregulates anti-inflammatory genes. Interestingly, these genes are reportedly expressed in prostate cancer tissues/cell lines. Further studies are required to elucidate the mechanism, including analyses using human prostate tissues.

Increased estrogen levels altered microRNA expression in prostate and plasma of rats dosed with sex hormones.

BACKGROUND: Elevated estrogen (E) levels caused by aging or exposure to endocrine disrupting chemicals are related to prostate disease development. Sixty to seventy percent of prostate cancer or benign prostatic hyperplasia patients are over the age of 65, while prostatitis is likely to occur in men under 45 years. MicroRNAs currently represent a class of distinctive biological indicators to be used for clinical disease diagnosis and treatment monitoring. This study aims to identify microRNAs that could serve as potential biomarkers for prostate disorders induced by elevated E levels according to their altered expression in prostate or plasma. MATERIALS AND METHODS: Groups of Sprague Dawley rats (offspring) were dosed with estradiol benzoate (EB) on postnatal days 1, 3, and 5, and subcutaneously implanted with tubes containing testosterone (T)/E on postnatal day 90. Expression levels of prostate and plasma microRNAs were evaluated using microRNA microarray and validated via qRT-PCR. The expression levels of the potential targeted genes of a set of identified microRNAs were also examined by qRT-PCR. RESULTS: Postnatal administration of EB, T, and E elevated serum E levels with decreased serum T levels in rats. Chronic inflammation was observed in the dorsolateral prostate. Significant changes in expression levels of several microRNAs (rno-miR-146-5p, rno-miR-329-3p, and rno-miR-126a-3p) in the dorsolateral prostate and of a microRNA (rno-miR-329-3p) in the plasma were found in the dosed rats. The target gene expression levels of the altered microRNAs also changed accordingly. CONCLUSION: Chronic inflammation in the dorsolateral prostate of rats dosed with EB, T, and E resulted in deregulated expression in a set of microRNAs whose target genes were related to tumor growth or abnormal proliferation. Our findings suggest the identified microRNAs and their target genes the potential use as biomarkers to predict prostate cancer development. Validation using human samples is warranted.

Gene expression profiling of cultured mouse testis fragments treated with ethinylestradiol.

The assessment of xenobiotic-induced testicular toxicity is important in drug development. Nonetheless, in vitro models to test drugs and chemicals that may cause testicular toxicity are lacking, requiring the continued use of animal models for those studies. We previously evaluated an in vitro mouse testis organ culture system using ethinylestradiol (EE), a well-studied testicular toxicant, and demonstrated a dose-dependent relationship between adverse effects to germ cell differentiation and increasing EE concentrations. However, we terminated that study after 20 days of culture due to oxygen deficiency during germ cell differentiation. Therefore, in the current study, we aimed to identify gene(s) with potential for supporting the histopathological evaluations of testicular toxicity using in vitro testis organ culture system. We cultured testis fragments obtained from mice at postnatal day (PND) 5 in α-Minimal Essential Medium containing 40 mg/mL AlbuMAX™ I and treated them with 0.01 or 1 nM EE on day 1 of culture. On day 20, we collected testis fragments for RNA sequencing analysis and quantitative polymerase chain reaction (qPCR). We found that phospholipase C, zeta 1 and testis-specific serine kinase 4 genes, that are involved in spermatogenesis and predominantly expressed in the testis, were significantly reduced in testis fragments treated with the highest concentration of EE. Also, cytochrome P450, family 26, subfamily b, polypeptide 1 (Cyp26b1) and interleukin 16 (Il16) were up-regulated in the highest EE-treated groups. Further studies are needed to confirm the variations of these gene expression using other testicular toxicants.

Metabolomics-based pathway changes in testis fragments treated with ethinylestradiol in vitro.

BACKGROUND: There is a need to develop in vitro models to test drugs and chemicals that induce toxicity in the male reproductive system. We have evaluated an in vitro mouse testis organ culture model capable of producing viable, fertilization-proven sperm as a possible toxicity test model. Although this in vitro model was limited to round spermatid differentiation, histopathology observations could still be performed. Liquid chromatography/mass spectrometry analysis (LC/MS)-based metabolomics was used to measure metabolome changes of chemically treated in vitro testis fragments. METHODS: On Postnatal Day 5, C57BL/6J mouse testes were divided into four fragments, which were placed onto a 1.5% agarose gel cube and cultured in α-MEM including 0.4% AlbuMAX I (Day 0). On Day 1 of culture, testis fragments were treated with 0 (control), 0.01, or 1 nM ethinylestradiol (EE). On Day 20 of culture, the testis fragments were collected for LC/MS and histology analysis. RESULTS: Several metabolites involved in glycogen metabolism and glycolysis pathways (uridine diphosphate-glucose, glucose phosphate, and pyruvate), in the tricarboxylic acid cycle pathway (oxaloacetate and aspartate), and in the arginine and proline metabolism (arginine and spermine) were significantly altered in the 1 nM EE treated group compared to the control group. The metabolite changes were associated with an increase in percentage of seminiferous tubules with round spermatids as well as dose-dependent dead cells. CONCLUSION: These findings suggest that EE treatment may cause testicular toxicity by affecting glycogen metabolism and energy pathways. To confirm these findings, further experiments will be necessary using other testicular toxicants.

Evaluation of an in vitro mouse testis organ culture system for assessing male reproductive toxicity.

BACKGROUND: Development of an in vitro system capable of producing mature sperm remains a challenging goal, with only few successes reported. Such a system, could be used to test agents for potential toxicity to the male reproductive system; to explore this, we exposed immature mouse testis fragments in culture to ethinylestradiol (EE), a well-known testicular toxicant in vivo. METHODS: Testis fragments from postnatal day 5 mice were cultured in Albumax I medium. After 24 hr of culture, fragments were treated with 0.01, 0.1 or 1 nM EE, then harvested after 20 days in culture and examined for histology or gene expression measures by quantitative PCR. RESULTS: There was substantial variability between fragments in the degree of spermatogenesis observed. The percentage of seminiferous tubules containing any dead germ cells increased as a result of EE exposure in a dose dependent fashion. This was accompanied with a decreased percentage of tubules with round spermatids. Expression of estrogen receptor 1, cytochrome P450, family 11, subfamily a, and polypeptide 1 also was reduced, depending on the dose. CONCLUSION: These gene expression changes in the testis fragments are similar to those seen after animals have been exposed to EE. Gene expression changes in testis fragments are encouraging, but the variability across samples will need to be reduced for this in vitro system to become a generally applicable method for assessing testicular toxicants.

Evaluation of Culture Time and Media in an In Vitro Testis Organ Culture System.

BACKGROUND: The complexity of spermatogenesis makes development of appropriate in vitro testis models challenging. A novel in vitro mouse testis culture system has been reported but not yet evaluated as an alternative model for male reproductive toxicity testing. We assessed the effects of media composition on sperm differentiation and testis morphology of cultured mouse testis fragments. METHODS: Testes from postnatal day 5 B6:CBA-Tg(Acrv1-EGFP)2727Redd/J male mice were cultured in knockout serum replacement (KSR) or Albumax I (Albumax) medium. Enhanced green fluorescent protein (EGFP) expression was examined on days 35, 42, 45, and 49 of culture. Histology and flow cytometry were performed for testis morphology and spermatid differentiation. RESULTS: EGFP signals were first observed in round spermatids on day 22 of culture (corresponding to postnatal day 27) and were observed until the end of culture, indicating testis-specific protein expression. A-kinase anchor protein 4 expression, a marker of elongated spermatid (step 15-16) occurred earlier in explants cultured in KSR than Albumax medium (typically day 35 and after day 42 of culture, respectively). The percentage of seminiferous tubules with elongated spermatid was higher in Albumax than KSR medium from days 45 to 49 of culture. CONCLUSION: Albumax medium may facilitate or support better morphology and spermatid production than KSR medium. Further studies need to improve spermatid production and refinement of this in vitro testis culture system that may be useful as a supplement to current male reproductive toxicity testing or an alternative model in cases where in vivo testing may be unfeasible. Birth Defects Research 109:465-474, 2017. © 2017 Wiley Periodicals, Inc.

Bundled postconditioning therapies improve hemodynamics and neurologic recovery after 17 min of untreated cardiac arrest.

OBJECTIVE: Ischemic postconditioning (stutter CPR) and sevoflurane have been shown to mitigate the effects of reperfusion injury in cardiac tissue after 15min of ventricular fibrillation (VF) cardiac arrest. Poloxamer 188 (P188) has also proven beneficial to neuronal and cardiac tissue during reperfusion injury in human and animal models. We hypothesized that the use of stutter CPR, sevoflurane, and P188 combined with standard advanced life support would improve post-resuscitation cardiac and neurologic function after prolonged VF arrest. METHODS: Following 17min of untreated VF, 20 pigs were randomized to Control treatment with active compression/decompression (ACD) CPR and impedance threshold device (ITD) (n=8) or Bundle therapy with stutter ACD CPR+ITD+sevoflurane+P188 (n=12). Epinephrine and post-resuscitation hypothermia were given in both groups per standard protocol. Animals that achieved return of spontaneous circulation (ROSC) were evaluated with echocardiography, biomarkers, and a blinded neurologic assessment with a cerebral performance category score. RESULTS: Bundle therapy improved hemodynamics during resuscitation, reduced need for epinephrine and repeated defibrillation, reduced biomarkers of cardiac injury and end-organ dysfunction, and increased left ventricular ejection fraction compared to Controls. Bundle therapy also improved rates of ROSC (100% vs. 50%), freedom from major adverse events (50% vs. 0% at 48h), and neurologic function (42% with mild or no neurologic deficit and 17% achieving normal function at 48h). CONCLUSIONS: Bundle therapy with a combination of stutter ACD CPR, ITD, sevoflurane, and P188 improved cardiac and neurologic function after 17min of untreated cardiac arrest in pigs. All studies were performed with approval from the Institutional Animal Care Committee of the Minneapolis Medical Research Foundation (protocol #12-11).

Role for toll-like receptor 4 in TNF-alpha secretion by murine macrophages in response to polysaccharide Krestin, a Trametes versicolor mushroom extract.

Woody fungi and yeast preparations show promise in cancer treatment by activating anti-tumor immune responses. Macrophages (J774A.1) were treated with PSK, Reishi extract, scleroglucan or vehicle control. Pre-incubation with TLR4 blocking antibody inhibited TNF-alpha secretion by both J774A.1 cells and primary splenocytes but had inconclusive effect on scleroglucan-induced secretion of TNF-alpha. PSK induced TNF-alpha and IL-6 secretion by wild type but not by TLR4-deficient peritoneal macrophages. We conclude that constituents from PSK act as ligands for TLR4 receptors leading to induction of TNF-alpha and IL-6 inflammatory cytokines. Receptor-mediated differences may be due to structural differences in beta glucans or non-glucan constituents.

WL-276, an antagonist against Bcl-2 proteins, overcomes drug resistance and suppresses prostate tumor growth.

Patients with hormone-refractory prostate cancer (HRPC) have an estimated median survival of only 10 months because of acquired drug resistance, urging the need to develop therapies against the drug-resistant HRPC phenotype. Accumulating evidence suggests that overexpressing antiapoptotic Bcl-2 family proteins is at least partially responsible for the development of drug resistance among HRPC patients. Antagonizing the antiapoptotic Bcl-2 family proteins, therefore, is one potential approach to circumventing drug resistance in HRPC. WL-276 was developed as a small-molecule antagonist against antiapoptotic Bcl-2 family proteins, with binding potency comparable to (-)-gossypol. Overexpressing Bcl-2 or Bcl-X(L) failed to confer resistance to WL-276. WL-276 also effectively induced apoptosis in PC-3 cells. In addition, three PC-3 cell lines with acquired drug resistance against standard cancer chemotherapies were more sensitive to WL-276 than the parent PC-3 cell line. The increased cytotoxicity toward drug-resistant PC-3 cells shows the clinical potential of WL-276 against HRPC that is resistant to conventional therapies. The anticancer activity of WL-276 was manifested in its suppression of PC-3-induced prostate tumor growth in vivo. The selective toxicity of WL-276 against drug-resistant PC-3 cells and its in vivo suppression of PC-3 prostate tumor growth suggest that WL-276 is a promising lead candidate for the development of Bcl-2 antagonists against drug-resistant HRPC.

Induction of apoptosis by antisense CK2 in human prostate cancer xenograft model.

Protein serine/threonine kinase CK2 (formerly casein kinase 2) is a ubiquitous protein kinase that plays key roles in cell growth, proliferation, and survival. We have shown previously that its molecular down-regulation induces apoptosis in cancer cells in culture. Here, we have employed a xenograft model of prostate cancer to extend these studies to determine whether antisense CK2alpha evokes a similar response in vivo. A single dose of antisense CK2alpha oligodeoxynucleotide given directly into the PC3-LN4 xenograft tumor in nude mouse induced a dose- and time-dependent tumor cell death in vivo. The tumor was completely resolved at the higher tested dose of the antisense. Cell death was due to apoptosis and correlated with a potent down-regulation of the CK2alpha message and loss of CK2 from the nuclear matrix in the xenograft tissue as well as in cancer cells in culture. These observations accorded with several of the earlier studies indicating that loss of CK2 from the nuclear matrix is associated with induction of apoptosis. Comparison of the effects of antisense CK2alpha oligodeoxynucleotide on cancer versus normal or noncancer cells showed that the concentration of antisense CK2alpha that elicited extensive apoptosis in tumor cells in culture or xenograft tumors in vivo had a relatively small or minimal effect on noncancer cells in culture or on normal prostate gland subjected to orthotopic injection of antisense oligodeoxynucleotide in vivo. The basis for the difference in sensitivity of cancer versus noncancer cells to antisense CK2alpha is unknown at this time; however, this differential response under similar conditions of treatment may be significant in considering the potential feasibility of targeting the CK2 signal for induction of apoptosis in cancer cells in vivo. Although much further work will be needed to establish the feasibility of targeting CK2 for cancer therapy, to our knowledge, this is the first report to provide important new evidence as an initial "proof of principle" for the potential application of antisense CK2alpha in cancer therapy, paving the way for future detailed studies of approaches to targeting CK2 in vivo to induce cancer cell death.