The effect of black cohosh extract and risedronate coadministration on bone health in an ovariectomized rat model.

Preparations of black cohosh extract are sold as dietary supplements marketed to relieve the vasomotor symptoms of menopause, and some studies suggest it may protect against postmenopausal bone loss. Postmenopausal women are also frequently prescribed bisphosphonates, such as risedronate, to prevent osteoporotic bone loss. However, the pharmacodynamic interactions between these compounds when taken together is not known. To investigate possible interactions, 6-month-old, female Sprague-Dawley rats underwent bilateral ovariectomy or sham surgery and were treated for 24 weeks with either vehicle, ethinyl estradiol, risedronate, black cohosh extract or coadministration of risedronate and black cohosh extract, at low or high doses. Bone mineral density (BMD) of the femur, tibia, and lumbar vertebrae was then measured by dual-energy X-ray absorptiometry (DEXA) at weeks 0, 8, 16, and 24. A high dose of risedronate significantly increased BMD of the femur and vertebrae, while black cohosh extract had no significant effect on BMD individually and minimal effects upon coadministration with risedronate. Under these experimental conditions, black cohosh extract alone had no effect on BMD, nor did it negatively impact the BMD-enhancing properties of risedronate.

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.

Alzheimer's disease: a step closer to understanding type 3 diabetes in African Americans.

Alzheimer's disease (AD) is the fourth leading cause of death in the United States and the most common cause of adult-onset dementia. Recent results suggest an increased prevalence and severity in African Americans compared to Caucasians. Our understanding of the potential mechanism(s) underlying this ethnicity difference is limited. We previously described ethnicity-related differences in levels of neurodegenerative proteins and cytokines/chemokines in the BA21 region of African Americans and Caucasians with AD. Here, similar multiplex assays were used to examine those endpoints in patient postmortem cerebrospinal fluid (CSF). Additionally, we measured levels of C-peptide, ghrelin, gastric inhibitory polypeptide (GIP), glucagon-like peptide-1 (GLP-1), glucagon, insulin, leptin, PAI-1, resistin, and visfatin using a human diabetes 10-plex assay. The cytokine and chemokine assays revealed that levels of 26 chemokines or cytokines differed significantly with ethnicity, and three of those were significantly associated with gender. The neurodegenerative disease panel indicated that levels of soluble RAGE were significantly elevated in African Americans compared to Caucasians. All measures in the diabetes disease panel assay were significantly elevated in African Americans: ghrelin, GIP, GLP-1, glucagon, insulin, and visfatin. Through peripheral sample analysis, these results provide further evidence that ethnicity is critically involved in the manifestation of AD.

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.

Evaluation of the Performance of Lipidyzer Platform and Its Application in the Lipidomics Analysis in Mouse Heart and Liver.

Lipids play important roles in cell signaling, energy storage, and as major structural components of cell membranes. To date, little work has been conducted to show the extent of tissue specificity of lipid compositions. Here, the recently acquired Lipidyzer platform was employed in this pilot study: (i) to assess the performance of the Lipidyzer platform, (ii) to explore lipid profiles in liver and cardiac tissue in mice, (iii) to examine sex-specific differences in lipids in the liver tissue, and (iv) to evaluate biological variances in lipidomes present in animals. In total, 787 lipid species from 13 lipid classes were measured in the liver and heart. Lipidomics data from the Lipidyzer platform were very reproducible with the coefficient of variations of the quality control (QC) samples, ∼10%. The total concentration of the cholesterol esters (CE) lipid class, and specifically CE(16:1) and CE(18:1) species, showed sex differences in the liver. Cardiac tissue had higher levels of phospholipids containing docosahexaenoic acid, which could be related to heart health status and function. Our results demonstrate the usefulness of the Lipidyzer platform in identifying differences in lipid profile at the tissue level and between male and female mice in specific tissues.

Increased inflammation in BA21 brain tissue from African Americans with Alzheimer's disease.

Chronic neuroinflammation is strongly associated with AD and altered peripheral and central levels of chemokines and cytokines have been frequently described in those with AD. Given the increasing evidence of ethnicity-related differences in AD, it was of interest to determine if those altered chemokine and cytokine levels are ethnicity-related. Because African Americans exhibit a higher incidence of AD and increased symptom severity, we explored chemokine and cytokine concentrations in post-mortem brain tissue from the BA21 region of African Americans and Caucasians with AD using multiplex assays. IL-1ß, MIG, TRAIL, and FADD levels were significantly increased in African Americans while levels of IL-3 and IL-8 were significantly decreased. Those effects did not interact with gender; however, overall levels of CCL25, CCL26 and CX3CL1 were significantly decreased in women. The NLRP3 inflammasome is thought to be critically involved in AD. Increased activation of this inflammasome in African Americans is consistent with the current results.

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.

Neurodegenerative Markers are Increased in Postmortem BA21 Tissue from African Americans with Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) presents with an earlier onset age and increased symptom severity in African Americans and Hispanics. OBJECTIVE: Although the prevalence of plaques and tangles may not exhibit ethnicity-related differences, levels of neurodegenerative proteins have not been described. METHODS: Here, levels of five proteins (i.e., S100B, sRAGE, GDNF, Aß40, and Aß42) and the Aß42/Aß40 ratio were measured in postmortem samples of the middle temporal gyrus (BA21) from age-matched African Americans and Caucasians with AD (n = 6/gender/ethnicity). RESULTS: S100B levels were increased 17% in African Americans (p < 0.003) while sRAGE was mildly decreased (p < 0.09). Aß42 levels were increased 121% in African Americans (p < 0.02), leading to a 493% increase in the Aß42/Aß40 ratio (p < 0.002). Analysis of GDNF levels did not indicate any significant effects. There were no significant effects of gender and no significant ethnicity with gender interactions on any analyte. Effect size calculations indicated "medium" to "very large" effects. CONCLUSION: S100B is typically elevated in AD cases; however, the increased levels in African Americans here may be indicative of increased severity in specific populations. Increased Aß42/Aß40 ratios in the current study are compatible with increased disease severity and might indicate increased AD pathogenesis in African Americans. Overall, these results are compatible with a hypothesis of increased neuroinflammation in African Americans with AD.

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.

Transcriptomics analysis of early embryonic stem cell differentiation under osteoblast culture conditions: Applications for detection of developmental toxicity.

The mouse embryonic stem cell test (mEST) is a promising in vitro assay for predicting developmental toxicity. In the current study, early differentiation of D3 mouse embryonic stem cells (mESCs) under osteoblast culture conditions and embryotoxicity of cadmium sulfate were examined. D3 mESCs were exposed to cadmium sulfate for 24, 48 or 72h, and whole genome transcriptional profiles were determined. The results indicate a track of differentiation was identified as mESCs differentiate. Biological processes that were associated with differentiation related genes included embryonic development and, specifically, skeletal system development. Cadmium sulfate inhibited mESC differentiation at all three time points. Functional pathway analysis indicated biological pathways affected included those related to skeletal development, renal and reproductive function. In summary, our results suggest that transcriptional profiles are a sensitive indicator of early mESC differentiation. Transcriptomics may improve the predictivity of the mEST by suggesting possible modes of action for tested chemicals.

Systems view of adipogenesis via novel omics-driven and tissue-specific activity scoring of network functional modules.

The investigation of the complex processes involved in cellular differentiation must be based on unbiased, high throughput data processing methods to identify relevant biological pathways. A number of bioinformatics tools are available that can generate lists of pathways ranked by statistical significance (i.e. by p-value), while ideally it would be desirable to functionally score the pathways relative to each other or to other interacting parts of the system or process. We describe a new computational method (Network Activity Score Finder - NASFinder) to identify tissue-specific, omics-determined sub-networks and the connections with their upstream regulator receptors to obtain a systems view of the differentiation of human adipocytes. Adipogenesis of human SBGS pre-adipocyte cells in vitro was monitored with a transcriptomic data set comprising six time points (0, 6, 48, 96, 192, 384 hours). To elucidate the mechanisms of adipogenesis, NASFinder was used to perform time-point analysis by comparing each time point against the control (0 h) and time-lapse analysis by comparing each time point with the previous one. NASFinder identified the coordinated activity of seemingly unrelated processes between each comparison, providing the first systems view of adipogenesis in culture. NASFinder has been implemented into a web-based, freely available resource associated with novel, easy to read visualization of omics data sets and network modules.

Developing osteoblasts as an endpoint for the mouse embryonic stem cell test.

The mouse Embryonic Stem cell Test (EST) using cardiomyocyte differentiation is a promising in vitro assay for detecting potential embryotoxicity; however, the addition of another differentiation endpoint, such as osteoblasts, may improve the predictive value of the test. A number of variables such as culture conditions and starting cell number were investigated. A 14 day direct plating method of D3 mouse embryonic stem cells (mESCs) was used to test the predictivity of osteoblast differentiation as an endpoint in the EST. Twelve compounds were tested using the prediction model developed in the ECVAM validation study. Eight of the compounds selected from the EST validation study served as model compounds; four additional compounds known to produce skeletal defects were also tested. Our results indicate comparable chemical classification between the validated cardiomyocyte endpoint and the osteoblast endpoint. These results suggest that differentiation to osteoblasts may provide confirmatory information in predicting embryotoxicity.

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.