Evidence for Feedback Regulation Following Cholesterol Lowering Therapy in a Prostate Cancer Xenograft Model.

BACKGROUND: Epidemiologic data suggest cholesterol-lowering drugs may prevent the progression of prostate cancer, but not the incidence of the disease. However, the association of combination therapy in cholesterol reduction on prostate or any cancer is unclear. In this study, we compared the effects of the cholesterol lowering drugs simvastatin and ezetimibe alone or in combination on the growth of LAPC-4 prostate cancer in vivo xenografts. METHODS: Proliferation assays were conducted by MTS solution and assessed by Student's t-test. 90 male nude mice were placed on a high-cholesterol Western-diet for 7 days then injected subcutaneously with 1 × 105 LAPC-4 cells. Two weeks post-injection, mice were randomized to control, 11 mg/kg/day simvastatin, 30 mg/kg ezetimibe, or the combination and sacrificed 42 days post-randomization. We used a generalized linear model with the predictor variables of treatment, time, and treatment by time (i.e., interaction term) with tumor volume as the outcome variable. Total serum and tumor cholesterol were measured. Tumoral RNA was extracted and cDNA synthesized from 1 ug of total RNA for quantitative real-time PCR. RESULTS: Simvastatin directly reduced in vitro prostate cell proliferation in a dose-dependent, cell line-specific manner, but ezetimibe had no effect. In vivo, low continuous dosing of ezetimibe, delivered by food, or simvastatin, delivered via an osmotic pump had no effect on tumor growth compared to control mice. In contrast, dual treatment of simvastatin and ezetimibe accelerated tumor growth. Ezetimibe significantly lowered serum cholesterol by 15%, while simvastatin had no effect. Ezetimibe treatment resulted in higher tumor cholesterol. A sixfold induction of low density lipoprotein receptor mRNA was observed in ezetimibe and the combination with simvastatin versus control tumors. CONCLUSIONS: Systemic cholesterol lowering by ezetimibe did not slow tumor growth, nor did the cholesterol independent effects of simvastatin and the combined treatment increased tumor growth. Despite lower serum cholesterol, tumors from ezetimibe treated mice had higher levels of cholesterol. This study suggests that induction of low density lipoprotein receptor is a possible mechanism of resistance that prostate tumors use to counteract the therapeutic effects of lowering serum cholesterol. Prostate 77:446-457, 2017. © 2016 Wiley Periodicals, Inc.

Got worms? Perinatal exposure to helminths prevents persistent immune sensitization and cognitive dysfunction induced by early-life infection.

The incidence of autoimmune and inflammatory diseases has risen dramatically in post-industrial societies. "Biome depletion" - loss of commensal microbial and multicellular organisms such as helminths (intestinal worms) that profoundly modulate the immune system - may contribute to these increases. Hyperimmune-associated disorders also affect the brain, especially neurodevelopment, and increasing evidence links early-life infection to cognitive and neurodevelopmental disorders. We have demonstrated previously that rats infected with bacteria as newborns display life-long vulnerabilities to cognitive dysfunction, a vulnerability that is specifically linked to long-term hypersensitivity of microglial cell function, the resident immune cells of the brain. Here, we demonstrate that helminth colonization of pregnant dams attenuated the exaggerated brain cytokine response of their offspring to bacterial infection, and that combined with post-weaning colonization of offspring with helminths (consistent with their mothers treatment) completely prevented enduring microglial sensitization and cognitive dysfunction in adulthood. Importantly, helminths had no overt impact on adaptive immune cell subsets, whereas exaggerated innate inflammatory responses in splenic macrophages were prevented. Finally, helminths altered the effect of neonatal infection on the gut microbiome; neonatal infection with Escherichia coli caused a shift from genera within the Actinobacteria and Tenericutes phyla to genera in the Bacteroidetes phylum in rats not colonized with helminths, but helminths attenuated this effect. In sum, these data point toward an inter-relatedness of various components of the biome, and suggest potential mechanisms by which this helminth might exert therapeutic benefits in the treatment of neuroinflammatory and cognitive disorders.

The effect of carbohydrate restriction on prostate cancer tumor growth in a castrate mouse xenograft model.

BACKGROUND: No- and low-carbohydrate diets delay tumor growth compared to western diet (WD) in prostate cancer (PCa) xenograft studies. The effect of these diets in concert with androgen deprivation is unknown. METHODS: A total of 160 male SCID mice were injected with 1× 10(5) LAPC-4 human PCa cells. Of these, 150 mice were castrated and randomized to an ad libitum WD or fed via a paired-feeding protocol with a no-carbohydrate ketogenic diet (NCKD), 10% carbohydrate diet, or 20% carbohydrate diet. The remaining 10 mice were not castrated and were fed an ad libitum WD. The mice were sacrificed once volumes reached 1,000 mm3 and survival tested using the log-rank test. Serum from the median surviving 8 mice/group was assayed for insulin, IGF-1, and IGFBP-3. RESULTS: Body weights were roughly equal among groups. The 10 non-castrated mice experienced accelerated tumor growth. Among castrated mice, WD had the most rapid tumor growth; 20% carbohydrate diet the slowest (P = 0.046). Survival was not significantly different among the various carbohydrate restricted groups (P = 0.51). When pooled, there was a non-significant trend (P = 0.11) in improved survival among the carbohydrate restricted diets versus WD. No significant difference in serum insulin, IGF-1, and IGFBP-3 levels was noted among all groups at pre-randomization or at sacrifice. CONCLUSIONS: A 20% carbohydrate diet slowed tumor growth versus a WD. Though the benefit of carbohydrate restriction was somewhat less than in prior studies in non-castrate mice, these data still suggest diets achievable in humans may play a role in PCa management.

Carbohydrate restriction and lactate transporter inhibition in a mouse xenograft model of human prostate cancer.

UNLABELLED: What's known on the subject? and What does the study add? It is known that both lactate inhibition and carbohydrate restriction inhibit tumour growth. What is unknown is whether the two work synergistically together. This study adds that though the combination of lactate inhibition and carbohydrate restriction did not synergistically slow tumour growth in our model, we confirmed that carbohydrate restriction started after tumour inoculation slowed tumour growth. Moreover, lactate inhibition resulted in changes in the tumour microenvironment that may have implications for future metabolic targeting of prostate cancer growth. OBJECTIVE: To determine if a no-carbohydrate ketogenic diet (NCKD) and lactate transporter inhibition can exert a synergistic effect on delaying prostate tumour growth in a xenograft mouse model of human prostate cancer. MATERIALS AND METHODS: 120 nude athymic male mice (aged 6-8 weeks) were injected s.c. in the flank with 1.0 × 10(5) LAPC-4 prostate cancer cells. • Mice were randomized to one of four treatment groups: Western diet (WD, 35% fat, 16% protein, 49% carbohydrate) and vehicle (Veh) treatment; WD and mono-carboxylate transporter-1 (MCT1) inhibition via α-cyano-4-hydroxycinnamate (CHC) delivered through a mini osmotic pump; NCKD (84% fat, 16% protein, 0% carbohydrate) plus Veh; or NCKD and MCT1 inhibition. • Mice were fed and weighed three times per week and feed was adjusted to maintain similar body weights. • Tumour size was measured twice weekly and the combined effect of treatment was tested via Kruskal-Wallis analysis of all four groups. Independent effects of treatment (NCKD vs WD and CHC vs Veh) on tumour volume were tested using linear regression analysis. • All mice were killed on Day 53 (conclusion of pump ejection), and serum and tumour sections were analysed for various markers. Again, combined and independent effects of treatment were tested using Kruskal-Wallis and linear regression analysis, respectively. RESULTS: There were no significant differences in tumour volumes among the four groups (P= 0.09). • When testing the independent effects of treatment, NCKD was significantly associated with lower tumour volumes at the end of the experiment (P= 0.026), while CHC administration was not (P= 0.981). However, CHC was associated with increased necrotic fraction (P < 0.001). CONCLUSIONS: Differences in tumour volumes were observed only in comparisons between mice fed a NCKD and mice fed a WD. • MCT1 inhibition did not have a significant effect on tumour volume, although it was associated with increased necrotic fraction.

Effect of intermittent fasting with or without caloric restriction on prostate cancer growth and survival in SCID mice.

INTRODUCTION: Caloric restriction (CR) delays cancer growth in animals, though translation to humans is difficult. We hypothesized intermittent fasting (i.e., intermittent extreme CR), may be better tolerated and prolong survival of prostate cancer (CaP) bearing mice. METHODS: We conducted a pilot study by injecting 105 male individually-housed SCID mice with LAPC-4 cells. When tumors reached 200 mm(3), 15 mice/group were randomized to one of seven diets and sacrificed when tumors reached 1,500 mm(3): Group 1: ad libitum 7 days/week; Group 2: fasted 1 day/week and ad libitum 6 days/week; Group 3: fasted 1 day/week and fed 6 days/week via paired feeding to maintain isocaloric conditions to Group 1; Group 4: 14% CR 7 days/week; Group 5: fasted 2 days/week and ad libitum 5 days/week; Group 6: fasted 2 day/week and fed 5 days/week via paired feeding to maintain isocaloric conditions to Group 1; Group 7: 28% CR 7 days/week. Sera from mice at sacrifice were analyzed for IGF-axis hormones. RESULTS: There were no significant differences in survival among any groups. However, relative to Group 1, there were non-significant trends for improved survival for Groups 3 (HR 0.65, P = 0.26), 5 (0.60, P = 0.18), 6 (HR 0.59, P = 0.16), and 7 (P = 0.59, P = 0.17). Relative to Group 1, body weights and IGF-1 levels were significantly lower in Groups 6 and 7. CONCLUSIONS: This exploratory study found non-significant trends toward improved survival with some intermittent fasting regimens, in the absence of weight loss. Larger appropriately powered studies to detect modest, but clinically important differences are necessary to confirm these findings.

Effect of isocaloric low fat diet on prostate cancer xenograft progression in a hormone deprivation model.

PURPOSE: Previous mouse studies suggesting that low fat diets slow prostate cancer growth often used corn oil (omega-6), which enhances prostate cancer growth, as the primary fat. Using a saturated fat based diet we previously found no significant difference in tumor growth between low and high fat fed SCID mice (Taconic Farms, Hudson, New York) xenografted with LAPC-4 cells. Whether similar results would hold in a castration model is unclear. MATERIALS AND METHODS: A total of 80 male SCID mice were fed a Western diet (40% fat and 44% carbohydrate) and injected with LAPC-4 human prostate cancer cells. When tumors were 200 mm(3), the mice were castrated and randomized to an isocaloric Western or a low fat diet (12% fat and 72% carbohydrate). Animals were sacrificed when tumors were 1,000 mm(3). Serum was collected and assayed for prostate specific antigen, insulin, insulin-like growth factor 1 and insulin-like growth factor binding protein 3. Tumors were assayed for total and phosphorylated Akt. RESULTS: Mouse weight was equivalent in the 2 groups. Overall dietary group was not significantly associated with survival (log rank p = 0.32). There were no statistically significant differences in prostate specific antigen (p = 0.53), insulin-like growth factor axis parameters (each p >0.05) or p-Akt-to-t-Akt ratios (p = 0.22) between the groups at sacrifice. CONCLUSIONS: In this xenograft model we found no difference in tumor growth or survival between low fat vs Western fed mice when the fat source was saturated fat. These results conflict with those of other studies in which corn oil was used to show that low fat diets delay prostate cancer growth, suggesting that fat type may be as important as fat amount in the prostate cancer setting.

Immunization enhances the natural antibody repertoire.

The role of immunization in the production of antibodies directed against immunogens is widely appreciated in laboratory animals and in humans. However, the role of immunization in the development of "natural antibodies" has not been investigated. Natural antibodies are those antibodies present without known history of infection or immunization, and react to a wide range of targets, including "cryptic" self-antigens that are exposed upon cell death. In this study, the ability of immunization to elicit the production of natural antibodies in laboratory rats was evaluated. Laboratory rats were immunized with a series of injections using peanut extracts (a common allergen), a high molecular weight protein conjugated to hapten (FITC-KLH), and a carbohydrate conjugated to hapten (DNP-Ficall). Significantly greater binding of antibodies from immunized animals compared to controls was observed to numerous autologous organ extracts (brain, kidney, liver, lung, prostate, and spleen) for both IgM and IgG, although the effect was more pronounced for IgM. These studies suggest that immunization may have at least one unforeseen benefit, enhancing networks of natural antibodies that may be important in such processes as wound repair and tumor surveillance. Such enhancement of natural antibody function may be particularly important in Western society, where decreased exposure to the environment may be associated with a weakened natural antibody repertoire.

Thalidomide enantiomers: determination in biological samples by HPLC and vancomycin-CSP.

Thalidomide is a racemate with potentially different pharmacokinetics and pharmacodynamics of the component (+)-(R)- and (-)-(S)-thalidomide enantiomers. As part of a project on the adjunctive effects of thalidomide and cytotoxic agents, a method for the chiral separation and quantitation of thalidomide was developed and validated. Thalidomide in relevant serum and tissue homogenate samples was stabilized by buffering with an equal volume of citrate-phosphate buffer (pH 2, 0.2M), and stored at -80 degrees C pending assay. The thalidomide enantiomers, extracted from the samples with diethyl ether, were well separated on a chiral HPLC column of vancomycin stationary phase and a mobile phase of 14% acetonitrile in 20 mM ammonium formate adjusted to pH 5.4; their concentrations were determined with phenacetin as internal standard at 220 nm detection. Over a thalidomide concentration range of 0.1-20 microg/ml, assay precision was 1-5% (CV) for both enantiomers, and calibration curves were linear with all correlation coefficients being >0.99. The estimated limit of quantification for both enantiomers was 0.05 microg/ml with 0.2-0.6 ml serum samples. Thalidomide in rat and human serum, acidified and stored as described above, was found to be chemically and chirally stable over 1 year. The method has been successfully applied to serum samples from human patients undergoing thalidomide treatment for mesothelioma, and to serum, blood and tissue samples from a laboratory rodent model using transplanted 9l gliosarcoma. Enantioselectivity in thalidomide pharmacokinetics has been found, thereby reinforcing the need for considering the relevance of chirality in thalidomide pharmacology.

Increased biodiversity in the environment improves the humoral response of rats.

Previous studies have compared the immune systems of wild and of laboratory rodents in an effort to determine how laboratory rodents differ from their naturally occurring relatives. This comparison serves as an indicator of what sorts of changes might exist between modern humans living in Western culture compared to our hunter-gatherer ancestors. However, immunological experiments on wild-caught animals are difficult and potentially confounded by increased levels of stress in the captive animals. In this study, the humoral immune responses of laboratory rats in a traditional laboratory environment and in an environment with enriched biodiversity were examined following immunization with a panel of antigens. Biodiversity enrichment included colonization of the laboratory animals with helminths and co-housing the laboratory animals with wild-caught rats. Increased biodiversity did not apparently affect the IgE response to peanut antigens following immunization with those antigens. However, animals housed in the enriched biodiversity setting demonstrated an increased mean humoral response to T-independent and T-dependent antigens and increased levels of "natural" antibodies directed at a xenogeneic protein and at an autologous tissue extract that were not used as immunogens.

Prospective polysomnographic analysis of obstructive sleep apnea in down syndrome.

OBJECTIVES: To investigate obstructive sleep apnea (OSA) in a consecutively encountered, nonselected population of young patients with Down syndrome using standard overnight polysomnography and to determine the effects of therapy. METHODS: In a population of patients seen for routine developmental evaluations, 9 boys and 10 girls were studied using standard overnight polysomnography. RESULTS: Using pediatric standards, OSA was found in 79% of the subjects (95% confidence interval, 54%-94%), with a median apnea index of 3 events per hour (interquartile range, 2-5), a median apnea-hypopnea index of 6 events per hour (interquartile range, 3-8), and a median arterial oxygen saturation (SaO2) low point of 88% (interquartile range, 84%-90%). Higher body mass index was significantly associated with a higher apnea index and a lower SaO2 level, and there was a significant inverse relationship between age and the lowest SaO2 value as well as a possible association between sleep-related symptoms at the time of diagnosis and the lowest SaO2 value. In addition, patients with OSA had a significantly higher movement arousal index than those without OSA. CONCLUSIONS: Using rigid polysomnographic standards, this pilot study revealed OSA in a high percentage of young subjects with Down syndrome and an association between OSA and obesity, age, and poor sleep quality. These findings justify larger and more detailed population studies to further define clinical factors that are concomitant with OSA in Down syndrome and to improve therapy.

Low-carbohydrate diets and prostate cancer: how low is "low enough"?

Previous studies indicate that carbohydrate intake influences prostate cancer biology, as mice fed a no-carbohydrate ketogenic diet (NCKD) had significantly smaller xenograft tumors and longer survival than mice fed a Western diet. As it is nearly impossible for humans to consume and maintain NCKD, we determined whether diets containing 10% or 20% carbohydrate kcal showed similar tumor growth as NCKD. A total of 150 male severe combined immunodeficient mice were fed a Western diet ad libitum, injected with the human prostate cancer cell line LAPC-4, and then randomized 2 weeks later to one of three arms: NCKD, 10% carbohydrate, or 20% carbohydrate diets. Ten mice not injected were fed an ad libitum low-fat diet (12% fat kcal) serving as the reference in a modified-paired feeding protocol. Mice were sacrificed when tumors reached 1,000 mm(3). Despite consuming extra calories, all mice receiving low-carbohydrate diets were significantly lighter than those receiving a low-fat diet (P < 0.04). Among the low-carbohydrate arms, NCKD-fed mice were significantly lighter than the 10% or 20% carbohydrate groups (P < 0.05). Tumors were significantly larger in the 10% carbohydrate group on days 52 and 59 (P < 0.05), but at no other point during the study. Diet did not affect survival (P = 0.34). There were no differences in serum insulin-like growth factor-I or insulin-like growth factor binding protein-3 at sacrifice among the low-carbohydrate arms (P = 0.07 and P = 0.55, respectively). Insulin was significantly lower in the 20% carbohydrate arm (P = 0.03). LAPC-4 xenograft mice fed a low-carbohydrate diet (10-20% carbohydrate kcal) had similar survival as mice consuming NCKD (0% carbohydrate kcal).