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The inherent abilities of natural killer (NK) cells to recognize and kill target cells place them among the first cells with the ability to recognize and destroy infected or transformed cells. Cancer cells, however, have mechanisms by which they can inhibit the surveillance and cytotoxic abilities of NK cells with one believed mechanism for this: their ability to release exosomes. Exosomes are vesicles that are found in abundance in the tumor microenvironment that can modulate intercellular communication and thus enhance tumor malignancy. Recently, our lab has found cancer cell exosomes to contain the inhibitor of apoptosis (IAP) protein survivin to be associated with decreased immune response in lymphocytes and cellular death. The purpose of this study was to explore the effect of survivin and lymphoma-derived survivin-containing exosomes on the immune functions of NK cells. NK cells were obtained from the peripheral blood of healthy donors and treated with pure survivin protein or exosomes from two lymphoma cell lines, DLCL2 and FSCCL. RNA was isolated from NK cell samples for measurement by PCR, and intracellular flow cytometry was used to determine protein expression. Degranulation capacity, cytotoxicity, and natural killer group 2D receptor (NKG2D) levels were also assessed. Lymphoma exosomes were examined for size and protein content. This study established that these lymphoma exosomes contained survivin and FasL but were negative for MHC class I-related chains (MIC)/B (MICA/B) and TGF-β. Treatment with exosomes did not significantly alter NK cell functionality, but extracellular survivin was seen to decrease natural killer group 2D receptor (NKG2D) levels and the intracellular protein levels of perforin, granzyme B, TNF-α, and IFN-γ.

Alfonso M Durán,1 Lorena M Salto,1 Justin Câmara,1 Anamika Basu,1 Ivette Paquien,1 W Lawrence Beeson,1,2 Anthony Firek,3 Zaida Cordero-MacIntyre,1,2 Marino De León1 1Center for Health Disparities and Molecular Medicine, Department of Basic Sciences, Loma Linda University School of Medicine, Loma Linda, CA, USA; 2Center for Nutrition, Healthy Lifestyle and Disease Prevention, School of Public Health, Loma Linda University, Loma Linda, CA, USA; 3Comparative Effectiveness and Clinical Outcomes Research Center, Riverside University Health System Medical Center, Moreno Valley, CA, USA Purpose: To determine whether dietary supplementation with omega-3 polyunsaturated fatty acids (PUFAs) reduces neuropathic pain symptoms in Mexican-Americans with type 2 diabetes.Methods: Forty volunteers with type 2 diabetes enrolled in the “En Balance-PLUS” program, which provided weekly nutrition–diabetes education and daily supplementation with 1,000 mg docosahexaenoic acid (DHA)–200 mg eicosapentaenoic acid over 3 months. The study assessed self-reported neuropathic pain symptoms pre/postintervention using the short-form McGill Pain Questionnaire (SF-MPQ), monitored clinical laboratory values at baseline and 3 months, and performed baseline and 3-month metabolomic analysis of plasma samples.Results: A total of 26 participants self-reported neuropathic pain symptoms at baseline. After 3 months of omega-3 PUFA supplementation, participants reported significant improvement in SF-MPQ scores (sensory, affective, and visual analogue scale; P

Background The incidence of thyroid cancer is increasing worldwide, and there is an emerging need to develop accurate tools for diagnosis. Fine needle aspiration biopsy has greatly improved evaluation of thyroid nodules, but challenges with indeterminate lesions remain in up to 25% of biopsies. Novel tissue biomarkers may assist in improved nodule characterization. Microcalcifications occurring in thyroid cancers suggest proteins involved in bone formation may play a role in thyroid carcinogenesis. We evaluated the expression of the known osteogenic protein, Enigma, in thyroid cancer as a candidate oncoprotein and role in carcinogenesis based on association with other known oncoproteins such as bone morphogenetic protein-1 (BMP-1). Methods The expression of both Enigma and BMP-1 were evaluated by immunohistochemistry (IHC) in an equal number of benign (n = 120) and different histological subtypes of malignant (n = 120) human archival thyroid nodules with and without calcification. The colocalization of Enigma with BMP-1 was evaluated by confocal microscopy using the BZ analyzer. Results Enigma was strongly expressed in thyroid cancer tissue with a higher immunoreactive score in advanced thyroid cancer compared to less advanced and benign nodules. Enigma was localized either in cytoplasm or nucleus depending on the histological subtypes. Higher expression of Enigma was associated with the tumor size and lymph node involvement. There was clear and strong colocalization signal of Enigma and that of BMP-1. Expression of Enigma occurred without regard to calcification in cancer tissue. Conclusion Enigma may serve as an oncoprotein marker, identifying benign from malignant thyroid tissue on FNA. Enigma may have a role in carcinogenesis of thyroid cancer independent of tissue calcification, possibly in relation to interaction with BMP-1.

Purpose. En Balance, a culturally sensitive diabetes education program, improves glycemic control in Hispanics with type 2 diabetes. The program emphasized diet, physical activity, and other factors important for glycemic control. However, the individual contributions of these education factors are unclear. The purpose of this study is to assess the contribution of physical activity to the success of En Balance in improving the health of Mexican Americans with type 2 diabetes. Methods. A retrospective study was conducted with plasma samples collected pre- and post-3-month study. Samples from 58 (18 males and 40 females) Hispanic subjects with type 2 diabetes were analyzed for the concentration of kynurenines, known to decrease in response to exercise. After three months, health outcomes for the active group (decreased kynurenines) and the rest of the cohort were evaluated by paired Wilcoxon signed-rank test. Results. Half of the subjects had increased kynurenine levels at the end of the educational program. We found that the subjects in the active group with decreased kynurenine concentrations displayed statistically greater improvements in fasting blood glucose, A1C, cholesterol, and triglycerides despite weight loss being higher in the group with increased kynurenine concentrations. Conclusions. En Balance participants with decreased kynurenine levels had significantly improved glycemic control. These data suggest that physical activity significantly contributes to the success of the En Balance education program. This analysis indicates that diabetes public health educators should emphasize the benefit of physical activity on glycemic control even in the absence of major weight loss.

IGF2 is essential in breast differentiation, lactation, tumor growth, and in breast cancer (BC) development and progression. This growth factor also inhibits apoptosis and promotes metastasis and chemoresistance, contributing to more aggressive tumors. We previously demonstrated that IGF2 protein levels are higher in BC tissues from African American women than in Caucasian women. We also showed that high IGF2 protein levels are expressed in normal breast tissues of African American women while little or no IGF2 was detected in tissues from Caucasian women. Others showed that decreased DNA methylation of the IGF2 gene leads to different BC clinical features. Thus, we designed this study to determine if differentially methylated regions of the IGF2 gene correspond to IGF2 protein expression in paired (Normal/Tumor) breast tissues and in BC cell lines. Methylation analysis was performed using Sodium Bisulphite Analysis and Methylation Sensitive Restriction Enzyme digestion methods. Our results show that a unique site in the INS-IGF2 region is hypermethylated in normal breast and hypomethylated in breast cancer. We designated this region the DVDMR. Furthermore, the methylation levels in the DVDMR significantly correlated with IGF2 protein levels. This novel DMR consists of 257bp localized in the INS-IGF2 gene. We propose that methylation of DVDMR represents a novel epigenetic biomarker that determines the levels of IGF2 protein expression in breast cancer. Since IGF2 promotes metastasis and chemoresistance, we propose that IGF2 levels contribute to BC aggressiveness. Validation of IGF2 as a biomarker will improve diagnosis and treatment of BC patients.

Background and Aim Docosahexaenoic acid (DHA) exhibits neuroprotective properties and has been shown to preserve nerve cells following trauma and ischemic injury. Recently, we showed that DHA pretreatment improved locomotion and reduced neuropathic pain after acute spinal cord injury in adult rats. These improvements were associated with an increase in the levels of AKT in spinal cord injury neurons. In this study, we investigate the implication of PI3K/AKT and mTOR pathway in DHA‐mediated protection of primary cultured Schwann cells (pSC) undergoing palmitic acid‐induced lipotoxicity (PA‐LTx). Methods Primary cultured Schwann cells were treated with PA (PA:BSA, 2:1) in the presence or absence of DHA (1–200 µM) for 24–48 hr. Cell viability was determined by crystal violet staining and nuclear morphology was examined using Hoechst staining. Results We found that pSC cultures exposed to palmitic acid (PA) overload showed chromatin condensation, a decrease in cell viability and an inhibition of AKT phosphorylation in a time‐dependent manner. Next, pSC exposed to PA overload were treated with DHA. The data show that co‐treatment with DHA inhibited the loss of cell viability and apoptosis caused by PA. Moreover, treatment with DHA inhibited chromatin condensation, significantly stimulated p‐AKT phosphorylation under PA‐LTx condition, and DHA alone increased AKT phosphorylation. Additionally, when these pSC cultures were treated with PI3K inhibitors LY294002 and, BKM120 and mTOR inhibitors Torin 1 (mTORC1/mTORC2), but not rapamycin (mTORC1), the protective effects of DHA were not observed. Conclusion These findings suggest PI3K/AKT and mTORC2 kinase pathways are involved in the protective function (s) of DHA in PA‐induced Schwann cell death.

Writing is a critical skill for graduate students, but few studies in the literature describe how it is supported in the training of biomedical graduate students. The Initiative for Maximizing Student Development program at Loma Linda University aims to develop this important skill in its students through an integrated, structured writing intervention. Specifically, the program hired a writing specialist who taught writing seminars, facilitated writing and publishing workshops, and mentored students in one-on-one writing conferences. Doctoral students in the program, primarily underrepresented minority students with some not having English as a first language, all exhibited writing apprehension and blocking behaviors. The percentage of students graduating, publishing, and entering science careers, all of which require writing, is high. To yield insight into how this intervention worked, we conducted in-depth interviews of six of the earliest graduates, derived themes, analyzed data from pre- and post-assessments, and described their publication records. Participating students increased their writing confidence, adopted productive writing strategies, decreased writing anxiety and blocking behaviors, and published successfully.

African American (AA)/Black men are more likely to develop aggressive prostate cancer (PCa), yet less likely to be screened despite guidelines espousing shared decision-making regarding PCa screening and prostate-specific antigen (PSA) testing. Given the documented racial disparities in PCa incidence and mortality, engaging interactions with physicians are especially important for AA/Black men. Thus, this study evaluated occurrence of physician–patient conversations among AA/Black men, and whether such conversations were associated with PCa knowledge. We also quantified the serum PSA values of participants who had, and had not, discussed testing with their physicians. Self-identified AA/Black men living in California and New York, ages 21–85, donated blood and completed a comprehensive sociodemographic and health survey ( n = 414). Less than half (45.2%) of participants had discussed PCa screening with their physicians. Multivariate analyses were used to assess whether physician–patient conversations predicted PCa knowledge after adjusting for key sociodemographic/economic and health-care variables. Increased PCa knowledge was correlated with younger age, higher income and education, and having discussed the pros and cons of PCa testing with a physician. Serum PSA values were measured by ELISA. Higher-than-normal PSA values were found in 38.5% of men who had discussed PCa screening with a physician and 29.1% who had not discussed PCa screening. Our results suggest that physician–AA/Black patient conversations regarding PCa risk need improvement. Encouraging more effective communication between physicians and AA/Black men concerning PCa screening and PSA testing has the potential to reduce PCa health disparities.

Background Diabetes mellitus (DM) is considered a serious affliction that highly augments the risk for serious health complications. Evidence is suggesting a role of n-3 polyunsaturated fatty acids...

Traumatic spinal cord injury (SCI) results in debilitating autonomic dysfunctions, paralysis and significant sensorimotor impairments. A key component of SCI is the generation of free radicals that contributes to the high levels of oxidative stress observed. This study investigates whether dietary supplementation with the antioxidant vitamin E (alpha-tocopherol) improves functional recovery after SCI. Female adult Sprague-Dawley rats were fed either with a normal diet or a dietary regiment supplemented with vitamin E (51 IU/g) for eight weeks. The rats were subsequently exposed either to a contusive SCI or sham operation, and evaluated using standard functional behavior analysis. We report that the rats that consumed the vitamin E-enriched diet showed an accelerated bladder recovery and significant improvements in locomotor function relative to controls, as determined by residual volumes and Basso, Beatie, and Bresnaham BBB scores, respectively. Interestingly, the prophylactic dietary intervention did not preserve neurons in the ventral horn of injured rats, but it significantly increased the numbers of oligodendrocytes. Vitamin E supplementation attenuated the depression of the H-reflex (a typical functional consequence of SCI) while increasing the levels of supraspinal serotonin immunoreactivity. Our findings support the potential complementary use of vitamin E to ameliorate sensory and autonomic dysfunctions associated with spinal cord injury, and identified promising new cellular and functional targets of its neuroprotective effects.

African American women suffer higher incidence and mortality of triple negative breast cancer (TNBC) than Caucasian women. TNBC is very aggressive, causing the worst clinical outcome. We previously demonstrated that tumors from these patients express high IGF-II and exhibit high activation of the IGF signaling pathways. IGF-II gene expression is imprinted (monoallelic), promotes tumor progression, and metastasis and regulates Survivin, a TNBC prognostic marker. Since BC mortality has increased among young Vietnamese women, we analyzed 48 (paired) TNBC samples from Vietnamese patients to assess IGF-II expression. We analyzed all samples by qrtPCR for identification of IGF-II heterozygosity and to determine allelic expression of the IGF-II gene. We also analyzed the tissues for proIGF-II and Survivin by RT-PCR and Western blotting. A total of 28 samples displayed IGF-II heterozygosity of which 78% were biallelic. Tumors with biallelic IGF-II gene expression exhibited the highest levels of proIGF-II and Survivin. Although 100% of these tissues corresponding normal samples were biallelic, they expressed significantly lower levels of or no proIGF-II and Survivin. Thus, IGF-II biallelic gene expression is differentially regulated in normal versus tumor tissues. We propose that intratumoral proIGF-II is dependent on the IGF-II gene imprinting status and it will promote a more aggressive TNBC.

Background Understanding how biological factors contribute to prostate cancer (PCa) health disparities requires mechanistic functional analysis of specific genes or pathways in pre-clinical cellular and animal models of this malignancy. The 22Rv1 human prostatic carcinoma cell line was originally derived from the parental CWR22R cell line. Although 22Rv1 has been well characterized and used in numerous mechanistic studies, no racial identifier has ever been disclosed for this cell line. In accordance with the need for racial diversity in cancer biospecimens and recent guidelines by the NIH on authentication of key biological resources, we sought to determine the ancestry of 22RV1 and authenticate previously reported racial identifications for four other PCa cell lines. Methods We used 29 established Ancestry Informative Marker (AIM) single nucleotide polymorphisms (SNPs) to conduct DNA ancestry analysis and assign ancestral proportions to a panel of five PCa cell lines that included 22Rv1, PC3, DU145, MDA-PCa-2b, and RC-77T/E. Results We found that 22Rv1 carries mixed genetic ancestry. The main ancestry proportions for this cell line were 0.41 West African (AFR) and 0.42 European (EUR). In addition, we verified the previously reported racial identifications for PC3 (0.73 EUR), DU145 (0.63 EUR), MDA-PCa-2b (0.73 AFR), and RC-77T/E (0.74 AFR) cell lines. Conclusions Considering the mortality disparities associated with PCa, which disproportionately affect African American men, there remains a burden on the scientific community to diversify the availability of biospecimens, including cell lines, for mechanistic studies on potential biological mediators of these disparities. This study is beneficial by identifying another PCa cell line that carries substantial AFR ancestry. This finding may also open the door to new perspectives on previously published studies using this cell line.

Palmitic acid (PA) and other saturated fatty acids are known to stimulate pro-inflammatory responses in human immune cells via Toll-like receptor 4 (TLR4). However, the molecular mechanism responsible for fatty acid stimulation of TLR4 remains unknown. Here, we demonstrate that PA functions as a ligand for TLR4 on human monocyte derived dendritic cells (MoDCs). Hydrophobicity protein modeling indicated PA can associate with the hydrophobic binding pocket of TLR4 adaptor protein MD-2. Isothermal titration calorimetry quantified heat absorption that occurred during PA titration into TLR4/MD2, indicating that PA binds to TLR4/MD2. Treatment of human MoDCs with PA resulted in endocytosis of TLR4, further supporting the function of PA as a TLR4 agonist. In addition, PA stimulated DC maturation and activation based on the upregulation of DC costimulatory factors CD86 and CD83. Further experiments showed that PA induced TLR4 dependent secretion of the pro-inflammatory cytokine IL-1β. Lastly, our experimental data show that PA stimulation of NF-κB canonical pathway activation is regulated by TLR4 signaling and that reactive oxygen species may be important in upregulating this pro-inflammatory response. Our experiments demonstrate for the first time that PA activation of TLR4 occurs in response to direct molecular interactions between PA and MD-2. In summary, our findings suggest a likely molecular mechanism for PA induction of pro-inflammatory immune responses in human dendritic cells expressing TLR4.

Purpose This study was designed to assess the feasibility of culturally and language-sensitive diabetes education as a way to increase physical activity and to improve health/diabetes management in a group of Spanish-speaking Hispanics in the Inland Empire region of Southern California. Methods En Balance is a culturally sensitive diabetes education program designed for Spanish-speaking Hispanic adults. The 3-month educational intervention assessed 16 males and 23 females living in Riverside and San Bernardino counties of Southern California. Baseline and 3-month evaluations of physical activity were assessed using the validated Arizona Activity Frequency Questionnaire. Results After 3 months on the En Balance program, there was a significant increase in moderate intensity physical activity energy expenditure (M = 368 ± 894 kcal/day, P < 0.01) and high intensity physical activity energy expenditure (M = 405 ± 2569 kcal/day, P = 0.05) compared to baseline and significant reductions in A1C (−0.90%, P = 0.01), total cholesterol (−13.44 mg/dl, P = 0.01), LDL cholesterol (−10.28 mg/dl, P = 0.03), and waist circumference (−1.52 cm, P = 0.04). Conclusion En Balance program resulted in significant mean increases in both moderate and high intensity physical activity energy expenditure among this group of Hispanic diabetic participants, indicating that despite a general pattern of low physical activity in this group, an intervention that stresses both nutrition and exercise in culturally sensitive ways can positively impact participant’s physical activity levels as well as impact nutritional changes.

The lens epithelium–derived growth factor p75 (LEDGF/p75) is a transcription coactivator that promotes resistance to oxidative stress- and chemotherapy-induced cell death. LEDGF/p75 is also known as the dense fine speckles autoantigen of 70 kDa (DFS70) and has been implicated in cancer, HIV-AIDS, autoimmunity, and inflammation. To gain insights into mechanisms by which LEDGF/p75 protects cancer cells against stress, we initiated an analysis of its interactions with other transcription factors and the influence of these interactions on stress gene activation. We report here that both LEDGF/p75 and its short splice variant LEDGF/p52 interact with MeCP2, a methylation-associated transcriptional modulator, in vitro and in various human cancer cells. These interactions were established by several complementary approaches: transcription factor protein arrays, pull-down and AlphaScreen assays, coimmunoprecipitation, and nuclear colocalization by confocal microscopy. MeCP2 was found to interact with the N-terminal region shared by LEDGF/p75 and p52, particularly with the PWWP-CR1 domain. Like LEDGF/p75, MeCP2 bound to and transactivated the Hsp27 promoter (Hsp27pr). LEDGF/p75 modestly enhanced MeCP2-induced Hsp27pr transactivation in U2OS osteosarcoma cells, whereas this effect was more pronounced in PC3 prostate cancer cells. LEDGF/p52 repressed Hsp27pr activity in U2OS cells. Interestingly, siRNA-induced silencing of LEDGF/p75 in U2OS cells dramatically elevated MeCP2-mediated Hsp27pr transactivation, whereas this effect was less pronounced in PC3 cells depleted of LEDGF/p75. These results suggest that the LEDGF/p75–MeCP2 interaction differentially influences Hsp27pr activation depending on the cellular and molecular context. These findings are of significance in understanding the contribution of this interaction to the activation of stress survival genes. Mol Cancer Res; 10(3); 378–91. ©2012 AACR.

BACKGROUND Lens epithelium-derived growth factor p75 (LEDGF/p75) is a stress survival transcription co-activator and autoantigen that is overexpressed in tumors, including prostate cancer (PCa). This oncoprotein promotes resistance to cell death induced by oxidative stress and chemotherapy by mechanisms that remain unclear. To get insights into these mechanisms we identified candidate target stress genes of LEDGF/p75 using pathway-specific gene expression profiling in PCa cells. METHODS A “Human oxidative stress and antioxidant defense” qPCR array was used to identify genes exhibiting significant expression changes in response to knockdown or overexpression of LEDGF/p75 in PC-3 cells. Validation of array results was performed by additional qPCR and immunoblotting. RESULTS Cytoglobin (CYGB), Phosphoinositide-binding protein PIP3-E/IPCEF-1, superoxidase dismutase 3 (SOD3), thyroid peroxidase (TPO), and albumin (ALB) exhibited significant transcript down- and up-regulation in response to LEDGF/p75 knockdown and overexpression, respectively. CYGB gene was selected for further validation based on its emerging role as a stress oncoprotein in human malignancies. In light of previous reports indicating that LEDGF/p75 regulates peroxiredoxin 6 (PRDX6), and that PRDXs exhibit differential expression in PCa, we also examined the relationship between these proteins in PCa cells. Our validation data revealed that changes in LEDGF/p75 transcript and protein expression in PCa cells closely paralleled those of CYGB, but not those of the PRDXs. CONCLUSIONS Our study identifies CYGB and other genes as stress genes potentially regulated by LEDGF/p75 in PCa cells, and provides a rationale for investigating their role in PCa and in promoting resistance to chemotherapy- and oxidative stress-induced cell death. Prostate 72:597–611, 2012. © 2011 Wiley Periodicals, Inc.

Genes for the fatty acid binding proteins (FABP) family encode small 14-15 kDa cytosolic proteins and can be regulated during type 2 diabetes mellitus (T2DM) and obesity. This study compared association of single nucleotide polymorphisms (SNPs) in FABP1-5 with T2DM in different ethnic groups. Associations with T2DM of SNPs in these proteins were assessed in African American (AA), non-Hispanic White (NHW), and Hispanic American (HA) individuals. A total of 650 DNA samples were genotyped; control samples were obtained from Coriell's North American Human Variation Panel Repository (NAHVP) of apparently healthy individuals and T2DM cases were taken from the American Diabetes Association GENNID Study. The rs454550 SNP of FABP5 showed a significant association with T2DM in NHW (OR: 9.03, 95% CI: 1.13-71.73, p=0.014). Our analysis also identified a new FABP5 SNP (nSNP) that showed a significant association with T2DM in NHW (OR: 0.44, 95% CI: 0.19-0.99, p=0.045) and AA (OR: 0.17, 95% CI: 0.03-0.80, p=0.016). The Ala54Thr FABP2 polymorphism was significantly associated with T2DM in HA individuals only (OR: 1.85, 95% CI: 1.05-3.27, p=0.032). All other FABP SNPs did not show association with T2DM. These findings suggest a potential distinct role(s) of SNPs in FABP5 and FABP2 genes in T2DM in different populations.

Purpose The purpose of this study was to assess dietary intake habits of Mexican American Hispanic adults participating in the En Balance diabetes education program. Methods En Balance is a 3-month culturally sensitive diabetes education intervention for Spanish-speaking Hispanics. Of the 46 participants enrolled, 39 mainly Mexican American Hispanic adults with type 2 diabetes completed the En Balance program. Participants lived in the Riverside and San Bernardino counties of California, and all participants completed the program by June 2008. Dietary intake was assessed at baseline and at 3 months using the validated Southwest Food Frequency Questionnaire. Results Clinically important decreases in glycemic control and serum lipid levels were observed at the end of the 3-month program. The baseline diet was characterized by a high intake of energy (2478 ± 1140 kcal), total fat (87 ± 44 g/day), saturated fat (28 ± 15 g/day), dietary cholesterol (338 ± 217 mg/day), and sodium (4236 ± 2055 mg/ day). At 3 months, the En Balance group mean intake of dietary fat (P = .045) and dietary cholesterol ( P = .033) decreased significantly. Low dietary intakes of docosahexaenoic acid, eicosapentaenoic acid, and vitamin E were also observed in these adults with type 2 diabetes. Conclusions The En Balance program improved glycemic control and lipid profiles in a group of Hispanic diabetic participants. En Balance also promoted decreases in dietary fat and dietary cholesterol intake.

BACKGROUND. The peroxiredoxins (PRDXs) are emerging as regulators of antioxidant defense, apoptosis, and therapy resistance in cancer. Because their significance in prostate cancer (PCa) is unclear, we investigated their expression and clinical associations in PCa. METHODS. Transcript expression of PRDX1–6 in PCa was evaluated in cancer gene microarray datasets, whereas protein expression was evaluated by immunoblotting in prostate cell lines, and by immunohistochemistry (IHC) in prostate tissue microarrays (TMAs) containing tumor (n ¼ 80) and control (n ¼ 17) tissues. PRDX3 was also analyzed in TMAs containing PCa tissues from African-American and Caucasian patients (n ¼ 150 per group). PRDX expression was correlated with patients’ clinicopathologic characteristics. RESULTS. Analysis of PRDX expression in cancer microarray datasets revealed consistent upregulation (tumor vs. normal) of PRDX3 and 4. All PRDXs exhibited elevated protein expression in PCa cell lines, compared with non-tumor cells. IHC revealed significant overexpression of PRDX3 and 4 in PCa, associated with age, increased prostate specific antigen (PSA), tumor stage, or Gleason score. High PRDX3 staining was associated with early age and elevated Gleason score at time of radical prostatectomy in African-American but not in Caucasian patients with PCa. PSA recurrence free survival in patients with low PRDX3 tumor expression was significantly longer in Caucasians compared to African-Americans, but no difference was detected for high expression. CONCLUSIONS. PRDXs exhibit differential expression in prostate tumors, with PRDX3 and 4 consistently upregulated. Their role in PCa development, and their potential as biological determinants of PCa health disparities and novel therapeutic targets, deserve further investigation. Prostate 71: 755–765, 2011. # 2010 Wiley-Liss, Inc.

Purpose—The purpose of this study was to evaluate the effects of a culturally sensitive diabetes education program for Hispanics with type 2 diabetes. Methods—This study is a prospective cohort study to test the impact of a comprehensive diabetes education program on blood glucose control on Hispanics with type 2 diabetes. The educational program focused on maintaining glycemic control and general aspects of managing diabetes and complications. The study participants were recruited by flyers placed in Hispanic markets and in ambulatory care clinics. A total of 34 Hispanic male and female subjects with type 2 diabetes participated in the study. The concentrations of glucose, insulin, hemoglobin A1c (HbA1c), total cholesterol, triglycerides, low-density lipoprotein and high-density lipoprotein (HDL) cholesterol were analyzed at baseline and at 3 months. Results—A significant mean change was observed for HbA1c, fasting plasma glucose, cholesterol/ HDL ratio, and HDL after 3 months of education compared with baseline. There were significant reductions in weight, total fat, percent fat, trunk fat, and waist-to-hip ratio compared with baseline. After 3 months, subjects showed a significant positive correlation between changes in body mass index and insulin and weight, total fat, trunk fat, and fat free mass and insulin. Conclusions—A culturally sensitive program conducted in Spanish had a significant impact on important clinical parameters in Hispanic subjects with diabetes in a relatively short time period. The study demonstrates the importance of designing education intervention studies that are sensitive to cultural diversity, particularly in at-risk diabetic subjects.

Apoptotic cell death has been proposed to play a role in the neuronal loss observed following traumatic injury in the CNS and PNS. The present study uses an in vitro tissue culture model to investigate whether free fatty acids (FFAs), at concentrations comparable to those found following traumatic brain injury, trigger cell death. Nerve growth factor (NGF)-differentiated PC12 cells exposed to oleic and arachidonic acids (2 : 1 ratio FFA/BSA) showed normal cell survival. However, when cells were exposed to stearic and palmitic acids, there was a dramatic loss of cell viability after 24 h of treatment. The cell death induced by stearic acid and palmitic acid was apoptotic as assessed by morphological analysis, and activation of caspase-8 and caspase-3-like activities. Western blotting showed that differentiated PC12 cells exposed to stearic and palmitic acids exhibited the signature apoptotic cleavage fragment of poly (ADP-ribose) polymerase (PARP). Interestingly, blockade of caspase activities with the pan-caspase inhibitor z-VAD-fmk failed to prevent the cell death observed induced by palmitic or stearic acid. RT-PCR and RNA blot experiments showed an up-regulation of the Fas receptor and ligand mRNA. These findings are consistent with our hypothesis that FFAs may play a role in the cell death associated with trauma in the CNS and PNS.

High levels of serum long chain saturated fatty acids (LCSFAs) have been associated with inflammation in type 2 diabetes. Dietary SFAs can promote inflammation, the secretion of IgG antibodies, and secretion of the proinflammatory cytokine IL-1β. This study characterizes anti-LCSFA IgG antibodies from patients with type 2 diabetes. Serum samples from several cohorts with type 2 diabetes were analyzed for the presence of anti-LCSFA IgG, the cytokine IL-1β, and nonesterified fatty acids. Anti-LCSFA IgG was isolated from patient samples and used forin vitrocharacterization of avidity and specificity. A cohort participating inEn Balance, a diabetes health education program that improved diabetes management, tested positive for anti-LCSFA IgG. Following the 3-month program, the cohort showed a significant reduction in anti-LCSFA IgG levels. Anti-LCSFA antibodies isolated from these patients demonstrated high avidity, were specific for long chain SFAs, and correlated with serum fatty acids in patients with managed type 2 diabetes. Interestingly, anti-LCSFA IgG neutralized PA-induced IL-1βsecretion by dendritic cells. Our data shows that nonesterified SFAs are recognized by IgG antibodies present in human blood. The identification of anti-LCSFA IgG antibodies in human sera establishes a basis for further exploration of lipid induced immune responses in diabetic patients.

African American women suffer higher incidence and mortality of triple negative breast cancer (TNBC) than Caucasian women. TNBC is very aggressive, causing the worst clinical outcome. We previously demonstrated that tumors from these patients express high IGF-II and exhibit high activation of the IGF signaling pathways. IGF-II gene expression is imprinted (monoallelic), promotes tumor progression, and metastasis and regulates Survivin, a TNBC prognostic marker. Since BC mortality has increased among young Vietnamese women, we analyzed 48 (paired) TNBC samples from Vietnamese patients to assess IGF-II expression. We analyzed all samples by qrtPCR for identification of IGF-II heterozygosity and to determine allelic expression of the IGF-II gene. We also analyzed the tissues for proIGF-II and Survivin by RT-PCR and Western blotting. A total of 28 samples displayed IGF-II heterozygosity of which 78% were biallelic. Tumors with biallelic IGF-II gene expression exhibited the highest levels of proIGF-II and Survivin. Although 100% of these tissues corresponding normal samples were biallelic, they expressed significantly lower levels of or no proIGF-II and Survivin. Thus, IGF-II biallelic gene expression is differentially regulated in normal versus tumor tissues. We propose that intratumoral proIGF-II is dependent on the IGF-II gene imprinting status and it will promote a more aggressive TNBC.

Long-chain omega-3 polyunsaturated fatty acids (LC-O3PUFAs) exhibit therapeutic potential for the treatment and prevention of the neurological deficits associated with spinal cord injury (SCI). However, the mechanisms implicated in these protective responses remain unclear. The objective of the present functional metabolomics study was to identify and define the dominant metabolic pathways targeted by dietary LC-O3PUFAs. Sprague-Dawley rats were fed rodent purified chows containing menhaden fish oil-derived LC-O3PUFAs for 8 weeks before being subjected to sham or spinal cord contusion surgeries. We show, through untargeted metabolomics, that dietary LC-O3PUFAs regulate important biochemical signatures associated with amino acid metabolism and free radical scavenging in both the injured and sham-operated spinal cord. Of particular significance, the spinal cord metabolome of animals fed with LC-O3PUFAs exhibited reduced glucose levels (−48 %) and polar uncharged/hydrophobic amino acids (less than −20 %) while showing significant increases in the levels of antioxidant/anti-inflammatory amino acids and peptides metabolites, including β-alanine (+24 %), carnosine (+33 %), homocarnosine (+27 %), kynurenine (+88 %), when compared to animals receiving control diets (p < 0.05). Further, we found that dietary LC-O3PUFAs impacted the levels of neurotransmitters and the mitochondrial metabolism, as evidenced by significant increases in the levels of N-acetylglutamate (+43 %) and acetyl CoA levels (+27 %), respectively. Interestingly, this dietary intervention resulted in a global correction of the pro-oxidant metabolic profile that characterized the SCI-mediated sensorimotor dysfunction. In summary, the significant benefits of metabolic homeostasis and increased antioxidant defenses unlock important neurorestorative pathways of dietary LC-O3PUFAs against SCI.

Spinal cord injury (SCI) remains a devastating cause of lifetime medical comorbidities in modern society. Unfortunately, only a few preventive strategies have been shown to prevent the profound pat...

Objective: The purpose of this study was to determine the association of selected nutrients (protein, calcium, phosphorus and vitamin D) and BMD in Hispanic subjects. Methods: This was a prospective three-month uncontrolled intervention pilot study. A culturally sensitive diabetes education program was conducted to determine the clinical impact on glycemic control in Hispanics subjects with type 2 diabetes. Besides glycemic control, BMD measured by fan beam DXA with Hologic QDR 4500-A software version 8.1A and dietary intake measured by a validated food frequency questionnaires. Paired t-tests and Wilcoxon sign rank test used to compare the change after intervention, while multiple linear regression used to determine the association between BMD and selected nutrients. Results: The study had 39 subjects, 59 % females, mean age 54 years old and 28.2 % had low BMD (T-score -1 and less). Results showed there was no significant difference of T-score (p=0.314), energy, protein, calcium, phosphorous, vitamin D, ...

Epithelial/epidermal fatty acid-binding protein (E-FABP) is induced in peripheral neurons during nerve regeneration and is found at high levels in central neurons during neuronal migration and development. Furthermore, E-FABP expression is required for normal neurite outgrowth in PC12 cells treated with nerve growth factor (NGF). The present study examined whether E-FABP plays a role in retinal ganglion cell (RGC) differentiation and axon growth. Rat retinal tissues from embryonic (E) and postnatal (P) development through adulthood were examined using immunocytochemical labeling with E-FABP and growth-associated protein 43 (GAP-43) antibodies. E-FABP colocalized with GAP-43 at E14 through P10. At E14, E-FABP immunoreactivity was confined to the somas of GAP-43-positive cells in the ganglion cell layer, but it was localized to their axons by E15. The axons in the optic nerve were GAP-43-positive and E-FABP-negative on E15, but the two proteins were colocalized by E18. Retinal cultures at E15 confirmed that E-FABP and GAP-43 colocalize in RGCs. Postnatally, labeling was present between P1 and P10 but decreased at older ages and was minimally present or absent in adult animals. Western immunoblotting revealed that at E18, P1, and P10 E-FABP levels were at least fourfold greater than those in the adult. By P15, protein levels were only twofold greater, with adult levels reached by P31. Furthermore, E-FABP could be reinduced during axon regeneration. Dissociated P15 retinal cells cultured in the presence of brain-derived neurotrophic factor, ciliary neurotrophic factor, and basic fibroblast growth factor exhibited sixfold more GAP-43 and E-FABP double-positive RGCs (cell body and axons) than controls. Moreover, all GAP-43-immunoreactive RGCs were also positive for E-FABP. Taken together, these results indicate the following: 1) E-FABP is expressed in RGCs as they reached the ganglion cell layer and 2) E-FABP plays a functional role in the elaboration of RGC axons in both development and regeneration. J. Neurosci. Res. 66:396–405, 2001. © 2001 Wiley-Liss, Inc.

The present study compares the temporal-spatial expression and tissue localization of the rat epidermal type fatty acid binding protein (E-FABP) (DA11/C-FABP/S-FABP/LEBP/KLBP) in the developing rat central nervous system (CNS). In situ hybridization (ISH) and immunocytochemistry (ICC) studies demonstrate that mRNA E-FABP and protein are expressed at high levels during neurogenesis, neuronal migration, and terminal differentiation. Migrating pyramidal cells in the cerebral cortex, Purkinje cells and deep nuclear neurons in the cerebellum, and neurons in the olfactory bulb and retina exhibited a strong E-FABP-like immunoreactivity (E-FABP-LI) throughout the entire process of differentiation and migration. The levels of E-FABP mRNA and protein were dramatically higher in prenatal and early postnatal neurons, as compared to adult neurons. The E-FABP antibody immunoreacted with growing neurites, and nuclear and cytoplasmic regions of neurons. The intracellular multiregional pattern of localization of E-FABP and its differential temporal expression during development, are consistent with its proposed role in transporting long chain free fatty acids and/or other hydrophobic ligands during neuronal differentiation and axon growth.

Epidermal fatty acid-binding protein (E-FABP/FABP5/DA11) binds and transport long-chain fatty acids in the cytoplasm and may play a protecting role during neuronal injury. We examined whether E-FABP protects nerve growth factor-differentiated PC12 cells (NGFDPC12 cells) from lipotoxic injury observed after palmitic acid (C16:0; PAM) overload. NGFDPC12 cells cultures treated with PAM/bovine serum albumin at 0.3 mM/0.15 mM show PAM-induced lipotoxicity (PAM-LTx) and apoptosis. The apoptosis was preceded by a cellular accumulation of reactive oxygen species (ROS) and higher levels of E-FABP. Antioxidants MCI-186 and N-acetyl cysteine prevented E-FABP's induction in expression by PAM-LTx, while tert-butyl hydroperoxide increased ROS and E-FABP expression. Non-metabolized methyl ester of PAM, methyl palmitic acid (mPAM), failed to increase cellular ROS, E-FABP gene expression, or trigger apoptosis. Treatment of NGFDPC12 cultures with siE-FABP showed reduced E-FABP levels correlating with higher accumulation of ROS and cell death after exposure to PAM. In contrast, increasing E-FABP cellular levels by pre-loading the cells with recombinant E-FABP diminished the PAM-induced ROS and cell death. Finally, agonists for PPARβ (GW0742) or PPARγ (GW1929) increased E-FABP expression and enhanced the resistance of NGFDPC12 cells to PAM-LTx. We conclude that E-FABP protects NGFDPC12 cells from lipotoxic injury through mechanisms that involve reduction of ROS. Epidermal fatty acid-binding protein (E-FABP) may protect nerve cells from the damaging exposure to high levels of free fatty acids (FA). We show that E-FABP can neutralize the effects of reactive oxygen species (ROS) generated by the high levels of FA in the cell and protect PC12 cells from lipotoxic injuries common in Type 2 diabetes neuropathy. Potentially, E-FABP gene up-regulation may be mediated through the NFkB pathway and future studies are needed to further evaluate this proposition.

Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) confer multiple health benefits and decrease the risk of neurological disorders. Studies are needed, however, to identify promising cellular targets and to assess their prophylactic value against neurodegeneration. The present study (1) examined the efficacy of a preventive diet enriched with ω-3 PUFAs to reduce dysfunction in a well-established spinal cord injury (SCI) animal model and (2) used a novel metabolomics data analysis to identify potential neurolipidomic targets. Rats were fed with either control chow or chow enriched with ω-3 PUFAs (750 mg/kg/day) for 8 weeks before being subjected to a sham or a contusion SCI operation. We report new evidence showing that rats subjected to SCI after being pre-treated with a diet enriched with ω-3 PUFAs exhibit significantly better functional outcomes. Pre-treated animals exhibited lower sensory deficits, autonomic bladder recovery, and early improvements in locomotion that persisted for at least 8 weeks after trauma. We found that SCI triggers a robust alteration in the cord PUFA neurolipidome, which was characterized by a marked docosahexaenoic acid (DHA) deficiency. This DHA deficiency was associated with dysfunction and corrected with the ω-3 PUFA-enriched diet. Multivariate data analyses revealed that the spinal cord of animals consuming the ω-3 PUFA-enriched diet had a fundamentally distinct neurolipidome, particularly increasing the levels of essential and long chain ω-3 fatty acids and lysolipids at the expense of ω-6 fatty acids and its metabolites. Altogether, dietary ω-3 PUFAs prophylaxis confers resiliency to SCI mediated, at least in part, by generating a neuroprotective and restorative neurolipidome.

We evaluated the changes in apolipoproteins, glycemic status, and body composition after 3 months using a culturally sensitive diabetes education program, En Balance, in diabetic Hispanics.Thirty-four (9 males, 25 females) Hispanic diabetics participated in the En Balance program over three months. Body composition was determined by dual energy X-ray absorptiometry (DXA), fasting plasma glucose (FPG), A1c, and apolipoproteins (Apo) measured after 3 months participation. Differences were analyzed using paired t testing and relationships between changes in Apo, A1c, total cholesterol, body mass index and body composition by Spearman correlations.Completion of En Balance resulted in a significant reduction in weight (80.31 +/- 1.97 kg vs 81.25 +/- 17.97 kg, P = .015), FPG (143.21 +/- 57.8 mg/dL vs 166.41 +/- 65.9 mg/dL P = .003), and A1c (7.08 +/- 1.6% vs 7.87 +/- 2.0%, P =.001). DXA demonstrated reduction in total fat (29.54 +/- 10.0 kg vs 30.24 +/- 11.80 kg, P =.001) and trunk fat (15.09 +/- 5.6 kg vs 16.87 +/- 5.4 kg, P = .001). High density lipoprotein significantly increased (48.85 +/- 11.4 vs 44.65 +/- 8.8, P = .002) and total serum cholesterol/high density lipoprotein ratio decreased (3.87 +/- .98 vs 4.35 +/- 1.0, P = .001). There were significant correlations at three months between changes in Apo A1 and A2 (r = .559, P.001), Apo E and total cholesterol (r = .746, P.001), between A1c and FPG (r = .563, P = .001) and BMI and body weight (r = .732, P.001).The En Balance program improved body composition, A1c, FPG, total cholesterol/HDL ratio and HDL. If these trends can be sustained, En Balance may serve as a unique educational paradigm for improving type 2 diabetes in Hispanics.

Currently, few interventions have been shown to successfully limit the progression of secondary damage events associated with the acute phase of spinal cord injury (SCI). Docosahexaenoic acid (DHA, C22:6 n-3) is neuroprotective when administered following SCI, but its potential as a pretreatment modality has not been addressed. This study used a novel DHA pretreatment experimental paradigm that targets acute cellular and molecular events during the first week after SCI in rats. We found that DHA pretreatment reduced functional deficits during the acute phase of injury, as shown by significant improvements in Basso-Beattie-Bresnahan (BBB) locomotor scores, and the detection of transcranial magnetic motor evoked potentials (tcMMEPs) compared to vehicle-pretreated animals. We demonstrated that, at 7 days post-injury, DHA pretreatment significantly increased the percentage of white matter sparing, and resulted in axonal preservation, compared to the vehicle injections. We found a significant increase in the survival of NG2+, APC+, and NeuN+ cells in the ventrolateral funiculus (VLF), dorsal corticospinal tract (dCST), and ventral horns, respectively. Interestingly, these DHA protective effects were observed despite the lack of inhibition of inflammatory markers for monocytes/macrophages and astrocytes, ED1/OX42 and GFAP, respectively. DHA pretreatment induced levels of Akt and cyclic AMP responsive element binding protein (CREB) mRNA and protein. This study shows for the first time that DHA pretreatment ameliorates functional deficits, and increases tissue sparing and precursor cell survival. Further, our data suggest that DHA-mediated activation of pro-survival/anti-apoptotic pathways may be independent of its anti-inflammatory effects.

Lens epithelium-derived growth factor p75 (LEDGF/p75) is a stress survival transcription co-activator and autoantigen that is overexpressed in tumors, including prostate cancer (PCa). This oncoprotein promotes resistance to cell death induced by oxidative stress and chemotherapy by mechanisms that remain unclear. To get insights into these mechanisms we identified candidate target stress genes of LEDGF/p75 using pathway-specific gene expression profiling in PCa cells.A "Human oxidative stress and antioxidant defense" qPCR array was used to identify genes exhibiting significant expression changes in response to knockdown or overexpression of LEDGF/p75 in PC-3 cells. Validation of array results was performed by additional qPCR and immunoblotting.Cytoglobin (CYGB), Phosphoinositide-binding protein PIP3-E/IPCEF-1, superoxidase dismutase 3 (SOD3), thyroid peroxidase (TPO), and albumin (ALB) exhibited significant transcript down- and up-regulation in response to LEDGF/p75 knockdown and overexpression, respectively. CYGB gene was selected for further validation based on its emerging role as a stress oncoprotein in human malignancies. In light of previous reports indicating that LEDGF/p75 regulates peroxiredoxin 6 (PRDX6), and that PRDXs exhibit differential expression in PCa, we also examined the relationship between these proteins in PCa cells. Our validation data revealed that changes in LEDGF/p75 transcript and protein expression in PCa cells closely paralleled those of CYGB, but not those of the PRDXs.Our study identifies CYGB and other genes as stress genes potentially regulated by LEDGF/p75 in PCa cells, and provides a rationale for investigating their role in PCa and in promoting resistance to chemotherapy- and oxidative stress-induced cell death.

OBJECTIVES: Diabetes mellitus and obesity are prevalent in the Hispanic community. This group has not benefited greatly from diabetes interventions due to cultural, language and financial constraints. We designed a prospective cohort study to determine the clinical impact on adiposity and glycemic control in Hispanics with type 2 diabetes. RESEARCH DESIGN AND METHODS: The program conducted in Spanish by a multidisciplinary team of health care providers focused on improving glycemic control and complications through cultural lifestyle changes. Outcomes were changes in glycemic control by fasting insulin, glucose and HbA1c, body composition and selected adipokines, adiponectin, leptin and ghrelin. Body composition was measured by dual energy x-ray absorptiometry. Changes from baseline at three months were compared using paired t-tests and with Spearman's correlations. RESULTS: Glycemic control improved by HbA1c (7.9% ± 2.0% vs 7.1% ± 1.7%; P =0.001), and fasting glucose (166.4 ± 66.0 mg/dl vs 143.2 ± 57.9 mg/dl; P = 0.003). Body weight (81.3 ± 17.9 kg vs 80.3 ± 18.0 kg; P = 0.002), waist circumference (101.6 ± 13.4 cm vs 99.1 ± 12.7 cm; P = 0.015), and truncal fat (16.5 ± 5.7 kg vs 15.9 ± 5.6 kg; P = 0.001) decreased. Only leptin (19.6 ± 15.0 ng/ml vs 16.3 ± 12.7 ng/ml; P = 0.002) was reduced and related to change in body weight (r = 0.392; P = 0.022). CONCLUSIONS: Our program significantly improved glycemic control and decreased obesity in diabetic Hispanic subjects. The early benefits on glycemic control may be related to reductions in leptin through loss of adipose tissue. Success in impacting diabetes and related complications can occur in a culturally focused and multidisciplinary context.

Lipid overload resulting in lipotoxicity is prominent in a number of chronic diseases and has been associated with cellular dysfunction and cell death. This study characterizes palmitic acid-induced lipotoxicity (PA-LTx) in Schwann cell cultures grown in normal and high glucose concentrations. The study shows for the first time that Schwann cell (SC) cultures exposed to elevated levels of PA exhibit a dose- and time-dependent loss in cell viability. Hoescht and Annexin V/7AAD staining confirmed cell death through apoptosis and the lipotoxic effect was more dramatic in SC cultures grown under high glucose conditions. The first indication of cellular dysfunction in treated SC cultures was a decrease in Ca(++) levels in the endoplasmic reticulum (ER, [Ca(++)](ER)) observed five minutes following the initial challenge with PA. This decrease in [Ca(++) ](ER) was followed by a significant increase in the expression of ER stress signature genes CHOP, Xbp1 and GRP78. The early ER stress response induced by PA-LTx was followed by a strong mitochondrial membrane depolarization. Flow cytometry using 2', 7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA) showed an increase in oxidative stress within three to six hours after PA treatment. Treatment of cultures undergoing PA-LTx with the calcium chelator BAPTA-AM and the anti-oxidant MCI-186 significantly reversed the lipotoxic effect by decreasing the generation of ROS and significantly increasing cell viability. We conclude that lipotoxicity in Schwann cells results in cellular dysfunction and cell death that involves a robust ER stress response, mitochondrial dysfunction and an augmented state of cellular oxidative stress (ASCOS).

Lipotoxicity, which is triggered when cells are exposed to elevated levels of free fatty acids, involves cell dysfunction and apoptosis and is emerging as an underlying factor contributing to various pathological conditions including disorders of the central nervous system and diabetes. We have shown that palmitic acid (PA)-induced lipotoxicity (PA-LTx) in nerve growth factor-differentiated PC12 (NGFDPC12) cells is linked to an augmented state of cellular oxidative stress (ASCOS) and apoptosis, and that these events are inhibited by docosahexanoic acid (DHA). The mechanisms of PA-LTx in nerve cells are not well understood, but our previous findings indicate that it involves ROS generation, mitochondrial membrane permeabilization (MMP), and caspase activation. The present study used nerve growth factor differentiated PC12 cells (NGFDPC12 cells) and found that lysosomal membrane permeabilization (LMP) is an early event during PA-induced lipotoxicity that precedes MMP and apoptosis. Cathepsin L, but not cathepsin B, is an important contributor in this process since its pharmacological inhibition significantly attenuated LMP, MMP, and apoptosis. In addition, co-treatment of NGFDPC12 cells undergoing lipotoxicity with DHA significantly reduced LMP, suggesting that DHA acts by antagonizing upstream signals leading to lysosomal dysfunction. These results suggest that LMP is a key early mediator of lipotoxicity, and underscore the value of interventions targeting upstream signals leading to LMP for the treatment of pathological conditions associated with lipotoxicity.

Background Hormone-refractory prostate cancer (HRPC) is characterized by poor response to chemotherapy and high mortality, particularly among African American men when compared to other racial/ethnic groups. It is generally accepted that docetaxel, the standard of care for chemotherapy of HRPC, primarily exerts tumor cell death by inducing mitotic catastrophe and caspase-dependent apoptosis following inhibition of microtubule depolymerization. However, there is a gap in our knowledge of mechanistic events underlying docetaxel-induced caspase-independent cell death, and the genes that antagonize this process. This knowledge is important for circumventing HRPC chemoresistance and reducing disparities in prostate cancer mortality. Results We investigated mechanistic events associated with docetaxel-induced death in HRPC cell lines using various approaches that distinguish caspase-dependent from caspase-independent cell death. Docetaxel induced both mitotic catastrophe and caspase-dependent apoptosis at various concentrations. However, caspase activity was not essential for docetaxel-induced cytotoxicity since cell death associated with lysosomal membrane permeabilization still occurred in the presence of caspase inhibitors. Partial inhibition of docetaxel-induced cytotoxicity was observed after inhibition of cathepsin B, but not inhibition of cathepsins D and L, suggesting that docetaxel induces caspase-independent, lysosomal cell death. Simultaneous inhibition of caspases and cathepsin B dramatically reduced docetaxel-induced cell death. Ectopic expression of lens epithelium-derived growth factor p75 (LEDGF/p75), a stress survival autoantigen and transcription co-activator, attenuated docetaxel-induced lysosomal destabilization and cell death. Interestingly, LEDGF/p75 overexpression did not protect cells against DTX-induced mitotic catastrophe, and against apoptosis induced by tumor necrosis factor related apoptosis inducing ligand (TRAIL), suggesting selectivity in its pro-survival activity. Conclusion These results underscore the ability of docetaxel to induce concomitantly caspase-dependent and independent death pathways in prostate cancer cells. The results also point to LEDGF/p75 as a potential contributor to cellular resistance to docetaxel-induced lysosomal destabilization and cell death, and an attractive candidate for molecular targeting in HRPC.

Lipotoxicity involves a series of pathological cellular responses after exposure to elevated levels of fatty acids. This process may be detrimental to normal cellular homeostasis and cell viability. The present study shows that nerve growth factor-differentiated PC12 cells (NGFDPC12) and rat cortical cells (RCC) exposed to high levels of palmitic acid (PA) exhibit significant lipotoxicity and death linked to an "augmented state of cellular oxidative stress" (ASCOS). The ASCOS response includes generation of reactive oxygen species (ROS), alterations in the mitochondrial transmembrane potential, and increase in the mRNA levels of key cell death/survival regulatory genes. The observed cell death was apoptotic based on nuclear morphology, caspase-3 activation, and cleavage of lamin B and PARP. Quantitative real-time PCR measurements showed that cells undergoing lipotoxicity exhibited an increase in the expression of the mRNAs encoding the cell death-associated proteins BNIP3 and FAS receptor. Cotreatment of NGFDPC12 and RCC cells undergoing lipotoxicity with docosahexaenoic acid (DHA) and bovine serum albumin (BSA) significantly reduced cell death within the first 2 hr following the initial exposure to PA. The data suggest that lipotoxicity in NGFDPC12 and cortical neurons triggers a strong cell death apoptotic response. Results with NGFDPC12 cells suggest a linkage between induction of ASCOS and the apoptotic process and exhibit a temporal window that is sensitive to DHA and BSA interventions.

Epidermal fatty acid-binding protein (E-FABP), a member of the family of FABPs, exhibits a robust expression in neurons during axonal growth in development and in nerve regeneration following nerve injury. This study examines the impact of E-FABP expression in normal neurite extension in differentiating pheochromocytoma cell (PC12) cultures supplemented with selected long chain free fatty acids (LCFFA). We found that E-FABP binds to a broad range of saturated and unsaturated LCFFAs, including those with potential interest for neuronal differentiation and axonal growth such as C22:6n-3 docosahexaenoic acid (DHA), C20:5n-3 eicosapentaenoic acid (EPA), and C20:4n-6 arachidonic acid (ARA). PC12 cells exposed to nerve growth factor (NGFDPC12) exhibit high E-FABP expression that is blocked by mitogen-activated protein kinase kinase (MEK) inhibitor U0126. Nerve growth factor-differentiated pheochromocytoma cells (NGFDPC12) antisense clones (NGFDPC12-AS) which exhibit low E-FABP expression have fewer/shorter neurites than cells transfected with vector only or NGFDPC12 sense cells (NGFDPC12-S). Replenishing NGFDPC12-AS cells with biotinylated recombinant E-FABP (biotin-E-FABP) protein restores normal neurite outgrowth. Cellular localization of biotin-E-FABP in NGFDPC12 was detected mostly in the cytoplasm and in the nuclear region. Treatment of NGFDPC12 with DHA, EPA, or ARA further enhances neurite length but it does not trigger further induction of TrkA or MEK phosphorylation or E-FABP mRNA observed in differentiating PC12 cells without LCFFA supplementation. Significantly, DHA and EPA neurite stimulating effects are higher in NGFDPC12-S than in NGFDPC12-AS cells. These findings are consistent with the scenario that neurite extension of differentiating PC12 cells, including further stimulation by DHA and EPA, requires sufficient cellular levels of E-FABP.

We are interested in the role cyclic AMP may play as a mediator of growth factor-induced gene expression in regenerating peripheral nerves. As a first step, we investigated mRNA levels and protein synthesis in intact frog dorsal root ganglia (DRG) treated with the adenylate cyclase activator, forskolin in vitro. Forskolin (10-7 M) increased intraganglionic cyclic AMP concentration 3-fold within 40 min of application and maintained this concentration through 60 min, when the drug was withdrawn. Addition of forskolin to isolated DRG neurons for 1 h increased the incorporation of [3H]leucine into TCA-insoluble material beginning 12 h after the withdrawal of forskolin. Axonally transported labeled material was increased almost 2-fold by 12 h. The effect of forskolin could be blocked by the simultaneous addition of actinomycin D, but not if actinomycin D was added 1 h later. Northern and dot-blot analysis of RNA extracted from the treated ganglia indicated that an mRNA coding for an α-subunit of tubulin was increased by treatment with forskolin. 2D PAGE also demonstrated an increase in an α-subunit of tubulin. An increase in neuronal cyclic AMP appears to selectively increase the production of specific proteins and may contribute to the production of macromolecules involved in the initiation and stimulation of axonal regeneration.

Cyclodextrins (CDs) are used to deliver hydrophobic molecules in aqueous environments. Methyl-beta-cyclodextrin (MbetaCD), a member of this family of molecules, has been proposed to be a good carrier to deliver fatty acids to cells in culture. This report focuses on studying the in vitro effects of MbetaCD on nerve growth factor-differentiated PC12 (NGFDPC12) cells, a tissue culture model to study neuronal survival and differentiation. The main findings are: (1) NGFDPC12 cells have normal viability when exposed to 0.12% MbetaCD but showed a significant loss in cell viability at higher concentrations; (2) NGFDPC12 cells exposed to 0.25% MbetaCD exhibit nuclear condensation, blebbing and apoptotic bodies, and whole cell lysates exhibited an increase in caspase-3-like activity and high levels of Bax and Bcl-X(L) protein expression compared to control. Cultures treated with 0.25% MbetaCD also showed cleavage of normal 21-kDa Bax protein into a 18-kDa fragment. (3) Experiments using 0.12% MbetaCD to deliver oleic acid did not affect cell viability, in contrast NGFDPC12 cultures in which 0.25% MbetaCD concentration is used exhibited similar loss of cell viability as observed with 0.25% MbetaCD alone. Treating these cultures with caspase-3 inhibitor z-VAD-fmk did not protect the cells from MbetaCD toxic effects. (4) Immortalized Schwann cells (iSC) exposed to MbetaCD 0.12% did not show loss of cell viability while 0.25% MbetaCD triggered a significant toxicity but with a different dose and time course dynamic than NGFDPC12 cells. Thus, NGFDPC12 or iSC cell cultures exposed to 0.12% MbetaCD exhibits normal viability while higher concentrations increase in cell death and apoptosis.