Cell-free DNA methylation analysis as a marker of malignancy in pleural fluid.

Diagnosis of malignant pleural effusion (MPE) is made by cytological examination of pleural fluid or histological examination of pleural tissue from biopsy. Unfortunately, detection of malignancy using cytology has an overall sensitivity of 50%, and is dependent upon tumor load, volume of fluid assessed, and cytopathologist experience. The diagnostic yield of pleural fluid cytology is also compromised by low abundance of tumor cells or when morphology is obscured by inflammation or reactive mesothelial cells. A reliable molecular marker that may complement fluid cytology for the diagnosis of malignant pleural effusion is needed. The purpose of this study was to establish a molecular diagnostic approach based on pleural effusion cell-free DNA methylation analysis for the differential diagnosis of malignant pleural effusion and benign pleural effusion. This was a blind, prospective case-control biomarker study. We recruited 104 patients with pleural effusion for the study. We collected pleural fluid from patients with: MPE (n = 48), indeterminate pleural effusion in subjects with known malignancy or IPE (n = 28), and benign PE (n = 28), and performed the Sentinel-MPE liquid biopsy assay. The methylation level of Sentinel-MPE was markedly higher in the MPE samples compared to BPE control samples (p < 0.0001) and the same tendency was observed relative to IPE (p = 0.004). We also noted that the methylation signal was significantly higher in IPE relative to BPE (p < 0.001). We also assessed the diagnostic efficiency of the Sentinel-MPE test by performing receiver operating characteristic analysis (ROC). For the ROC analysis we combined the malignant and indeterminate pleural effusion groups (n = 76) and compared against the benign group (n = 28). The detection sensitivity and specificity of the Sentinel-MPE test was high (AUC = 0.912). The Sentinel-MPE appears to have better performance characteristics than cytology analysis. However, combining Sentinel-MPE with cytology analysis could be an even more effective approach for the diagnosis of MPE. The Sentinel-MPE test can discriminate between BPE and MPE. The Sentinel-MPE liquid biopsy test can detect aberrant DNA in several different tumor types. The Sentinel-MPE test can be a complementary tool to cytology in the diagnosis of MPE.

Cell-Free DNA Methylation Analysis as a Marker of Malignancy in Pleural Fluid.

Background: Diagnosis of malignant pleural effusion (MPE) is made by cytological examination of pleural fluid or histological examination of pleural tissue from biopsy. Unfortunately, detection of malignancy using cytology has an overall sensitivity of 50%, and is dependent upon tumor load, volume of fluid assessed, and cytopathologist experience. The diagnostic yield of pleural fluid cytology is also compromised by low abundance of tumor cells or when morphology is obscured by inflammation or reactive mesothelial cells. A reliable molecular marker that may complement fluid cytology malignant pleural effusion diagnosis is needed. The purpose of this study was to establish a molecular diagnostic approach based on pleural effusion cell-free DNA methylation analysis for the differential diagnosis of malignant pleural effusion and benign pleural effusion. Results: This was a blind, prospective case-control biomarker study. We recruited 104 patients with pleural effusion for the study. We collected pleural fluid from patients with: MPE (n = 48), PPE (n = 28), and benign PE (n = 28), and performed the Sentinel-MPE liquid biopsy assay. The methylation level of Sentinel-MPE was markedly higher in the MPE samples compared to BPE control samples (p < 0.0001) and the same tendency was observed relative to PPE (p = 0.004). We also noted that the methylation signal was significantly higher in PPE relative to BPE (p < 0.001). We also assessed the diagnostic efficiency of the Sentinel-MPE test by performing receiver operating characteristic analysis (ROC). For the ROC analysis we combined the malignant and paramalignant groups (n = 76) and compared against the benign group (n = 28). The detection sensitivity and specificity of the Sentinel-MPE test was high (AUC = 0.912). The Sentinel-MPE appears to have better performance characteristics than cytology analysis. However, combining Sentinel-MPE with cytology analysis could be an even more effective approach for the diagnosis of MPE. Conclusions: The Sentinel-MPE test can discriminate between BPE and MPE. The Sentinel-MPE liquid biopsy test can detect aberrant DNA in several different tumor types. The Sentinel-MPE test can be a complementary tool to cytology in the diagnosis of MPE.

Liquid biopsy, using a novel DNA methylation signature, distinguishes pancreatic adenocarcinoma from benign pancreatic disease.

We tested the ability of a novel DNA methylation biomarker set to distinguish metastatic pancreatic cancer cases from benign pancreatic cyst patients and to monitor tumor dynamics using quantitative DNA methylation analysis of cell-free DNA (cfDNA) from blood samples. The biomarkers were able to distinguish malignant cases from benign disease with high sensitivity and specificity (AUC = 0.999). Furthermore, the biomarkers detected a consistent decline in tumor-derived cfDNA in samples from patients undergoing chemotherapy. The study indicates that our liquid biopsy assay could be useful for management of pancreatic cancer patients.

Evaluation of the effects of the T-type calcium channel enhancer SAK3 in a rat model of TAF1 deficiency.

The TATA-box binding protein associated factor 1 (TAF1) is part of the TFIID complex that plays a key role during the initiation of transcription. Variants of TAF1 are associated with neurodevelopmental disorders. Previously, we found that CRISPR/Cas9 based editing of the TAF1 gene disrupts the morphology of the cerebral cortex and blunts the expression as well as the function of the CaV3.1 (T-type) voltage gated calcium channel. Here, we tested the efficacy of SAK3 (ethyl 8'-methyl-2', 4-dioxo-2-(piperidin-1-yl)-2'H-spiro [cyclopentane-1, 3'-imidazo [1, 2-a] pyridine]-2-ene-3-carboxylate), a T-type calcium channel enhancer, in an animal model of TAF1 intellectual disability (ID) syndrome. At post-natal day 3, rat pups were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 CRISPR/Cas9 viruses. At post-natal day 21, the rat pups were given SAK3 (0.25 mg/kg, p.o.) or vehicle for 14 days (i.e. till post-natal day 35) and then subjected to behavioral, morphological, and molecular studies. Oral administration of SAK3 (0.25 mg/kg, p.o.) significantly rescued locomotion abnormalities associated with TAF1 gene editing. SAK3 treatment prevented the loss of cortical neurons and GFAP-positive astrocytes observed after TAF1 gene editing. In addition, SAK3 protected cells from apoptosis. SAK3 also restored the Brain-derived neurotrophic factor/protein kinase B/Glycogen Synthase Kinase 3 Beta (BDNF/AKT/GSK3ß) signaling axis in TAF1 edited animals. Finally, SAK3 normalized the levels of three GSK3ß substrates - CaV3.1, FOXP2, and CRMP2. We conclude that the T-type calcium channel enhancer SAK3 is beneficial against the deleterious effects of TAF1 gene-editing, in part, by stimulating the BDNF/AKT/GSK3ß signaling pathway.

The investigation of the T-type calcium channel enhancer SAK3 in an animal model of TAF1 intellectual disability syndrome.

T-type calcium channels, in the central nervous system, are involved in the pathogenesis of many neurodegenerative diseases, including TAF1 intellectual disability syndrome (TAF1 ID syndrome). Here, we evaluated the efficacy of a novel T-type Ca2+ channel enhancer, SAK3 (ethyl 8'-methyl-2', 4-dioxo-2-(piperidin-1-yl)-2'H-spiro [cyclopentane-1, 3'-imidazo [1, 2-a] pyridine]-2-ene-3-carboxylate) in an animal model of TAF1 ID syndrome. At post-natal day 3, rat pups were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 CRISPR/Cas9 viruses. At post-natal day 21 animals were given SAK3 (0.25 mg/kg, p.o.) or vehicle up to post-natal day 35 (i.e. 14 days). Rats were subjected to behavioral, morphological, electrophysiological, and molecular studies. Oral administration of SAK3 (0.25 mg/kg, p.o.) significantly rescued the behavior abnormalities in beam walking test and open field test caused by TAF1 gene editing. We observed an increase in calbindin-positive Purkinje cells and GFAP-positive astrocytes as well as a decrease in IBA1-positive microglia cells in SAK3-treated animals. In addition, SAK3 protected the Purkinje and granule cells from apoptosis induced by TAF-1 gene editing. SAK3 also restored the excitatory post synaptic current (sEPSCs) in TAF1 edited Purkinje cells. Finally, SAK3 normalized the BDNF/AKT signaling axis in TAF1 edited animals. Altogether, these observations suggest that SAK3 could be a novel therapeutic agent for TAF1 ID syndrome.

DNA methylation biomarkers discovered in silico detect cancer in liquid biopsies from non-small cell lung cancer patients.

Identification of cancer-specific methylation of DNA released by tumours can be used for non-invasive diagnostics and monitoring. We previously reported in silico identification of DNA methylation loci specifically hypermethylated in common human cancers that could be used as epigenetic biomarkers. Using DNA methylation specific qPCR we now clinically tested a group of these cancer-specific loci on cell-free DNA (cfDNA) extracted from the plasma fraction of blood samples from healthy controls and non-small cell lung cancer (NSCLC) patients. These DNA methylation biomarkers distinguish lung cancer cases from controls with high sensitivity and specificity (AUC = 0.956), and furthermore, the signal from the markers correlates with tumour size and decreases after surgical resection of lung tumours. Presented observations suggest the clinical value of these DNA methylation biomarkers for NSCLC diagnostics and monitoring. Since we successfully validated the biomarkers using independent DNA methylation data from multiple additional common carcinoma cohorts (bladder, breast, colorectal, oesophageal, head and neck, pancreatic or prostate cancer) we predict that these DNA methylation biomarkers will detect additional carcinoma types from plasma samples as well.

TAF1-gene editing alters the morphology and function of the cerebellum and cerebral cortex.

TAF1/MRSX33 intellectual disability syndrome is an X-linked disorder caused by loss-of-function mutations in the TAF1 gene. How these mutations cause dysmorphology, hypotonia, intellectual and motor defects is unknown. Mouse models which have embryonically targeted TAF1 have failed, possibly due to TAF1 being essential for viability, preferentially expressed in early brain development, and intolerant of mutation. Novel animal models are valuable tools for understanding neuronal pathology. Here, we report the development and characterization of a novel animal model for TAF1 ID syndrome in which the TAF1 gene is deleted in embryonic rats using clustered regularly interspaced short palindromic repeats (CRISPR) associated protein 9 (Cas9) technology and somatic brain transgenesis mediated by lentiviral transduction. Rat pups, post-natal day 3, were subjected to intracerebroventricular (ICV) injection of either gRNA-control or gRNA-TAF1 vectors. Rats were subjected to a battery of behavioral tests followed by histopathological analyses of brains at post-natal day 14 and day 35. TAF1-edited rats exhibited behavioral deficits at both the neonatal and juvenile stages of development. Deletion of TAF1 lead to a hypoplasia and loss of the Purkinje cells. We also observed a decreased in GFAP positive astrocytes and an increase in Iba1 positive microglia within the granular layer of the cerebellum in TAF1-edited animals. Immunostaining revealed a reduction in the expression of the CaV3.1 T-type calcium channel. Abnormal motor symptoms in TAF1-edited rats were associated with irregular cerebellar output caused by changes in the intrinsic activity of the Purkinje cells due to loss of pre-synaptic CaV3.1. This animal model provides a powerful new tool for studies of neuronal dysfunction in conditions associated with TAF1 abnormalities and should prove useful for developing therapeutic strategies to treat TAF1 ID syndrome.

A novel variant in TAF1 affects gene expression and is associated with X-linked TAF1 intellectual disability syndrome.

We investigated the genome of a 5-year-old male who presented with global developmental delay (motor, cognitive, and speech), hypotonia, possibly ataxia, and cerebellar hypoplasia of unknown origin. Whole genome sequencing (WGS) and mRNA sequencing (RNA-seq) were performed on a family having an affected proband, his unaffected parents, and maternal grandfather. To explore the molecular and functional consequences of the variant, we performed cell proliferation assays, quantitative real-time PCR (qRT-PCR) array, immunoblotting, calcium imaging, and neurite outgrowth experiments in SH-SY5Y neuroblastoma cells to compare the properties of the wild-type TATA-box-binding protein factor 1 (TAF1), deletion of TAF1, and TAF1 variant p.Ser1600Gly samples. The whole genome data identified several gene variants. However, the genome sequence data failed to implicate a candidate gene as many of the variants were of unknown significance. By combining genome sequence data with transcriptomic data, a probable candidate variant, p.Ser1600Gly, emerged in TAF1. Moreover, the RNA-seq data revealed a 90:10 extremely skewed X-chromosome inactivation (XCI) in the mother. Our results showed that neuronal ion channel genes were differentially expressed between TAF1 deletion and TAF1 variant p.Ser1600Gly cells, when compared with their respective controls, and that the TAF1 variant may impair neuronal differentiation and cell proliferation. Taken together, our data suggest that this novel variant in TAF1 plays a key role in the development of a recently described X-linked syndrome, TAF1 intellectual disability syndrome, and further extends our knowledge of a potential link between TAF1 deficiency and defects in neuronal cell function.

Modulation of breast cancer cell viability by a cannabinoid receptor 2 agonist, JWH-015, is calcium dependent.

INTRODUCTION: Cannabinoid compounds, both nonspecific as well as agonists selective for either cannabinoid receptor 1 (CB1) or cannabinoid receptor 2 (CB2), have been shown to modulate the tumor microenvironment by inducing apoptosis in tumor cells in several model systems. The mechanism of this modulation remains only partially delineated, and activity induced via the CB1 and CB2 receptors may be distinct despite significant sequence homology and structural similarity of ligands. METHODS: The CB2-selective agonist JWH-015 was used to investigate mechanisms downstream of CB2 activation in mouse and human breast cancer cell lines in vitro and in a murine mammary tumor model. RESULTS: JWH-015 treatment significantly reduced primary tumor burden and metastasis of luciferase-tagged murine mammary carcinoma 4T1 cells in immunocompetent mice in vivo. Furthermore, JWH-015 reduced the viability of murine 4T1 and human MCF7 mammary carcinoma cells in vitro by inducing apoptosis. JWH-015-mediated reduction of breast cancer cell viability was not dependent on Gαi signaling in vitro or modified by classical pharmacological blockade of CB1, GPR55, TRPV1, or TRPA1 receptors. JWH-015 effects were calcium dependent and induced changes in MAPK/ERK signaling. CONCLUSION: The results of this work characterize the actions of a CB2-selective agonist on breast cancer cells in a syngeneic murine model representing how a clinical presentation of cancer progression and metastasis may be significantly modulated by a G-protein-coupled receptor.

Flexner 2.0-Longitudinal Study of Student Participation in a Campus-Wide General Pathology Course for Graduate Students at The University of Arizona.

Faculty members from the Department of Pathology at The University of Arizona College of Medicine-Tucson have offered a 4-credit course on enhanced general pathology for graduate students since 1996. The course is titled, "Mechanisms of Human Disease." Between 1997 and 2016, 270 graduate students completed Mechanisms of Human Disease. The students came from 21 programs of study. Analysis of Variance, using course grade as the dependent and degree, program, gender, and year (1997-2016) as independent variables, indicated that there was no significant difference in final grade (F = 0.112; P = .8856) as a function of degree (doctorate: mean = 89.60, standard deviation = 5.75; master's: mean = 89.34, standard deviation = 6.00; certificate program: mean = 88.64, standard deviation = 8.25), specific type of degree program (F = 2.066, P = .1316; life sciences: mean = 89.95, standard deviation = 6.40; pharmaceutical sciences: mean = 90.71, standard deviation = 4.57; physical sciences: mean = 87.79, standard deviation = 5.17), or as a function of gender (F = 2.96, P = .0865; males: mean = 88.09, standard deviation = 8.36; females: mean = 89.58, standard deviation = 5.82). Students in the physical and life sciences performed equally well. Mechanisms of Human Disease is a popular course that provides students enrolled in a variety of graduate programs with a medical school-based course on mechanisms of diseases. The addition of 2 new medically oriented Master of Science degree programs has nearly tripled enrollment. This graduate level course also potentially expands the interdisciplinary diversity of participants in our interprofessional education and collaborative practice exercises.

An updated h-index measures both the primary and total scientific output of a researcher.

The growing interest in scientometry stems from ethical concerns related to the proper evaluation of scientific contributions of an author working in a hard science. In the absence of a consensus, institutions may use arbitrary methods for evaluating scientists for employment and promotion. There are several indices in use that attempt to establish the most appropriate and suggestive position of any scientist in the field he/she works in. A scientist's Hirsch-index (h-index) quantifies their total effective published output, but h-index summarizes the total value of their published work without regard to their contribution to each publication. Consequently, articles where the author was a primary contributor carry the same weight as articles where the author played a minor role. Thus, we propose an updated h-index named Hirsch(p,t)-index that informs about both total scientific output and output where the author played a primary role. Our measure, h(p,t) = h(p),h(t), is composed of the h-index h(t) and the h-index calculated for articles where the author was a key contributor; i.e. first/shared first or senior or corresponding author. Thus, a h(p,t) = 5,10 would mean that the author has 5 articles as first, shared first, senior or corresponding author with at least 5 citations each, and 10 total articles with at least 10 citations each. This index can be applied in biomedical disciplines and in all areas where the first and last position on an article are the most important. Although other indexes, such as r- and w-indexes, were proposed for measuring the authors output based on the position of researchers within the published articles, our simpler strategy uses the already established algorithms for h-index calculation and may be more practical to implement.

The S100P/RAGE signaling pathway regulates expression of microRNA-21 in colon cancer cells.

S100P signaling through the receptor for advanced glycation end-products (RAGE) contributes to colon cancer invasion and metastasis, but the mechanistic features of this process are obscure. Here, we investigate whether activation of S100P/RAGE signaling regulates oncogenic microRNA-21 (miR-21). We show that exogenous S100P up-regulates miR-21 levels in human colon cancer cells, whereas knockdown of S100P results in a decrease of miR-21. Furthermore, blockage of RAGE with anti-RAGE antibody suppresses S100P induction of miR-21. In addition, we found that S100P induction of miR-21 expression involves ERK and is suppressed by the MEK inhibitor U0126. Also, S100P treatment stimulates the enrichment of c-Fos, and AP-1 family members, at the miR-21 gene promoter.

How microRNAs influence both hereditary and inflammatory-mediated colon cancers.

MicroRNAs have emerged as important post-translational regulators of gene expression and are involved in several physiological and pathological states including the pathogenesis of human colon cancers. In regards to tumor development, microRNAs can act as oncogenes or tumor suppressors. Two hereditary predispositions (i.e., Lynch syndrome and familial adenomatous polyposis) contribute to the development of colon cancer. In addition, individuals who suffer from inflammatory bowel diseases such as Crohn's disease or ulcerative colitis have a higher risk of developing colon cancer. Here, we discuss the occurrence of the deregulated expression of microRNAs in colon cancer that arise as a result of hereditary predisposition and inflammatory bowel disease.

S100P/RAGE signaling regulates microRNA-155 expression via AP-1 activation in colon cancer.

Accumulating evidence indicates that elevated S100P promotes the pathogenesis of cancers, including colon cancer. S100P exerts its effects by binding to and activating the Receptor for Advance Glycation End-products (RAGE). The effects of up-regulated S100P/RAGE signaling on cell functions are well documented. Despite these observations, little is known about the downstream targets of S100P/RAGE signaling. In the present study, we demonstrated for the first time that activation of RAGE by S100P regulates oncogenic microRNA-155 (miR-155) expression through Activator Protein-1 (AP-1) stimulation in colon cancer cells. Ectopic S100P up-regulated miR-155 levels in human colon cancer cells. Conversely, knockdown of S100P resulted in a decrease in miR-155 levels. Exogenous S100P induced miR-155 expression, but blockage of the RAGE with anti-RAGE antibody suppressed the induction of miR-155 by exogenous S100P. Attenuation of AP-1 activation through pharmacological inhibition of MEK activation or genetic inhibition of c-Jun activation using dominant negative c-Jun (TAM67) suppressed miR-155 induction by exogenous S100P. Also, S100P treatment stimulated the enrichment of c-Fos, an AP-1 family member, at the miR-155 host gene promoter site. Finally, a functional study demonstrated that miR-155 knockdown decreases colon cancer cell growth, motility, and invasion. Altogether, these data demonstrate that the expression of miR-155 is regulated by S100P and is dependent on RAGE activation and stimulation of AP-1.

Disease modification of breast cancer-induced bone remodeling by cannabinoid 2 receptor agonists.

Most commonly originating from breast malignancies, metastatic bone cancer causes bone destruction and severe pain. Although novel chemotherapeutic agents have increased life expectancy, patients are experiencing higher incidences of fracture, pain, and drug-induced side effects; furthermore, recent findings suggest that patients are severely undertreated for their cancer pain. Strong analgesics, namely opiates, are first-line therapy in alleviating cancer-related pain despite the severe side effects, including enhanced bone destruction with sustained administration. Bone resorption is primarily treated with bisphosphonates, which are associated with highly undesirable side effects, including nephrotoxicity and osteonecrosis of the jaw. In contrast, cannabinoid receptor 2 (CB(2) ) receptor-specific agonists have been shown to reduce bone loss and stimulate bone formation in a model of osteoporosis. CB(2) agonists produce analgesia in both inflammatory and neuropathic pain models. Notably, mixed CB(1) /CB(2) agonists also demonstrate a reduction in ErbB2-driven breast cancer progression. Here we demonstrate for the first time that CB(2) agonists reduce breast cancer-induced bone pain, bone loss, and breast cancer proliferation via cytokine/chemokine suppression. Studies used the spontaneously-occurring murine mammary cell line (66.1) implanted into the femur intramedullary space; measurements of spontaneous pain, bone loss, and cancer proliferation were made. The systemic administration of a CB(2) agonist, JWH015, for 7 days significantly attenuated bone remodeling, assuaged spontaneous pain, and decreased primary tumor burden. CB(2) -mediated effects in vivo were reversed by concurrent treatment with a CB(2) antagonist/inverse agonist but not with a CB(1) antagonist/inverse agonist. In vitro, JWH015 reduced cancer cell proliferation and inflammatory mediators that have been shown to promote pain, bone loss, and proliferation. Taken together, these results suggest CB(2) agonists as a novel treatment for breast cancer-induced bone pain, in which disease modifications include a reduction in bone loss, suppression of cancer growth, attenuation of severe bone pain, and increased survival without the major side effects of current therapeutic options.

MicroRNA-101 (miR-101) post-transcriptionally regulates the expression of EP4 receptor in colon cancers.

PURPOSE: Expression of the PGE2 receptor, EP4, is up-regulated during colorectal carcinogenesis. However the mechanism leading to deregulation of the EP4 receptor is not known. The present study was conducted to investigate the regulation of EP4 receptor by miRNAs. EXPERIMENTAL DESIGN: We analyzed 26 colon cancers (i.e. 15 adenocarcinomas and 9 adenomas) and 16 normal colon specimens for EP4 receptor expression by immunohistochemistry. A bioinformatics approached identified putative microRNA binding sites with the 3'-UTR of the EP4 receptor. Both colon cancer cell lines and tumor specimens were analyzed for miR-101 and EP4 expression by qRT-PCR and Western analysis respectively and simultaneously in situ hybridizations was used to confirm our results. In vitro and in vivo assays were used to confirm our clinical findings. RESULTS: We observed an inverse correlation between the levels of miR-101 and EP4 receptor protein. Transfection of LS174T cells with miR-101 significantly suppressed a luciferase reporter containing the EP4 receptor-3'-UTR. In contrast, a mutant EP4 receptor-3'-UTR construct was unaffected. Ectopic expression of miR-101 markedly reduced cell proliferation and motility. Co-transfection of EP4 receptor could rescue colon cancer cells from the tumor suppressive effects of miR-101. Moreover, the pharmacologic inhibition of EP4 receptor signaling or silencing of EP4 receptor phenocopied the effect of miR-101. This is the first study to show that the EP4 receptor is negatively regulated by miR-101. CONCLUSIONS: These data provide new insights in the modulation of EP-4 receptor expression at the post-transcriptional level by miR-101 and suggests therapeutic strategies against miR-101 targets may be warranted.

Redox agents modulate neuronal activity and reproduce physiological aspects of neuronal aging.

The high oxygen consumption and post-mitotic nature of the central nervous system (CNS) makes it particularly susceptible to oxidative stress, the impact of which is widely regarded as a root cause of functional impairment of the aging brain in vertebrates and invertebrates alike. Using an invertebrate model system we demonstrate that the lipid soluble antioxidant α-tocopherol can both reverse 2,2-azobis(2-methylpropion-amidine) dihydrochloride (AAPH) induced decline in excitability in young neurons as well as restore the electrical activity and excitability of aged neurons not unlike the level of their younger equivalents. Furthermore, using two analogs of α-tocopherol where either the acyl chain has been removed (Trolox) or the hydroxyl group of the chromanol ring has been methylated we were able to assert that the restorative effect of α-tocopherol requires both insertion into the plasma membrane as well as an active OH group. Thus, our results indicate peroxidation is an important modulator of neuronal excitability as well as support the growing body of evidence suggesting α-tocopherol's actions may extend well beyond its established role as a lipid domain preventative antioxidant.

Inhibition of p38-MAPK signaling pathway attenuates breast cancer induced bone pain and disease progression in a murine model of cancer-induced bone pain.

BACKGROUND: Mechanisms driving cancer-induced bone pain are poorly understood. A central factor implicated to be a key player in the process of tumorigenesis, osteoclastogenesis and nociception is p38 MAPK. We determined the role of p38 MAPK in a mouse model of breast cancer induced bone pain in which mixed osteolytic and osteoblastic remodeling occurs. RESULTS: In cancer-treated mice, acute as well as chronic inhibition of p38 MAPK with SB203580 blocked flinching and guarding behaviors in a dose-dependent manner whereas no effect on thresholds to tactile stimuli was observed. Radiographic analyses of bones demonstrated that chronic inhibition of p38 MAPK reduced bone loss and incidence of spontaneous fracture in cancer-treated mice. Histological analysis of bones collected from mice treated with the p38 MAPK inhibitor showed complete absence of osteoblastic growth in the intramedullary space as well as significantly reduced tumor burden. CONCLUSIONS: Blockade of non-evoked pain behaviors but not hypersensitivity suggests differences in the underlying mechanisms of specific components of the pain syndrome and a possibility to individualize aspects of pain management. While it is not known whether the role of p38 MAPK signaling can be expanded to other cancers, the data suggest a need for understanding molecular mechanisms and cellular events that initiate and maintain cancer-induced bone pain for effective management for both ongoing pain as well as breakthrough pain.

Mechanisms for the activity of heterocyclic cyclohexanone curcumin derivatives in estrogen receptor negative human breast cancer cell lines.

Estrogen receptor (ER)-negative breast cancer is an aggressive form that currently requires more drug treatment options. Thus, we have further modified cyclohexanone derivatives of curcumin and examined them for cytotoxicity towards ER-negative human breast cancer cells. Two of the analogs screened elicited increased cytotoxic potency compared to curcumin and other previously studied derivatives. Specifically, 2,6-bis(pyridin-3-ylmethylene)-cyclohexanone (RL90) and 2,6-bis(pyridin-4-ylmethylene)-cyclohexanone (RL91) elicited EC(50) values of 1.54 and 1.10 µM, respectively, in MDA-MB-231 cells and EC(50) values of 0.51 and 0.23 in SKBr3 cells. All other new compounds examined were less potent than curcumin, which elicited EC(50) values of 7.6 and 2.4 µM in MDA-MB-231 and SKBr3 cells, respectively. Mechanistic analyses demonstrated that RL90 and RL91 significantly induced G(2)/M-phase cell cycle arrest and apoptosis. RL90 and RL91 also modulated the expression of key cell signaling proteins, specifically, in SKBr3 cells, protein levels of Her-2, Akt, and NFκB were decreased in a time-dependent manner, while activity of stress kinases JNK1/2 and P38 MAPK were increased. Signaling events in MDA-MB-231 cells were differently implicated, as EGFR protein levels were decreased and activity of GSK-3ß transiently decreased, while ß-catenin protein level and activity of P38 MAPK, Akt, and JNK1/2 were transiently increased. In conclusion replacement of the phenyl group of cyclohexanone derived curcumin derivatives with heterocyclic rings forms a class of second-generation analogs that are more potent than both curcumin and other derivatives. These new derivatives provide a platform for the further development of drugs for the treatment of ER-negative breast cancer.