Comorbidities in a Nationwide, Heterogenous Population of Veterans with Interstitial Cystitis/Bladder Pain Syndrome.

OBJECTIVE: To examine the prevalence of comorbid conditions in a nationwide population of men and women with IC/BPS utilizing a more heterogeneous sample than most studies to date. METHODS: Using the Veterans Affairs Informatics and Computing Infrastructure, we identified random samples of male and female patients with and without an ICD-9/ICD-10 diagnosis of IC/BPS. Presence of comorbidities (NUAS [chronic fatigue syndrome, fibromyalgia, irritable bowel syndrome, migraines], back pain, diabetes, and smoking) and psychosocial factors (alcohol abuse, post-traumatic stress disorder, sexual trauma, and history of depression) were determined using ICD-9 and ICD-10 codes. Associations between these variables and IC/BPS status were evaluated while adjusting for the potential confounding impact of race/ethnicity, age, and gender. RESULTS: Data was analyzed from 872 IC/BPS patients (355 [41%] men, 517 [59%] women) and 558 non-IC/BPS patients (291 [52%] men, 267 [48%] women). IC/BPS patients were more likely than non-IC/BPS patients to have a greater number of comorbidities (2.72+/-1.77 vs 1.73+/-1.30, P < 0.001), experience one or more NUAS (chronic fatigue syndrome, fibromyalgia, irritable bowel syndrome, and migraines) (45% [388/872] vs. 18% [101/558]; P < 0.001) and had a higher prevalence of at least one psychosocial factor (61% [529/872] v. 46% [256/558]; P < 0.001). Differences in the frequencies of comorbidities between patients with and without IC/BPS were more pronounced in female patients. CONCLUSION: These findings validate the findings of previous comorbidity studies of IC/BPS in a more diverse population.

Pharmacokinetic-pharmacodynamic correlations in the development of ginger extract as an anticancer agent.

Anticancer efficacy of ginger phenolics (GPs) has been demonstrated in various in vitro assays and xenograft mouse models. However, only sub-therapeutic plasma concentrations of GPs were detected in human and mouse pharmacokinetic (PK) studies. Intriguingly, a significant portion of GPs occurred as phase II metabolites (mainly glucuronide conjugates) in plasma. To evaluate the disposition of GPs and understand the real players responsible for efficacy, we performed a PK and tissue distribution study in mice. Plasma exposure of GPs was similar on day 1 and 7, suggesting no induction or inhibition of clearance pathways. Both free and conjugated GPs accumulated in all tissues including tumors. While non-cytotoxicity of 6-ginerol glucuronide precluded the role of conjugated GPs in cell death, the free forms were cytotoxic against prostate cancer cells. The efficacy of ginger was best explained by the reconversion of conjugated GPs to free forms by ß-glucuronidase, which is over-expressed in the tumor tissue. This previously unrecognized two-step process suggests an instantaneous conversion of ingested free GPs into conjugated forms, followed by their subsequent absorption into systemic circulation and reconversion into free forms. This proposed model uncovers the mechanistic underpinnings of ginger's anticancer activity despite sub-therapeutic levels of free GPs in the plasma.

Preclinical Development of a Nontoxic Oral Formulation of Monoethanolamine, a Lipid Precursor, for Prostate Cancer Treatment.

Purpose: Most currently available chemotherapeutic agents target rampant cell division in cancer cells, thereby affecting rapidly dividing normal cells resulting in toxic side-effects. This nonspecificity necessitates identification of novel cellular pathways that are reprogrammed selectively in cancer cells and can be exploited to develop pharmacologically superior and less toxic therapeutics. Despite growing awareness on dysregulation of lipid metabolism in cancer cells, targeting lipid biosynthesis is still largely uncharted territory. Herein, we report development of a novel nontoxic orally deliverable anticancer formulation of monoethanolamine (Etn) for prostate cancer by targeting the Kennedy pathway of phosphatidylethanolamine (PE) lipid biosynthesis.Experimental Design: We first evaluated gastrointestinal tract stability, drug-drug interaction liability, pharmacokinetic, and toxicokinetic properties of Etn to evaluate its suitability as a nontoxic orally deliverable agent. We next performed in vitro and in vivo experiments to investigate efficacy and mechanism of action.Results: Our data demonstrate that Etn exhibits excellent bioavailability, gastrointestinal tract stability, and no drug-drug interaction liability. Remarkably, orally fed Etn inhibited tumor growth in four weeks by approximately 67% in mice bearing human prostate cancer PC-3 xenografts without any apparent toxicity. Mechanistically, Etn exploits selective overexpression of choline kinase in cancer cells, resulting in accumulation of phosphoethanolamine (PhosE), accompanied by downregulation of HIF-1α that induces metabolic stress culminating into cell death.Conclusions: Our study provides first evidence for the superior anticancer activity of Etn, a simple lipid precursor formulation, whose nontoxicity conforms to FDA-approved standards, compelling its clinical development for prostate cancer management. Clin Cancer Res; 23(14); 3781-93. ©2017 AACR.

Ginger augmented chemotherapy: A novel multitarget nontoxic approach for cancer management.

Cancer, referred to as the 'disease of civilization', continues to haunt humanity due to its dreadful manifestations and limited success of therapeutic interventions such as chemotherapy in curing the disease. Although effective, chemotherapy has repeatedly demonstrated inadequacy in disease management due to its debilitating side effects arising from its deleterious nonspecific effects on normal healthy cells. In addition, development of chemoresistance due to mono-targeting often results in cessation of chemotherapy. This urgently demands development and implementation of multitargeted alternative therapies with mild or no side effects. One extremely promising strategy that yet remains untapped in the clinic is augmenting chemotherapy with dietary phytochemicals or extracts. Ginger, depository of numerous bioactive molecules, not only targets cancer cells but can also mitigate chemotherapy-associated side effects. Consequently, combination therapy involving ginger extract and chemotherapeutic agents may offer the advantage of being efficacious with reduced toxicity. Here we discuss the remarkable and often overlooked potential of ginger extract to manage cancer, the possibility of developing ginger-based combinational therapies, and the major roadblocks along with strategies to overcome them in clinical translation of such inventions. We are optimistic that clinical implementation of such combination regimens would be a much sought after modality in cancer management.

Cholesterol-induced changes in hippocampal membranes utilizing a phase-sensitive fluorescence probe.

The function of membrane receptors in the nervous system depends on physicochemical characteristics of neuronal membranes such as membrane order and phase. In this work, we have monitored the changes in hippocampal membrane order and related parameters by cholesterol and protein content utilizing a Nile Red-based phase-sensitive fluorescent membrane probe NR12S. Since alteration of membrane cholesterol is often associated with membrane phase change, the phase-sensitive nature of NR12S fluorescence becomes useful in these experiments. Our results show that fluorescence spectroscopic parameters such as emission maximum, anisotropy, and lifetime of NR12S display characteristic dependence on membrane cholesterol content. Interestingly, cholesterol-dependent red edge excitation shift is displayed by NR12S under these conditions. Hippocampal membranes exhibited reduction in liquid-ordered phase upon cholesterol depletion. These results provide insight into changes in hippocampal membrane order in the overall context of cholesterol and protein modulation.

Location, dynamics and solvent relaxation of a Nile Red-based phase-sensitive fluorescent membrane probe.

Fluorescent membrane probes offer the advantage of high sensitivity, suitable time resolution, and multiplicity of measurable parameters, and provide useful information on model and cell membranes. In this paper, we have explored the location, dynamics, and solvent relaxation characteristics of a novel Nile Red-based phase-sensitive probe (NR12S). Unlike Nile Red, NR12S enjoys unique orientation and location in the membrane, and is localized exclusively in the outer leaflet of the membrane bilayer. By analysis of membrane depth using the parallax approach, we show that the fluorescent group in NR12S is localized at the membrane interface, a region characterized by slow solvent relaxation. Our results show that NR12S exhibits REES (red edge excitation shift), consistent with its interfacial localization. More interestingly, REES of NR12S displays sensitivity to the membrane phase. In addition, fluorescence emission maximum, anisotropy, and lifetime of NR12S are dependent on the membrane phase. We envision that NR12S may prove to be a useful probe in future studies of complex natural membranes.

Cholesterol biosynthesis and homeostasis in regulation of the cell cycle.

The cell cycle is a ubiquitous, multi-step process that is essential for growth and proliferation of cells. The role of membrane lipids in cell cycle regulation is not explored well, although a large number of cytoplasmic and nuclear regulators have been identified. We focus in this work on the role of membrane cholesterol in cell cycle regulation. In particular, we have explored the stringency of the requirement of cholesterol in the regulation of cell cycle progression. For this purpose, we utilized distal and proximal inhibitors of cholesterol biosynthesis, and monitored their effect on cell cycle progression. We show that cholesterol content increases in S phase and inhibition of cholesterol biosynthesis results in cell cycle arrest in G1 phase under certain conditions. Interestingly, G1 arrest mediated by cholesterol biosynthesis inhibitors could be reversed upon metabolic replenishment of cholesterol. Importantly, our results show that the requirement of cholesterol for G1 to S transition is absolute, and even immediate biosynthetic precursors of cholesterol, differing with cholesterol merely in a double bond, could not replace cholesterol for reversing the cell cycle arrest. These results are useful in the context of diseases, such as cancer and Alzheimer's disease, that are associated with impaired cholesterol biosynthesis and homeostasis.

Membrane cholesterol stabilizes the human serotonin(1A) receptor.

A number of recently solved crystal structures of G-protein coupled receptors reveal the presence of closely associated cholesterol molecules in the receptor structure. We have previously shown the requirement of membrane cholesterol in the organization, dynamics and function of the serotonin(1A) receptor, a representative G-protein coupled receptor. In this work, we explored the role of membrane cholesterol in the stability of the human serotonin(1A) receptor. Analysis of sensitivity of the receptor to thermal deactivation, pH, and proteolytic digestion in control, cholesterol-depleted and cholesterol-enriched membranes comprehensively demonstrate that membrane cholesterol stabilizes the serotonin(1A) receptor. We conclude that these results could have potential implications in future efforts toward crystallizing the receptor.

Cholesterol depletion modulates detergent resistant fraction of human serotonin(1A) receptors.

Insolubility of membrane components in non-ionic detergents such as Triton X-100 at low temperature is a widely used biochemical criterion to identify, isolate and characterize membrane domains. In this work, we monitored the detergent insolubility of the serotonin(1A) receptor in CHO cell membranes and its modulation by membrane cholesterol. The serotonin(1A) receptor is an important member of the G-protein coupled receptor family. It is implicated in the generation and modulation of various cognitive, behavioral and developmental functions and serves as a drug target. Our results show that a significant fraction (∼28%) of the serotonin(1A) receptor resides in detergent-resistant membranes (DRMs). Interestingly, the fraction of the serotonin(1A) receptor in DRMs exhibits a reduction upon membrane cholesterol depletion. In addition, we show that contents of DRM markers such as flotillin-1, caveolin-1 and GM1 are altered in DRMs upon cholesterol depletion. These results assume significance since the function of the serotonin(1A) receptor has previously been shown to be affected by membrane lipids, specifically cholesterol. Our results are relevant in the context of membrane organization of the serotonin(1A) receptor in particular, and G-protein coupled receptors in general.

Comparative analysis of calcium spikes upon activation of serotonin(1A) and purinergic receptors.

Calcium signaling represents one of the most important signaling cascades in cells and regulates diverse processes such as exocytosis, muscle contraction and relaxation, gene expression and cell growth. G protein-coupled receptors (GPCRs) are the most important family of receptors that activate calcium signaling. Since calcium signaling regulates a large number of physiological responses, it is intriguing that how changes in cytosolic calcium levels by a wide range of stimuli lead to signal-specific physiological responses in the cellular interior. In order to address this issue, we have analyzed temporal calcium profiles induced by two GPCRs, the serotonin(1A) and purinergic receptors. In this work, we have described a set of parameters for the analysis of calcium transients that could provide novel insight into mechanisms responsible for maintaining signal specificity by shaping calcium transients. An interesting feature of calcium signaling that has emerged from our analysis is that the profile of individual transients in a calcium response could play an important role in maintaining downstream signal specificity. In summary, our analysis offers a novel approach to identify differences in calcium response patterns induced by various stimuli.

Reorganization of the actin cytoskeleton upon G-protein coupled receptor signaling.

The actin cytoskeleton is involved in a multitude of cellular responses besides providing structural support. While the role of the actin cytoskeleton in cellular processes such as trafficking and motility has been extensively studied, reorganization of the actin cytoskeleton upon signaling by G-protein coupled receptors (GPCRs) represents a relatively unexplored area. The G-protein coupled receptor superfamily is an important protein family in mammals, involved in signal transduction across membranes. G-protein coupled receptors act as major signaling hubs and drug targets. The serotonin(1A) receptor is a representative member of the G-protein coupled receptor superfamily and plays a crucial role in the generation and modulation of various cognitive, developmental and behavioral functions. In order to monitor the changes in the actin cytoskeleton upon serotonin(1A) receptor signaling in a quantitative manner, we developed an approach based on high magnification imaging of F-actin in cells, followed by image reconstruction. Our results suggest that the actin cytoskeleton is reorganized in response to serotonin(1A) receptor signaling. In addition, we show that reorganization of the actin cytoskeleton is strongly dependent on adenosine 3',5'-cyclic monophosphate level, and is mediated by the activation of protein kinase A. Our results are consistent with the possibility of a feedback mechanism involving the actin cytoskeleton, adenosine 3',5'-cyclic monophosphate level and the serotonin(1A) receptor.

Membrane organization and dynamics of the serotonin1A receptor in live cells.

The G-protein coupled receptor (GPCR) superfamily is one of the largest classes of molecules involved in signal transduction across the plasma membrane. The serotonin(1A) receptor is a representative member of the GPCR superfamily and serves as an important target in the development of therapeutic agents for neuropsychiatric disorders such as anxiety and depression. In the context of the pharmacological relevance of the serotonin(1A) receptor, the membrane organization and dynamics of this receptor in the cellular environment assume relevance. We have highlighted results, obtained from fluorescence microscopy-based approaches, related to domain organization and dynamics of the serotonin(1A) receptor. A fraction of serotonin(1A) receptors displays detergent insolubility, monitored using green fluorescent protein, that increases upon depletion of membrane cholesterol. Fluorescence recovery after photobleaching measurements with varying bleach spot sizes show that lateral diffusion parameters of serotonin(1A) receptors in normal cells are consistent with models describing diffusion of molecules in a homogenous membrane. Interestingly, these characteristics are altered in cholesterol-depleted cells. Taken together, we conclude that the serotonin(1A) receptor exhibits dynamic confinement in the cellular plasma membranes. Progress in understanding GPCR organization and dynamics would result in better insight into our overall understanding of GPCR function in health and disease.

Identification of a red-emitting fluorescent ligand for in vitro visualization of human serotonin 5-HT(1A) receptors.

The 5-HT(1A) receptor subtype is the most thoroughly studied serotonin receptor subtype. We report here the design, synthesis and characterization of two new fluorescent ligands for the 5-HT(1A) receptor. The new 1-arylpiperazine-based red-emitting fluorescent compound 6 displayed good binding affinity at the 5-HT(1A) receptor (K(i)=35 nM) and was able to label specifically the human 5-HT(1A) receptor stably expressed in CHO cells visualized using confocal laser scanning microscopy.

Differential effects of cholesterol and desmosterol on the ligand binding function of the hippocampal serotonin(1A) receptor: implications in desmosterolosis.

Cholesterol is a unique molecule in terms of high level of in-built stringency, fine tuned by natural evolution for its ability to optimize physical properties of higher eukaryotic cell membranes in relation to biological functions. We previously demonstrated the requirement of membrane cholesterol in maintaining the ligand binding activity of the hippocampal serotonin(1A) receptor. In order to test the molecular stringency of the requirement of cholesterol, we depleted cholesterol from native hippocampal membranes followed by replenishment with desmosterol. Desmosterol is an immediate biosynthetic precursor of cholesterol in the Bloch pathway differing only in a double bond at the 24th position in the alkyl side chain. Our results show that replenishment with desmosterol does not restore ligand binding activity of the serotonin(1A) receptor although replenishment with cholesterol led to significant recovery of ligand binding. This is in spite of similar membrane organization (order) in these membranes, as monitored by fluorescence anisotropy measurements. The requirement for restoration of ligand binding activity therefore appears to be more stringent than the requirement for the recovery of overall membrane order. These novel results have potential implications in understanding the interaction of membrane lipids with this important neuronal receptor in diseases such as desmosterolosis.

Exploring the organization and dynamics of hippocampal membranes utilizing pyrene fluorescence.

The organization and dynamics of cellular membranes in the nervous system are crucial for the function of neuronal membrane receptors and signal transduction. Previous work from our laboratory has established hippocampal membranes as a convenient natural source for studying neuronal receptors. In this paper, we have monitored the organization and dynamics of hippocampal membranes and their modulation by cholesterol using pyrene fluorescence. The apparent dielectric constant experienced by pyrene in hippocampal membranes turns out to be approximately 20+/-3, depending on the experimental condition. Our results show that the polarity of the hippocampal membrane is increased upon cholesterol depletion, as monitored by changes in the ratio of pyrene vibronic peak intensities (I1/I3). This is accompanied by an increase in lateral diffusion, measured as an increase in the pyrene excimer/monomer ratio. These results are relevant in understanding the complex organization and dynamics of hippocampal membranes and could have implications in neuronal diseases characterized by defective cholesterol metabolism.