The purpose of this study was to determine the effects of down-regulation of Aquaporin 1 (AQP1) and Aquaporin 5 (AQP5) on cell proliferation and migration in human corneal endothelial (HCEC) and human corneal epithelial (CEPI17) cell lines, respectively.
AQP1 and AQP5 were down regulated using siRNA following lipofectamine-mediated transfection in corneal endothelial and epithelial cells, respectively. Down-regulation was confirmed using RT–PCR, indirect immunofluorescence, and immunoblot analysis. Total internal reflection fluorescence (TIRF) microscopy was used to detect cell surface aquaporin expression. Cell proliferation was determined by SRB (sulfrodamine B) assay. Cell migration was determined by in vitro wound healing and migration assay.
In HCEC cells, AQP1 was localized to the cytosol as well as cell membrane and its down-regulation resulted in decreased cell proliferation and migration with a significant decrease in phosphorylated ERK (pERK). In CEPI17 cells AQP5 protein expression was also localized to cytosol as well as cell membrane. AQP5 down-regulation resulted in an increase in proliferation and cell migration with no significant difference in pERK.
AQP1 plays a role in HCEC proliferation and migration via the ERK signaling pathway and therefore may have significant implications in corneal endothelial dysfunction whereas; AQP5 may play an indirect role in human corneal epithelial cell proliferation and migration.
Cardiac toxicity is the foremost reason for drug discontinuation from development to clinical evaluation and post market surveillance [Fung 35:293-317, 2001; Piccini 158:317-326 2009]. The Food and Drug Administration (FDA) has rejected many potential pharmaceutical agents due to QT prolongation effects. Since drug development and FDA approval takes an enormous amount of time, money and effort with high failure rates, there is an increased focus on rescuing drugs that cause QT prolongation. If these otherwise safe and potent drugs were formulated in a unique way so as to mitigate the QT prolongation associated with them, these potent drugs may get FDA approval for clinical use. Rescuing these compounds not only benefit the patients who need them but also require much less time and money thus leading to faster clinical translation. In this study, we chose curcumin as our drug of choice since it has been shown to posses anti-tumor properties against various cancers with limited toxicity. The major limitations with this pharmacologically active drug are (a) its ability to prolong QT by inhibiting the hERG channel and (b) its low bioavailability. In our previous studies, we found that lipids have protective actions against hERG channel inhibition and therefore QT prolongation.
Results of the manual patch clamp assay of HEK 293 cells clearly illustrated that our hybrid nanocurcumin formulation prevented the curcumin induced inhibition of hERG K+ channel at concentrations higher than the therapeutic concentrations of curcumin. Comparing the percent inhibition, the hybrid nanocurcumin limited inhibition to 24.8% at a high curcumin equivalent concentration of 18 μM. Liposomal curcumin could only decrease this inhibition upto 30% only at lower curcumin concentration of 6 μM but not at 18 μM concentration.
Here we show a curcumin encapsulated lipopolymeric hybrid nanoparticle formulation which could protect against QT prolongation and also render increased bioavailability and stability thereby overcoming the limitations associated with curcumin.
QT prolongation; hERG; Curcumin; Nanoparticle; Hybrid
The Texas Center for Health Disparities, a National Institute on Minority Health and Health Disparities Center of Excellence, presents an annual conference to discuss prevention, awareness education and ongoing research about health disparities both in Texas and among the national population. The 2013 Texas Conference on Health Disparities brought together experts, in research, patient care and community outreach, on the “Intersection of Smoking, Human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS) and Cancer”. Smoking, HIV/AIDS and cancer are three individual areas of public health concern, each with its own set of disparities and risk factors based on race, ethnicity, gender, geography and socio-economic status. Disparities among patient populations, in which these issues are found to be comorbid, provide valuable information on goals for patient care. The conference consisted of three sessions addressing “Comorbidities and Treatment”, “Public Health Perspectives”, and “Best Practices”. This article summarizes the basic science, clinical correlates and public health data presented by the speakers.
Cancer; health disparities; human immunodeficiency virus/acquired immunodeficiency syndrome; smoking
The neuropathic side-effects of trauma, stroke or therapeutic radiation of the brain for life-threatening neoplastic diseases are the result of damage to normal tissues resulting in defects in cognition and memory. Based upon published preclinical data of curcumin activity application of parenteral curcumin formulations may prove to be to be promising chemotherapy for disorders following neuropathic insults. Studies in in vitro and animal models suggest curcumin may be an effective remediative agent for brain damage. The initial steps in curcumin development for clinical applications to neuropathic disorders are formulating it for intravenous administration, determining the formulated product passes the blood–brain barrier and reaches therapeutic amounts in damaged areas in the brain with tolerable safety. Following intravenous administration of liposomal curcumin, polymeric nanocurcumin and polylactic glycolic acid co-polymer (PLGA)–curcumin in rats, these formulations were observed to cross the blood–brain barrier using a sensitive HPLC assay. All three formulations localized in specific sites in the brain without observable adverse events. One hour following intravenous injection of 5 mg/kg nanocurcumin, or 20 mg/kg PLGA–curcumin, or liposomal curcumin, up to 0.5% of the injected material localized in the brain stem, the striatum, and the hippocampus with varied accumulation and clearance rates.
These data indicate that curcumin does localize in putative damaged brain tissues and suggest therapeutic trials be explored with all three formulations in animal models with pre- and post traumatic states.
Intravenous curcumin; blood; brain barrier; post-traumatic sequelae; hippocampus; striatum; brain stem
There are striking disparities in health status, access to health care, and risk factors among racial and ethnic minorities and the general population in Texas. The disparities are multifactorial comprising genetic, sociocultural, and environmental variables. The Texas Center for Health Disparities (TCHD), a NIMHD Center of Excellence (COE), aims to prevent, reduce, and eliminate health disparities in the communities through research, education, and community-based programs. As part of the center's outreach activities, an annual conference is organized to build awareness and knowledge on health disparities. The overall theme for the 2012 conference was “Battling Breast Cancer Disparities: Frontline Strategies”. The scientific program consisted of three sessions: “Breakthroughs in Breast Cancer”, “Triple Negative Breast Cancer,” and “Hormone Resistant Breast Cancer” featuring different aspects of bench-research from molecular biology, proteomics, and genetics to the clinical aspects such as detection, diagnosis, and finally to community-based approaches. This article summarizes the proceedings of the meeting providing salient strategies and best practices presented by the speakers.
Breast cancer; conference; disparities; proceedings; triple negative breast cancer
Nanoparticle based delivery of anticancer drugs have been widely investigated. However, a very important process for Research & Development in any pharmaceutical industry is scaling nanoparticle formulation techniques so as to produce large batches for preclinical and clinical trials. This process is not only critical but also difficult as it involves various formulation parameters to be modulated all in the same process.
In our present study, we formulated curcumin loaded poly (lactic acid-co-glycolic acid) nanoparticles (PLGA-CURC). This improved the bioavailability of curcumin, a potent natural anticancer drug, making it suitable for cancer therapy. Post formulation, we optimized our process by Reponse Surface Methodology (RSM) using Central Composite Design (CCD) and scaled up the formulation process in four stages with final scale-up process yielding 5 g of curcumin loaded nanoparticles within the laboratory setup. The nanoparticles formed after scale-up process were characterized for particle size, drug loading and encapsulation efficiency, surface morphology, in vitro release kinetics and pharmacokinetics. Stability analysis and gamma sterilization were also carried out.
Results revealed that that process scale-up is being mastered for elaboration to 5 g level. The mean nanoparticle size of the scaled up batch was found to be 158.5 ± 9.8 nm and the drug loading was determined to be 10.32 ± 1.4%. The in vitro release study illustrated a slow sustained release corresponding to 75% drug over a period of 10 days. The pharmacokinetic profile of PLGA-CURC in rats following i.v. administration showed two compartmental model with the area under the curve (AUC0-∞) being 6.139 mg/L h. Gamma sterilization showed no significant change in the particle size or drug loading of the nanoparticles. Stability analysis revealed long term physiochemical stability of the PLGA-CURC formulation.
A successful effort towards formulating, optimizing and scaling up PLGA-CURC by using Solid-Oil/Water emulsion technique was demonstrated. The process used CCD-RSM for optimization and further scaled up to produce 5 g of PLGA-CURC with almost similar physicochemical characteristics as that of the primary formulated batch.
Scale up; Optimization; PLGA nanoparticles; Cancer; Response surface methodology (RSM); Curcumin C3 complex; Central composite design (CCD)
Reduced levels of global DNA methylation are associated with genomic instability and are independent predictors of cancer risk. Little is known about the environmental determinants of global DNA methylation in peripheral blood. We examined the association between demographic and lifestyle factors and levels of global leukocyte DNA methylation in 161 cancer-free subjects enrolled in the North Texas Healthy Heart Study aged 45–75 years in 2008. We used in-person interviews for demographics and lifestyle factors, a self-administrated Block food frequency questionnaire for diet, and bioelectrical impedance analysis and CT-scan for body composition. We measured genomic DNA methylation using bisulfite conversion of DNA and pyrosequencing for LINE-1. Body composition measures including body mass index, waist circumference, areas of subcutaneous fat and visceral fat, percent of fat mass and fat-free mass were not associated with global genomic DNA methylation after controlling the effect of age, gender and race/ethnicity. Instead, female gender was significantly associated with a reduced level of global methylation (β = −2.77, 95% CI: −4.33, −1.22). Compared to non-Hispanic whites, non-Hispanic blacks (β = −2.02, 95% CI: −3.55, −0.50) had significantly lower levels of global methylation. No association was found with age, cigarette smoking, alcohol drinking and dietary intake of nutrients in one-carbon metabolism. Global leukocyte DNA methylation differs by gender and race/ethnicity, suggesting these variables need to be taken into consideration in studies of global DNA methylation as an epigenetic marker for cancer.
gender; race/ethnicity; DNA methylation
Changes in DNA methylation may represent an intermediate step between the environment and human diseases. Little is known on whether behavioral risk factors may modify gene expression through DNA methylation. To assess whether DNA methylation is associated with different levels of physical activity, we measured global genomic DNA methylation using bisulfite-converted DNA and real-time PCR (MethyLight) for LINE-1 in peripheral blood of 161 participants aged 45–75 years enrolled in the North Texas Healthy Heart Study and levels of physical activity using an accelerometer (Actigraph GT1M Monitor). We found that individuals with physical activity 26–30 min/day had a significantly higher level of global genomic DNA methylation compared to those with physical activity ≤10 min/day (β = 2.52, 95% CI: 0.70, 4.35). However, the association was attenuated and became statistically insignificant after multivariate adjustment (β = 1.24, 95% CI: −0.93, 3.40). There were some suggestions of a positive association between physical activity and global genomic DNA methylation in non-Hispanics (β = 1.50, 95% CI: −0.08, 3.08) that warrants further investigation.
DNA methylation; physical activity; peripheral blood
Prostate cancer is a leading cause of death among men in the United States, and currently early diagnosis and appropriate treatment remain key approaches for patient care. Molecularly prostate cancer cells carry multiple perturbations that generate malignant phenotype capable of uncontrolled growth, survival, and invasion-metastasis to other organs. These alterations are acquired both by genetic and epigenetic changes in tumor cells resulting in the activation of growth factor receptors, signaling proteins, kinases, transcription factors and coregulators, and multiple proteases required for the progression of the disease. Recent advances provide novel insights into the molecular functions of these oncogenic activators, implicating potential therapeutic targeting opportunities for the treatment of prostate cancer.
Prostate cancer; oncogene; PI3K-Akt; proteases; signaling pathways; growth factor receptors
MMP-9 enzyme recognizes a peptide sequence Lys-Gly-Pro-Arg-Ser-Leu-Ser-Gly-Lys and cleaves the peptide into two parts. We synthesized a dual fluorophore beacon consisting of 5-FAM and Cy5 dyes. The fluorescence emission of the fluorescein moiety is dramatically quenched by Cy5 molecule due to Förster Resonance Energy Transfer (FRET) and the fluorescence of Cy5 is strongly enhanced. Upon addition of MMP-9 enzyme, the fluorescence of 5-FAM intensifies and Cy5 decreases. The control MMP-2 enzyme does not cause any changes in either 5-FAM or Cy5 fluorescence. We believe that our observation will help in early detection of elevated MMP-9 levels under disease conditions.
MMP-9; fluorescence; FRET; labeled peptide
The changes in gene expression profile as prostate cancer progresses from an androgen-dependent disease to an androgen-independent disease are still largely unknown.
We examined the gene expression profile in the LNCaP prostate cancer progression model during chronic treatment with Casodex using cDNA microarrays consisting of 2305 randomly chosen genes.
Our studies revealed a representative collection of genes whose expression was differentially regulated in LNCaP cells upon treatment with Casodex. A set of 15 genes were shown to be highly expressed in Casodex-treated LNCaP cells compared to the reference sample. This set of highly expressed genes represents a signature collection unique to prostate cancer since their expression was significantly greater than that of the collective pool of ten cancer cell lines of the reference sample. The highly expressed signature collection included the hypoxia-related genes membrane metallo-endopeptidase (MME), cyclin G2, and Bcl2/adenovirus E1B 19 kDa (BNIP3). Given the roles of these genes in angiogenesis, cell cycle regulation, and apoptosis, we further analyzed their expression and concluded that these genes may be involved in the molecular changes that lead to androgen-independence in prostate cancer.
Our data indicate that one of the mechanisms of Casodex action in prostate cancer cells is induction of hypoxic gene expression.
Annexin I, one of the 20 members of the annexin family of calcium and phospholipid-binding proteins, has been implicated in diverse biological processes including signal transduction, mediation of apoptosis and immunosuppression. Previous studies have shown increased annexin I expression in pancreatic and breast cancers, while it is absent in prostate and esophageal cancers.
Data presented here show that annexin I mRNA and protein are undetectable in 10 out of 12 B-cell lymphoma cell lines examined. Southern blot analysis indicates that the annexin I gene is intact in B-cell lymphoma cell lines. Aberrant methylation was examined as a cause for lack of annexin I expression by treating cells 5-Aza-2-deoxycytidine. Reexpression of annexin I was observed after prolonged treatment with the demethylating agent indicating methylation may be one of the mechanisms of annexin I silencing. Treatment of Raji and OMA-BL-1 cells with lipopolysaccharide, an inflammation inducer, and with hydrogen peroxide, a promoter of oxidative stress, also failed to induce annexin I expression. Annexin I expression was examined in primary lymphoma tissues by immunohistochemistry and presence of annexin I in a subset of normal B-cells and absence of annexin I expression in the lymphoma tissues were observed. These results show that annexin I is expressed in normal B-cells, and its expression is lost in all primary B-cell lymphomas and 10 of 12 B-cell lymphoma cell lines.
Our results suggest that, similar to prostate and esophageal cancers, annexin I may be an endogenous suppressor of cancer development, and loss of annexin I may contribute to B-cell lymphoma development.
Annexin I; immunohistochemistry; oxidative stress; methylation; gene expression
Annexin II heavy chain (also called p36, calpactin I) is lost in prostate cancers and in a majority of prostate intraepithelial neoplasia (PIN). Loss of annexin II heavy chain appears to be specific for prostate cancer since overexpression of annexin II is observed in a majority of human cancers, including pancreatic cancer, breast cancer and brain tumors. Annexin II exists as a heterotetramer in complex with a protein ligand p11 (S100A10), and as a monomer. Diverse cellular functions are proposed for the two forms of annexin II. The monomer is involved in DNA synthesis. A leucine-rich nuclear export signal (NES) in the N-terminus of annexin II regulates its nuclear export by the CRM1-mediated nuclear export pathway. Mutation of the NES sequence results in nuclear retention of annexin II.
Annexin II localized in the nucleus is phosphorylated, and the appearance of nuclear phosphorylated annexin II is cell cycle dependent, indicating that phosphorylation may play a role in nuclear entry, retention or export of annexin II. By exogenous expression of annexin II in the annexin II-null LNCaP cells, we show that wild-type annexin II is excluded from the nucleus, whereas the NES mutant annexin II localizes in both the nucleus and cytoplasm. Nuclear retention of annexin II results in reduced cell proliferation and increased doubling time of cells. Expression of annexin II, both wild type and NES mutant, causes morphological changes of the cells. By site-specific substitution of glutamic acid in the place of serines 11 and 25 in the N-terminus, we show that simultaneous phosphorylation of both serines 11 and 25, but not either one alone, prevents nuclear localization of annexin II.
Our data show that nuclear annexin II is phosphorylated in a cell cycle-dependent manner and that substitution of serines 11 and 25 inhibit nuclear entry of annexin II. Aberrant accumulation of nuclear annexin II retards proliferation of LNCaP cells.
Alternative survival pathways are commonly seen to be upregulated upon inhibition of receptor tyrosine kinases (RTK), including Her-2. It is established that treatment with Herceptin leads to selective overexpression and activation of epidermal growth factor receptor (EGFR) and Src which further contributes to oncogenesis in Herceptin resistant and triple negative breast cancer (TNBC) patients. Here, we show a co-regulated upregulation in the expression of Annexin A2 (AnxA2), a known substrate of Src and one of the regulators of EGFR receptor endocytosis, in Herceptin resistant and Her-2 negative breast cancer. Immunohistochemical expression analysis revealed a reciprocal regulation between Her-2 and AnxA2 in breast cancer clinical samples as well as in cell lines as confirmed by protein and RNA analysis. The siRNA and Herceptin mediated downregulation/inhibition of Her-2 in Her-2 amplified cells induced AnxA2 expression and membrane translocation. In this study we report a possible involvement of AnxA2 in maintaining constitutively activated EGFR downstream signaling intermediates and hence in cell proliferation, migration and viability. This effect was consistent in Herceptin resistant JIMT-1 cells as well as in Her-2 negative breast cancer. The siRNA mediated AnxA2 downregulation leads to increased apoptosis, decreased cell viability and migration. Our studies further indicate the role of AnxA2 in EGFR-Src membrane bound signaling complex and ligand induced activation of downstream signaling pathways. Targeting this AnxA2 dependent positive regulation of EGFR signaling cascade may be of therapeutic value in Her-2 negative breast cancer.
Prostate cancer (PCa) incidences vary with genetic, geographical and ethnic dietary background of patients while angiogenesis is modulated through exquisite interplay of tumor-stromal interactions of biological macromolecules. We hypothesized that comprehensive analysis of four biomarkers modulating angiogenesis in PCa progression in two diverse populations might explain the variance in the incidence rates.
Immunohistochemical analysis of 42 PCa biopsies reveals that though Anx-II expression is lost in both the Indian and American population with Gleason scores (GS) ranging between 6 and 10, up to 25 % of cells in the entire high grade (GS > 8) PD PCa samples from US show intense focal membrane staining for Anx-II unlike similarly graded specimens from India. Consistent with this observation, the prostate cancer cell lines PC-3, DU-145 and MDA PCa 2A, but not LNCaP-R, LNCAP-UR or MDA PCa 2B cell lines, express Anx-II. Transcriptional reactivation of Anx-II gene with Aza-dC could not entirely account for loss of Anx-II protein in primary PCa. Cyclooxygenase-2 (COX-2) was moderately expressed in most of high grade PIN and some MD PCa and surrounding stroma. COX-2 was not expressed in PD PCa (GS ~7–10), while adjacent smooth muscles cells stained weakly positive. Decorin expression was observed only in high grade PIN but not in any of the prostate cancers, atrophy or BPH while stromal areas of BPH stained intensively for DCN and decreased with advancing stages of PCa. Versican expression was weak in most of the MD PCa, moderate in all of BPH, moderately focal in PD PC, weak and focal in PIN, atrophy and adjacent stroma.
Expression of pro- and anti-angiogenic modulators changes with stage of PCa but correlates with angiogenic status. Focal membrane staining of Anx-II reappears in high grade PCa specimens only from US indicating differential expression of Anx-II. COX-2 stained stronger in American specimens compared to Indian specimens. The sequential expression of DCN and VCN in progressive stages was similar in specimens from India and USA indicating no population-based differences. The mechanistic and regulatory role of Anx-II in PCa progression warrants further investigation.