Selective preservation of protein kinase C-ζ in the chemoprevention of azoxymethane-induced colonic tumors by piroxicam

AbstractWhile nonsteroidal anti-inflammatory drugs have been shown to exert preventive effects against the development of colonic tumors in humans and in chemically-induced tumors in animal models, the mechanism(s) involved in this phenomenon is unclear. We have recently demonstrated that one such agent, piroxicam, when supplemented (75 ppm) in the diets of rats administered azoxymethane, reduced the incidence of rats bearing tumors. To date, the effects of piroxicam on protein kinase C, a family of serine/threonine kinases which may be intimately involved in the colonic malignant transformation process, have not been examined. It was, therefore, of interest to determine whether piroxicam altered the expression of one or more isoforms of this kinase in these tumors. The present studies demonstrate that dietary piroxicam selectively preserved the expression of protein kinase C-ζ in azoxymethane-induced tumors; suggesting that this is at least one mechanism involved in this agent's chemopreventive actions in this organ

Risk of colon cancer in hereditary non-polyposis colorectal cancer patients as predicted by fuzzy modeling: Influence of smoking

AIM: To investigate whether a fuzzy logic model could predict colorectal cancer (CRC) risk engendered by smoking in hereditary non-polyposis colorectal cancer (HNPCC) patients. METHODS: Three hundred and forty HNPCC mismatch repair (MMR) mutation carriers from the Creighton University Hereditary Cancer Institute Registry were selected for modeling. Age-dependent curves were generated to elucidate the joint effects between gene mutation (hMLH1 or hMSH2), gender, and smoking status on the probability of developing CRC. RESULTS: Smoking significantly increased CRC risk in male hMSH2 mutation carriers (P \u3c 0.05). hMLH1 mutations augmented CRC risk relative to hMSH2 mutation carriers for males (P \u3c 0.05). Males had a significantly higher risk of CRC than females for hMLH1 non smokers (P \u3c 0.05), hMLH1 smokers (P \u3c 0.1) and hMSH2 smokers (P \u3c 0.1). Smoking promoted CRC in a dose-dependent manner in hMSH2 in males (P \u3c 0.05). Females with hMSH2 mutations and both sexes with the hMLH1 groups only demonstrated a smoking effect after an extensive smoking history (P \u3c 0.05). CONCLUSION: CRC promotion by smoking in HNPCC patients is dependent on gene mutation, gender and age. These data demonstrate that fuzzy modeling may enable formulation of clinical risk scores, thereby allowing individualization of CRC prevention strategies

Nanoscale changes in chromatin organization represent the initial steps of tumorigenesis: a transmission electron microscopy study

BACKGROUND: Nuclear alterations are a well-known manifestation of cancer. However, little is known about the early, microscopically-undetectable stages of malignant transformation. Based on the phenomenon of field cancerization, the tissue in the field of a tumor can be used to identify and study the initiating events of carcinogenesis. Morphological changes in nuclear organization have been implicated in the field of colorectal cancer (CRC), and we hypothesize that characterization of chromatin alterations in the early stages of CRC will provide insight into cancer progression, as well as serve as a biomarker for early detection, risk stratification and prevention. METHODS: For this study we used transmission electron microscopy (TEM) images of nuclei harboring pre-neoplastic CRC alterations in two models: a carcinogen-treated animal model of early CRC, and microscopically normal-appearing tissue in the field of human CRC. We quantify the chromatin arrangement using approaches with two levels of complexity: 1) binary, where chromatin is separated into areas of dense heterochromatin and loose euchromatin, and 2) grey-scale, where the statistics of continuous mass-density distribution within the nucleus is quantified by its spatial correlation function. RESULTS: We established an increase in heterochromatin content and clump size, as well as a loss of its characteristic peripheral positioning in microscopically normal pre-neoplastic cell nuclei. Additionally, the analysis of chromatin density showed that its spatial distribution is altered from a fractal to a stretched exponential. CONCLUSIONS: We characterize quantitatively and qualitatively the nanoscale structural alterations preceding cancer development, which may allow for the establishment of promising new biomarkers for cancer risk stratification and diagnosis. The findings of this study confirm that ultrastructural changes of chromatin in field carcinogenesis represent early neoplastic events leading to the development of well-documented, microscopically detectable hallmarks of cancer

Novel UV Spectrophotometer Methods for Quantitative Estimation of Concensi (Amlodipine 10mg and Celecoxib 200mg) Using Hydrotropic Solubilizing Agents

Two simple, accurate, novel, safe and precise methods were developed for the simultaneous estimation of poorly water-soluble drugs Amlodipine besylate (AMD) and Celecoxib (CLX) in a in-house formulation using 2M sodium benzoate as a hydrotropic solution. AMD and CLX show maximum absorbances at 243 and 255 nm, respectively. Sodium benzoate did not show any absorbance above 225 nm and thus no interference in the estimation of drugs was seen. AMD and CLX follow Beer’s law in the concentration range of 2-10mg/ml and 10-50mg/ml (r2 = 0.9992 and 0.9995). Method-A employs the simultaneous equation method using 243 and 255 nm as two analytical wavelengths; method-B employs the absorption ratio method, which uses 243 and 250nm as two analytical wavelengths for estimation of AMD and CLX. The mean percent label claims of in-house formulation were found to be 98.74± 0.912and 99.22± 1.012in method-1, 97.89± 0.872and 99.49± 0.903 in method 2 for AMD and CLX, respectively. The developed methods were validated according to ICH guidelines and values of accuracy, precision and other statistical analysis were found to be in good accordance with the prescribed values therefore both methods can be used for routine monitoring of AMD and CLX in industry in the assay of bulk drug and pharmaceutical formulation. Keywords: Amlodipine besylate, Celecoxib, Simultaneous equation method; Absorption ratio method, Hydrotropic solubilizing agent

Rectal Optical Markers for In-vivo Risk Stratification of Premalignant Colorectal Lesions.

Purpose: Colorectal cancer remains the second leading cause of cancer deaths in the U.S. despite being eminently preventable by colonoscopy via removal of premalignant adenomas. In order to more effectively reduce colorectal cancer mortality, improved screening paradigms are needed. Our group pioneered the use of low coherence enhanced backscattering (LEBS) spectroscopy to detect the presence of adenomas throughout the colon via optical interrogation of the rectal mucosa. In a previous ex-vivo biopsy study of 219 patients, LEBS demonstrated excellent diagnostic potential with 89.5% accuracy for advanced adenomas. The objective of the current cross-sectional study is to assess the viability of rectal LEBS in-vivo. Experimental Design: Measurements from 619 patients were taken using a minimally invasive 3.4 mm diameter LEBS probe introduced into the rectum via anoscope or direct insertion, requiring ~1 minute from probe insertion to withdrawal. The diagnostic LEBS marker was formed as a logistic regression of the optical reduced scattering coefficient μs∗ and mass density distribution factor D. Results: The rectal LEBS marker was significantly altered in patients harboring advanced adenomas and multiple non-advanced adenomas throughout the colon. Blinded and cross-validated test performance characteristics showed 88% sensitivity to advanced adenomas, 71% sensitivity to multiple non-advanced adenomas, and 72% specificity in the validation set. Conclusions: We demonstrate the viability of in-vivo LEBS measurement of histologically normal rectal mucosa to predict the presence of clinically relevant adenomas throughout the colon. The current work represents the next step in the development of rectal LEBS as a tool for colorectal cancer risk stratification

Quantification of nanoscale density fluctuations by electron microscopy: probing cellular alterations in early carcinogenesis *

Abstract Most cancers are curable if they are diagnosed and treated at an early stage. Recent studies suggest that nanoarchitectural changes occur within cells during early carcinogenesis and that such changes precede microscopically evident tissue alterations. It follows that the ability to comprehensively interrogate cell nanoarchitecture (e.g., macromolecular complexes, DNA, RNA, proteins and lipid membranes) could be critical to the diagnosis of early carcinogenesis. We present a study of the nanoscale mass-density fluctuations of biological tissues by quantifying their degree of disorder at the nanoscale. Transmission electron microscopy images of human tissues are used to construct corresponding effective disordered optical lattices. The properties of nanoscale disorder are then studied by statistical analysis of the inverse participation ratio (IPR) of the spatially localized eigenfunctions of these optical lattices at the nanoscale. Our results show an increase in the disorder of human colonic epithelial cells in subjects harboring early stages of colon neoplasia. Furthermore, our findings strongly suggest that increased nanoscale disorder correlates with the degree of tumorigenicity. Therefore, the IPR technique provides a practicable tool for the detection of nanoarchitectural alterations in the earliest stages of carcinogenesis. Potential applications of the technique for early cancer screening and detection are also discussed

Depletion of Transmembrane Mucin 4 (Muc4) Alters Intestinal Homeostasis in a Genetically Engineered Mouse Model of Colorectal Cancer

Mucins are components of the mucus layer overlying the intestinal epithelial cells, which maintains physiological homeostasis. Altered mucin expression is associated with disease progression. Expression of MUC4 decreases in colorectal cancer (CRC); however, its functional role and implications in the intestinal pathology in CRC are not studied well. Therefore, we generated a genetically engineered Muc4 knockout (Muc4-/-) CRC mouse model by crossing with Muc4-/- and Apcflox/flox mice in the presence of colon-specific inducible Cre. We observed that deficiency of Muc4 results in an increased number of macroscopic tumors in the colon and rectal region and leads to poor survival. Further, the absence of Muc4 was associated with goblet cell dysfunction where the expression of intestinal homeostasis molecules (Muc2 and Fam3D) was downregulated. Next, we also observed that loss of Muc4 showed reduced thickness of mucus layer, leading to infiltration of bacteria, reduction in anti-microbial peptides, and upregulation of pro-inflammatory cytokines. Further, Apc gene mutation results in activation of the Wnt/β-catenin signaling pathway that corroborated with an increased nuclear accumulation of β-catenin and activation of its target genes: cyclin D1 and c-Myc in Muc4-/- mice was observed. We conclude that the presence of Muc4 is essential for intestinal homeostasis, reduces tumor burden, and improves overall survival

Cooperation Between Systemic and Mucosal Antibodies Induced by Virosomal Vaccines Targeting HIV-1 Env: Protection of Indian Rhesus Macaques Against Low-Dose Intravaginal SHIV Challenges.

A virosomal vaccine inducing systemic/mucosal anti-HIV-1 gp41 IgG/IgA had previously protected Chinese-origin rhesus macaques (RMs) against vaginal SHIVSF162P3 challenges. Here, we assessed its efficacy in Indian-origin RMs by intramuscular priming/intranasal boosting (n=12/group). Group K received virosome-P1-peptide alone (harboring the Membrane Proximal External Region), Group L combined virosome-rgp41 plus virosome-P1, and Group M placebo virosomes. Vaccination induced plasma binding but no neutralizing antibodies. Five weeks after boosting, all RMs were challenged intravaginally with low-dose SHIVSF162P3 until persistent systemic infection developed. After SHIV challenge #7, six controls were persistently infected versus only one Group L animal (vaccine efficacy 87%; P=0.0319); Group K was not protected. After a 50% SHIV dose increase starting with challenge #8, protection in Group L was lost. Plasmas/sera were analyzed for IgG phenotypes and effector functions; the former revealed that protection in Group L was significantly associated with increased binding to FcγR2/3(A/B) across several time-points, as were some IgG measurements. Vaginal washes contained low-level anti-gp41 IgGs and IgAs, representing a 1-to-5-fold excess over the SHIV inoculum's gp41 content, possibly explaining loss of protection after the increase in challenge-virus dose. Virosomal gp41-vaccine efficacy was confirmed during the initial seven SHIV challenges in Indian-origin RMs when the SHIV inoculum had at least 100-fold more HIV RNA than acutely infected men's semen. Vaccine protection by virosome-induced IgG and IgA parallels the cooperation between systemically administered IgG1 and mucosally applied dimeric IgA2 monoclonal antibodies that as single-agents provided no/low protection - but when combined, prevented mucosal SHIV transmission in all passively immunized RMs

Precision Medicine for CRC Patients in the Veteran Population: State-of-the-Art, Challenges and Research Directions.

Colorectal cancer (CRC) accounts for ~9% of all cancers in the Veteran population, a fact which has focused a great deal of the attention of the VA\u27s research and development efforts. A field-based meeting of CRC experts was convened to discuss both challenges and opportunities in precision medicine for CRC. This group, designated as the VA Colorectal Cancer Cell-genomics Consortium (VA4C), discussed advances in CRC biology, biomarkers, and imaging for early detection and prevention. There was also a discussion of precision treatment involving fluorescence-guided surgery, targeted chemotherapies and immunotherapies, and personalized cancer treatment approaches. The overarching goal was to identify modalities that might ultimately lead to personalized cancer diagnosis and treatment. This review summarizes the findings of this VA field-based meeting, in which much of the current knowledge on CRC prescreening and treatment was discussed. It was concluded that there is a need and an opportunity to identify new targets for both the prevention of CRC and the development of effective therapies for advanced disease. Also, developing methods integrating genomic testing with tumoroid-based clinical drug response might lead to more accurate diagnosis and prognostication and more effective personalized treatment of CRC