Dose-response effect of ferulic acid against nicotine-induced tissue damage and altered lipid levels in experimental rats: a pathohistological evaluation

In the present study, ferulic acid (FA), a naturally occurring nutritional compound, was investigated for its protective effect against nicotine-induced toxicity in a dose dependent manner. The toxicity was induced by subcutaneous injection of nicotine at a dose of 2.5 mg/kg body weight (b.w.) for 22 weeks in female albino Wister rats. Simultaneously, rats were treated with FA at three different doses (10, 20 and 40 mg/kg b.w.) via intragastric intubations for 22 weeks. At the end of the experiment, circulatory marker enzymes (i.e. lactate dehydrogenase and alkaline phosphatase) and tissue (lung, liver and kidney) lipid levels (i.e. cholesterol, free fatty acids, triglycerides and phospholipids) were analysed, which were significantly increased in the nicotine-treated group, whereas FA treatment positively modulated these levels. FA at a dose of 20 mg/kg b.w. was found to be more effective when compared with the other two doses. The lung, liver and kidney excised from the different groups were fixed and stained to perform histological assessments. The protective effect of FA on histological observations confirms that 20 mg/kg b.w. of FA modulates the deleterious effects of nicotine. The results suggest that FA exerts its inhibitory action against nicotine-induced tissue damage through its antioxidant and antihyperlipidemic properties.     

Awareness of Mouth Cancer Among Adult Dental Patients Attending the Kuwait University Dental School Clinic

In Kuwait, the age-standardized incidence rate (per 100,000) for oral cancer is 1.5 and the mortality rate is 0.4. Early detection of oral cancer combined with appropriate treatment greatly improves the chances of cure and the quality of life. However, little is known about patient awareness of this disease and the ability to identify early signs, particularly among high-risk groups. Hence, the aim of this study is to assess dental patients’ awareness and knowledge of mouth cancer and beliefs and perceptions about risk factors. A self-administered questionnaire was used to collect information from a convenience sample of outpatients attending the dental admission clinic. The questionnaire included questions to ascertain information on socio-demographic characteristics, knowledge of risk factors, and signs of oral cancer as well as sources of information regarding the same. Data were analyzed using the Statistical Package for the Social Sciences for Windows 19.0. A total of 160 questionnaires were distributed out of which 136 completed questionnaires were returned and used for the study. The mean knowledge score for oral cancer risk factors was found to be 5.2 ± 2.7 out of ten while that of signs and symptoms was 3.4 ± 2.7 out of eight. When the knowledge of risk factors of oral cancer was taken into consideration along with variables, significant difference was seen only in sex with women having better knowledge (p = 0.03). Knowledge about signs and symptoms of oral cancer revealed a highly significant difference with the level of education (p = 0.03). Family, friends, and colleagues were mentioned as the main source of information regarding oral cancer. Our findings suggest that knowledge regarding oral cancer risk factors, signs, and symptoms was found to be lacking among the dental patients which emphasizes the need for patient education at the dental centers as well as public awareness programs.     

Stromal epigenetic dysregulation is sufficient to initiate mouse prostate cancer via paracrine Wnt signaling

Carcinomas most often result from the stepwise acquisition of genetic alterations within the epithelial compartment. The surrounding stroma can also play an important role in cancer initiation and progression. Given the rare frequencies of genetic events identified in cancer-associated stroma, it is likely that epigenetic changes in the tumor microenvironment could contribute to its tumor-promoting activity. We use Hmga2 (High-mobility group AT-hook 2) an epigenetic regulator, to modify prostate stromal cells, and demonstrate that perturbation of the microenvironment by stromal-specific overexpression of this chromatin remodeling protein alone is sufficient to induce dramatic hyperplasia and multifocal prostatic intraepithelial neoplasia lesions from adjacent naïve epithelial cells. Importantly, we find that this effect is predominantly mediated by increased Wnt/β-catenin signaling. Enhancement of Hmga2-induced paracrine signaling by overexpression of androgen receptor in the stroma drives frank murine prostate adenocarcinoma in the adjacent epithelial tissues. Our findings provide compelling evidence for the critical contribution of epigenetic changes in stromal cells to multifocal tumorigenesis.Prostate cancer is the leading nondermatologic malignancy for males in many developed countries (1). Primary prostate cancer often presents as a multifocal malignant disorder, consisting of multiple disparate tumor clones with distinct histological features and heterogeneous biological behaviors (2). The interfocal heterogeneity is generally believed to be caused by genetic or epigenetic alterations occurring synchronously or metachronously in the epithelia during cancer evolution. However, an aberrant tumor microenvironment may provide “field effects” (3) to facilitate the development of multiclonal neoplastic lesions.Increasing evidence demonstrates that genetic alterations in individual constituents of the mesenchyme can disrupt epithelial homeostasis, triggering tumor formation from neighboring epithelial cells (4, 5). Targeted inactivation of TGF-beta receptor type-2 in mouse fibroblasts led to prostatic intraepithelial neoplasia (PIN) and squamous cell carcinoma of the forestomach (4). Previous work from our laboratory has shown that enhanced mesenchymal expression of FGF10 induces the formation of PIN or prostate adenocarcinoma (5). Van Dyke’s group showed that cancer cells impose selective pressure on the surrounding stroma, which stimulates the clonal expansion of cancer-associated fibroblasts (CAFs) and in turn promotes tumor progression (6). Moreover, cancer cells can influence their microenvironment by recruiting bone marrow-derived inflammatory cells and inducing profound changes in the extracellular matrix (ECM), which fuels tumor survival, growth, invasion, and metastasis (7).Reciprocal communication between cancer cells and their microenvironment is evident during tumor evolution; however, the mechanisms for the phenotypic and molecular changes in CAFs remain uncertain. Despite certain genetic alterations found in stromal cells from various cancers (8), the absence of detectable genetic changes has been described in CAFs from breast and ovarian carcinomas, accompanied by dramatic changes in expression of genes encoding secreted or cell surface proteins (9, 10). Despite their tumor-promoting function, it has been reported that CAFs generally lack cell-intrinsic oncogenic properties (11). These findings raise the possibility that epigenetic changes including DNA methylation, histone modifications, and chromatin remodeling may contribute to the tumor-promoting trait of CAFs. Using methylation-specific digital karyotyping, Hu et al. (12) showed that numerous genomic loci were differentially methylated in the stroma from normal breast tissue and breast carcinomas, suggesting that altered DNA methylation may be one mechanism for phenotypic and molecular changes in CAFs. However, little is known regarding how stromal epigenetic alterations occur during tumor–mesenchymal interactions. More importantly, the functional consequences of these epigenetic changes in the mesenchyme on tumorigenesis remain unclear.Given the biological similarities between embryonic development and cancer progression, it is postulated that several pathways involved in the epithelial–mesenchymal interactions during prostate development could be inappropriately reactivated during tumorigenesis (13). Recent studies of gene expression profiles in embryonic stem cells (ESCs), adult stem cells, and various human cancers reveal that the ESC-like gene signature is activated in diverse epithelial cancers including prostate cancer and is associated with poor differentiation status and an unfavorable outcome (14, 15). However, because most of these microarray data were generated from bulk tumor tissues, it is unclear whether the activated ESC-like program is expressed in cancer stem cells, the entire malignant epithelia, or in the surrounding stroma. It remains unknown how these embryonic regulatory networks reawaken during cancer evolution. In light of the recent findings about reprogramming of adult fibroblasts to pluripotency with defined factors (16, 17) and the intriguing contribution of epigenetic remodeling to reprogramming efficiency (18), we hypothesized that epigenetic modifications in the stromal cells that reverse their chromatin status and growth-promoting potential toward the embryonic state could be one of the mechanisms for activation of the ESC-like program in cancers. In this study, we chose Hmga2 (High-mobility group AT-hook 2), a downstream target of Lin28 (19), to test this hypothesis.Hmga2 is a member of the high mobility group family of nonhistone chromatin remodeling proteins implicated in embryonic development, tumorigenesis, and self-renewal of stem cells (20, 21). By binding to AT-rich DNA and interacting with histone-modifying enzymes and other proteins in enhanceosomes and chromatin, Hmga2 functions as a global epigenetic regulator (22, 23). Using a dissociated prostate regeneration approach (24), we examined the contribution of Hmga2-modified stroma to prostate cancer initiation and progression and found that overexpression of stromal Hmga2 was extraordinarily potent and led to the development of multifocal PIN in a paracrine Wnt-dependent manner. Enhancement of Hmga2-induced paracrine signaling by elevated expression of androgen receptor (AR) in the stroma resulted in frank carcinoma in the adjacent epithelial tissues, suggesting that changes in the microenvironment are sufficient to drive epithelial cancer progression.     

Regulation of intracellular calcium levels and urokinase activity in MDA MB 231 cells by quercetin

BACKGROUND: The common plant bioflavonoid, quercetin, is cytotoxic to various tumor cell lines, particularly breast cancer, by affecting the protein-kinase-C-dependent signal pathways and by cell cycle regulation. However, its role in breast cancer metastasis has not been studied so far. Increased uPA activity is evident in highly metastatic breast cancer, which is calcium dependent. METHODS: MDA MB 231 cells were treated with various concentrations of quercetin (15-45 microg/ml). The cytotoxic effect of quercetin was assessed by MTT assay and DNA fragmentation analysis. Intracellular calcium levels were measured using Fura-2, a specific Ca2+ fluorescence indicator. Calcium uptake and release in cells treated with quercetin were measured using radioactive 45Ca2+. Urokinase enzyme activity was assayed by a casein zymogram. RESULTS: Quercetin elicited dose- and time-dependent cytotoxicity as evidenced by the MTT assay. The maximum effect was observed at 48 h with a quercetin concentration of (45 microg/ml). DNA agarose gel electrophoresis showed dose-dependent DNA fragmentation on quercetin treatment. Quercetin caused significant depletion of cytosolic calcium levels and decreased calcium uptake from the intracellular stores. Casein zymogram showed a marked reduction of urokinase activity as evident by clear lysis bands on a dark background on treatment with quercetin. CONCLUSION: Quercetin was found to exhibit cytotoxicity in the highly invasive breast cancer cell line MDA MB 231 in a dose- and time-dependent manner. Quercetin inhibited calcium dependent urokinase activity and hence may prove to be an effective antimetastatic agent.     

Do native and polymeric alpha1-antitrypsin activate human neutrophils in vitro?

BACKGROUND: alpha(1)-Antitrypsin (AAT)-Z deficiency is a risk factor for the development of COPD. Compared to wild-type M, AAT-Z has an increased tendency to polymerize, rendering it inactive as a serine proteinase inhibitor. It has been demonstrated that wild-type M- and Z-deficiency AAT polymers are chemotactic for human neutrophils. However, our own studies dispute a proinflammatory role for polymerized AAT-M and AAT-Z, suggesting rather that they are predominantly antiinflammatory, exhibiting inhibitory effects on lipopolysaccharide-stimulated human monocyte activation. The discrepancies between these observations prompted us to re-examine the effects of AAT. METHODS AND RESULTS: The effects of native and polymerized AAT-M and AAT-Z with varying levels of endotoxin contamination (0.08 to 2.55 endotoxin units [EU]/mg protein) on human neutrophil chemotaxis and interleukin (IL)-8 release, in vitro, were evaluated. Neither native nor polymerized (M- or Z-deficient) AAT contaminated with low levels of endotoxin (/= 0.88 EU/mg protein), and significant chemotaxis occurred when AAT was spiked with either endotoxin or zymosan. In support, native and polymeric AAT-M with low endotoxin contamination completely inhibited neutrophil IL-8 release triggered by the zymosan, while AATs with high endotoxin contamination strongly induced IL-8 release and did not inhibit zymosan-stimulated IL-8 release. CONCLUSIONS: The proinflammatory effects of native and polymeric AAT may be critically dependent on the presence of other cell activators, bacterial or otherwise, while pure preparations of AAT appear to exert predominantly antiinflammatory activity.     

Evaluating the radioprotective effect of hesperidin in the liver of Swiss albino mice

Amongst calcium channel blockers, amlodipine is known to have unique cardioprotective activities likely attributable to its capacity to increase nitric oxide (NO) release from endothelial cells (EC). Because endothelial NO synthase (eNOS), the main source of NO in EC is known to be inhibited by caveolin-1 (Cav-1), the purpose of this study is to investigate the possibility that amlodipine can modulate eNOS interaction with Cav-1. Using cultured EC, we confirm that amlodipine potentiates vascular endothelial growth factor (VEGF)-induced NO release. eNOS trafficking to specialized plasma membrane microdomains, which is essential to eNOS signaling, is unaffected by amlodipine. However, glutathione s-transferase (GST) pulldown assays reveal that amlodipine can prevent binding of native, acylated eNOS complexes to the active domain of Cav-1 in a concentration-dependent fashion, suggesting that amlodipine has an antagonistic effect on the native eNOS/Cav-1 signaling complex. Moreover, experiments performed in a reconstituted cell line confirm that amlodipine's effect on NO release is highly selective for the eNOS/Cav-1 interaction. To our knowledge, these data are the first to demonstrate a direct effect of amlodipine on the eNOS/Cav-1 protein complex and support the concept of developing novel therapies specifically aimed at modulating the eNOS/Cav-1 interaction to improve endothelial function in cardiovascular diseases.     

Oral traumatic granuloma: report of a case and review of literature

Abstract –  Traumatic granuloma is an uncommon condition considered to be a benign, reactive lesion that usually affects the tongue. The exact pathogenesis implicated in the development of this lesion is not clear. However, trauma has been found to be a contributing factor in a majority of the cases. Clinically, it often presents as an ulceration or an indurated submucosal mass. Microscopically, it is characterized by a diffuse polymorphic cell infiltrate composed predominantly of eosinophils extending deep into the submucosa causing degeneration of the underlying muscle. Recognition of the lesion is important because it often mimics oral squamous cell carcinoma. But traumatic granuloma is self-limiting and tends to resolve spontaneously. This paper describes a case of traumatic granuloma on the dorsal surface of tongue in a 62-year-old woman. The clinical aspects, pathogenesis and histopathology of this uncommon lesion are discussed with an emphasis on its benign, self-limiting nature.     

Influence of ferulic acid on nicotine-induced lipid peroxidation, DNA damage and inflammation in experimental rats as compared to N-acetylcysteine

We examined the effect of ferulic acid (FA), a naturally occurring phenolic compound on lipid peroxidation and endogenous antioxidant status, DNA damage and inflammation in nicotine-administered Wistar rats. The effect of FA against nicotine toxicity was compared with N-acetylcysteine (NAC), a well-known antioxidant. Lung toxicity was induced by subcutaneous injection of nicotine at a dose of 2.5mg/kg body weight (5 days a week, for 22 weeks) and FA and NAC were given simultaneously by intragastric intubation for 22 weeks. Seventy two Wistar rats were divided into six groups: (i) control, (ii) nicotine, (iii) nicotine+FA (iv), nicotine+NAC, (v) FA and (vi) NAC. At the end of the experimental period, cellular damage was assessed by measuring the activities of lactate dehydrogenase and alkaline phosphatase in plasma, which were significantly elevated in nicotine-administered rats when compared with control group. Enhanced lipid peroxidation (evaluated by measuring the thiobarbituric acid reactive substances and hydroperoxides) was accompanied by a significant decrease in the endogenous antioxidant status viz., superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione in circulation, lung and liver of nicotine-treated rats when compared with control group. DNA single strand breaks (evaluated by comet assay) and frequency of micronuclei were significantly increased in peripheral blood of nicotine-treated rats when compared with control. Our Western blot analysis showed a significant increase in the expression of cyclooxygenase-2 and NF-kappaB in lung and liver of nicotine-treated rats. FA and NAC co-treated rats showed a significant decrease in the activities of circulatory lactate dehydrogenase and alkaline phosphatase, the levels of lipid peroxidative markers (in circulation, lung and liver), DNA single stranded breaks (comet parameters), micronuclei frequency (in the whole blood) and expression of cyclooxygenase-2 and Nf-kappaB (in lung and liver tissues), and significant increase in antioxidant status (in circulation, lung and liver). The protection of FA against nicotine-induced toxicity was merely equal to the effect of NAC. FA and NAC treatment alone did not produce any damage to control rats. Thus, we propose that FA exerts protective effect against nicotine toxicity by modulating the lipid peroxidation, inflammation, DNA damage and endogenous antioxidant status.     

Dose-response effect of ellagic acid on circulatory antioxidants and lipids during alcohol-induced toxicity in experimental rats

Ellagic acid (EA) is a naturally occurring polyphenolic compound that exhibits antioxidative, anti-inflammatory, anti-hyperlipidaemic and anticarcinogenic activities in a wide range of assays both in vitro and in vivo. It occurs in various foods such as strawberries, grapes, walnuts, etc. The aim of this study was to assess the effect of ellagic acid on alcohol-induced changes in the circulatory antioxidative status, micronutrients and lipid levels in a dose-dependent fashion. Female albino Wistar rats weighing 150-170 g were used to assess the effects of EA against alcohol-induced damage. Three different concentrations of EA (30, 60 and 90 mg/kg body weight) were tested against 20% alcohol via intragastric administration. At the end of the experimental duration of 45 days, we evaluated endogenous antioxidants: both enzymatic (superoxide dismutase, catalase and glutathione peroxidase) and non-enzymatic (vitamin C and E, and reduced glutathione) status, micronutrients, viz. copper and zinc, and lipids: cholesterol, triglycerides, free fatty acids and phospholipids in the circulation. The body weight gain of both alcohol-fed rats and EA-treated rats were also inferred. EA significantly inhibits alcohol-induced toxicity by improving body weight, restoring antioxidant status, modulating micronutrients and attenuating the lipid levels in the circulation. The greatest inhibitory effect was observed with 60 mg/kg body weight of EA in all the biochemical assessments. The results support the hypothesis that EA at the concentration of 60 mg/kg body weight decreases the intensity of alcohol-induced toxicity and could be developed as a potential drug for alcohol abuse in the near future.