Molecular pathway for (-)-epigallocatechin-3-gallate-induced cell cycle arrest and apoptosis of human prostate carcinoma cells

Epigallocatechin-3-gallate (EGCG), the major polyphenolic constituent present in green tea, is a promising chemopreventive agent. We recently showed that green tea polyphenols exert remarkable preventive effects against prostate cancer in a mouse model and many of these effects are mediated by the ability of polyphenols to induce apoptosis in cancer cells [Proc. Natl. Acad. Sci. USA 98 (2001) 10350]. Earlier, we showed that EGCG causes a G0/G1 phase cell cycle arrest and apoptosis of both androgen-sensitive LNCaP and androgen-insensitive DU145 human prostate carcinoma cells, irrespective of p53 status [Toxicol. Appl. Pharmacol. 164 (2000) 82]. Here, we provide molecular understanding of this effect. We tested a hypothesis that EGCG-mediated cell cycle dysregulation and apoptosis is mediated via modulation of cyclin kinase inhibitor (cki)-cyclin-cyclin-dependent kinase (cdk) machinery. As shown by immunoblot analysis, EGCG treatment of LNCaP and DU145 cells resulted in significant dose- and time-dependent (i) upregulation of the protein expression of WAF1/p21, KIP1/p27, INK4a/p16, and INK4c/p18, (ii) down-modulation of the protein expression of cyclin D1, cyclin E, cdk2, cdk4, and cdk6, but not of cyclin D2, (iii) increase in the binding of cyclin D1 toward WAF1/p21 and KIP1/p27, and (iv) decrease in the binding of cyclin E toward cdk2. Taken together, our results suggest that EGCG causes an induction of G1 phase ckis, which inhibits the cyclin-cdk complexes operative in the G0/G1 phase of the cell cycle, thereby causing an arrest, which may be an irreversible process ultimately leading to apoptotic cell death. This is the first systematic study showing the involvement of each component of cdk inhibitor-cyclin-cdk machinery during cell cycle arrest and apoptosis of human prostate carcinoma cells by EGCG.     

Ultrastructural and hormonal changes in rat cauda epididymal spermatozoa induced by Boswellia papyrifera and Boswellia carterii

Boswellia papyrifera and Boswellia carterii diffuses smoke polluting air that adversely affects indoor environment that certainly harm human health. Therefore, this study aims at ascertaining the effect of these plants on gonadal hormones and molecular changes in rat spermatozoa. The animals were exposed to 4 g/kg body weight of B. papyrifera and B. carterii daily for 120 days along with suitable controls. Significant decreases in FSH, LH and testosterone levels were evidenced, along with a reduction of protein, sialic acid, and carnitine levels. In sperm physiology, sperm count, motility, speed decrease, whereas sperm anomalies increase. TEM observation indicates morphological changes in plasma and acrosomal membranes, cytoplasmic droplet in the tail region, vacuolated, and disorganization of the mitochondrial sheath. These findings demonstrate that B. papyrifera and B. carterii smoke affects the process of sperm formation and maturation, which indicates the detrimental effects of these plants on the reproductive system.     

Expression of the gene for large subunit of m-calpain is elevated in skeletal muscle from Duchenne muscular dystrophy patients

Calpain is an intracellular nonlysosomal protease involved in essential regulatory or processing functions of the cell, mediated by physiological concentrations of Ca²⁺. However, in an environment of abnormal intracellular calcium, such as that seen in Duchenne muscular dystrophy (DMD), calpain is suggested to cause degeneration of muscle owing to enhanced activity. To test whether the reported increase in calpain activity in DMD results fromde novo synthesis of the protease, we have assessed the quantitative changes in mRNA specific for m-calpain. mRNA isolated from DMD and control muscle was analysed by dot blot hybridization using a cDNA probe for the large subunit of m-calpain. Compared to control a four-fold increase in specific mRNA was observed in dystrophic muscle. This enhanced expression of the m-calpain gene in dystrophic condition suggests that the reported increase in m-calpain activity results fromde novo synthesis of protease and underlines the important role of m-calpain in DMD.     

Adiponectin gene variants and the risk of coronary artery disease in patients with type 2 diabetes

Patients with type 2 diabetes (T2D) are more susceptible to develop cardiovascular complications than non-diabetic subjects. Several studies have indicated a role of adiponectin gene in the increased coronary artery disease (CAD) risk in T2D patients. The data however is limited and have been inconsistent. In this study we examined the association of SNP45T>G and SNP276G>T of adiponectin gene with CAD risk in T2D patients in a Saudi population. A total of 418 type 2 diabetic patients were randomly recruited in this study from the RIYADH COHORT. Of the total diabetes patients, 123 were also diagnosed to have CAD, while the rest were control subjects. Anthropometric, clinical and biochemical parameters were measured by standard procedures. Genotyping of polymorphisms was carried out by PCR–RFLP analysis. Genotype distribution of SNP45T>G was significantly (P = 0.005) different between control and CAD subjects, while the distribution of SNP276G>T genotypes was comparable between the subjects. The SNP45T>G was significantly associated with risk of CAD [OR (95% CI), 4.7 (1.6–13.5), P < 0.003] but not SNP276G>T [OR (95% CI), 1.02 (0.53–1.9), P > 0.05]. The association of SNP45T>G with CAD risk remained significant even after adjusting for potential confounding factors [OR (95% CI), 7.2 (1.1–45.9), P < 0.05]. The SNP45T>G of adiponectin gene is an independent risk factor for CAD in T2D patients in a Saudi population. These findings support a role for adiponectin gene in the increased CAD risk in diabetes patients and are consistent with genetic heterogeneity in the association between adiponectin gene and coronary artery disease.     

Induction of CYP1A1, CYP1A2, CYP1B1, increased oxidative stress and inflammation in the lung and liver tissues of rats exposed to incense smoke

Incense smoke is increasingly being recognized as a potential environmental contaminant and is linked to malignant and non-malignant respiratory diseases. The detoxification of environmental contaminants including polycyclic aromatic hydrocarbons (PAHs) involves the induction of cytochrome P-450 family enzymes (CYPs) by PAHs. However, the detoxification of PAHs also results in the generation of reactive and unstable intermediary metabolites which are implicated in the oxidative stress, DNA damage, and inflammation. It is unclear whether CYPs are similarly induced by incense smoke, which incidentally contains substantial amounts of PAHs. Here, we examined the impact of long-term incense smoke exposure on the induction of CYPs in male Wister Albino rats. Incense smoke exposure significantly induced the expression of CYP1A1, CYP1A2, and CYP1B1 mRNAs in both lung and liver tissues. The extent of CYP1A1 and CYP1B1 induction was significantly higher in the liver compared to that in the lung, while that of CYP1A2 was greater in the lung than in liver. Incense smoke exposure also increased malondialdehyde and reduced glutathione levels in lung and liver tissues, and the catalase activity in the liver tissues to significant levels. Furthermore incense smoke exposure led to a marked increase in TNF-α and IL-4 levels. The data demonstrate for the first time the capacity of incense smoke to induce CYP1 family enzymes in the target and non-target tissues. Induction of CYPs increased oxidative stress and inflammation appear to be intimately linked to promote the carcinogenesis and health complications in people chronically exposed to incense smoke.     

Innate immune responses in murine pleural mesothelial cells: Toll-like receptor-2 dependent induction of beta-defensin-2 by staphylococcal peptidoglycan

The innate immune response is mediated in part by pattern recognition receptors including Toll-like receptors (TLRs). The pleural mesothelial cells (PMCs) that line the pleural surface are in direct contact with pleural fluid and accordingly carry the risk of exposure to infiltrating microorganisms or their components in an event of a complicated parapneumonic effusion. Here we show that murine primary PMCs constitutively express TLR-1 through TLR-9 and, upon activation with peptidoglycan (PGN), mouse PMC produce antimicrobial peptide beta-defensin-2 (mBD-2). Treatment of PMCs with staphylococcal PGN, a gram-positive bacterial cell wall component and a TLR-2 agonist, resulted in a significant increase in TLR-2 and mBD-2 expression. Silencing of TLR-2 expression by small interfering RNA led to the downregulation of PGN-induced mBD-2 expression, thereby establishing causal relationship between the activation of TLR-2 receptor and mBD-2 production. PMCs exposed to PGN showed increased p38 MAPK activity. In addition, PGN-induced mBD-2 expression was attenuated by SB203580, a p38 MAPK inhibitor, underlining the importance of p38 MAPK in mBD-2 induction. Inhibition of erk1/erk2 or phosphatidylinositol 3-kinase did not block PGN-induced mBD-2 expression in PMC. PGN-activated PMC-derived mBD-2 significantly killed Staphylococcus aureus, and mBD-2-neutralizing antibodies blunted this antimicrobial activity. Taken together, these data indicate that PMCs may contribute to host innate immune defense upon exposure to gram-positive bacteria or their products within the pleural space by upregulating TLR-2 and mBD-2 expression.     

Machine learning algorithm-based risk prediction model of coronary artery disease

Molecular Biology Reports - In view of high mortality associated with coronary artery disease (CAD), development of an early predicting tool will be beneficial in reducing the burden of the...     

Molecular insights into the association of obesity with breast cancer risk: relevance to xenobiotic metabolism and CpG island methylation of tumor suppressor genes

Obesity, genetic polymorphisms of xenobiotic metabolic pathway, hypermethylation of tumor suppressor genes, and hypomethylation of proapoptotic genes are known to be independent risk factors for breast cancer. The objective of this study is to evaluate the combined effect of these environmental, genetic, and epigenetic risk factors on the susceptibility to breast cancer. PCR–RFLP and multiplex PCR were used for the genetic analysis of six variants of xenobiotic metabolic pathway. Methylation-specific PCR was used for the epigenetic analysis of four genetic loci. Multifactor dimensionality reduction analysis revealed a significant interaction between the body mass index (BMI) and catechol-O-methyl transferase H108L variant alone or in combination with cytochrome P450 (CYP) 1A1m1 variant. Women with “Luminal A” breast cancer phenotype had higher BMI compared to other phenotypes and healthy controls. There was no association between the BMI and tumor grade. The post-menopausal obese women exhibited lower glutathione levels. BMI showed a positive association with the methylation of extracellular superoxide dismutase (r = 0.21, p < 0.05), Ras-association (RalGDS/AF-6) domain family member 1 (RASSF1A) (r = 0.31, p < 0.001), and breast cancer type 1 susceptibility protein (r = 0.19, p < 0.05); and inverse association with methylation of BNIP3 (r = ?0.48, p < 0.0001). To conclude based on these results, obesity increases the breast cancer susceptibility by two possible mechanisms: (i) by interacting with xenobiotic genetic polymorphisms in inducing increased oxidative DNA damage and (ii) by altering the methylome of several tumor suppressor genes.     

The Q192R polymorphism of the paraoxonase 1 gene is a risk factor for coronary artery disease in Saudi subjects

Paraoxonase-1 (PON1) is a HDL-bound antioxidant enzyme that protects LDL from oxidative modification. Discovery of the antioxidant properties of PON1 led to extensive research on its role in the initiation and progression of atherosclerosis. The Q192R (rs662; A/G) polymorphism, which results in the glutamine to arginine substitution at position 192, of the PON1 gene has been linked to increased atherosclerosis risk in several but not all population studies. Besides genetic factors, environmental variables and ethnicity have been implicated as factors responsible for the ambiguity in relating the PON1 gene with atherosclerotic risk. Here, we tested the association of the Q192R polymorphism with coronary artery disease (CAD) in Saudi ethnic subjects taking environmental factors into consideration. The genomic DNA samples from 121 angiographically confirmed CAD cases and 108 normal healthy control subjects were genotyped by PCR–RFLP analysis. The distribution of QQ, QR, and RR genotypes was significantly different between cases and controls (p < 0.005). The RR genotype was associated with CAD risk independently of several established risk factors including age, gender, smoking, obesity, and diabetes (OR 2.2, 1.4–7.4, p < 0.01). Genotype-based stratification of demographic and biochemical data revealed that the RR genotype has proatherogenic properties. This study, thus, identifies the Q192R polymorphism as an additional risk factor for CAD in the Saudi population and suggests that it may have prognostic value. The negative effect of this genetic variant is presumably due to the diminished ability of the RR variant genotype of PON1 to blunt LDL oxidation.     

Long-term exposure to incense smoke alters metabolism in Wistar albino rats

The burning of incense is an important source of indoor air pollution in Asia. We assessed the effect of long‐term exposure to incense smoke on the body weight and levels of circulating glucose, triglycerides, total cholesterol, HDL‐cholesterol, insulin, adiponectin and leptin in Wistar albino rats. Two groups of rats were used. First group (n = 12) was exposed daily to incense smoke for 4 months at the rate of 4 g day^?1 in the exposure chamber. Another group of rats (n = 12), was used as non‐exposed control. Blood samples were collected from all animals after 4, 8, 12 and 16 weeks of exposure. Serum glucose, triglycerides, total cholesterol and HDL‐cholesterol, LDL‐cholesterol insulin, adiponectin and leptin were measured. Our results showed that incense smoke exposure was associated with decreased weight gain and the adverse metabolic changes of increased triglycerides and decreased HDL‐cholesterol concentrations. Exposure to incense was also associated with a transient increase of leptin levels. Taken together, these data suggest that incense smoke influences metabolism adversely in rats. The effect of incense smoke on human health and the underlying mechanisms need to be studied further. Copyright © 2011 John Wiley & Sons, Ltd.