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.     

Occludin phosphorylation: identification of an occludin kinase in brain and cell extracts as CK2

In epithelial and endothelial cells, tight junctions limit paracellular flux of ions, proteins and other macromolecules. However, mechanisms regulating tight junction function are not clear. Occludin, a tight junction protein, undergoes phosphorylation changes in several situations but little is known about occludin kinases. A recombinant C-terminal fragment of occludin is a substrate for a kinase in crude extracts of brain. This activity was purified about 10000-fold and identified as CK2 (casein kinase 2) by peptide mass fingerprinting, immunoblotting and mutation of CK2 sites within the occludin sequence. CK2 is therefore a candidate kinase for regulation of occludin phosphorylation in vivo.     

Smurf1 facilitates myogenic differentiation and antagonizes the bone morphogenetic protein-2-induced osteoblast conversion by targeting Smad5 for degradation

Controlled proteolysis mediated by Smad ubiquitination regulatory factors (Smurfs) plays a crucial role in modulating cellular responses to signaling of the transforming growth factor-beta (TGF-beta) superfamily. However, it is not clear what influences the selectivity of Smurfs in the individual signaling pathway, nor is it clear the biological function of Smurfs in vivo. Using a mouse C2C12 myoblast cell differentiation system, which is subject to control by both TGF-beta and bone morphogenetic protein (BMP), here we examine the role of Smurf1 in myogenic differentiation. We show that increased expression of Smurf1 promotes myogenic differentiation of C2C12 cells and blocks the BMP-induced osteogenic conversion but has no effect on the TGF-beta-induced differentiation arrest. Consistent with an inhibitory role in the BMP signaling pathway, the elevated Smurf1 markedly reduces the level of endogenous Smad5, whereas it leaves unaltered that of Smad2, Smad3, and Smad7, which are components of the TGF-beta pathway. Adding back Smad5 from a different source to the Smurf1-overexpressing cells restores the BMP-mediated osteoblast conversion. Finally, by depletion of the endogenous Smurf1 through small interfering RNA-mediated RNA interference, we demonstrate that Smurf1 is required for the myogenic differentiation of C2C12 cells and plays an important regulatory role in the BMP-2-mediated osteoblast conversion.     

Peroxisome proliferator-activated receptor-gamma agonist 15-deoxy-Delta(12,14)-prostaglandin J(2) ameliorates experimental autoimmune encephalomyelitis

Peroxisome proliferator-activated receptors (PPAR) are members of a nuclear hormone receptor superfamily that includes receptors for steroids, retinoids, and thyroid hormone, all of which are known to affect the immune response. Previous studies dealing with PPAR-gamma expression in the immune system have been limited. Recently, PPAR-gamma was identified in monocyte/macrophage cells. In this study we examined the role of PPAR-gamma in experimental autoimmune encephalomyelitis (EAE), an animal model for the human disease multiple sclerosis. The hypothesis we are testing is whether PPAR-gamma plays an important role in EAE pathogenesis and whether PPAR-gamma ligands can inhibit the clinical expression of EAE. Initial studies have shown that the presence of the PPAR-gamma ligand 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ2) inhibits the proliferation of Ag-specific T cells from the spleen of myelin basic protein Ac(1-11) TCR-transgenic mice. 15d-PGJ2 suppressed IFN-gamma, IL-10, and IL-4 production by both Con A- and myelin basic protein Ac(1-11) peptide-stimulated lymphocytes as determined by ELISA and ELISPOT assay. Culture of encephalitogenic T cells with 15d-PGJ2 in the presence of Ag reduced the ability of these cells to adoptively transfer EAE. Examination of the target organ, the CNS, during the course of EAE revealed expression of PPAR-gamma in the spinal cord inflammatory infiltrate. Administration of 15d-PGJ2 before and at the onset of clinical signs of EAE significantly reduced the severity of disease. These results suggest that PPAR-gamma ligands may be a novel therapeutic agent for diseases such as multiple sclerosis.     

Effect of NO synthase inhibition on cardiovascular and pulmonary dysfunction in a porcine short-term model of endotoxic shock

In a porcine model of endotoxic shock, we evaluated the circulatory and respiratory effects of NO synthase (NOS) blockade. Twenty anaesthetised pigs were divided into three groups and studied for 240 min after induction of endotoxic shock with lipopolysaccharides of Escherichia coli (LPS). After 180 min of endotoxic shock, one group (n = 6) received aminoguanidine, another group (n = 6) received N(G)-nitro-L -arginine methyl ester (L -NAME) and a third group (n = 8) received only LPS. A sham group (n = 3) was also studied. LPS decreased systemic arterial pressure and cardiac output (CO) and increased mean pulmonary arterial pressure (MPAP), pulmonary vascular resistance (PVR) and heart rate. Significant changes were also observed in compliance (-18.4%) and resistance (+33.6%) of the respiratory system. Aminoguanidine did not modify LPS-dependent effects, while, after L -NAME, a significant increase in MPAP, PVR and SVR and a decrease in CO were observed. In conclusion, aminoguanidine does not play a significant cardiocirculatory and pulmonary role in the short-term dysfunction of endotoxic shock, while L -NAME has a detrimental effect on haemodynamics, suggesting a protective role of constitutive NO production at vascular level during the early stages of endotoxaemia.     

DELLA Proteins and GA Signalling in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Gibberellin (GA) is a classical plant hormone involved in many aspects of plant growth and development. A family of five homologs called the DELLA proteins, comprised of GAI, RGA, RGL1, RGL2 and RGL3, were recently found to act as critical GA signal mediators in Arabidopsis. Reports have shown that GAI and RGA are coupled together to repress stem elongation growth whereas RGL2 is a major negative regulator of seed germination. GA down-regulates DELLA proteins through protein degradation likely via the proteasome pathway. The conserved and functionally important DELLA domain is responsible for protein stability in response to GA.     

Immunohistochemical expression of pi class glutathione S-transferase and alpha-fetoprotein in hepatocellular carcinoma and chronic liver disease

OBJECTIVE: To determine whether tumor marker pi glutathione transferase (GST-pi) is expressed in hepatocellular carcinoma (HCC) and other chronic liver diseases and to compare its expression with that of alpha-fetoprotein (AFP). STUDY DESIGN: Samples used were formalin-fixed, paraffin-embedded liver tissues: normal (n = 3), chronic hepatitis B (n = 15), cirrhosis (n = 15) and HCC (n = 30). The expression of AFP and GST-pi was detected by using immunohistochemistry with the peroxidase-antiperoxidase method. AFP immunoreactivity was based on the cytoplasm of the hepatocytes, while GST-pi immunoreactivity was based on the nuclei of hepatocytes. RESULTS: In normal liver tissues, AFP was not expressed. However, there was strong staining of GST-pi in bile duct epithelium cells and weak staining in hepatocytes. Our results showed higher AFP immunoreactivity in cases of HCC (36.7%) as compared to cirrhosis (6.7%) and hepatitis B (0%), whereas GST-pi immunoreactivity was lower in cases of HCC (53.3%) as compared to cases of cirrhosis (100.0%) and hepatitis B (93.3%). Percent sensitivity of AFP determination for HCC was 36.7% as compared to 53.3% for GST-pi, thus making GST-pi a more sensitive marker for detection of HCC. This study showed a significant relationship between the intensity and percentage of cells stained in hepatitis B, cirrhosis and HCC for GST-pi immunoreactivity (P < .001, .001 and .05, respectively) but not for AFP (P > .05). Statistical analysis showed that there was no significant relationship between expression of AFP and GST-pi in cirrhosis and HCC cases. Hepatitis B virus infection in HCC cases showed a positive rate of 46.7%, with AFP staining positively in 42.9% of tissues and GST-pi staining positively in 57.1% of tissues. CONCLUSION: AFP is a diagnostic but rather insensitive tissue marker for HCC. However, the absence of AFP in benign chronic liver disease makes this marker useful in differentiating between HCC and other chronic liver diseases, whereas GST-pi can be used as a diagnostic marker for HCC as well as in detecting other chronic liver diseases.