Caspase-3 inhibits the growth of breast cancer cells independent of protease activity

In this study, the MCF-7 breast cancer cells that lack caspase-3 were transfected with a wild type (WT) or mutant caspase-3 cDNA. Expression of the WT, but not of the mutant, caspase-3 was associated with increased caspase activity and susceptibility to staurosporine (STS)-induced apoptosis. Both derivatives displayed inhibition of cell growth compared with vector control cells. Growth inhibition was associated with increased expression of the cyclin dependent kinase (CDK) inhibitor p27Kip1 in the WT, but not in the mutant caspase-3 expressing cells. Cyclin D1 expression level was not affected by caspase-3 expression. Phosphorylation of the Akt protein was decreased in both WT and mutant caspase transfected cells, although Akt expression level remained unchanged. These results suggest that caspase-3 might have biological functions independent of its protease activity and that its loss might contribute to tumor development by increasing the growth potential of cancer cells.     

A protective effect of the synthetic coumarine derivative Cloricromene against DNB-colitis in the rat

Biologic therapies, namely antibodies against tumor necrosis factor-alpha (TNF- alpha) or its receptors, have been recently introduced for the treatment of patients with inflammatory bowel disease (IBD). In the present study the effects of cloricromene, an agent with known antithrombotic actions and with demonstrated anti-TNF- alpha activity were investigated in a rat model of experimental colitis induced with dinitrobenzenesulphonic acid (DNB)/ethanol. We investigated three experimental groups: (i) sham-colitis with vehicle-treatment (controls, n = 6), (ii) colitis with vehicle-treatment (saline, 0.1 ml s.c., daily) (DNB-V, n = 7), (iii) colitis with cloricromene-treatment (10 mg/kg/day s.c.; DNB-C, n = 8). After 7 days, the weight gain, colon wet weight, macroscopic damage score, coagulation parameters, colon mucosal myeloperoxidase activity (MPO), and tissue concentrations of TNF- alpha and of macrophage inhibitory peptide-2 (MIP-2) were assessed. The macroscopic damage scores, colon wet weights, and tissue MIP-2 levels were significantly increased in untreated and in cloricromene-treated rats compared with controls. Cloricromene treatment was associated with a minor body weight loss (p < 0.025) and significantly reduced tissue concentrations of MPO and TNF-alpha (p < 0.02, both). Blood coagulation parameters were not affected by treatment. In the DNB-model treatment with cloricromene effectively reduces tissue levels of TNF- alpha and of myeloperoxidase, whereas MIP-2 concentrations were not influenced. Blood coagulation parameters remained unchanged indicating safety of treatment. Since biological therapies frequently fail to improve disease course of IBD, other therapies with similar targets should be further investigated.     

Crucial role of granulocytic myeloid-derived suppressor cells in the regulation of central nervous system autoimmune disease

There is a need in autoimmune diseases to uncover the mechanisms involved in the natural resolution of inflammation. In this article, we demonstrate that granulocytic myeloid-derived suppressor cells (G-MDSCs) abundantly accumulate within the peripheral lymphoid compartments and target organs of mice with experimental autoimmune encephalomyelitis prior to disease remission. In vivo transfer of G-MDSCs ameliorated experimental autoimmune encephalomyelitis, significantly decreased demyelination, and delayed disease onset through inhibition of encephalitogenic Th1 and Th17 immune responses. Exposure of G-MDSCs to the autoimmune milieu led to up-regulation of the programmed death 1 ligand that was required for the G-MDSC-mediated suppressive function both in vitro and in vivo. Importantly, myeloid-derived suppressor cells were enriched in the periphery of subjects with active multiple sclerosis and suppressed the activation and proliferation of autologous CD4(+) T cells ex vivo. Collectively, this study revealed a pivotal role for myeloid-derived suppressor cells in the regulation of multiple sclerosis, which could be exploited for therapeutic purposes.     

DHA induces apoptosis by altering the expression and cellular location of GRP78 in colon cancer cell lines

n-3 polyunsaturated fatty acids exert growth-inhibitory and pro-apoptotic effects in colon cancer cells. We hypothesized that the anti-apoptotic glucose related protein of 78kDa (GRP78), originally described as a component of the unfolded protein response in endoplasmic reticulum (ER), could be a molecular target for docosahexaenoic acid (DHA) in these cells. GRP78 total and surface overexpression was previously associated with a poor prognosis in several cancers, whereas its down-regulation with decreased cancer growth in animal models. DHA treatment induced apoptosis in three colon cancer cell lines (HT-29, HCT116 and SW480), and inhibited their total and surface GRP78 expression. The cell ability to undergo DHA-induced apoptosis was inversely related to their level of GRP78 expression. The transfection of the low GRP78-expressing SW480 cells with GRP78-GFP cDNA significantly induced cell growth and inhibited the DHA-driven apoptosis, thus supporting the essential role of GRP78 in DHA pro-apoptotic effect. We suggest that pERK1/2 could be the first upstream target for DHA, and demonstrate that, downstream of GRP78, DHA may exert its proapoptotic role by augmenting the expression of the ER resident factors ERdj5 and inhibiting the phosphorylation of PKR-like ER kinase (PERK), known to be both physically associated with GRP78, and by activating caspase-4. Overall, the regulation of cellular GRP78 expression and location is suggested as a possible route through which DHA can exert pro-apoptotic and antitumoral effects in colon cancer cells.     

Relative influence of the adeno-associated virus (AAV) type 2 p5 element for recombinant AAV vector site-specific integration

The p5 promoter region of the adeno-associated virus type 2 (AAV-2) rep gene has been described as essential for Rep-mediated site-specific integration (RMSSI) of plasmid sequences in human chromosome 19. We report here that insertion of a full-length or minimal p5 element between the viral inverted terminal repeats does not significantly increase RMSSI of a recombinant AAV (rAAV) vector after infection of growth-arrested or proliferating human cells. This result suggests that the p5 element may not improve RMSSI of rAAV vectors in vivo.     

Rho/ROCK/actin signaling regulates membrane androgen receptor induced apoptosis in prostate cancer cells

In this study we describe a novel Rho small GTPase dependent pathway that elicits apoptotic responses controlled by actin reorganization in hormone-sensitive LNCaP- and hormone insensitive DU145-prostate cancer cells stimulated with membrane androgen receptor selective agonists. Using an albumin-conjugated steroid, testosterone-BSA, we now show significant induction of actin polymerization and apoptosis that can be reversed by actin disrupting agents in both cell lines. Testosterone-BSA triggered RhoA/B and Cdc42 activation in DU145 cells followed by stimulation of downstream effectors ROCK, LIMK2 and ADF/destrin. Furthermore, dominant-negative RhoA, RhoB or Cdc42 mutants or pharmacological inhibitors of ROCK inhibited both actin organization and apoptosis in DU145 cells. Activation of RhoA/B and ROCK was also implicated in membrane androgen receptor-dependent actin polymerization and apoptosis in LNCaP cells. Our findings suggest that Rho small GTPases are major membrane androgen receptor effectors controlling actin reorganization and apoptosis in prostate cancer cells.     

Cardiovascular effects produced by choline injected into the lateral cerebral ventricle of the unanesthetized rat

Intracerebroventricular (icv) injection of choline (50–150 μg) causes a transient increase in blood pressure and a more prolonged decrease in heart rate (HR) in conscious rats. The bradycardia results from a centrally mediated increase in vagal tone. The cardiovascular effects do not appear to involve endogenous brain acetylcholine since there is no significant difference in the responses induced by choline before and after icv injection of hemicholinium-3. Intracerebroventricular ventricular injection of atropine or mecamylamine, alone, failed to influence the choline effect. However, atropine and mecamylamine, given together, abolished the reduction of HR, but still failed to modify the pressor response. The changes in blood pressure and HR appear to be due to effects of choline on post-synaptic receptors in different brain regions.