Inhibition of caspase-9 through phosphorylation at Thr 125 by ERK MAPK

Many pro-apoptotic signals activate caspase-9, an initiator protease that activates caspase-3 and downstream caspases to initiate cellular destruction. However, survival signals can impinge on this pathway and suppress apoptosis. Activation of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) pathway is associated with protection of cells from apoptosis and inhibition of caspase-3 activation, although the targets are unknown. Here, we show that the ERK MAPK pathway inhibits caspase-9 activity by direct phosphorylation. In mammalian cell extracts, cytochrome c-induced activation of caspases-9 and -3 requires okadaic-acid-sensitive protein phosphatase activity. The opposing protein kinase activity is overcome by treatment with the broad-specificity kinase inhibitor staurosporine or with inhibitors of MEK1/2. Caspase-9 is phosphorylated at Thr 125, a conserved MAPK consensus site targeted by ERK2 in vitro, in a MEK-dependent manner in cells stimulated with epidermal growth factor (EGF) or 12-O-tetradecanoylphorbol-13-acetate (TPA). Phosphorylation at Thr 125 is sufficient to block caspase-9 processing and subsequent caspase-3 activation. We suggest that phosphorylation and inhibition of caspase-9 by ERK promotes cell survival during development and tissue homeostasis. This mechanism may also contribute to tumorigenesis when the ERK MAPK pathway is constitutively activated.     

Characterization of Mycolytic Enzymes of Bacillus Strains and Their Bio-Protection Role Against Rhizoctonia solani in Tomato

Four antagonists bacteria namely, Bacillus megaterium MB3, B. subtilis MB14, B. subtilis MB99 and B. amyloliquefaciens MB101 were able to produce chitinase, β-1,3-glucanase and protease in different range with the presence of Rhizoctonia solani cell wall as a carbon source. Amplification of chitinase (chiA) gene of 270 bp and β-1, 3-glucanase gene of 415 bp was given supportive evidence at molecular level of antibiosis. After in vitro screening, all antagonists were tested against R. solani under greenhouse conditions. Root treatment of Bacillus strains showed superior defense during pathogen suppression in terms of chitinase, glucanase, peroxidase, poly phenol oxidase, phenylalanine ammonia-lyase activity and total phenolic content in leaves of tomato. All these enzymes accumulated high in tomato leaves as compared to roots. Pathogenesis-related proteins and defense-related enzymes accumulation was directly correlated with plant protection and greenhouse results indicated that B. amyloliquefaciens MB101- and B. subtilis MB14-treated plants offered 69.76 and 61.51 % disease reductions, respectively, over the infected control. These results established that these organisms have the potential to act as biocontrol agents. This study could be highlighted a mutual importance of liquid formulation of antagonistic Bacillus spp. against root associated sclerotia former pathogen R. solani.     

Monoallelic and Biallelic Variants in EMC1 Identified in Individuals with Global Developmental Delay,Hypotonia, Scoliosis,and Cerebellar Atrophy

The paradigm of a single gene associated with one specific phenotype and mode of inheritance has been repeatedly challenged. Genotype-phenotype correlations can often be traced to different mutation types, localization of the variants in distinct protein domains, or the trigger of or escape from nonsense-mediated decay. Using whole-exome sequencing, we identified homozygous variants in EMC1 that segregated with a phenotype of developmental delay, hypotonia, scoliosis, and cerebellar atrophy in three families. In addition, a de novo heterozygous EMC1 variant was seen in an individual with a similar clinical and MRI imaging phenotype. EMC1 encodes a member of the endoplasmic reticulum (ER)-membrane protein complex (EMC), an evolutionarily conserved complex that has been proposed to have multiple roles in ER-associated degradation, ER-mitochondria tethering, and proper assembly of multi-pass transmembrane proteins. Perturbations of protein folding and organelle crosstalk have been implicated in neurodegenerative processes including cerebellar atrophy. We propose EMC1 as a gene in which either biallelic or monoallelic variants might lead to a syndrome including intellectual disability and preferential degeneration of the cerebellum.     

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.     

Receptor-dependent and receptor-independent endocannabinoid signaling: A therapeutic target for regulation of cancer growth

The endocannabinoid system comprises the G-protein coupled CB1 cannabinoid receptor (CB1R) and CB2 cannabinoid receptor (CB2R), their endogenous ligands (endocannabinoids), and the enzymes responsible for their synthesis and catabolism. Recent works have revealed several important interactions between the endocannabinoid system and cancer. Moreover, it is now well established that synthetic small molecule cannabinoid receptor agonist acting on either CB1R or CB2R or both exerts anti-cancer effects on a variety of tumor cells. Recent results from many laboratories reported that the expression of CB1R and CB2R in prostate cancer, breast cancer, and many other cancer cells is higher than that in corresponding non-malignant tissues. The mechanisms by which cannabinoids acting on CB1R or CB2R exert their effects on cancer cells are quite diverse and complex. Further, several studies demonstrated that some of the anti-proliferative and apoptotic effects of cannabinoids are mediated by receptor-independent mechanisms. In this minireview we provide an overview of the major findings on the effects of endogenous and/or synthetic cannabinoids on breast and prostate cancers. We also provide insight into receptor independent mechanisms of the anti-cancer effects of cannabinoids under in vitro and in vivo conditions.