Manipulation of cellular redox parameters for improving therapeutic responses in B-cell lymphoma and multiple myeloma

Developing novel combined-modality therapeutic approaches based on understanding of the involvement of redox biology in apoptosis of malignant cells is a promising approach for improving clinical responses in B-cell lymphoma and multiple myeloma. Therapeutic modalities that generate reactive oxygen species (i.e., radiation, photodynamic therapy, and specific chemotherapeutic drugs) have been shown to be selectively cytotoxic to malignant B-cells. In this review, we will discuss agents that induce apoptosis in B-cell tumors by oxidative stress. Subsequently, a novel biochemical rationale (based on fundamental differences in cancer vs. normal cell oxidative metabolism) for combining oxidative stressors with radiotherapy and chemotherapy, that may lead to designing of more effective treatment strategies for B-cell malignancies, will be discussed. Besides providing potential curative benefit, such novel therapies could also selectively target and inhibit the emergence of drug-resistance in tumor cells, which is a major determinant of treatment failure in many B-cell malignancies.     

Both LRP5 and LRP6 receptors are required to respond to physiological Wnt ligands in mammary epithelial cells and fibroblasts

A canonical Wnt signal maintains adult mammary ductal stem cell activity, and this signal requires the Wnt signaling reception, LRP5. However, previous data from our laboratory have shown that LRP5 and LRP6 are co-expressed in mammary basal cells and that LRP6 is active, leading us to question why LRP6 is insufficient to mediate canonical signaling in the absence of LRP5. Here, we show that at endogenous levels of LRP5 and LRP6 both receptors are required to signal in response to some Wnt ligands both in vitro (in mouse embryonic fibroblasts and mammary epithelial cells) and in vivo (in mammary outgrowths). This subgroup of canonical ligands includes Wnt1, Wnt9b, and Wnt10b; the latter two are expressed in mammary gland. In contrast, the ligand commonly used experimentally, Wnt3a, prefers LRP6 and requires just one receptor regardless of cellular context. When either LRP5 or LRP6 is overexpressed, signaling remains ligand-dependent, but the requirement for both receptors is abrogated (regardless of ligand type). We have documented an LRP5-6 heteromer using immiscible filtration assisted by surface tension (IFAST) immunoprecipitation. Together, our data imply that under physiological conditions some Wnt ligands require both receptors to be present to generate a canonical signal. We have designed a model to explain our results based on the resistance of LRP5-6 heteromers to a selective inhibitor of E1/2-binding Wnt-LRP6 interaction. These data have implications for stem cell biology and for the analysis of the oncogenicity of LRP receptors that are often overexpressed in breast tumors.     

Molecular cloning and expression profile of snow trout GPDH gene in response to abiotic stress

Glycerol-3-phosphate dehydrogenase (GPDH) gene possibly plays a key role for cold acclimation process in snow trout during winter months when water temperature goes down to 4–5?°C. In this study, 1,012?bp nucleotide fragment of GPDH gene was obtained from two snow trout species (Schizothorax richardsonii and S. niger; family: Cyprinidae), distributed in several Himalayan rivers. The gene encoded a protein of 334 amino acids. The encoded protein sequence was very similar to GPDH of Danio rerio (94.36?%) using BLASTx searches. In S. richardsonii the qRT-PCR showed highest expression in muscle tissue followed by liver and also revealed 19 fold gene expression in liver tissue under cold (5?°C) in comparison with warm (15?°C) condition. The elevated expression levels of GPDH cDNA on cold treatment furthermore suggest that GPDH plays a role in stress related responses in S. richardsonii. The phylogenetic analysis showed that the two snow trout species GPDH share the same clade with characterized GPDHs from other teleost fishes suggesting a common evolutionary origin and a similar catalytic function. In addition, the Ka/Ks ratios of these sequences suggested that they are under purifying selection. Moreover, the expression profile of GPDH gene among co generic species of genus Schizothorax showed that GPDH cDNA expression was highest in S. richardsonii and lowest in S. esocinus which gives an indication of species specific adaptation in relation to different geographical areas.     

Proteomic analysis of survival of Rhodococcus jostii RHA1 during carbon starvation

Rhodococcus jostii RHA1, a catabolically diverse soil actinomycete, is highly resistant to long-term nutrient starvation. After 2 years of carbon starvation, 10% of the bacterial culture remained viable. To study the molecular basis of such resistance, we monitored the abundance of about 1,600 cytosolic proteins during a 2-week period of carbon source (benzoate) starvation. Hierarchical cluster analysis elucidated 17 major protein clusters and showed that most changes occurred during transition to stationary phase. We identified 196 proteins. A decrease in benzoate catabolic enzymes correlated with benzoate depletion, as did induction of catabolism of alternative substrates, both endogenous (lipids, carbohydrates, and proteins) and exogenous. Thus, we detected a transient 5-fold abundance increase for phthalate, phthalate ester, biphenyl, and ethyl benzene catabolic enzymes, which coincided with at least 4-fold increases in phthalate and biphenyl catabolic activities. Stationary-phase cells demonstrated an ～250-fold increase in carbon monoxide dehydrogenase (CODH) concurrent with a 130-fold increase in CODH activity, suggesting a switch to CO or CO(2) utilization. We observed two phases of stress response: an initial response occurred during the transition to stationary phase, and a second response occurred after the cells had attained stationary phase. Although SigG synthesis was induced during starvation, a ΔsigG deletion mutant showed only minor changes in cell survival. Stationary-phase cells underwent reductive cell division. The extreme capacity of RHA1 to survive starvation does not appear to involve novel mechanisms; rather, it seems to be due to the coordinated combination of earlier-described mechanisms.     

Mn‐catalase (Alr0998) protects the photosynthetic,nitrogen‐fixing cyanobacterium Anabaena PCC7120 from oxidative stress

Role of the non‐haem, manganese catalase (Mn‐catalase) in oxidative stress tolerance is unknown in cyanobacteria. The ORF alr0998 from the Anabaena PCC7120, which encodes a putative Mn‐catalase, was constitutively overexpressed in Anabaena PCC7120 to generate a recombinant strain, AnKat⁺. The Alr0998 protein could be immunodetected in AnKat⁺ cells and zymographic analysis showed a distinct thermostable catalase activity in the cytosol of AnKat⁺ cells but not in the wild‐type Anabaena PCC7120. The observed catalase activity was insensitive to inhibition by azide indicating that Alr0998 protein is indeed a Mn‐catalase. In response to oxidative stress, the AnKat⁺ showed reduced levels of intracellular ROS which was also corroborated by decreased production of an oxidative stress‐inducible 2‐Cys‐Prx protein. Treatment of wild‐type Anabaena PCC7120 with H₂O₂ caused (i) RNA degradation in vivo, (ii) severe reduction of photosynthetic pigments and CO₂ fixation, (iii) fragmentation and lysis of filaments and (iv) loss of viability. In contrast, the AnKat⁺ strain was protected from all the aforesaid deleterious effect under oxidative stress. This is the first report on protection of an organism from oxidative stress by overexpression of a Mn‐catalase.     

Induction of TRAIL- and TNF-alpha-dependent apoptosis in human monocyte-derived dendritic cells by microfilariae of Brugia malayi

Dysregulation of professional APC has been postulated as a major mechanism underlying Ag-specific T cell hyporesponsiveness in patients with patent filarial infection. To address the nature of this dysregulation, dendritic cells (DC) and macrophages generated from elutriated monocytes were exposed to live microfilariae (mf), the parasite stage that circulates in blood and is responsible for most immune dysregulation in filarial infections. DC exposed to mf for 24-96 h showed a marked increase in cell death and caspase-positive cells compared with unexposed DC, whereas mf exposure did not induce apoptosis in macrophages. Interestingly, 48-h exposure of DC to mf induced mRNA expression of the proapoptotic gene TRAIL and both mRNA and protein expression of TNF-alpha. mAb to TRAIL-R2, TNF-R1, or TNF-alpha partially reversed mf-induced cell death in DC, as did knocking down the receptor for TRAIL-R2 using small interfering RNA. The mf also induced gene expression of BH3-interacting domain death agonist and protein expression of cytochrome c in DC; mf-induced cleavage of BH3-interacting domain death agonist could be shown to induce release of cytochrome c, leading to activation of caspase 9. Our data suggest that mf induce DC apoptosis in a TRAIL- and TNF-alpha-dependent fashion.     

The ketogenic diet increases mitochondrial glutathione levels

The ketogenic diet (KD) is a high-fat, low carbohydrate diet that is used as a therapy for intractable epilepsy. However, the mechanism(s) by which the KD achieves neuroprotection and/or seizure control are not yet known. We sought to determine whether the KD improves mitochondrial redox status. Adolescent Sprague–Dawley rats (P28) were fed a KD or control diet for 3 weeks and ketosis was confirmed by plasma levels of β-hydroxybutyrate (BHB). KD-fed rats showed a twofold increase in hippocampal mitochondrial GSH and GSH/GSSG ratios compared with control diet-fed rats. To determine whether elevated mitochondrial GSH was associated with increased de novo synthesis, the enzymatic activity of glutamate cysteine ligase (GCL) (the rate-limiting enzyme in GSH biosynthesis) and protein levels of the catalytic (GCLC) and modulatory (GCLM) subunits of GCL were analyzed. Increased GCL activity was observed in KD-fed rats, as well as up-regulated protein levels of GCL subunits. Reduced CoA (CoASH), an indicator of mitochondrial redox status, and lipoic acid, a thiol antioxidant, were also significantly increased in the hippocampus of KD-fed rats compared with controls. As GSH is a major mitochondrial antioxidant that protects mitochondrial DNA (mtDNA) against oxidative damage, we measured mitochondrial H₂O₂ production and H₂O₂-induced mtDNA damage. Isolated hippocampal mitochondria from KD-fed rats showed functional consequences consistent with the improvement of mitochondrial redox status i.e. decreased H₂O₂ production and mtDNA damage. Together, the results demonstrate that the KD up-regulates GSH biosynthesis, enhances mitochondrial antioxidant status, and protects mtDNA from oxidant-induced damage.