Vasu Punj

Bacterial redox protein azurin, tumor suppressor protein p53, and regression of cancer

The use of live bacteria in the treatment of cancer has a long and interesting history. We report the use of a purified bacterial redox protein, azurin, that enters human cancer (melanoma UISO-Mel-2) cells and induces apoptosis. The induction of apoptosis occurs readily in melanoma cells harboring a functional tumor suppressor protein p53, but much less efficiently in p53-null mutant melanoma (UISO-Mel-6) cells. A redox-negative mutant form of azurin (M44K/M64E) demonstrates much less cytotoxicity to the UISO-Mel-2 cells than the wild-type protein. Azurin has been shown to be internalized in UISO-Mel-2 cells and is localized predominantly in the cytosol and in the nuclear fraction. In the p53-null UISO-Mel-6 cells, azurin is localized only in the cytosol. Thus, intracellular trafficking of azurin to the nucleus is p53-dependent. Azurin forms a complex with p53, thereby stabilizing it and raising its intracellular level in cytosolic, mitochondrial, and nuclear fractions. Corresponding to an increasing level of p53, an inducer of apoptosis, the level of Bax also increases in mitochondria, allowing significant release of mitochondrial cytochrome c into the cytosol, thus initiating the onset of apoptosis. The M44K/M64E mutant form of azurin, deficient in cytotoxicity, is also deficient in forming a complex with p53 and is less efficient in stabilizing p53 than wild-type azurin. Azurin has been shown to allow regression of human UISO-Mel-2 tumors xenotransplanted in nude mice and may potentially be used in cancer treatment.

Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast cancer

Azurin, a copper-containing redox protein released by the pathogenic bacterium Pseudomonas aeruginosa, is highly cytotoxic to the human breast cancer cell line MCF-7, but is less cytotoxic toward p53-negative (MDA-MB-157) or nonfunctional p53 cell lines like MDD2 and MDA-MB-231. The purpose of this study was to investigate the underlying mechanism of the action of bacterial cupredoxin azurin in the regression of breast cancer and its potential chemotherapeutic efficacy. Azurin enters into the cytosol of MCF-7 cells and travels to the nucleus, enhancing the intracellular levels of p53 and Bax, thereby triggering the release of mitochondrial cytochrome c into the cytosol. This process activates the caspase cascade (including caspase-9 and caspase-7), thereby initiating the apoptotic process. Our results indicate that azurin-induced cell death stimuli are amplified in the presence of p53. In vivo injection of azurin in immunodeficient mice harboring xenografted human breast cancer cells in the mammary fat pad leads to statistically significant regression (85%, P=0.0179, Kruskal–Wallis Test) of the tumor. In conclusion, azurin blocks breast cancer cell proliferation and induces apoptosis through the mitochondrial pathway both in vitro and in vivo, thereby suggesting a potential chemotherapeutic application of this bacterial cupredoxin for the treatment of breast cancer.

The Bacterial Redox Protein Azurin Induces Apoptosis in J774 Macrophages through Complex Formation and Stabilization of the Tumor Suppressor Protein p53

ABSTRACT Two redox proteins, azurin and cytochrome c551 elaborated by Pseudomonas aeruginosa, demonstrate significant cytotoxic activity towards macrophages. Azurin can enter macrophages, localize in the cytosol and nuclear fractions, and induce apoptosis. Two redox-negative mutants of azurin have less cytotoxicity than does wild-type (wt) azurin. Azurin has been shown to form a complex with the tumor suppressor protein p53, a known inducer of apoptosis, thereby stabilizing it and enhancing its intracellular level. A higher level of reactive oxygen species (ROS), generated during treatment of macrophages with wt azurin, correlates with its cytotoxicity. Treatment with some ROS-removing antioxidants greatly reduces azurin-mediated cytotoxicity, thus demonstrating a novel virulence property of this bacterial redox protein.

Internalization of bacterial redox protein azurin in mammalian cells: entry domain and specificity.

Azurin is a member of a group of copper‐containing redox proteins called cupredoxins. Different cupredoxins are produced by different aerobic bacteria as agents of electron transfer. Recently, we demonstrated that azurin enters into J774 and several types of cancer cells leading to the induction of apoptosis. We now demonstrate that azurin is internalized in J774 or cancer cells in a temperature‐dependent manner. Azurin shows preferential entry into cancer compared with normal cells. An 28‐amino‐acid fragment of azurin fused to glutathione S‐transferase (GST) or the green fluorescent protein (GFP), which are incapable of entering mammalian cells by themselves, can be internalized in J774 or human melanoma or breast cancer cells at 37°C, but not at 4°C. Competition experiments as well as studies with inhibitors such as cytochalasin D suggest that azurin may enter cells, at least in part, by a receptor‐mediated endocytic process. The 28‐amino‐acid peptide therefore acts as a potential protein transduction domain (PTD), and can be used as a vehicle to transport cargo proteins such as GST and GST–GFP fusion proteins. Another member of the cupredoxin family, rusticyanin, that has also been shown to enter J774 and human cancer cells and exert cytotoxicity, does not demonstrate preferential entry for cancer cells and lacks the structural features characteristic of the azurin PTD.

NOTCH reprograms mitochondrial metabolism for proinflammatory macrophage activation

Metabolic reprogramming is implicated in macrophage activation, but the underlying mechanisms are poorly understood. Here, we demonstrate that the NOTCH1 pathway dictates activation of M1 phenotypes in isolated mouse hepatic macrophages (HMacs) and in a murine macrophage cell line by coupling transcriptional upregulation of M1 genes with metabolic upregulation of mitochondrial oxidative phosphorylation and ROS (mtROS) to augment induction of M1 genes. Enhanced mitochondrial glucose oxidation was achieved by increased recruitment of the NOTCH1 intracellular domain (NICD1) to nuclear and mitochondrial genes that encode respiratory chain components and by NOTCH-dependent induction of pyruvate dehydrogenase phosphatase 1 (Pdp1) expression, pyruvate dehydrogenase activity, and glucose flux to the TCA cycle. As such, inhibition of the NOTCH pathway or Pdp1 knockdown abrogated glucose oxidation, mtROS, and M1 gene expression. Conditional NOTCH1 deficiency in the myeloid lineage attenuated HMac M1 activation and inflammation in a murine model of alcoholic steatohepatitis and markedly reduced lethality following endotoxin-mediated fulminant hepatitis in mice. In vivo monocyte tracking further demonstrated the requirement of NOTCH1 for the migration of blood monocytes into the liver and subsequent M1 differentiation. Together, these results reveal that NOTCH1 promotes reprogramming of mitochondrial metabolism for M1 macrophage activation.

Kaposi's sarcoma associated herpesvirus-encoded viral FLICE inhibitory protein (vFLIP) K13 suppresses CXCR4 expression by upregulating miR-146a

Kaposis sarcoma (KS)-associated herpesvirus (KSHV)-encoded viral FLICE inhibitory protein (vFLIP) K13 is a potent activator of the nuclear factor-κB (NF-κB) pathway. In this study, we show that infection with KHSV and ectopic expression of K13, but not its NF-κB-defective mutant, suppressed the expression of CXCR4. Suppression of CXCR4 by KSHV and K13 was associated with upregulated expression of miR-146a, a microRNA that is known to bind to the 3′-untranslated region of CXCR4 mRNA. Reporter studies identified two NF-κB sites in the promoter of miR-146a that were essential for its activation by K13. Accordingly, ectopic expression of K13, but not its NF-κB-defective mutant or other vFLIPs, strongly stimulated the miR-146a promoter activity, which could be blocked by specific genetic and pharmacological inhibitors of the NF-κB pathway. Finally, expression of CXCR4 was downregulated in clinical samples of KS and this was accompanied by an increased expression of miR-146a. Our results show that K13-induced NF-κB activity suppresses CXCR4 through upregulation of miR-146a. Downregulation of CXCR4 expression by K13 may contribute to KS development by promoting premature release of KSHV-infected endothelial progenitors into the circulation.

Nanoparticles in the treatment and diagnosis of neurological disorders: untamed dragon with fire power to heal

UNLABELLED The incidence of neurological diseases of unknown etiology is increasing, including well-studied diseases such as Alzhiemers, Parkinsons, and multiple sclerosis. The blood-brain barrier provides protection for the brain but also hinders the treatment and diagnosis of these neurological diseases, because the drugs must cross the blood-brain barrier to reach the lesions. Thus, attention has turned to developing novel and effective delivery systems that are capable of carrying drug and that provide good bioavailability in the brain. Nanoneurotechnology, particularly application of nanoparticles in drug delivery, has provided promising answers to some of these issues in recent years. Here we review the recent advances in the understanding of several common forms of neurological diseases and particularly the applications of nanoparticles to treat and diagnose them. In addition, we discuss the integration of bioinformatics and modern genomic approaches in the development of nanoparticles. FROM THE CLINICAL EDITOR In this review paper, applications of nanotechnology-based diagnostic methods and therapeutic modalities are discussed addressing a variety of neurological disorders, with special attention to blood-brain barrier delivery methods. These novel nanomedicine approaches are expected to revolutionize several aspects of clinical neurology.

Genome-wide profiling identifies a DNA methylation signature that associates with TET2 mutations in diffuse large B-cell lymphoma

The discovery that the Ten-Eleven Translocation (TET) hydroxylases cause DNA demethylation has fundamentally changed the notion of how DNA methylation is regulated. Clonal analysis of the hematopoetic stem cell compartment suggests that TET2 mutations can be early events in hematologic cancers and recent investigations have shown TET2 mutations in diffuse large B-cell lymphoma. However, the detection rates and the types of TET2 mutations vary, and the relation to global methylation patterns has not been investigated. Here, we show TET2 mutations in 12 of 100 diffuse large B-cell lymphomas with 7% carrying loss-of-function and 5% carrying missense mutations. Genome-wide methylation profiling using 450K Illumina arrays identified 315 differentially methylated genes between TET2 mutated and TET2 wild-type cases. TET2 mutations are primarily associated with hypermethylation within CpG islands (70%; P<0.0001), and at CpG-rich promoters (60%; P<0.0001) of genes involved in hematopoietic differentiation and cellular development. Hypermethylated loci in TET2 mutated samples overlap with the bivalent (H3K27me3/H3K4me3) silencing mark in human embryonic stem cells (P=1.5×10−30). Surprisingly, gene expression profiling showed that only 11% of the hypermethylated genes were down-regulated, among which there were several genes previously suggested to be tumor suppressors. A meta-analysis suggested that the 35 hypermethylated and down-regulated genes are associated with the activated B-cell-like type of diffuse large B-cell lymphoma in other studies. In conclusion, our data suggest that TET2 mutations may cause aberrant methylation mainly of genes involved in hematopoietic development, which are silenced but poised for activation in human embryonic stem cells.

Coat-Tether Interaction in Golgi Organization

Biogenesis of the Golgi apparatus is likely mediated by the COPI vesicle coat complex, but the mechanism is poorly understood. Modeling of the COPI subunit betaCOP based on the clathrin adaptor AP2 suggested that the betaCOP C terminus forms an appendage domain with a conserved FW binding pocket motif. On gene replacement after knockdown, versions of betaCOP with a mutated FW motif or flanking basic residues yielded a defect in Golgi organization reminiscent of that occurring in the absence of the vesicle tether p115. Indeed, betaCOP bound p115, and this depended on the betaCOP FW motif. Furthermore, the interaction depended on E(19)E(21) in the p115 head domain and inverse charge substitution blocked Golgi biogenesis in intact cells. Finally, Golgi assembly in permeabilized cells was significantly reduced by inhibitors containing intact, but not mutated, betaCOP FW or p115 EE motifs. Thus, Golgi organization depends on mutually interacting domains in betaCOP and p115, suggesting that vesicle tethering at the Golgi involves p115 binding to the COPI coat.

Pluripotency factor-mediated expression of the leptin receptor (OB-R) links obesity to oncogenesis through tumor-initiating stem cells

Misregulation of a pluripotency-associated transcription factor network in adult tissues is associated with the expansion of rare, highly malignant tumor-initiating stem cells (TISCs) through poorly understood mechanisms. We demonstrate that robust and selective expression of the receptor for the adipocyte-derived peptide hormone leptin (OB-R) is a characteristic feature of TISCs and of a broad array of embryonic and induced pluripotent stem cells and is mediated directly by the core pluripotency-associated transcription factors OCT4 and SOX2. TISCs exhibit sensitized responses to leptin, including the phosphorylation and activation of the pluripotency-associated oncogene STAT3 and induction of Oct4 and Sox2, thereby establishing a self-reinforcing signaling module. Exposure of cultured mouse embryonic stem cells to leptin sustains pluripotency in the absence of leukemia inhibitory factor. By implanting TISCs into leptin-deficient ob/ob mice or into comparably overweight Leprdb/db mice that produce leptin, we provide evidence of a central role for the leptin-TISC–signaling axis in promoting obesity-induced tumor growth. Differential responses to extrinsic, adipocyte-derived cues may promote the expansion of tumor cell subpopulations and contribute to oncogenesis.