Thambi Dorai

Clonal Isolation of Different Strains of Mouse Mammary Tumor Virus-Like DNA Sequences from Both the Breast Tumors and Non-Hodgkin's Lymphomas of Individual Patients Diagnosed with Both Malignancies

Purpose: In a previous study, we had detected the presence of mouse mammary tumor virus (MMTV)-like envelope (ENV) gene sequences in both the breast tumors and non-Hodgkin’s lymphoma tissue of two of our breast tumor patients who had been diagnosed simultaneously with both malignancies. The aim of this study was to determine if MMTV-like DNA sequences are present in the breast tumors and non-Hodgkin’s lymphomas of additional patients suffering from both malignancies and if so to characterize these sequences in detail. Experimental Design: DNA was extracted from formalin-fixed, paraffin-embedded tissue sample blocks of breast tumors and non-Hodgkin’s lymphomas from patients suffering from both malignancies. A 250-bp region of the MMTV ENV gene and a 630-bp region of the MMTV long terminal repeat (LTR) open reading frame (ORF) that encodes the MMTV superantigen (sag) gene were amplified by PCR from the isolated DNA. Amplified products were analyzed by Southern blotting, cloned, and sequenced. Results: MMTV-like ENV and LTR sequences were detected in both the breast tumors and non-Hodgkin’s lymphomas of 6 of 12 patients suffering from both malignancies. A novel mutant of the MMTV ENV gene was identified in these patients. Characterization of the MMTV-like LTR highly variable sag sequences revealed total or nearly total identity to three distinct MMTV proviruses from two different branches of the MMTV phylogenetic tree. Conclusions: The presence of MMTV-like ENV and LTR sequences in both the breast tumors and non-Hodgkin’s lymphomas of 6 additional patients suggests a possible involvement of these sequences in these two malignancies. MMTV-like LTR sequence homology to different MMTV proviruses revealed the presence of more than one strain of MMTV-like sequences in each individual suggesting the possibility of multiple infections in these patients.

Amelioration of renal ischemia-reperfusion injury with a novel protective cocktail.

PURPOSE Extended warm ischemia during partial nephrectomy can lead to considerable renal injury. Using a rat model of renal ischemia we examined the ability of a unique renoprotective cocktail to ameliorate warm ischemia-reperfusion injury. MATERIALS AND METHODS A warm renal ischemia model was developed using 60 Sprague-Dawley® rats. The left renal artery was clamped for 40 minutes, followed by 48 hours of reperfusion. A renoprotective cocktail of a mixture of specific growth factors, mitochondria protecting biochemicals and Manganese-Porphyrin (MnTnHex-2-PyP(5+)) was given intramuscularly at -24, 0 and 24 hours after surgery. At 48 hours the 2 kidneys were harvested and examined with hematoxylin and eosin, and periodic acid-Schiff stains. Protein and gene expression were also analyzed to determine ischemia markers and the antioxidant response. RESULTS Compared to ischemic controls, kidneys treated with the renoprotective cocktail showed significant reversal of morphological changes and a significant decrease in the specific ischemic markers lipocalin-2, mucin-1 and galectin-3. Quantitative reverse transcriptase-polymerase chain reaction revealed up-regulation of several antioxidant genes in treated animals. CONCLUSIONS According to histopathological and several molecular measures our unique renoprotective cocktail mitigated ischemia-reperfusion injury.

Role of Carbonic Anhydrases in the Progression of Renal Cell Carcinoma Subtypes: Proposal of a Unified Hypothesis

Renal cell carcinoma (RCC) has the highest rate of occurrence within the US when compared with other countries. Recent advances in the basic research and molecular diagnostics of this malignancy have revealed that RCC is not a single disease, but it is a mixture of several types of malignancies with unique molecular mechanisms and pathological attributes. RCC is now divided into clear cell carcinoma (80% of all kidney cancers), papillary type 1 and papillary type 2 neoplasms (10–15% of all RCC patients) and RCC with chromophobic and oncocytic features, called the Birt-Hogg-Dube (BHD) subtype, in roughly 5% of all patients. Apart from these, neoplasms such as the tuberous sclerosis (TSC) syndrome may occur with a mixed pathological features with a renal presentation. In this review, molecular evidence, both direct and indirect, published so far on all these RCC subtypes have been analyzed to find out whether there is any common thread that could run through these disparate malignancies that happen to occur in a single organ, i.e., the kidney. We believe that the role played by the expression and certain non-traditional activities of the cabonic anhydrase (CA) family members, along with the differing levels of hypoxia induced within these tumors may be the most common denominators. Evidence is presented focusing on how the CA family members could participate in the genesis and progression of each and every one of these RCC subtypes and how their function could be influenced by hypoxia, activities of receptor type protein tyrosine kinases and certain other pre-disposing factors. These rationalizations point towards a unified hypothesis that may help explain the occurrence of all these RCC subtypes in a molecular manner. We hope that these analyses would a) stimulate further studies aimed toward a better understanding of the role played by carbonic anhydrases in RCC subtypes and b) would pave way to a better and rationally designed therapies to interfere with their function to benefit patients with RCC and possibly other cancers.

ω-Amidase: an underappreciated, but important enzyme in L-glutamine and L-asparagine metabolism; relevance to sulfur and nitrogen metabolism, tumor biology and hyperammonemic diseases

In mammals, two major routes exist for the metabolic conversion of l-glutamine to α-ketoglutarate. The most widely studied pathway involves the hydrolysis of l-glutamine to l-glutamate catalyzed by glutaminases, followed by the conversion of l-glutamate to α-ketoglutarate by the action of an l-glutamate-linked aminotransferase or via the glutamate dehydrogenase reaction. However, another major pathway exists in mammals for the conversion of l-glutamine to α-ketoglutarate (the glutaminase II pathway) in which l-glutamine is first transaminated to α-ketoglutaramate (KGM) followed by hydrolysis of KGM to α-ketoglutarate and ammonia catalyzed by an amidase known as ω-amidase. In mammals, the glutaminase II pathway is present in both cytosolic and mitochondrial compartments and is most prominent in liver and kidney. Similarly, two routes exist for the conversion of l-asparagine to oxaloacetate. In the most extensively studied pathway, l-asparagine is hydrolyzed to l-aspartate by the action of asparaginase, followed by transamination of l-aspartate to oxaloacetate. However, another pathway also exists for the conversion of l-asparagine to oxaloacetate (the asparaginase II pathway). In this pathway, l-asparagine is first transaminated to α-ketosuccinamate (KSM), followed by hydrolysis of KSM to oxaloacetate by the action of ω-amidase. One advantage of both the glutaminase II and the asparaginase II pathways is that they are irreversible, and thus are important in anaplerosis by shuttling 5-C (α-ketoglutarate) and 4-C (oxaloacetate) units into the TCA cycle. In this review, we briefly mention the importance of the glutaminase II and asparaginase II pathways in microorganisms and plants. However, the major emphasis of the review is related to the importance of these pathways (especially the common enzyme component of both pathways—ω-amidase) in nitrogen and sulfur metabolism in mammals and as a source of anaplerotic carbon moieties in rapidly dividing cells. The review also discusses a potential dichotomous function of ω-amidase as having a role in tumor progression. Finally, the possible role of KGM as a biomarker for hyperammonemic diseases is discussed.

Kynurenine Aminotransferase III and Glutamine Transaminase L Are Identical Enzymes that have Cysteine S-Conjugate β-Lyase Activity and Can Transaminate l-Selenomethionine

Background: KAT I and GT-Kidney (K) are identical enzymes that β-eliminate and transaminate Se-methyl-l-selenocysteine (MSC). Results: KAT III and GT-Liver (L) are identical and metabolize l-selenomethionine (SM). Conclusion: MSC and SM are transaminated to seleno-keto acids, recognized HDAC inhibitors, by KAT/GT enzymes. Significance: Anticancer efficacy of MSC and SM depends in part on tissue expression of KAT/GT enzymes. Three of the four kynurenine aminotransferases (KAT I, II, and IV) that synthesize kynurenic acid, a neuromodulator, are identical to glutamine transaminase K (GTK), α-aminoadipate aminotransferase, and mitochondrial aspartate aminotransferase, respectively. GTK/KAT I and aspartate aminotransferase/KAT IV possess cysteine S-conjugate β-lyase activity. The gene for the former enzyme, GTK/KAT I, is listed in mammalian genome data banks as CCBL1 (cysteine conjugate beta-lyase 1). Also listed, despite the fact that no β-lyase activity has been assigned to the encoded protein in the genome data bank, is a CCBL2 (synonym KAT III). We show that human KAT III/CCBL2 possesses cysteine S-conjugate β-lyase activity, as does mouse KAT II. Thus, depending on the nature of the substrate, all four KATs possess cysteine S-conjugate β-lyase activity. These present studies show that KAT III and glutamine transaminase L are identical enzymes. This report also shows that KAT I, II, and III differ in their ability to transaminate methyl-l-selenocysteine (MSC) and l-selenomethionine (SM) to β-methylselenopyruvate (MSP) and α-ketomethylselenobutyrate, respectively. Previous studies have identified these seleno-α-keto acids as potent histone deacetylase inhibitors. Methylselenol (CH3SeH), also purported to have chemopreventive properties, is the γ-elimination product of SM and the β-elimination product of MSC catalyzed by cystathionine γ-lyase (γ-cystathionase). KAT I, II, and III, in part, can catalyze β-elimination reactions with MSC generating CH3SeH. Thus, the anticancer efficacy of MSC and SM will depend, in part, on the endogenous expression of various KAT enzymes and cystathionine γ-lyase present in target tissue coupled with the ability of cells to synthesize in situ either CH3SeH and/or seleno-keto acid metabolites.

The administration of renoprotective agents extends warm ischemia in a rat model.

BACKGROUND AND PURPOSE Extended warm ischemia time during partial nephrectomy leads to considerable renal injury. Using a rat model of renal ischemia, we examined the ability of a unique renoprotective cocktail to ameliorate warm ischemia-reperfusion injury and extend warm ischemia time. MATERIALS AND METHODS A warm renal ischemia model was developed using Sprague-Dawley rats, clamping the left renal artery for 40, 50, 60, and 70 minutes, followed by 48 hours of reperfusion. An improved renoprotective cocktail referred to as I-GPM (a mixture of specific renoprotective growth factors, porphyrins, and mitochondria-protecting amino acids) was administered -24 hours, 0 hours, and +24 hours after surgery. At 48 hours, both kidneys were harvested and examined with hematoxylin and eosin and periodic acid-Schiff stains for the analysis of renal tubular necrosis. Creatinine, protein, and gene expression levels were also analyzed to evaluate several ischemia-specific and antioxidant response markers. RESULTS I-GPM treated kidneys showed significant reversal of morphologic changes and a significant reduction in specific ischemic markers lipocalin-2, galectin-3, GRP-78, and HMGB1 compared with ischemic controls. These experiments also showed an upregulation of the stress response protein, heat shock protein (HSP)-70, as well as the phosphorylated active form of the transcription factor, heat shock factor (HSF)-1. In addition, quantitative RT-PCR analyses revealed a robust upregulation of several antioxidant pathway response genes in I-GPM treated animals. CONCLUSIONS By histopathologic and several molecular measures, our unique renoprotective cocktail mitigated ischemia-reperfusion injury. Our cocktail minimized oxidative stress in an ischemic kidney rat model while at the same time protecting the global parenchymal function during extended periods of ischemia.

Role of Glutamine Transaminases in Nitrogen, Sulfur, Selenium, and 1-Carbon Metabolism

Glutamine metabolism is largely controlled by two enzyme pathways: 1) Conversion of glutamine to glutamate catalyzed by glutaminases, followed by conversion of glutamate to α-ketoglutarate by a glutamate-linked aminotransferases (or by the action of glutamate dehydrogenase); and 2) conversion of glutamine to α-ketoglutaramate (KGM) catalyzed by glutamine-utilizing transaminases (aminotransferases), followed by conversion of KGM to α-ketoglutarate by the action of ω-amidase. The former pathway has been well documented and intensively studied for over 60 years, whereas only recently has research focused on the latter pathway, its importance in homeostasis and the control of anaplerotic metabolites. The glutamine transaminases are of fundamental importance 1) as repair enzymes (salvage of α-keto acids), 2) in nitrogen and sulfur homeostasis (closure of the methionine salvage pathway), 3) in 1-carbon metabolism, and 4) in metabolism of seleno amino acids. As a result of their broad substrate specificity the two principal mammalian glutamine transaminases (i.e. glutamine transaminases L and K) have also been characterized as kynurenine aminotransferases (KAT I and KAT III, respectively), responsible for the production of neuroactive kynurenate. The glutamine transaminases are also active with a variety of sulfur- and selenium-containing amino acids. Some of the products derived from the transamination of these amino acids may also be neuroactive (e.g. certain sulfur-containing cyclic ketimines) as well as chemopreventive (e.g. the α-keto acids derived from seleno amino acids). Of relevance to human health and disease, the glutamine transaminases may contribute to the bioactivation (toxification) of halogenated alkenes (and possibly other xenobiotic electrophiles), some of which are environmental contaminants. Finally, the role of the glutamine transaminases and ω-amidase in cancer biology has been little studied. However, the “glutamine addiction” of many tumors suggests that the glutamine transaminases together with ω-amidase may have a fundamental and influential role in regulating cancer progression

NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis

In the course of studies aimed at the role of oxidative stress in the development of metastatic potential in the LNCaP‐C4‐2B prostate cancer progression model system, we found a relative decrease in the level of expression of the cytoplasmic nicotinamide riboside: quinone oxidoreductase (NQO2) and an increase in the oxidative stress in C4‐2B cells compared to that in LNCaP or its derivatives C4 and C4‐2. It was also found that C4‐2B cells specifically shed large extracellular vesicles (LEVs) suggesting that these LEVs and their cargo could participate in the establishment of the osseous metastases. The level of expression of caveolin‐1 increased as the system progresses from LNCaP to C4‐2B. Since NQO2 RNA levels were not changed in LNCaP, C4, C4‐2, and C4‐2B, we tested an altered cellular distribution hypothesis of NQO2 being compartmentalized in the membrane fractions of C4‐2B cells which are rich in lipid rafts and caveolae. This was confirmed when the detergent resistant membrane fractions were probed on immunoblots. Moreover, when the LEVs were analyzed for membrane associated caveolin‐1 as possible cargo, we noticed that the enzyme NQO2 was also a component of the cargo along with caveolin‐1 as seen in double immunofluorescence studies. Molecular modeling studies showed that a caveolin‐1 accessible site is present in NQO2. Specific interaction between NQO2 and caveolin‐1 was confirmed using deletion constructs of caveolin‐1 fused with glutathione S‐transferase (GST). Interestingly, whole cell lysate and mitochondrial preparations of LNCaP, C4, C4‐2, and C4‐2B showed an increasing expression of glutaminase (GLS, kidney type). The extrusion of LEVs appears to be a specific property of the bone metastatic C4‐2B cells and this process could be inhibited by a GLS specific inhibitor BPTES, suggesting the critical role of a functioning glutamine metabolism. Our results indicate that a high level of expression of caveolin‐1 in C4‐2B cells contributes to an interaction between caveolin‐1 and NQO2 and to their packaging as cargo in the shed LEVs. These results suggest an important role of membrane associated oxidoreductases in the establishment of osseous metastases in prostate cancer.

Abstract B003: NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis

Advanced-stage prostate cancer is characterized by osseous metastases whose establishment involves the dynamic interplay of factors and exchange of cellular contents by constituents of the tumor bony microenvironment. Among the factors that transport cellular cargoes and facilitate the transmission of signaling complexes for establishment of the bone metastatic lesions are large extracellular vesicles (LEVs), also known as large oncosomes. Information on the contents of LEVs and the mechanisms by which LEVs are formed and regulated is incomplete. In the course of studies aimed at elucidating the mechanisms of bony metastases using the LNCaP-C4-2B prostate cancer progression model, we show that the expression and cellular distribution of nicotinamide riboside: quinone oxidoreductase 2 (NQO2) and glutaminase (GLS, kidney type) both participate in the induction of membrane-localized oxidative stress and the formation of LEVs. The salient features of our findings are the following: The LNCaP model system exhibits gradually increasing oxidative stress levels as it progresses to the bone metastatic derivative C4-2B, while at the same time showing a progressive decrease in the expression level of NQO2. Reverse transcription PCR studies showed that NQO2 mRNA levels were unchanged between LNCaP and its metastatic derivatives C4, C4-2 and C4-2B. As a control, the expression level of the analogous enzyme NADP(H):quinone oxidoreductase 1 (NQO1) was also unchanged. These results suggested the possibility that the NQO2 enzyme in the bone metastatic C4-2B derivative could be localized elsewhere, possibly at an extracellular location. Further studies showed that a fraction of the NQO2 enzyme was associated with the detergent resistant membrane fraction of the C4-2B prostate cancer cells. Molecular modeling studies showed that the NQO2 enzyme had two caveolin-1 and NQO1 enzyme had one caveolin-1 binding sites. While the caveolin-1 binding site of NQO1 is buried inside the molecule and hence inaccessible to caveolin-1, one of the two binding sites available in NQO2 was external and accessible to caveolin-1. We have validated the specific interaction between NQO2 and caveolin-1 using deletion constructs of caveolin-1 fused with glutathione S-transferase (GST). Mitochondrial preparations of LNCaP, C4, C4-2, and C4-2B cells showed increasing level of expression of the GLS enzyme, suggesting a correlation between increased oxidative stress, glutaminase activity, and metastatic potential. Moreover, we found that the metastatic C4-2B derivative extruded LEVs in which the enzyme NQO2 and caveolae specific caveolin-1 could be visualized as cargo using double immunofluorescence studies. The extrusion of these LEVs could be inhibited by the GLS specific inhibitor BP-TES. Characteristically, curcumin was also found to inhibit the extrusion of these LEVs. This naturally occurring plant-based compound was shown earlier to inhibit prostate cancer bone metastasis in vivo using the C4-2B model. These results indicate a role for the NQO2 enzyme at the membrane level, possibly increasing the oxidative stress in a localized manner by lipid peroxidation. These results also suggest that a glutamine/GLS-mediated metabolic reprogramming in the LNCaP model system as it progresses toward the bone metastatic C4-2B is an integral component of the force driving the metastatic process. Based on the results of our studies, a combinatorial strategy using antimetastatic therapies such as nitrogen-containing bisphosphonates (NBPs) and anti-GLS therapy or, alternatively, a naturally occurring plant compound-based anti-NQO2 and anti-GLS therapy, could be considered for treating advanced prostate cancer patients with bone metastatic complications. Citation Format: Thambi Dorai, Ankeeta B. Shah, Faith Summers, Rajamma Mathew, Jing Huang, Tze-chen Hsieh, Joseph M. Wu. NRH:quinone oxidoreductase 2 (NQO2) and glutaminase (GLS) both play a role in large extracellular vesicles (LEV) formation in preclinical LNCaP-C4-2B prostate cancer model of progressive metastasis [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B003.

Sweetening of glutamine metabolism in cancer cells by Rho GTPases through convergence of multiple oncogenic signaling pathways

More than 60 years ago, Otto Warburg showed that cancer cells exhibit enhanced glycolysis accompanied by greatly elevated levels of lactate secretion, even in the presence of normal levels of oxygen (1). Warburg suggested that cancer cells arise from normal cells in a two-phase process: phase 1 is “injury” to the respiratory machinery (i.e., mitochondria), followed in phase 2 by enhanced “fermentation” (i.e., production of lactate from glucose) in the protoplasm (i.e., cytosol) (1).