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1.  Nitrosylcobalamin Potentiates the Anti-Neoplastic Effects of Chemotherapeutic Agents via Suppression of Survival Signaling 
PLoS ONE  2007;2(12):e1313.
Background
Nitrosylcobalamin (NO-Cbl) is a chemotherapeutic pro-drug derived from vitamin B12 that preferentially delivers nitric oxide (NO) to tumor cells, based upon increased receptor expression. NO-Cbl induces Apo2L/TRAIL-mediated apoptosis and inhibits survival signaling in a variety of malignant cell lines. Chemotherapeutic agents often simultaneously induce an apoptotic signal and activation of NF-κB, which has the undesired effect of promoting cell survival. The specific aims of this study were to 1) measure the anti-tumor effects of NO-Cbl alone and in combination with conventional chemotherapeutic agents, and to 2) examine the mechanism of action of NO-Cbl as a single agent and in combination therapy.
Methodology
Using anti-proliferative assays, electrophoretic mobility shift assay (EMSA), immunoblot analysis and kinase assays, we demonstrate an increase in the effectiveness of chemotherapeutic agents in combination with NO-Cbl as a result of suppressed NF-κB activation.
Results
Eighteen chemotherapeutic agents were tested in combination with NO-Cbl, in thirteen malignant cell lines, resulting in a synergistic anti-proliferative effect in 78% of the combinations tested. NO-Cbl pre-treatment resulted in decreased NF-κB DNA binding activity, inhibition of IκB kinase (IKK) enzymatic activity, decreased AKT activation, increased caspase-8 and PARP cleavage, and decreased cellular XIAP protein levels.
Conclusion
The use of NO-Cbl to inhibit survival signaling may enhance drug efficacy by preventing concomitant activation of NF-κB or AKT.
doi:10.1371/journal.pone.0001313
PMCID: PMC2117345  PMID: 18074035
2.  Suppression of NF-κB Survival Signaling by Nitrosylcobalamin Sensitizes Neoplasms to the Anti-tumor Effects of Apo2L/TRAIL* 
The Journal of biological chemistry  2003;278(41):39461-39469.
We have previously demonstrated the anti-tumor activity of nitrosylcobalamin (NO-Cbl), an analog of vitamin B12 that delivers nitric oxide (NO) and increases the expression of tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) and its receptors in human tumors. The specific aim of this study was to examine whether NO-Cbl could sensitize drug-resistant melanomas to Apo2L/TRAIL. Antiproliferative effects of NO-Cbl and Apo2L/TRAIL were assessed in malignant melanomas and non-tumorigenic melanocyte and fibro-blast cell lines. Athymic nude mice bearing human melanoma A375 xenografts were treated with NO-Cbl and Apo2L/TRAIL. Apoptosis was measured by TUNEL and confirmed by examining levels and activity of key mediators of apoptosis. The activation status of NF-κB was established by assaying DNA binding, luciferase reporter activity, the phosphorylation status of IκBα, and in vitro IKK activity. NO-Cbl sensitized Apo2L/TRAIL-resistant melanoma cell lines to growth inhibition by Apo2L/TRAIL but had minimal effect on normal cell lines. NO-Cbl and Apo2L/TRAIL exerted synergistic anti-tumor activity against A375 xenografts. Treatment with NO-Cbl followed by Apo2L/TRAIL induced apoptosis in Apo2L/TRAIL-resistant tumor cells, characterized by cleavage of caspase-3, caspase-8, and PARP. NO-Cbl inhibited IKK activation, characterized by decreased phosphorylation of IκBα and inhibition of NF-κB DNA binding activity. NO-Cbl suppressed Apo2L/TRAIL- and TNF-α-mediated activation of a transfected NF-κB-driven luciferase reporter. XIAP, an inhibitor of apoptosis, was inactivated by NO-Cbl. NO-Cbl treatment rendered Apo2L/TRAIL-resistant malignancies sensitive to the anti-tumor effects of Apo2L/TRAIL in vitro and in vivo. The use of NO-Cbl and Apo2L/TRAIL capitalizes on the tumor-specific properties of both agents and represents a promising anti-cancer combination.
doi:10.1074/jbc.M306111200
PMCID: PMC2080861  PMID: 12881518
3.  Effect of Inositol Hexakisphosphate Kinase 2 on Transforming Growth Factor β-activated Kinase 1 and NF-κB Activation* 
The Journal of biological chemistry  2007;282(21):15349-15356.
We previously showed that inositol hexakisphosphate kinase 2 (IHPK2) functions as a growth-suppressive and apoptosis-enhancing kinase during cell stress. Overexpression of IHPK2 sensitized ovarian carcinoma cell lines to the growth-suppressive and apoptotic effects of interferon β (IFN-β), IFN-α2, and γ-irradiation. Expression of a kinase-dead mutant abrogated 50% of the apoptosis induced by IFN-β. Because the kinase-dead mutant retained significant response to cell stressors, we hypothesized that a portion of the death-promoting function of IHPK2 was independent of its kinase activity. We now demonstrate that IHPK2 binds to tumor necrosis factor (TNF) receptor-associated factor (TRAF) 2 and interferes with phosphorylation of transforming growth factor β-activated kinase 1 (TAK1), thereby inhibiting NF-κB signaling. IHPK2 contains two sites required for TRAF2 binding, Ser-347 and Ser-359. Compared with wild type IHPK2-transfected cells, cells expressing S347A and S359A mutations displayed 3.5-fold greater TAK1 activation following TNF-α. This mutant demonstrated a 6 –10-fold increase in NF-κB DNA binding following TNF-α compared with wild type IHPK2-expressing cells in which NF-κB DNA binding was inhibited. Cells transfected with wild type IHPK2 or IHPK2 mutants that lacked S347A and S359A mutations displayed enhanced terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling staining following TNF-α. We believe that IHPK2-TRAF2 binding leads to attenuation of TAK1- and NF-κB-mediated signaling and is partially responsible for the apoptotic activity of IHPK2.
doi:10.1074/jbc.M700156200
PMCID: PMC2048714  PMID: 17379600
4.  Inositol hexakisphosphate kinase 2 sensitizes ovarian carcinoma cells to multiple cancer therapeutics 
Oncogene  2002;21(12):1882-1889.
We recently identified inositol hexakisphosphate kinase 2 (IP6K2) as a positive regulator of apoptosis. Overexpression of IP6K2 enhances apoptosis induced by interferon-β (IFN-β) and cytotoxic agents in NIH-OVCAR-3 ovarian carcinoma cells. In this study, we contrast and compare IFN-β and radiation-induced death, and show that IP6K2 expression sensitizes tumor cells. Unirradiated NIH-OVCAR-3 cells transfected with IP6K2 formed fewer colonies compared to unirradiated vector-expressing cells. IP6K2 overexpression caused increased radiosensitivity, evidenced by decreased colony forming units (CFU). Both IFN-β and radiation induced caspase 8. IFN-β, but not γ-irradiation, induced TRAIL in NIH-OVCAR-3 cells. Gamma irradiation, but not IFN-β, induced DR4 mRNA. Apoptotic effects of IFN-β or γ-irradiation were blocked by expression of a dominant negative mutant death receptor 5 (DR5Δ) or by Bcl-2. Caspase-8 mRNA induction was more pronounced in IP6K2-expressing cells compared to vector-expressing cells. These data suggest that overexpression of IP6K2 enhances sensitivity of some ovarian carcinomas to radiation and IFN-β. IP6K2 may function to enhance the expression and/or function of caspase 8 and DR4 following cell injury. Both IFN-β and γ-irradiation induce apoptosis through the extrinsic, receptor-mediated pathway, IFN-β through TRAIL, radiation through DR4, and both through caspase 8. The function of both death inducers is positively regulated by IP6K2.
doi:10.1038/sj/onc/1205265
PMCID: PMC2043497  PMID: 11896621
inositol hexakisphosphate kinase; apoptosis; radiation; interferon
5.  Inositol Hexakisphosphate Kinase 2 Mediates Growth Suppressive and Apoptotic Effects of Interferon-β in Ovarian Carcinoma Cells* 
The Journal of biological chemistry  2001;276(27):24965-24970.
Interferons (IFNs) regulate the expression of genes that mediate their antiviral, antitumor, and immuno-modulatory actions. We have previously shown that IFN-β suppresses growth of human ovarian carcinoma xenografts in vivo and induces apoptosis of ovarian carcinoma cells in vitro. To investigate mechanisms of IFN-β-induced apoptosis we employed an antisense technical knockout approach to identify gene products that mediate cell death and have isolated several regulators of interferon-induced death (RIDs). In this investigation, we have characterized one of the RIDs, RID-2. Sequence analysis revealed that RID-2 was identical to human inositol hexakisphosphate kinase 2 (IP6K2). IP6K2 is post-transcriptionally induced by IFN-β in ovarian carcinoma cells. A mutant IP6K2 with substitutions in the putative inositol phosphate binding domain abrogates IFN-β-induced apoptosis. These studies identify a novel function for IP6K2 in cell growth regulation and apoptosis.
doi:10.1074/jbc.M101161200
PMCID: PMC2025680  PMID: 11337497
6.  Effects of Interferon β on Transcobalamin II-Receptor Expression and Antitumor Activity of Nitrosylcobalamin 
Background
The ubiquitous plasma membrane transcobalamin II receptor (TC II-R) mediates uptake of cobalamin (Cbl; vitamin B12), an essential micronutrient. Tumors often require more Cbl than normal tissue, and increased Cbl uptake may result from increased TC II-R expression. To examine whether Cbl could therefore be used as a carrier molecule to target a chemotherapy drug, we tested an analogue of Cbl with nitric oxide as a ligand, nitrosylcobalamin (NO-Cbl). Because interferon β (IFN-β) has antitumor effects and increases expression of some membrane receptors, we examined whether it may enhance the effects of NO-Cbl.
Methods
Antiproliferative effects of NO-Cbl were assessed in 24 normal and cancer cell lines. Xenograft tumors of human ovarian cancer NIH-OVCAR-3 cells were established in athymic nude mice, and tumor growth was monitored after treatment with NO-Cbl and IFN-β, both individually and concomitantly. TC II-R expression and apoptosis was monitored in vitro and in vivo. RNA protection assays and mitochondrial membrane potential assays were used to distinguish the extrinsic and intrinsic apoptotic pathways, respectively.
Results
Cancer cell lines were more sensitive to NO-Cbl (with ID50s [the dose that inhibits growth by 50%] as low as 2 µM) than normal cell lines (with ID50s of 85–135 µM). Single-agent NO-Cbl and IFN-β treatment of NIH-OVCAR-3 xenografts induced tumor regression, whereas combination treatment induced tumor eradication. IFN-β treatment increased TC II-R expression in vitro and uptake of [57Co]cobalamin in vivo. Compared with NIH-OVCAR-3 cells treated with NO-Cbl, cells treated with NO-Cbl and IFN-β were more apoptotic and expressed higher mRNA levels of various apoptosis-associated genes. No changes in mitochondrial membrane potential were observed in cells treated with NO-Cbl.
Conclusion
NO-Cbl inhibited tumor growth in vivo by activating the extrinsic apoptotic pathway. The increased expression of TC II-R induced by IFN-β resulted in enhanced antitumor effects with NO-Cbl both in vitro and in vivo.
PMCID: PMC2020433  PMID: 12096086
7.  Hepatitis B virus precore mutation and fulminant hepatitis in the United States. A polymerase chain reaction-based assay for the detection of specific mutation. 
Journal of Clinical Investigation  1994;93(2):550-555.
Hepatitis B virus (HBV) variants with precore mutation(s) resulting in the absence of HBeAg production have been associated with the occurrence of fulminant hepatitis in Japan, Israel, and southern Europe, where the prevalence of this HBV strain appears common. In areas such as United States, where HBV infection is not endemic, the role of this mutant virus in fulminant hepatitis is unknown. We developed an amplification refractory mutation detection system to detect specifically the presence of the G to A mutation at nucleotide position 1898, which is the most frequently observed mutation resulting in a precore stop codon. In addition, this method provided a quantitative measurement of the relative ratio of one strain to the other. Using this system, we tested HBV strains for the presence of the stop codon mutation in sera from 40 cases of fulminant hepatitis B occurring in the United States. Serum HBV DNAs from 28 patients were analyzed successfully. A mixture of wild-type and mutant strains in various ratios were observed in 15 patients, wild type exclusively in 11, and mutant exclusively in 2. Four of these patients had undergone liver transplantation for HBV-associated cirrhosis and developed fulminant HBV-associated hepatitis after transplantation. Pre- and posttransplant serum samples from one patient were analyzed: a mixture of wild-type and mutant HBV strains was detected in both samples. Our study demonstrated that both wild-type and mutant HBV strains are associated with fulminant hepatitis, and that in some patients in the United States, factors other than precore mutations contribute to the development of fulminant hepatitis.
Images
PMCID: PMC293877  PMID: 8113393

Results 1-7 (7)