Vitamin B12b Research Articles

The SHARON trial: A study of melphalan, BCNU, hydroxocobalamin, ascorbic acid, and autologous stem cell rescue for metastatic pancreatic cancer with an inherited BRCA mutation.

TPS774 Background: SHARON is a phase 1, single-arm trial to assess the safety and preliminary efficacy of 2 cycles of melphalan, BCNU, hydroxocobalamin (vitamin B12b), dose-escalated vitamin C, and autologous stem cells in patients with stage IV pancreatic cancer and an inherited, deleterious BRCA1/2 mutation. Cancers that evolve in patients with a germline BRCA1/ 2 mutation generally have loss or inactivation of the wild type allele, which results in hypersensitivity to melphalan. The melphalan dose-response is log-linear: doubling the melphalan area under the curve (AUC) doubles the log reduction in clonogenic survival. The melphalan AUC that can safely be achieved with melphalan followed by stem cell rescue has the potential to provide major log reductions in cancer cell survival in BRCA-deficient cancers. However, reversion mutations in BRCA can cause resistance to DNA-crosslinking agents such as melphalan. Studies have shown that oxidizing cellular glutathione (GSH) to glutathione disulfide (GSSG) can inhibit DNA repair and hypersensitize wild-type cancer cells to melphalan. In this trial, BCNU, vitamin B12b, and vitamin C are used to increase cancer cell GSSG levels. Methods: Eligibility criteria include stage IV pancreatic cancer, an inherited deleterious BRCA1/2 mutation, life expectancy of at least 6 months, measurable or non-measurable disease, and suitability for apheresis, chemotherapy, and stem cell infusion. Prior treatment with PARP inhibitors is allowed. Stem cells are collected by apheresis. Participants receive 2 cycles of melphalan 100 mg/m2, BCNU 150 mg/m2, vitamin B12b 1.45 g/m2, and vitamin C 3, 6, or 8 g/m2 on Day −2. Stem cell infusion administered on day 0. A 2 + 4 dose-escalation schedule is employed for vitamin C. Adverse reactions summarized by CTCAE 5.0. The primary endpoint is safety, and secondary endpoints are objective response rate (overall and for metastatic lesions) and overall and progression-free survival. Amendment pending to include stage IV, BRCA-related, HER2-negative breast cancer and PALB2 mutations. The trial is enrolling at Memorial Sloan Kettering Cancer Center and Massachusetts General Hospital. Clinical trial information: NCT04150042 .

Abstract 2032: Vitamin B12 augments Ca2+ dependent cytotoxicity of diethyldithiocarbamate through excessive ER-vacuolization, oxidative stress and mitochondrial depolarization in MCF-7 cells in culture

Abstract Coordinated organization of intracellular organelles into functional networks is vital for cells. Mitochondrial and endoplasmic reticulum (ER) interaction in regulation of cell Ca2+ homeostasis is crucial for cell death and survival. Function of these intracellular organelles involved in two-way regulation of the status of major cell metabolites including protein, lipid exchange and intracellular Ca2+ homeostasis. Disulfiram and its derivatives recently attracted attention as a potential anticancer treatment (Kona et al., 2011). Combined use of hydroxycobalamin (vitamin B12b) and diethyldithiocarbamate (DDTC) demonstrated synergistic effect (combination index=0.25) and enhanced the cytotoxic effect of DDTC in number of transformed cell lines in culture (Solovieva et al., 2020). In the present work using MCF-7, human breast cancer cells in culture we demonstrate that combined action of DDTC and B12b resulted in induction of UPRER manifested by dilation of ER cisterns, increased level of polyubiquitinated proteins, and increased expression of CHOP. Confocal microscopy revealed disorganization of mitochondrial network and ER and treatment of these cells with B12b/DDTC and accompanied by mitochondrial depolarization (~50% after 6 h of exposure to drugs). Treatments also induced changes in morphology of mitochondrial network, and within 6 h of exposure, mitochondria demonstrated large amplitude swelling, characteristic feature of mitochondrial permeability transition pore opening. Drug exposure induced mitochondrial oxidative stress and increase in superoxide anion level (monitored with MitoSOX) and increased content of mitochondrial Ca2+ (xRhod-1) which almost doubled following treatment with B12b/DDTC. Short-time exposure of MCF-7 cells to mixture of drugs in Ca2+-free incubation media (2.5 mM EGTA) resulted in generation of significantly smaller size of vacuoles as compared with control. Similarly, pre-loading of cells with intracellular Ca2+ chelator (BAPTA, 5 µM, 30 min, 37oC) or inhibition of IP3 receptor(s) with 2-APB, delayed the drug-induced vacuolization. Taken together, our data indicate that the mechanism of B12b/DDTC-induced cytotoxicity in human MCF-7 cancer cell lines involves mitochondrial oxidative stress, activation of MPT pore and Ca2+ overload. Support of Funding Information: Russian Science Foundation № 19-75-20145 Citation Format: Marina E. Solovieva. Vitamin B12 augments Ca2+ dependent cytotoxicity of diethyldithiocarbamate through excessive ER-vacuolization, oxidative stress and mitochondrial depolarization in MCF-7 cells in culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2032.

Vitamin B12b Enhances the Cytotoxicity of Diethyldithiocarbamate in a Synergistic Manner, Inducing the Paraptosis-Like Death of Human Larynx Carcinoma Cells.

We have shown that hydroxycobalamin (vitamin B12b) increases the toxicity of diethyldithiocarbamate (DDC) to tumor cells by catalyzing the formation of disulfiram (DSF) oxi-derivatives. The purpose of this study was to elucidate the mechanism of tumor cell death induced by the combination DDC + B12b. It was found that cell death induced by DDC + B12b differed from apoptosis, autophagy, and necrosis. During the initiation of cell death, numerous vacuoles formed from ER cisterns in the cytoplasm, and cell death was partially suppressed by the inhibitors of protein synthesis and folding, the IP3 receptor inhibitor as well as by thiols. At this time, a short-term rise in the expression of ER-stress markers BiP and PERK with a steady increase in the expression of CHOP were detected. After the vacuolization of the cytoplasm, functional disorders of mitochondria and an increase in the generation of superoxide anion in them occurred. Taken together, the results obtained indicate that DDC and B12b used in combination exert a synergistic toxic effect on tumor cells by causing severe ER stress, extensive ER vacuolization, and inhibition of apoptosis, which ultimately leads to the induction of paraptosis-like cell death.

The mechanism of cytotoxic action of acetylcysteine in combination with vitamin B12b

The mechanism of cytotoxic action of acetylcysteine in combination with vitamin B12b

Prooxidant and cytotoxic action of N-acetylcysteine and glutathione in combinations with vitamin B12b

We studied the prooxidant and cytotoxic action of thiols N-acetylcystein (NAC) and glutathione (GSH) combined with vitamin Bl2b. The synergism of action of the thiols and Bl2b resulted in human carcinoma cell damage was found. It was shown that GSH and NAC in physiological doses combined with Bl2b caused the initiation of apoptosis. It was established that prooxidant action of the thiols combined with vitamin Bl2b, i. e. generation and accumulation of hydrogen peroxide in culture medium, led to intracellular oxidative stress and injury of cell redox system. These effects were completely abolished by nonthiol antioxidants catalase and pyruvate. The chelators of iron phenanthroline and deferoxamine did not suppress the H2O2 accumulation in culture medium but significantly inhibited the cell death induced by the thiols combined with Bl2b. Therefore, the thiols GSH and NAC widely used as antioxidants, in combination with vitamin Bl2b show prooxidant characteristics and induce, with the participation of intracellular iron, apoptotic HEp-2 cell death.

Prooxidant and Cytotoxic Effects of Thiols Combined with Vitamin B12b

Prooxidant and Cytotoxic Effects of Thiols Combined with Vitamin B12b

Oxidative stress in HEp-2 human laryngeal carcinoma cells induced by combination of vitamins B12b and C.

Incubation of human laryngeal epidermoid carcinoma HEp-2 cells with hydroxocobalamin (vitamin B12b) and ascorbic acid (vitamin C) for 1 h initiated oxidative stress accompanied by damage to mitochondria and increase in intracellular oxidative activity. Studies of the kinetics of these processes showed that the increase in intracellular H2O2 activity and mitochondrial damage are more likely a result, but not the cause of cell apoptosis during the first hour of their incubation with vitamins B12b and C.

Combined treatment with vitamin B12b and ascorbic acid causes in vitro DNA degradation in tumor cells.

Incubation of Ehrlich ascites carcinoma and HEp-2 human epidermoid laryngeal carcinoma cells with hydroxycobalamin (vitamin B12b) and ascorbic acid induced generation and accumulation of double-stranded DNA fragments (23,000 b.p. and longer) in cells. The same vitamins alone in the same concentrations produced no such effects. DNA degradation in HEp-2 cells caused by long-term (4 h) incubation with 5-25 microM hydroxycobalamin and ascorbic acid (1:10-1:40 molar ratio) at 37 degrees C was comparable with that induced by gamma-irradiation in a dose of 150 Gy at 4 degrees C.

Combined vitamins Bl2b and C induce the glutathione depletion and the death of epidermoid human larynx carcinoma cells HEp-2.

The combination of hydroxocobalamin (vitamin B12b) and ascorbic acid (vitamin C) can cause the death of tumor cells at the concentrations of the components at which they are nontoxic when administered separately. This cytotoxic action on epidermoid human larynx carcinoma cells HEp-2 in vitro is shown to be due to the hydrogen peroxide generated by the combination of vitamins B12b and C. The drop in the glutathione level preceding cell death was found to be the result of combined action of the vitamins. It is supposed that the induction of cell death by combined action of vitamins B12b and C is connected to the damage of the cell redox system.

Influence of the Axial Alkyl Ligand on the Reduction Potential of Alkylcob(III)alamins and Alkylcob(III)yrinates

AbstractThe irreversible‐reduction potentials of 26 alkylcob(III)alamins (RCblIII 1a–z) and 26 alkylcob(III)yrinates (R‘Cby’III; 2a–z) (Ep 1a–z and Ep 2a–z, resp.) have been measured in situ by single‐scan voltammetry of hydroxocob(III)alamin hydrochloride (vitamin B12b‐ HCl; 1) or heptamethyl cob(II)yrinate perchlorate (ClO4‘Cby’II; 2) in presence of the corresponding alkyl halides (RX;3a–z) in DMF. The reduction potentials of alkylcobalt complexes exhibiting half‐life times as short as a few seconds become measurable by this technique. Thermodynamic cycles prove that the observed reduction potentials are closely related to the standard reduction potentials E°(RCoIII + e−⇆R⋅ + CoI). Electron‐withdrawing groups and/or an increased degree of substitution at the Co‐bound C‐atom in RCblIII and, R‘Cby’III shift Ep(1a–z) and Ep (2a–z) towards positive potentials. Linear correlations have been found between Ep(1a–z) (Ep(2a–z)) of RCblIII (R‘Cby’III) and the pKa of RH (or the Taft σ*‐ or the Hammett σ‐values of R) within each class of R, i. e. MeCblIII (Me‘Cby’III), primary RCblIII (R‘Cby’III) and secondary RCblIII (R‘Cby’III). The correlations allow to distinguish between electronic effects of the Co‐bound alkyl residues and their steric interactions with the corrin side chains. The correlations have further been used to visualize the light‐induced formal insertion of an olefin into the Co, C‐bond of an alkylcobalamin (Scheme 2, 1a→1u), a key step in the vitamin‐B12‐catalized C, C‐bond formation.

Vitamin B-12b as an antidote for over-sedation

Several observations have led the author to conclude that massive injections of vitamin B-12b (hydroxocobalamin) will prove an excellent antidote for patients overdosed with sedatives/tranquilizers and/or alcohol.

Vitamin B-12: Placebo or neglected therapeutic tool?

Patients with normal serum vitamin B-12 levels were given injections of hydroxocobalamin (vitamin B-12b) interspersed with injections of sterile water. Those who felt better with B-12b had additional injections to establish the amount of B-12b that gave the maximum feeling of well-being. Serum vitamin B-12 tests were performed to record the level at which the patients felt best. On the same day they took a Multiphasic Personality Inventory (MMPI), an objective computerized psychological test. Hydroxocobalamin injections were then discontinued for 5 to 7 days. The patients took another serum vitamin B-12 test and another MMPI. With the higher vitamin B-12 levels, the MMPI patterns were at or closer to normal. With lower serum vitamin B-12 levels, MMPI patterns showed much more emotional distress. The author suspects that vitamin B-12 dependency disorders are common and are neglected by the medical profession.

Effect of vitamin B12b injections on the growth of young Merino sheep

In two experiments near Robe in the south-east of South Australia, on soil highly prone to the occurrence of cobalt deficiency in sheep, the effect of injections of vitamin B12b (hydroxocobalamin) on grossly cobalt-deficient weaner sheep and suckling lambs was compared with the effect of oral doses of cobalt as liquid drenches or ruminal pellets. All the supplements used were effective in treating cobalt-deficient weaned sheep or preventing the onset of cobalt deficiency in lambs. A single injection of vitamin B12b (1 mg), under the conditions of these experiments, satisfied the requirements of lambs for 14 weeks and cobalt-deficient weaner sheep for up to 40 weeks. The use of vitamin B12b in preventing or correcting cobalt deficiency in sheep in the field is discussed. Serum vitamin B12b concentration was not a reliable indicator of the vitamin B12b status of young sheep which had been given single injections of vitamin B12b. Vitamin B12b, deficiency in sheep was associated withdecreased concentrations of blood haemoglobin and of glucose, total proteins, urea and cholesterol in blood serum. The activities of serum aspartate transminase and alkaline phosphatase did not appear to be affected by the vitamin B12b status of the sheep.

The diagnosis and treatment of vitamin B12 deficiency in young lambs.

Abstract Extract In the course of some earlier experiments, it was observed that vitamin B12 deficiency in 2 hoggets was corrected by injection of hydroxocobalamin (vitamin B12b) (E. D. Andrews, pers. comm., 1972). Since this form of therapy offered a convenient and effective means of treating young lambs suffering from vitamin B12 deficiency, more detailed investigations were desirable. It has been known for some time that lambs on the cobalt-deficient area at the Ministry of Agriculture and Fisheries Kaitoke Farm lose condition after weaning. Abnormal amounts of methylmalonic acid (MMA) had been found in the urine of some of these lambs, indicating a state of vitamin B12 deficiency Andrews et al, (1970 Andrews, E. D., Hogan, K. G., Stephenson, B. J., White, D. A. and Elliott, D. C. 1970. Cobalt and thiabendazole live-weight responses in grazing sheep, and their relation to the urinary excretion of methylmalonic acid. N.Z. Jl agric. Res., 13: 950–965. [Taylor & Francis Online], [Web of Science ®] , [Google Scholar]).

Isolation of Coenzyme B12 from Liver

The identity of the major cobamide in liver has not been satisfactorily established. Cyanocobalamin (vitamin B12, 5,6dimethylbenzimidazolylcobamide cyanide), which has been obtained in crystalline form with liver as a source material (1, 2), is certainly a very minor component of the cobalamins in liver. Analyses of liver concentrates, both by a paper chromatographicbioautographic method (3) and by a chemical method (4), have shown that cyanocobalamin is usually not present in detectable amounts and seldom constitutes as much as 10% of the total cobalamin. The preparation of cyanocobalamin from liver probably was made possible by the presence of cyanide in the charcoal used in the isolation process. Examination of a liver extract by the chromatographic-bioautographic method (3) indicated that hydroxocobalamin (vitamin B12b, 5,6-dimethylbenzimidazolylcobamide hydroxide) was the most abundant cobalamin. This compound was also obtained in pure form from liver (1, 5). However, the recent discovery of the cobamide coenzymes in bacteria (6,7) and the demonstration that they constitute a large part of the total cobamides in these organisms (8,9) raised the possibility that coenzyme Bit (5,6-dimethylbenzimidazolylcobamide coenzyme, DMBC coenzyme, DBC coenzyme) may be a constituent of liver. Since coenzyme B12, like the adenylcobamide coenzyme (lo), is relatively unstable and is readily converted to hydroxocobalamin or cyanocobalamin by treatment with light or cyanide,’ respectively, the inadvertent decomposition of the coenzyme, if present in liver extracts, could account for previous observations on the occurrence of the two cobalamin vitamins. The present paper reports some observations on the occurrence and abundance of a cobamide coenzyme, which has been identified as coenzyme Blz, in livers of rabbit, chicken, sheep, and man. A brief report of the occurrence of coenzyme Blz in rabbit liver has already appeared (7).

The Vitamin B12 Activity of Natural Material with Particular Reference to Cod Liver Protein

Investigations concerning the stability of a group of factors contained in cod liver residue and displaying vitamin B12 activity have led to the conclusion that under the conditions prevailing during the rendering process, as developed at the Atlantic Fisheries Experimental Station, little or no deactivation occurs. This is in sharp contrast with what occurs under more drastic conditions such as used in the alkali-digestion process.The study of the microbiological method used for the determination of vitamin B12 activity has shown that one step of the analytical procedure, namely, that phase during which the extraction of the sample is performed, is responsible for anomalous results. Deactivation occurs during this stage to an extent which varies according to the previous history of the material. The addition of thioglycolic acid or ascorbic acid during this period does not effectively protect the material unless culture medium is added as well. Medium alone is inefficient. Sodium bisulphite on the other hand exerts protection even in the absence of medium. This effect of sodium bisulphite applies not only to cod liver residue, but also to materials of diverse origin, mammalian viscera included. The data obtained show that vitamin B12 itself is not involved; presumably vitamin B12a and vitamin B12b are, although there is no direct evidence of their presence in cod liver.As a protection during extraction, 0.08 per cent to 0.1 per cent of sodium bisulphite calculated on the amount of wet material appeared to be optimum.

Stability of Vitamin B12 and B12b

Stability of Vitamin B12 and B12b

Cobalamin (vitamin B12) and the intrinsic factor of Castle.

Excerpt Vitamin B12, originally isolated by Rickes and associates1in the United States and by Lester Smith2in England, is now known to be but one of a group of related compounds for which the gener...

Observations on the Etiologic Relationship of Achylia Gastrica to Pernicious Anemia: XIII. Hematopoietic Activity of Vitamin B12a (Vitamin B12b)

Abstract1. Vitamin B12a, derived from vitamin B12 by catalytic hydrogenation, is as potent a hematopoietic agent as vitamin B12 when administered parenterally to patients with pernicious anemia in relapse.2. The hematopoietic activity of vitamin B12a, like that of vitamin B12, and of vitamin B12b, is potentiated by simultaneous oral administration with normal human gastric juice.3. Observations on one patient suggest that vitamin B12a, like vitamin B12, will arrest the progress of subacute degeneration of the spinal cord in pernicious anemia.

The magnetic susceptibility of vitamin B12 and B12b.

The magnetic susceptibility of vitamin B12 and B12b.