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`ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY
`
`Editorial Board:
`
`NATHAN BACK, State University of New York at Buffalo
`
`NICHOLAS R. DI LUZIO, Tulane University School of Medicine
`
`EPHRAIM KATCHALSKI-KATZIR, The Weizmann Institute of Science
`
`DAVID KR1TCHEVSKY, tVistar Institute
`
`ABEL LAJTHA, Rockland Research Institute
`
`RODOLFO PAOLETTI, University of Milan
`
`Recent Volumes in this Series
`
`Volume 201
`L1POPROTEIN DEFICIENCY SYNDROMES
`Edited by Aubie Angel and Jiri Frohlich
`
`Volume 202
`INFECTIONS IN THE IMMUNOCOMPROMISED HOST
`Laboratory Diagnosis and Treatment
`Edited by Paul Actor, Alan Evangelista, James Poupard, and Eileen Hinks
`
`Volume 203
`EXCITATORY AMINO ACIDS AND EPILEPSY
`Edited by Robert Schwarcz and Yehezkel Ben-Ari
`
`Volume 204
`NEUROBIOLOGY OF CENTRAL D~-DOPMAINE RECEPTORS
`Edited b~y George R. Breese and Ian Creese
`
`Volume 205
`MOLECULAR AND CELLULAR ASPECTS OF REPRODUCTION
`Edited by Dharam S. Dhindsa and Om P. Bahl
`
`Volume 206
`ESSENTIAL NUTRIENTS IN CARCINOGENESIS
`Edited by Lionel A, Poirier, Paul M. Newberne, and
`Michael W. Pariza
`
`Volume 207
`THE MOLECULAR AND CELLULAR BIOLOGY OF FERTILIZATION
`Edited by Jerry L. Hedriek
`
`Volume 208
`PHOSPHATE AND MINERAL HOMEOSTASIS
`Edited by Shaul G. Massry, Michel Olmer, and
`Eberhard Ritz
`
`A Continuation Order Plan is available for this series, A continuation order will bring delivery of each new volume
`immediately upon publication. Volumes are billed only upon actual shipment. For further information please contact
`the publisher.
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`22
`
`THE ROLE OF VITAMIN BI2 AND FOLATE IN CARCINOGENESIS*
`
`Victor Herbert
`
`Department of Medicine
`Mount Sinai School of Medicine
`New York, New York, and
`Hematology and Nutrition Laboratory
`Bronx Veterans Administration Medical Center
`Bronx, New York 10468
`
`ABSTRACT
`
`The roles of vitamin BI2 and folate in carcinogenesis are largely
`extensions of and linked to their roles in normal metabolism, particularly
`l-carbon unit metabolism. A possible key area may be hypomethylation to
`"switch on" genes and methylation to "switch them off." Some vitamin
`analogues may act as antivitamins in these reactions~ as may some vitamin-
`binding proteins° Others may act as specific delivery proteins. Using
`appropriate radioactive substrates and suspensions of vitamin-dependent
`normal and malignant cells, it may be possible to work out their positive
`and negative control of DNA synthesis.
`
`INTRODUCTION
`
`The roles of vitamin BI2 and folate in carcinogenesis are largely
`extensions of and linked to their roles in normal metabolism. One key
`area is the conversion of homocysteine to methionine (methyl homocysteine)o
`This process is dependent on folate delivering its l-carbon unit to vitamin
`BI2, which then becomes methyl-Bl2 and transfers that methyl unit to homo-
`cysteine (Fig. I)o Newberne et alo1 recently reviewed the role of the
`lipotropes choline and methionine and related factors in oncogenesis,
`including the impaired hormonal and cell-mediated immunity in folate-
`deficient humans and animals, and they pointed out the synergism between
`3
`2
`high fat die.ts and methyl deprivation. Poirier reviewed the protective
`effect of methionine against hepatocarcinogenesis, and Farber4 discussed
`the carcinogenesis promotion effect of the ethyl analogue of methionine,
`ethionineo
`
`ABBREVIATIONS: azaC = 5-azacytidine; TC II = transcobalamin II; PGA =
`pteroglutamic acid; SAM = S__-adenosylmethionine; dU = deoxyuridine;
`AIDS = acquired immunodeficiency syndrome; dThd = thymidine; PHA =
`phytohemagglutinin Ao
`*Supported in part by the Research Service of the UoS. Veterans
`Administration and U.S. Public Health Service Grant AM35709.
`
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`AMe = S-adenosylmethionine,
`1 =serine hydroxymelhyllranslerase
`2 = methylene THF reduclase
`3 = homocysleine transmelhylase (melhyllransferase)
`4 = thymidylale synlhelase
`
`(The numbers represenl enzymes)
`
`DNA
`
`Thymldylate
`
`DHF
`
`5 = |ormiminolransferase
`THF = telrahydrofolate
`DHF = dihydrololale
`B,, = reduced Vitamin
`
`Formlmlno THF
`
`Glutamate
`
`Formlmlnoglutamate ~
`
`Serlne ~ Cystathlonine
`
`F Homocystelne
`
`MethyI-B,~
`
`~
`Enzyme
`
`AMe
`
`Urocanate
`
`,~
`
`Histldlne
`
`=lne
`
`D~oxyurldylate
`
`Methl~nlne
`
`Enzyme
`
`N~,,OMethyleneTHF ,~--~-~----I..~N,MethylTHF
`
`Fig.
`
`Biochemical interrelationships between vitamin BI2 and folate in
`human metabolism.
`
`In a series of painstaking studies, Poirier’s group5 determined that,
`over a 76-week period, dietary methyl deficiency markedly promoted liver
`carcinogenesis and exhibited complete carcinogenic activity in this organ
`in the rat. They showed this in rats fed methyl-deflcient, amino acid-
`deficient diets. When the diets were also devoid of folic acid and
`vitamin B12, the diethylnitrosamine-initiated rats died within 23 experi-
`mental weeks, before developing hepatocellular carcinoma, but all had
`livers containing hepatocytes of atypical appearance and, particularly
`at the 2 higher dosages of diethylnitrosamine, a cirrhotic pseudonodular
`architecture. They also found neoplastic conversion of rat liver
`epithelial cells in culture by ethionine and ~-adenosylethionine.6
`
`Krumdieck7 reviewed the literature pertaining to the role of folate
`deficiency in facilitating carcinogenesis through 1982 and Eto8 has
`
`carried the subject through the beginning of 1985. 9F~ate is essential
`in the biosynthesis of both purines and pyrimidines ’ and therefore is
`required by all dividing cells. The conversion of deoxyuridylate to
`thymidylate (methyldeoxyuridylate) is folate- and B1o-dependent, involving
`.~ 9-11
`these 2 vitamins in a key step in DNA synthesls (Fig. i)o
`These
`biochemical facts underlie the chromosom~l abnormalities~that characterize
`human clinical deficiency of vitamin BI2~ and/or folate.12’13 ~4wide
`range of chemical carcinogens inhibit DNA methylation in vitro. It has
`been suggested that deficiency of folate or vitamin BI2 or any cause of
`failure to methylate DNA and/or RNA can activate malignancy by hypomethyl-
`ating oncogenes, leading to such gene expression and/or gene amplications,
`and that methylating oncogenes can inhibit malignancy by making them
`
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`dormanto3’15 This is similar to the concept of "relaxed co~rol" of RNA
`synthesis, discussed B decades ago by Borek and co-workerso v They noted
`that when an organism auxotrophic for methionine is deprived of methionine,
`it loses its ability to suppress synthesis of RNA, which is then synthe-
`sized more rapidly; they tied that observation to methylation of RNAo We
`speculated that vitamin BI2 or folate deficienc~ could produce such
`5
`"relaxed control," - and we noted more recently ~hat folate, vitamin BI2,
`and their antagonists could be involved in the control of normal gene
`expression if in fact hypomethylation of DNA "switches on" normal genes
`and methylation "switches them off.’’18 Although the evidence of this
`process is significant but inconclusive, one would expect that hypomethyla-
`tioa of the DNA or RNA of oncogenes would activate them and methylation
`would inactivate them. Perhaps some of the second cancers that develop
`after successful antimetabolic chemotherapy are due to the same chemo-
`therapy that directly destroys an active cancer, demethylating an oncogene
`of a dormant cancer.
`
`Gene amplification is a mechanism for tumor resistance to anti-
`metabolites.19 One can speculate that it may also be a mechanism to aid
`in tumor proliferation by, for example, producing gene2~mplification of
`the hepatic Phas@ I enzymes that activate carcinogens°
`
`Gautsch and Wilson21 found that de novo methylation of the input
`provirus occurs in embryonal carcinoma cells but not in permissive, differ-
`entiated teratocarcinoma. Harrison et alo22 demonstrated a 3-way correla-
`tion between tumorigenicity, trisomy for 3q, and specific demethylation,
`suggesting that decreased DNA methylation may be involved both in differ-
`entiation and in tumorigenicity and that the antileukemia drug azaC may
`induce chromosomal aberrations as well as altering DNA methylation.
`Altering DNA methylation is just one of the varied effects of azaC on
`cellular metabolism.23-25 The drug reduces DNA methylation and induces
`theoretically therapeutically valuable differentiation of human promyelo-
`eytic leukemia cells (HL-60) in culture, although this induction is less
`effective than that brought about in these cells by dimethylsulfoxide and
`L-ethionine.26 Anderson and colleagues25 found that azaC selectively
`hypomethylates fetal glo~n genes, supporting work by Ley et al. and
`Charache and associates.
`
`Patients with neoplasms excrete el~a~d levels of certain methylated
`bases in their urine, and Borek s group - has been attempting to corre-
`late the quantity of such excretion with the de~ree of tumor activity.
`Gross’s group, in collaboration with our group,15,31,32 were unable to
`show any reproducible inhibitory effect of 5-methylcytidine on the
`development of presumably RNA virus-induced transplanted L2C leukemia-
`lymphoma in guinea pigs; this appears to be an animal analogue to human
`leukemia-lymphoma of RNA virus etiologyo33’34 After we switched to
`5-iodocytidine, whichseemed more promising, both grouP~5UnSUCCessfully
`sought funding targeted to continue this work. Gross’s recent dramatic
`report of reduction in the incidence (i.eo, the initial development) of
`radiation-induced tumors in rats after restriction of caloric food intake
`has been associated with renewed funding. His group previously noted that
`restriction of food intake will not significantly influence the growth or
`progress of established tumors in mice.32 ~nerican Cancer Society
`statistics suggest an increased frequency of malignancy in obese persons
`(Lo Gross, personal communication).
`
`The roles of vitamin BI2 and folate in carcinogenesis are not at the
`simple level at which serum vitamin levels correlate with extent of
`disease. No correlation has been found between seru~6folate and vitamin
`B12 levels and the extent of small cell lung cancer. However, a corre-
`lation may exist between levels of certain naturally occurring folate and
`
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`BI2 analogues in serum and/or tissues and malignancy° Some folate and BI2
`analogues may not only be vitamln-inactive for humans~ but they may
`facilitate carcinogenesis or inhibit it by blocking normal vitamin action
`or in other ways° We now know, for example~ that what is assayed as "B12"
`in serum by most microbiological and radloassays is in fact a mixture of
`cobalamins and non~Qbalamin corrinoids (Figo^~);~7 this is also true of
`the B12 in tissues~8 and multivitamin pills.3~O Enormous amounts of this
`
`A
`
`A=Analogues (i.e., cobalamins plus
`all other corrinoids)
`
`B~=Cobalamins (biologically active Bt~)
`Cbi=Cobinamide (a corrinoid)
`
`IF=Intrinsic factor
`
`R= R-binders
`
`Serum BI2o The "serum B12" consists of the sum of "true B12"
`(i.e., hydroxocobalamin and other cobalamins biologically active
`for humans), designated BI2, plus the serum content of other
`corrlnoids (i.eo, molecules that have the heme-like corrin
`nucleus of cobalamin but differ from cobalamin in part or all
`of the rest of their structure), designated A (analogues). ~Pure
`gastric IF (intrinsic factor) binds only cobalamins, whereas R
`binders (the BI2 binders ubiquitous in body fluids, including
`saliva, serum, cerebrospinal fluid, bile, and urine) bind all
`corrinoids (cobalamin + analogues)° The brackets identify the
`portions of total serum corrinoids bound by each of 4 different
`commercially available B12 radioassay binders. The mixture
`(IF + R + ~ Cbi) binds a little more than BI2 alone and, there-
`fore, more than pure IF does, when the amount (~) of preadded
`cobinamide exceeds the cobalamin-binding capacity of the mixture
`by about 100-fold; if the excess reaches 1,000-fold~ there will
`be some binding to IF despite its specificity for cobalamins,
`and the mixture will therefore bind a little less than true B12.
`Because of the specificity of its IF portion for cobalamin, (IF
`+ R) binds a bit less than the total corrinoids that are bound
`by pure Ro
`Important note. Confusion can arise when results are
`reported as "serum vitamin BI2 levels," which are actually serum
`total corrinoid levels, but the range of normal given with the
`report is for cobalamins rather than total corrinoids. For maxi-
`mum reliability, each laboratory should devel’op its own range of
`normal for whatever serum BI2 assay(s) it uses and should not use
`the range of normal determined in a different laboratory~ because
`minor differences in methodology such as pH produce different
`values° Reprinted with permission from refo 45.
`
`Fig. 2~
`
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`Table I. Cobalamin Content Versus Total Corrinoid Content
`of Spirulinaa
`
`Corrinoid Content, pg
`
`Radioassay
`
`Total
`BI2 Claim Lactobacillus Euglena
`on Bottle leichmanii
`gracilis Corrinoids Cobalamin Analogues
`
`6 pg
`
`1.24
`
`0.615
`
`1.63
`
`0°09
`
`1.54
`
`aAll values are for 6 tablets, which is the daily dose recommended
`by the manufacturer, the Earthrise Company. See refo 38.
`
`BI2 have been found in the "health food" ~pirulina (Table i),41’42 in human
`colon, and lesser amounts in many foods.4~ Nome of these analogues are
`40 4~
`antimetabolites; their role in inhibiting or promoting carcinogenesis,
`or even as direct carcinogens, remains to be determined, as do their
`levels in serum, tissues, bile, and colo~ of patients with and without
`various neoplasms° Furthermore, just as serum iron i~ attached to protein
`in both a "delivery" form (on transferrin) in equilibrium with parenchvmal
`iron and a "storage" form (ferriti~ in equil~rium with storage iron,~5
`the same may prove true for folate~ and B12. ° Loss of BI2 from TC II
`appears to be an earlier indicator ~ parenchymal BI2 deficiency than
`clear reduction of total serum B12,~ suggesting that BI2 on TC II is
`equilibrated with bioavailable tissue BI2, whereas the greater amount of
`BI2 on TC (I + Ill) is not. A recent review of the macromolecules involved
`in the assimilation and transport of cobalamin discusses their known
`functions.
`
`FOLATE DEFICIENCY
`
`Some years ago, Heller and his associates50 observed that folate
`deficiency increased normal hemoglobin production in a patient with sickle
`cell trait. Since higher normal hemoglobin production means lower sickle
`cell hemoglobin, this was a desirable phenomenon. However, treatment
`with folic acid produced the undesirable result of lowering the normal
`hemoglobin.
`
`Recognizing that the primary role of folate is in transferring
`l-carbon units, one of which is the methyl unit, Heller and others focused
`on cytidine, the most heavily methylated of the 4 bases of the genetic
`code (the other sometimes methylated base is guanine). In 1982, they
`reported that azaC selectively activates the gene for fetal hemoglobin
`synthesis in patients with beta-thalassemia and sickle cell diseaseo18’51
`They raised the possibility that this activation ~esults from the incorpor-
`ation of azaC as cytidine into the gene for hemoglobin, which, with its 5
`position blocked, cannot take on the methyl group made available in folate-
`(and vitamin BI2-) dependent reactions and thus was not methylated. It
`was suggested that failure to methylate the cytidine of the gene for fetal
`hemoglobin prevented it from becoming dormant, and it was activated to
`produce fetal hemoglobin° Whether or not the bases that make up the
`genetic code are methylated plays a major role in gene expression.52’53
`Normally, 4-5% of cytosine residues in human DNA are methylatedo54 Whether
`oncogene expression can similarly be prevented by increasing ordinarily
`
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`
`vitamin B1o-dependent and folate-dependent l-carbon transfer to the genetic
`code of t~ oncogene has been the subject of subsequent studies.21’22,24-26
`
`The B vitamin, folic acid, was isolated in 1943: Stokstad Durified
`PGA and Pfiffner and associates crystallized folate from livero55 Within
`a year, Leuchtenberger and associates at Mount Sinai Hospital56’57 reported
`that a form of this vitamin called folic acid concentrate [later to be
`known as’ oxidized folate triglutamate (teropterin)] inhibited the growth
`of transplanted sarcoma 180 in mice~ This material and similar crystalline
`oxidized triglutamate known as fermentation Lactobacillus casei factor
`produced complete, regression of single spontaneous breast cancers in
`mice.58 One wonders if oxidized folate triglutamate could methylate
`sarcoma 180 and mouse breast cancer onco~eneso Leuchtenberger’s first
`paper stated that Pollack and associates59 had reported that fermentation
`L. casei factor (folic acid in triglutamate form60) was present in human
`and rat cancers at higher levels than inositol and at much higher levels
`than biotin or pyridoxineo
`
`Laszlo and Leuchtenberger61 reported that inositol (then believed
`to be a B vitamin for humans but more recently shown to be a B vitamin
`only for some bacteria, since it is synthesized adequately by humans62)
`inhibited animal tumor growth~ but other B vitamins did not° Vitamin B12
`was yet to be discovered.
`
`Lewisohn et al.63 reported that not only did oxidized folate mono-
`glutamate (liver L. casei factor from Lederle Laboratory) not inhibit
`spontaneous breast cancers in mice, but it actually produced a more rapid
`growth of the primary tumors and a significant increase in lung metastases°
`Here was evidence that one of the monoglutamate forms of the vitamin
`promoted tumor growth but that one of the triglutamate forms for the same
`vitamin inhibited the growth of the same tumor. Recent studies were aimed
`at determining whether oxidized folate monoglutamate may inhibit methyla-
`tion of oncogenes, thereby allowing their expression, and whether trigluta-
`mates can do the opposite. Folate triglutamates are frequently more
`
`
`a r Folate t iglutam te
`n 64,65 active than monoglutamates in methylation in ma .
`
`has much greater affinity than folate monoglutamate for milk folate-binding
`protein,66 which appears to be a folate delivery protein.67 Gene amplifi-
`cation can lead to overproduction of certain proteins, including transport
`and delivery proteins as well as enzymes. Chabner’s group68 isolated a
`methotrexate-resistant mutant that could not polyglutamate methotrexate,
`causing the drug to leak out of the cell.
`
`These and other studies suggest that one form of a vitamin can be a
`growth promoter~ by acting as a coenzyme, while another form can attach
`to the same apoenzyme or other ligand, such as a vitamin-transportlng
`protein, and interfere with the reaction, just as a key with a missing
`tooth can fit into a lock but will not turn° Different foKms and major
`parts of the same basic vitamin structure~ that is, analogues and
`congeners, exist in nature and are synthesizable; some are antagonists
`or antivitamins~ some of which can be created from vitamins by only
`slightly warping their structure°
`
`Farber et al°60 gave pteroyltriglutamic acid (teropterin) and
`pteroyldiglutamic acid (diopterin), both Synthesized by Yo SubbaRow and
`his associates at Lederle Laboratories, to 90 patients with various
`malignancies, noting that "in general~ adult patients experienced
`improvement in energy, appetite, sense of well beingooomight be ascribed
`to improved morale from frequent visits, more medical attention°.°" They
`also reported inconstant temporary decreases in size of metastases in
`some tumors and degeneration and necrosis in others° The observation by
`Welch69 of rapid deterioration of adult patients with chronic myeloge-
`
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`
`nous leukemia given folic acid led to the earliest attempts at therapy in
`2 patients by limiting the availability of folate through the use of
`x-methyl folio acid, coupled with succinylsulfathathioazole and low-folate
`diets~ Both patients went into remission, stopped following the low-folate
`diets, and quickly relapsed.
`
`It is apocryphally alleged that Farber was also giving the oxidized,
`stable Nharmaceutical form of folic acid (ioe., PGA) to children with
`lymphoproliferative malignancies (lymphocytic leukemia and lymphoma)
`until one of his residents collected sufficient data to suggest that the
`children receiving this new vitamin were dying faster than those children
`not receiving it. This observation allegedly led Farber to ask Lederle
`to create a warped folic acid molecule that would interfere with folate
`metabolism in the malignant cells; this was done by adding an NH2 group,
`thereby creating aminopterin. A second alteration, methylation in the i0
`position, created methotrexate, still one of our most potent anticancer
`agents, particularly effective against childhood lymphoproliferative
`disorders and trophoblastic malignancies.
`
`Rapidly growing neoplastic tissue consumes folate so rapidly that
`folate deficiency megaloblastosis can occur in the host cellso70,71
`There is also evidence that tumor growth may be slowed by any form of
`vitamin BI2 deficiency, including inadequate absorption or elevated levels
`in ser~n of a vitamin BI2 binder that does not deliver the vitamin to
`tumor tissue72 but does deliver it to the liver in a calcium-dependent
`fashion.70’73-76 Granulocytes and liver are major sources of serum-
`binding proteins for both vitamin B12 and folic acid that tightly bind
`those vitamins; malignancies of granulocytes and liver theoretically can
`repress themselves by releasing large amounts of these binders, which
`could tie up supplies of the vitamins and prevent vitamin delivery to,
`and nourishment of, the malignancyo47’77
`
`Oxidized folate (such as PGA, the stable pharmaceutical form of the
`vitamin) is not per se metabolically active and may even be neurotoxic;
`it has a structural similarity to Dilantino78 PGA can produce seizures
`in patients wtih epilepsy by blocking the protective action of Dilantin,
`79
`as Butterworth s group has shown. Folic acid and Dilantin compete for
`80
`"ntestinal absorption, and they probably also compete at the brain cell,
`i
`where Dilantin may interfere with ATPase~ as in the gut.81 There appears
`to be a one-way transport system to remove noxious oxidized folates from
`the nervous system and a one-way transport system to deliver useful~
`reduced folate into the nervous system;82 this information has been used
`to successfully treat with folinic acid a child with congenital folate
`malabsorption unresponsive to folic acid.83’84
`
`Similarly, there may be a one-~Y8~ransport system to remove noxious
`vitamin BI2 analogues from the body ’ and another transport system to
`deliver helpful forms of the vitamin to normal tissues, as7~e first
`
`reported for human serum delivery of vitamin BI2 to liver. Recent
`evidence suggests that vitamin Blo analogues in human tissues may arise
`primarily from human colon flora.~3
`
`The transport systems have different affinities for different forms
`of vitamin BI2 and folic acid and different delivery ability for helpful
`and noxious forms of the vitamins with respect to different normal cells
`46,47
`ut n 86
`and possibly also tumor cells° S herla d noted that folate
`deficiency produces fragile chromosomes° Das12 has noted that folate
`therapy will not correct folate deficiency in circulating human lymphocytes
`or their chromosomes for i-2 months after the start of therapy, whereas
`deficiency is corrected in bone marrow cells within 6 hours after the
`start of folate therapy° Fragile chromosomes can persist in lymphocytes
`
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`
`for 1 or 2 months after the beginning of replacement therapy for folate
`deficiency, because circulating lymphocytes are impervious to nutrients
`such as the B vitamins until they are triggered to make DNA by a virus
`or lectino12’87 The role of fragile chromosomes in carcinogenesis was
`recently reviewedo88
`
`Vitamin BI2 is not vitamin active in its stable pharmaceutical form,
`cyanocsbalamino The cyanide must be removed for vitamin function to
`occur. Even an active form of the vitamin, hydroxocobalamin, can block
`vitamin BI2 metabolism by competing with adenosylcobalamin for the binding9
`site on the adenosylcobalamin-dependent enzyme methyl malonyl-CoA mutaseo
`Conversely, the oncogenic potential of absorbed cyanide from cassava and
`other foods90 may be muted by inactivation of the cyanide by the metaboli-
`cally active form of vitamin BI2, hydroxocobalamino Indeed, anesthesiolo-
`gists have used massive doses of hydroxocobalamin to reverse cyanide
`toxicity by soakin~ up the cyanide from the nitroprusside used in open-
`heart operationso9~
`
`Butterworth92 noted that naturally occurring folate analogues such
`as pteroic acid may be lethal for rats and can displace folate from human
`tissues and flush it out in the urineo93 It remains to be determined
`whether there is a folate analogue, such as pteroic acid, that will
`selectively flush folate or vitamin BI2 out of tumor tissue or selectively
`deliver folate or vitamin BI2 to tumor ~i~ue to potentiate fluoropyrimi-
`dine or other drug antltumor activltyo9 ’
`
`Human serum, Dilantin, and methotrexate have been reported to inhibit
`pteroyl monoglutamate and methyl tetrahydrofolate uptake by human bone
`marrow cells in vitro, but 2-deoxyglucose, an antagonist of glucose, will
`enhance such uptakeo96 It would be worthwhile to determine whether simul-
`taneous administration of folate and 2-deoxyglucose are more harmful to
`tumor cells than to normal ones.
`
`A folate-free diet was given for more than 4 months, with no clinical
`benefit, to 7 patients with disseminated cancers° The folate levels in
`97
`tumor, liver, and blood all declined at the same rate. On the other
`hand, Whitehead and colleagues98 noted disappearance of megaloblastosis
`in cervical epithelial cells with folate therapy of women taking oral
`cont 99
`raceptives, and Butterworth et alo reported improvement in apparent
`cervical dysplasia in oral contraceptive users treated with folic acid.
`Longo and co-workersI00 noted human selective folate deficiency in the
`lymphocyte cell line after 4 months of oral contraceptive administration;
`lymphocytes and cervical epithelium may be similarly selectively folate
`deprived. Selective folate deficiency in one cell line and not another
`was first noted in 1962 in the first case of deliberately produced dietary
`folate deficiency in a volunteer, whose intestinal biopsy showed normal
`epithelial cells when his bone marrow had become megaloblasticoTM
`Presumably, the intestine epithelial cells took up the traces of folate
`in the folate-deficient diet to sustain their own normality~ leaving
`little or none to be absorbed and delivered to the bone marrow.
`
`Selective delivery of nutrients and antimet~,bolites to one cell line
`and not another is generally a function of selective transport and delivery
`protein and hlgh-affinity receptorsoI02
`
`VITAMIN BI2
`
`Shortly after Minot and Murphy reported that liver therapy cured
`pernicious anemia, an achievement for which they shared a Nobel prize
`with Whipple, Minot reported the development of a malignancy (polycythemia
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`vera) occurring immediately after the start of treatment for pernicious
`anemia. There were other sporadic reports of myeloproliferative disorders
`associated with therapy for pernicious anemia with liver extract and
`subsequently with pure vitamin BI2o The small number of such cases
`implies coincidence rather than cause and effect but does not prove it.
`
`Warped vitamin BI2 molecules (the anilide and ethylamide of vitamin
`B12, synthesized by Eo Lester Smith of Glaxo Laboratories) were first used
`a quarter of a century ago1~~ the treatment of myelogenous leukemia refrac-
`tory to all other therapy. One woman given the anilide of vitamin BI2
`appeared to go into complete remission, with her bone marrow aspirate
`changing from florid myelogenous leukemia to a fibrotic picture. However~
`she continued to have a small number of leukemic cells in her peripheral
`blood° When she died of pneumonia 6 months later, her marrow was largely
`fibrotic. ~ether this one case was cause and effect or coincidence is
`impossible to determine.
`
`At the Great Ormond Street Hospital for Sick Children in London~
`104
`Bodian found that megadoses of vitamin B prg~ced remission in neuro-
`12. Io~ . . ,
`blastoma in children, but Sawltsky and Desposlto dld not confirm thls
`in 103 children. Bodian believed that megadoses of vitamin produced
`maturation of the tumor~ but spontaneous maturation and spontaneous
`remission can occur in neuroblastoma with no therapy.
`
`Day et aloI06 and Ostryanina107 reported that vitamin B12 enhanced the
`carcinogenic effect of !-dimethylaminoazobenzene and 3 other carcinogens in
`rats consuming a methionine-deficient diet. Conversely, chemically induced
`tumors of the liver, colon, and esophagus are enhanc~SbY diets deficient
`
`in folic acid~ vitamin B]2, choline, and methionineo Poirier et alo5’6
`have elegantly and laboriously delineated this phenomenon°
`
`Folic acid and vitamin BI2 can prove useful in those tumors that grow
`more rapidly as more of these vitamins are supplied, because the tumor
`cells can be stimulated into the DNA synthesis phase in which a number of
`cancer chemotherapy agents exert their deadly effects. Those ag~t~5can
`be used in a sequence right after folic acid and/or vitamin BI2.~"’~
`
`37
`Analogues of vitamin BI2 appe~ to bel~lquitous in human se~m;
`red blood cells, liver, an~.~rain; bile; ~ multivitamin pills;~ a wide
`variety of microorganisms;~i and human colon content and feces.~ Two
`analogues from multivitamin pills have been reported to block vitamin BI2
`metabolism in normal human cells in vitroo40 The highest known pill
`content of~@nalogue of vitamin BI2 is in the health food fad pill
`spirulina.~I Such analogues must be evaluated for their a~lity to block
`vitamin BI2 metabolism in both normal and malignant cells. The ~ero-
`hepatic circulation functions to get rid of vitamin B12 analogues,~ a
`teleologic suggestion that such analogues may have undesirable effects°
`
`The anesthetic gas nitrous oxide can produce acute vitamin BI2
`deficiency, as was first made clear by Amess et aloIII Nitrous oxide
`produces a greater than 95% reduction in liver vitamin B~o in the fruit
`bat.I12 Scott and Weir in DublinI13 and Metz’s group in~outh AfricaI14
`showed that methionine can prevent the nerve damage produced by nitrous
`oxide-induced vitamin BI2 deficiency in primates and the fruit bat~
`respectively° Chanarin et al.I15 suggested that formyl folate monogluta-
`mate, with its formate derived from methylthioribose, is the preferred
`substrate for the polyglutamate forms of folate that normal cells prefer-
`entially use° Studies of the effects of nitrous oxide potentiation of
`cytotoxic drugs on tumors in experimental animals, with and without
`methionine "rescue" of normal cells, are of great interest. Kroes and
`associatesI16,117 showed that nitrous oxide retards leukemic proliferation
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`in a transplantable acute nonlymphocytic leukemia in BN rats and enhanced
`this cytostatic effect with cycloleucine, which inactivates methionine
`adenosyltransferase and thereby inhibits the formation of SAM. Cycloleucine
`reversal of dU suppression was greater than the sum of the reversals
`produced by either gas or drug alone.
`
`Differences in ability of normal versus tumor cells to take up
`various nutrients and antinutrients are being sought in efforts to kill
`tumor cells selectively. Normal cells treated with nitrous oxide to
`destroy vitamin BI2 are better rescued by methionine than by SAM, even
`though SAM is the active form, because methionine much more readily crosses
`normal cell wallsoI15 The relative ability of these 2 rescue agents to
`cross tumor versus normal cell walls requires further study°
`
`Because folic acid and vitamin B12 are intimately related to the
`synthesis of DNA, lack of either damages DNA synthesis. The primary