INFLUENCE OF METHYLCOBALAMIN ON THE
`
`ANTINEOPLASTIC ACTIVITY OF METHOTREXATE
`
`F. G. Arsenyan, N. V. Myasishcheva,
`Z. P. Sof’ina, M. O. Raushenbakh,
`I. P. Rudakova, E. G. Chauser,
`and A. M. Yurkevich
`
`UDC 615,277.3.015.2 : 615.355:577.
`152.611’.133
`
`One of the possible ways of increasing the selectivity of the action of chemotherapeutic substances on
`tumor cells is the combined use of preparations, taking the peculiarities of the mechanism of their action into
`account. A new trend in this field is the use of cobalamin derivatives in combination with definite antineo-
`plastic preparations.
`
`The special significance of methylcobalaminwas first noted in the case of impaired coba!amin metab-
`olism in leukemia patients. An analysis of the functional activity of cobalamin coenzymes in the organism,
`in comparison with the effectiveness of combined cytostatic therapy, has shown that the clinical course of the
`process in acute leukemia with an increased content of hydroxy- and methylcobalamins in the blood is less
`favorable [1]. The results obtained were evidence of the important role of methylcobalamin in metabolic
`processes as a coenzyme of methionine synthetase (EC 2.1.1.13)-a key link in the control of the synchronized
`action of cobalamins in compounds of folic acid in processes of cell proliferation [1-2].
`
`A study of the morphofunctional state of the hemopoietic system of animals under conditions of intensive
`cobalamin metabolism in the organism confirmed the fact that at a high concentration of cobalamin coenzymes,
`the rate of proliferation of ceils of the hemopoietic tissue increases. In the spleens of healthy mice, in the
`case of prolonged administration of methylcobalamin, hyperplssia of the lymphoid elements, an increase in the
`number of DNA- synthesizing cells, and an increase in their mitotic index were noted. The s tability of the
`periods of the mitotic cycle of spleen lymphocytes in the presence of an increase in the size of the proliferative
`
`TABLE 1. Stimulating Effects of Methyl-
`cobalamin on the Growth of Transplantable
`Tumors of Mice
`
`I I Incr~as~ in tumor
`
`I , ~ I volume after ad-
`
`~.~ I ~n~m-at~on of
`] me~ylcoba~
`[ % of con~ol
`
`~ne of
`
`~ day* x ~ day*
`
`Ca-755
`
`BDF~
`
`F~
`
`Fi
`AKATOL BALB, c
`RShM-5 CBA
`Sarcoma 37 SHK
`
`10
`
`5~
`500
`
`10
`~o
`~
`
`~"1,
`
`~-10~
`
`+45 [+~ +~0
`+77 +~Y ~5~
`
`+1~6[~-37 ~33
`o
`+4r ] o
`o
`+~r ] o
`
`* Period after transplantation of tumor.
`tP > 0.05, in all remaining cases P < 0.05.
`Note. Here and in Table 2: the preparation
`was administered on the second and sixth
`days after transplantation of the tumor.
`A "plus" sign denotes stimulation of
`tumor growth.
`
`Oncological Scientific Center of the Academy of Medical Sciences of the USSR. Scientific-Industrial
`Vitamin Combine, Moscow. Translated from Khimiko-Farmatsevticheskii Zhurna~, Vol. 12, No. 10, pp. 49-54,
`October, 1978. Original article submitted April 3, 1978.
`
`0091-150X/78/1210-1299 S07.50© 1979 Plenum Publishing Corporation
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`TABLE 2. Results of Combined Action of Methylcobalamin and Mcthotrexate on the Growth of Ca- 755 (BDF1)
`
`Preparations
`
`Dose of preparation
`
`10 mg/kg
`10 Dg/kg
`10 g/kg
`
`Methotrexate
`Ylethylcobalamin
`Methylcobalamin
`
`÷
`
`methotrexate
`Methylcobalamin
`
`+
`methotrexate
`
`Inhibition of tumor
`growth* after course of
`administration of prep-
`arations, % of control
`
`lst- 2nd
`day~
`
`94
`+ 180
`
`7-Sth
`day~
`
`51
`+ 65
`
`Increase in
`lifetime of
`animals, %
`
`195
`0
`
`10 mg/k4~ (simultaneously)
`10 ~glkg
`
`10/zg/kg (methotrexate was ad-
`ministrated 6 h after methyl-
`cobalamin}
`
`94
`
`76
`
`6O
`
`+ 36
`
`+ 62
`
`215
`
`*Average results of five series of experiments.
`~Period after transplantation of tumor.
`SP> 0.05; in all remaining cases P < 0.05.
`evaluated relative to methotrexate.
`
`In the case of combined influence, the results obtained were
`
`pool made it possible to conclude that the intensified lwolifcral.i(m of hemopoietic cells under these con-
`ditions is due to an increase in the number of cells entering the mitotic cycle [3- 5]. Further experimental
`investigations revealed the active role of methylcobalamin not only in processes of proliferation of cells of
`the hemopoietic tissue. An analogous influence on proliferative activity (an increase in the fraction of cells
`labeled with ~H]thymidine and an increase in the mitotic index) has also been detected in various periods of
`culturing of embryonic human fibroblasts in media with a high methylcobalamin concentration [6- 7].
`
`In view of the fact that normal cells of adult animals, embryonic and tumor cells differ in their ability
`to respond to the inducing influence of cobalamins, it was necessary to evaluate the action of methylcobalamin
`on processes of growth of various types of tumors.
`
`The stimulating influence of cyanooobalamin on the growth and development of certain transplantable
`(sarcoma 45, Guerin carcinoma, Walker oarcinosarcoma, sarcoma 180, Lewis sarcoma, etc.) and induced
`tumors is evidently due to its conversion to cobalamin coenzymes in the animal organism. Methyloobalamin
`and adenosylcobalamin have been detected in spleen ce.lls of mice with La Leukemia, as well as in leukemia
`L-1210 and Ehrlich’s asoites carcinoma cells [8-10].
`
`The aggregate of clinico-experimentaldatathus determined the advisability of the search for effective
`antagonists of oobalamins for the blocking of certain cobalamin-dependent reactions. In view of the activating
`influence of methylcobalamin on methionine synthetase and the increase in the total pool of tetrahydrofolic
`acid (THFA) in the cells, regardless of the folate reductase system, the greatest attention is attracted by
`antagonists of methylcobalamin [11-13]. In our investigations using methyloobalamin antagonists to lower the
`methionine synthetase activity, we succeeded in slowing down the processes of growth of bacterial and em-
`bryonic cells, as well as certain types of tumors [6, 14]. It was al~o shown that the antineoplastio activity of
`methotrexate - aspecifie inhibitor of folate reductase - increases when it is used An combination with methionine
`synthetase inhibitors [14].
`
`In addition, there is still another possibility of enhancing the antineoplastic activity of methotrexate
`with cobalamins. The prerequisite for this means of combined influenco with methotrexate was experimental
`data showing the ability of cobalamins to stimulate processes of proliferation and to increase the number of
`DNA-synthesizing cells, most sensitive to methotrexate, in the population [15, 4].
`
`The present communication presents the results of the combined action of methylcobalamin and metho-
`trexate on various transplantable tumors in animals.
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`EXPERIMENTAL
`
`The e~periments were conducted on mice of the Cs~BL, CBA, and BALB/c lines, the hybrids, BDFi-
`(Cs~BL× DBA/2), Ft(C58BL x CBA) and SHK mice, obtained from the nursery of the Academy of Medical -
`Sciences of the USSR. In the experiments we used 420 mice, weighing 20-25 g.
`
`The action of methylcobalamin was studied on solid tumors: adenocarcinoma of the mammary gland
`(Ca-755), cancer of the cervix (RShM-5), adenocarcinoma of the intestine (AKATOL), sarcoma 37~ as well
`as on leukemia L- 1210, according to the procedure used in the laboratory [16, 14].
`
`Ylethylcobalamin (CH3Cbl), synthesized according to the method of [17], was injected intramuscularly
`in doses of 10 and 500 Dgi%g twice at 96-h intervals or 500 Dg/k~g daily for five days.
`
`Methotrexate (from Lederle) was used in a dose of 10 mg/kg intraperitoneally twice at a 96-h interval.
`
`In part of the experiments, methylcobalamin chloropalladate (CH3Cbl. PdCI3; I) and dibromide-4-[[[[[[1-
`methylpyridino- 4- amino]phenyl]amino]carbouyl]phenyl]amino]- 6- amino- 1-methykluinoline (NSC- 176319;
`II), which we obtained from the National Cancer Institute of the United States according to the program of
`cooperation between the USSR and the US in the field of chemotherapy of tumors [18], were used as methtonine
`synthetase inhibitors.
`
`Complex I~ synthesized at the AH-Union Vitamin Scientific Research Institute [19], was administered
`perorally in a dose of 250 mg/kg; the quinolinium derivative II was administered intraperitoneally in a dose
`of 5 mg/kg twice at a 96-h interval.
`
`The treatment of the animals was begun 48 h after transplantation of the tumor. The antineoplastic
`effect was estimated directly after the end of the course of therapy and at various periods over the subsequent
`life of the animals.
`
`The criteria of effectiveness were the percent inhibition of tumor growth, calculated according to its
`volume, and the increase in the lifetime of the animals. The data obtained were subjected to statistical treat-
`ment according to the Student method.
`
`RESULTS AND DISCUSSION
`
`From the data that we obtsined it follows that methylcobalamin substantially stimulates the growth of
`transplantable tumors: Ca-755, AKATOL, and to a lesser degree RShNI-5 and sarcoma 37 (Table 1).
`
`The intensity of tumor growth depended on the line of experimental animals, the frequency of administra-
`tion, and the concentration of methylcobalamin. The greatest stimulatIng effect on growth of the tumor Ca- 755
`was noted in the case of two administrations of the preparation in a dose of 10 #g/kg after transplantation of
`the tumor into the hybrids BDFl (+180%), and to a lesser degree for mice of the pure line Cs~BL (+75%). In
`F1 hybrids, a substantial intensification of tumor growth was detected in the case of five administrations of
`methylcobalamin in a dose of 500/zg/a~go The stimulation of the growth of Ca- 755 and AKATOL was followed
`for a period of two to three weeks, whereas in mice with sarcoma 37 and RShM-5, it was noted only directly
`after the end of the course of administration of the preparation. In mice of the pure line (Cs~BL), intensified
`tumor growth was observed for a longer period (2-3 weeks after transplantation of the tumor) than in hybrids.
`For precisely this reason, in subsequent investigations of the actionof methyleobalamin and its analogs on the
`cell kinetics of Ca- 755, we used mice of the C~TBL line.
`
`In the case of simultaneous administration of methotrexate and methylcobalamin, an intensification of
`their inhibiting effect on tumor growth was observed (L-1210, Ca-755, RShM-5). The lifetime of animals
`with leukemia L-1210 was increased by 78% in this case, whereas in the case of isolated administration of
`methotrexate the increase was onl~ 55%. The most rapid results were obtained for adenocarcinoma of the
`mammary gland (Table 2). In this case the combination of methetrexate with methylcobalamin increased the
`lifetime of the animals by 60 %, which was three times as great as the effect of methotrexate alone. On the
`8th to 14th days after the end of the combined course of therapy with methylcobalamin and methotrexate, the
`inhibition of tumor growth was 76-40%, respectively, whereas methotrexate alone had practically no activity
`at the same periods (51-0%).
`
`It is known that as solid tumors grow, the number of cells in the resting phase in them increases sub-
`stantially, and the sensitivity of the tumors to cyclospecific preparations decreases appreciably [20]. Evidently
`the sensitivity of the tumor to methotrexate can be substantially increased by administering methylcobalamin,
`
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`I
`I
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`F g. 1
`Fig. 2
`Fig. 1. Combined action of m~thylcobalamin and m~hot~ex~te on
`growth of RShM-5. Along x-axis: period after administration of
`prep’aration (in days); along "y-axis: average volume of tumor (in
`mm~). The arrows indicate t~he time of administration of the prep-
`arations. 1) Control; 2) methotrexate; 3) methylcobalamin+
`methotrex~te.
`
`Fig. 2. Combined action of methylcoba~min, methion~.e synthetase
`inhibitors, and methotrexate on growth of Ca-755. Along x-axis:
`periods after administration of preparations (in days); along y-
`axis: average volume of tumor (in ram3). 1) Control; 2) metho-
`trexate; 3) CH3Cbl +NSC-176319 + CH3Cbl ¯ PdCI~ + methotrexate;
`4) NSC -176319 +CH~Cbl- PdC]~ + methotrexate.
`
`which increases the total pool of DNA- synthesizing cells. To test this hypothesis we used transplautable mouse
`cancer of the cervix (RShM-5). This tumor is characterized by slow growth, which makes it possible to
`administer a longer course of therapy and to evaluate the antineoplastic effect in long-term periods after
`transplautation. The experimental results confirmed our hypothesis. The average volume of the tumor in the
`control group of animals on the llth day after transpl~tation of RShl~I-5 exceeded the initial volume, when
`treatment of the animals was begun (7th day after transplantation), by 5.2-fold. In animals that received only
`methotrexate, the volume of the tumor at the same periods was increased 3.5-fold, and in the case of joint
`administration with methylcobalamin, there was only a 1.7-fold increase. Repeated combined administrations
`of the preparations (each four days) led to an inhibition of tumor growth in longer term periods as well. Thus,
`on the 17th day after transplantation, the average volume of the tumors in the control group exceeded the orig-
`inal volume by 22.7-fold, in the group of animals treated with methotrexate by 12.7-fold, and in the group of
`mice that received methylcob~l~min and methotrexate by 7.6-fold (Fig. 1).
`
`The interval between administration of methylcob~in and methotrexate is of vital importance. Ac-
`cording to the data obtained, with increasing time between administrations of the preparations, a partial or
`total loss of activity of methotrexate is noted, and in certain cases even the appearance of an effect of stimula-
`tion (see Table 2). Thus, for example, the inhibition of growth of Ca- 755 on the 7th day after the end of treat-
`ment with methotrexate was 69%. And yet, when methylcobalamin was preliminarily administered (6 h before
`the use of methotrexate), a total loss of activity of methotrexate was observed. The weakening of the anti-
`neoplastic activity of methotrexate is especially pronounced in the hybrids BDF1. As was shown, precisely
`in mice of this line, methylcobalamin induced the greatest stimulation of tumor growth. In Ft hybrids with
`the absence of a stimulating effect, in the case of its isolated use, the combined inD_uence did not lead to any
`weakening of the methotre~ate activity. An appreciable decrease in the antineoplastic activity of methotrexate
`after preliminary administration of mcthylcobalamin is evidently due to activation of the cobalamin-dependent
`methionine synthetase system and an increase in the total pool of the TGFA of the cells. This is confirmed by
`the results of the combined action of methotrexate and inhibitors of methionine synthetase against a background
`of preliminarily administered methylcobalamin (Fig. 2). The joint influence of methylcobalamin chloropal-
`ladate, the quinolinium derivative, aud methotrexate substantially exceeds the activity of a combination of the
`same preparations with methylcobalamin. Thus, the inhibition of growth of Ca- 755 on the 14th day after the
`end of therapy of mice that received methotrexate and complexes II and I was 85%, whereas in the case of
`combined influence of the three inhibitors with methylcobalamin it was only 61%. The increase in the lifetime
`of the auimals in these groups was 30% (P < 0.05) and 15% (P > 0.05), respectively.
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`Thus, in our investigations the stimulating action of methylcobalamin on the growth of certain solid
`tumors in mice was demonstrated for the first time. The results of our investigations permit an explanation
`of the decrease in the therapeutic effect of a number of alkylatingpreparations (sarcolysin, thioTEPA, embitol,
`and novembitol) in the case of their simultaneous use with cyanocobalamin [21-23]. The stimulating effect of
`methylcobalamin on solid tumors is clearly correlated with recent investigations, in which a significant in-
`crease in the methylcobalamin content in the rat liver was revealed after the administration of a chemical
`carcinogen and in certain transplantable Morris hepatomas [24]. It is important to note that the frequency
`of development of hemoblastoses of mice and of the simultaneous influence of methylcobalamin with endog-
`enous blastomogens also increases significantly [25]. The aggregate of the indicated experimental data
`thereby confirms the involvement of methylcobalamin in processes of proliferation of tumor cells of various
`histogenesis.
`
`In discussing the mechanism of the combined action of methylcobalamin with methotrexate, in our
`opinion, we should consider two possible aspects. In view of the fact that cobalamins promote the entry of
`the basic transport form of folic acid (methyl- THFA) into cells, and there is a common pathway of active
`transport of methyl-THFA and methotrexate into cells [26, 27], it can be assumed that methylcobalamin also
`influences the transport of methotrexate. At present there are no data in the literature on the mechanism
`of the penetration of methotrexate into Ca- 755 cells. However, under our conditions of influence, at a
`physiological level of methyl-THFA in the blood of the animals and the therapeutic concentration of metho-
`trexate, evidently the possibility of facilitated penetration of the latter into the tumor cells is realistic.
`
`Vitally important factors in the combined influence are activation of the methionine synthetase reaction
`and an increase in the number of DNA-synthesizing tumor cells, i.e., those most sensitive to methotrexate,
`under the influence of methylcobalamin. This may play a deciding role in the increase in the antineoplastic
`activity of methotrexate when it is administered simultaneously with methylcobalamin. The data that we ob-
`tained at present on the study of the cell kinetics of Ca- 755 under the influence of methylcobalamin confirm
`this premise.
`
`LITERATURE CITED
`
`1. N.V. Myasishcheva, Characteristics of Metabolism of B-12 Compounds (Cobalamins) in Leukemia
`Doctoral Dissertation [in Russian], Moscow (1972).
`2. H. Sauer and L. Jaenicke, Blur, ~ 321-327 (1974).
`3. O.D. Golenko and N. V. Myasishcheva, Probl. Gematol., No. 5, 24-28 (1971).
`4. O.D. Golenko, Influence of Cobalamins (B-12 compounds) on the Morphofunctional State of Tissues of
`the Hemopoietic System. Candidate’s Dissertation [in Russian], Moscow (1975).
`5. N.V. Myasishcheva, O. D. Golenko, and M. O. Raushenbakh, in: The Role of Endogenous Factors in the
`Development of Leukemias [in Russian], Moscow (1974), pp. 151-169.
`6. N.V. Myasishcheva, O. D. Golenko, L. E. Kuznetsova, et aL, Vopr. Med. Khim., No. 5, 622.-628 (1977).
`7. H. Ashe, B. R. Clark, F. Chu, et al., Biochem. Biophys. Res. Commun., ~ 417-425 (1974).
`8. Yu. V. Vares and N. V. Myasishcheva, Vopr. Med. Khim., No. 5, 681-684 (1977).
`9. R.A. Gains, E. M. Ryel, and L. M. Meyer, Prec. Soc. Exp. Biol. (N. Y.), 149, 384-388 (1975).
`10. R. Peirce, A. Tsulmsa, and B. A. Cooper, Biochim. Biophys. Acta, 381, 348-358 (1975).
`11.
`J.H. Mangum, K. Byron, B. K. Murray, ct al., Biochemistry (Wash.), 8~ 3496-3499 (1969).
`12. G.T. Burke, J. H. Mangum, and J. D. Brodie, Biochemistry (Wash.), ~ 3079-3085 (1971).
`13. F.M. Huennekens, P. M. DiGrolomo, K. Fujii, ct al., Adv. Enzyme Regul., ~ 187-205 (1975).
`14. Z.P. Sofina, N. V. Myasishcheva, F. G. Arsenyan, et aL, Vesti, AMI-I SSSR, No. 5, 36-39 (1978).
`15. O.D. Golenko, N. V. Myasishcheva, M. O. Raushenbakh, et al., Vopr. Med. Khim., No. 5, 549-554 (1974).
`16. Z.P. Sol’inn, Vopr. Onkol., No. 4, 82-96 (1976).
`17. W. Friedrich and J. P. Nordmeyer, Z. Naturforsch, 2~4b, 588-596 (1969).
`18. G.J. Atwell and B. F. Cain, J. Med. Chem., 1_~6, 673-678 (1973).
`19. E.G. Chauser, I. P. Rudakova, and A. M. Yurkevich, Zh. Obshch. Khim., ~ 360-365 (1976).
`20. O.S. Frankfurt, Cell Mechanisms of Tumor Chemotherapy [in Russian], Moscow (1976).
`21. R.A. Alimov, Influence of Certain Stimulators of Hemopoiesis on the Biological Activity of Alkylating
`Antineoplastic Compounds. Candidate’ s Dissertation [in Russian], Tashkent (1964).
`22. Z.P. Bulkina and P. R. Polyak, Vopr. Onkol., No. 4, 70-75 (1968).
`23. G.F. Dyadyusha and Z. P. Bulkina, in: Materials of the Sixth Republican Conference of Oncologists of
`the Lithuanian SSR [in Russian], Vil’nyus (1964), p. 194.
`24. J.C. LinneI~ E. V. Quadros, D. M. Matthews, et al., Cancer Res., ~ 2975-2978 (1977).
`
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`
`L A. Kudryavtsev, O. D. Golenko, and I~. V. Myasishcheva, Probl. GematoL, No. 3, 26-29 (1978).
`25.
`26.
`G. Tisman and V. Herbert, Blood, ~ 465-469 (1973).
`27. D. Goldman, Cancer Chemother. Rep., 6~ Pt. 3, 63-72 (1975).
`
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