L-Canavanine, the principal nonprotein
amino acid of certain leguminous plants, is a potent L-arginine antimetabolite.
This natural product has demonstrative antineoplastic activity against
a number of human cancers. Recent studies with MIAPaCa-2 and CFPAC have
established canavanine's potential anticancer potential against these human
pancreatic adenocarcinomas. Canavanine has promise as a lead compound in
the development of a chemotherapeutic agent for the treatment of human
pancreatic carcinoma, but it has not been adequately investigated. Greater
study of canavanine and its derivatives is needed to fully realize the
experimental and therapeutic value of this naturally-occurring non-protein
amino acid, and to obtain a chemotherapeutic agent of clinical value in
treating human carcinomas.
L-Canavanine is a potent arginine antimetabolite that bears strong structural analogy to its protein amino acid counterpart:
Replacement of the terminal methylene group of arginine with oxygen produces this nonprotein amino acid distinguished by a guanidinooxy moiety that has a pKa value of 7.04 and an isoelectric point near neutrality (Boyar and Marsh, 1982). The pKa of 10.48 for the guanidino group of L-arginine produces a much more basic amino acid with Io = 12.48 (Greenstein and Winitz, 1963). Therefore, at physiological conditions, arginine is essentially fully protonated whereas canavanine is not.
As a subtle structural mimic of L-arginine, canavanine can function in all enzymic reactions for which arginine is a substrate (Rosenthal, 1977). Therefore, canavanine potentially can inhibit any enzyme-directed reaction employing arginine as the preferred substrate. Arguably, canavanine's most adverse effect results from its activation and aminoacylation to the cognate tRNAArg by the arginyl-tRNA synthetase of canavanine-sensitive organisms (Allende and Allende, 1964; Mitra and Mehler, 1967). Incorporated into the nascent polypeptide chain, the decreased basicity of canavanine relative to arginine can affect residue interaction and thereby disrupt the tertiary and/or quaternary interactions essential for establishing the requisite three dimensional conformation of a given protein.
Biochemical Basis for Canavanine's Antimetabolic Properties
To determine the biochemical and biological consequence of canavanyl protein formation, a number of canavanine-containing proteins, obtained from various insect sources, were examined in detail. In the initial study, canavanine was provided to gravid females of the locust, Locusta migratoria migratorioides [Orthoptera], whose ovarian mass had been removed surgically on the day of adult emergence. This surgical procedure resulted in a pronounced accumulation of vitellogenin, an important hemolymph storage protein. Analysis of canavanyl vitellogenin, purified from hemolymph of these canavanine-treated locusts, shows that on average 18 of the 200 arginyl residues of vitellogenin or about 1 in 225 amino acids are replaced by canavanine. This modest level of replacement is nevertheless sufficient to elicit a dramatic alteration in protein structure that is shown most effectively by electrophoretic analysis. The altered fragmentation pattern of canavanyl vitellogenin, relative to the native macromolecule, undoubtedly reflects the profound change in vitellogenin structure resulting from canavanine incorporation (Rosenthal et al., 1989a).
These disparate forms of vitellogenin were also treated with chemicals able to react with surface-exposed amino acid residues to form novel amino acids. For example, treatment with cyanate carbamylates reactive lysyl residues and converts them to homocitrullyl. About one quarter of the lysyl residues of native vitellogenin do not react with cyanate; presumably, these are inaccessible and buried within the interior of the macromolecule. These residues are readily carbamylated in canavanyl vitellogenin. A similar chemical approach establishes that nearly twice as many surface-exposed tyrosine residues are acetylated as compared to the native protein. These experiments establish that canavanine incorporation into vitellogenin alters the three dimensional conformation of the protein, a property essential for normal function (Rosenthal et al., 1989a).
Microbial infection or mechanical injury to larvae of the fly, Phormia terranovae [Diptera] induces a family of protective, antibacterial proteins known trivially as the diptericins (Keppi et al., 1986). If canavanine is provided at the time of mechanically injury, it is incorporated into all of these protective proteins (Rosenthal et al., 1989b). This assimilation causes a total loss of detectable antibacterial activity for nearly all the diptericins; only a single diptericin—diptericin A retains demonstrable biological activity. This insectan investigation provides compelling experimental evidence that canavanine incorporation into a protein can impair protein function.
Canavanine-mediated impairment in function was also demonstrated with a catalytic protein, lysozyme (EC 22.214.171.124), that is induced by injection of fragments of the cell wall of Micrococcus lutea into larvae of the tobacco hornworm, Manduca sexta [Sphingidae]. If these larvae are provided canavanine when challenged, it is readily incorporated into de novo-synthesized lysozyme. The ratio of canavanine to arginine in this aberrant lysozyme is 1:3.8; assay of canavanyl lysozyme discloses a 48% loss in catalytic activity. This study provided the first demonstration of the ability of canavanine, through aberrant protein formation, to adversely affect the catalytic activity of an enzyme.
The importance of aberrant protein formation in the expression of canavanine's antimetabolic effect is also supported convincingly by a radically different experimental approach that determines how frequently canavanine replaces arginine in the proteins of various insects, i.e. the substitution error frequency (SEF). Manduca sexta larvae incorporate about 3.5% of the administered radiolabeled canavanine into hemolymphic proteins after 24 h. This results in a SEF that varies according to the insectan tissue but is about I in 2 for proteins of the larval body wall and musculature (Rosenthal et al., 1987). The bruchid beetle, Caryedes brasiliensis [Coleoptera], an inhabitant of the neotropic forests of Costa Rica, develops from larva to adult in the canavanine-laden seeds of Dioclea megacarpa [Fabaceae]. The woody pericarp of D. megacarpa houses seeds that can contain as much as 13% canavanine by dry weight (Rosenthal, 1983). The weevil, Sternechus tuberculatus [Curculionidae], oviposits on the pericarp of Canavalia brasiliensis [Fabaceae]; larvae, foraging within the fruit, are sustained by seeds that also store appreciable canavanine. These two seed predators are examples of insects that are adapted biochemically to canavanine (Bleiler et al., 1988) and can therefore tolerate, even flourish with this normally poisonous natural product (Rosenthal, 1983). The SEF for C. brasiliensis is 1 in 365 while that of S. tuberculatus is 1 in 500-1,000. The tobacco budworm, Heliothis virescens [Noctuidae], does not consume canavanine-containing plants; however, it is naturally resistant to this potentially toxic allelochemical (Berge et al., 1986; Melangeli et al., 1997). This aggressive generalist herbivore exhibits a SEF of 1 in 65. Thus, canavanine-adapted and canavanine-resistant insects minimize or avoid canavanyl protein formation while canavanine-sensitive insects readily incorporated this arginine antagonist.
Article of Human Diet
At present, few canavanine-containing seeds are part of the human diet, but this is changing as the worldwide demand for reasonably priced, high quality protein increases. Jack bean seeds, Canavalia ensiformis (L.) DC. [Fabaceael, which contain about 2.5% canavanine by dry weight, and sword bean, Canavalia gladiata [Fabaceae], storing about 1.4% canavanine by dry weight (Rosenthal and Nkomo, 1997), are important table legumes, particularly in Asia and parts of the tropics and Africa. In North America, the most heavily consumed canavanine-containing plant is alfalfa, Medicago sativa [Fabaceae]. Canavanine is the preponderant nonprotein amino acid of the seed where it accounts for 1.5% of the dry matter; the sprout is also canavanine-rich since it can accumulate as much as 2.4% canavanine by dry weight (Rosenthal and Nkomo, 1997).
Canavanine's Antineoplastic Activity
In 1958, Kruse and McCoy reported that canavanine competed with arginine in meeting the growth requirements of Walker carcinosarcoma 256 cells. In a subsequent study, Kruse et al. (1959) demonstrated that canavanine was incorporated into the proteins of these cancer cells, and that the diminution in the amount of arginine in the protein hydrolysate equaled the canavanine content. This report established for the first time that canavanine specifically replaced arginine in de novo-synthesized tumor proteins. Schachtele and Rogers (1965) employed the same experimental approach to demonstrate the incorporation of canavanine into the proteins of Escherichia coli. These initial observations were confirmed by experiments conducted with M. sexta larvae that were injected with L-[guanidinooxy-14C]canavanine (Dahlman and Rosenthal, 1976). Enzymatic degradation of the radiolabeled canavanine, isolated from the insectan hydrolysate, demonstrated unequivocally that the hydrolysate 14carbon was derived from radiolabeled canavanine.
Numerous descriptive studies have documented
that exposure of a particular organism to canavanine adversely affected
a basic property or functional parameter of one or more of its enzymes.
For example, the normally soluble
b-galactosidase of E. coli exhibited diminished activity, and sedimented readily with the 10,000 xg pellet after exposing the bacterium to canavanine (Prouty et al., 1972; Rosenthal, 1977).
Several studies of canavanine's antineoplastic activity have been conducted. Naha et al. (1980) demonstrated that canavanine selectively inhibited DNA replication in epithelial monkey kidney cells possessing a transformed phenotype, as compared to their counterpart that remained "contact-inhibited" with a temperature change from 33 to 39.5oC.
A detailed study of canavanine's antineoplastic activity was conducted with mice bearing L1210 leukemic cells (Green et al., 1980). These workers reported that DNA synthesis was reduced to only 9% of the control level, as assessed by [3H]thymidine incorporation, after 12-hourly i.p. injections of 20 mg canavanine per injection. A dramatic attenuation in DNA synthesis of 86% of the control level was also achieved when the infected mice were infused s.c. with canavanine continuously for 1 d at a rate of 20 mg h-1 after an initial i.p. injection of 20 mg canavanine. At an optimal dose of 18 g kg-1, the median life span of the cancerous mice increased by 44%. This investigation was of great significance because it demonstrated that canavanine could mediate its toxic effect not only at the level of protein function, but also through its ability to disrupt DNA replication.
In an important follow-up study, Green and Ward (1983) reported that canavanine enhanced significantly the efficacy of g -irradiation of cultured HT-29 cells, a human tumor cell line. The lethal effect of this radiation was augmented both when canavanine was provided prior to as well as after g -irradiation. These workers provided convincing experimental evidence for their contention that canavanine's lethal effect was manifested preferentially in rapidly proliferating cells—a property often essential for chemotherapeutic efficacy. The experimental efforts of Green and his collaborators did not distinguish between the possibility that canavanine affected nucleic acid turnover directly as compared to its acting by affecting the activity of one or more proteins essential to maintaining DNA replication.
Canavanine also affected the growth of a rat colonic carcinoma in male Fischer rats (Thomas et al., 1986). This tumor, which became palpable in 8-10 d, had a doubling time of 3 to 4 d. Canavanine, administered by s.c. injection into the flank opposite the tumor site, was provided initially after the tumor attained a volume of 500-1,000 m3 (Figure 1). Providing 2.0 g kg-1 canavanine for 5 d produce a tumor vs. control of 23%; after 9 d, this value fell to 14%. Canavanine's efficacy was enhanced when the dose was increased to 3.0 g kg-1. At this dose, the percentage of regression was –13% after 5 d, and –8% after 9 d. These negative values reflect tumor regression. The loss in tumor volume, expressed as percentage of regression, was 22% for the 3 g kg-1 daily for 5 d, and 60% in the 3 g kg-1 daily for 9 d treatment groups.
These promising findings with canavanine had the drawback that the treated rats lost weight. Canavanine's cumulative toxicity resulted in about a 15% diminution in body weight after 5 treatment days. This finding led to an examination of the relationship of caloric deprivation to tumor growth reduction, and established that canavanine-directed curtailment of tumor growth was not caused by reduced food intake. Most importantly, canavanine-dependent weight loss was fully reversible. This investigation instigated efforts to develop canavanine derivatives with an enhanced therapeutic index while diminishing body weight loss.
Canavanine's Cytotoxic Effect On Human Pancreatic Cells
Toxicological study of canavanine metabolism in the male Fischer rat revealed that L-[guanidinooxy-14C]canavanine was assimilated preferentially by proteins of the pancreas. Radiolabeled canavanine incorporation into these proteins was 10-times that of liver, brain, and muscle tissues, and 5-times that of the proteins of most other body organs (Thomas and Rosenthal, 1987a). Given the pronounced assimilation of canavanine into pancreatic proteins, we examined the effect of canavanine on the growth of MIAPaCa-2, a human pancreatic adenocarcinorna cell line (Swaffar et al., 1994). When these cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 0.4 mM arginine, the 50% inhibitory concentration (IC50) for canavanine was 2 mM. Most importantly, the IC50 value fell precipitously to 10.0 µM when the arginine content of the culture medium was reduced to 0.4 µM.
This study was important because it demonstrated canavanine's marked cytotoxic activity against a human pancreatic cancer cell line. Moreover, it provided an excellent experimental system for evaluating novel canavanine derivatives on a small scale prior to conducting whole animal studies. Swaffar et al. (1994) also demonstrated that canavanine-mediated inhibition of MIAPaCa-2 cell growth was reversible by arginine up to 12 hr post treatment, but then became irreversible. This finding, while not presently explicable, was important because it demonstrated that conditions existed where canavanine's anti-cancer effect would not be reversed.
Having established canavanine's antineoplastic activity against MIAPaCa-2 cells, Swaffer et al. (1995) asked the salient question of how canavanine functioned in combination with 5-fluorouracil (5-FU), the preferred drug presently utilized for treating pancreatic carcinoma. In this companion study, canavanine's efficacy as a chemotherapeutic agent was assessed in combination with 5-FU. At a fixed molar ratio of 1:1, over the range of 0.06 to 1.0 mM, both drugs exhibited greater cytotoxic effects against MIAPaCa-2 cells relative to single administration of these drugs. The IC50 values for canavanine and 5-FU were 0.060 and 0.363, respectively; in combination, the IC50 value fell to 0.021.
As part of this investigation, the interaction of canavanine and 5-FU was also assessed with the colonic carcinoma of adult male Fischer rats described above (Swaffar et al., 1995). Providing canavanine, either at 1.0 g kg-1 or 2.0 g kg-1 daily for 5 d in combination with 5-FU increased significantly the anti-tumor activity of either drug alone (Figure 2).
These investigations demonstrated the value of employing canavanine in combination with drugs presently employed in treating pancreatic and colonic cancer. Thus, the development of a canavanine derivative as an effective drug is not limited only to its independent use, but also may prove more valuable when provided in combination with an established anticancer drug.
Canavanine's Accumulative Toxicity
Analysis of canavanine catabolism in the adult rat demonstrated that hepatic argjnase (EC 126.96.36.199) fostered the hydrolysis of L-canavanine to yield L-canaline and urea, this reaction pathway was the principal basis for canavanine catabolism in this mammal (Thomas and Rosenthal, 1987b).
Thus, it is reasonable to propose that administration of L-canavanine to a human would result in the formation of L-canaline, a highly toxic nonprotein amino acid that is a powerful inhibitor of pyridoxal phosphate-dependent enzymes. Rahiala et al (1971) were the first to recognize that a direct reaction occurred between canaline and the vitamin B6 moiety of an enzyme by postulating: "...that canaline probably inhibits pyridoxal phosphate-containing enzymes by its nonenzymic, irreversible and stoichiometric binding with pyridoxal phosphate."
The ability of canaline to inactivate an enzyme by forming a stable, covalently-linked oxime was demonstrated' directly with L-[U-14C]canaline which reacted with ornithine aminotransferase (EC 188.8.131.52) of larval Manduca sexta to yield an enzyme-bound, radiolabeled canaline-pyridoxal phosphate oxime (Rosenthal and Dahlman, 1990). In addition, canaline's facile ability to form oximes means that canaline can scavenge such essential 2-oxo-containing metabolites as pyruvate, oxaloacetate, and 2-oxoglutarate to deplete carboxylic acid reserves and carbon skeleton required for amino acid synthesis. Finally, canaline also possesses significant antineoplastic and antiproliferative properties (Rosenthal, 1998).
Canavanine Derivatives As Chemotherapeutic Agents
The intrinsic toxicity of canavanine would be decreased significantly if it failed to function as a substrate for hepatic degradation via the action of arginine. Production of a simple ester of canavanine, such as methyl-L-canavanine, provided a derivative that was not an effective substrate for rat arginase and therefore could not elicit the adverse biological effects caused by canaline. In addition, this ester possessed enhanced hydrophobicity relative to canavanine, and might therefore enjoy enhanced cellular uptake. Intracellular esterases should release the parent compound through hydrolysis and thereby increase canavanine's bioavailability.
To test this hypothesis, the methy, ethyl, n-propyl, isopropyl, n-butyl, and n-octyl esters of L-canavanine were synthesized and evaluated for cytotoxicity against MIAPaCa-2 cells (NaPhuket et al., 1997). While the methyl, ethyl n-propyl, and isopropyl esters of canavanine exhibited slightly improved growth-inhibitory activity against MIAPaCa-2 cells, the n-butyl and n-octyl esters of canavanine dramatically attenuated cellular growth (Figure 3). This conclusion was firmly confirmed by bioevaluation of the growth inhibiting properties of these canavanine derivatives in terminal instar M. sexta larvae (Rosenthal et al., 1997). The increased potency of these latter esters may reflect their enhanced lipophilicity and greater membrane penetrational properties. None of the free alcohols, except n-octanol, possessed significant growth-inhibiting activity. Only at concentrations above 2.5 mM did octanol exhibit significant activity against MIAPaCa-2 cells.
The experimental efforts outlined in this review detail studies that demonstrate the significant antineoplastic activity of canavanine. Recently completed experiments probing 14C-radiolabeled canavanine uptake (0.26% of the administered dose) by MIAPaCa-2 cells revealed that 70% of the cellular canavanine was found in the proteins, the remainder was accounted for in the cytosol; 95% of the 14carbon found in proteins of the treated cells was canavanine (NaPhuket, 1997). In contrast, only 58% of the 14carbon of the cytosol was canavanine.
The author raises for thought the interesting possibility that canavanine's antineoplastic activity might arise from the formation of structurally aberrant, dysfunctional, canavanine-containing proteins by the cancer cells and that they may be unique to these cells. The findings outlined in this review support the contention that canavanine has marked potential as a lead compound in the development of a chemotherapeutic agent for the treatment of human pancreatic carcinoma. Particularly noteworthy are certain ester derivatives of canavanine, which might provide an efficacious drug capable of eliciting little if any body weight loss while enhancing the therapeutic index. Esterification of the carboxyl group of canavanine with longer-chained alcohols such as butanol and octanol represent structural modification of canavanine that augments significantly the growth-inhibiting properties of the parent compound against MIAPaCa-2 cells.
Further study of canavanine and its derivatives could lead to chemotherapeutic agents of clinical value in treating human carcinomas. Additional experimental effort is warranted to realize the experimental and therapeutic value of this unusual amino acid antimetabolite of higher plants.
Allende, C.C., and Allende, J. E. (1964). Purification and substrate specificity of arginyl-ribonucleic acid synthetase from rat liver. J. Biol. Chem. 239:1102-1106.
Berge, M.A., Rosenthal, G.A., and Dahlman, D.L (1986). Tobacco budworm, Heliothis virescens [Noctuidae] resistance to L-canavanine, a protective allelochemical. Pest. Biochem. Physiol. 25:319-326.
Bleiler, J., Rosenthal, G.A., and Janzen, D. H. (1988). Biochemical ecology of canavanine-eating seed predators. Ecol. 69:427-433.
Boyar, A., and Marsh, R. E. (1982). L-Canavanine, a paradigm for the structures of substituted guanidines. J. Am. Chem. Soc. 104:1995-1998.
Dahlman, D.L., and Rosenthal, G.A. (1976). Further studies on the effect of L-canavanine on the tobacco hornworm, Manduca sexta (L.) (Sphingidae) J. Insect Physiol. 22:265-271.
Green, M.H., Brooks, T.L., Mendelsohn, J., and Howell, S.B., (1980). Antitumor activity of L-canavanine against L1210 murine leukemia. Cancer Res. 40: 535-537.
Green, M. H., and Ward, J. F. (1983). Cancer Res. Enhancement of human tumor cell killing by L-canavanine in combination with g -radiation. Cancer Res. 43: 4180-4182.
Greenstein, J.P., and Winitz, M. (1963). Chemistry of the Amino Acids, Vols. 1-3, Wiley & Sons, New York.
Keppi, E., Zachary, D., Robertson, M., Hoffmann, D., and Hoffmann, J. (1986). Induced antibacterial proteins in the hemolymph of Phormia terranovae [Diptera]. Purification and possible origin of one protein. Insect Biochem. 16:395-399.
Kruse, Jr., P.F., and McCoy, T.A. (1958). The competitive effect of canavanine on utilization of arginine in growth of Walker carcinosarcoma 256 cells in vitro. Cancer Res. 18:279-282.
Kruse, Jr., P.F., White, P.B., Carter, H.A.,.and McCoy, T.A. (1959). Incorporation of canavanine into protein of Walker carcinosarcoma 256 cells cultured in vitro. Cancer Res. 19:122-125.
Melangeli, C., Rosenthal, G.A, and Dahlman, D.L. (1997). The biochemical basis for the tolerance of Heliothis virescens to L-canavanine Proc. NatI. Acad. Sci. USA 94:2255-2260.
Mitra, S.K., and Mahler, A.H. (1967). The arginyl transfer ribonucleic acid synthetase of Escherichia coli. J. Biol. Chem. 242:5490-5494.
Naha, P.M., Silcock, J. M., and Fellows, L (1980). An experimental model for selective inhibition of proliferating cells by chemotherapeutic agents. Cell Biol. Inter Rept. 4: 155-166.
NaPhuket, S. L-Canavanine and its Derivatives as Potential Chemotherapeutic Agents for Pancreatic Cancer: Synthesis, Structure-Activity Relationships And Metabolic Studies. Ph.D. Thesis, University of Kentucky, 1997.
NaPhuket, S.,Trifonov, L.S., Crooks. P.A., Rosenthal, G.A., Freeman, J.W., and Strodel W.E. (1997). Synthesis and structure-activity relationships of some antitumor congeners of L-canavanine. Drug Devel. Res. 40:325-332.
Prouty, W.F., Karnovsky, M.J., and Goldberg, A.L. (1972). Degradation of abnormal proteins in Escherichia coli. J. Biol. Chem. 250:1112-1122.
Rahiala, E.-L., Kekomaki, M.K., Janne, J., Raina, A., and Raiha, N.C.R., (1971). Inhibition of pyridoxal enzymes by L-canaline Biochim. biophys. Acta 227:337-343.
Rosenthal, G.A. (1977). The biological effects and mode of action of L-canavanine, a structural analogue of L-arginine. Q. Rev. Biol. 52:155-178.
Rosenthal, G.A. (1983). The adaptation of a beetle to a poisonous plant. Sci. Amer. 249:164-171.
Rosenthal, G.A., and Dahlman, D.L., (1990). Interaction of L-canaline with ornithine aminotransferase of the tobacco hornworm, Manduca sexta [Sphingidae]. J. Biol. Chem. 265:868-873
Rosenthal, G.A., and Nkomo, P. (1997). Unpublished data.
Rosenthal, G.A., Lambert, J., and Hoffmann, D. (1989a). L-Canavanine incorporation into protein can impair macromolecular function. J. Biol. Chem. 264:9768-9771.
Rosenthal, G.A., Reichhart, J.-M., and Hoffmann, J.A. (1989b). L-Canavanine incorporation into vitellogenin and macromolecular conformation. J. Biol. Chem. 264: 13693-13696.
Rosenthal, G.A., Berge, M.A., Bleiler, J.A., and Rudd, T. (1987). Avoidance of aberrant protein production and an organism's ability to utilize or tolerate L-canavanine. Experientia 43:558-561.
Schachtele, C.F., and Rogers, P. (1965). Canavanine death in Escherichia coli. J. Mol. Biol. 34:843-860.
Swaffar, D.S., Ang, C.Y., Desai, P.B., and Rosenthal, G.A. (1994). Inhibition of the growth of human pancreatic cancer cells by the arginine antimetabolite, L-canavanine. Cancer Research 54:6045-6048.
Swaffar, D.S., Choo, Y.A.., Desai, P.B., Rosenthal, G.A., Thomas, D.A., John, W.J., and Crooks, P.A., (1995). Combination therapy with 5-flurouracil and L-canavanine: in vitro and in vivo studies. Anti-Cancer Res. 6:586-593
Thomas, D.A., Rosenthal, G.A., Gold, D.V., and Dickey, K. (1986). Growth inhibition of a rat colon tumor by L-canavanine. Cancer Research 46:2898-2903
Thomas, D.A., and Rosenthal, G.A., (1987a) Toxicity and pharmacokinetics of the nonprotein amino acid L-canavanine in the rat. Toxicology & Appl. Pharm. 91:395-405.
Thomas, D.A., and Rosenthal, G.A., (1987b) Metabolism of L-[guanidinooxy-14C]-canavanine in the rat. Toxicology & App/. Pharm. 91:406-414.
LEGEND TO THE FIGURES
Figure 1. The effect of canavanine on tumor growth in male Fischer 344 rats. The rats were administered canavanine, 2.0 ( ), 3-0 ( ) g kg-1 for 9 d. Control animals ( ) received 0.95% (w/v) NaCl. The standard error bar was omitted if it fell within the area occupied by the data point, n=5 + SEM.
Figure 2. Evaluation of the combined effect of canavanine and 5-FU on colonic tumor growth in male Fischer rats. Tumor growth was evaluated after 5 daily s.c. injections of: 1.0 g kg-1 canavanine ( ) 2.0 g kg-1, canavanine ( ) 35 mg kg-1 5-FU ( ) 35 mg kg-1 5-FU + 1.0 g kg-1 canavanine ( ) and 35 mg kg-1 5-FU + 2.0 g kg-1 canavanine ( ). The control animals ( ) received 0.95% (w/v) NaCl. Each value is the mean (n = 5) + SEM.
Figure 3. Comparison of the IC50 value for canavanine and some of its esters.