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 MlAPaCa-2 and CFPAC have established canavanine's potent anticancer potential against these human pancreatic adenocarcinomas. Canavanine has real potential 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 agents of clinical value in treating human carcinomas.
INTRODUCTION
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 (1). The pKa of 10.48 for the guanidino group of L-arginine produces a much more basic amino acid with an Io = 12.48 (2). Therefore, at physiological conditions, arginine is essentially fully protonated-canavanine is not.
As a subtle structural mimic of L-arginine, canavanine can function in all enzymic reactions for which arginine is a substrate (3). 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 (4,5). 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 to establishing the requisite three-dimensional conformation of a given protein. A series of detailed biochemical investigations, focusing on structurally aberrant, canavanine-containing enzymes, established unequivocally that canavanine assimilation can alter protein conformation and adversely affect normal biological function and biochemical activities (6-8).
Article of Human Diet
At present, few canavanine-containing seeds are part of the human diet, but this is changing as the world-wide demand for reasonably priced, high quality protein increases. Jack bean seeds (Canavalia ensiformis), which contain about 2.5% canavanine by dry weight, and sword bean, (Canavalia gladiata), storing about 1.4% canavanine by dry weight (9), are important table legumes, particularly in Asia and parts of the tropics and Africa. In North America, the most abundantly consumed canavanine-containing plant is alfalfa, Medicago sativa. Canavanine is the preponderant nonprotein amino acid of the seed where 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 (9).
Biochemical Basis For Canavanine's Toxicity
In 1958, Kruse and McCoy (10) reported that canavanine competed with arginine in meeting the growth requirements of Walker carcinosarcoma 256 cells. In a subsequent study, Kruse et al. (11) demonstrated that canavanine was incorporated into the proteins of these cancer cells, and that the diminution 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 (12) 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 larvae of the tobacco hornworm, Manduca sexta, which were injected with L-[guanidinooxy-14C]canavanine (13). Enzymatic degradation of the radiolabeled canavanine of 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 10,000g pellet after exposing the bacterium to canavanine (14).
These descriptive studies did not address in a systematic manner the biochemical consequences of canavanine incorporation on protein function. As mention previously, to overcome this deficiency, a series of biochemical investigations were conducted with the tobacco hornworm, Manduca sexta, a canavanine-sensitive insect. These studies established that canavanine incorporation dramatically altered three-dimensional protein conformation, and affect adversely its biological or enzymatic activity (6-8).
Canavanine's Antineoplastic Activity
Several studies of canavanine's antineoplastic activity have been conducted. Naha et al. (15) 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" from a temperature change from 33 to 39.5°C.
A detailed study of canavanine's antineoplastic activity was conducted with mice bearing L1210 leukemic cells (16). These workers reported that DNA synthesis fell 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 18g kg-1, the median life span of the cancerous mice increased by 44% (16). 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 (17) 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 essential to chemotherapeutic efficacy (17). 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 (18). This tumor, which became palpable in 8-10 d, possessed 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).
Figure 1. The effect of canavanine on tumor growth in male Fischer 344 rats. The rats were administered canavanine, 2.0 ( square), 3.0 (triangle ) g kg-1 for 9 d. Control animals (circle ) received 0.95% (w/v) NaCI. The standard error bar was omitted if it fell within the area occupied by the data point, n=5 + SEM.
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 3.0g 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 3g kg-1 daily for 5 d, and 60% in the 3g 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 (18). 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 (19). Given the pronounced assimilation of canavanine into pancreatic proteins, we examined the effect of canavanine on the growth of MIA PaCa-2, a human pancreatic adenocarcinoma cell line (20). 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 (Figure 2).
Figure 2. The growth of MlAPaCa-2 cells exposed to canavanine. MlAPaCa-2 cells were exposed to the indicated amount of canavanine for 3 d in culture medium containing 0.4 mM arginine. Each point represents the mean of 10 independent determinations. The standard error bar was omitted when it fell within the area occupied by the data point.
Most importantly, the IC50 value fell precipitously to 10.0 mM when the arginine content of the culture medium was reduced to 0.4mM (Figure 3)
Figure 3. The effect of canavanine on the growth of MlAPaCa-2 cells. MlAPaCa-2 cells were exposed to the indicated amount of canavanine in DMEM medium (square) or 0.04 mM arginine-containing, DMEM medium (circle).
This study was of considerable importance 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. (20) 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 were not be reversed.
Combination Therapy.
Having established canavanine's antineoplastic activity against MlAPaCa-2 cells, Swaffer et al. (21) 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 (Figure 4).
Figure 4. Analysis of canavanine, 5-FU and combined drug administration on the cytotoxicity to MlAPaCa-2 cells. The growth of MlAPaCa-2 cells was evaluated in arginine-reduced medium (0.4 mM) after exposure for 3 d to the indicated level of 5-FU (triangle) canavanine (circle) or a 1:1 ratio of each drug (square). The mean values were obtained from 4 independent experiments; the SEM is shown only when it exceeded the area occupied by the data point.
At a fixed molar ratio of 1:1, over the range of 0.06 to 1.0mM, both drugs exhibited greater cytotoxic effects against MlAPaCa-2 cells relative to single administration of these drugs. The IC50 value for canavanine was 0.060 and 0.363 for 5-FU; in combination, the IC50 value fell to 0.021(21). 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 (21). Providing canavanine, either at 1.0g kg-1' or 2.0g kg-1' daily for 5 d in combination with 5-FU increased significantly the anti-tumor activity of either drug alone (Figure 5).
Figure 5. 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: 1.0g kg-1 canavanine (open, triangle up) 2.0g kg-1, canavanine (closed, square) 35mg kg-1 5-FU (closed, circle) 35mg kg-1 5-FU + 1.0g kg-1 canavanine (open, triangle down) and 35mg kg-1 5-FU + 2.0g kg-1 canavanine (open, circle). The control animals (open, square) received 0.95% (w/v) NaCI. Each value is the mean (n = 5) + SEM.
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 a well established anticancer drug.
Canavanine's Accumulative Toxicity
Analysis of canavanine catabolism in the adult rat demonstrated that hepatic arginase (EC 3.4.1.5) 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 (22).
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 (23) 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 2.6.1.13) of larval Manduca sexta to yield an enzyme-bound, radiolabeled canaline-pyridoxal phosphate oxime (24). 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 tricarboxylic acid reserves and carbon skeleton required for amino acid synthesis.
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 1-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, butyl, and octyl esters of L-canavanine were synthesized and evaluated for cytotoxicity against MlAPaCa-2 cells (Na Phuket et al (25). While the methyl, ethyl and n-propyl esters of canavanine exhibited improved growth-inhibitory activity against MlAPaCa-2 cells, the n-butyl and n-octyl esters of canavanine dramatically attenuated cellular growth (Figure 6).
Figure 6. Comparison of the IC50 value for canavanine and some of its esters.
The experimental efforts outlined in this review detail studies that demonstrate the significant antineoplastic activity of canavanine. Their results 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 for canavanine.
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 non-protein amino acid.
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