L-Canavanine potentiates the cytotoxicity of doxorubicin and cisplatin in arginine deprived human cancer cells

Introduction

Twenty-two protein amino acids (including selenocysteine and pyrrolysine) are common to all organisms. However, more than 700 amino acids exist in nature, especially in plants, which are structural analogues of protein amino acids and termed non-protein amino acids (NPAA). Some of them, such as those from the legume genus Lathyrus, are toxic and cause neurological disorders in both man and animals (Bell, 2003; Wink & Wyk, 2008). Many NPAAs act as antimetabolites when integrated into proteins, because some aminoacyl tRNA synthetases do not discriminate between a protein amino acid and its corresponding NPAA. When incorporated into a protein, very often such a protein will assume a different 3D structure than the original protein and therefore loses its bioactivity. This property provides plants, which produce NPAAs, with a defence mechanism against herbivores, microbes, and viruses, and competing plants (Bell, 2003). Some NPAAs, such as L-canavanine, L-canaline, β-aminopropionitrile (BAPN), azaserine, and mimosine are able to inhibit the growth of some cancer cell lines (Bondareva et al., 2009; Chung et al., 2012; Jang et al., 2002; Lyons, Sant & Christopherson, 1990; Rosenkranz & Wink, 2007; Rosenthal, 1997).

L-canavanine (L-CAV), first isolated in 1929 from Canavalia ensiformis (Fabaceae), is a structural homologue of L-arginine (L-ARG). When L-CAV is incorporated into a protein instead of L-ARG during ribosomal protein biosynthesis, the protein structure and function are altered, often leading to an inhibition of cell growth and causing cellular death (Bence, Adams & Crooks, 2003). Some studies have revealed that L-CAV is able to inhibit the proliferation of some tumour cell types, such as pancreatic, lung, colon, and leukemic cancers, both in vitro and in vivo (Green et al., 1980; Swaffar et al., 1994; Thomas et al., 1986). Moreover, L-CAV is cytotoxic for both parental and multidrug-resistant human tumour cells (Worthen et al., 1998). In order to be effective, L-CAV needs to be applied over a long period and at a high dosage, since at low dosages L-CAV may not efficiently compete with L-ARG for incorporation into proteins. Therefore, a clinical potential of L-CAV could best be realized in an adjuvant or drug combination setting.

To date, only one combinatorial study of L-CAV has been carried out. A combination of L-CAV and 5-fluoruracil (5-FU) in human pancreatic carcinoma (MIA PaCa-2) in a molar ratio of 1:1 (L-CAV : 5-FU) in arginine-rich media showed a synergistic effect. An increase of 5-FU in the combination, however, reduced the synergism (Swaffar et al., 1995). A reduction of endogenous L-ARG levels, for example via activation of arginine deiminase (ADI-PEG 20), has been regarded as a novel approach developed to target cancers which are auxotrophic for arginine (Feun et al., 2008). However, arginine deiminase is highly antigenic and has a short half-life, so it needs high dosages to achieve a significant effect. In large amounts, it induces overexpression of argininosuccinate synthase (ASS), a key enzyme in the synthesis of L-ARG in certain melanoma cell lines, leading to drug resistance (Lind, 2004). This study aims to investigate the cytotoxicity of a combination of L-CAV with the chemotherapeutic drugs, doxorubicin (DOX) or cisplatin (CIS), in two types of cancer cell lines, those with ASS overexpression (HeLa and Caco-2 cells) and those with low ASS levels (pancreatic cells MIA PaCa-2 and BxPC-3 cells and hepatocellular carcinoma Hep G2 and SK-HEP-1 cells). Both of the drugs, DOX and CIS are DNA targeting-drugs with different mode of action. DOX has two proposed mechanisms in cancer cells: 1) intercalation into the DNA and disruption of topoisomerase-II mediated DNA repair and 2) production of free radicals, which disturbs cellular membrane, DNA and proteins (Thorn et al., 2011). Unlike DOX, CIS binds to DNA and forms crosslink and DNA adducts. The DNA adduct activates several signalling transduction pathways such as ATR, p53, p73 and MAPK and results in the activation of apoptosis (Florea & Buesselberg, 2011).

One focus of the experiments was to find out whether the combinations of L-CAV with DOX or CIS are additive or synergistic. Our hypothesis is that L-CAV application may result in a synergistic dosage reduction of DOX and CIS in L-ARG deprived cancer cells and enhance their cytotoxicity.

Methods

Results

Expression levels of ASS

Quantitative real time PCR was performed to evaluate the expression of argininosuccinate synthase (ASS) at the mRNA level in the 6 cancer cell lines used in this study. Figure 1 illustrates that HeLa and Caco-2 expressed higher ASS levels as compared with pancreatic cells (MIA PaCa-2 and BxPC-3) and hepatocellular carcinoma (Hep G2 and SK-HEP-1). SK-HEP-1 expressed the lowest level of ASS among 5 other cell lines. This finding is in agreement with other studies using RT-PCR and western blot analysis showing that pancreatic and hepatocellular cancer cells express low levels of ASS (Bowles et al., 2008; Feun et al., 2008; Liu et al., 2014).

Combinations of L-CAV with DOX or CIS

L-CAV was combined with DOX or CIS using two different approaches, i.e., in constant or non-constant ratios, as developed by Chou (2006). Constant ratio combinations were carried out employing three different molar ratios of L-CAV and DOX or CIS. The first molar ratio of combination was based on the IC₅₀ value of each drug; the second was chosen by increasing the toxicity of L-CAV to the value of IC₇₀ and decreasing the toxicity of DOX or CIS to the value of IC₃₀; and the third one was an additional combination designed to observe the interaction pattern of a combination according to the presence of L-CAV. For non-constant ratios a constant non-toxic value of IC₁₀ or IC₃₀ of L-CAV was employed, while the concentration DOX or CIS covered the whole concentration range.

Figures 2, 3, and 4 illustrates the results of the combination of L-CAV with DOX and CIS, whose cytotoxicity could be substantially enhanced by all combination treatments. Detailed results of these combinations are documented in Table 3 (DOX) and Table 4 (CIS). The combination of L-CAV and DOX (both IC₅₀ concentration) is not significantly different to the combination in which the IC₇₀ dosage of L-CAV and IC₃₀ dosage of DOX were employed in HeLa, Caco-2, MIA PaCa-2, and BxPC-3 cells. However, a significant difference was observed in both hepatocellular carcinomas, Hep G2 and SK-HEP-1 cells (Table 3). When L-CAV was combined with CIS, IC₅₀ and IC₇₀ concentrations of L-CAV were significantly different in the cell lines (except SK-HEP-1), as summarized in Table 4.

In order to interpret the nature of combinations, the corresponding CI values were calculated (Tables 3 and 4). The combinations of L-CAV with DOX were synergistic in the six cell lines; only the IC₇₀:IC₃₀ combination of L-CAV with DOX in Hep G2 cells showed a nearly additive effect (Table 3). The combinations of L-CAV with CIS are illustrated in Table 4. Most of the combinations are synergistic, with the exception of the IC₅₀:IC₅₀ combination in MIA PaCa-2 cells, which suggested a slight antagonism.

In a second set of experiments with HeLa (overexpressing ASS) and MIA PaCa-2 and SK-HEP-1 cells (low ASS expression), a non-constant ratio was used in which L-CAV was applied at a constant IC₁₀ or IC₃₀ value, whereas DOX and CIS were serially diluted. Table 5 is a summary of these combinations, clearly showing that all combinations were synergistic in nature.

Table 5:

IC₅₀ values (µM) of doxorubicin (DOX) and cisplatin (CIS) alone and in combination with L-canavanine (L-CAV), using non-constant ratio combinations.

N°	Cell	IC50 value of DOX or CIS Alone (IC50 ± SD)	Combination	IC50 value of DOX or CIS in Combination (IC50 ± SD)	CI	Interpretation
1	HeLa	4.07 ± 0.26	IC10 of L-CAV and DOX	0.11 ± 0.03	0.11	Strong synergism
2			IC30 of L-CAV and DOX	0.0043 ± 0.0015	0.38	Synergism
3		54.07 ± 12.25	IC10 of L-CAV and CIS	0.30 ± 0.09	0.09	Very strong synergism
4			IC30 of L-CAV and CIS	0.066 ± 0.02	0.38	Synergism
5	MIA PaCa-2	22.25 ± 3.35	L-CAV IC10 and DOX	2.44 ± 0.28	0.14	Strong synergism
6			L-CAV IC30 and DOX	0.62 ± 0.06	0.27	Strong synergism
7		97.20 ± 20.34	L-CAV IC10 and CIS	88.80 ± 5.44	0.81	Moderate synergism
8			L-CAV IC30 and CIS	14.57 ± 2.85	0.37	Synergism
9	SK-HEP-1	11.09 ± 0.82	L-CAV IC10 and DOX	0.32 ± 0.09	0.28	Strong synergism
10			L-CAV IC30 and DOX	0.31 ± 0.08	0.61	Synergism
11		77.89 ± 17.77	L-CAV IC10 and CIS	20.72 ± 2.16	0.44	Synergism
12			L-CAV IC30 and CIS	4.56 ± 1.31	0.63	Synergism

DOI: 10.7717/peerj.1542/table-5

Note:

Data from three independent experiments (n = 3) are represented as mean ± SD.

The dosage reduction index (DRI) of DOX and CIS, calculated from 6 different cell lines, is illustrated in Figs. 5 and 6. The higher DRI value for HeLa and Caco-2 cells indicates that both cell lines are more sensitive to the combination treatment as compared with pancreatic cells (MIA PaCa-2 and BxPC-3) and hepatocellular carcinoma cells (Hep G2 and SK-HEP-1). A dosage reduction of CIS was observed to be superior as compared with DOX in some cancer cell lines.

Discussion

Based on the mode of action, L-CAV is categorized as an antimetabolite, as it inhibits the metabolism of arginine (L-ARG), to which it is structurally related. The only difference between these two structures is the replacement of the methylene bridge (-CH2- unit) in L-ARG with an oxygen in L-CAV, making it less basic. L-CAV can be incorporated into the protein because arginyl-tRNA synthetase is unable to distinguish between L-ARG and L-CAV. Some studies have reported that L-CAV replaces L-ARG in the protein in Walker carcinoma 256 cells (Kruse et al., 1959), Chinese hamster ovary cells (Laszlo & Li, 1993), HeLa S-3 cells (Wheatley & Robertson, 1981), and Hep G2 cells (Redman, Avellino & Yu, 1983), and that an incorporation leads to the alteration of protein conformation (Rosenthal, Reichhart & Hoffmann, 1989) in murine leukemia virus, moloney murine sarcoma virus (Murphy & Arlinghaus, 1980), and Hep G2 cells (Redman, Avellino & Yu, 1983). However, because L-CAV is structurally related to L-ARG, a high concentration of L-CAV is required to outcompete L-ARG, which is normally present as an essential amino acid in good quantities.

In this study, to enhance the efficacy of L-CAV, the concentration of L-ARG was reduced in the cell culture media. In clinical application, deprivation of L-ARG can be achieved via arginine deiminase (ADI) which has high affinity to arginine and catalyzes arginine to citrulline and ammonia. The application of L-ARG deprivation has been optimized using polyethylene glycol (PEG) technology to diminish immunogenicity, increase bioavailability, and selectively degrade arginine, therefore resulting in tumour cell death (Feun et al., 2008). In phase I/II clinical trials, a weekly schedule of intramuscular application of ADI-PEG 20 resulted in 25% and 47% (complete and partial) response in patients with advanced melanoma and hepatocellular carcinoma (Izzo et al., 2004; Ascierto et al., 2005). Clinical trial with several hundred patients with cancer has revealed that ADI-PEG 20 is feasible and safe. However, a concern shall be taken in several patients in whom arginine deprivation can cause contraindication related to T cell immunity and cardiac function, such as in patients with an urea-cycle disorders, active or treated infections (TB and HIV), immunodeficiency and cardiovascular disease (Delage et al., 2010). In addition, arginine deprivation can also induce ASS expression in certain melanoma cell lines which possibly lead to drug resistance (Feun et al., 2008; Savaraj et al., 2007). Combination of L-ARG deprivation (i.e. ADI) with other cytotoxic agent or targeted agent can reduce the side effect, for instances hyperuricaemia in the case of hepatocelular carcinoma, other side effects such as pain in the injection side and also the possibility of drug resistance (Delage et al., 2010). Combination of L-ARG deprivation with L-ARG analogue, L-CAV is a rational approach. In addition the combination with chemotherapeutic drugs, such as DOX and CIS will enhance the anticancer activity while reducing the toxicity and side effects.

Application of L-CAV may compete with metabolic reactions in which arginine is the substrate of reaction, such as polyamine synthesis. A study using rat bearing colon tumour treated with L-CAV alone showed a body weight loss after certain dose and schedule of administration (Thomas et al., 1986). Combination of L-CAV with other agents could reduce the L-CAV dose, diminish the body weight loss (Thomas and Rosenthal, 1987) and reduce the possible immunotoxic effect, as seen in peripheral blood mononuclear cells (Bence et al., 2002). The potency of L-CAV in drug combination, for instance with other chemotherapeutic drugs is still little studied. Only a study conducted by Swaffar and co-workers has reported a potential combination of L-CAV and classical antimetabolite, 5-fluoruracil in human pancreatic cancer cells (Swaffar et al., 1995).

In this study, drug combinations were evaluated in 6 different cancer cell lines, which differed in the expression of argininosuccinate synthase (ASS). Some studies have investigated the importance of ASS deficiency and the effect of L-ARG deprivation in human cancers (Bowles et al., 2008; Dillon et al., 2004; Scott et al., 2000; Sugimura et al., 1992). As confirmed in this study, HeLa and Caco-2 cells exhibit a higher ASS expression as compared to both pancreatic cancer cells (MIA PaCa-2 and BxPC-3) and hepatocellular carcinoma cells (Hep G2 and SK-HEP-1) (Fig. 1). The cytotoxicity of DOX was found to be lower in HeLa, Bx-PC3, Hep G2 and SK-HEP-1 treated in AFM as compared to ARM. Deprivation of ARG concentration in the culture media of HeLa, Bx-PC3, Hep G2 and SK-HEP-1 disturbed cell metabolism and may affect the active transport protein, which related to the reduction of uptake kinetic to DOX, known as a drug substrate, belonging to the adenosine triphosphate (ATP) binding cassette (ABC) transporters (Katayama, Noguchi & Sugimoto, 2014). Thus, a reduction of DOX cytotoxicity was observed in those cells with ARG deprivation. On the other hand, an increased cytotoxity of CIS was noted in HeLa, Bx-PC3, Hep G2 and SK-HEP-1 when the cells were treated in AFM as compared to ARM. Some authors have reported that cisplatin resistance in some cancer cells is due to the rapid efflux of CIS (Wang & Lippard, 2005). This result suggests that L-ARG deprivation alone is not sufficient to enhance the cytotoxicity of DOX and CIS. But L-CAV could potentiate the cytotoxicity of DOX and CIS, especially in cells expressing high levels of ASS as compared to the cells with reduced ASS levels (Tables 3 and 4).

How can this difference be explained? Cells expressing ASS also show high levels of arginyl-tRNA synthetase (Kim, You & Hwang, 2011). Arginyl-tRNA synthetase is an enzyme which uses L-ARG as a substrate to form the complex L-arginyl-tRNA(Arg), necessary in ribosomal protein synthesis. L-CAV acts as an alternative substrate for arginyl-tRNA synthetase; thus, in cells expressing high ASS, higher levels of arginyl-tRNA synthetase may increase the chance of L-CAV incorporation into a protein in place of L-ARG. Enzymes which become functionless may include the battery of DNA repair enzymes or proteins involved in drug metabolism (including ABC transporters), which often reduce the toxicity of chemotherapeutic drugs. As a consequence, such tumour cells will become more sensitive to DOX and CIS when treated with L-CAV in combination.

In Caco-2 and MIA PaCa-2, the cytotoxicity of DOX and CIS in IC₅₀ values did not show a significant difference in ARM and AFM. When a combination of L-CAV and DOX or CIS is performed in ARM, we assume that L-CAV can potentiate the cytotoxicity of drugs in both cell lines, optimally using sequence–dependent administration (L-CAV following the chemotherapeutic drugs) instead of simultaneous-dependent administration as used in this study. Administration of L-CAV at the first order for a certain period may efficiently compete with the presence of L-ARG and resulting the sensitivity of cancer cells to the chemotherapeutic drugs.

Scientific reason why different tumor cell lines possess different levels of ASS expression remains unclear. Previous studies indicate that ASS regulation occurs at pre-translational levels (Husson et al., 2003) and it can be influenced by multiple factors and may be tissue specific. Further investigation is necessary to identify the dose reduction index in the cells where ASS activity is decreased. For instance, using pharmacological inhibition of ASS such as fatty acids; insulin and growth hormone in liver tissue (Feun et al., 2008) or by silencing of ASS expression to understand in detail the role of ASS protein and related signalling pathway that can be influenced due to the application of L-CAV.

Conclusions

In conclusion, the mechanism underlying the synergistic interaction between L-CAV and two DNA interacting drugs could be explained by L-ARG deprivation, which facilitates L-CAV incorporation into proteins in place of L-ARG. Incorporation of L-CAV disturbs the correct 3D-structure of target proteins and thus leads to protein dysfunction or prevention of repair of DNA damage. This condition may sensitize cancer cells for DOX and CIS. This study has revealed an interesting potentiation of L-CAV in combination with chemotherapeutic drugs, specific DNA-targeting drugs in L-ARG auxotrophic and non-auxotrophic cancer cell lines.

In vivo study employing an exact dose level and schedule of administration in combination with the chemotherapeutic drugs is warranted to understand whether drug combination works in vivo. The mechanism of combination, complex protein signaling pathways and virtually metabolic reactions associated with L-ARG utilizing pathway requires a further investigation.

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