A mixture of amino acids and other small molecules present in the serum suppresses the growth of murine and human tumors in vivo

International Journal of Cancer A mixture of amino acids and other small molecules presentin the serum suppresses the growth of murine and humantumors in vivo ´ kos Schulcz2 and Tam
as Cz€omp€oly1 1 Immunal Ltd., Cancer Research and Product Development Laboratory, H-7630 P
ecs, Finn u. 1/1., Hungary2 Department of Experimental Pharmacology, National Institute of Oncology, H-1122 Budapest, R
ath Gy€orgy u. 7-9., Hungary Previously we have hypothesized that the small molecules which are selectively accumulated in cancer cells might participate in a non-immunological antitumor surveillance mechanism. We demonstrated earlier that a mixture of experimentally selected substances (‘‘active mixture'', AM: L-arginine, L-histidine, L-methionine, L-phenylalanine, L-tyrosine, L-tryptophan, L-ascorbate, D-biotin, pyridoxine, riboflavin, adenine, L(-)malate) possesses a selective toxic effect in vitro on a variety of tumor cell lines, and we have shown that the AM selectively induces apoptosis of cancer cells in vitro. To explore the in vivo significance ofour earlier findings we examined the antitumor effect of AM in Colon 26 murine colorectal adenocarcinoma, B16 murine melanoma, MXT murine mammary carcinoma, S180 murine sarcoma, P388 murine lymphoid leukemia, HL-60 human promyeloid leukemia, PC-3 human prostate carcinoma, and HT-29 human colon carcinoma tumor models. Treatment of tumor bearing mice with AM inhibited the growth of the tumors investigated, with an inhibitory effect ranging from 40 to 69%. The AM had a comparable antitumor effect with 5-fluorouracil and cisplatin in the Colon-26 tumor model, and combined treatment with AM and 5-fluorouracil or cisplatin resulted in an enhanced tumor growth inhibitory effect. The AM induced apoptosis through the mitochondrial pathway and induced G1 arrest in PC-3 cells and increased the number of apoptotic cells in PC-3 xenografts. These findings suggest that the AM might offer an interesting perspective in the treatment of cancer and in combination with other treatments may offer hope for a more effective cancer therapy.
Cancer cells formed despite the nonimmune safeguards All multicellular living organisms have different mechanisms should be eliminated by the next stage of host protection.
that reduce the otherwise high probability of cancer develop- Although, there are indications for the existence of an inter- ment. In higher vertebrates at least three types of nonim- cellular surveillance (microenvironmental control of tu- mune surveillance take part in the first line defense against mor),1,2 the main role in the second line of defense is tumors. In addition to the two major forms of surveillance: ascribed to the immune system, the only defense mechanism the genetic (DNA repair, checkpoint control) and the intra- which has components in the circulatory system. However, cellular (largely apoptosis related) surveillance, recent findings accumulating evidence indicates that tumors use multiple suggest the existence of the epigenetic surveillance (stringency mechanisms to evade the effect of the immune surveillance.
of chromatin imprinting).1,2 As a result of their mechanisms In addition immunoediting can lead from immune surveil- the role of these first line defense systems is to prevent can- lance to tumor escape. Consequently, the immune system fre- cer cell formation.
quently fails to eliminate cancer cells, and its action is mainlyrestricted to the virus induced tumors.1–7 Considering the above described limitations in the effi- ciency of immune surveillance in tumor control and the mul- Key words: antitumor effect, amino acids, vitamins, apoptosis, tiple mechanisms used by tumors to evade the effect of the immune system, together with the fact that despite these lim- Additional Supporting Information may be found in the online itations tumors do not develop in the majority of people, version of this article.
motivated us to examine the possibility that besides the well Grant sponsor: Immunal Ltd established immunological and nonimmunological surveil- DOI: 10.1002/ijc.27756 lance additional defense mechanism(s) might operate to pre- History: Received 1 Jun 2011; Revised 2 Jul 2012; Accepted 9 Jul vent the development of tumors.
2012; Online 1 Aug 2012 We hypothesized that the components of this additional Correspondence to: Gyula Kulcs
ar, Immunal Ltd., Cancer Research defense mechanism (a ‘‘surveillance'') might be in the circula- and Product Development Laboratory, Finn u. 1/1, H-7630, P
ecs, tory system and turned our attention to those small substan- Hungary, Tel: þ3672525010, Fax: þ3672525011, ces (amino acids, monosacharides, nucleobases, etc.) present in the serum which are differentially taken up by tumor and Int. J. Cancer: 000, 000–000 (2012) V Antitumor effect of a mixture of amino acids and small molecules Tumor cells have increased glucose uptake and in addition accumulate other molecules such as amino acids and vitamins to higher levels than non-tumor cells. The authors hypothesized that this accumulation represents a tumor defense mechanism and tested the effect of a mixture of these substances called Active Mixture in murine and human xenograft tumor models.
Active Mixture induced apoptosis of tumor cells both in vitro and in vivo and effectively diminished tumor growth in a varietyof tumor models offering an interesting new perspective on tumor therapy.
normal cells.8 Since the elevated uptake of glucose and mM L(-)-malic acid, 72.64 mM L-phenylalanine, 51.66 mM increased glycolitic activity of cancer cells has been first L-arginine, 73.47 mM L-histidine, 1.38 mM L-tyrosine, 20.11 reported by Otto Warburg,9 it has been shown that in addi- mM L-methionine, 14.69 mM L-tryptophan, 0.06 mM D-bio- tion to glucose many molecules (amino acids, vitamins) are tin, 1.02 mM pyridoxine hydrochloride, 2.49 mM adenine accumulated in cancer cells.10–12 The accumulation of these hydrochloride, 0.41 mM riboflavin-50-phosphate, and 23.39 substances by cancer cells is utilized in positron emission to- mM L-ascorbic acid. The solution was prepared by reconsti- mography,13 and targeting strategies has been started to tuting of Culevit powder for solution (manufactured by emerge on the basis of amino acid and vitamin accumula- Human Serum and Pharmaceutical Manufacturing Company, tion.14,15 In recent years it became increasingly clear that ollo†, Hungary for Immunal Ltd., Budapest, Hungary).
there are a significant number of common signaling pathways The CM used for the in vivo experiment has the following regulating both cellular metabolism and cell proliferation.16 composition: 32.07 mM succinic acid disodium salt, 72,64 Taking into account that many molecules in the living system mM L-valine, 51.66 mM L-asparagine, 73.47 mM L-serine, have more than one fundamentally different role we assumed 1.38 mM L-alanine, 20.11 mM glycine, 14.69 mM L-proline, that some of the accumulated substances besides their usual 0.06 mM thiamin hydrochloride, 1.02 mM folic acid sodium role in metabolism might participate in a defense system ca- salt, 2.49 mM hypoxanthine, 0.41 mM D-pantothenic acid pable of killing emerging cancer cells.
hemicalcium salt, 23.39 mM niacin.
Previously we have substantiated our hypothesis by exper- On the basis of a 25-fold in vivo dilution factor (200 ll imentally selecting substances present in the serum whose injected mixture/5 ml of extracellular fluid volume22) the mixture (‘‘active mixture,'' AM) showed a selective toxic concentrations of the components of the in vitro used AM effect in vitro on a variety of tumor cell lines.17,18 We have and CM were calculated with division of the in vivo used also demonstrated by several methods that the AM selectively concentrations by 25.
induce apoptosis of cancer cells in vitro.19,20 Recently we All chemicals, media, and materials used in this study have shown that combination of the AM with various cyto- were purchased from Sigma (Budapest, Hungary) except oth- static agents or irradiation results in an increased cytotoxic erwise indicated.
effect in vitro.21 In this study we investigated the in vivo antitumor effect Cell lines, tumors and animals of the AM alone or in combination with cytostatic agents. In The description of cell lines, tumors and animals is provided this article we provide evidence that the AM has a significant in Supporting Information Materials and Methods.
tumor inhibitory effect in vivo, treatment with AM increasesthe antitumor activity of cytostatic agents, and induces apo-ptosis both in vitro and in vivo. In addition we demonstrate Evaluation of antitumor activity of the active mixture in that the AM induces apoptosis via the mitochondrial path- syngeneic mouse tumor models way, and influences the proliferation of cancer cell by induc- P388 lymphoid leukemia (1 x 107 cells/mouse) were injected ing G1 arrest.
subcutaneously (s.c.) into the flank of BD2F1 mice. Tissuefragments (3–4 mm, app. 25 mg in weight) of Colon 26 ade- nocarcinoma and S180 sarcoma were transplanted s.c. into Material and Methods the flank of BALB/c mice. Tissue fragments of MXT hor- mone sensitive mammary carcinoma and B16 melanoma The selection of the components of the ‘‘active mixture'' were transplanted s.c. into the flank of BD2F1 mice. The (AM) and ‘‘control mixture'' (CM) has been described previ- treatments were started on the first day after tumor inocula- ously,17,18 a brief description is provided in Supporting Infor- tion. The AM was given i.p. daily in a volume of 0.2 ml (in mation Materials and Methods. The AM used for the in vivo the case of dose dependence experiment 0.2, 0.1 or 0.05 ml) experiments was formulated on the basis of the above men- at 1-hr intervals eight times a day for 10 consecutive days (or tioned results17,18 with consideration of unavoidable practical for 17 days as indicated). Cisplatin was injected i.p. once a aspects (the rate of excretion, the solubility, the stability, the day on Days 1, 5 and 9 at a dose of 2.5 mg/kg. 5-FU was pharmaceutical grade and the price, etc. of the components).
administered i.p. once a day for 5 days after tumor inocula- This ‘‘practical'' AM has the following composition: 32.07 tion at a dose of 25 mg/kg. Control mice were injected with Int. J. Cancer: 000, 000–000 (2012) V Table 1. Antitumor activity of the AM in mouse syngeneic and potential and mitochondrial mass, western-blot, cell division human xenograft tumor models tracking, cell cycle analysis and quantitative RT-PCR (QPCR) Tumor volume (mm3) is provided in Supporting Information Materials and Methods.
Statistical analysis Mouse syngeneic models Statistical analysis was performed by either unpaired Stu- dent's t-test or ANOVA followed by Bonferroni test, as indi- cated. p values less than 0.05 were considered statistically sig- nificant. Statistical analyses were performed with OriginPro7 Human xenograft models Antitumor activity of the AM in various mouse syngeneic and human xenograft tumor models We studied the antitumor activity of the AM on various syngeneic mouse tumor models (Colon 26 adenocarcinoma,B16 melanoma, MXT breast carcinoma, S180 sarcoma, Note: Treatment was initiated on the first day (mouse syngeneicmodels) or on the seventh day (human xenograft models) after tumor P388 lymphoid leukemia) and on human xenograft tumor inoculation. Tumor volumes measured at the end of the 10 day long models (HL-60 human promyeloid leukemia, the PC-3 treatment period are shown. Values represent mean 6 SEM. P values human prostate carcinoma and the HT-29 human colon were calculated by unpaired t-test.
carcinoma). In case of the syngeneic mouse models treat- saline. The tumor growth inhibition (TGI) was monitored by ment with AM for 10 days produced a significant growth measuring the tumor volume with a digital caliper. Tumor inhibitory effect ranging from 55 to 69%. The growth of volume (V) was calculated by the formula of V ¼ a2 x b x p/ the human xenograft tumors was also significantly inhibited 6 where ''a'' and ''b'' stand for the shortest and the longest (TGI: 40%) (Table 1). On the basis of body weight meas- diameter of the tumor, respectively.23 All animal procedures urements performed on every second day we observed no were performed in accordance with published guidelines on toxic effect (data not shown). Using the P388 lymphoid the welfare of animals in experimental neoplasia,24 therefore leukemia model the antitumor activity of the AM was the animals were decapitated when the volume of the tumors found to be dose dependent and could be sustained during reached or exceeded
2,000 mm3. The protocols were an extended treatment period (Supporting Information Fig.
approved by the Ethical Committee of Animal Experiments S1). We also examined the effect of chronic AM exposure of the National Institute of Oncology, Budapest, Hungary.
using the PC-3 tumor model. In these experiments we usedcells instead of tissue fragments for tumor inoculation andwe changed the dosage regimen (0.2 ml of the AM was Evaluation of antitumor activity of the active mixture in given i.p. daily at 2-hr intervals four times a day). Accord- human xenograft tumor models ing to our results the TGI was lower at the early phase of Tissue fragments (3–4 mm, app. 25 mg in weight) of HL-60 the long-term experiments than in our previous short term promyeloid leukemia, PC-3 human prostate carcinoma and experiments; however, the TGI measured at the end of the HT-29 human colon carcinoma were transplanted s.c. into experiment reached the same level as in the short term the intrascapular region of CB17/ICR-Prkdcscid mice. For the experiments (TGI at day 42: 41%, Fig. 1). To exclude any long-term experiments 5x105 PC-3 cells were injected s.c.
potential nonspecific effect we performed an experiment into the left flank. To spare animals, in the experiments with with a control mixture (CM) which had the same osmolar- CM five mice per group (bearing 2 tumors/animal) were ity as the AM and contained similar but ineffective small treated. The AM and CM was given i.p. daily in a volume of molecules as it is described in the ‘‘Supporting Information 0.2 ml at 1-hr intervals eight times a day for 10 consecutive Materials'' section. We have found that the CM had no days (or for 16 days as indicated). To reduce the potential effect on the growth of PC-3 xenografts (Supporting Infor- distress on the animals the dosage regimen was changed in mation Fig. S2).
the long-term experiments: the AM was given i.p. daily in avolume of 0.2 ml at 2-hr intervals four times a day for 30 The antitumor effect of the AM is comparable with 5-FU in consecutive days. Control mice were injected with saline the Colon 26 colon carcinoma model except for one experiment in which CM was also used. The We have found that treatment of the Colon 26 adenocarci- evaluation of TGI was performed as described for the synge- noma with the AM or 5-FU inhibited the tumor growth with neic mouse tumor models.
comparable efficacy. The TGI at the end of the 10 day long The description of the cell growth assay, annexin V stain- treatment period was 57% and 47% (p < 0.001 for both) for ing, TUNEL assay, measurement of mitochondrial membrane the AM and 5-FU, respectively. Concomitant administration Int. J. Cancer: 000, 000–000 (2012) V

Antitumor effect of a mixture of amino acids and small molecules Figure 1. The tumor growth inhibitory effect of the AM issustainable during the long term treatment of PC-3 xenografts.
Mice were treated with AM started from the 13th day after tumorinoculation (arrow). Error bars represent SEM. *p < 0.05 (Student'st-test).
of the AM and 5-FU produced a 65% TGI (p < 0.001 vs.
control, Fig. 2a). At the end of the treatment period tumorvolumes of the group which received combined treatmentshowed no statistically significant difference when comparedwith groups treated with 5-FU alone or the AM alone. How-ever, a sustained TGI was observed in the combined treat-ment group after the termination of treatment, and the dif-ference between the tumor volumes of the group treated with5-FU only and the group which received the combined treat-ment became significant.
Figure 2. Combination of the AM with 5-FU or cisplatin increasesthe TGI in the Colon 26 colon carcinoma model. (a) Mice weretreated with 5-FU, with AM, or with 5-FU and AM. *p < 0.05, **p < Combination of the AM and cisplatin increases the TGI in 0.001 vs. control; #p < 0.001 vs. 5-FU (ANOVA). B, Mice were the Colon 26 colon carcinoma model treated with cisplatin, with AM, or with cisplatin and AM. *p <0.05, ** Next we assessed the antitumor effect of the AM in compari- p < 0.001 vs. control; #p < 0.001 vs. cisplatin and vs. AM (ANOVA). Arrows indicate the last days of treatment. Error bars son with cisplatin. Treatment of Colon 26 adenocarcinoma represent SEM.
with AM seemed to produce a higher TGI than cisplatin(AM: 57%, p < 0.001 vs. control, cisplatin: 31%, p < 0.001vs. control), however the difference between these treatment any potential nonspecific effect we applied a CM correspond- groups was not significant. Combination of the AM with cis- ing to the composition of the in vitro used AM in all in vitro platin resulted in a 73% (p < 0.001) TGI (Fig. 2b). It is im- portant to note that significant difference was observed In a series of in vitro experiments we have found that between the antitumor effect of the single and combined treatment with AM, but not with CM inhibits the growth of treatments. Moreover, in case of the combined treatment the PC-3 cells and induces apoptosis as evidenced by phosphati- tumor volume decreased during the treatment period, and a dylserine externalization (Supporting Information Fig. S3).
statistically significant TGI was maintained even after treat- To correlate our in vitro findings with the in vivo antitu- ment termination.
mor activity of the AM we performed an in vivo experimentin which we assessed the extent of apoptosis in the tumor The AM induces apoptosis of PC-3 cells both in vitro and tissue with a TUNEL assay. We have found that treatment of PC-3 tumor xenografts with AM produced a 55% TGI at We sought to investigate the mechanism underlying the in the end of the 16 days long treatment period (Fig. 3a), and vivo antitumor activity of the AM. We have selected the PC- the number of apoptotic cells in the treated group showed a 3 human androgen independent prostate carcinoma model 2.5-fold increase compared with the control group (Figs. 3b for further in vitro and in vivo investigations. To rule out Int. J. Cancer: 000, 000–000 (2012) V

Figure 3. Treatment with AM inhibits tumor growth and inducesapoptosis in the PC-3 human prostate carcinoma model. (a) Mice Figure 4. AM activates the mitochondrial pathway of apoptosis in were treated with AM started from the first day after tumor PC-3 cells. (a) Percentage of cells with low DWm as measured by inoculation. Arrow indicate the last day of treatment. Error bars JC-1. *p < 0.05, **p < 0.001 (ANOVA). (b) Percentage of cells with represent SEM. *p < 0.001 (Student's t-test). (b) Representative decreased mitochondrial mass as measured by NAO. *p < 0.001 images of TUNEL assay. Bars represent 100 lm. (c), Percentage of (ANOVA). (c) Western-blot analysis of protein lysates from cells TUNEL positive cells. Data are presented as mean 6 SEM of nine treated with AM or CM. þ Lysate from Jurkat cells treated with 25 sections. *p < 0.001 (Student's t-test).
lM etoposide. Data are presented as mean 6 SEM of threeindependent experiments (panels a and b) or representative ofthree independent experiments (panel c).
The AM reduces the mitochondrial membrane potential(DWm), decreases mitochondrial mass, and activatescaspase-9 increased the percentage of cells with low DWm as measured To study the mechanism of apoptosis induction we investi- by JC-1 (Fig. 4a), and with low mitochondrial mass as meas- gated the effect of AM on DWm and mitochondrial mass in ured by acridine orange 10-nonyl bromide (NAO) (Fig. 4b).
PC-3 cells. We found that treatment with AM significantly To further examine the mechanism of apoptosis we Int. J. Cancer: 000, 000–000 (2012) V

Antitumor effect of a mixture of amino acids and small molecules Figure 5. AM inhibits proliferation, induces G1 arrest, and influences gene expression in PC-3 cells. (a) Cell division tracking of CM or AMtreated cells with CFSE. The mean fluorescence intensity (MFI) is plotted against the treatment lengths. (b) Cell cycle analysis of cellstreated with AM using propidium iodide staining. (c) Quantitative RT-PCR analysis of gene expression in AM treated PC-3 cells. (d), As in c,but LNCaP cells were treated. Data are presented as mean 6 SEM of three independent experiments. *p < 0.05, **p < 0.001 (ANOVA).
investigated whether caspase-3, 8 and 9 are activated.
in the percentage of cells in S and G2/M phase (Fig. 5b). The According to our results caspase-9 and caspase-3 are acti- percentage of cells with hypodiploid DNA content (sub-G1) vated after 6–24 hr of treatment (Fig. 4c), while we found no was increased after 48 and 72 hr of treatment (Fig. 5b). These evidence of the processing of caspase-8 (data not shown).
results suggest that upon treatment with AM the cells are transiently arrested in G1; however, additional experimentsare needed to clarify whether the apoptotic cells originate The AM inhibits proliferation and induces G1 arrest from the G1-arrested population or become apoptotic during in PC-3 cellsTo further explore the mechanism of cell growth inhibition the S/G2 transition. The CM had no influence on the cell we performed cell division tracking with the dye carboxy- cycle distribution of PC-3 cells.
fluorescein succinimidyl ester (CFSE). We found that treat-ment with AM significantly inhibited the dilution of CFSE The AM influences the expression of genes involved in (Fig. 5a). The average doubling time calculated from the apoptosis and cell cycle regulation kinetics of the intensity decay was increased from 20.6 6 2.8 Next we measured the transcript levels of certain proapop- hr (CM treated cells) to 28.6 6 4.0 hr (AM treated cells).
totic genes, anti-apoptotic genes, genes involved in NF-jB Next we examined whether the slower rate of cell division signaling, and genes regulating the cell cycle by QPCR after is associated with alterations in the cell-cycle phase distribu- various periods of treatment with AM or CM. Since the tion. Cell-cycle analysis showed that treatment with AM for expression of many of the above genes is regulated by P53, 24 hr causes G1-arrest, which is accompanied by a decrease these experiments were carried out in both PC-3 (mutated Int. J. Cancer: 000, 000–000 (2012) V p53) and LNCaP (wild type p53) cells. Treatment of PC-3 Moreover, the AM induced apoptosis in PC-3 cells and xeno- cells with AM increased the expression of the BH3-only pro- grafts, which are reported to have a frame shift mutation in tein encoding PUMA (11.9-fold, p < 0.001), NOXA (5.7-fold, p < 0.001), and BIM (2.5-fold, p < 0.05) compared with The tumor growth inhibitory effect observed in human tu- untreated control (Fig. 5c). Interestingly, the expression level mor xenografts seemed to be slightly lower when compared of the anti-apoptotic protein encoding cIAP2 seemed to be with the growth inhibition measured in syngeneic mouse also increased (4.3-fold, p < 0.001). Among the caspase tumors (Table 1). This could be explained by the different encoding genes only the expression level of CASP5 was treatment schedules: in case of the human xenografts treat- increased (27.4-fold, p < 0.001), while among the NF-jB sig- ment was initiated when the tumors became detectable at naling related genes the expression level was increased in the Day 7 after inoculation, while treatment in the mouse tumor case REL (2.5-fold, p < 0.001), RELA (2.1-fold, p < 0.001), models was started at Day 1 after tumor inoculation. This and RELB (3.6-fold, p< 0.001). The expression level of caused a shift in the ratio of AM components to tumor mass, CDKN1A was increased to 17.4-fold (p < 0.001) (Fig. 5c).
which in case of the human xenografts resulted in a lower Treatment with CM had no effect on the expression of genes relative amount of AM components. The decrease of the rela- investigated (data not shown). Essentially similar results were tive amount of AM was more pronounced when the mice obtained when LNCaP cells were treated with AM (Fig. 5d).
had two tumors/animal and the treatment was initiated whenthe tumors became detectable. Consequently, the effect of AM decreased slightly further but it remained significant In this study we demonstrated that a mixture of amino acids, (Supporting Information Fig. S2). This is in agreement with vitamins and other small molecules (AM) has antitumor ac- our results which indicate that the antitumor activity of the tivity in both murine and human xenograft tumor models.
AM is dose dependent. In addition, when treatment of PC-3 Our in vivo studies show that AM inhibits the growth of Co- xenografts has been initiated on the first day after tumor lon 26 murine colorectal adenocarcinoma, B16 murine mela- inoculation the inhibition of tumor growth was comparable noma, MXT murine mammary carcinoma, S180 murine sar- with the growth inhibition observed in the mouse tumor coma, P388 murine lymphoid leukemia, HL-60 human models (Fig. 3a).
promyeloid leukemia, PC-3 human prostate carcinoma and We have previously shown that combination of AM and HT-29 human colon carcinoma with a TGI ranging from 40 various cytostatic agents (doxorubicin, etoposide, mitoxan- trone, 5-FU, vinblastine, mitomycin and cytarabine) increases We have shown earlier that the AM, but not the individ- the in vitro inhibitory effect on the growth of a number of ual components induced apoptosis of tumor cell lines.19 Here tumor cell lines (K562, Jurkat, A20, MCF7, HeLa).21 Here we we showed that the AM is capable to inhibit the growth of a demonstrated that the AM has a comparable antitumor effect wide range of murine and human tumors, induces apoptosis with 5-FU and cisplatin in the Colon-26 tumor model. More- of PC-3 human prostate carcinoma cells in vitro, and over, combination of AM and 5-FU or cisplatin enhances the increases the number of apoptotic cells in PC-3 xenografts.
in vivo tumor growth inhibitory effect, which could provide The fact that the antitumor activity of the AM could also be rationale for the combined use of AM and cytostatic agents demonstrated in CB17/ICR-Prkdcscid mice indicates that the in clinical practice. Furthermore, the tumor growth inhibitory tumor inhibitory effect of the AM is not dependent on func- effect of the AM proved to be sustainable over a long-term tional T and B cells. It has been reported that the inhibition treatment period, which in our view also supports the poten- of arginase I and L-arginine supplementation inhibits the tial use of AM in cancer therapy.
growth of Lewis lung carcinoma, however the inhibitory To further investigate the mechanism of tumor growth in- effect could not be demonstrated in immunodeficient mice.25 hibition we performed additional in vitro experiments with Thus the mechanism of the in vivo antitumor effect of the AM on PC-3 cells. It seems that the AM induces apoptosis AM seems to be different from that of arginase I inhibition.
via the mitochondrial pathway, since treatment with AM These data support a direct tumor inhibitory effect of the causes mitochondrial depolarization, decreases the mitochon- AM, though participation of the components of innate drial mass, and activates caspase-9 and caspase-3. However, immune mechanisms which are functional despite of the scid according to our results inhibition of cell proliferation and/or mutation could not be excluded. Thus further experiments changes in the cell-cycle distribution could also be involved performed with the same syngeneic tumor model and identi- in the cell growth inhibitory effect of the AM.
cal administration schedule using various immunodeficient To gain further insight into the mechanism of apoptosis and immunocompetent hosts are needed to clarify the role of induction and G1 arrest we quantified the expression level of immune mechanisms in the antitumor activity of the AM.
selected genes. According to our results treatment with AM According to our results the antitumor activity of the AM increases the expression of PUMA, NOXA and BIM. These is not dependent on P53 function, since inhibition of tumor pro-apoptotic members of the Bcl-2 family are among the growth could also be demonstrated in tumor models which effectors of mitochondrial apoptosis and considered to be pri- have deleted or mutated P53 gene (P388, HL-60, HT-29).
mary targets of P53; however, P53-independent induction of Int. J. Cancer: 000, 000–000 (2012) V Antitumor effect of a mixture of amino acids and small molecules these genes is also reported.27,28 This is in agreement with phenylalanine availability affects the invasion related signal- our findings, which show that the expression levels of these ing pathways, modulates the metastatic phenotype, causes cell genes were elevated in both PC-3 and LNCaP cells. The cycle arrest, and induces apoptosis in melanoma and prostate induction of these genes supports our functional data, and cancer cell lines in vitro.31–33 In addition the in vivo metasta- these results collectively point toward the induction of apo- sis or tissue infiltration inhibiting effect of tyrosine and phe- ptosis through the mitochondrial pathway. Surprisingly the nylalanine restriction has also been demonstrated in murine expression level of the anti-apoptotic gene cIAP2 was also melanoma, leukemia, lung carcinoma and hepatocarcinoma elevated in both cell lines. Though this finding is seemingly models.34–36 These in vivo data are not necessarily in contra- contradictory to the apoptosis inducing effect of the AM, it diction with our results, since they seem to demonstrate the is possible that the transcriptional activation of cIAP2 is inhibition of the metastatic processes, while the AM appear compensated by the elevated expression of the pro-apoptotic to exert a direct antitumor effect through the induction of genes, and the net effect is the apoptosis of the cell. This apoptosis. We think that their and our studies represent two hypothesis is supported by the work of Bednarski et al.29 different approaches to the problem using the same starting who showed that doxorubicin induces cIAP2 in sarcoma point, namely that the accumulation of many substances is cells, while the net effect of doxorubicin treatment was the increased by cancer cells. In theory both their depletion and apoptosis of the sarcoma cells (though in their case the our ‘‘overloading'' strategy could be valid.
compensatory effect was due to the downregulation of other In conclusion, we demonstrated that the AM has an in anti-apoptotic genes). The elevated expression of the cyclin- vivo antitumor effect, and the induction of apoptosis dependent kinase inhibitor CDKN1A supports the G1-arrest through the mitochondrial pathway plays a role in this causing effect of the treatment with AM. Elevated CDKN1A tumor growth inhibitory effect. In addition to apoptosis expression was found in both cell lines tested, which is con- induction the AM also slows the proliferation and induces sistent with the fact that CDKN1A could be induced either G1 arrest of PC3-cells. The strength of the antitumor effect in a P53-dependent or a P53-independent fashion.30 These of the AM is comparable with that of cytostatic agents, in vitro findings collectively indicate that treatment with and the combined treatment inhibits tumor growth more AM in addition to apoptosis induction through the mito- effectively than the single treatments. These findings to- chondrial pathway also slows the proliferation rate and gether suggest that the usage of AM might offer an inter- causes G1 arrest in PC-3 cells. However, further experi- esting perspective for new therapies in the treatment of ments are needed to clarify the functional relationship of cancer without side effects and in combination with other these effects.
treatments may offer hope for a more effective cancer The selective accumulation of amino acids and other small molecules by cancer cells in theory provides a possibility to interfere with the metabolic activity of the malignant cells by The authors thank Dr. L
aszl
o Lex for continuous support and help, Dr. P
eter decreasing the availability of the substances which are taken Balogh for help in TUNEL analysis, and Dr. J
ov
ari for help in the up in increased amounts. Indeed there are reports which long term in vivo experiments. Potential conflict of interest: Gyula Kulcs
ar demonstrate that the restriction of tyrosine, methionine and owns a 30% share in Immunal Ltd.
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