Creatine & Cancer

Dr. Theo Wallimann, Prof. Emeritus, Institute of Cell Biology, ETH Zürich-Hönggerberg, 8093 Zürich, Switzerland
Private: Schürmattstrasse 23, CH-8962 Bergdietikon Tel.: +41-(0)44-740-70-47, Fax: +41-(0)44-741-30-08
E-mail: theo.wallimann@cell.biol.ethz.ch
Internet:http://www.mhs.biol.ethz.ch/about-us/emeriti-formermembers/wallimann.html

The antitumor activity of Creatine and Creatine analogs, such as Phosphocreatine or Cyclocreatine, has been recognized since a number of years. Numerous studies investigating the effects of Creatine and Creatine analogues on a variety of different cancer cells, either in cell cultures (in vitro) or implanted into experimental animals (in vivo), have been published by different groups of scientists in peer-reviewed scientific journals over the last decades. Creatine-depletion or Creatine-replacement with Creatine analogues, especially beta-guanidino-propionic acid (beta-GPA) or Cyclocreatine have consistently shown significant effects on cancer cell proliferation (inhibiton of growth and survival of different types of cancer cells, among them many types of human cancer cells, either freshly explanted or establishe cell lines (see numerous refs. below) and in addition rendered these cancer cells more sensitive (by several orders of magnitude) to chemotherapy and/or radiation treatment (see Teicher et al.1995), inhibited cell cycle progression (Martin et al. 1994) and inhibited stimulated motility (potential for metastasis) (Mulvaney 1998).

In the some of these studies, Creatine has either shown similar, less pronounced or no anti-cancer cell growth and proliferation effects, but in none of the studies reported has Creatine ever been shown to enhance cancer cell growth in vitro or in vivo in a statistically significant manner. This is strong evidence that Creatine does not promote the growth of cancer cells nor enhance cancer cell survival in vivo, Furthermore, there is no evidence that orally-taken Creatine would act as a mutagenic or carcinogenic compound or that Creatine-supplementation would increase the risk of cancer, or generate cancer.

The latter has been claimed in a recent report by the French “Agencie Francais de Securite Sanitaire des Aliments” report of Jan. 24rd 2001, see URL: http://www.afssa.fr/actualites/index.asp?mode=actu&ladate=&id_theme=1086&id_info=3022
This report has been mistaken world-wide as the outcome of a French epidemiological study showing that creatine would cause cancer, while it turned out to be a rather embarrassing, unprofessional review of the existing literature on the effects of creatine in sports and its possible side effects.
Creatine and much more so its decay product, Creatinine, which is present at high concentrations in meat to be consumed are indeed known to be precursors of carcinogenic products that are formed only at high temperatures (200-250o C), e.g. when meat is broiled, grilled or barbequed (for a review see Wyss M. and Kaddurah-Daouk R. 2000, Physiol. Rev. 80, 1107-1125). These well known mostly creatinine-derived carcinogens seem to be responsible for the possible health hazards going with heavily broiled / grilledmeat. However, this latter fact has nothing to do with oral supplementation of pure creatine powder. Therefore, these two situations should not be related.

The work by Ohira and Inoue (1995) (see below), which is heavily cited in the French “Agencie Francais de Securite Sanitaire des Aliments” report “concerning the effects and safety of creatine” has been mis-interpreted by this agency as indicating that creatine would increase cancer growth, while in fact, the Japanese researchers (Ohira and Inoue 1995; and Ohira et al.1991) stress the point that replacement or withdrawl, by beta-GPA (a Creatine analogue), of cellular creatine in Ehrlich ascites tumor cells leads to a significantly lower proliferation of these tumor cells. The addition of 1% creatine to the food given to Nude mice that had been injected with equal numbers of Ehrlich ascites tumor cells, compared to control food without extra creatine, led to a slight decrease in tumor volume, a slight decrease in total cell counts (both statistically not significant ) (Fig.2), but to a significant loss in viability of these cancer cell in the creatine group (Fig. 3 B) and to strong anti-cancer effect by beta-GPA (Ohira et al. 1991). The 1% creatine concentration given to these animals would correspond to a daily intake of approximately 30-50 grams of Creatine per day for an adult person. If in the same animal model system beta-GPA or creatine were injected directly into the tumor volume, beta-GPA had again a significant inhibitory effect on cancer growth, whereas Creatine supplementation "tended to enhance the growth of these Ehrlich ascites tumor cells" as Ohira and Inoue (1995) state in their abstract. Looking, however, at the actual data, the statistical significance is not convincing and therefore one cannot conclude that creatine would enhance cancer growth. When investigating the effects of beta-GPA and creatine on isolated Ehrlich ascites tumor cells in vitro in cell cultures, these authors found that the cells grew much better in their original ascites fluid environment with no supplement than in serum-free medium or in serum supplemented with creatine.

An important control, cells grown in ascites fluid plus added creatine is missing in their work. In those cultures with serum, Creatine had no effect, but In serum-free cultures Creatine had a markedly positiv effect on the growth of the Ehrlich ascites tumor cells in vitro, which, however, is not astonishing since many other normal, not cancerous cells, e.g. muscle cells (Pulido et al. 1998, FEBS Letters 439, 357-362) or neuronal cells (Brewer and Wallimann 2000, J. Neurochemistry 1968-1978) also grow better in medium containing creatine or even get protected against cellular stress by creatine (see the above references).
This is one of the reasons why creatine attracted so much attention lately as a possible therapeutic intervention for neuromusclular (Tarnopolsky and Martin 1999, Neurology 52, 854-857; Walter et al. 2000, Neurology 54, 1848-1850) and neurodegenerative diseases (Klivenyi et al. 1999, Nature Medicine 5, 347-350) (for review see Wallimann et al. 1999, in: Guanidino Compunds, Mori et al. eds. Blackwell sceince Asia pty ltd. pp 117-129). This simple, cheap and safe nutritional supplement has shown to afford remarkable neuroprotective effects in vitro (Wallimann and Brewer, 2000, J. Neurochemistry 74, 1968-1978; Brustovetsky et al. 2001, J. Neurochemistry 76, 425-434), in situ (Carter et al. 1995, J. Neurochem. 64, 2692-2699; Wilken et al. 1998, Pediatric Research 43, 8-14) and in vivo (Holtzman et al. 1998, Pediatric Research 44, 410-414; Ferrante et al. 2000, J. Neuroscience 20, 4389-4397; Malcom et a. 2000, Brain Research 860, 195-198; Sullivan et al. 2000, Amm. Neurol. 48, 723-729).
In discordance with the in vivo results, addition of creatine to the Ehrlich ascites cells, cultured in normal serum, did not lead to increased viability of these cancer cells and neither did beta-GPA show an inhibitory action anymore on cell viability, as seen in vivo (Figs. 4 and 5 of Ohira and Inoue 1995). Interestingly, the earlier work of the same group (Ohira et al. 1991), which contains results that are contradicting in part the 1995 paper, by showing no enhancing effects at all of creatine on the very same Ehrlich ascites tumor cells in vivo (Fig 1), was not cited in the French report.
Thus again, to conclude from these data that creatine enhances cancer growth is absolutely untenable, especially in light of the majority of reports (see below) that provide clear evidence for either neutral or even clear-cut anti-cancer cell-growth and cell-viability effects of creatine on a variety of freshly explanted cancer cells or on established cancer cell lines, a number of both derived from human origin.

Provided that the recommended dosages, which are entirely within the realm of nutritional supplementation (except for the short loading phase where a somewhat higher dosage is used) are not exceeded, creatine is considered as a safe and valuable nutritional supplement for sports, rehabilitation and for persons who have to deal with the ever increasing physical and psychological demands of every days life, as well as for seniors and elderly people. This opinion is shared by US food and nutritional organisations, as for example by the Council for Responsible Nutrition Washington DC, USA, which officially declared the French creatine-cancer scare unfounded based on the existing scientific literature. For details, please, consult the following home pages: http://www.mmusa.com/creatine-supplement-cancer.php

Some of the most important publications concerning this topic are listed below, some of them with complete and some with shortened abstracts.

G. Ara, L.M. Gravelin, R. Kaddurah-Daouk, B. A. Teicher.
Antitumor Activity of Creatine Analogs Produced by Alterations in Pancreatic Hormones and Glucose Metabolism.
In vivo 1998, 12, 223-232.
Abstract: Animal study on rats bearing the 13762 mammary carcinoma. Intravenously administration of Creatine analogs Phosphocreatine, Cyclocreatine or *-guanidinopropionic acid on days 4 through 8 and 14 through 18 post tumor implantation leads to a tumor growth delay up to 15 days. The antitumor activity of the Creatine analogs results from three effects: a) interference with energy balance in the malignant cells through the Creatine kinase system, b) inhibition of the secretion of the positive growth factor insulin from the pancreatic beta-cells, and c) increased secretion of the growth inhibitory factor somatostatin from the pancreatic delta-cells.

G. Bergnes, W. Yuan, V.S. Khandekar, M.M. O'Keefe, K.J. Martin, B.A. Teicher, R. Kaddurak-Daouk.
Creatine and Phosphocreatine Analogs: Anticancer Activity and Enzymatic Analysis.
Oncology Research 1996, 8, 121-130.
Abstract: in vitro study on the cytotoxicity to human ME-180 cervical carcinoma, the MCF-7 breast adenocarcinoma and the HAT-29 colon adenocarcinoma cell lines at low mM concentrations. Creatine was active against the colon HAT-29 line and slightly active against the MCF-7 line. Of the Creatine analogs tested, cyclocreatine and phosphinic cyclocreatine were the most potent cytotoxic agents. Of Phosphocreatine analogs Phosphocreatine itself and phospho-cyclocreatine were most potent cytotoxic agents. In vivo evaluation showed a good correlation to in vitro results: significant tumor growth delay for Phosphocreatine, phosphocyclocreatine, cyclocreatine and phosphinic cyclocreatine (comparable to that seen with standard cancer chemotherapeutic drugs).

N.M. Hoosein, Martin KJ, Abdul M, Logothetis CJ, Kaddurah-Daouk R.
Antiproliferative effects of cyclocreatine on human prostatic carcinoma cells.
Anticancer Res 1995, 15(4),1339-1342.
Creatine kinase (CK; EC 2.7.3.2) isoenzymes and their substrates have an important function in cellular energy generation and utilization. The brain isoform (CK-BB) has been implicated in cellular transformation processes involving the oncogenic products the Ela virus and the p53 tumor suppressor gene. Cyclocreatine, an analogue of creatine, has been previously shown to inhibit the growth of a broad spectrum of cancer cells derived from solid tumors. Results reported herein indicate an increased level of creatine kinase activity in human prostate carcinoma cell lines and inhibitory effects of cyclocreatine alone and in combination with adriamycin on the growth of these cells in vitro and in vivo, in immune-deprived mice. Our results suggest the possible use of cyclocreatine in the treatment of prostatic carcinoma.

K.S. Jeong, S.J. Park, C.S. Lee, T.W. Kim, S.H. Kim, S.Y. Ryu, B.H. Williams, R.L. Veech, Y.S. Lee.
Effects of cyclocreatine in rat hepatocarcinogenesis model.
Anticancer. Res. 2000, 20, 1627-33.
Abstract: Cyclocreatine inhibits the degrees of GST-P-positive cells and apoptosis and is active against hepatocarcinogenesis in rat models. This result points out the unique nature of an anticancer agent that inhibits progression of chemically induced hepatocarcinogenesis of rats.

C.A. Kristensen, N. Askenasy, R.K. Jaln, A.P. Koretsky.
Creatine and cyclocreatine treatment of human colon adenocarcinoma xenografts: 31P and 1H magnetic resonance spectroscopic studies.
British Journal of Cancer 1999, 79, 278-285.
Abstract: Study on the antitumor effect of Creatine and Cyclocreatine in relation to drug accumulation, energy metabolism, tumor water accumulation and toxicity. Intratumoral substrate concentrations induced a similar decrease in growth rate in Nude mice carrying a human colon adenocarcinoma expressing CK activity, indicating that both substrates (Creatine and Cyclocreatine) were equally potent in tumor growth inhibition. In vivo, these agents did not induce excessive water accumulation and had no systemic effects on the mice (weight loss, hypoglycaemia) that may have caused growth inhibition.
Note: in this study Creatine was equally potent as an anti-cancer growth agent as Cyclocreatine, albeit at somewhat higher concentratons than the latter. It is conclueded that the anti-proliferative effect of the above CK substrates was not related to energy deficiency, but was associated with acidosis.

J.W. Lillie, M.O'Keefe, H. Valinski, H.A. Hamlin, Jr., M.L. Varban, R. Kaddurah-Daouk.
Cyclocreatine (1-Carboxymethyl-2-iminoimidazolidine) inhibits growth ofa broad spectrum of cancer cells derived from solid tumors.
Cancer Research 1993, 53, 3172-3178.
Abstract: In an effort to investigate the role of creatine kinase and its substrates in malignancy we have tested the effect of cyclocreatine [1-carboxymethyl-2-iminoimidazolidine (CCr)] on the growth of tumor cells in vitro and in vivo. CCr is phosphorylated by creatine kinase to yield a synthetic phosphagen [(N-phosphorylcyclocreatine (CCr approximately P)] with thermodynamic and kinetic properties distinct from those of creatine phosphate. Weshow that CCr accumulates as CCr approximately P in tumor cells expressing a high level of creatine kinase, and that the accumulation of this phosphagen is detrimental to tumor cell growth. Tumor cell lines expressing a low level of creatine kinase accumulate much less CCr approximately P, and consequently are growth inhibited only at higher concentrations of CCr. When these resistant cells are transfected with a creatine kinase B expression vector, they express creatine kinase, accumulate CCr approximately P, and are growth inhibited. In vivo, in nude mouse xenografts, the rate of growth of a high creatine kinase expressing tumor cell line is inhibited in animals fed 1% CCr. Our results indicate that CCr inhibits the growth of tumor cells in vitro and in vivo.

N. Maril, Degani H, Rushkin E, Sherry AD, Cohn M.
Kinetics of cyclocreatine and Na(+) cotransport in human breast cancer cells: mechanism of activity.
Am J Physiol, 1999, 277(4 Pt 1):C708-C716
Abstract The growth-inhibitory effect of cyclocreatine (CCr) and the kinetics of CCr and Na(+) cotransport were investigated in MCF7 human breast cancer cells and its adriamycin-resistant subline with use of (31)P- and (23)Na-NMR spectroscopy. The growth-inhibitory effect in the resistant line occurred at a lower CCr concentration and was more pronounced than in the wild-type line. This correlated with an approximately 10-fold higher affinity of CCr to the transporter in the resistant line. The passive diffusion coefficient of CCr was also higher in the resistant line by three- to fourfold. The transport of CCr was accompanied by a rapid increase in intracellular Na(+). This increase was found to depend on the rate of CCr transport and varied differently with CCr concentration in the two cell lines. It is proposed that the cotransport of CCr and Na(+) followed by increased Na(+) concentration, together with the accumulation of the highly charged phosphocyclocreatine, are responsible for cell swelling and death.
Note: This paper shows that creatine also had a clear effect on cancer cell swelling and killing those cells, albeit much lower than that obtained with cyclocreatine.

K.J. Martin, E.R. Winslow, M. O?Keefe, V.S Khandekar, A. Hamlin, J.W. Lillie, Kaddurah-Daouk, R.
Specific targeting of tumor cells by the creatine analog cyclocreatine.
International Journal of Oncology1996, 9, 993-999.
Abstractd: Cyclocreatine (CCr), a CK substrate analog was shown to be cytotoxic to a broad spectrum of solid tumors. We have measured and compared the CK activity and CCr sensitivity of 49 transformed and non-transformed cell lines. Tumor cell lines highest in CK and most sensitive to CCr were derived from prostate, small cell lung and neuronal tissue. The hematopoetic tumor lines tested were generally low in CK and all were resistant to CCr. Fourteen non-transformed cell lines were examined and all were resistant to the compound including six with high levels of CK. Thus, CCr preferentially targeted tumor cells. Further, CCr inhibited tumor cell proliferation more efficiently than macromolecular synthesis indicating that, rather than exerting a general effect on energy metabolism, CCr may act on a specific pathway involved in controlling tumor cell proliferation.
Note: this paper holds arguments against the notion that if antagonists of creatine would inhibit cancer growth then creatine should do the contrary by promoting it.

K.J. Martin, S.-F. Chen, G.M. Clark, D. Degen, M. Wajima, D.D. von Hoff, R. Kaddurah-Daouk.
Evaluation of Creatine Analogues as a New Class of Anticancer Agents Using Freshly Explanted Human Tumor Cells.
Journal of the National Cancer Institute 1994, 86, 608-613.
Abstract: The Creatine analogues, cyclocreatine and homocyclocreatine, effectively reduced colony formation of freshly explanted human tumor cells. The mechanism of action from those compounds seems to differ from those of standard chemotherapeutics.

K.J. Martin, E.R. Winslow, R. Kaddurah-Daouk.
Cell Cycle Studies of Cyclocreatine, a New Anticancer Agent.
Cancer Research 1994, 54, 5160-5165.
Abstract: Investigation of the effects of Cyclocreatine on proliferation, viability, and cell progression. Cyclocreatine demonstrated components of both cytostatic and cyctotoxic activity and caused general block of progression out of all phases of the cell cycle.

E.E. Miller, A.E. Evans, M. Cohn.
Inhibition of rate of tumor growth by Creatine and Cyclocreatine.
Proc. Natl. Acad. Sci. USA 1993, 90, 3304-3308.
Abstract: Growth rate inhibition of subcutaneously implanted tumors results from feeding rats and athymic nude mice diets containing 1% cyclocreatine or 1%, 2%, 5%, or 10% creatine. The tumors studied included rat mammary tumors (Ac33tc in Lewis female rats and 13762A in Fischer 344 female rats), rat sarcoma MCI in Lewis male rats, and tumors resulting from the injection of two human neuroblastoma cell lines, IMR-5 and CHP-134, in athymic nude mice. Inhibition was observed regardless of the time experimental diets were administered, either at the time of tumor implantation or after the appearance of palpable tumors. For mammary tumor Ac33tc, the growth inhibition during 24 days after the implantation was approximately 50% for both 1% cyclocreatine and 1% creatine, and inhibition increased as creatine was increased from 2% to 10% of the diet. For the other rat mammary tumor (13762A), there was approximately 35% inhibition by both 1% cyclocreatine and 2% creatine. In the case of the MCI sarcoma, the inhibitory effect appeared more pronounced at earlier periods of growth, ranging from 26% to 41% for 1% cyclocreatine and from 30% to 53% for 1% creatine; there was no significant difference in growth rate between the tumors in the rats fed 1% and 5% creatine. The growth rate of tumors in athymic nude mice, produced by implantation of the human neuroblastoma IMR-5 cell line, appeared somewhat more effectively inhibited by 1% cyclocreatine than by 1% creatine, and 5% creatine feeding was most effective. For the CHP-134 cell line, 33% inhibition was observed for the 1% cyclocreatine diet and 71% for the 5% creatine diet. In several experiments, a delay in appearance of tumors was observed in animals on the experimental diets. In occasional experiments, neither additive inhibited tumor growth rate for the rat tumors or the athymic mouse tumors.
Note: In all these tumors, mammary tumors, rat sarcoma and neuroblastoma dietary creatine alone had a remarkabe inhibitory effect on cancer growth in vivo.

P.T. Mulvaney, M.L Stracke., S.W.Nam, E. Woodhouse, M. O?Keefe, T.Clair, L.A. Liotta, R. Kaddurah- Daouk, E. Schiffmann.
Cyclocreatine inhibits stimulated motility in tumor cells possessing creatine kinase.
Int. J. Cancer 1998, 78, 46-52.
Abstract: Cyclocreatine (CCr) inhibits the stimulated motility o tumor cells wich posses creatine kinase. Human melanoma cells, transfected with a creatine kinase gene, showed an 80-90% reduction in chemotactic response to type iV collagen when incubated overnight in the presence of 10mM CCr. This inhibitory effect of CCr can only be partially reversed by addition of creatine. Further experiments utilizing type IV collagen as attractant demonstrated that CCr in hibited the chemokinetic and the haptotactic responses and the in vitro invasion of these melanoma cells through Matrigel coated membranes. In addition, motility stimulation of these cells bei either autotaxin or fibronectin was markedly inhibited by CCr. DU-145 prostatic tumor cells, expressing endogenous CK, also have a reduced motility response to either autotaxin or epidermal growth factor-induced motility in the presence of CCr indicating that this compound can also lower the metastatic potential of cancer cells.

Y.Ohira , and Inoue N.
Effects of creatine and beta-guanidinopropionic acid on the growth of Ehrlich ascites tumor cells: i.p. injection and culture study.
Biochim Biophys Acta 1995, 1243(3):367-372
Abstract Growth of Ehrlich ascites tumor (EAT) cells in the abdominal space of mice or in cell culture was studied in response to i.p. injection or addition, respectively, of creatine or creatine analogue beta-guanidinopropionic acid (beta-GPA). The increase in body weight of the mice due to cancer growth was less in the beta-GPA-injected than in the creatine- or sham-injected group. The volume of abdominal ascites and total cell counts at 11th day after implantation of EAT cells was significantly less in the beta-GPA than in the other groups. The proliferation rate of EAT cells in the beta-GPA group was 27% and 35% of the creatine- and sham-injected groups, respectively. Supplementation of creatine tended to enhance the growth of EAT cells. The creatine concentration in ascites fluid was approximately 4-times greater than in blood plasma of sham-injected control mice. But the creatine content in EAT cells was significantly reduced to approximately 50% in response to beta-GPA injection. Cell culture without creatine caused a significant decrease in viability. The viability was improved, however, by addition of either creatine or serum into the medium. By contrast, it was not significantly increased by addition of serum alone which caused only a minor elevation of the creatine level (23 microM). It is suggested that EAT cell growth isinhibited by lowering the availability of creatine in association with some unknown factors in serum or ascites fluid.

Y.Ohira , Ishine S, Inoue N, Yunoki K.
Reduced growth of Ehrlich ascites tumor cells in creatine depleted mice fed beta-guanidinopropionic acid.
Biochim Biophys Acta 1991,1097(2),117-122.
Abstract: The effect of implantation of Ehrlich ascites tumor (EAT) cells on creatine distribution was investigated. It was also studied how depletion of creatine by feeding creatine-analogue beta-guanidinopropionic acid (beta-GPA) affects the growth of EAT cells in mice. Enhanced mobilization of creatine from host tissues to EAT cells against a greater concentration gradient was observed. The creatine (but not creatinine) level in blood plasma was lowered to 22% of the normal value by beta-GPA feeding alone and assimilation of 14C-creatine into EAT cells was inhibited. The growth of EAT cells was significantly reduced and the duration of survival of mice after implantation of EAT cells was extended when the creatine concentration was decreased. A decrease in daily food consumption and the degree of muscle atrophy after implantation of EAT cells was less in beta-GPA than control groups. In the creatine-depleted mice, the rate of increase in total EAT cell number and the volume of abdominal ascites were approximately half of the control values, and more dead EAT cells were observed. These results suggest that supplementation of beta-GPA inhibits creatine transfer to EAT cells and reduces the growth of cancer cells.

Schiffenbauer YS, Tempel C, Abramovitch R, Meir G, Neeman M.
Cyclocreatine accumulation leads to cellular swelling in C6 glioma multicellular spheroids: diffusion and one-dimensional chemical shift nuclear magnetic resonance microscopy.
Cancer Res 1995, 55(1):153-158.
Abstract: Cyclocreatine, an analogue of creatine, inhibits tumor cell proliferation in vitro and in vivo. The effects of cyclocreatine in large C6 glioma multicellular spheroids were mapped here by magnetic resonance microscopy. Diffusion-weighted images of C6 glioma spheroids resolved the bright viable rim and the dark necrotic center. Sequential sets of diffusion images, following cyclocreatine administration, showed increasing self-diffusion coefficients of the intracellular water in the viable rim (0.49 x 10(-5) cm2/s for untreated spheroids, 0.62 x 10(-5) cm2/s after 48 h perfusion with 20 mM cyclocreatine). This fact correlated with cellular swelling apparent in histological sections. The radial distribution of cyclocreatine and soluble lipids across perfused C6 spheroids was measured by one-dimensional chemical shift imaging. Cyclocreatine accumulation was prominent throughout the viable cell layer, with no cyclocreatine accumulation in the necrotic center. In both cyclocreatine-treated and control spheroids the lipid signal was highest in the necrotic center and lower in the inner viable cell layer.

Schiffenbauer YS, Meir G, Cohn M, Neeman M.
Cyclocreatine transport and cytotoxicity in rat glioma and human ovarian carcinoma cells: 31P-NMR spectroscopy.
Am J Physiol 1996 Jan;270(1 Pt 1):C160-9
Abstract: Cyclocreatine (CY), an analogue of creatine, inhibits tumor growth in vivo and proliferation of tumor cells in vitro. The goal of this study was to probe the mechanism of CY transport and cytotoxicity in C6 rat glioma cells and OC238 human ovarian carcinoma cells (creatine kinase activities of 0.16 and 0.016 units/mg protein, respectively). In both cell lines, CY significantly inhibited cell growth with no effect on membrane integrity and on the content of nucleoside triphosphates. An intrinsic 31P-nuclear magnetic resonance (31P-NMR) signal of phosphocreatine, as well as accumulation of phosphocyclocreatine (PCY) after addition of CY, was observed for C6 glioma but not for the OC238 cells. Transport of CY in C6 glioma showed Michaelis-Menten kinetics for an active sodium-dependent component. Transport was reduced more than fivefold in low-glucose medium. The toxicity of CY to C6 glioma cells may be due to PCY accumulation and cellular swelling. Another mechanism must be invoked to explain CY effects on the human ovarian cancer cells in which no PCY accumulation could be detected and no cellular swellin was observed.

L. Schimmel, V.S. Khandekar, K.J. Martin, T. Riera, C. Honan, D.G. Shaw, R. Kaddurah-Daouk.
The Synthetic Phosphagen Cyclocreatine Phosphate Inhibits the Growth of a Broad Spectrum of Solid Tumors.
Anticancer Research 1996, 16, 375-380.
Abstract: BACKGROUND: The brain isoform of creatine kinase (CKBB), an enzyme involved in energy metabolism, has been implicated in cellular transformation process. Cyclocreatine (CCr), a creatine kinase (CK) substrate analogue, was shown to inhibit the growth of a broad spectrum of solid tumors expressing high levels of CK. Cyclocreatine phosphate (CCrP) generated by CK, was proposed to be the active form responsible for growth inhibition. MATERIALS AND METHODS: We synthesized CCrP and tested its cellular uptake and anti tumor activity in stem cell assays and in athymic mouse models. RESULTS: CCrP seems to be taken up by cells and inhibitsthe growth of solid tumors with high levels of CK. CCr and CCrP have similar specificity and potency. CONCLUSION: The observation that only high-CK cell lines were responsive to CCrP, similar to CCr, indicates that the enzyme requirement was not bypassed. We propose that CK is a target for CCrP, and is involved in mediating its antiproliferative activity.

B.A. Teicher, K. Menon, D. Northey, J. Liu, D.W. Kufe, R. Kaddurah-Daouk.
Cyclocreatine in cancer chemotherapy.
Cancer Chemother. Pharmacol. 1995, 35, 411-416.
Abstract: Cyclocreatine, an analog of creatine, is an efficient substrate for creatine kinase, but its phosphorylated form is a poor phosphate donor in comparison with creatine phosphate. Cyclocreatine was not very cytotoxic upon 24 h of exposure of human SW2 small-cell lung cancer cells to concentrations of up to 5 mM. However, combinations of cyclocreatine (0.5 mM, 24 h) with each of four antitumor alkylating agents, cis-diamminedichloroplatinum(II), melphalan, 4-hydroperoxy-cyclo-phosphamide, and carmustine, resulted in additive to greater-than-additive cytotoxicity toward exponentially growing SW2 cells. The greatest levels of synergy were seen at higher concentrations of 4-hydroperoxycyclophosphamide and carmustine as determined by isobologram analysis. In vivo cyclocreatine (0.5 or 1 g/kg) was more effective if given i.v. rather than i.p. The longest tumor-growth delays, up to 10 days, were produced by extended regimens of cyclocreatine. Cyclocreatine was an effective addition to therapy with standard anticancer agents including cis-diamminedichloroplatinum(II), cyclophosphamide, Adriamycin, or 5-fluorouracil. No additional toxicity was observed when 10 days of cyclocreatine treatment was given with full standard-dose regimens of each drug. The resultant increases in tumor-growth delay were 1.7- to 2.4-fold as compared with those obtained for each of the drugs alone. These results indicate that cyclocreatine may be an effective single agent and an effective addition to combination chemotherapy regimens.

Kornacker M, Schlattner U, Wallimann T, Verneris MR, Negrin RS, Kornacker B, Staratschek-Jox A, Diehl V, Wolf J.
Hodgkin disease-derived cell lines expressing ubiquitous mitochondrial creatine kinase show growth inhibition by cyclocreatine treatment independent of apoptosis.
Int. J. Cancer 2001 Nov15;94(4):513-519
Abstract: Ubiquitous mitochondrial creatine kinase (uMtCK), a key enzyme in energy metabolism, was identified by differential display PCR to be specifically overexpressed in L1236, the first cell line of definite Hodgkin origin. RT-PCR confirmed overexpression of uMtCK in the L1236 cell line and the absence of cytosolic B-CK, which is co-expressed with MtCK physiologically. Cyclocreatine (cCr), whose phosphorylated form is a very poor substrate for CK, inhibited proliferation of the L1236 cell line nearly entirely. This inhibition by cCr was partially reversed by competition with creatine, which by itself, however, had no effect, whatsoweve, on proliferation of the L1236 cell line. Although these results support a role of CK activity in the inhibitory action of cCr, it remains open whether the cCr effect is due to its inhibition of CK-linked energy metabolism or if alternative mechanisms have to be considered. Because the anti-proliferative effect of cCr was not due to induction of apoptosis, in contrast to most other anticancer agents, treatment with the creatine analogue cCr may represent an advantageous therapeutic approach for cells resistant to programmed cell death.

NOTOX Safety & Environmental Research B.V., Hambakenwetering 3, P.O. Box 3476, 5203 DL 's-Hertogenbosch, The Netherlands.
Evaluation of the mutagenic activity of Creapure * (Creatine Monohydrate, AlzChem-Degussa-Trostberg AG, Germany) in the salmonella typhimurium reverse mutation assay with independent repeat (Ames test), 1997.
Abstract: Evaluation of Creatine Monohydrate for its ability to induce reverse mutations in a gene of histidine-requiring Salmonella typhimurium bacterial strains according to the OECD principles of Good Laboratory Practice. Creatine Monohydrate did not induce a dose-related increase in the number of relevant colonies. Creatine Monohydrate is not mutagenic in the Salmonella typhimurium mutation assay.