Creatine and Creatinine Metabolism - Physiological Reviews

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1156 MARKUS WYSS AND RIMA KADDURAH-DAOUK Volume 80 siveness to cCr in a rat syngeneic model (641). Dietary cCr at 1% for 3–4 wk inhibited the growth of two rat mammary adenocarcinomas (Ac33tc and 13762A) as well as of MCI sarcoma by 20–50%. Cr at very high concentrations also inhibited the growth of some of the tumors, although in several other studies (60) (see also below), it was inactive. To optimize the therapeutic potency of cCr, different dosages, routes of administration, and schedules were compared in the rat mammary tumor 13762 (991). The antitumor activity was scored as difference in the number of days required for treated tumors to reach a volume of 500 mm 3 as compared with untreated animals. Tumor growth delay was more pronounced at the higher cCr dosage (1.0 vs. 0.5 g � kg �1 � day �1 ) and when cCr was administered intravenously rather than intraperitoneally. The earlier the cCr administration was initiated, the better growth inhibition was noted. The tumor growth delay with cCr was comparable to that achieved with clinically used anticancer drugs such as the antitumor alkylating agents CDDP [cis-diamminedichloroplatinum(II) � cisplatin] and cyclophosphamide, the antitumor antibiotic adriamycin, or the antimetabolite 5-fluorouracil. Because cCr seems to be working through a unique mechanism of action, it might act synergistically with other agents. cCr was therefore tested both in vitro and in vivo in combination with several standard chemotherapeutic agents (991). The human small cell lung carcinoma cell line SW2 was only marginally responsive to cCr in vitro. When SW2 cells were incubated for 24 h with cCr and, additionally, during the fifth hour of cCr treatment with either one of four antitumor alkylating agents, CDDP, melphalan, 4-hydroperoxycyclophosphamide, or carmustine, a greater than additive killing of SW2 cells was observed (Fig. 14A). The antitumor alkylating agents used alone at the same concentrations were only moderately effective. In the rat mammary carcinoma 13762 model in vivo, cCr given in combination with either CDDP, cyclophosphamide, adriamycin, or 5-fluorouracil resulted in longer tumor growth delays than those achieved with any of the anticancer drugs alone (Fig. 14B). Favorably, no evidence of increased general toxicity was noted. The effect of combination therapies was also tested with cCr and adriamycin on the human prostate tumor cell line LNCaP both in vitro and in vivo (379). Survival curves determined in cell culture revealed that LNCaP cells are only moderately sensitive to adriamycin, with 35% growth inhibition at a concentration of 8.4 �M. The combination of cCr and adriamycin produced additive to synergistic inhibition of cell proliferation, with the greatest effects being seen at the highest concentrations tested. For example, effects 10-fold greater than expected for additivity were observed for the combination of 6 mM cCr and 8.4 �M adriamycin. In vivo, cCr as a single agent FIG. 14. Synergistic antitumor activity of cCr in vitro (A) and in vivo (B). A: human SW2 small cell lung carcinoma cells were treated with either cyclophosphamide (4-HC) or cisplatin (CDDP) alone (F) orin combination with cCr (Œ). The shaded area represents the surviving fraction expected in case of an additivity of the antitumor effects. B: growth delay of rat 13762 mammary carcinoma produced by anticancer drugs in the presence or absence of cCr given intravenously. Tumor growth delay is defined as the difference in the number of days required for treated tumors to reach a volume of 500 mm 3 as compared with untreated controls. [Modified from Teicher et al. (991).] significantly inhibited the growth of LNCaP xenografts even when the tumor size was large. In contrast, adriamycin was not effective against larger tumors. Combined administration of cCr and adriamycin produced better tumor growth inhibition than either drug alone. To more clearly understand the underlying basis of the cCr effects, the responsive ME-180 cervical carcinoma cell line was treated with a range of cCr concentrations, and the cell cycle distribution was examined after 0, 8, 16, and 24 h of drug treatment (602). FACS analysis revealed no major alterations in cell cycle distribution. A minor twofold accumulation in G 2-M was seen after 16 h but was not sustained. When synchronized ME-180 cells were analyzed, progression out of each phase (G 1,S,orM)was significantly reduced within the first 8hoftreatment with cCr. With continued treatment, progression was blocked. These results suggest that the predominant effect of the drug is to block progression out of all phases of the cell cycle.
July 2000 CREATINE AND CREATININE METABOLISM 1157 To determine whether cCr was cytotoxic to cells during a specific phase of the cell cycle, ME-180 cells were blocked in G 1, S, or M. The synchronizing agent was then removed, and cells were allowed to grow in the presence or absence of cCr for 4 days. These experiments revealed that cCr is a phase-specific cytotoxic agent that kills cells preferentially in the S phase of the cell cycle. Preliminary studies showed no evidence for apoptotic cell death in response to treatment with cCr for up to 4 days. From these findings, it can be concluded that the tumor growth inhibition exerted by cCr is due in part to both cytostatic and cytotoxic effects and that cCr causes a general block of progression out of all phases of the cell cycle. It has been proposed that such a general effect on cell cycle progression is a result of impacting a fundamental cellular target such as the rate of ATP synthesis. Compounds with anticancer activity that have been reported to block general cell cycle progression in some cell lines include interferon-� (431) and genistein, a tyrosine kinase inhibitor (1032). Both compounds act through cell signaling pathways and are likely to have many effects on tumor cells. The unique mechanism of antitumor activity of cCr coupled with its general effect on cell cycle progression may potentially explain why it acts synergistically with other anticancer chemotherapeutic agents that are cell cycle stage specific. Remarkably, normal cell lines that express high levels of CK such as brain and cardiac and skeletal muscle cells do not seem to be growth inhibited by cCr (603). The cytosolic CK isoenzymes have been observed to associate with the cellular cytoskeleton. Evidence suggesting an association between CK on one hand and microtubules and intermediate filaments on the other hand has been provided by immunolocalization, in vitro binding, and functional studies (for references, see Ref. 601). Microtubules are known to be critical for many vital interphase functions, including cell shape, motility, attachment, intracellular transport, and cell signaling pathways. On the basis of these observations, the effect of cCr on the organization of microtubules in interphase cancer cells was investigated (601). Treatment of the cCr-responsive human tumor cell lines ME-180 and MCF-7 (see above) for 38 or 48 h with the minimum concentration of cCr that prevented proliferation caused the microtubules to become more randomly organized, an effect most apparent at the periphery of ME-180 cervical carcinoma cells. The microtubule changes were accompanied morphologically by cell flattening and by loss of the cell’s bipolar shape. To address the mechanism causing altered microtubule structure, ME-180 and MCF-7 cells were challenged for 1 h with nocodazole, an agent that induces rapid depolymerization of microtubules similar to effects seen with colchicine. cCr induced the formation of an aberrant new population of microtubules that was more stable when challenged with nocodazole than were normal microtubules. These microtubules were short, randomly organized, and apparently not associated with the centrosome. For studying microtubule repolymerization, microtubules of ME-180 cervical carcinoma cells were dissociated by exposing the cells to nocodazole. This drug was then removed, and microtubules were allowed to repolymerize. The presence of cCr during the periods of preincubation, nocodazole treatment, and repolymerization gave rise to a more extensive array of microtubules than in the absence of cCr. The newly polymerized microtubules appeared to originate from the centrosome. Interestingly, nontransformed cell lines that express low levels of CK and are not growth inhibited by cCr also lacked an effect of cCr on microtubule dynamics in nocodazole challenge experiments. This suggests a correlation between tumor growth inhibition, CK activity, the buildup of PcCr, and effects on microtubule dynamics, and that the antiproliferative activity of cCr may be due, at least in part, to its effects on microtubules. This assumption is supported by the fact that cCr induces microtubule stabilization after approximately the same period of time required for the inhibition of cell cycle progression, i.e., around 13 h. cCr may therefore represent the first member of a second class of anticancer agents, in addition to the taxanes, that increases the stability of microtubules. Taxol, a member of the taxanes, stabilizes microtubules by binding directly to tubulin and lowering its critical concentration (see Refs. 179, 593). Like cCr, it induces the formation of microtubules that do not originate at the centrosome. Nevertheless, there are some differences between the effects of cCr and taxol. Although taxol stabilizes existing interphase microtubules, cCr seems to induce the formation of what appeared to be newly formed and highly stable microtubules. In addition, taxol induces extensive arrays of microtubules aligned in parallel bundles, an effect not noted for cCr. A synergistic tumorkilling effect was seen when cCr was combined with taxol (601). This further demonstrates that cCr and taxol have different modes of action. The effect of cCr to induce the formation of stable microtubules may be hypothesized to be a result of decreasing the rate of ATP production via CK, which may secondarily affect the activity of proteins that regulate microtubule dynamics in tumor cells. Metabolic inhibitors that deplete ATP such as 2-deoxyglucose protect microtubules against depolymerization. It has been proposed that local changes in ATP concentration inhibit the phosphorylation of microtubule-associated proteins (MAP) which, in turn, control microtubule dynamics (63, 279). In addition, ATPases such as katanin (626) participate in microtubule disassembly and may be targets for cCr. Further experiments are needed to evaluate the effects of
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