Creatine and Creatinine Metabolism - Physiological Reviews

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1154 MARKUS WYSS AND RIMA KADDURAH-DAOUK Volume 80 D. Use of Cr Analogs as Antitumor Agents The cytosolic BB-CK isoenzyme is expressed in a wide range of tissues such as brain, intestine, uterus, kidney, or prostate. It is also induced in a variety of tumors, including neuroblastoma, small cell lung carcinoma, colon and rectal adenocarcinoma, as well as breast and prostate carcinoma (see Refs. 41, 529, 559, 1157). In cancer patients, elevated B-CK expression is associated mainly with untreated or progressive metastatic disease. In several studies, elevated CK levels were an adverse prognostic indicator. The gene encoding B-CK is subject to regulation by both an oncogene and a tumor suppressor gene. A remarkable similarity was identified between the human B-CK promoter and the adenovirus E2E promoter region (152). This region of the E2E gene has two overlapping promoter elements that are induced by the adenovirus oncogenic product E1a. A series of cotransfection and infection experiments in tissue culture demonstrated that both the enzymatic activity and the mRNA level of B-CK are induced by E1a (446). Mutational analysis revealed that the transforming domains of the E1a protein are required for B-CK induction. The tumor suppressor gene p53 has been suggested to regulate normal cell growth by activating transcription of genes whose products suppress growth and tumor formation. Conceivably, p53 may also repress genes whose products function to initiate or sustain accelerated growth. As a matter of fact, B-CK expression is elevated in human small cell lung carcinomas, many of which contain mutations in p53 alleles. The effects of wild-type or mutant p53 on the expression of rat B-CK and M-CK were therefore determined in transient transfection experiments (1161). Wild-type p53 repressed the B-CK promoter in HeLa cells (cervical carcinoma cells transformed by human papilloma virus type 18), but not in CV-1 monkey kidney cells. Conversely, p53 activated the M-CK promoter in CV-1 but not in HeLa cells. Coexpression of the E6 protein from human papilloma virus type 16, which is known to promote p53 degradation, blocked the p53mediated modulation of CK expression. These findings suggest that p53 exerts its effects through association with corepressors or coactivators that are distinctly expressed in different cell types (see, e.g., Ref. 981). Furthermore, they indicate that CK isoenzymes are among the few cellular genes that may be targets of p53 in vivo. In a series of missense mutants with alterations in conserved region II of p53, the ability of the mutants to transactivate M-CK and/or to transrepress B-CK correlated well with their ability to inhibit transformation of rat embryonic fibroblasts by adenovirus E1a or activated Ras. Taken together, the regulation of B-CK expression by p53, E1a, and by a variety of hormones and components of signal transduction pathways suggests that this enzyme of cellular energy metabolism is important for the metabolic events that take place during or after oncogenic activation. If the CK system is involved in tumor growth through regulation of ATP production or modulation of as yet unknown processes, then molecules that disturb this system may have an impact on tumor growth or progression. Many Cr and PCr analogs that decrease the rate of ATP production through CK were synthesized, catalytically characterized, and evaluated for antitumor activity. Several were shown to be active both in vitro and in vivo against a broad spectrum of solid tumors characterized by high levels of CK expression (60) (see below). Therefore, the CK system emerges as a novel and interesting target for drug design, and Cr analogs potentially emerge as a new class of cancer chemotherapeutics that work through a unique mechanism of action. cCr is an interesting lead compound. Its antitumor activity has been studied extensively. In vitro, there seems to exist a correlation between the CK activity of a tumor and its responsiveness to cCr (Fig. 13). Tumor cell lines expressing a high level of CK were inhibited by cCr, with 50% inhibition of their ability to form colonies in soft agar (CD 50) being achieved at cCr concentrations of 2–8 mM (559). On the other hand, tumor cell lines expressing low levels of CK were inhibited only at 10-fold higher concentrations of cCr or not at all. cCr was also evaluated as an antitumor agent in a colony-forming system against freshly explanted human tumor cells (600). This was thought to more truly represent human disease than established cell lines that had been in culture for many years and hence underwent many changes. CK activity was evaluated in 192 tumor samples from 166 patients and ranged from 0.001 to 1.524 U � (mg protein) �1 , with a median of 0.042 U � (mg protein) �1 . The highest CK levels were found in mesotheliomas as well as in small cell lung and brain tumors, whereas the lowest levels were observed in kidney cancers, lymphomas, and non-small cell lung carcinomas. Interestingly, tumors with very low CK activity were not able to form colonies in soft agar, suggesting that the enzyme might be required for tumor establishment. The effect of cCr was evaluated on 51 of the 192 tumor samples. At a concentration of 6.7 mM, cCr inhibited colony formation of 21% of these tumors. Activity was noted against bladder, brain, kidney, lung, lymphatic, ovarian, pancreatic, and uterine cancers. Standard chemotherapeutic agents were tested on the same tumor samples. No relationship was seen between tumor samples sensitive to cCr and those sensitive to standard chemotherapeutics such as alkylating agents, antimetabolites, DNA intercalators, platinum compounds, topoisomerase II inhibitors, and tubulin interacting agents, thus supporting that cCr works through a unique mechanism of action. The specificity of cCr in vitro against tumor cell lines
July 2000 CREATINE AND CREATININE METABOLISM 1155 expressing high levels of CK may be a result of the accumulation of cCr or of the synthetic phosphagen PcCr. Four tumor cell lines expressing high levels of CK, namely, ME-180 (human metastatic cervical carcinoma), DU145 (human prostate carcinoma), MCF-7 (breast adenocarcinoma), and OVCAR-3 (human ovarian adenocarcinoma), were sensitive to cCr and accumulated substantial amounts of PcCr [0.33–0.63 �mol � (mg cellular protein) �1 ] (559). In contrast, two cell lines expressing low levels of CK, U-87 MG (human glioblastoma/astrocytoma) and A2058 (human melanoma), were resistant to cCr and had undetectable levels of PcCr. All six cell lines, however, accumulated between 5 and 27 �mol cCr � (mg cellular protein) �1 . To further test the correlation between accumulation of the synthetic phosphagen PcCr and growth inhibition, the unresponsive, low CK expressing melanoma cell line A2058 (see above) was transformed into a high CK expressing line, A2058–055. This was accomplished by transfecting the cells with a plasmid containing the human B-CK gene linked to the cytomegalovirus IE promoter and enhancer (559). The engineered line expressed �2,000 times the CK activity of the parent line A2058 and accumulated a large pool of the phosphagen PcCr. Remarkably, A2058–055, but not the parent line, was growth inhibited by cCr. These findings suggest that accumulation of the synthetic phosphagen PcCr is essential for inhibition of tumor growth. Metastasis, the primary feature in fatal tumor pro- FIG. 13. Correlation between the CK activity of tumor cell lines and their responsiveness to cyclocreatine (cCr) (IC 50 of cCr for inhibition of colony formation of the cell lines in soft agar). For a description of the cell lines, see Lillie et al. (559). gression, involves cell motility as a major determinant step (562, 953). Tumor cell motility is an energy-requiring process and depends on glycolysis. In in vitro studies in the high CK expressing cell lines A2058–055 and DU145, cCr was found to inhibit motility in response to different stimuli (676). In low CK expressing cell lines (A2058 and A2058–032), on the other hand, motility was not or only partially inhibited. Cr had no effect on the motility of the four cell lines when given alone but reversed the inhibition of motility by cCr. These results suggest that 1) the CK system may not only be important in solid tumor growth, but could also play an important role in the spread and invasion of these tumors, and that 2) cCr affects a key step utilized by several motility-stimulating systems. The in vitro findings were complemented by evaluating the antiproliferative activity of cCr in vivo in a human tumor xenograft nude mouse model (559, 641). The growth of tumors derived from freshly injected ME-180 cells was significantly inhibited in athymic mice by feeding a diet supplemented with 1% cCr compared with animals fed a control diet (149 vs. 506 mm 3 by day 36). cCr also inhibited the growth of established (�50 mm 3 )ME- 180 cell-derived tumors, as evidenced by tumor volumes on day 44 of 465 and 1,033 mm 3 for cCr-fed and control mice, respectively. Similarly, dietary cCr inhibited the growth of human neuroblastoma cell line-derived tumors in nude mice by up to 46%. A variety of rat tumors were evaluated for respon-
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