Creatine and Creatinine Metabolism - Physiological Reviews

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