[CANCER RESEARCH 61, 8703– 8711, December 15, 2001]
Synergistic Cytotoxicity Exhibited by Combination Treatment of Selective Retinoid
Ligands with Taxol (Paclitaxel)
Valerie Vivat-Hannah,
1
Dan You, Cheryl Rizzo, Jean-Paul Daris, Philippe Lapointe, F. Christopher Zusi,
Anne Marinier, Matthew V. Lorenzi, and Marco M. Gottardis
Department of Oncology Drug Discovery, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543-4000 [V. V. H., D. Y., C. R., M. V. L., M. M. G.];
Wallingford Discovery Chemistry, Bristol-Myers Squibb, Wallingford, Connecticut 03492-1996 [C. F. Z.]; and Drug Discovery Chemistry, Bristol-Myers Squibb, Candiac
(Quebec), J5R 1J1 Canada [J. P. D., P. L., A. M.]
ABSTRACT
The focus of this study was to develop retinoic acid receptor (RAR)
RAR/ selective agonists with anticancer efficacy and reduced toxicity
associated with RAR activity. In these studies, we report the identifica-
tion and characterization of high-affinity RAR/ selective agonists with
limited RAR activity. These compounds inhibited human tumor cell line
proliferation with similar efficacy to that observed for a pan-RAR agonist.
However, for most tumor cell lines, the efficacy of these compounds was
restricted to the micromolar range. To determine whether the RAR/
selective agonists could be additive or synergistic with existing agents, we
investigated the effects of combining RAR/ selective agonists with
various cytotoxic agents. Our results showed that the / selective reti-
noids dramatically lowered the effective dose of Taxol needed to induce
cytotoxicity of a wide range of tumor cell lines. This synergy was specific
to tubulin-modifying agents and could not be observed with a variety of
other cytotoxic agents of diverse function. Examination of pathways com-
mon to Taxol and retinoid signaling revealed that this synergy was related
in part to effects on Bcl-2 expression/phosphorylation as well as the
activity of the c-Jun NH
2
-terminal kinase and activator protein-1. In
contrast, the tubulin polymerization induced by Taxol was not further
affected by cotreatment with a variety of retinoid receptor ligands. These
observations indicate that potent RAR/ selective agonists may be of
therapeutic benefit in combination with Taxol therapy.
INTRODUCTION
The retinoids are biologically active derivatives of vitamin A that
can regulate the proliferation, differentiation, and apoptosis of a wide
variety of both normal and malignant cells (1–3). The natural retinoids
primarily consist of ATRA
2
and 9-cis retinoic acid, which are pro-
cessed from vitamin A through its irreversible oxidation (4, 5). In
addition, several synthetic ligands have been designed that retain
many of the biological properties of the natural ligands (6, 7). The
biological activities of the retinoids are mediated by two classes of
nuclear receptors, the RARs and the RXRs, each of which comprise
three isotypes designated , , and (reviewed in Ref. 8). RAR and
RXR act as ligand-dependent transcription factors, which can modu-
late target gene expression by binding as RAR/RXR heterodimeric
complexes to a specific RAR element DNA sequence (9, 10). The
activity of the RAR/RXR heterodimer can be repressive or stimula-
tory, depending upon its apo (unliganded) or holo (ligand-bound)
conformation, respectively. The apo conformation of RAR/RXR
heterodimers allows protein interactions with large corepressor
multiprotein complexes that contain nuclear corepressor and his-
tone deacetylases and act to maintain the chromatin in a condensed
transcriptionally inactive state to repress target gene expression (11–
13). In contrast, the binding of agonist ligand to RARs is associated
with a conformational transition of the ligand binding domain (14),
resulting in the destabilization of the corepressor complex and simul-
taneous recruitment of coactivators, including p160 protein family,
CBP/p300, and the multiprotein complexes thyroid hormone receptor
associated proteins (TRAP), Vitamin D
3
receptor interacting proteins
(DRIP), activator recruited cofactor (ARC) (11). Some of these fac-
tors contain histone acetyltransferase activity, allowing the deconden-
sation of the chromatin required for the subsequent link of the holo-
RAR/RXR heterodimer with the basal transcriptional complex to
initiate target gene expression (11, 13).
ATRA and synthetic retinoid receptor ligands have been shown to
promote tumor regression in a number of animal models of carcino-
genesis and have shown efficacy in patients afflicted with acute
promyelocytic leukemia (15, 16). Among the three RAR isotypes, the
RAR2 isoform has been associated with the tumor-suppressive ef-
fects of the retinoids. RAR2 arises from alternative promoter use of
the RAR gene promoter P2 (8), which contains a strong RAR
element, suggesting that RAR expression is up-regulated by retinoic
acid and places RAR2 as a potential target gene underlying the
growth-suppressive effects of retinoic acid. Furthermore, a large num-
ber of reports have described the loss of the RAR2 expression during
the early phases of cancer development including breast and lung
tumors (17–19). In contrast, the expression of RAR and RAR as
well as RXR are mostly unaltered in malignant tissues. Consistent
with a role in modulating cell growth, RAR gene inactivation in
several cell types has led to the loss of retinoic acid-dependent growth
arrest (20, 21). In contrast, the induction of RAR expression in
different tumor cell lines has been shown to be associated with a
strong enhancement of retinoic acid responsiveness (22, 23). These
observations highlight RAR as an important mediator of retinoid
action and suggest that its modulation may be effective in reducing
tumor cell growth.
A growing interest has focused recently on increasing the antitumor
activity of the retinoids through combination with other types of
agents, including IFN- (24), tumor necrosis factor-related apoptosis-
inducing ligand (25), HDAC inhibitors (26 –28), as well as chemo-
therapeutic agents (29 –32). For instance, in cell lines isolated from
patients afflicted with acute myeloid leukemia, HDAC inhibitors
potentiate the differentiation effects of the retinoids and show activity
in retinoid-resistant acute myeloid leukemia cells (26). The molecular
mechanisms underlying the effect of these agents are related to the
derepression of the retinoid receptor transduction pathway (26). For
instance, in breast cancer cells, HDAC inhibitors have been shown to
restore the retinoic acid-inducible expression of RAR, independently
of the methylation status of the RAR gene promoter (33). In contrast,
the mechanisms supporting the synergistic effects observed in com-
bination treatments using the retinoids and IFN-, or with chemother-
apeutic agents such as cisplatin or Taxol, remain largely undefined.
In the present study, we report the identification of selective
Received 5/1/01; accepted 10/16/01.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
18 U.S.C. Section 1734 solely to indicate this fact.
1
To whom requests for reprints should be addressed, at Bristol-Myers Squibb, Phar-
maceutical Research Institute, Oncology Drug Discovery, P. O. Box 4000, K19-04,
Princeton, NJ 08543-4000. Phone: (609) 252-4313; Fax: (609) 252-6051; E-mail: valerie.
vivat@bms.com.
2
The abbreviations used are: ATRA, all-trans retinoic acid; RAR, retinoic acid
receptor; RXR retinoid X receptor; HDAC, histone deacetylase; JNK, c-Jun NH
2
-terminal
kinase; AP, activator protein; PMA, phorbol 12-myristate 13-acetate; FBS, fetal bovine
serum; tk, thymidine kinase.
8703
Research.
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