Cancer Letters

Cancer Letters

Volume 268, Issue 2, 18 September 2008, Pages 260-271
Cancer Letters

Loss of XIAP sensitizes colon cancer cells to PPARγ independent antitumor effects of troglitazone and 15-PGJ2

https://doi.org/10.1016/j.canlet.2008.04.003Get rights and content

Abstract

We investigated whether the anticancer effect of a combination of XIAP down-regulation and PPAR γ activation on colon cancer is PPARγ receptor dependent. HCT116-XIAP+/+ cells and HCT116-XIAP−/− cells were treated with troglitazone or 15-deoxy-Δ12,14-prostaglandin J2 (15-PGJ2) with or without prior exposure to PPARγ inhibitor GW9662. Cell proliferation and apoptosis was evaluated. Athymic mice carrying HCT116-XIAP−/− cells-derived tumors were treated with troglitazone in the presence or absence of GW9662. Inhibition of cell proliferation and induction of apoptosis by troglitazone and 15-PGJ2 were more prominent in HCT116-XIAP−/− cells. PPARγ ligand-induced growth inhibition, apoptosis, caspase and PARP cleavage could not be blocked by GW9662. Troglitazone significantly retarded growth of xenograft tumors and this effect was not blocked by GW9662. Marked apoptosis and an up-regulation of E-cadherin were observed in xenograft tumor tissues, and GW9662 did not affect these effects. Thus, a combination of XIAP down-regulation and PPARγ ligands exert a significant anticancer effect in colon cancer via a PPARγ independent pathway.

Introduction

XIAP (X-linked inhibitor of apoptosis protein) is a member of the IAP (inhibitor of apoptosis protein) family that functions as a potent endogenous inhibitor of apoptosis [1], [2], [3], [4], [5]. Selectively targeting cancer cells for apoptosis can be a promising approach for cancer therapy. Recent studies have indicated that XIAP may have a great potential in cancer gene therapy [6]. However, as cancer development is such a complex biological process involving numerous genes and pathways that regulate apoptosis, proliferation, and angiogenesis, targeting XIAP alone may not be potent enough for cancer control. Consequently, combinational therapeutic approaches may worth exploring [7].

Peroxisome proliferator-activated receptor gamma (PPARγ) is an important member of a superfamily of nuclear hormone receptors. It functions as a transcription factor and is widely expressed in many human tissues including but not limited to adipose tissues, normal colonic mucosa, duodenal mucosa, endothelial cells, liver, and small intestine [8], [9], [10], [11]. Binding of PPARγ ligands to PPARγ receptor leads to a heterodimerization of PPARγ with the retinoid X receptor (RXR) which then binds to peroxisome response elements (PPRE), leading to transcription of downstream target genes. Studies have showed that ligand-induced activation of PPARγ induces cell differentiation and apoptosis in a variety of cancers including colon cancer cells [12], [13], [14], [15], [16], [17]. Some natural PPARγ ligands such as conjugated linoleic acid have been showed to inhibit peritoneal metastasis of gastrointestinal cancers [18], [19]. Activation of PPAR γ has also been reported to suppress angiogenesis in cancers [20]. These results highly suggest that PPARγ ligands may be potentially useful as anticancer or chemoprevention agents for colon cancer. However, using PPARγ ligands as a monotherapy for cancer only have limited efficacy. Furthermore, the effect of PPAR γ ligands on colon cancer is rather controversial. Activation of PPAR γ by its ligands was found to promote the growth of colon cancer in some studies [21], [22], [23]. More recently, large body of evidence indicates that specific ligands for PPARγ may exert their effect independent of PPARγ receptor [13], [24], [25], [26], [27], [28], [29]. The fact that the same PPARγ ligand may exert different effect in different subtypes of a particular cell line [30], or different PPARγ ligands exert different effects in the same cell line [31], [32] further suggest that mechanisms other than PPARγ receptor may be responsible for the anticancer effect of PPARγ ligands.

We were interested in exploring whether combinational targeting XIAP and PPARγ would offer a better therapeutic benefit for colon cancer. In our recent studies, we observed that a combination of XIAP down-regulation and PPARγ activation exhibited a stronger anticancer effect in colon cancer [33].

In this study, we aimed to elucidate the molecular mechanisms by which the combination of XIAP down-regulation and PPARγ activation exerts anticancer effects against colon cancer. In particular, we will evaluate whether the anticancer effect is dependent upon PPARγ receptor.

Section snippets

Chemicals, reagents, and cell lines

Troglitazone, 15-deoxy-Δ12,14-prostaglandin (15-PGJ2), GW9662, and PPARγ Transcription Factor Assay Kit were purchased from Cayman Chemicals (Ann Arbor, MI). Human colon cancer cell lines HCT116 cells with wild type and XIAP knockout phenotypes (HCT116-XIAP+/+, HCT116-XIAP−/−) were kindly provided by Professor Bert Vogelstein (Howard Hughes Medical Institute & Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins Medical Institutions, USA) [34]. All culture-related materials were

Expression of XIAP and PPARγ in colon cancer tissues by immunohistochemistry and Western blot

In the 2 microarray slides containing 140 pairs of colon cancer tissues and matched non-cancerous tissues, immunohistochemical staining showed that XIAP was positive in 41% of colon cancer tissues and 9% of non-cancerous tissues. Among the XIAP positive colon cancer tissues, 63% had strong expression (defined as score  3). For PPARγ, non-cancerous and colon cancer tissues exhibited similar expression positivity: 42.9% vs 42.6%, respectively. Among these cases, 61% of cancer tissues and 21% of

Discussion

In this study, we found that the expression levels of XIAP and PPARγ in cancer tissues exceeded that of the matched non-cancerous colonic tissues, but the PPARγ transcriptional activity is significantly reduced in cancer tissues. This novel finding may suggest that a reduced activity of PPARγ may be related to colon cancer development. This finding is consistent with the previous results by others that PPARγ may act as a tumor suppressor gene [12], [38], [39]. The observed over-expression of

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