Troglitazone activates p21Cip/WAF1 through the ERK pathway in HCT15 human colorectal cancer cells

doi:10.1016/S0304-3835(01)00869-2

Cancer Letters

Volume 179, Issue 2, 28 May 2002, Pages 185-195

https://doi.org/10.1016/S0304-3835(01)00869-2 Get rights and content

Abstract

In this study, we identified a new mechanism for the anti-proliferation of HCT15 colorectal cancer cells by troglitazone (TRO). Treating HCT15 cells with 20 μM of TRO transiently increased extracellular signal regulated kinase (ERK) activity within 15 min, and this subsequently induced p21^Cip/WAF1 cell cycle regulator and localized in the nucleus. Raf-1 modification and MEK activation also occurred after TRO treatment, and Elk-1-dependent trans-reporter gene expression was concomitantly induced. The induction and nuclear localization of p21^Cip/WAF1 by TRO were blocked by PD98059 pre-treatment, which suggested a role for the ERK pathway in p21^Cip/WAF1 activation. TRO inhibited BrdU incorporation and no BrdU incorporation was observed in most p21^Cip/WAF1-activated cells. Therefore, TRO regulates the proliferation of HCT15 cells at least partly by a mechanism involving the activation of p21^Cip/WAF1.

Introduction

Troglitazone (TRO) is a member of the family of thiazolidinediones (TZDs), which are orally active drugs that improve insulin sensitivity in both animals and humans with insulin resistance [1], [2]. TRO binds to and activates peroxisome proliferator-activated receptor γ (PPARγ), a member of the ligand-activated nuclear receptor family [3], [4]. Activated PPARγ regulates transcription of target genes, which are mainly involved in fatty acid and lipid metabolism of fat cells, and are associated with the differentiation of fat cells [5]. However, recent studies found that TRO also inhibits growth of human breast and prostate cancers, myeloid leukemia, and vascular smooth muscle cells [6], [7], [8], [9]. A role of TRO in the growth regulation of colorectal cancer cells was also identified and related to the activation of the PPARγ [10]. However, a no-regulatory mechanism of cell growth by TRO independent of PPARγ has been identified. A recent study also found that TRO induced cyclin-dependent kinase inhibitor p21^Cip/WAF1, and this resulted in the suppression of the growth of a myeloid leukemia cell line [8]. However, the p21^Cip/WAF1 induction by TRO has not been reported in solid tumors, and the mechanism of p21^Cip/WAF1 induction has not been determined. The p21^Cip/WAF1 induction is closely related to the growth arrest of many different cell types, including colorectal cancer cells [11], [12], and this is dependent on the activation of either p53 tumor suppressor [13] or the extracellular signal regulated kinase (ERK) pathway [14], [15], [16]. In addition, high aberrant ERK activities were observed in normal colonic epithelial cells, as compared with the paired tumors, and this might be involved in growth regulation [17]. Therefore, the ERK pathway is likely to be involved in the growth arrest, as well as the proliferation of cells [14], [15], [16].

In this study, we found that TRO induces p21^Cip/WAF1 cell cycle regulator in HCT15 human colorectal cancer cells, and further identified a mechanism for the induction and nuclear localization of p21^Cip/WAF1 by TRO. The p21^Cip/WAF1 activation is related to the anti-proliferation of colorectal cancer cells, and this provides an alternative mechanism for the growth regulation of HCT15 colorectal cancer cells by TRO, which is independent of PPARγ.

Section snippets

Materials

HCT15 (CCL-225) and HT29 (HTB-38) human colorectal cell lines were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Dulbecco's modified Eagle's medium (DMEM), McCoy's 5A medium, fetal bovine serum (FBS), antibiotics and Lipofectamin plus reagent were purchased from Life Technologies, Inc. (Grand Island, NY, USA). Phospho-ERK and phospho-MEK antibodies were obtained from New England Biolabs Inc. (Beverly, MA, USA), and ERK antibody from Stratagene (La Jolla, CA,

TRO transiently increases ERK activity and subsequently induces p21^Cip/WAF1 cell cycle regulator

The ERK pathway, which is also often called the MAPK pathway, is an important signaling route in cell proliferation, and aberrant activation of the MAPK pathway by genetic alterations of the signaling components often results in the development of cancers [20], [21], [22]. However, recent studies have also shown that the activation of the MAPK pathway plays a role in the growth arrest of the cells, and that this is related to the activation of the cell cycle regulator, p21^Cip/WAF1 [14], [15],

Discussion

The p21^Cip/WAF1 is a cell cycle regulator, which is involved in the anti-proliferation of cells by inhibiting cell cycle progression at the G1 to S phase; moreover, the induction of p21^Cip/WAF1 is a good indicator of growth arrest in colorectal cancer cells [11], [12]. In the present study, we identified a mechanism for the activation (induction and nuclear localization) of p21^Cip/WAF1 by TRO in HCT15 cells, and related this to cell growth. Transient activation of the ERK pathway appeared to be

Acknowledgements

We would like to thank Drs G. Johnson and J.H. Kim for providing kinase inactive MEK and dominant inhibitory Raf-1 kinase construct, respectively. This work was supported by the following grants to K.-Y.C.: the 1999 Korean National Cancer Control Program, Ministry of Health and Welfare, Korea; grant 1999-1-212-001-5 from the basic research program, and Research Center for Biomedical Resources of the Korean Science and Engineering Foundation. J.-A.K., K.-S.P. and H.-I.K. are recipients of a

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Troglitazone activates p21Cip/WAF1 through the ERK pathway in HCT15 human colorectal cancer cells

Abstract

Introduction

Section snippets

Materials

TRO transiently increases ERK activity and subsequently induces p21Cip/WAF1 cell cycle regulator

Discussion

Acknowledgements

J. Biol. Chem.

Cell

Biochem. Biophys. Res. Commun.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Cell

Cell

J. Biol. Chem.

J. Biol. Chem.

Bioorg. Med. Chem. Lett.

Improvement in glucose tolerance and insulin resistance in obese subjects treated with troglitazone

N. Engl. J. Med.

Thiazolidinediones in the treatment of insulin resistance and type II diabetes

Diabetes

The structure–activity relationship between peroxisome proliferator-activated receptor gamma agonism and the antihyperglycemic activity of thiazolidinediones

J. Med. Chem.

Troglitazone activates p21^Cip/WAF1 through the ERK pathway in HCT15 human colorectal cancer cells

TRO transiently increases ERK activity and subsequently induces p21^Cip/WAF1 cell cycle regulator