Growth regulation of human colon cancer cells by epidermal growth factor and 1,25-dihydroxyvitamin D3 is mediated by mutual modulation of receptor expression

doi:10.1016/S0959-8049(98)00267-6

European Journal of Cancer

Volume 34, Issue 13, December 1998, Pages 2119-2125

https://doi.org/10.1016/S0959-8049(98)00267-6 Get rights and content

Abstract

The human colon adenocarcinoma-derived cell line Caco-2 was used as a model system to study the interaction of epidermal growth factor (EGF) and 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) in control of colorectal cancer cell growth. The mitogenic stimulus of EGF was rapidly transduced via apical and basal membrane receptors alike into elevation of c-myc expression, causing a shift of Caco-2 cells from the G₀/G₁ into the S phase of the cell cycle. The stimulatory effect of EGF on cell division was effectively counteracted by 1,25(OH)₂D₃: the presence of the steroid hormone prevents the negative effect of EGF on vitamin D receptor abundance and concurrently minimises ligand-occupied EGF receptor numbers on both sides of Caco-2 cell monolayers. Our data suggest that EGF and 1,25-(OH)₂D₃ actions on mutual receptor levels represent a specific feature of the potent antimitogenic effect of the steroid hormone on colon cancer cells.

Introduction

Epidermal growth factor (EGF) is a 6000 kDa polypeptide which is produced predominantly in the gastrointestinal tract by salivary glands, Brunner’s glands and Paneth cells. The peptide hormone is released into the digestive fluids as well as into the circulation and, hence, can locally as well as systemically regulate cell growth and function in a variety of tissues[1]. In the gastrointestinal tract, EGF inhibits acid secretion by gastric parietal cells[2]and, importantly, stimulates ornithine decarboxylase[3], DNA synthesis and, thus, proliferation of small and large intestinal epithelial cells[4]. There is also evidence that EGF plays a significant role in autocrine growth stimulation of colon tumour cells[5].

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) inhibits proliferation and induces differentiated cell functions in normal and, notably, also in malignant cells[6]. In this respect, vitamin D compounds have been shown, for example, to inhibit effectively cell growth and to support phenotypic redifferentiation of the human colon cancer cell line, Caco-27, 8, 9. The effects of 1,25(OH)₂D₃ on cell growth and differentiation are mediated by binding to the nuclear vitamin D receptor (VDR) which then acts as a transactivation factor for various genes.

Regulation of receptor abundance may be an important mechanism for modulating the extent of target cell responsiveness to hormones and growth factors. In this respect, Krishnan and Feldman[10]showed that in mouse fibroblasts and human breast cancer cells, VDR gene expression can be upregulated by serum and growth factors. EGF has also been found to increase VDR in the small intestine of the neonatal rat[11]. Conversely, 1,25(OH)₂D₃ has been reported to modulate EGF receptor (EGFR) expression, although in opposite directions depending on the cell type involved. In human T-47D breast cancer cells, growth inhibition induced by 1,25(OH)₂D₃ is associated with a decrease in EGFR numbers[12], whereas Desprez and colleagues[13]reported an increase in EGFR expression as the result of 1,25(OH)₂D₃ treatment in BT-20 breast cancer cells. Because no respective data are available for human colon cancer cells, we investigated the action and interaction of EGF and 1,25(OH)₂D₃ in growth control and mutual receptor regulation using the human colon adenocarcinoma-derived cell line Caco-2 as a model system.

The cell line Caco-2, despite its malignant origin, has retained the potential for spontaneous redifferentiation in culture[14]. Caco-2 cells also display two distinct classes of EGFR, at the apical and basolateral plasma membrane, respectively15, 16and, importantly, express the VDR at the mRNA and protein level17, 18.

Section snippets

Materials

1,25(OH)₂D₃ was a gift from Hoffmann-LaRoche (Basle, Switzerland). EGF (tissue culture grade) was obtained from Sigma (Deisenhofen, Germany). An anti-c-myc monoclonal antibody was purchased from Oncogene Science (Cambridge, Massachusetts, U.S.A.). The anti-VDR monoclonal antibody was from Chemicon (Temecula, California, U.S.A.).

Cell culture

Caco-2 cells (provided by A. Quaroni, Cornell University, Ithaca, New York, U.S.A.) were routinely cultured in Costar vented tissue culture flasks at 37°C in a

EGF affects proliferation and c-myc expression

The time-course of the mitogenic effect of EGF on Caco-2 cells is illustrated in Fig. 1, showing the relationship between [³H]thymidine incorporation into DNA, shifts in cell cycle distribution and expression of c-myc mRNA and of nuclear c-myc protein. Within 6 h after the addition of EGF, c-myc mRNA was increased 2-fold and then sharply declined to a level still above that of untreated controls (Fig. 1a). Nuclear c-myc protein also showed a transient change inasmuch as its initial value

Discussion

The present study strongly suggests that, regardless of whether the stimulatory effect of EGF on the growth of human colon adenocarcinoma-derived Caco-2 cells is elicited via luminal or basolateral EGFR activation, it eventually involves upregulation of the cell cycle regulatory protein c-myc. This would facilitate Caco-2 cells to enter into and progress through the G₁ phase of the cell cycle in response to EGF.

Until now the mechanism underlying the antimitogenic action of 1,25(OH)₂D₃ in Caco-2

Acknowledgements

These investigations were supported by grants from the Austrian Science Foundation (Project nos. P 09917-MED and P 10133-MED). from the Herzfelder Foundation, from the Oncology Research Fund and from the Anton Dreher Memorial Fund (Project no. 225/93) of the University of Vienna Medical School. Drs Wolfgang Hulla and Wei-Min Tong are presently affiliated with the International Agency for Research on Cancer, 150 cours Albert Thomas, 69008 Lyon, France. Dr W. Hulla acknowledges gratefully

References (27)

H.S Cross et al.
Antiproliferative effect of 1,25-dihydroxyvitamin D3 and its analogs on human colon adenocarcinoma cells (Caco-2): influence of extracellular calcium
Biochem Biophys Res Comm
(1991)
P.Y Desprez et al.
1,25-dihydroxyvitamin D3 increase epidermal growth factor receptor gene expression in BT-20 breast carcinoma cells
Biochem Biophys Res Commun
(1991)
A.R Giuliano et al.
Characterization of the vitamin D receptor from the Caco-2 human colon carcinoma cell line: effect of cellular differentiation
Arch Biochem Biophys
(1991)
I.J Hidalgo et al.
Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability
Gastroenterology
(1989)
H.S Cross et al.
Growth regulation of human colon adenocarcinoma-derived cells by calcium, vitamin D and epidermal growth factor
J Nutr
(1995)
N Falette et al.
Increased epidermal growth factor receptor level in breast cancer cells treated by 1,25-dihydroxyvitamin D3
Mol Cell Endocrinol
(1989)
C Garland et al.
Dietary vitamin D and calcium and risk of colorectal cancer: a 19-year prospective study in men
Lancet
(1985)
H.S Cross et al.
Vitamin D metabolism in human colon adenocarcinoma-derived Caco-2 cells: expression of 25-hydroxyvitamin D3-1α-hydroxylase activity and regulation of side-chain metabolism
J Steroid Biochem Mol Biol
(1997)
G Carpenter
Epidermal growth factor
Handb Exp Pharmacol
(1981)
J.M Bower et al.
The inhibition of gastric acid secretion by epidermal growth factor
Experimentia (Basel)
(1975)

E.J Feldman et al.

Epidermal growth factor stimulates ornithine decarboxylase activity in the digestive tract of the mouse

Proc Soc Exp Biol Med

(1978)

L.A Scheving et al.

Circadian phase-dependent stimulatory effects of epidermal growth factor on deoxyribonucleic acid synthesis in the duodenum, jejunum, ileum, caecum, colon and rectum of the adult male mouse

Endocrinology

(1980)

Gross ME, Zorbas MA, Daniels YJ, et al. Cellular growth response to epidermal growth factor in colon carcinoma cells...

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Original PaperGrowth regulation of human colon cancer cells by epidermal growth factor and 1,25-dihydroxyvitamin D3 is mediated by mutual modulation of receptor expression

Abstract

Introduction

Section snippets

Materials

Cell culture

EGF affects proliferation and c-myc expression

Discussion

Acknowledgements

Biochem Biophys Res Comm

Biochem Biophys Res Commun

Arch Biochem Biophys

Gastroenterology

J Nutr

Mol Cell Endocrinol

Lancet

J Steroid Biochem Mol Biol

Epidermal growth factor

Handb Exp Pharmacol

The inhibition of gastric acid secretion by epidermal growth factor

Experimentia (Basel)

Epidermal growth factor stimulates ornithine decarboxylase activity in the digestive tract of the mouse

Proc Soc Exp Biol Med

Circadian phase-dependent stimulatory effects of epidermal growth factor on deoxyribonucleic acid synthesis in the duodenum, jejunum, ileum, caecum, colon and rectum of the adult male mouse

Endocrinology

Original Paper
Growth regulation of human colon cancer cells by epidermal growth factor and 1,25-dihydroxyvitamin D₃ is mediated by mutual modulation of receptor expression