Cancer Letters

Cancer Letters

Volume 164, Issue 1, 10 March 2001, Pages 15-23
Cancer Letters

The retinoid fenretinide inhibits proliferation and downregulates cyclooxygenase-2 gene expression in human colon adenocarcinoma cell lines

https://doi.org/10.1016/S0304-3835(00)00714-XGet rights and content

Abstract

Fenretinide [N-(4–Hydroxyphenyl)retinamide, 4-HPR] (10−10–10−6 M) treatment of HT-29 human colon cancer cells for 24–72 h significantly inhibited their growth. Using HCT-15 cells, 4-HPR had limited inhibitory effects on cell proliferation over the same concentration range and time period. The inhibitory effects of 4-HPR on cell growth in HT-29 cells were markedly reduced in the presence of exogenously added prostaglandins (PGs), suggesting a possible role for inhibition of PG synthesis as a mechanism for 4-HPR's antiproliferative effects. Inhibition of PGE2 production was caused by 4-HPR in a concentration-dependent manner and decreased COX-2 but not COX-1 mRNA levels; this is the first indication that 4-HPR selectively inhibits COX-2 gene expression. Our findings suggest a possible mechanism for the chemopreventive and anti-proliferative effects of 4-HPR.

Introduction

Retinoids are a group of compounds, either natural or synthetic, that are derived from vitamin A or retinol, which play an important role in growth, reproduction, differentiation and immune response, actions which are mediated via nuclear receptors [1], [2], [3], [4]. Retinoids, evaluated extensively in in vitro studies of cellular proliferation, have been used to both protect from and treat several cancers in various animal models of carcinogenesis [5], [6], [7], [8], [9], [10]. However, the use of retinoids as chemotherapeutic agents has been limited by their toxicity, particularly liver damage when given chronically in high doses [11]. The synthetic retinoid, fenretinide [N-(4-hydroxyphenyl)retinamide, 4-HPR] is very promising because of its potential biological activity and decreased hepatotoxicity [12], [13], [14]. This agent has been used in chemopreventive clinical trials of breast cancer [15]. The mechanism(s) by which retinoids exert their anti-neoplastic effect is not clearly established; proposed mechanisms include potentiation of an antitumor immune response [1], [16], anti-promotion activity [17], and induction of apoptosis [18], [19], [20].

Prostaglandins (PGs) in general, and PGE2 in particular, have been widely implicated in tumor growth [21], [22]. Not only their concentration is markedly increased in colon cancer [23], [24], but also nonsteroidal anti-inflammatory drugs (NSAIDs), which inhibit their production, reduce the incidence of and mortality from colon cancer by about 50% [25], [26], [27].

The mechanism(s) by which NSAIDs reduce the risk of colorectal cancer is not well understood. However, because NSAIDs inhibit cyclooxygenase (COX) enzyme activity, hence reducing PG levels, this has been proposed as a possible mechanism of their action [22], [28], [29], [30], [31]. There are at least two COX activities expressed in humans, COX-1, which is constitutively expressed and COX-2, which is mitogen-inducible (reviewed in Ref. [32]). In human colorectal cancer COX-2 but not COX-1 gene expression is elevated [33].

Since 4-HPR is a potent inhibitor of PG biosynthesis by inhibiting COX enzyme activity [34], we elected to examine the effects of this agent on cell proliferation, apoptosis and differentiation in HT-29 and HCT-15 colon carcinoma cell lines, which constitute a useful experimental system to study components of the eicosanoid pathway including COX. As we have demonstrated, HT-29 cells express both COX-1 and COX–2, produce several PGs including PGE2, PGF and PGI2, and they respond appropriately to exogenous stimulation of PG synthesis. In contrast, HCT-15 cells express neither COX isoform, produce no PGs and fail to respond to exogenous stimulation of PG synthesis [35]. We have also demonstrated that when HT-29 cells were stimulated by phorbol esters, it was the expression of COX-2 that was induced and not of COX-1 [36]. The results of this investigation are presented here.

Section snippets

Cell lines

The human colon adenocarcinoma cell lines HT–29 (ATCC HTB 38) and HCT-15 (ATCC CCL 225) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). HT–29 cells were cultured in McCoy's 5A medium and HCT-15 cells in RPMI 1640 (Cellgro, Mediatech, Herndon, VA). All media were supplemented with 10% FBS (Gemini Bioproducts, Inc., Calabasas, CA), non-essential amino acids, penicillin (50 U/ml), and streptomycin (50 μg/ml) (Life Technologies Inc., Gaithersburg, MD). Plain medium

Effect of 4-HPR on cell number in colon cancer cell lines

Fig. 1 shows the concentration- and time-response results for 4-HPR in HT-29 and HCT-15 cells. In HT-29 cells at 24 h, compared to controls 4-HPR caused a decrease in cell number of 25±1% at 2×10−10 M and 2×10−8 M (P<0.05), and 50±1% at 2×10−6 M (P<0.01). Treatment of HT-29 cells with 2×10−10 M, 2×10−8 M and 2×10−6 M 4-HPR for 48 h caused a significant decrease in cell growth of 31±1%, 50±2%, and 63±2%, respectively (all at P<0.01). At 72 h, the reduction in the cell number was 5±1% for 2×10−10

Discussion

Our data demonstrate that the retinoid 4-HPR (fenretinide) decreases significantly the number of cultured colon cancer cells; this effect is primarily an antiproliferative effect. In addition, our findings substantiate a complex effect of 4-HPR on the prostanoid pathway (inhibition of PG synthesis and decrease in the steady-state mRNA levels of only COX-2), and indicate that this effect may or may not be required for its cell kinetic effect.

These findings represent an initial approach to the

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