Fenretinide inhibits phorbol ester-induced cyclooxygenase-2 expression in human colon adenocarcinoma cells

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Fenretinide inhibits phorbol ester-induced cyclooxygenase-2 expression in human colon adenocarcinoma cells ARTICLE in BIOCHEMICAL SOCIETY TRANSACTIONS · SEPTEMBER 1997 Impact Factor: 3.19 · DOI: 10.1042/bst025457s · Source: PubMed

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Biochemical Society Transactions (1997) 25 4578

88

Fencetinide inhibits pborbol ester-induced cyclooxygenase-2 expression in human colon adenocarcinomn cells.

ELIAS T. ALPRANDIS *, GREOGORY MERRITT t, FRANCESCO PRADA *, BASIL RIGAS 7,and KHOSROW KASHFI *go

Divisions of Di estive Diseases, New York Hospital- *Cornell Medical Center, Medical Center of Queens. and qThe Rockefeller University, New Yo& NY 10021, U.S.A.

!

Retinoids, a group of natural or synthetic analogs of vitamin A have been used to treat and to protect against a variety of cancers in different animal models of carcinogenesis[l-31. However, the use of retinoids as chemotherapeutic agents has been limited due to toxicity of these agents, particularly liver damage when given in high doses [4]. A relatively new syntheticretinoid, Femtinide (4HPR) is very promising because of its biological activity and decreased hepatotoxicity [5,6]. The mechanism(s) by which retinoids exert their anticarcinogenic effect is not clearly established. Other agents such as NSAIDs that have been shown to protect against colon cancer in animals and humans [7,8] may do so in part by inhibiting cyclooxygenase(COX). There are at least two COX activities expressed in humans. COX-1 is constitutively expressed and COX-2 which is mitogen-inducible [9] has an elevated gene expression in human colorectal cancer [lo]. Inhibition of COX mluces PGE2 synthesis which is important in tumorgenesis [7]. Phorbol esters (PMA) which promote tumor formation also increase PGE2 synthesis [ l l ] . With this background, we evaluated the potential link between 4-HPR, PMA treatment, PGE2 synthesisand COX gene expression. Human colon adenocarcinomacells (HT-29) were cultured in McCoy's 5A medium supplemented with 10% FBS,non-essential amino acids, penicillin (50 Ulml), and streptomycin (50 pglml). Plain medium was used for experiments in which prostaglandin effects were studied. Cells were grown as monolayers in 63 cm2 plates and were incubated at 37"C, in 5 % C02 and 90% relative humidity. PGE2 was measured using a monoclonal Enzyme Immunoassay Kit with the cells being incubated for 6-24 hrs f different concentration of 4-HPR and PMA. For evaluation of COX-1 and COX-2 gene expression, equal quantities (500 ng) of mRNA from control an4 treated HT-29 cells were reversetranscribed and amplitled using an RT-PCR kit, using the appro riate primers and following the instructions of the manuracturer as described previously [ 121. Following the amplification, an aliquot of reaction mixture was fractionated by electrophoresis on a 2% agarose gel. After taking a photograph under UV light. the appropriatebands on the gels were quantified using a densitometer. As a control, actin mRNA was also reversetranscribed and ampWicd. Data are presented as means f SEM for at least 3 diffenmt sets of plates and treatment groups. Table 1 shows a dose dependent inhibition of PGE2 production by 4-HPR in control cells after 24 hrs of treatment. The experiment was repeated under the conditions given in Figure 1 showing that PMA at a concentration of 50 ng/mL caused approximately a 2-fold increape in FGE2 levels in thesecells. Pretreatment of the cells with 2 pM CHPR inhibited the inductive effects of PMA. Figure 2 shows that treatment of these cells with 4-HPR had no effect on COX-1 mRNA levels where as COX-2 mRNA levels were significantly decreased. PMA treatment had no effect on COX-1 mRNA but significantly increased COX-2 mRNA levels. The increase in COX-2 mRNA due to PMA treatment was blocked by pretreating the cells with 4-HPR. The results presented here show that changes in COX-2 mRNA are in parallel with changes in PGE2 synthesis both of which are down regulated by 4-HPR. Moreover. PMA which is a known tumor promoter induces both PGE2 and COX-2 mRNA levels. Once again these effects were blocked by 4-HPR suggesting a possible* mechanism for the chemopreventive effects of this . . :d4-HPR, N-(4-hydroxyphenyl)retinamide also known as Fenretinide; COX, cyclooxygenase; PGE2, ProstaglandinE2; NSAIDs, nonsteroidalanti-inflammatory drugs; PMA, pborbol 12-myristate 13-acetate. $To whom aN correspondenceshould be addressed at The Rockgfkller Universiry.

1. -t f4-HpBLM 0 (control) 10-10

10-8 10-6

of Fem&&b on PGQJ,ev&

. ..

1050f 200

700 f 85 390 f 50* 50 f 10*

3358 63 f 5 95f3

*P c 0.05 compared to control, Student's t-test .

Figure 1. Effect of Pretreatment with Fencetinide on Phorbol Ester-Induced PGE2 Levels

n

0 Control I4-HPR IPMA m 4-HPRPMA

T

Treatment

HT-29 cells (3 x 106) were incubated with control medium f 4HPR (2 pM) for 24 hrs after which the cells were washed and fresh culture medium added as follows: control, 4-HPR (2 pM), and PMA (50 ng/mL) to both control and 4-HPR preincubated cells. Culture medium was removed after 6 hr and assayed for PGE2 levels. The cells were then washed and used for mRNA isolation. The RT-FCR product was evaluated by desitomhy, the results of which are shown below. Each treatment group had its own appropriate control which was set at an arbitrary value of 100%.This experiment was repeated once and the results were of the values &p,yn.

Figure 2. Effect of Fencetinide and Phorbol Ester on Cyclooxygenase mRNA

0 Control

m cox-1 Ic o x - 2 m cox-1 cox-2 cox-2

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