Retinoids and Cancer: Antitumor Effect of ATRA and of a New Derivative of Retinoic Acid, IIF, on Colon Carcinoma Cell Lines CaCo-2 and HT-29

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1 Retinoids and Cancer: Antitumor Effect of ATRA and of a New Derivative of Retinoic Acid, IIF, on Colon Carcinoma Cell Lines CaCo-2 and HT-29 G.BARTOLINI 1, K.AMMAR 1, B. MANTOVANI 1, F.SCANABISSI 1, A.M.FERRERI 2, P.ROCCHI 2 and M.ORLANDI 1 1 Dipartimento di Biologia Evoluzionistica Sperimentale, via Selmi 3, Bologna; 2 Dipartimento di Patologia Sperimentale, Sezione di Cancerologia, viale Filopanti 22, Bologna, Italy Abstract. Vitamin A and its metabolic forms, like all-trans retinoic acid (ATRA), are used with promising results in the treatment of many tumors. Two major problems in the clinical use of retinoids are that the doses needed for successful treatment are often toxic, leading to "hypervitaminosis A syndrome" and that patients often develop drug resistance. In order to find compounds that can overcome these problems, many new derivatives of retinoids have been synthesized and tested. Here we present a study on the effect of a new derivative of retinoic acid, IIF (pat.wipo W0 00/17143), on growth and differentiation of two colon carcinoma cell lines, CaCo-2 and HT-29, with different degrees of tumorigenicity, the second one being more undifferentiated. The effect of IIF was compared with that of ATRA, whose antitumoral action on colon cancer cells and other tumoral cells is widely described in the literature. Besides exerting a strong antiproliferative effect, even higher than that of ATRA, IIF induced cellular differentiation, as demonstrated by the appearance of morphological (domes and microvilli formation) and biochemical (alkaline phosphatase induction) markers. Therefore, these findings indicate the new retinoid IIF as a possible candidate in the treatment of colon cancer. Vitamin A and its metabolic forms, like all-trans retinoic acid (ATRA) and 9-cis retinoic acid (9-cis RA), are used with promising results in the treatment of many tumors (1,2), but the doses needed for successful treatment are often toxic, leading to "hypervitaminosis A syndrome" (3); in addition, patients often develop resistance to the drug Correspondence to: G. Bartolini, Dipartimento di Biologia Evoluzionistica Sperimentale, Via Selmi 3, Bologna, Italia. Tel: , Fax: , giovanna.bartolini@unibo.it Key Words: Retinoids, colon cancer, CaCo-2, HT-29, IIF, cell differentiation. (4). In order to find compounds that can overcome these problems, many new derivatives of retinoids have been synthesized and tested, including IIF, whose antitumoral action is examined in this paper. The HT-29 and CaCo-2 cell lines are human colon adenocarcinoma cell lines. The HT-29 line is more undifferentiated, while the CaCo-2 line spontaneously differentiates into enterocytes at confluency in culture. Differentiation is expressed by a number of morphological and biochemical markers, among which are the appearence of an actin-based brush border cytoskeleton (5), expression of brush border enzyme markers (6,7) and development of cell polarity with dome formation (8). Domes are the result of fluid transport by the monolayer and demonstrate that the intercellular junctions, characteristic of epithelial cells, have been established. ATRA exerts a strong antiproliferative and prodifferentiating effect on colon cancer cells (1,9,10). In previous works we described a strong antiproliferative and prodifferentiating effect of a new derivative of retinoic acid, IIF (pat. WIPO W0 00/17143), on the leukemic cell line HL-60 (11) and in the neuroblastoma TS12 line (12). Here we present a study on the effect of IIF on the growth and differentiation of two colon carcinoma cell lines, CaCo-2 and HT-29, with different degrees of tumorigenicity. The effect of IIF was compared to that of ATRA, whose antitumoral action on colon cancer cells and other tumoral cells is widely described in literature (1,9,10). Besides exerting a strong antiproliferative effect, IIF induced cellular differentiation, as demonstrated by the appearance of morphological (domes and microvilli formation) and biochemical (alkaline phosphatase induction) markers. Materials and Methods The human colonic tumor cell lines CaCo-2 and HT-29 ( gift of Dr. M. Chiricolo and F. Dall Olio, Dept. of Exp. Pathology, University of Bologna, Italy), were maintained in DMEM (Sigma) supplemented /2004 $

2 Figure 1. Growth pattern of CaCo-2 cells in the presence of different doses (ÌM) of IIF (top) and ATRA (bottom) added every other day. Results are expressed as a percentage of the controls. Each bar represents the mean (±SE) of 8 replicate wells from 4 independent experiments. Figure 3. Light micrographs of domes in CaCo-2 cells in the presence of 10ÌM IIF, added every other day, after 8 days (a) and 14 days (b) of culture. Duplicate determinations were performed in each experiment. The results are from one experiment representative of three. Bar: 100 Ìm. Figure 2. Growth pattern of HT-29 cells in the presence of different doses (ÌM) of IIF (top) and ATRA (bottom) added every other day. Results are expressed as a percentage of the controls. Each bar represents the mean (±SE) of 8 replicate wells from 4 independent experiments. with FCS (GIBCO, Grand Island, NY, USA), 15% for CaCo-2 and 10% for HT-29, 2mM glutamine, gentamicin (0.05 mg/ml) and amphotericin (0.25 Ìg/ml) at 37ÆC in a humidified atmosphere containing 5% CO 2. The cells were refed three times a week. Experiments were performed on cells seeded in T-25 flasks at a density of 8 x 10 3 /cm 2 or, for the proliferation tests, in 96-well plates at a density of 1 x 10 3 / well. IIF (pat. WIPO W0 00/17143) and ATRA (Sigma) were dissolved in propylene glycol and ethanol, respectively. The cells were treated with the compounds as indicated and control cells were treated with equivalent amounts of glycol and ethanol. Cell viability was estimated by trypan blue dye exclusion. The cells were mycoplasma free. Cell proliferation was evaluated by a method based on the reduction of MTT taken as an index of the number of metabolically active cells and the results were expressed as a percentage of the controls. This method eliminates the problems due to the tendency of cells to form aggregates after trypsinization (13). The presence of domes was observed under light microscopy; the cells were fixed by methanol and stained with May Grunwald- Giemsa staining solution (Sigma). Samples for scanning electron microscopy (SEM) being processed through a graded ethyl alcohol series, through hexamethyldisilazane overnight were then fixed on stubs with double-sided adhesive tape and vacuum-coated with gold, for 3 minutes at 30 ma, before observation with a JEOL JSM5200 scanning electron microscope. Alkaline phosphatase activity was determined by the ALP Kine test (Sclavo Diagnostics, Siena, Italy). Enzyme activity was expressed in units: 1 unit is equivalent to the micromoles of substrate hydrolyzed per minute. 1780

3 Bartolini et al: Antitumoral Effect of Retinoids on Colon Cancer Cells Figure 4. SEM micrographs of CaCo-2 cells after 30 days from seeding, treated every other day (down) or not (up) with10 ÌM IIF. Bar: 10 Ìm Figure 5. Alkaline phosphatase activity in CaCo-2 (top) and HT-29 (bottom) treated or not for two weeks with different doses of IIF (ÌM) added every other day. Specific activity values are the mean (±SE) of 2 replicates of 4 experiments. Results We compared the antiproliferative effect of IIF with that of ATRA in CaCo-2 and HT-29 cell lines. Different doses of the compounds were added to the cells every other day. In CaCo-2 (Figure 1) after five days of treatment IIF (30ÌM) led to a decrease of 90%, while ATRA (40ÌM) had a lesser effect (60% decrease). In HT-29 (Figure 2), the treatment gave similar results, with IIF being more effective than ATRA. In order to evaluate whether the antiproliferative effect of IIF was accompanied by morphological and biochemical changes indicating that cells undergo differentiation, we then analyzed some markers typical of colonic cell maturation. The development of cell polarity is accompanied by the formation of domes. In Figure 3 CaCo-2 cells treated with IIF 10 ÌM are shown. After 8 days from seeding (Figure 3 a), confluence was complete and many domes started to be evident. After 14 days (Figure 3 b), the dome size was strongly increased. Both dome size and number were increased in treated samples with respect to controls. In particular, by day 14 the number of domes was increased 3.5-fold passing from 240/T-25 flask in controls to 840/T-25 flask in IIF-treated cells flask (not shown). Another marker of differentiation in CaCo-2 cells is the formation of microvilli. SEM analysis of CaCo-2 cells treated with IIF 10 ÌM revealed, after 30 days from confluence, numerous granular elevations on top of the cell surface that probably reflected microvilli (Figure 4 b). The elevations could not be seen in untreated cells (Figure 4 a). In addition, IIF-treated cells were flat and polygonal, while control cells were round. Upon reaching confluence CaCo-2 cells spontaneously assemble a brush border (14,15) and express brush-border membrane-associated hydrolases, typical of a differentiated phenotype (16). Among these hydrolases, we studied alkaline phosphatase (ALP), whose induction is significantly correlated with cell differentiation (17,18). In CaCo-2, following treatment with IIF 10 ÌM, ALP specific activity 1781

4 was doubled with respect to controls after 2 weeks from seeding (Figure 5 top). In HT-29 the ALP activity was much lower with respect to that found in CaCo-2 and doubled when IIF 50 ÌM was added (Figure 5 bottom). ALP activity was partially inhibited in the presence of l-leucine 5 mm that is a specific inhibitor of CaCo-2 ALP, thus confirming the specificity of the test (data not shown). Discussion In previous work we have described a strong antiproliferative and prodifferentiating effect of a new derivative of retinoic acid, IIF, on the neuroblastoma cell line TS12 (12) and on the leukemic cell line HL-60 and we demonstrated that IIF binds RXRÁ receptor, which is the receptor of 9-cis RA (11). In this study we evaluated the effect of IIF on the growth and differentiation of two colon carcinoma cell lines, CaCo- 2 and HT-29, that show different degrees of tumorigenicity. The effect of IIF was compared with that of ATRA, whose antitumoral action on colon cancer cells and other tumoral cells has been widely described in the literature (1,2,9,10). IIF exerted an antiproliferative effect, higher than that observed with ATRA, both in CaCo-2 and in HT-29 cells, even if more pronounced in HT-29 cells, which are more undifferentiated. Our results were in accordance with those obtained by Mc Cormack et al. (19) who studied the effect of retinoic acid on CaCo-2 cells and found that maximum inhibition of growth occurred between day 7 and 9 after plating, which was the time at which confluence was reached and domes were formed, signalling the beginning of differentiation. In CaCo-2, differentiation is expressed by a number of morphological and biochemical markers, among which is the development of cell polarity with dome formation (8). Domes are the result of fluid transport by the monolayer and demonstrate that the intercellular junctions, characteristic of epithelial cells, have been established. We demonstrated that IIF treatment, after 14 days from cell seeding, strongly increased dome number and size with respect to controls, demonstrating that the compound influenced the normal process of CaCo-2 differentiation that follows cell confluency. Another important marker of differentiation in CaCo-2 cells is the formation of microvilli in cells that have reached confluence (20). SEM analysis of CaCo-2 after 30 days from confluence revealed, besides different cell shape, the presence of numerous granular elevations on top of the cell surface, but only in cells treated with IIF. This observation was in contrast with what has been reported by other authors (6), who have described the appearence of microvilli also in untreated cells after a few days from confluency and could be explained by the fact that we employed a different cell clone. Reaching confluence or under the influence of modifications of the culture medium, CaCo2 and HT-29 cells assemble a brush border (14) and express brush border membrane-associated hydrolases, typical of a differentiated phenotype (16,18) like ALP. Treatment with IIF strongly increased ALP specific activity with respect to controls especially in CaCo-2 cells. It is interesting that IIF at the dose 10 ÌM, which was not particularly active in reducing proliferation when given for short periods, had strong prodifferentiating activity in long-term treatment, as demonstrated by the appearance of microvilli and of increased ALP activity. In conclusion, the data concerning the new derivative of retinoic acid IIF (pat. WIPO W0 00/17143), confirmed that, in CaCo-2 and HT29 cells, besides exerting a strong antiproliferative effect, it induced cellular differentiation, as demonstrated by the appearance of morphological and biochemical markers. Therefore, IIF can be indicated as a possible candidate in the treatment of colon cancer. Acknowledgements The authors thank Ms L. Di Pietrangelo for her expert photographical assistance. The present work was supported by grants from Program "Differenziamento Cellulare", University of Bologna, Italy, to G.B. References 1 Miller WH Jr: The emerging role of retinoids and retinoic acid metabolism blocking agents in the treatment of cancer. Cancer 83(8): , Brtko J and Thalhamer J: Renaissance of the biologically active vitamin A derivatives: established and novel directed therapies for cancer and chemoprevention. Curr Pharm Des 9(25): , Frankel SR, Eardley A, Lauwers G, Weiss M and Warrell RP Jr: The retinoic acid syndrome in acute promyelocytic leukemia. 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