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Retinol Inhibits the Growth of All-Trans-Retinoic Acid–Sensitive and All-Trans-Retinoic Acid–Resistant Colon Cancer Cells through a Retinoic Acid Receptor–Independent Mechanism

¹ Institute for Cell and Molecular Biology and ² Division of Nutritional Sciences, Department of Human Ecology, University of Texas at Austin, Austin, Texas

Requests for reprints: Michelle A. Lane, Division of Nutritional Sciences, Department of Human Ecology, University of Texas at Austin, Gearing Hall Room 115, Mail Stop A2700, Austin, TX 78712. Phone: 1-512-232-9410; Fax: 1-512-471-5844; E-mail: mlane{at}mail.utexas.edu.

	Abstract

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Retinol (vitamin A) is thought to exert its effects through the actions of its metabolite, all-trans-retinoic acid (ATRA), on gene transcription mediated by retinoic acid receptors (RAR) and retinoic acid response elements (RARE). However, retinoic acid resistance limits the chemotherapeutic potential of ATRA. We examined the ability of retinol to inhibit the growth of ATRA-sensitive (HCT-15) and ATRA-resistant (HCT-116, SW620, and WiDR) human colon cancer cell lines. Retinol inhibited cell growth in a dose-responsive manner. Retinol was not metabolized to ATRA or any bioactive retinoid in two of the cell lines examined. HCT-116 and WiDR cells converted a small amount of retinol to ATRA; however, this amount of ATRA was unable to inhibit cell growth. To show that retinol was not inducing RARE-mediated transcription, each cell line was transfected with pRARE-chloramphenicol acetyltransferase (CAT) and treated with ATRA and retinol. Although treatment with ATRA increased CAT activity 5-fold in ATRA-sensitive cells, retinol treatment did not increase CAT activity in any cell line examined. To show that growth inhibition due to retinol was ATRA, RAR, and RARE independent, a pan-RAR antagonist was used to block RAR signaling. Retinol-induced growth inhibition was not alleviated by the RAR antagonist in any cell line, but the antagonist alleviated ATRA-induced growth inhibition of HCT-15 cells. Retinol did not induce apoptosis, differentiation or necrosis, but affected cell cycle progression. Our data show that retinol acts through a novel, RAR-independent mechanism to inhibit colon cancer cell growth.

	Introduction

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Colorectal cancer is currently the third leading cause of death due to cancer in the United States. Retinoids, a group of compounds consisting of vitamin A (retinol), its natural metabolites, and several synthetic compounds, have been shown to act as cancer chemopreventive agents (for reviews, see refs. 1–3). In rats, retinol, 9-cis-retinoic acid, and 4-(hydroxyphenyl)retinamide can inhibit the formation of carcinogen-induced aberrant crypt foci, a precursor to colon cancer, as well as colon tumors (4–7). Retinyl palmitate was recently shown to inhibit high-fat diet–induced aberrant crypt foci (7). Additionally, several in vitro studies indicate that retinoids have potent antiproliferative effects on colon cancer cell lines and may hold potential for both chemoprevention and chemotherapy of colon cancer.

In almost all of the above studies, the retinoid examined has been an isoform of retinoic acid or a synthetic retinoid such as 4-(hydroxyphenyl)retinamide. Although these compounds are effective at inhibiting all-trans-retinoic acid (ATRA)–sensitive cell growth, the use of exogenous ATRA to study the effects of vitamin A assumes that all of the biological phenomena attributed to retinol are due to ATRA. The diet contains very little ATRA (8). Rather, the diet contains vitamin A in two forms: (1) previtamin A carotenoids and (2) preformed vitamin A as retinol and retinyl esters. Retinyl esters are cleaved within the intestinal lumen to yield retinol. Therefore, human colonocytes are exposed primarily to retinol, the focus of this study. Within most cells, retinol is either esterified for storage or metabolized to ATRA. ATRA exerts its effects on cell growth and differentiation by binding to retinoic acid receptors (RAR) located in cell nuclei. RARs heterodimerize with retinoid X receptors (RXR) and bind to retinoic acid response elements (RARE) located in the regulatory regions of retinoid-responsive genes. When ATRA binds to the RAR member of the RAR/RXR heterodimer, gene transcription via RARE is induced (for review, see ref. 2).

Retinoic acid resistance is believed to be due to a defect in RAR, RARß, or RAR induction in response to ATRA (for review, see refs. 2, 9–11). Retinoic acid resistance occurs when tumors or tumor-derived cell lines cease to growth inhibit or differentiate in response to treatment with ATRA. Retinoic acid resistance is a common phenomenon and appears to arise spontaneously in numerous types of cancer and tumor-derived cell lines. The defective receptor varies with cell line but RARß expression is frequently lost.

The objective of the present study was to determine if retinol could inhibit the growth of both ATRA-sensitive and ATRA-resistant colon cancer cell lines in vitro. Because the ATRA-resistant cell lines lack one or more RARs, their use allowed us to determine the effects of retinol on cell growth, exclusive of the effects of ATRA. Our data show that retinol itself inhibits the growth of both ATRA-sensitive and ATRA-resistant colon cancer cells through an ATRA- and RAR-independent mechanism.

	Materials and Methods

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Cell culture. Three human colorectal adenoma cell lines, HCT-15, SW620, and WiDR, and one human colon carinoma cell line, HCT-116, were obtained from the American Type Culture Collection (Manassas, VA) and grown as recommended. HCT-15 cells were grown in MEM, HCT-116 in McCoy's medium, and SW620 and WiDR cells in DMEM in a humidified atmosphere at 37°C with 5% CO₂. All medium was supplemented with 10% FCS and antibiotics (1,000 units/mL penicillin and 1,000 µg/mL streptomycin). The experiment was repeated with each cell line grown in either supplemented DMEM or McCoy's medium. Medium type did not affect cell growth. Cells were seeded in 12-well culture dishes at a density of 1 x 10⁴ cells per well. The following day, the medium was removed and replaced with medium containing 0, 0.1, 1, or 10 µmol/L ATRA or all-trans-retinol (Sigma, St. Louis, MO). All retinoids were prepared as 10 mmol/L stocks in 100% ethanol. All treatments, including control, received equal volumes of ethanol vehicle and all retinoid manipulations were performed in the dark. All treatments were done in duplicate. Cells were harvested using trypsin and counted via hemocytometer every 24 hours for 4 days.

Retinoid extraction and high-performance liquid chromatography analysis. To examine retinol metabolism, cells were seeded in 60 mm dishes at the following densities to yield 60% to 80% confluence and maximum high-performance liquid chromatography (HPLC) detection sensitivity at the time of harvest: 5 x 10⁵ cells/dish for 24 hours, 2.5 x 10⁵ cells/dish for 48 hours, 1 x 10⁵ cells/dish for 72 hours, and 5 x 10⁴ cells/dish for 96 hours. Twenty-four hours after plating, cells were treated with 0, 1, and 10 µmol/L retinol for 24, 48, 72, or 96 hours. Sixteen hours before harvest, the culture medium was removed and replaced with medium containing 5% FCS and 50 nmol/L [³H]retinol (specific activity = 52.5 Ci/mmol). Cells and medium were harvested 2, 4, 8, and 16 hours after the addition of label as described previously (12). A control of labeling medium without cells was also incubated for 16 hours. F9 murine teratocarcinoma cells, treated with 1 µmol/L ATRA for 48 hours and incubated with 50 nmol/L [³H]retinol for 16 hours, were used as a positive control for 4-oxoretinol production (13). Retinoids were extracted and separated using a Waters Millennium HPLC system as described previously (14).

Cell transfection and chloramphenicol acetyltransferase assays. To examine the possibility that an undetected metabolite of retinol was activating RAR/RXR-mediated transcription, all cell lines were transiently transfected with pRARE-chloramphenicol acetyltransferase (CAT; generously provided by Dr. Dianne Soprano). Cells were seeded onto 12-well plates at a density of 1.75 x 10⁵ cells/well and incubated overnight in FCS-supplemented medium. The following day, cells were transfected using LipofectAMINE 2000 (Promega, Madison, WI) according to the protocol of the manufacturer with 1 µg of pRARE-CAT and 0.5 µg of pSV-ß-gal. Twenty-four hours later, the transfection medium was removed and the cells were treated with fresh medium containing 0, 1, and 10 µmol/L ATRA or retinol. The cells were harvested after treatment for 24 or 48 hours and assayed for ß-galactosidase (ß-Galactosidase Enzyme Assay System; Promega) and CAT (CAT Enzyme Assay System; Promega) activity as per instructions of the manufacturer. CAT activity was corrected for transfection efficiency using the ß-galactosidase activity.

Retinoic acid receptor antagonist assays. To determine if retinol was inhibiting cell growth via RAR, the pan-RAR antagonist, AGN 193109 was used to block RAR function. The RAR pan-antagonist was synthesized by Allergan, Inc. (Irvine, CA). Cell lines were plated at a density of 1 x 10⁴ in 12-well plates and allowed to attach overnight. The following day, HCT-15 cells were treated with 0 and 1 µmol/L ATRA or retinol with and without 10 µmol/L AGN 193109. HCT-116, SW620, and WiDR cells were treated with 0 and 1 µmol/L retinol with and without 10 µmol/L AGN 193109. Control cells received an equal volume of DMSO and ethanol vehicle. Cells were harvested after treatment for 48 hours (HCT-15) or 96 hours (HCT-116, SW620, and WiDR). All treatments were done in duplicate. The pharmacologic, 10 µmol/L, concentrations of ATRA and retinol were not examined because 100 µmol/L AGN 193109 was toxic to the cells.

Detection of apoptosis. Nuclear staining via 4',6-diamidino-2-phenylindole (DAPI) and flow cytometry analysis, described below, were used to determine if cell growth inhibition was due to apoptosis. For DAPI staining, cells were plated at 1 x 10⁴ cells per well in 12-well plates before treatment with 0, 1, and 10 µmol/L retinol. Cells incubated for 4 hours at 37°C with 4 µg/mL camptothecin served as the positive control for apoptosis. Both adherent and floating cells were harvested every 24 hours for 4 days. The cells were centrifuged and washed with PBS to remove all traces of media. Cells were then incubated with 2 µg/mL DAPI for 10 minutes at 37°C before counting at 400x magnification with an Olympus upright fluorescence microscope. To obtain cell counts, at least three different locations on each slide were used. Two hundred cells were counted at each location yielding a minimum of 600 cells counted per slide. Cells with segmented nuclei were scored as apoptotic.

Cellular differentiation. Alkaline phosphatase activity was used to determine if retinol was inhibiting cell growth by inducing cellular differentiation. All cell lines were plated on 60 mm dishes at a density of 5 x 10⁴ cells per plate. Twenty-four hours later, cells were treated with 0, 1, and 10 µmol/L retinol or 2 mmol/L sodium butyrate (positive control) for 96 hours. Alkaline phosphatase activity was determined as described previously (15). Alkaline phosphatase activity was measured by the conversion of p-nitrophenyl phosphate (19.8 mmol/L) to p-nitrophenol by 0.1 mL of cell lysate in 100 mmol/L glycine buffer, containing 1 mmol/L MgCl₂ (pH 10). Alkaline phosphatase enzyme activity was corrected for lysate protein content and expressed as percentage positive control.

Necrosis assays. Trypan blue exclusion assays were used to measure cell death. Briefly, an aliquot of the cells harvested for the growth curve assays was pelleted by centrifugation, resuspended in 0.5 mL HBSS, and incubated with an equal volume of 0.4% trypan blue solution (Sigma-Aldrich, St. Louis, MO) for 5 minutes at room temperature before counting with a hemocytometer. Blue cells were scored as necrotic.

Flow cytometry analysis. To determine if growth inhibition was due to cell cycle arrest and to confirm the absence of apoptosis through lack of a sub-G₁ peak, cells were seeded on 60 mm dishes at a density of 3 x 10⁵ (HCT15 and SW620) or 2 x 10⁵ (HCT-116 and WiDR) cells per dish to provide 50% to 60% confluence at the time of harvest. To synchronize, cells were plated in serum-free medium for 24 hours and treated with 1 (HCT-15) or 3 µg/mL (HCT-116, SW620, WiDR) aphidicholin for an additional 24 hours. The following day, the cells were washed with PBS and treated with fresh FCS-supplemented media containing 0, 1, and 10 µmol/L retinol. Cells were harvested, fixed in 70% ethanol overnight, and stained with 40 µg/mL propidium iodide as described previously (16). At least 10,000 cells were analyzed per sample using a FACSCalibur machine (Becton Dickinson, San Jose, CA). DNA content was determined using Modfit software version 3.0 (Verity Software House, Inc., Topsham, ME).

	Results

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Growth of all-trans-retinoic acid–resistant colon cancer cells is inhibited by retinol. The ability of retinol to inhibit cell growth was examined in three ATRA-resistant human colon cancer cell lines HCT-116 (17), SW620 (18), and WiDR (11). An ATRA-sensitive cell line, HCT-15, was chosen to serve as a positive control for the inhibitory effects of ATRA on colon cancer cell growth (19). Serum concentrations of retinol range from 0.5 to 2 µmol/L (20). Therefore, 0.1 µmol/L was selected to represent a subphysiologic, and 1 µmol/L a physiologic, concentration of retinol. The highest level, 10 µmol/L retinol, was used as a pharmacologic, but potentially therapeutically relevant, concentration. There is very little ATRA (4-14 nmol/L) in the serum (21, 22). ATRA levels were chosen to match the concentrations of retinol used and to reflect ATRA levels commonly found in the literature (9–11, 23).

After 96 hours of treatment, the growth of HCT-15 cells was inhibited by ATRA (Fig. 1A) as expected. In addition, HCT-15 cell growth was also inhibited by retinol in a dose-responsive manner. Cells treated with 10 µmol/L retinol exhibited the largest degree of inhibition to 36.7 ± 7.8% of control. HCT-116 cell growth was inhibited slightly by 0.1 and 1 µmol/L retinol but this decrease was not significant when compared with the same concentrations of ATRA (Fig. 1B). However, HCT-116 cell growth was significantly inhibited by 10 µmol/L retinol (37.5 ± 9.2% of control) when compared with 10 µmol/L ATRA (74.3 ± 4.7% of control). SW620 and WiDR cell growth was significantly inhibited by treatment with 0.1 and 1 µmol/L retinol for 96 hours when compared with ATRA (Fig. 1C and D), indicating that physiologic levels of retinol can inhibit the growth of ATRA-resistant cells. At 10 µmol/L concentrations, there was no significant difference in the ability of ATRA and retinol to inhibit SW620 and WiDR cell growth. The highest concentration of ATRA, 10 µmol/L, inhibited cell growth slightly in all cell lines examined (Fig. 1). These data show that retinol can inhibit the growth of both ATRA-sensitive and ATRA-resistant colon cancer cells.

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Figure 1. Retinol inhibits the growth of ATRA-resistant human colon cancer cells. HCT-15 (A) HCT-116 (B), SW620 (C), and WiDR (D) cells were seeded and treated with 0, 0.1, 1, or 10 µmol/L ATRA or retinol. All treatments were performed in duplicate. Cells were counted via hemocytometer daily for 4 days. Columns, mean for three experiments; bars, SE. Left, percentage growth inhibition exhibited by human colon cancer cell lines after 96 hours of treatment with increasing amounts of ATRA or retinol. Statistical analysis was done using t tests comparing ATRA to retinol for each concentration. *Significantly different from ATRA, P < 0.05. Right, growth rates of HCT-15 (A), HCT-116 (B), SW620 (C), and WiDR (D) cells grown for 4 days with increasing amounts of retinol.

No [³H]ATRA, 4-oxoretinol, or anhydroretinol was produced from [³H]retinol at any time point or treatment examined in HCT-15 and SW620 cells (Fig. 2). The absence of [³H]4-oxoretinol was also confirmed by Northern blot analysis that failed to show CYP261A mRNA expression in any of the four cell lines examined (data not shown). HCT-116 and WiDR cells did synthesize a small amount of [³H]ATRA from [³H]retinol (Fig 2). However, both control and retinol-treated cells metabolized [³H]retinol to [³H]ATRA. The time point displayed in Fig. 2 (48 hours of retinol treatment followed 8 hours of incubation with 50 nmol/L [³H]retinol) showed the largest concentration of 50 nmol/L [³H]ATRA synthesis by HCT-116 and WiDR cells of any time point examined. When corrected for cell number, HCT-116 cells treated with the vehicle control synthesized 0.15 nmol/L [³H]ATRA/million cells from 50 nmol/L [³H]retinol. Cells treated with 10 µmol/L retinol synthesized 0.55 nmol/L [³H]ATRA/million cells from 50 nmol/L [³H]retinol. Because the metabolism of 50 nmol/L [³H]retinol reflects the metabolism of 10 µmol/L retinol (12, 14, 24), we can assume that if HCT-116 cells were treated with 10 µmol/L retinol, 0.11 µmol/L of ATRA would be produced per million cells. There were 5.4 x 10⁶ cells on a duplicate plate of HCT-116 cells treated with 10 µmol/L retinol for 96 hours and included in the experiment shown in Fig. 2. As can be seen in Fig. 1B, HCT-116 cell growth is inhibited only slightly by 0.1 or 1 µmol/L ATRA when compared with control. The amount or [³H]ATRA synthesized from [³H]retinol by WiDR cells was even less than that synthesized by the HCT-116 cell line. Therefore, the small amount of ATRA produced by these cell lines when treated with 10 µmol/L retinol cannot be responsible for the decrease in cell number that occurs when these cells are treated with 10 µmol/L retinol.

View larger version (41K): [in this window] [in a new window] [Download PPT slide]   Figure 2. Retinol is not metabolized to bioactive compounds in HCT-15 and SW620 cells but is converted to retinoic acid by HCT-116 and WiDR cells. Cells were plated and allowed to attach for 24 hours before addition of medium containing 0, 1, or 10 µmol/L retinol. Cells were allowed to grow for 24, 48, 72, or 96 hours before the medium was removed and replaced with new medium containing 5% FCS and 50 nmol/L [3H]retinol. The concentration of nonradioactive retinol in 5% FCS was 50 nmol/L (14). Cells were allowed to incubate for another 2, 4, 8, or 16 hours before harvest. The retinoids were extracted from cell and medium samples and separated by HPLC as described (14). Data shown represent radiolabeled retinoids extracted from medium (A) or cells (B) of the indicated cell lines 48 hours after treatment with 0 or 10 µmol/L retinol followed by incubation for 8 hours with 50 nmol/L [3H]retinol. The identities of the retinoids were determined by coelution with known nonradiolabeled (cold) standards included in the samples. Changes in absorbance are recorded as changes in voltage by the FloOne software that generated these chromatographs and controls the liquid scintillation counter. Thus, the unit for the y-axis of "Standards" panel is volts. The slight difference in elution time between the cold standards and the 3H peaks is due to the transit time from the photodiode array to the scintillation counter. [3H]Retinol extracted from labeling medium at time zero is shown in the left column, one panel from the bottom. F9 murine teratocarcimona cells were treated with 1 µmol/L ATRA for 48 hours followed by incubation with [3H]retinol for 16 hours before retinoid extraction. The retinoids extracted from F9 murine teratocarcimona cells are included as a positive control for 4-oxoretinol production and are shown in the last panel of the right column. Retinoid standards are shown in the bottom panel of the left column and eluted as follows: 4-oxoretinol, 13.5 minutes; ATRA, 18.5 minutes; all-trans-retinol, 34.0 minutes; and anhydroretinol, 39.8 to 40.5 minutes. This experiment was repeated twice with similar results.

Figure 3A shows that treatment of ATRA-sensitive HCT-15 cells with both 1 and 10 µmol/L ATRA for 24 or 48 hours resulted in an increase in CAT activity to 4.98 ± 0.39-fold over control at 48 hours for cells treated with 10 µmol/L ATRA. Because HCT-15 cells were ATRA sensitive, we were surprised to find that treatment of HCT-15 cells with retinol did not increase CAT activity to >1.80 ± 0.06-fold over control at 48 hours for cells treated with 1 µmol/L retinol (Fig. 3A). However, this lack of CAT activity reflects the metabolism data (Fig. 2A-D), showing that HCT-15 cells do not metabolize retinol to ATRA.

View larger version (18K): [in this window] [in a new window] [Download PPT slide]   Figure 3. Retinol does not induce RARE-CAT reporter gene expression. HCT-15 (A), HCT-116 (B), SW620 (C), and WiDR (D) cells were transiently transfected with 1 µg pRARE-CAT and 0.5 µg pSV-ß-gal using LipofectAMINE 2000. Twenty-four hours following transfection, cells were treated with 0, 1, and 10 µmol/L ATRA or retinol. Cells were harvested 24 and 48 hours after treatment and CAT and ß-galactosidase assays were done. CAT activity was normalized for transfection efficiency using the ß-galactosidase activity. The CAT activity in control cells treated with the ethanol vehicle was set equal to one and all other values are expressed as fold induction. Columns, mean of three separate experiments; bars, SE.

Retinol is not acting through retinoic acid receptors to inhibit cell growth. To confirm that the growth inhibition exhibited by cells treated with retinol was not mediated by the retinoic acid/RAR/RARE retinoid signaling mechanism, all cell lines were treated with a RAR pan-antagonist, AGN 193109. This antagonist, when added at 10 times the concentration of agonist, blocks the ability of agonist to bind to RAR (25). HCT-15 cells treated with 1 µmol/L ATRA and 10 µmol/L AGN 193109 served as a positive control for the ability of AGN 193109 to block RAR-mediated cell growth inhibition. Because 1 µmol/L ATRA does not inhibit HCT-116, SW620, or WiDR cell growth, we did not test the effects of the combined treatment of AGN 193109 and ATRA in these cell lines. As shown in Fig. 4A, AGN 193109 blocked ATRA-induced growth inhibition in HCT-15 cells, as expected. However, AGN 193109 did not block growth inhibition due to retinol treatment in any of the four cell lines examined, including the ATRA-sensitive HCT-15 cell line (Fig. 4). The inability of AGN 193109 to block retinol-induced growth inhibition confirms the results of the metabolism and RARE reporter experiments, which also indicate that retinol is not acting via retinoic acid/RAR/RARE to affect cell growth even in the ATRA-sensitive, HCT-15 cell line. Unlike the ATRA-resistant cell lines, HCT-15 cells contain all of the cellular machinery required for induction of ATRA/RAR/RARE-mediated gene transcription and growth inhibition (11). As shown in Fig. 4A, ATRA is acting via this mechanism to inhibit the growth of HCT-15 cells. In contrast, retinol is acting via a novel, receptor-independent mechanism to inhibit the growth of both ATRA-resistant and, surprisingly, ATRA-sensitive human colon cancer cell lines.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Figure 4. Retinol does not act through RAR to inhibit cell growth. HCT-15 (A) cells were plated with 0 or 1 µmol/L ATRA or retinol with or without 10 µmol/L of the RAR pan-antagonist, AGN 193109, for 48 hours. HCT-116 (B), SW620 (C), and WiDR (D) were treated with 0 or 1 µmol/L retinol with or without 10 µmol/L AGN 193109 for 96 hours. All treatments were plated in duplicate. Columns, mean of two separate experiments; bars, SE.

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Figure 5. Retinol does not induce apoptosis or cellular differentiation in ATRA-resistant human colon cancer cell lines. HCT-15 (A), HCT-116 (B), SW620 (C), and WiDR (D) cells were plated and treated with 0, 1, or 10 µmol/L retinol as described in Materials and Methods. To measure apoptosis (left), floating and adherent cells were harvested every 24 hours, centrifuged, and washed with PBS to remove all traces of media. Cells were stained with 2 µg/mL DAPI for 10 minutes in 37°C before observation. To measure cellular differentiation (right), cells were plated as described and treated with 0, 1, or 10 µmol/L retinol or 2 mmol/L sodium butyrate for 96 hours. Alkaline phosphatase activity was determined as described (15) by the conversion of p-nitrophenyl phosphate to p-nitrophenol at 410 nm. Alkaline phosphatase enzyme activity is expressed as percentage positive control. Columns, mean for three independent experiments; bars, SE.

View larger version (27K): [in this window] [in a new window] [Download PPT slide]   Figure 6. Retinol alters cell cycle progression but does not induce apoptosis. HCT-15 (A), HCT-116 (B), SW620 (C), and WiDR (D) cells were synchronized with serum starvation for a total of 48 hours and the addition of aphidicholin during the last 24 hours of serum starvation. Following synchronization, fresh medium containing 10% FCS and 0 (left) or 10 µmol/L retinol (right) was added for 24 hours before fixation and staining with propidium iodide. At least 10,000 cells were analyzed per sample using a FACSCalibur machine. Absence of a sub–G0-G1 indicates that apoptosis was not induced by retinol treatment. Dark gray, G0-G1 and G2-M; hatched, S phase. One representative experiment of two is shown.

To ensure that retinol was not inducing necrosis, trypan blue dye exclusion assays were performed on the adherent cells used for the growth curve experiments described in Fig. 1. The percentage of cells that stained with trypan blue dye varied between 0.1% and 7% and no consistent pattern was exhibited under any treatment condition at any time point (data not shown). Therefore, necrosis is not responsible for the growth inhibition exhibited by colon cancer cells treated with retinol.

Retinol affects cell cycle progression. Treatment with 10 µmol/L retinol increased the percentage of cells in G₀-G₁ while decreasing the percentage of cells in S phase in the HCT-15, SW620, and WiDr cell lines (Fig. 6A, C, and D). Treatment with retinol decreased the percentage of HCT-15 cells in G₂-M, slightly increased the percentage of HCT-116 and SW620 cells in G₂-M, and notably increased the percentage of WiDr cells in G₂-M. In contrast, the percentage of HCT-116 cells in G₀-G₁ was not affected by retinol (Fig. 6B), but retinol decreased the percentage of HCT-116 cells in S phase. As can be seen in Fig. 1D, control HCT-116 cells continued to divide in a linear manner, whereas HCT-116 cells treated with 10 µmol/L retinol ceased to divide between 24 and 48 hours of treatment. This result, when considered in light of the absence of apoptosis, differentiation, and necrosis in the HCT-116 cell line despite strong growth inhibition by retinol, may indicate that retinol acts to slow the overall rate of cell division and increase the generation time of this cell line.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study shows that retinol inhibits the growth of both ATRA-sensitive and ATRA-resistant human colon cancer cell lines. We provide three lines of evidence that retinol is acting independent of the established ATRA/RAR/RARE retinoid signaling pathway. The first line of evidence indicates that retinol is not metabolized to bioactive compounds, such as ATRA, in two of the four cell lines examined. The remaining two cell lines synthesized only small amounts of ATRA from retinol. Second, we show that retinol does not activate RARE-mediated gene transcription. Finally, we present evidence that a RAR antagonist blocks the ability of ATRA to inhibit the growth of ATRA-sensitive HCT-15 cells, as expected, but does not block the ability of retinol to inhibit the growth of any cell line examined. The most surprising outcome of this study is that retinol is not acting through a RAR-dependent pathway in ATRA-sensitive HCT-15 cells. Therefore, even in the presence of functioning RAR, retinol does not inhibit cell growth by the actions of its metabolite ATRA, because this metabolite is not present in ATRA-sensitive HCT-15 cells (Fig. 2).

The ability of retinol to inhibit colon cancer cell growth is particularly interesting given that colon cancer cell lines produce little or no ATRA (Fig. 2). This finding is supported in a recent study by Jette et al. (26) that used Northern blot analysis to show that colon cancer cell lines, including HCT-116, lack retinol dehydrogenases 5 and L, and therefore the ability to synthesize ATRA. The metabolism of retinol by colon cancer cells was not examined in the study by Jette et al. (26). In contrast, our data shows that the HCT-116 cell line is capable of synthesizing very small amounts of ATRA from retinol (Fig. 2). This discrepancy is perhaps due to the ability of HPLC to detect extremely small amounts of [³H]retinoids compared with the relative lack of sensitivity of Northern blot analysis.

4-Oxoretinol and anhydroretinol are two naturally occurring retinoids capable of inhibiting cell growth. 4-Oxoretinol acts via RARs (13), much like ATRA, whereas anhydroretinol acts via a receptor-independent cytosolic mechanism to inhibit cell growth (27, 28). Neither compound was formed from retinol by any of the cell lines we examined (Fig. 2). We cannot eliminate the possibility that an unknown bioactive metabolite of retinol was formed that existed only briefly or was not detected by our HPLC protocol. However, the inability of retinol to induce CAT activity in cells transfected with a pRARE-CAT construct (Fig. 3) as well as the inability of a pan-RAR antagonist to block the effects of retinol on cell growth (Fig. 4) support the metabolism data.

We chose to use the pan-RAR antagonist, AGN 193109, to block RARs because this compound exhibits a high affinity for RAR (25). Although a genetic approach would have been more specific, the dominant-negative RAR construct available is activated by retinol (29), making it inappropriate for this study. Therefore, we included a positive control, showing that AGN 193109 blocks ATRA-induced growth inhibition in the HCT-15, ATRA-sensitive cell line (Fig. 3A), to indicate that AGN 193109 is functioning to block RAR-mediated growth inhibition.

Retinoids have been previously shown to inhibit cancer cell growth by increasing cellular differentiation, inducing apoptosis, or causing cell cycle arrest. With respect to colon cancer, retinoids tend to induce tumor apoptosis both in vitro (11, 30, 31) and in vivo (4, 32, 33). The cell lines examined in this study showed no apoptosis in response to retinol treatment (Figs. 5, left, and 6). In contrast, retinol induced G₀-G₁ arrest in three of the cell lines examined (Fig. 6). Although retinol failed to increase the percentage of cells in G₀-G₁ in the HCT-116 cell line, growth inhibition in these cells could be due to an overall increase in generation time because retinol does decrease cell growth (Fig. 1B). The differing responses between the HCT-116 cell line and the other three may reflect the heterogenicity of these cell lines, tumor stage (carcinoma versus adenoma), and presence or absence of various proteins in each cell line, e.g., adenomatous polyposis coli or p53.

Retinoids tend to induce cell cycle arrest by blocking the G₁-S phase transition (for review, see ref. 1). Unfortunately, the effect of retinoids on cell cycle regulatory proteins seems to be cell type specific (1). For example, in carcinogen-exposed immortalized human bronchial epithelial cells, ATRA-induced G₁ arrest is associated with decreased cyclin D1 protein levels due to ubiquitin-mediated degradation of cyclin D1 (34, 35). In contrast, ATRA-induced G₁ arrest in MCF-7 breast cancer cells is associated with decreased pRB phosphorylation, whereas cyclin D1, p21^WAF1/CIP1, cdk4, and cdk6 activity either does not change or decreases slightly, depending on the study (36–38).

This study shows that retinol is acting independent of the RAR to inhibit colon cancer cell growth. Previously, retinoids have been shown to exert their receptor-independent effects via interactions with protein kinase C (39), F-actin (40), c-Raf kinase (28), regulating mitochondrial membrane potential (41), generating reactive oxygen species (42), increasing intracellular ceramide levels (43), activating c-Jun NH₂-terminal kinase (44), inducing ubiquitin-dependent proteolysis (34, 35), and affecting mitogen-activated protein kinase (45, 46), phosphatidylinositol 3-kinase (PI3K)/Akt (47), and epidermal growth factor receptor signaling (48). The Hammerling lab has shown that the retroretinoids, retinol, and ATRA can bind protein kinase C and affect its redox activation (39). In contrast to our present study, they speculate that retinol antagonizes anhydroretinol and increases cell survival by binding to c-Raf and augmenting its response to reactive oxygen species generated during UV irradiation; however, the link between cell growth and c-Raf activation was not directly examined (28). Because anhydroretinol induces apoptosis, we do not expect retinol to affect cell growth by interacting with c-Raf or any of the other pathways listed above that induce apoptosis.

In conclusion, this study shows that retinol acts through a novel mechanism to inhibit the growth of both ATRA-sensitive and ATRA-resistant colon cancer cells by affecting cell cycle progression. Resistance to ATRA is a common phenomenon and limits the use of retinoic acid derivatives as chemotherapy. We speculate that retinol, or a derivative of it, may prove an effective therapy to treat colorectal cancer.

	Acknowledgments

 
Grant support: American Cancer Society grant RSG-03-233-01-CNE.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Dr. Lorraine Gudas (Department of Pharmacology, Weill Medical College of Cornell University, New York, NY) for the gift of the 4-oxoretinol standards, Dr. Ulrich Hammerling (Immunology Program, Memorial Sloan Kettering Cancer Center, New York, NY) for providing the anhydroretinol standard, Dr. Dianne Soprano (Temple University, Philadelphia, PA) for the pRARE-CAT construct, and Dr. Rosh Chandraratna (formerly at Allergan Pharmaceuticals, Irvine, CA; currently at Vitae Pharmaceuticals, Irvine, CA) for the RAR-pan antagonist, AGN 193109.

Received 5/10/05. Revised 7/29/05. Accepted 8/16/05.

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