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-3 fatty acid–mediated inhibition of colon carcinogenesis
Address correspondence to Alan P. Fields, Sealy Center for Cancer Cell Biology, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-1048. Tel.: (409) 747-1935. Fax: (409) 747-1938. E-mail: afields{at}utmb.edu
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Abstract |
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 Top Abstract IntroductionResults and discussionMaterials and methodsReferences |
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-3 fatty acids inhibit colonic PKCßII activity in vivo and block PKCßII-mediated hyperproliferation, enhanced carcinogenesis, and repression of TGFßRII expression in the colonic epithelium of transgenic PKCßII mice. These data indicate that dietary -3 fatty acids prevent colon cancer, at least in part, through inhibition of colonic PKCßII signaling and restoration of TGF-ß responsiveness.
Key Words: protein kinase C; colon carcinogenesis; -3 fatty acids; transforming growth factor ß; hyperproliferation
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Introduction |
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TopAbstractIntroductionResults and discussionMaterials and methodsReferences |
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-3 polyunsaturated fatty acids, particularly eicosapentaenoic acid (c20:5, n-3) and docosahexaenoic acid (c22:6, n-3), is associated with reduced colon cancer incidence (Bang et al., 1976; Caygill and Hill, 1995; Caygill et al., 1996). Biochemical studies confirm that -3 fatty acids have potent chemopreventive effects on carcinogen-induced colon cancer in rodents (Deschner et al., 1990; Reddy et al., 1991; Chang et al., 1997). Despite the compelling epidemiological and biochemical evidence demonstrating the efficacy of -3 fatty acids in colon cancer chemoprevention (Rose and Connolly, 1999), little is known about how these dietary lipids mediate their protective effects. -3 fatty acids can inhibit the colonic epithelial hyperproliferation observed during colon carcinogenesis, suggesting that proliferative signaling is an important target of their chemopreventive effects (Anti et al., 1994, 1997; Latham et al., 1999).
Elevated colonic protein kinase C ßII (PKCßII)* expression and activity correlate with colon carcinogenesis in both rodents and humans (Murray et al., 1999; Gokmen-Polar et al., 2001). Elevated PKCßII expression in the colonic epithelium of transgenic mice induces hyperproliferation and enhances susceptibility to azoxymethane (AOM)-induced colon carcinogenesis (Murray et al., 1999), demonstrating a direct role for PKCßII in colon carcinogenesis. Based on these data, we hypothesized that -3 fatty acids block colon carcinogenesis by interfering with proliferation via inhibition of PKCßII signaling. To test this hypothesis, we evaluated the effect of a diet high in -3 fatty acids on colonic PKCßII activity and signaling. Dietary -3 fatty acids inhibited PKCßII activity and suppressed PKCßII-mediated hyperproliferation and colon carcinogenesis in vivo. In addition, our data demonstrate that PKCßII represses transforming growth factor ß receptor type II (TGFßRII) expression in vitro and in vivo, and that this repression is reversed by dietary -3 fatty acids.
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Results and discussion |
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TopAbstractIntroductionResults and discussionMaterials and methodsReferences |
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-3 fatty acids (Holian and Nelson, 1992; Davidson et al., 2000; Seung Kim et al., 2001). Therefore, we assessed the relationship between colonic PKCßII activity and dietary -3 fatty acids during AOM-induced colon carcinogenesis in vivo. Cytosolic and membrane extracts from colonic epithelia of male Sprague-Dawley rats fed an -6 fatty acid diet (15% maize oil by weight) or -3 fatty acid diet (3.5% maize oil, 11.5% fish oil) were subjected to immunoblot analysis for PKCßII (Fig. 1). A significant decrease in the level of membrane-associated PKCßII was observed in the colonic epithelium of animals fed -3 fatty acids, whereas cytosolic PKCßII levels were similar in both diet groups. The membrane association of conventional PKC isozymes, including PKCßII, is widely accepted as an indirect measure of PKC enzyme activation in vivo, because it is not technically feasible to directly determine the activity of the enzyme in situ. Our results are consistent with the direct inhibition of PKCßII activity by -3 fatty acids in vitro (Holian and Nelson, 1992).
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-3 fatty acids on AOM-mediated carcinogenesis in transgenic PKCßII mice. Transgenic PKCßII mice fed a diet rich in -6 fatty acids exhibited enhanced colon carcinogenesis, as evidenced by increased numbers of preneoplastic lesions, aberrant crypt foci (ACF) (Fig. 2 A). In contrast, a diet rich in -3 fatty acids inhibited the enhanced colon carcinogenesis characteristic of transgenic PKCßII mice. Thus, dietary -3 fatty acids block PKCßII activation and attenuate the procarcinogenic effects of PKCßII in the colon in vivo.
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-3 fatty acids suppress carcinogen-induced hyperproliferation (Latham et al., 1999). Therefore, we assessed the effect of dietary -3 fatty acids on the hyperproliferative phenotype of transgenic PKCßII mice (Fig. 2 B). Whereas transgenic PKCßII mice exhibited hyperproliferation when fed an -6 fatty acid diet, an -3 fatty acid diet selectively suppressed this hyperproliferative phenotype (Fig. 2 B). These data demonstrate that dietary -3 fatty acids suppress the hyperproliferative phenotype observed in transgenic PKCßII mice and provide a plausible mechanism for the chemopreventive effects of dietary -3 fatty acids on PKCßII-mediated colon carcinogenesis. To gain insight into how PKCßII enhances colon carcinogenesis, we established rat intestinal epithelial (RIE)-1 cells that stably express PKCßII (Fig. 3). Parental RIE-1 cells, which, like most cells of the intestinal epithelium, express little or no PKCßII, were infected with a retrovirus containing the full-length human PKCßII cDNA (Fig. 3 A). Comparative gene microarray analysis of RIE-1 and RIE/PKCßII cells was used to identify potential targets for PKCßII. Among the genes identified through this analysis, TGFßRII expression was found to be markedly repressed in RIE/PKCßII cells, when compared with RIE-1 cells. These results were confirmed by Western blotting using a specific antibody to TGFßRII (Fig. 3 A). Given the importance of TGF-ß signaling in colonic epithelial cell proliferation and differentiation, and the frequent loss of TGF-ß responsiveness during colon carcinogenesis (Markowitz and Roberts, 1996), we assessed whether this change in TGFßRII expression leads to an alteration in the well-documented response of the RIE-1 cells to TGF-ß (Ko et al., 1995). RIE-1 and RIE/PKCßII cells were transfected with a reporter plasmid containing a TGF-ß–responsive element from the tissue plasminogen activator promoter linked to luciferase (Wrana et al., 1992). RIE-1 cells respond to TGF-ß by activation of this TGF-ß–dependent reporter construct (Fig. 3 B). However, RIE/PKCßII cells showed a diminished transcriptional response to TGF-ß consistent with repression of TGFßRII expression. Cotransfection of the TGF-ß reporter plasmid with a plasmid containing R4TD202, a constitutively active mutant of TGFßRI (Feng and Derynck, 1996), resulted in comparable increases in transcriptional activity in both RIE-1 and RIE/PKCßII cells (Fig. 3 B), indicating that signaling downstream of the TGF-ß receptor is intact in both RIE-1 and RIE/PKCßII cells. Consistent with the transcriptional response to TGF-ß, RIE-1 cell proliferation was inhibited by TGF-ß as determined by a reduction in the number of bromodeoxyuridine (BrdU)-labeled cells after addition of TGF-ß1 to mid-log phase cultures (Fig. 3 C). RIE/PKCßII cells, however, were relatively insensitive to TGF-ß inhibition of BrdU labeling (Fig. 3 C). Although there was no detectable growth phenotype associated with expression of PKCßII in the absence of added TGF-ß (Fig. 3 D), RIE/PKCßII cells were much less sensitive to TGF-ß–mediated inhibition of proliferation (Fig. 3 E). Thus, expression of PKCßII leads to repression of TGFßRII and loss of TGF-ß responsiveness in RIE-1 cells.
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40 nM LY379196, consistent with the reported IC50 of LY379196 for PKCßII of 30 nM (Jirousek et al., 1996). It should also be noted that re-expression of TGFßRII is accompanied by reduced electrophoretic mobility of TGFßRII upon SDS-PAGE. Because TGFßRII is highly glycosylated, it is possible that this shift reflects changes in posttranslational processing of the protein. The significance of this observation to TGF-ß–mediated signaling is unclear. Nevertheless, these data provide direct evidence that PKCßII activity is responsible for repression of TGFßRII in these cells.
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-6 fatty acid diet were subjected to immunoblot (Fig. 5 A) and immunofluorescence analysis (Fig. 5 B, 1 and 4) using a specific TGFßRII antibody. Extracts from nontransgenic mice contained little PKCßII, but abundant TGFßRII, whereas those from transgenic PKCßII mice contained elevated PKCßII and a reduced level of TGFßRII (Fig. 5 A). Immunofluorescence analysis for TGFßRII confirmed these immunoblotting results (Fig. 5 B). Colonic crypts from nontransgenic mice fed an -6 fatty acid diet (Fig. 5 B, 4) exhibited abundant staining for TGFßRII. Staining was strongest in the upper third and along the luminal surfaces of the colonic crypts, consistent with the reported distribution of TGFßRII in the colonic epithelium (Guda et al., 2001). In contrast, colonic crypts from transgenic PKCßII mice fed an -6 fatty acid diet (Fig. 5 B, 1) expressed much lower levels of TGFßRII, confirming that TGFßRII expression was repressed by PKCßII in the colonic epithelium in vivo.
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-3 fatty acids inhibit PKCßII signaling, we predicted that dietary -3 fatty acids would restore TGFßRII expression in transgenic PKCßII mice. Indeed, transgenic PKCßII mice fed an -3 fatty acid diet showed levels of TGFßRII comparable to those seen in nontransgenic mice (Fig. 5 B, 2). Therefore, TGFßRII is a critical gene target of PKCßII-mediated repression in RIE-1 cells in vitro and in the colonic epithelium in vivo. Our data are consistent with the conclusion that PKCßII down-regulates TGFßRII expression, thereby inducing a TGF-ß–insensitive state. Because TGF-ß inhibits proliferation and promotes differentiation of intestinal epithelial cells (Lamprecht et al., 1989), PKCßII-mediated repression of TGFßRII imposes a hyperproliferative state that increases sensitivity to carcinogens, such as AOM. Dietary -3 fatty acids inhibit PKCßII activity and restore TGFßRII expression in the colonic epithelium of transgenic PKCßII mice, resulting in reversal of the hyperproliferative phenotype and attenuation of the enhanced colon carcinogenesis characteristic of transgenic PKCßII mice.
Our results provide a plausible link between chemopreventive dietary -3 fatty acids, colonic PKCßII activity, TGF-ß signaling, cellular proliferation, and susceptibility to colon cancer. Therefore colonic PKCßII, which is induced early during colon carcinogenesis, represents a novel and potentially highly effective target for chemopreventive therapy in colon cancer. Given the strong association of dietary -3 fatty acids with the chemoprevention of other epithelial cancers, including breast and prostate cancer (Rose and Connolly, 1999), as well as neurological conditions such as bipolar disorder (Seung Kim et al., 2001), it is possible that selective PKCßII inhibition could prove to be an important therapeutic modality in the treatment and chemoprevention of multiple epithelial cancers and central nervous system disorders. Likewise, because repression of TGFßRII expression has been documented in many cancer cell types, including gastric cancer, colon cancer, small cell lung cancer, esophageal cancer, hepatocellular carcinoma, squamous cell carcinoma, breast cancer, endometrial cancer, bladder cancer, and osteosarcoma (for review see Kim et al., 2000), it is possible that PKCßII inhibition, either through dietary modulation or pharmacological intervention, may be of therapeutic value for a broad range of major cancer types by restoring TGF-ß responsiveness in these tumors.
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Materials and methods |
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TopAbstractIntroductionResults and discussionMaterials and methodsReferences |
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Cell fractionation and PKCßII immunoblot analysis
3-wk-old Sprague-Dawley rats fed either an -6 fatty acid diet (15% maize oil by weight) or an -3 fatty acid diet (3.5% maize oil, 11.5% fish oil, by weight; Chang et al., 1997). After 1 wk on experimental diets, rats were injected with AOM (Chang et al., 1997). Animals were terminated at 16 wk after AOM injection. Colonic epithelial cell extracts from the distal colon were fractionated into membrane and cytosolic fractions and subjected to immunoblot analysis for PKCßII expression (Davidson et al., 2000). Band intensity was quantitated using the Bio-Rad Laboratories Fluor-s imaging system.
ACF analysis
6–8-wk-old transgenic PKCßII mice and nontransgenic littermates were maintained on either an -6 or -3 fatty acid diet (ad libitum) until termination of the experiment. 1 wk after introduction of the diet, mice received intraperitoneal injections of 10 mg/kg AOM 1x/wk for 4 wk. Mice were terminated 12 wk after the final AOM injection and the colons were analyzed for ACF formation (McLellan et al., 1991; Murray et al., 1999).
Colonic epithelial cell proliferation analysis
6–8-wk-old transgenic PKCßII mice and nontransgenic littermates were maintained on an -6 or -3 fatty acid diet for 18 d. 1 h before sacrifice, mice were injected with 50 mg/kg BrdU. Distal colon was isolated, fixed in 4% paraformaldehyde, and analyzed for proliferation as determined by BrdU labeling (Chang et al., 1997).
TGF-ß transcriptional response assay
RIE-1 and RIE/PKCßII cells were transiently transfected with 0.4 µg of 3TP/luc TGF-ß reporter plasmid (Wrana et al., 1992) plus 1 µg of vector expressing renilla luciferase under control of the thymidine kinase promoter as an internal control for transfection efficiency. TGF-ß1 (3 ng/ml) was added to cells. After 24 h of TGF-ß exposure, cellular extracts were prepared and assayed for firefly and renilla luciferase activity. Data are presented as firefly luciferase corrected for the level of renilla luciferase. Some cells were cotransfected with 0.1 µg of an expression vector encoding constitutively active TGF-ß chimeric receptor R4TD202 (Feng and Derynck, 1996) and assayed for TGF-ß reporter activity as described above.
Effect of PKCßII on RIE-1 cell proliferation and TGF-ß responsiveness
TGF-ß responsiveness of RIE-1 and RIE/PKCßII cells was assayed by measuring inhibition of BrdU incorporation. Midlog phase cells were treated with 3 ng/ml TGF-ß for 24 h followed by 1 mM BrdU for 30 min. Cells were then permeabilized and stained with anti-BrdU antibody using the BD PharMingen BrdU FlowKit®, according to the manufacturer's recommendations. Approximately 15,000 cells from each culture were analyzed using a Becton Dickinson flow cytometer. RIE-1 and RIE/PKCßII cells were plated at 105 cells per 6-cm dish. At 24-h intervals, three dishes from each line were harvested by trypsinization and the cells were counted using a hemocytometer. RIE-1 and RIE/PKCßII cells were plated in 96-well plates at 2 x 104 cells in 0.2 ml of medium ± 3 ng/ml TGF-ß1. At 24-h intervals, cell growth was assayed in triplicate wells by measuring OD570 after reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT).
Analysis of TGF-ß RII expression
Equal amounts of cell lysates from RIE-1 and RIE/PKCßII cells were subjected to immunoblot analysis using antibody specific for TGFßRII, PKCßII, or actin (Santa Cruz Biotechnology, Inc.). PKCßII transgenic mice and nontransgenic littermates were killed, the colonic epithelia were scraped, and 150 µg of the resulting protein extract was subjected to immunoblot analysis using the same antibodies listed above. Immunoblot images were captured using a Stratagene Eagle Eye imager. To assess the effect of PKCßII inhibition on TGFßRII protein expression, confluent cultures of RIE/PKCßII cells were placed in medium containing 0.2% FBS plus the concentrations of LY379196 indicated in the figure legend. All cultures contained 0.1% DMSO as a diluent control. After 2 d, total cellular protein was extracted and 50 µg of protein per lane was subjected to immunoblot analysis for TGFßRII expression. Chemiluminescent exposure was adjusted to conform to a linear response range, and data were quantified using a Lynx digital image processor.
Immunofluorescence analysis of TGFßRII expression
6-wk-old PKCßII transgenic mice and nontransgenic littermates were fed either an -3– or -6–containing diet ad libitum. After 18 d on the defined diets, the mice were killed. Distal colon was isolated, fixed in 4% paraformaldehyde, sectioned, and subjected to immunofluorescence analysis for TGFßRII expression. Images were captured using a CCD color digital camera and digital image capture software.
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Footnotes |
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Acknowledgments |
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This work was supported by National Institutes of Health grants CA81436 (A.P. Fields), CA24347 (E.A. Thompson), and CA59034 (R.S. Chapkin).
Submitted: 30 January 2002
Revised: 23 April 2002
Accepted: 24 April 2002
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