Protein kinase C betaII and TGFbetaRII in omega-3 fatty acid-mediated inhibition of colon carcinogenesis - PubMed (original) (raw)

Protein kinase C betaII and TGFbetaRII in omega-3 fatty acid-mediated inhibition of colon carcinogenesis

Nicole R Murray et al. J Cell Biol. 2002.

Abstract

Increasing evidence demonstrates that protein kinase C betaII (PKCbetaII) promotes colon carcinogenesis. We previously reported that colonic PKCbetaII is induced during colon carcinogenesis in rodents and humans, and that elevated expression of PKCbetaII in the colon of transgenic mice enhances colon carcinogenesis. Here, we demonstrate that PKCbetaII represses transforming growth factor beta receptor type II (TGFbetaRII) expression and reduces sensitivity to TGF-beta-mediated growth inhibition in intestinal epithelial cells. Transgenic PKCbetaII mice exhibit hyperproliferation, enhanced colon carcinogenesis, and marked repression of TGFbetaRII expression. Chemopreventive dietary omega-3 fatty acids inhibit colonic PKCbetaII activity in vivo and block PKCbetaII-mediated hyperproliferation, enhanced carcinogenesis, and repression of TGFbetaRII expression in the colonic epithelium of transgenic PKCbetaII mice. These data indicate that dietary omega-3 fatty acids prevent colon cancer, at least in part, through inhibition of colonic PKCbetaII signaling and restoration of TGF-beta responsiveness.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

Dietary ω-3 fatty acids block AOM-induced PKCβII activity. PKCβII expression was assessed in membrane and cytosolic fractions from colonic epithelial cell extracts from the distal colon of AOM-treated Sprague-Dawley rats fed either an ω-6 or an ω-3 fatty acid diet. Values represent means ± SEM, n = 5.

Figure 2.

Figure 2.

Dietary ω-3 fatty acids block PKCβII-enhanced colon carcinogenesis and colonic hyperproliferation. (A) Effect of dietary fat intake on AOM-induced ACF formation in transgenic PKCβII mice. Mice were terminated 12 wk after the final AOM injection and the colons analyzed for ACF formation as described previously (McLellan et al., 1991; Murray et al., 1999). Values represent means ± SEM, n = 6–18 animals/experimental group. (B) Mice were fed an ω-6 or ω-3 diet for 18 d. 1 h before sacrifice, mice were injected with 50 mg/kg BrdU and distal colon was isolated, fixed in 4% paraformaldehyde, and analyzed for proliferation as determined by BrdU labeling (Chang et al., 1997). Results respresent proliferative index relative to nontransgenic controls. Values represent means ± SEM, n = 4–5 animals/experimental group.

Figure 3.

Figure 3.

Characterization of TGF-β signaling in RIE/PKCβII cells. (A) Expression of PKCβII, TGFβRII, and actin in RIE-1 and RIE/PKCβII cells. RIE-1 cells were infected with pBABEpuro containing full-length human PKCβII or empty pBABEpuro vector. Puromycin-resistant cell populations were isolated and screened for expression of PKCβII, TGFβRII, and actin by immunoblotting. (B) Effect of PKCβII expression on TGF-β–induced transcription in RIE-1 cells. RIE-1 and RIE/PKCβII cells were transiently transfected with 0.4 μg of 3TP/luc TGF-β reporter plasmid plus 1 μg of TK/renilla as an internal control for transfection efficiency. TGF-β1 (3 ng/ml) was added to cells as indicated. Cellular extracts were prepared after 24 h of TGF-β exposure and assayed for firefly and renilla luciferase activity. In addition, some cells were cotransfected with an expression vector encoding constitutively active TGF-β chimeric receptor R4TD202. Data represent the mean ± SD from three independent experiments measuring firefly luciferase activity normalized to renilla luciferase activity. (C) Effect of PKCβII expression on TGF-β–mediated inhibition of BrdU incorporation in RIE-1 cells. TGF-β responsiveness of RIE-1 and RIE/PKCβII cells was assayed by measuring inhibition of BrdU incorporation. (D) Effect of PKCβII expression on RIE-1 cell proliferation. Growth of RIE-1 and RIE/PKCβII cells in growth medium in the absence of TGF-β was measured by hemocytometer count on a daily basis. Data represent the mean ± SD from three dishes. (E) Effect of PKCβII expression on TGF-β–mediated inhibition of RIE-1 cell proliferation. Cultures of RIE-1 and RIE/PKCβII cells were treated with 3 ng/ml TGF-β1, and cell growth was monitored by measuring OD570 after reduction of MTT. Data represent the mean ± SD from three wells.

Figure 4.

Figure 4.

The selective PKCβ inhibitor LY379196 restores TGFβRII expression in RIE/PKCβII cells. (A) RIE/PKCβII cells were treated with the indicated concentration of LY379196 for 48 h, and total cellular protein extracts were subjected to immunoblot analysis for TGFβRII expression. (B) Quantitative analysis of the immunoblot data shown in A.

Figure 5.

Figure 5.

ω-3 fatty acids restore colonic TGFβRII expression in PKCβII mice. (A) Immunoblot analysis of TGFβRII expression. PKCβII 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 antibody specific for TGFβRII, PKCβII, or actin (Santa Cruz Biotechnology, Inc.). (B) Immunofluorescence analysis of TGFβRII expression. PKCβII transgenic mice (1, 2, and 3) and nontransgenic littermates (4, 5, and 6) were fed (ad libitum) either an ω-3– (2, 3, 5, and 6) or ω-6– (1 and 4) containing diet beginning at 6 wk of age. After 18 d, mice were killed and distal colons were subjected to immunofluorescence analysis using a specific antibody to TGFβRII (1,2, 4, and 5) or secondary antibody only (3 and 6). All panels are at 400× magnification.

References

    1. Anti, M., F. Armelao, G. Marra, A. Percesepe, G.M. Bartoli, P. Palozza, P. Parrella, C. Canetta, N. Gentiloni, I. De Vitris, and G. Gasbarrini. 1994. Effects of different doses of fish oil on rectal cell proliferation in patients with sporadic colonic adenomas. Gastroenterology. 107:1709–1718. - PubMed
    1. Anti, M., G. Marra, F. Arlemao, A. Percesepe, and N. Gentiloni. 1997. Modulating effect of omega-3 fatty acids on the proliferative pattern of human colorectal mucosa. Adv. Exp. Med. Biol. 400B:605–610. - PubMed
    1. Bang, H.O., J. Dyerberg, and N. Hjorne. 1976. The composition of food consumed by Greenland Eskimos. Acta. Med. Scand. 200:69–73. - PubMed
    1. Bartsch, H., J. Nair, and R.W. Owen. 1999. Dietary polyunsaturated fatty acids and cancers of the breast and colorectum: emerging evidence for their role as risk modifiers. Carcinogenesis. 20:2209–2218. - PubMed
    1. Caygill, C.P.J., and M.J. Hill. 1995. Fish, n-3 fatty acids and human colorectal and breast cancer mortality. Eur. J. Cancer Prev. 4:329–332. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources