Chemoprevention of colon carcinogenesis by polyethylene glycol: suppression of epithelial proliferation via modulation of SNAIL/β-catenin signaling

Abstract

Polyethylene glycol (PEG) is one of the most potent chemopreventive agents against colorectal cancer; however, the mechanisms remain largely unexplored. In this study, we assessed the ability of PEG to target cyclin D1 -B-catenin -mediated hyperproliferation in the azoxymethane-treated rat model and the human colorectal cancer cell line, HT-29. Azoxymethane-treated rats were randomized to AIN-76A diet alone or supplemented with 5% PEG-8000. After 30 weeks, animals were euthanized and biopsies of aberrant crypt foci and uninvolved crypts were subjected to immunohistochemical and immunoblot analyses. PEG markedly suppressed both early and late markers of azoxymethane-induced colon carcinogenesis (fractal dimension by 80%, aberrant crypt foci by 64%, and tumors by 74%). In both azoxymethane-treated rats and HT-29 cells treated with 5% PEG-3350 for 24 hours, PEG decreased proliferation (45% and 52%, respectively) and cyclin D1 (78% and 56%, respectively). Because B-catenin is the major regulator of cyclin D1 in colorectal cancer, we used the T-cell factor (Tcf) -TOPFLASH reporter assay to show that PEG markedly inhibited B-catenin transcriptional activity. PEG did not alter total B-catenin expression but rather its nuclear localization, leading us to assess E-cadherin expression (a major determinant of B-catenin subcellular localization), which was increased by 73% and 71% in the azoxymethane-rat and HT-29 cells, respectively. We therefore investigated the effect of PEG treatment on levels of the negative regulator of E-cadherin, SNAIL, and observed a 50% and 75% decrease, respectively. In conclusion, we show, for the first time, a molecular mechanism through which PEG imparts its antiproliferative and hence profound chemopreventive effect.

Involvement of p21cip1/waf1 in the anti-proliferative effects of polyethylene glycol in colon carcinogenesis

“…As shown (Fig. 1) and consistent with our earlier report (12), AOM resulted in increased colonic epithelial proliferation compared to saline injected rats, as measured by epithelial BrdUrd incorporation. On the other hand PEG supplementation markedly reduced the BrdUrd incorporation in the AOM rats (by ~70%).…”

“…The efficacy of PEG was truly remarkable, resulting in a 90% reduction in tumors. These findings were corroborated by our group (12). To keep these results in perspective, PEG outperformed all other chemopreventive agents including well established agents such as NSAIDs.…”

“…To complement the effect of PEG in cell cycle arrest, we studied the effect of PEG on the proliferative index, as measured by PCNA incorporation, and found that PEG suppressed the cellular proliferation by about 80%. As we found that PEG decreased cellular proliferation and induced G 1 arrest in the cell cycle and inhibited cyclin D1 (our previous data) (12), Western blot analysis was performed to determine whether treatment of HT-29 cells with PEG alters cellular levels of cell cycle protein inhibitor Figure 3. PEG modulates p21 expression in HT-29.…”

“…Our group has previously shown that PEG treatment suppresses epithelial proliferation in the wellvalidated AOM-treated rat model (12). Because p21 cip1/waf1 has been shown to be an important regulator of cell cycle which is lost in early colon carcinogenesis (20), we assessed the effect of PEG treatment on this cell cycle regulator in the AOM-treated rat model.…”

“…Thus, novel agents with greater efficacy and lower toxicity profile are urgently warranted in any significant clinical setting. Polyethylene glycol (PEG) has recently received attention as a promising chemopreventive agent of a superior capacity with potent anti-proliferative properties in both cell culture and animal models of colon carcinogenesis (12)(13)(14). This promising effect has also been corroborated in a pilot clinical (case-control) study (15).…”