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. 2012 Sep;33(9):1726-35.
doi: 10.1093/carcin/bgs174. Epub 2012 May 11.

Chemopreventive sphingadienes downregulate Wnt signaling via a PP2A/Akt/GSK3β pathway in colon cancer

Ashok Kumar  1 , Ashok K Pandurangan, Fang Lu, Henrik Fyrst, Meng Zhang, Hoe-Sup Byun, Robert Bittman, Julie D Saba
Affiliations

Chemopreventive sphingadienes downregulate Wnt signaling via a PP2A/Akt/GSK3β pathway in colon cancer

Ashok Kumar et al. Carcinogenesis. 2012 Sep.

Abstract

Sphingadienes (SDs) derived from soy and other natural sphingolipids are cytotoxic to colon cancer cells via an Akt-dependent mechanism and reduce adenoma formation in Apc(Min/+) mice. Wnt signaling is fundamental to colon carcinogenesis and is the basis for spontaneous tumorigenesis in Apc(Min/+) mice and patients with familial adenomatous polyposis. In the present study, we investigated the impact of SDs on Wnt signaling. Oral SD administration reduced levels of active β-catenin and Wnt targets c-Myc and cyclin D1 in Apc(Min/+) mouse intestinal tissues. Colon cancer cells treated with SDs exhibited reduced Wnt transcriptional activity, as well as reduced nuclear β-catenin localization and subsequent reduction in active-β-catenin levels. Further, we observed a decrease in phosphorylated (inactive) GSK3β in SD-treated mice and colon cancer cells. Expression of constitutively active myristoylated-Akt or inactivation of GSK3β using LiCl attenuated SD-mediated inhibition of Wnt transcriptional activity and active-β-catenin levels. SDs exhibited additive effects with inhibitors of the phosphatidylinositol-3-kinase/Akt/mTOR pathway to induce cytotoxicity. Further, a combination regime of SDs and low-dose rapamycin decreased visible polyps in Apc(Min/+) mice and reduced the levels of Wnt target gene expression and mTOR target activation. SD-mediated inhibition of Akt and Wnt pathways and cytotoxicity in colon cancer cells was dependent upon the activity of protein phosphatase 2A, as shown by reversal of these effects by pretreatment with okadaic acid or calyculin A. Our cumulative findings indicate that SDs inhibit Wnt signaling through a protein phosphatase 2A/Akt/GSK3β-dependent mechanism that may contribute to their chemopreventive effects in intestinal tumorigenesis.

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Figures

Fig. 1.
Fig. 1.
SDs reduces Wnt signaling in Apc Min/+ intestines and downregulate Wnt transcriptional activity in colon cancer cells. (A–D) Apc Min/+ mice were administered 25mg/kg body weight/day SDs or vehicle (n = six/group). Mice were killed and intestinal tissues harvested on day 10. (A) Intestinal levels of activated β-catenin (Non-P-β-catenin), total β-catenin, c-Myc and cyclin D1 were measured in extracts prepared from non-tumor tissue by immunoblotting. (C) Microadenomas were removed from frozen sections of intestine by laser capture microdissection and polyp free sections were lysed in tissue homogenization buffer and subjected to immunoblotting as in panel A. (B and D) Quantification of blots were done by ImageJ software. Densitometric analysis for non-P-β-catenin, c-Myc, and cyclin D1 is shown as ratio to actin levels. Values are the average ± standard deviation. *P < 0.05 control versus SDs; **P < 0.01 control versus SDs. (E) SW480 and (F) DLD1 colon cancer cells were transfected with the TOPflash or FOPflash TCF/LEF reporter plus SV40 Renilla plasmid. Transfected cells were incubated with 20 µM SDs for 0–24h. Cells were then harvested and TCF/LEF reporter activity was measured. Data are presented as ratio of firefly luciferase over Renilla luciferase. Results are shown as average values ± SDEV of at least three independent experiments. P < 0.001 vehicle versus indicated time points.
Fig. 2.
Fig. 2.
SDs reduce active β-catenin and downstream targets of Wnt signaling in colon cancer cells. (A) HT29 and (B) SW480 cells were incubated with vehicle or 5–30 μM SDs for 3h. HT29 (C) and SW480 (D) cells were treated with vehicle or 20 µM SDs for the indicated times. Cells were harvested, and protein levels of non-P and total β-catenin, c-Myc, and cyclin D1 were evaluated by immunoblotting. Actin and GAPDH are loading controls.
Fig. 3.
Fig. 3.
SDs reduce nuclear localization of β-catenin in colon cancer cells. SW480 cells were grown in eight-chamber slides for 48h, and then were treated with vehicle or SDs for indicated times. Cells were fixed, permeablized, and stained with antibodies against non-P-β-catenin (A) or total β-catenin (B). Cells were counterstained with 4′,6-diamidino-2-phenylindole and images acquired using a fluorescence microscope with a 40× objective lens.
Fig. 4.
Fig. 4.
Suppression of Wnt signaling by SDs is mediated in an Akt- and GSK3β-dependent manner. Apc Min/+ mice were administered 25mg/kg body weight/day SDs or vehicle (n = six/group). Mice were killed and intestinal tissues harvested on day 10. Intestinal levels of phospho-GSK3β, total GSK3β, and actin were measured in extracts prepared from non-tumor tissue by immunoblotting (A) and quantification (B) by ImageJ software. Phospho-GSK3β levels are shown as a ratio to total GSK3β levels. *P < 0.05 control versus SDs. (C) HT29 cells were incubated with vehicle or 5–30 μM SDs for 3h. Cells were harvested, and protein levels of phospho-GSK3β, total GSK3β, phospho-Akt, and total Akt were evaluated by immunoblotting. Actin and GAPDH are loading controls. (D) DLD1 cells were transfected with green fluorescent protein or myristoylated-Akt (Myr-Akt) along with TOPflash TCF/LEF and Renilla luciferase reporter plasmids. After 48h, cells were treated 
with 20 μM SDs for 3h or left untreated. TCF/LEF and Renilla reporter activities were measured and shown as average of firefly luciferase/
Renilla luciferase activity ± SDEV from three independent experiments *P < 0.05 compared with green fluorescent protein+SD. (E) DLD1 and SW480 cells were treated with vehicle or 20 μM SDs in the presence or absence of LiCl (20mM) for 3h. Cells were harvested and protein levels of non-P-β-catenin, total β-catenin, and actin were determined by immunoblotting.
Fig. 5.
Fig. 5.
SDs exhibit additive effect with inhibitors of PI3K/mTOR pathway to inhibit intestinal tumorigenesis. Apc Min/+ mice were administered vehicle, 25mg/kg body weight/day SDs, 2.5mg/kg body weight/day rapamycin, or both (SDs + rapamycin; n = five/group). Mice were killed and intestinal tissues harvested on day 10. (A) Gross polyps were counted in the small intestines of Apc Min/+ mice. *P < 0.05 control versus SDs or control versus rapamycin; **P < 0.001 SDs + rapamycin versus control. (B–E) Intestinal levels of activated β-catenin (non-P-β-catenin), total β-catenin, c-Myc, and cyclin D1, phospho-p70 S6 kinase, and phospho-4EBP1 were measured in extracts prepared from non-tumor tissue by immunoblotting. (C and E) ratios of non-P-β-catenin, c-Myc, cyclin D1, phospho-p70 S6 kinase, and phospho-4EBP1 to actin were quantitated by densitometric analysis (n = 3) *P < 0.05 as compared with control; **P < 0.01 as compared with control.
Fig. 6.
Fig. 6.
SDs inhibit Akt and Wnt signaling in a PP2A-dependent manner. (A, B) SW480 cells were grown to 70% confluency, and then cells were serum starved for 20h. Cells were treated with a PP2A inhibitor, 100nM okadaic acid (OA) or 5 µM myrisolated PKCζ peptide inhibitor and 20 μM C18 (4,8) SDs alone or in combination. Then, cells were treated with 10nM IGF1 for 30min. Cell lysates were immunoblotted (A) with indicated antibodies and densitometric analysis (B) was shown for lanes treated with IGF1. (C) HT29 cells were transfected with the TOPflash TCF/LEF reporter plus SV40 Renilla plasmid. Transfected cells were preincubated with 50nM Okadaic acid (OA) in DMEM containing 2% FBS for 3h and treated 20 µM SDs for 3h. Cells were then harvested and TCF/LEF reporter activity was measured. Data are presented as ratio of firefly luciferase over Renilla luciferase. Results are shown as average values ± SDEV of at least three independent experiments. *P < 0.05 SD versus OA+SD. (D) DLD1 cells were grown in complete media to 70% confluency, then cells switched to DMEM media containing 2% FBS and pretreated with 10nM Okadaic acid (OA) and 1nM Calyculin A (Cal A) and cells were treated with 20 µM SD for 24h. Cell growth was determined by MTT assay. **P < 0.01 control versus SD, *P < 0.05 SD versus SD + OA or SD versus Cal A + SD.
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