Antiproliferative effect of sulindac in colonic neoplasia prevention: role of COOH-terminal Src kinase

Abstract

Although the nonsteroidal anti-inflammatory drugs (NSAID) protection against colorectal cancer is well established, the molecular mechanisms remain unclear. We show herein that induction of the tumor suppressor gene COOH-terminal Src kinase (Csk) by NSAID is important for their antiproliferative and hence chemopreventive effects. In the azoxymethane-treated rat model of experimental colon carcinogenesis, sulindac treatment markedly induced Csk with a corresponding increase in inhibitory phosphorylation of Src (Tyr(527)). Sulindac-mediated Csk induction was replicated in the human colorectal cancer cell line HT-29, with a corresponding suppression of both Src kinase activity (63% of vehicle; P < 0.05) and E-cadherin tyrosine phosphorylation (an in vivo Src target). To determine the importance of Csk in NSAID antiproliferative activity, we stably transfected a Csk-specific short hairpin RNA (shRNA) vector into HT-29 cells, thereby blunting the sulindac-mediated Csk induction. These transfectants were significantly less responsive to the antiproliferative effect of sulindac sulfide (suppression of proliferating cell nuclear antigen was 21 +/- 2.3% in transfectants versus 45 +/- 4.23% in wild-type cells), with a corresponding mitigation of the sulindac-mediated G(1)-S-phase arrest (S-phase cells 48 +/- 3.6% versus 14 +/- 2.8% of vehicle respectively). Importantly, the Csk shRNA cells had a marked decrease in the cyclin-dependent kinase inhibitor p21(cip/waf1), a critical regulator of G(1)-S-phase progression (49% of wild-type cells). Moreover, although sulindac-mediated induction of p21(cip/waf1) was 113% in wild-type HT-29, this induction was alleviated in the Csk shRNA transfectants (65% induction; P < 0.01). Thus, this is the first demonstration that the antiproliferative activity of NSAID is modulated, at least partly, through the Csk/Src axis.

Figure 1

Effect of sulindac on expression of Csk and p-Src-Tyr⁵²⁷ and BrdUrd incorporation in preneoplastic colonic mucosa in azoxymethane-treated rats. Colonic tissue sections from azoxymethane-treated male Fisher 344 rats (randomized to AIN76a diet with or without supplementation with 320 ppm sulindac) were subjected to immunohistochemical analysis as described in Materials and Methods. Sulindac-treated rats showed significant Csk induction with corresponding increase in the inhibitory Src-Tyr⁵²⁷ phosphorylation. Sulindac markedly suppressed cell proliferation assessed by BrdUrd incorporation. Representative images are shown in A. The immunohistochemical staining was scored on a four-point scale by an observer blinded to treatment groups. In the sulindac-treated azoxymethane-treated rats, there was a significant increase in Csk and p-Src-Tyr⁵²⁷ with decrease in BrdUrd incorporation (marker of proliferation) compared with unsupplemented rats (B). *, P < 0.05; **, P < 0.01 (n = 10).

Figure 2

Sulindac sulfide decreases Src activity. HT-29 cells were treated with either vehicle (DMSO) or sulindac sulfide (100 μmol/L) for 72 h. Src activity was measured through an immunoprecipitation kinase assay as detailed in Material and Methods. Compared with vehicle (DMSO), the cells treated with sulindac sulfide showed significant decrease in Src kinase activity (67% of control; P < 0.05).

Figure 3

NSAID responsiveness of wild-type HT-29 cells and the Csk knockdown construct. Wild-type HT-29 cells and the Csk shRNA construct were treated with either vehicle (DMSO) or sulindac sulfide (100 μmol/L) for 72 h and proteins were subjected to standard Western blotting techniques. Levels of Csk, p-Src-Tyr⁵²⁷ and PCNA, phosphorylated E-cadherin, and densitometric analysis in wild-type HT-29 cells (A) and Csk shRNA construct (B). Whereas sulindac sulfide caused marked Csk induction with corresponding increase in p-Src-Tyr⁵²⁷ and a notable decrease in PCNA in the wild-type HT-29 cells, the Csk shRNA construct showed only a modest Csk induction with minimal alterations in p-Src-Tyr⁵²⁷ and lesser degree of PCNA reduction. *, P < 0.05; **, P < 0.01. NS, nonsignificant.

Figure 4

Effect of sulindac sulfide on cell cycle in HT-29 cells and its Csk knockdown construct. Wild-type HT-29 cells and the Csk shRNA construct were incubated with either vehicle (DMSO) or sulindac sulfide (125 μmol/L) for 72 h. Cells were fixed in 70% ethanol, stained with propidium iodide, and analyzed for cell cycle distribution by flow cytometry. In wild-type HT-29 cells, sulindac sulfide treatment caused a striking reduction in the percentage of cells in S phase (14% of vehicle; P < 0.01) compared with only 52% decrease in S-phase cells in the Csk shRNA construct. *, P < 0.01.

Figure 5

Effect of sulindac sulfide on p21 expression. A, immunohistochemical detection of p21^cip/waf1 in azoxymethane-treated rats. Sulindac treatment caused a significant induction of the CDK inhibitor p21^cip/waf1 in azoxymethane-treated rats (200% of control rats; P = 0.0132). B, flow cytometric detection of p21^cip/waf1 in the wild-type HT-29 cells and the Csk shRNA cells. The levels of p21^cip/waf1 were detected by flow cytometry as described in Materials and Methods. Whereas sulindac sulfide caused a significant induction (213% of vehicle; P < 0.05) on p21^cip/waf1 in wild-type HT-29 cells, the Csk knockdown showed a lesser degree of p21 activation (165% of vehicle; P < 0.05).

Figure 6

Proposed mechanism for NSAID-mediated colon cancer chemoprevention via Csk/Src axis.