Curcumin suppresses proliferation of colon cancer cells by targeting CDK2

Abstract

Curcumin, the yellow pigment of turmeric found in Southeast Indian food, is one of the most popular phytochemicals for cancer prevention. Numerous reports have demonstrated modulation of multiple cellular signaling pathways by curcumin and its molecular targets in various cancer cell lines. To identify a new molecular target of curcumin, we used shape screening and reverse docking to screen the Protein Data Bank against curcumin. Cyclin-dependent kinase 2 (CDK2), a major cell-cycle protein, was identified as a potential molecular target of curcumin. Indeed, in vitro and ex vivo kinase assay data revealed a dramatic suppressive effect of curcumin on CDK2 kinase activity. Furthermore, curcumin induced G1 cell-cycle arrest, which is regulated by CDK2 in HCT116 cells. Although the expression levels of CDK2 and its regulatory subunit, cyclin E, were not changed, the phosphorylation of retinoblastoma (Rb), a well-known CDK2 substrate, was reduced by curcumin. Because curcumin induced cell-cycle arrest, we investigated the antiproliferative effect of curcumin on HCT116 colon cancer cells. In this experiment, curcumin suppressed HCT116 cell proliferation effectively. To determine whether CDK2 is a direct target of curcumin, CDK2 expression was knocked down in HCT116 cells. As expected, HCT116 sh-CDK2 cells exhibited G1 arrest and reduced proliferation. Because of the low levels of CDK2 in HCT116 sh-CDK2 cells, the effects of curcumin on G1 arrest and cell proliferation were not substantially relative to HCT116 sh-control cells. From these results, we identified CDK2 as a direct target of curcumin in colon cancer cells.

Fig. 1

Curcumin directly binds to CDK2 in an ATP-competitive manner. (A) The interaction between curcumin and several residues in the ATP binding pocket of CDK2. Curcumin forms hydrogen bonds with Lys33, Leu83, Asp86 and Asp145, respectively. Note: the α-helices are drawn as cylinders and the β-strands as arrows. Curcumin is shown in stick model and protein residues are shown in line model. (B) Curcumin directly binds with CDK2 in HCT116 colorectal cancer cells. The binding of curcumin with CDK2 in HCT116 cells was detected by immunoblotting with a specific CDK2 antibody: lane 1 (input control), whole-cell lysates from HCT116 cells; lane 2 (control), lysates from HCT116 cells; and lane 3, whole-cell lysates from HCT116 cells precipitated with curcumin-Sepharose 4B beads. (C) Curcumin competes with ATP for binding with CDK2. Active CDK2 was incubated with ATP at different concentrations (0, 1, 10, or 100 µM) with 50 µl of curcumin-Sepharose 4B beads or 50 µl of Sepharose 4B (as a negative control) beads in reaction buffer. After washing, the pulled-down CDK2 proteins were detected by Western blotting.

Fig. 2

Curcumin effectively suppresses CDK2 kinase activity in vitro compared with CDK1 or 4 kinase activity. The effect of curcumin on CDK2 (A) or CDK1 and CDK4 (B) in vitro kinase activity was examined as described in "Materials and Methods". Data are represented as means ± S.D. as determined from 3 independent experiments. The asterisk (*) indicates a significant (p < 0.001) decrease in kinase activity compared to untreated control.

Fig. 3

CDK2 is highly expressed in colon cancer cells and curcumin selectively suppresses HCT116 cell proliferation. (A) The CDK2 expression level is much higher in colon cancer cells (HCT116, HCT15, and DLD-1) compared to “normal” (see footnote) colon cells (HCEC). Cells were harvested and CDK2 expression was visualized using a specific antibody. Curcumin reduces proliferation of HCT116 (B), HCT15 (C), and DLD-1 (D) colon cancer cells. Each colon cancer cell line was seeded (1×10³ cells per well) in 96-well plates and then cells were treated with curcumin (0, 5, 10, 20 or 40 µM) at 6 h after seeding. Cell growth was determined at 1, 2, or 3 days using the MTS assay as described in "Materials and Methods". Data are represented as the means ± S.D. as determined from 3 independent experiments. The asterisk (*) indicates a significant (p < 0.001) decrease in activity compared to untreated control.

Fig. 4

Curcumin induces G₁ arrest in HCT116 colon cancer cells. (A) The effect of curcumin on cell cycle was investigated using flow cytometry. HCT116 colon cancer cells were cultured until they reached confluence in 96-well plates and then synchronized in G₀ phase by serum deprivation. After 9 h of treatment with curcumin (0, 10, 20, or 40 µM) or SU9516 (10 µM), cell cycle phases were analyzed. Data are represented as means ± S.D. as determined from 3 independent experiments and the asterisk (*) indicates a significant difference (p < 0.001) compared to untreated group. (B) To determine the effect of curcumin on cell cycle proteins, Western blotting was performed using specific antibodies. After synchronizing the cells for 12 h, cells were treated with the indicated concentration of curcumin for 30 min. Data are representative of 3 independent experiments that gave similar results.

Fig. 5

The effect of curcumin on cell cycle in sh-CDK2-transfected HCT116 colon cancer cells. (A) HCT116 cells stably expressing knockdown of CDK2 were established and G₁ phase was arrested in sh-CDK2-transfected HCT116 cells. Cell cycle was analyzed using flow cytometry. (B) The effect of curcumin on G₁ phase arrest was less in sh-CDK2-transfected HCT116 colon cancer cells compared to sh-Mock-transfected cells. After 9 h of curcumin treatment, cell cycle phases were analyzed. Data are shown as means ± S.D. as determined from 3 independent experiments and the asterisk (*) indicates a significant (p < 0.001) difference compared to sh-Mock-transfected HCT116 cells.

Fig. 6

The effect of curcumin on proliferation and cell cycle protein expression in sh-CDK2-transfected HCT116 colon cancer cells. (A) Proliferation was relatively lower in sh-CDK2-transfected HCT116 cells compared to sh-Mock-transfected HCT116 cells. HCT116 cells (1×10³ cells) were seeded in 96-well plates and after 72 h, proliferation was measured using the MTS assay as described in "Materials and Methods". (B) The effects of curcumin on cell proliferation were less in sh-CDK2-transfected HCT116 cells compared to sh-Mock-transfected cells. sh-Mock- and sh-CDK2-transfected HCT116 cells were treated with curcumin (0, 10, 20, or 40 µM) and growth was determined at 3 days using the MTS assay as described in "Materials and Methods". Data are represented as means ± S.D. as determined from 3 independent experiments. For A and B, the asterisk (*) indicates a significant difference (p < 0.001) compared to sh-Mock-transfected HCT116 cells. (C) Curcumin had no effect on cell cycle proteins in sh-CDK2-transfected HCT116 colon cancer cells. sh-Mock- and sh-CDK2-transfected HCT116 cells were treated with the indicated concentrations of curcumin for 30 min. Cell cycle protein expression was determined using specific antibodies. Data are representative of 3 independent experiments that gave similar results.