Multiple mechanisms are involved in 6-gingerol-induced cell growth arrest and apoptosis in human colorectal cancer cells

Abstract

6-Gingerol, a natural product of ginger, has been known to possess anti-tumorigenic and pro-apoptotic activities. However, the mechanisms by which it prevents cancer are not well understood in human colorectal cancer. Cyclin D1 is a proto-oncogene that is overexpressed in many cancers and plays a role in cell proliferation through activation by beta-catenin signaling. Nonsteroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) is a cytokine associated with pro-apoptotic and anti-tumorigenic properties. In the present study, we examined whether 6-gingerol influences cyclin D1 and NAG-1 expression and determined the mechanisms by which 6-gingerol affects the growth of human colorectal cancer cells in vitro. 6-Gingerol treatment suppressed cell proliferation and induced apoptosis and G(1) cell cycle arrest. Subsequently, 6-gingerol suppressed cyclin D1 expression and induced NAG-1 expression. Cyclin D1 suppression was related to inhibition of beta-catenin translocation and cyclin D1 proteolysis. Furthermore, experiments using inhibitors and siRNA transfection confirm the involvement of the PKCepsilon and glycogen synthase kinase (GSK)-3beta pathways in 6-gingerol-induced NAG-1 expression. The results suggest that 6-gingerol stimulates apoptosis through upregulation of NAG-1 and G(1) cell cycle arrest through downregulation of cyclin D1. Multiple mechanisms appear to be involved in 6-gingerol action, including protein degradation as well as beta-catenin, PKCepsilon, and GSK-3beta pathways.

Figure 1

6-Gingerol suppresses cell proliferation, induces apoptosis, and arrests at G₁ cell cycle in human colorectal cancer cells. (A) Human colorectal cancer cells were treated with indicated concentration of 6-gingerol for 72 h. Cell growth was measured using CellTiter96 Aqueous One Solution Cell Proliferation Assay. Values are expressed as mean ± SD of four replicates. (Left panel) HCT-116 cells were treated with the indicated concentration of 6-gingerol and cell growth rate was determined. (Right panel) Four different human colorectal cancer cells were treated with 6-gingerol and measured cell growth. The data represent % of reduction compared to vehicle treatment. (B) Human colorectal cancer cells were treated with 200 μM of 6-gingerol for 72 h. The cells were stained with Annexin V-FITC and propidium iodide (BD Biosciences). Apoptosis was quantified by flow cytometry as described in Materials and Methods section. Values are expressed as mean ± SD of three replicates. (C) HCT-116, SW480, and LoVo cells were treated with 200 μM of 6-gingerol for 72 h. Cell cycle distribution was analyzed by FACS analysis of propidium iodide-stained cells. Values are expressed as mean ± SD of three replicates. *P<0.05; **P<0.01; ***P<0.001 versus vehicle-treated cells.

Figure 2

6-Gingerol suppresses cyclin D1 expression and increases NAG-1 expression in HCT-116 cells. (A) HCT-116 cells were treated with the indicated concentration of 6-gingerol for 24 h. Total cell lysates were harvested, and subsequently, 30 μg of total cell lysates were subjected to 14% SDS–PAGE. Cyclin D1, cyclin D3, CDK4, p21, p27, NAG-1, p53, and actin antibodies were probed. The results shown here are representative of three independent experiments. (B) HCT-116 cells were treated with the indicated concentrations of 6-gingerol for 24 h, and then RT-PCR (cyclin D1) or Northern blot analysis (NAG-1) was performed as described in Materials and Methods section. The GAPDH and 28S/18S shown represent a loading control. (C) HCT-116 cells were treated with 200 μM of 6-gingerol at indicated time points. Total cell lysates were harvested, and Western analysis was performed for cyclin D1, NAG- 1, and Actin antibodies. (D) HCT-116, SW480, HT-29, LoVo, and Caco-2 cells were grown as described in Materials and Methods section, and treated with 200 μM of 6-gingerol for 24 h. Western analysis was performed for cyclin D1, NAG-1, and Actin antibodies. (E and F) HCT-116 cells were grown in a glass slide chamber and then treated with 200 μM of 6-gingerol for 24 h. The cells were incubated with a specific antibody for cyclin D1 (1:250) or NAG-1 (1:250) overnight. Cyclin D1 and NAG-1 were detected by using rhodamine conjugate (red) and Alexa Fluor 488 conjugate (green), respectively, and visualized by fluorescence microscopy as described in Materials and Methods section. The DAPI staining (blue) was used to visualize the nuclei of the cells. Magnifications correspond to 400×. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 3

6-Gingerol suppresses transactivation of the cyclin D1 gene through suppression of β-catenin activity and increases proteolysis of cyclin D1 protein. (A) HCT-116 cells were transfected with a reporter gene containing cyclin D1 promoter, and then the cells were treated with 200 μM of 6-gingerol for 24 h. Luciferase activity was measured as a ratio of firefly luciferase signal/renilla luciferase signal and is presented as a relative luciferase unit (RLU). The TCF binding site is shown as ■ [14], **P<0.01; ***P<0.001 versus vehicle-treated cells. (B) HCT-116 cells were treated with 200 μM of 6-gingerol for 24 h, and nuclear (N) and cytosol fractions (C) were isolated as described in Materials and Methods section. Western blot was performed for p-pRb (Ser⁷⁸⁰) and Actin antibodies. (C) HCT-116 cells were pretreated with vehicle or 200 μM of 6- gingerol for 1 h and then exposed to 10 μg/mL of CHX for the indicated time. Western analysis was performed for cyclin D1 and actin antibodies. (D) HCT-116 cells were pretreated with the indicated concentration of MG-132 for 30 min and then exposed to 200 μM of 6-gingerol for 24 h. Western analysis was performed for cyclin D1 and actin antibodies. (E) Human colorectal cancer cells were transfected with TOP-FLASH or FOP-FLASH constructs containing six copies of wild or mutated TCF binding sites. Then, the cells were treated with 200 μM of 6-gingerol for 24 h. Luciferase activity was measured as a ratio of TOP-FLASH over FOP-FLASH. *P<0.05; ***P<0.001 versus vehicle-treated cells. (F) SW480 cells were grown in a glass slide chamber and then treated with 200 μM of 6- gingerol for 24 h. The cells were incubated with a specific antibody for β-catenin (1:250) overnight, and β-catenin was detected by using rhodamine conjugate (red). The DAPI staining (blue) was used to visualize the nuclei of the cells (blue). Magnifications correspond to 600×. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 3

6-Gingerol suppresses transactivation of the cyclin D1 gene through suppression of β-catenin activity and increases proteolysis of cyclin D1 protein. (A) HCT-116 cells were transfected with a reporter gene containing cyclin D1 promoter, and then the cells were treated with 200 μM of 6-gingerol for 24 h. Luciferase activity was measured as a ratio of firefly luciferase signal/renilla luciferase signal and is presented as a relative luciferase unit (RLU). The TCF binding site is shown as ■ [14], **P<0.01; ***P<0.001 versus vehicle-treated cells. (B) HCT-116 cells were treated with 200 μM of 6-gingerol for 24 h, and nuclear (N) and cytosol fractions (C) were isolated as described in Materials and Methods section. Western blot was performed for p-pRb (Ser⁷⁸⁰) and Actin antibodies. (C) HCT-116 cells were pretreated with vehicle or 200 μM of 6- gingerol for 1 h and then exposed to 10 μg/mL of CHX for the indicated time. Western analysis was performed for cyclin D1 and actin antibodies. (D) HCT-116 cells were pretreated with the indicated concentration of MG-132 for 30 min and then exposed to 200 μM of 6-gingerol for 24 h. Western analysis was performed for cyclin D1 and actin antibodies. (E) Human colorectal cancer cells were transfected with TOP-FLASH or FOP-FLASH constructs containing six copies of wild or mutated TCF binding sites. Then, the cells were treated with 200 μM of 6-gingerol for 24 h. Luciferase activity was measured as a ratio of TOP-FLASH over FOP-FLASH. *P<0.05; ***P<0.001 versus vehicle-treated cells. (F) SW480 cells were grown in a glass slide chamber and then treated with 200 μM of 6- gingerol for 24 h. The cells were incubated with a specific antibody for β-catenin (1:250) overnight, and β-catenin was detected by using rhodamine conjugate (red). The DAPI staining (blue) was used to visualize the nuclei of the cells (blue). Magnifications correspond to 600×. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]

Figure 4

6-Gingerol activates NAG-1 expression via the PKCε and GSK-3β-dependent pathway. (A) HCT-116 cells were pretreated with the indicated concentration of RO-31-8220 for 30 min and then exposed to 200 μM of 6-gingerol for 24 h. Total cell lysates were harvested, and subsequently, 30 μg of total cell lysates were subjected to 14% SDS–PAGE. Cyclin D1, NAG-1, and Actin antibodies were probed. (B) HCT-116 cells were pretreated with 5 μM of RO-31-8220, 0.5 μM of Rottlerin, or 0.5 μM of Gö6983 for 30 min and then exposed to 200 μM of 6-gingerol for 24 h. Western analysis was performed for cyclin D1, NAG-1, and Actin antibodies. (C) HCT-116 cells were transfected with wild type (WT), or dominant negative PKCε expression vector (DN) as described previously [36]. The cells were then treated with 200 μM of 6-gingerol for 24 h. Western analysis was performed for hemagglutinin (HA), NAG-1, and Actin antibodies. (D) HCT-116 cells were transfected with control or PKCε siRNA as described in Materials and Methods section. Then, the cells were treated with 200 μM of 6-gingerol for 24 h. Western analysis was performed using 60 μg of total cell lyates for PKCε and 30 μg of total cell lysates for NAG-1 and Actin antibodies. (E) HCT-116 cells were pretreated with 20 mM of LiCl for 30 min and then exposed to 200 μM of 6-gingerol for 24 h. Western analysis was performed for cyclin D1, NAG-1, and actin antibodies. (F) HCT-116 cells were transfected with control or GSK-3 siRNA. Then, the cells were treated with 200 μM of 6-gingerol for 24 h. Western analysis was performed for GSK-3β, NAG-1, and actin antibodies.

Figure 5

Proposed mechanism by which 6-gingerol induces apoptosis and suppresses cell growth in human CRC. 6-Gingerol can activate GSK-3 and PKCε to increase NAG-1 expression and downregulate cyclin D1 by the inactivation of β-catenin signaling and increase of proteolysis. Upregulation of NAG-1 and downregulation of cyclin D1 by 6-gingerol results in the induction of apoptosis and cell cycle arrest in human colorectal cancer cells.