Yonsei Med J.  2006 Oct;47(5):688-697. 10.3349/ymj.2006.47.5.688.

[6]-Gingerol Induces Cell Cycle Arrest and Cell Death of Mutant p53-expressing Pancreatic Cancer Cells

• Park YJ
• Wen J
• Bang S
• Park SW
• Song SY

• KMID: 1381250
• DOI: http://doi.org/10.3349/ymj.2006.47.5.688

Abstract

[6]-Gingerol, a major phenolic compound derived from ginger, has anti-bacterial, anti-inflammatory and anti-tumor activities. While several molecular mechanisms have been described to underlie its effects on cells in vitro and in vivo, the underlying mechanisms by which [6]-gingerol exerts anti-tumorigenic effects are largely unknown. The purpose of this study was to investigate the action of [6]-gingerol on two human pancreatic cancer cell lines, HPAC expressing wild- type (wt) p53 and BxPC-3 expressing mutated p53. We found that [6]-gingerol inhibited the cell growth through cell cycle arrest at G1 phase in both cell lines. Western blot analyses indicated that [6]-gingerol decreased both Cyclin A and Cyclin-dependent kinase (Cdk) expression. These events led to reduction in Rb phosphorylation followed by blocking of S phase entry. p53 expression was decreased by [6]-gingerol treatment in both cell lines suggesting that the induction of Cyclin-dependent kinase inhibitor, p21(cip1), was p53-independent. [6]-Gingerol induced mostly apoptotic death in the mutant p53-expressing cells, while no signs of early apoptosis were detected in wild type p53-expressing cells and this was related to the increased phosphorylation of AKT. These results suggest that [6]-gingerol can circumvent the resistance of mutant p53- expressing cells towards chemotherapy by inducing apoptotic cell death while it exerts cytostatic effect on wild type p53- expressing cells by inducing temporal growth arrest.

Keyword

[6]-gingerol; pancreatic cancer; G1 phase; apoptosis; AKT

MeSH Terms

Tumor Suppressor Protein p53/*genetics/metabolism
Proto-Oncogene Proteins c-akt/genetics/metabolism
Pancreatic Neoplasms/*drug therapy
Mutation
Humans
Gene Expression Regulation, Neoplastic/drug effects
Fatty Alcohols/*pharmacology/therapeutic use
Drug Resistance, Neoplasm
Cell Proliferation/drug effects
Cell Line, Tumor
Cell Cycle/*drug effects
Apoptosis/*drug effects
Antineoplastic Agents/*pharmacology/therapeutic use

Figure

• 

  Fig. 1 Effect of [6]-gingerol on survival of BxPC-3 and HPAC cells. [6]-Gingerol inhibited growth of both BxPC-3 and HPAC cells in a dose-dependent manner. Cells were treated with different concentrations of [6]-gingerol. Control cells (empty circle) were treated with vehicle alone (0.1% DMSO). Cells were harvested at day 1, 3 and day 5 and cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as described in Materials and methods. Percent viability was calculated by comparison with controls. Values shown as the mean ± SEM obtained from four independent experiments.

• 

  Fig. 2 [6]-Gingerol induces cell cycle arrest in both BxPC-3 and HPAC cell lines. Exponentially growing cells were exposed to either 0.1% DMSO (control) or [6]-gingerol (400 µM) for 24, 48 or 72 hrs. Cells were then harvested, washed in PBS, and fixed in 70% ethanol. DNA content was evaluated with propidium iodide staining and fluorescence measured and analyzed as described in Materials and Methods. Data are representative of three independent experiments.

• 

  Fig. 3 Western blot analyses of the cell cycle regulatory proteins. Both BxPC-3 and HPAC were treated with 400 µM of [6]-gingerol, and harvested every 12 hr, and levels of Cyclins, Cdks, Rb and pRb were determined by Western blotting. The upper band of Rb indicates phosphorylated form. pRb antibody detects specifically on Ser-780 phophorylated Rb. Data are representative of three independent experiments. Actin was used as a standard for each sample.

• 

  Fig. 4 Effects of [6]-gingerol on the expression of p21cip and p53. Both BxPC-3 and HPAC were treated with 400 µM of [6]-ingerol, and harvested every 12 hr. The levels of p21cip1, a Cyclin dependent kinase inhibitor, and p53 changed by [6]-Gingerol in BxPC-3 and HPAC cells. Three replicate experiments were done with similar results.

• 

  Fig. 5 Differential mode of death induced by [6]-Gingerol in pancreatic cancer cells expressing wt versus mutant p53. Cells were treated with 400 µM of [6]-gingerol for 24, 28 and 72 hrs. Annexin-V and PI staining revealed increasing percentage of Annexin-positive and PI-positive cells with increasing time of [6]-gingerol in BxPC-3 cells. On the other hand, [6]-gingerol treatment on HPAC cells showed no changes in cell viability under the identical culture condition. The X and Y axis represents annexin V-FITC and Propidium Iodide (PI) fluorescence respectively. Population in lower-left part (Annexin V-FITC and PI negative) is viable, and lower-right part (Annexin V-FITC positive and PI negative) is undergoing apoptosis. Cells observed in Annexin V-FITC and PI positive (upper-right and upper-left parts) indicates either late stage of apoptosis or dead cells. The percentage of each part is calculated in the bottom table. The figure is representative of three independent experiments.

• 

  Fig. 6 Western bolt analysis for MPAK and PI3K/AKT pathways in [6]-gingerol-treated pancreatic cancer cells. The BxPC-3 (A) and HPAC (B) were incubated with 400 µM of gingerol for indicated times. Cells were lysed and the cell lysates (30 µg) were resolved by SDS-PAGE. AKT, ERK and JNK activation was analyzed by Western blotting with anti-phospho-AKT, anti-phosph-ERK, anti-phosph-JNK or anti-p85-specific antibodies as indicated. Equal protein loading was confirmed by probing the membranes with antibodies detecting the respective unphosphorylated proteins (ERK and AKT). Three replicate experiments were done with similar results.

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