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

Affiliations
  • 1Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, Seoul, Korea.
  • 2Internal Medicine and Institute of Gastroenterology, Yonsei University College of Medicine, Seoul, Korea. sysong@yumc.yonsei.ac.kr

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.


Cited by  1 articles

Prognostic Implications of Cyclin B1, p34cdc2, p27Kip1 and p53 Expression in Gastric Cancer
Dong-Hoon Kim
Yonsei Med J. 2007;48(4):694-700.    doi: 10.3349/ymj.2007.48.4.694.


Reference

1. Ahmad N, Feyes DK, Nieminen AL, Agarwal R, Mukhtar H. Green tea constituent epigallocatechin-3-gallate and induction of apoptosis and cell cycle arrest in human carcinoma cells. J Natl Cancer Inst. 1997; 89:1881–1886. PMID: 9414176.
Article
2. Liu XJ, Yang L, Mao YQ, Wang Q, Huang MH, Wang YP, et al. Effects of the tyrosine protein kinase inhibitor genistein on the proliferation, activation of cultured rat hepatic stellate cells. World J Gastroenterol. 2002; 8:739–745. PMID: 12174389.
Article
3. Pan MH, Chen WJ, Lin-Shiau SY, Ho CT, Lin JK. Tangeretin induces cell-cycle G1 arrest through inhibiting Cyclin-dependent kinases 2 and 4 activities as well as elevating Cdk inhibitors p21 and p27 in human colorectal carcinoma cells. Carcinogenesis. 2002; 23:1677–1684. PMID: 12376477.
Article
4. Agarwal R. Cell signaling and regulators of cell cycle as molecular targets for prostate cancer prevention by dietary agents. Biochem Pharmacol. 2000; 60:1051–1059. PMID: 11007941.
Article
5. Bhatia N, Agarwal C, Agarwal R. Differential responses of skin cancer-chemopreventive agents silibinin, quercetin, and epigallocatechin 3-gallate on mitogenic signaling and cell cycle regulators in human epidermoid carcinoma A431 cells. Nutr Cancer. 2001; 39:292–299. PMID: 11759294.
Article
6. Surh Y. Molecular mechanisms of chemopreventive effects of selected dietary and medicinal phenolic substances. Mutat Res. 1999; 428:305–327. PMID: 10518003.
Article
7. Surh YJ, Lee E, Lee JM. Chemoprotective properties of some pungent ingredients present in red pepper and ginger. Mutat Res. 1998; 402:259–267. PMID: 9675305.
Article
8. Suzuki F, Kobayashi M, Komatsu Y, Kato A, Pollard RB. Keishi-ka-kei-to, a traditional Chinese herbal medicine, inhibits pulmonary metastasis of B16 melanoma. Anticancer Res. 1997; 17:873–878. PMID: 9137420.
9. Park KK, Chun KS, Lee JM, Lee SS, Surh YJ. Inhibitory effects of [6]-gingerol, a major pungent principle of ginger, on phorbol ester-induced inflammation, epidermal ornithine decarboxylase activity and skin tumor promotion in ICR mice. Cancer Lett. 1998; 129:139–144. PMID: 9719454.
Article
10. Yoshimi N, Wang A, Morishita Y, Tanaka T, Sugie S, Kawai K, et al. Modifying effects of fungal and herb metabolites on azoxymethane-induced intestinal carcinogenesis in rats. Jpn J Cancer Res. 1992; 83:1273–1278. PMID: 1483942.
Article
11. Bode AM, Ma WY, Surh YJ, Dong Z. Inhibition of epidermal growth factor-induced cell transformation and activator protein 1 activation by [6]-gingerol. Cancer Res. 2001; 61:850–853. PMID: 11221868.
12. Lee E, Surh YJ. Induction of apoptosis in HL-60 cells by pungent vanilloids, [6]-gingerol and [6]-paradol. Cancer Lett. 1998; 134:163–168. PMID: 10025876.
Article
13. Mahady GB, Pendland SL, Yun GS, Lu ZZ, Stoia A. Ginger (Zingiber officinale Roscoe) and the gingerols inhibit the growth of Cag A+ strains of Helicobacter pylori. Anticancer Res. 2003; 23:3699–3702. PMID: 14666666.
14. Oya N. Chemoradiotherapy for pancreatic cancer: current status and perspectives. Int J Clin Oncol. 2004; 9:451–457. PMID: 15616875.
Article
15. Ridwelski K, Meyer F. Current options for palliative treatment in patients with pancreatic cancer. Dig Dis. 2001; 19:63–75. PMID: 11385253.
Article
16. Nio Y, Dong M, Uegaki K, Hirahara N, Minari Y, Sasaki S, et al. p53 expression affects the efficacy of adjuvant chemotherapy after resection of invasive ductal carcinoma of the pancreas. Anticancer Res. 1998; 18:3773–3779. PMID: 9854494.
17. King TC, Estalilla OC, Safran H. Role of p53 and p16 gene alterations in determining response to concurrent paclitaxel and radiation in solid tumor. Semin Radiat Oncol. 1999; 9(2):Suppl 1. 4–11. PMID: 10210535.
18. Xu ZW, Friess H, Buchler MW, Solioz M. Overexpression of Bax sensitizes human pancreatic cancer cells to apoptosis induced by chemotherapeutic agents. Cancer Chemother Pharmacol. 2002; 49:504–510. PMID: 12107556.
Article
19. Song SY, Meszoely IM, Coffey RJ, Pietenpol JA, Leach SD. K-Ras-independent effects of the farnesyl transferase inhibitor L-744,832 on Cyclin B1/Cdc2 kinase activity, G2/M cell cycle progression and apoptosis in human pancreatic ductal adenocarcinoma cells. Neoplasia. 2000; 2:261–272. PMID: 10935512.
20. Wallace-Brodeur RR, Lowe SW. Clinical implications of p53 mutations. Cell Mol Life Sci. 1999; 55:64–75. PMID: 10065152.
Article
21. Natsugoe S, Nakashima S, Matsumoto M, Xiangming C, Okumura H, Kijima F, et al. Expression of p21 WAF1/Cip1 in the p53-dependent pathway is related to prognosis in patients with advanced esophageal carcinoma. Clin Cancer Res. 1999; 5:2445–2449. PMID: 10499617.
22. Chang F, Lee JT, Navolanic PM, Steelman LS, Shelton JG, Blalock WL, et al. Involvement of PI3K/Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy. Leukemia. 2003; 17:590–603. PMID: 12646949.
Article
23. Kim SO, Kundu JK, Shin YK, Park JH, Cho MH, Kim TY, et al. [6]-Gingerol inhibits COX-2 expression by blocking the activation of p38 MAP kinase and NF-kappaB in phorbol ester-stimulated mouse skin. Oncogene. 2005; 24:2558–2567. PMID: 15735738.
24. Yang CS, Landau JM, Huang MT, Newmark HL. Inhibition of carcinogenesis by dietary polyphenolic compounds. Annu Rev Nutr. 2001; 21:381–406. PMID: 11375442.
Article
25. Quelle DE, Ashmun RA, Shurtleff SA, Kato JY, Bar-Sagi D, Roussel MF, et al. Overexpression of mouse D-type Cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev. 1993; 7:1559–1571. PMID: 8339933.
Article
26. Kranenburg O, van der Eb AJ, Zantema A. Cyclin D1 is an essential mediator of apoptotic neuronal cell death. EMBO J. 1996; 15:46–54. PMID: 8598205.
Article
27. Willis AC, Chen X. The promise and obstacle of p53 as a cancer therapeutic agent. Curr Mol Med. 2002; 2:329–345. PMID: 12108946.
Article
28. Nelson WG, Kastan MB. DNA strand breaks: the DNA template alterations that trigger p53-dependent DNA damage response pathways. Mol Cell Biol. 1994; 14:1815–1823. PMID: 8114714.
29. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell. 1997; 88:323–331. PMID: 9039259.
Article
30. Seeman S, Maurici D, Oliver M, de Formentel CC, Hainaut P. The tumor suppressor gene TP53: implications for cancer management and therpay. Crit Rev Clin Lab Sci. 2004; 41:551–583. PMID: 15603511.
31. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 1999; 13:1501–1512. PMID: 10385618.
Article
32. Coleman ML, Marshall CJ, Olson MF. Ras promotes p21(Waf1/Cip1) protein stability via a Cyclin D1-imposed block in proteasome-mediated degradation. EMBO J. 2003; 22:2036–2046. PMID: 12727871.
Article
33. Olson MF, Paterson HF, Marshall CJ. Signals from Ras and Rho GTPases interact to regulate expression of p21Waf1/Cip1. Nature. 1998; 394:295–299. PMID: 9685162.
Article
34. Liberto M, Cobrinik D. Growth factor-dependent induction of p21(CIP1) by the green tea polyphenol, epigallocatechin gallate. Cancer Lett. 2000; 154:151–161. PMID: 10806303.
Article
35. Cho SG, Choi EJ. Apoptotic signaling pathways: caspases and stress-activated protein kinases. J Biochem Mol Biol. 2002; 35:24–27. PMID: 16248966.
Article
36. Datta SR, Brunet A, Greenberg ME. Cellular survival: a play in three Akts. Genes Dev. 1999; 13:2905–2927. PMID: 10579998.
Article
37. Li Q, Zhu GD. Targeting serine/threonine protein kinase B/Akt and cell-cycle checkpoint kinases for treating cancer. Curr Top Med Chem. 2002; 2:939–971. PMID: 12171565.
38. Testa JR, Bellacosa A. AKT plays a central role in tumorigenesis. Proc Natl Acad Sci USA. 2001; 98:10983–10985. PMID: 11572954.
Article
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