Phenolic compounds comprise one of the largest and most ubiquitous groups of plant metabolites. They are formed to protect the plant from photosynthetic stress, reactive oxygen species (ROS), wounds, and herbivores.24
The most commonly contained ones in foods are flavonoids and phenolic substances. Hence, phenolic compounds take important parts of the human diet. In addition, current interest is raised up by many observations that dietary phenolic compounds have various activities such as antioxidant, anti-inflammation and anti-carcinogenesis.
In this present study, we first investigated the effects of -gingerol, a phenolic substance derived from ginger roots, on two pancreatic cancer cell lines. We found that -gingerol inhibited the cell growth, disrupted the cell cycle progression in both HPac cells (wild type p53) and BXPC-3 cells (mutant p53 protein), and also induced apoptosis in BxPC-3 cells. Interestingly, it is noticeable that normal cell showed highly resistance to the cytotoxic effects of -gingerol. RIE (rat intestinal epithelial cell) showed 50 percents growth inhibition at over 900 µM (data not shown) of -gingerol for 3 days treatment. This selectivity may be the great advantage of -gingerol for the therapeutic or preventative use. While there were some reports that various phenolic substances induce cell cycle arrest in some phases,1-5
this is the first report that reveals on -gingerol effect upon cell cycle in cancer cell lines. Western blot analyses indicated that -gingerol decreased the expression of Cyclin A and Cdks including Cdk2, Cdk4, and Cdk6 in BxPC-3. Also, Cyclin A and Cdk 6 expression levels were decreased in HPAC. Thus, the reduction of Cyclin or Cdk expressions results the blocking of Cyclin-Cdk complexes formation and that lowers the level of phospho-Rb. Since Rb proteins remain in unphosphorylated form, E2F cannot be activated and the cells fail to enter the S phase. Cyclin D1 might be the cause of apoptotic cell death when overexpressed.25,26
Alternatively, increase of Cyclin D1 in BxPC-3 cells may be a feedback response to drug-induced cell cycle arrest.
p53 is a tumor suppressor gene encoding a transcription factor. Its tumor-suppressive activity involves inhibition of cell proliferation through cell cycle arrest and/or apoptosis. Mutation in p53 occurs in more than half of human cancers.27
Cells harboring mutated p53 lose the ability to elicit the enzymatic DNA repair cascade, to inhibit cell proliferation and to induce programmed cell death, resulting in induction of uncontrolled proliferation and malignancy.28,29
Tumors consisting of mutant p53-expressing cells exhibit high resistance to radiation and chemotherapeutic drugs. Circumventing this abnormal resistance is a major challenge in cancer therapy.20,30
In the present study, we showed that -gingerol exerted its cytotoxic activy toward cancer cells harboring mutant p53. Based on in vitro
and in vivo
studies, the Cip/Kip family including p21Cip1
were initially thought to interfere with the activation of G1/S phase related Cyclin/Cdk complexes. The Cyclin-dependent kinase inhibitor p21cip1
is a major transcriptional target of the tumor-suppressor p53.21
BxPC-3 cells have point mutated p53 proteins and the HPAC cells have the wild type p53 proteins. While the basal levels of p53 were elevated in mutant p53-expressing cells as previously reported,20
the level of p53 is decreased by -gingerol in both cell lines. Thus, p21cip1
induction by -gingerol was not necessarily dependent on p53 wild-type status since we detected p21 induction in mutated p53 cells.
It has been reported Ras signaling through the Raf/MAPK pathway also elevates levels of p21cip1
in some cell types.31-33
However it is still unclear whether the over-expression of p21cip1
by -gingerol is related with Ras signaling activations. A number of phytochemicals, including EGCG,34
genestein and silymarin4
have been shown to induce cell cycle arrest accompanied by increased p21cip1
expression, an important Cdk inhibitor in G1 and S phase. -Gingerol also increased the level of p21cip1
in both cell lines. The overexpression of p21cip1
facilitated cell cycle arrest effects of -gingerol. Unlike other Cyclins, Cyclin D1 level was increased in BxPC-3. It is probably as a feedback response to G1 arrest. More recent study, however, has altered this view and revealed that even p21cip1
proteins are specific inhibitors of Cyclin E- and A-dependent Cdk2, they act as positive regulators of Cyclin D-dependent kinases.31
Thus, the increase of Cyclin D1 level in BxPC-3 cells may be the consequence of the overexpression of p21cip1
. However, it is unclear why there was no change of Cyclin D1 level in spite of G1 phase arrest and overexpression of p21cip1
in HPAC cells. The fact that -gingerol has different effects on the cell cycle in different cell lines is intriguing and suggests that the ability of -gingerol to affect the cell cycle may be dependent on other genetic alterations that the tumor harbors. For example, the levels of Cyclins, Cyclin-dependent kinases, their inhibitors, or the status of tumor suppressor genes such as p53 and Rb, that are all involved in cell cycle regulation, may determine whether a chemical inhibitor (drug) results in cell cycle arrest or not.
Exposure of mammalian cells to growth factors or genotoxic stress elicits a variety of cellular responses, including the activation of protein kinase cascades involving ERKs, stress-activated protein kinases (SAPK/JNK) and p38 MAPK.35
Therefore, we determined the effect of -gingerol on the activation of extracellular signal-regulatted protein kinase-1/2 (ERK1/2), p38 MAPK, and JNK, which are representative MAPKs involved in a wide array of cellular signaling cascade. PI3K/AKT pathway has an important role in preventing cells from undergoing apoptosis and contributing to the pathogenesis of malignancy.36
More recently evidences have suggested that this pathway is also associated with the regulation of cell cycle progression.22
The anti-cancer activities of  gingerol could be associated with a control of signal transduction of PI3K/AKT pathway and this led us to investigate the change of the survival pathway-associated proteins. In the both cells -gingerol could not affect the expression of the regulatory subunit of PI3K, p85α. However, -gingerol increased phosphorylation of AKT, which is regulated by PI3K, in only the HPAC cells. Activated AKT is known to promote the cell survival by anti-apoptoic mechanism.37
Additionally, activated AKT also phosphorylates and inactivates the proapoptotic protein, BAD.22,36,38
In HPAC cells, the increase of phospholated AKT might protect apoptosis, despite of cell cycle arrest by -gingerol. On the other hand, there was no change in phosphorylation of AKT by -gingerol-treated BxPC-3 cells and thus failed to counteract the gingerol-induced apoptotic cell death.
In conclusion, we describe experiments that show -gingerol induces apoptotic cell death in p53-mutant cancer cells. The death mechanism was characterized, revealing that -gingerol not only initiated cell cycle arrest but ultimately caused cell death through apoptosis. Thus, -gingerol, is capable of killing cancer cells expressing mutant p53, overcoming the phenotypic resistance to chemotherapy- and irradiation-induced cell death. These findings support the importance of studying -gingerol and gingerol-related compounds as anticancer agents that can potentially eradicate tumors resistant to radiation and to currently available chemotherapy.