Accumulating evidence demonstrates the inhibitory effects of 6-gingerol on the development of cancer [4
]. However, underlying mechanisms by which 6-gingerol affects human CRC have not been determined. In this study, we demonstrated that 6- gingerol inducedG1
cell cycle arrest and apoptosis in human colorectal cancer cells. 6-Gingerol suppressed cyclin D1 expression by inhibiting the transcriptional regulation and by activating proteolysis of cyclin D1. In addition, 6-gingerol activated NAG-1 expression through the activation of the PKCε and GSK3 pathways. These results demonstrate that 6-gingerol is a pungent compound that affects many pathways involved in tumorigenesis.
Colorectal tumorigenesis is affected by many signaling pathways including COX-2, β-catenin, p53, APC, and mismatch repair. 6-Gingerol uniformly inhibits cell proliferation in various human colorectal cancer models, including HCT-116, SW480, HT-29, LoVo, and Caco-2 cells, suggesting that suppression of cell growth by 6-gingerol is a common phenomenon in human CRC. However, the influence of 6-gingerol on apoptosis is inconsistent, inducing it in HCT-116, SW480, and LoVo cells, but not changing it in HT-29 and Caco-2 cells (). Because HT-29 and Caco-2 cells are COX- 2 expressing cells, while HCT-116, SW480, and LoVo cells are COX-2 negative cells [44
], it is likely that 6- gingerol-induced apoptosis is affected by COX-2 expression. Along with this finding, we do not exclude the possibility that inflammatory status may affect 6-gingerol’s effects. Therefore, further studies are needed to elucidate the involvement of COX-2 in NAG-1-mediated apoptosis by 6-gingerol.
6-Gingerol induced G1
arrest in HCT-116 cells and LoVo cells, but not in SW480. It is notable that HCT- 116 and LoVo cells are p53-WT while SW480 is p53 mutant [45
]. It has been known that inactivation of p53 results in chemoresistance by chemopreventive drugs in many types of cancer cells including colon cancer cell lines. Actually, colon tumor cell lines expressing wild-type p53 are more sensitive to 5-aza- CdR-mediated growth arrest [46
], and p53 mutation decreased sensitivity to the G1
/S interface of the cell cycle in prostate cancer cells [47
]. Thus, it is assumed that effect of 6-gingerol on cell cycle might be dependent on p53 status.
The effect of 6-gingerol on β-catenin/TCF-dependent gene transcription can be important for the 6- gingerol induced anti-tumorigenesis. The importance of this effect is clearly indicated by the reduced expression of cyclin D1, a protein that plays an important role in cell-cycle transitions. 6-Gingerol led to a sustained suppression of cyclin D1 levels, a result consistent with the inhibition of G1
to S transitions and hypophosphorylation of p-Rb in HCT-116 cells. Our results indicate that there are multiple mechanisms by which 6-gingerol represses cyclin D1 expression. One of these mechanisms is through transcriptional regulation. The mRNA of cyclinD1 was decreased by 6-gingerol, and the cyclin D1 promoter assay, as determined by the reporter gene, indicated that the 6-gingerol response element is located between −163 and +130 of the 5′ region of the cyclin D1 promoter. Indeed, it has been reported that the TCF/LEF site is located in this region of the promoter and plays an important role in β-catenin-dependent transcriptional regulation of cyclin D1 [14
]. In our studies, 6-gingerol decreased localization of β-catenin, which is followed by the inhibition of cyclin D1 expression at the transcriptional level. Another mechanism to suppress cyclin D1 expression is the activation of proteasome degradation. Our results are similar to those previously reported with curcumin [38
], retinoic acid [39
], and troglitazone [40
], which downregulate cyclin D1 by proteolysis. This bidirectional downregulation of cyclin D1 supports previous reports that curcumin suppresses cyclin D1 transactivation as well as activates proteolysis [38
GSK-3β is one of the primary target genes of the PI3K/AKT pathway and mediates apoptotic signals. In our previous study, transfection of specific GSK-3 siRNA blocked the induction of NAG-1 by the PI3K inhibitor [43
], and GSK3 is the critical mediator of NAG-1 regulation by conjugated linoleic acid and LY294002 [29
]. The current study supports that GSK-3β acts as an upstream target of NAG-1 expression and is responsible for NAG-1 induction by 6- gingerol. In addition, we found that PKCε is another mediator of NAG-1 induction by 6-gingerol. PKC constitutes a family of serine-threonine kinases, which are classified into three major groups based on their structure and activation mechanisms: conventional (α, βI, βII, γ), novel (δ, ε, η, θ, μ), and atypical (ζ, λ) [17
]. Of the novel PKC isoforms, colon carcinoma cell lines express predominantly the PKCε and PKCδ isoforms [48
]. Although PKCδ mediates NAG-1 expression in prostate cancer cells [42
], our studies using PKC inhibitors and interferences of PKCε showed that PKCε at least partially mediates NAG-1 expression by 6-gingerol. PKCε’s effects on apoptosis and tumorigenesis are controversial. PKCε has been known to stimulate proliferation and inhibit apoptosis in prostate [18
], glioma [19
], and squamous carcinoma cells [20
]. On the other hand, PKCε mediates the ability of vitamin D3 to prevent adenomas in colon tumors [21
], and overexpression of PKCε sensitized LNCaP cells to induce apoptosis [22
]. In addition, inactivation of PKCε using dominant- negative transfection has showed marked resistance to apoptosis in thyroid carcinoma cells [23
]. Thus, the effect of PKCε on apoptosis may differ in tissues and cell types. In this study, we have shown that PKCε plays a pivotal role in 6-gingerol-induced NAG-1 expression, probably resulting in the induction of 6-gingerol-induced apoptosis.
The suppression of cyclin D1 and induction of NAG-1 by 6-gingerol were observed at 150 and 200 μM concentrations. This is in agreement with other observations that at least 300 μMof 6-gingerol was required to induce apoptosis [7
]. It is believed that a higher concentration of 6-gingerol is required for inducing apoptosis in CRC and leukemia, but lower concentrations (30–50 μMof 6-gingerol) were effective to inhibit angiogenesis in endothelial cells [9
] and skin carcinoma [5
]. In addition, the large intestinal epithelia, including colon and rectum, could be highlighted as logical target tissue to further explore the relationship between 6-gingerol and colorectal cancer prevention. Investigation of ginger accumulation in the hindgut would serve to expand our understanding of the role of 6-gingerol in prevention of colorectal cancers. The efficacy of some metabolites of 6-gingerol will be particularly interesting because 6-gingerol in rats is biotransformed to various metabolites by gut flora and liver enzyme when orally administered [49
]. In this regard, it is important that future studies expand beyond in vitro cell culture models to relevant in vivo models, addressing absorption and distribution of 6-gingerol and its metabolites.
In conclusion, the current study provides information on cellular events of pro-apoptotic and antitumorigenic activity by 6-gingerol. As depicted in , 6-gingerol inhibits the transcription of cyclin D1 by suppression of β-catenin translocation into the nucleus and subsequent lowered β-catenin signaling in the nucleus. 6-Gingerol also leads to an increase of cyclin D1 proteolysis through proteosomal degradation. Downregulation of cyclin D1 results in suppression of Rb phosphorylation, resulting in cell growth arrest. In addition, 6-gingerol upregulates NAG-1 expression through GSK-3β and PKCε-mediated pathways. The resulting NAG-1 activation induces an apoptosis signaling pathway in colorectal cancer cells.
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 (more ...)