[6]-Gingerol induces caspase-dependent apoptosis and prevents PMA-induced proliferation in colon cancer cells by inhibiting MAPK/AP-1 signaling

Abstract

We report mechanism-based evidence for the anticancer and chemopreventive efficacy of [6]-gingerol, the major active principle of the medicinal plant, Ginger (Zingiber officinale), in colon cancer cells. The compound was evaluated in two human colon cancer cell lines for its cytotoxic effect and the most sensitive cell line, SW-480, was selected for the mechanistic evaluation of its anticancer and chemopreventive efficacy. The non-toxic nature of [6]-gingerol was confirmed by viability assays on rapidly dividing normal mouse colon cells. [6]-gingerol inhibited cell proliferation and induced apoptosis as evidenced by externalization of phosphatidyl serine in SW-480, while the normal colon cells were unaffected. Sensitivity to [6]-gingerol in SW-480 cells was associated with activation of caspases 8, 9, 3 &7 and cleavage of PARP, which attests induction of apoptotic cell death. Mechanistically, [6]-gingerol down-regulated Phorbol Myristate Acetate (PMA) induced phosphorylation of ERK1/2 and JNK MAP kinases and activation of AP-1 transcription factor, but had only little effects on phosphorylation of p38 MAP kinase and activation of NF-kappa B. Additionally, it complemented the inhibitors of either ERK1/2 or JNK MAP kinase in bringing down the PMA-induced cell proliferation in SW-480 cells. We report the inhibition of ERK1/2/JNK/AP-1 pathway as a possible mechanism behind the anticancer as well as chemopreventive efficacy of [6]-gingerol against colon cancer.

Figure 1. Cytotoxic effects of [6]-gingerol on SW-480 colon cancer cells.

(A) Phase contrast microscopy images of morphological changes in SW-480 cells when treated with indicated concentrations of [6]-gingerol for 72 h. (B) Relative cell viability of SW-480 cells after [6]-gingerol treatment, determined by MTT assay and expressed as percentage of the untreated control. 5000 cells/well of SW-480 cells were treated with the indicated concentration of [6]-gingerol for 72 h prior to MTT assay.(C) Relative cell viability of HCT-116 cells after [6]-gingerol treatment. The results are represented as mean of triplicate experiments ± standard deviation (SD).

Figure 2.Comparison. Comparison of cytotoxic effects of [6]-gingerol on SW-480 colon cancer cells with mouse normal intestinal epithelial cells (IECs).

(A) Phase contrast microscopy images of IECs treated with indicated concentrations of [6]-gingerol for 72 h. (B) Time dependent cytotoxic effects of [6]-gingerol on SW-480 and IECs at 48, 72 and 96 h. 5000 cells/well of SW-480 and 10,000 cells/well of IECs were treated for 48 to 96 h with indicated doses of [6]-gingerol prior to MTT assay. The results are represented as mean ± SD from triplicate experiments.

Figure 3. Effects of [6]-gingerol on induction of apoptosis in SW-480 cells and IECs.

(A, upper panel) SW-480 cells were treated with indicated concentrations of [6]-gingerol for 16 h and stained for Annexin V/propidium iodide (PI) positivity. Green-fluorescence indicates Annexin V binding to the damaged cell membrane during early apoptosis and the red fluorescence indicates PI binding to the exposed DNA during late apoptosis. (A, lower panel) Graphical representation of the percentage of Annexin V/PI positive SW-480 cells in relation to [6]-gingerol concentration. (B, upper panel) IECs were treated with up to 500 µM of [6]-gingerol for 16 h before performing Annexin V-PI staining. Treatment with 100 µM 5-FU was used as positive control for apoptosis induction. (B, lower panel) Representative histogram of the percentage of Annexin/PI positive IECs following [6]-gingerol/5-FU treatment. The results are represented as mean from triplicate experiments ± SD.

Figure 4. Effects of [6]-gingerol on activation of caspases and PARP cleavage in SW-480 cells.

SW-480 cells (10⁶ cells/well) were treated with the indicated concentrations of [6]-gingerol for 48 h and the whole cell extracts were Western blotted on to PVDF membrane. The activation of caspases and PARP cleavage were detected by probing the blotted membrane with antibodies against Caspase-3, 7, 8, 9 and against PARP. The blots were developed using Enhanced Chemiluminescence (ECL). The relative fold differences of bands with control treatments were quantified from volume analysis of the bands using Biorad- Quantity One software. β-actin served as the loading control in each case.

Figure 5. Effect of [6]-gingerol on PMA-induced phosphorylation of MAP kinases and activation of AP-1 and NF-kappaB in SW-480 cells.

(A) Overnight grown SW-480 cells were treated with 50 ng/ml of PMA for different time intervals (0–120 min) and the whole cell lysate was immunoblotted onto PVDF membrane, probed using antibodies against phospho-ERK1/2, phospho-JNK and phospho-p38, developed by ECL. The kinetics of PMA-induced phosphorylation of these MAP kinases were followed from this blot (B) SW-480 cells were pre-treated with 200 µM [6]-gingerol before treating with 50 ng/ml of PMA for 30 min and the whole cell lysates were immunoblotted, probed and detected as above. The relative fold difference of bands with control treatments are indicated below each lane. β-actin served as the loading control in each case. (C) SW-480 cells, pre-treated with indicated concentrations of [6]-gingerol for 2 h, were treated with PMA (50 ng/ml) for 30 min. The nuclear extracts from each treatment were analysed for the activation of AP-1 or (D) NF-kappaB, by performing the transcriptional binding assay on isotope labelled AP-1/NF-kappaB specific DNA binding probe and analysing it on electrophoretic mobility shift assay (EMSA). The arrowhead in each case indicates complexes between AP-1/NF-kappaB and DNA probe.

Figure 6. Effects of [6]-gingerol on PMA-induced cell proliferation in SW-480 in presence of inhibitors of MAP kinases and NF-kappaB.

(A) Overnight grown SW-480 cells were pre-treated with inhibitors (U0126, SP600125, SB203580 for ERK1/2, JNK and p38 MAP kinases, respectively or SN50 for NF-kappaB) for 1 h and then treated with [6]-gingerol for 2 h, followed by exposure to PMA for 48 h, before performing cell viability assay using MTT. The relative viability of SW-480 cells in each treatment represented as percentage of untreated control. The values presented are mean of triplicate experiments ± SD. Total lysates from cells treated with combination of [6]-gingerol with (B) U0126 and (C) SP600125 inhibitors and from appropriate control treatments were Western blotted and probed with anti-caspase-3 antibody and the blots were developed using ECL. Beta-actin served as the loading control.