Terpenoids from Zingiber officinale (Ginger) Induce Apoptosis in Endometrial Cancer Cells through the Activation of p53

Novel strategies are necessary to improve chemotherapy response in advanced and recurrent endometrial cancer. Here, we demonstrate that terpenoids present in the Steam Distilled Extract of Ginger (SDGE) are potent inhibitors of proliferation of endometrial cancer cells. SDGE, isolated from six different batches of ginger rhizomes, consistently inhibited proliferation of the endometrial cancer cell lines Ishikawa and ECC-1 at IC50 of 1.25 mg/ml. SDGE also enhanced the anti-proliferative effect of radiation and cisplatin. Decreased proliferation of Ishikawa and ECC-1 cells was a direct result of SDGE-induced apoptosis as demonstrated by FITC-Annexin V staining and expression of cleaved caspase 3. GC/MS analysis identified a total of 22 different terpenoid compounds in SDGE, with the isomers neral and geranial constituting 30–40%. Citral, a mixture of neral and geranial inhibited the proliferation of Ishikawa and ECC-1 cells at an IC50 10 mM (2.3 mg/ml). Phenolic compounds such as gingerol and shogaol were not detected in SDGE and 6-gingerol was a weaker inhibitor of the proliferation of the endometrial cancer cells. SDGE was more effective in inducing cancer cell death than citral, suggesting that other terpenes present in SDGE were also contributing to endometrial cancer cell death. SDGE treatment resulted in a rapid and strong increase in intracellular calcium and a 20–40% decrease in the mitochondrial membrane potential. Ser-15 of p53 was phosphorylated after 15 min treatment of the cancer cells with SDGE. This increase in p53 was associated with 90% decrease in Bcl2 whereas no effect was observed on Bax. Inhibitor of p53, pifithrin-a, attenuated the anti-cancer effects of SDGE and apoptosis was also not observed in the p53neg SKOV-3 cells. Our studies demonstrate that terpenoids from SDGE mediate apoptosis by activating p53 and should be therefore be investigated as agents for the treatment of endometrial cancer

SDGE induces apoptosis in endometrial cancer cells.

<p>Ishikawa cells were treated with 250 ng/ml of SDGE for 30 min, 2 h, 4 h, and 16 h. After incubation with SDGE, cell survival was determined by labeling the cells with FITC-conjugated FITC-Annexin V and propidium iodide (A). The cells were analyzed by flow cytometry and cell death and apoptosis were identified as the events that were single positive for FITC-Annexin V (lower right quadrant) or double positive for both FITC-Annexin V and proidium iodide (upper right quadrant). SDGE-induced apoptotic cell death in the endometrial cancer cells was confirmed by detecting cleaved caspase 3 in western blot analysis (B). An increase in cleaved caspase 3 levels was observed when the Ishikawa cells were treated with SDGE (250 ng/ml) for 0, 24, 48, and 72 h. Data in A and B is representative of results obtained in three separate experiments,</p

SDGE mediates endometrial cancer cell apoptosis through the activation of p53.

<p>After treating Ishikawa cells with SDGE (250 ng/ml) for the designated time points, the cells were harvested and lysates were analyzed by Western blotting for expression of Bcl-2 and Bax (A). Phosphorylation of Ser-15 of p53 was monitored in Ishikawa cells treated with SDGE (250 ng/ml) (B). Ishikawa cells were incubated with SDGE in the presence or absence of pifithrin-α for 18 h. Control Ishikawa cells were not exposed to either SDGE or pifithrin-α. (C) Cells were harvested and apoptosis was measured by flow cytometry after staining with FITC-Annexin V and propidium iodide. Apoptosis in the p53^neg SKOV-3 cells after treatment with SDGE (250 ng/ml) was also measured by the FITC-Annexin V assay (D) or by monitoring the levels of cleaved Caspase3 by Western blotting (E) at the designated time intervals.</p

Citral inhibits proliferation of endometrial cancer cells.

<p>MTT assays were conducted to determine the effect of 6-gingerol (A and B) and citral (C and D) on the proliferation of Ishikawa and ECC-1 cells. Each data point is a mean of 16 individual readings. Based on this data the IC₅₀ of citral was calculated to be 15–25 µM.</p

Terpenes are the major components of SDGE identified by GC-MS analysis.

<p>Three separate preparations of SDGE, two from the US (termed Wisconsin 1 and 2) and one from India were separated on a non-polar gas chromatography column. The compounds separating on this column were identified by mass spectrometry. Compounds identified through this analysis along with their relative abundances are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053178#pone-0053178-t001" target="_blank">Table 1</a>.</p

SDGE inhibits the proliferation of endometrial cancer cell lines.

<p>Effect of SDGE on cell proliferation was determined by conducting MTT assays. The cells Ishikawa (A), and ECC-1 (B) were incubated with the depicted concentrations of SDGE for 24, 48, and 72 h. Optical density at 570 nm was determined to quantify the number of live cells in the cultures. Control (Con) wells in all experiments were treated with DMSO, the vehicle control. Cumulative data for SDGE isolated from three different batches of ginger rhizomes is shown. Each data point in all figures is mean of 48 individual readings. Anti-proliferative effect produced by SDGE (2.5 µg/ml) in Ishikawa (C and E) and ECC-1 (D and F) cells was comparable to treatment of these two cell lines with radiation (C and D) or cisplatin (E and F). The cells were simultaneously treated with SDGE and radiation (4 Gy) or cisplatin (5 µM). MTT assays were conducted to determine the effect of these treatments on the proliferation of the Ishikawa and ECC-1 cell lines. Each bar in C–F represents a mean of eight replicates. *p<0.001, ^#p<0.05, and ^†p>0.05 (not significant).</p

SDGE increases intracellular calcium and decreases the mitochondrial membrane potential of endometrial cancer cells.

<p>Effect of SDGE on the intracellular calcium flux was determined by treating Indo-1 loaded Ishikawa cells (A). Immediately after addition of SDGE to the cell suspensions, the Ishikawa cells were flowed through the cytometer and increase in fluorescence was measured to detect calcium flux. Decrease in mitochondrial membrane potential was detected by loading Ishikawa cells with DiOC6 (B). SDGE or vehicle control was added to the cells. After 24 h of treatment, the cells were harvested and incubated with DiOC6 for 15 min. Fluorescence was measured to determine changes in mitochondrial membrane potential.</p

Terpenes identified by GC-MS in SDGE isolated from three separate batches of ginger.

<p>Terpenes identified by GC-MS in SDGE isolated from three separate batches of ginger.</p