Ginger inhibits growth in ovarian cancer cells as compared to non-transformed ovarian epithelial cells

Continuous exposure to ginger extract resulted in a marked reduction in cell growth after 1–5 days of exposure in A2780 ovarian cancer cells (Figure 1A, p < .0001 at all doses and time points). We tested additional ovarian cancer cell lines to determine if this was an effect unique to the A2780 ovarian cancer cells. Ginger treatment resulted in similar effects in all cell lines tested, including the chemoresistant cell lines SKOV3 and ES-2[39] (Figure 1B,C, p < .05 for all doses and time points). Untransformed human ovarian surface epithelial cells (HOSE) were minimally affected by ginger extract exposure at days 1 and 3, and showed some inhibition in growth by day 5 (Figure 1D, p > .05 for days 1 and 3, p < .05 for day 5). To confirm that ginger treatment inhibited cell growth, treated cells were analyzed by trypan blue exclusion as well. As expected, ginger treatment resulted in a profound inhibition of cell proliferation and growth at doses of 50 μg/ul and higher (Figure 2).

Figure 1 Continuous ginger exposure inhibits growth in ovarian cancer cells in vitro. A, B, C: The effect of ginger on growth of A2780, SKOV3 and ES2 ovarian cancer cell lines was assessed by using sulforhodamine B assays. Cells were incubated continuously with media containing ginger at the indicated concentrations and growth was assayed at Days 1, 3 and 5 of exposure. Ginger treated cells displayed significant growth inhibition as compared to control treated cells (p < .05 for all cell lines, all ginger concentrations). D: Human ovarian surface epithelial cells were treated with the indicated concentrations of ginger with minimal effect seen following days 1–3 of culture (p > .05). HOSE demonstrated some inhibition of growth by day 5 of treatment (p < .05). Data are presented as means ± S.D, and are representative of at least 3 independent experiments. Full size image

To determine whether lower doses of ginger could also inhibit cell growth, an extended range of concentrations was tested. In the A2780 and ES-2 cell lines, ginger concentrations of less than 50 μg/ml did not significantly impact cell growth, whereas in the SKOV3 cell line, some inhibition of cell growth was seen with ginger concentrations as low as 30 μg/ml (Figure 3 and data not shown).

Figure 2 Continuous ginger exposure inhibits growth in ovarian cancer cells in vitro. A, B, C: The effect of ginger on growth of A2780, SKOV3 and ES2 ovarian cancer cell lines was assessed using the trypan blue exclusion assay. Cells were incubated continuously with media containing ginger at the indicated concentrations and viable cells were counted on days Days 1, 3 and 5 of exposure. Ginger treated cells displayed significant growth inhibition as compared to control treated cells. Full size image

Figure 3 Low concentrations of ginger have minimal effect on ovarian cancer cell growth. A, B, C: The effect of ginger on growth of A2780, SKOV3 and ES2 ovarian cancer cell lines was assessed by using sulforhodamine B assays. Cells were incubated continuously with media containing ginger at the indicated concentrations and growth was assayed at Days 1, 3 and 5 of exposure. Using these concentrations of ginger, only SKOV3 cells displayed diminished cell growth. Full size image

6-Shogaol is the most active of the individual ginger components tested in ovarian cancer cells

Previous investigators have shown bio-activity of various individual ginger components in several tumor types [30, 40–43]. To determine the relative bio-activity in ovarian cancer, A2780 ovarian cancer cells were treated with 6-, 8- and 10-gingerol as well as 6-shogaol. In contrast to other published findings, we found that 6-, 8- and 10-gingerol had no effect on the growth or viability of ovarian cancer cells (p > .05 at all time points). Treating cells with whole ginger extract or 6-shogaol resulted in profound growth inhibition (Figure 4A, p < .05 at all time points for both ginger and 6-shogaol treated cells). Morphologically, cells treated with ginger appeared markedly growth inhibited, similar to cisplatin treated cells (Figure 5). Cells cultured with vehicle control (DMSO) continued to proliferate.

Figure 4 6-shogaol is the most effective individual ginger component in ovarian cancer cells. Transient ginger exposure results in a non-sustained decrease in proliferation of ovarian cancer cells. A: A2780 cells were treated for 24 hours with 75 μg/ml of ginger extract or 7.5 mM of each of the ginger standards. Media containing the indicated compounds was replenished at day 3. Growth was assayed via sulforhodamine B assays on Days 1, 3, 5, and 7. Data are presented as means ± S.D. B. A2780 cells were treated for 24 hours with indicated concentrations of ginger extract or 7.5 mM of 6-gingerol and 6-shogaol. Media containing the indicated compounds was washed off and replaced with complete media after 24 hours. Growth was assayed via sulforhodamine B assays on Days 1, 3, 5, and 7. Data are presented as means ± S.D. Full size image

Figure 5 Morphologic appearance of ginger treated ovarian cancer cells. A2780 ovarian cancer cells were incubated with DMSO solvent, ginger (75 μg/ml, replenished on day 3), or Cisplatin (2.5 μg/ml). Cells were examined by light microscopy at 1, 3, and 5 days of treatment. Full size image

We next determined if continuous exposure to individual ginger components was necessary to cause the growth inhibitory effect seen in ovarian cancer cells. Similar to the use of whole ginger root extract, continuous exposure to 6-shogaol was necessary to cause the growth inhibitory effect seen in ovarian cancer cells. We treated cells with ginger and individual ginger components for 24 hours, after which the cells were washed and media was changed. Once ginger or 6-shogaol was removed from the media, cell growth resumed (Figure 4B).

Ginger inhibits NF-κB in ovarian cancer cells

Because we found that ginger markedly suppressed ovarian cancer cell proliferation in vitro, and several genes that regulate proliferation are regulated by NF-κB, we hypothesized that ginger may mediate its anti-neoplastic activity in ovarian cancer cells though modulation of this pathway. Constitutive activation of NF-κB has been described in many tumor types including ovarian cancer[9], suggesting that targeting NF-κB may have an anti-neoplastic effect in this tumor type. Natural products such as ginger, or ginger components such as zerumbone can inhibit NF-κB in other cell types [16, 44, 45]. We chose two chemoresistant ovarian cancer cell lines (CaOV3 and SKOV3) to evaluate the effect of ginger treatment on activation of NF-κB. As shown in Figure 6, treatment with ginger extract resulted in a significant inhibition of NF-κB activation in CaOV3 and SKOV3 cell lines

Figure 6 Ginger inhibits NF-kB activation in ovarian cancer cells. Representative ovarian cancer cell lines with high endogenous NF-kB activation, (A) CaOV3 and (B) SKOV3 were transfected with an NF-κB-dependent reporter plasmid (pBVIx-Luc). Cells were treated with DMSO (vehicle control) or ginger (75 μg/ml). NF-κB activation was determined by measuring relative luciferase activity 48 hours after treatment. Luciferase activity is reported as arbitrary relative light units (mean +/- S.D.) Ginger treatment resulted in inhibition of NF-κB activation (p < .05 for both cell lines). Representative data is shown. Full size image

Ginger Inhibits IL-8 and VEGF Secretion in Ovarian Cancer Cells

IL-8 can function as a paracrine and/or autocrine growth factor in some tumor types, and the secretion of IL-8 protein from tumor cells themselves is thought to be crucial for these effects[46, 47]. In ovarian cancer patients, elevated IL-8 expression has been found in ascites as well as in serum[33]. Furthermore, IL-8 has been shown to stimulate proliferative growth in ovarian cancer cells in vitro[35]. Because IL-8 secretion is thought to be regulated in part by NF-κB, and ginger can clearly inhibit NF-κB in ovarian cancer cells, we hypothesized that ginger could also inhibit IL-8 secretion. Using a representative panel of ovarian cancer cell lines, we found that A2780 and CaOV3 cells produced negligible amounts of IL-8 (<0.05 pg/ml), whereas the cell lines ES-2 and SKOV3 had high constitutive expression of IL-8 (Figure 7A). Treatment with ginger resulted in significant inhibition of IL-8 production in the ES-2 and SKOV3 cell lines (p < .05 for both cell lines).

Figure 7 Ginger inhibits VEGF and IL-8 in ovarian cancer cells. Ovarian cancer cells were cultured with DMSO (vehicle control) or Ginger (75 μg/ml) for 48 hours. Production of the angiogenic factor s IL-8 (A) and VEGF (B) were assayed using ELISA assays. (A.) Only ES-2 and SKOV3 cells expressed high IL-8 levels at Baseline, and ginger treatment resulted in a significant decrease in IL-8 production (p < .05 for both cell lines). (B.) VEGF production was reduced in all cell lines following ginger treatment (p = .19, .18, .007, and .07 for A2780, CaOV3, ES-2 and SKOV3 cells respectively). Full size image

VEGF, the most important inducer of angiogenesis, is also under transcriptional control of NF-κB[9]. Serum VEGF levels as well as tumor expression of VEGF are associated with poor prognosis in ovarian cancer patients [31], and inhibition of VEGF function using Avastin™ has shown promise in the treatment of ovarian cancer patients[48]. Because ginger treatment resulted in inhibition of NF-κB, we next sought to determine whether ginger could similarly inhibit VEGF in ovarian cancer cells. In all cell lines tested, there was high endogenous production of VEGF, and ginger treatment resulted in inhibition of VEGF secretion. Inhibition of VEGF secretion was most evident in the ES-2 cell line (p = .007), as compared to the other cell lines tested (p = .19, .18, and .07 for A2780, CaOV3, and SKOV3 respectively, Figure 7B).