Gemcitabine combined with gum mastic causes potent growth inhibition and apoptosis of pancreatic cancer cells

Abstract

Aim: To investigate the antiproliferative and apoptotic effects of gemcitabine combined with gum mastic and the underlying mechanisms in human pancreatic cancer cell lines.

Methods: Cell proliferation and apoptosis were examined using the methyl thiazolyl tetrazolium (MTT) assay and propidium iodine staining, respectively. The expression of Bcl-2, Bax, NF-kappaB p65 subunit, and IkappaBalpha protein was measured using Western blotting.

Results: Gemcitabine 0.01-100 microg/mL inhibited cell proliferation and induced apoptosis in both pancreatic cancer BxPC-3 and COLO 357 cells. Gum mastic 40 microg/mL significantly potentiated the antiproliferative and apoptotic effects of gemcitabine 10 microg/mL after 72-h treatment. When cells were treated with gemcitabine in combination with gum mastic, the IkappaBalpha level was increased, whereas NF-kappaB activation was blocked; the expression of Bax protein was substantially increased, but Bcl-2 protein was down-regulated.

Conclusion: Gemcitabine combined with gum mastic causes potent apoptosis in pancreatic cancer cells. The combination may be an effective therapeutic strategy for pancreatic cancer.

Figure 1

The inhibition of cell proliferation by either gemcitabin (0.01–100 μg/mL) or gum mastic (10–50 μg/mL) alone (A) or the combination (B). The concentration of gemcitabine (10 μg/mL) and gum mastic (40 μg/mL) used in Figure 1B is based on the result from Figure 1A. Results are representative of 3 independent experiments. ^cP<0.01.

Figure 2

Effect of gemcitabine and gum mastic on cell apoptosis. BxPC-3 (4×10⁵ cells per well) and COLO 357 cells (3×10⁵ cells per well) were incubated with either gemcitabine (10 μg/mL) or gum mastic (40 μg/mL) or in combination. After 48 h, cell apoptosis was examined by Annexin V-PI double-labeling and FACS analysis. ^bP<0.05, ^cP<0.01 vs gemcitabine or gum mastic alone.

Figure 3

NF-κB activation can be inhibited by combined treatment with gemcitabine and gum mastic. (A)Western blot analysis for NF-κB in nuclear extracts of BxPC-3 cells treated with 10 μg/mL gemcitabine at different time points. (B) Western blot analysis for NF-κB p65 subunit in nuclear extracts of both BxPC-3 and COLO 357 cells after 48 h treatment with cell medium (lane 1), gum mastic (lane 2), gemcitabine (lane 3) or their combination (lane 4). β-actin protein was used as an internal control. Densitometric measurement for NF-κB p65 protein levels was normalized to internal control, respectively, and expressed as a relative value.

Figure 4

The treatment of gemcitabine combined with gum mastic alters the expression of Bcl-2, Bax, and IκBα. The expression of Bcl-2, Bax, and IκBα was analyzed by Western blot. BxPC-3 cells were treated with cell medium, 40 μg/mL of gum mastic, 10 μg/mL of gemicitabine or their combination for 48 h. β-actin was used as an internal control. Densitometric measurement for these proteins levels was normalized to internal control, respectively, and expressed as a relative value.