Inhibitory effect of baicalein combined with gemcitabine in human pancreatic cancer cell lines

Abstract

Pancreatic cancer is an aggressive disease with a particularly poor prognosis contributing to a substantial percentage of cancer-associated mortality rates. In the present study, the combination treatment of baicalein (BAI) and gemcitabine (GEM) was investigated to examine whether it inhibited the growth of the human CFPAC-1 pancreatic cancer cell line in vitro and in vivo. The cytotoxic interactions between BAI and GEM in human pancreatic cancer cell lines were determined using MTT assays, and the effect of the two agents on apoptosis was detected using Hoechst 33258 staining and annexin V/7-AAD. The protein levels of Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), caspase-3, poly ADP ribose polymerase (PARP) and survivin were detected using western blot analysis. Furthermore, the expression levels of Bax, Bcl-2, caspase-3 and survivin in tumor tissues were detected using immunohistochemistry. The results demonstrated that following GEM treatment, the growth of CFPAC-1 cells and xenografts in nude mice were inhibited, and the expression levels of Bcl-2 and survivin were downregulated, whilst the expression levels of Bax, caspase-3 and PARP were upregulated. These effects were enhanced with the use of BAI in combination with GEM. The mechanism underlying the anti-tumor effect of BAI combined with GEM may be associated with the induction of cell apoptosis and the inhibition of proliferation. To the best of our knowledge, this is the first evidence of the efficacy of BAI against pancreatic cancer and may provide the potential clinical evidence for the use of this drug combination for the treatment of patients with pancreatic cancer.

Keywords: apoptosis; baicalein; gemcitabine; pancreatic cancer.

Figure 1.

Inhibitory effect of GEM combined with BAI treatment on the cell viability of the human CFPAC-1 and PANC-1 pancreatic cancer cell lines in vitro. (A) MTT assay revealed that an increased BAI concentration (3.2–100 µM) significantly decreased cell viability in CFPAC-1 cells compared with that in the control. BAI significantly enhanced the inhibitory effect of GEM treatment in (B) CFPAC-1 and (C) and PANC-1 cell lines. *P<0.05, ***P<0.001 and ****P<0.0001 vs. the control or with comparisons indicated by lines. GEM, gemcitabine; BAI, baicalein.

Figure 2.

BAI augments apoptosis induced by GEM in human CFPAC-1 and PANC-1 pancreatic cancer cell lines. Morphological analysis of nuclear chromatin was performed using Hoechst 33258 staining in CFPAC-1 cells treated with (A) 1.6 µM or (B) 6.25 µM BAI, in addition to 0.125 µg/ml GEM alone or in combination for 48 h. (C) PANC-1 cells were treated with 6.25 µM BAI and 0.125 µg/ml GEM alone or in combination for 48 h using Hoechst 33258 staining. Early and late apoptotic events in the cells were determined using Annexin V/7-AAD staining and flow cytometry staining in CFPAC-1 cells treated with (D) 1.6 µM or (E) 6.25 µM BAI, in addition to 0.125 µg/ml GEM alone or in combination for 48 h. (F) PANC-1 cells were treated with 6.25 µM BAI and 0.125 µg/ml GEM alone or in combination for 48 h using Annexin V/7-AAD staining. Images were captured using a light microscope at a magnification of ×20. ***P<0.001 and ****P<0.0001 with comparisons indicated by lines. GEM, gemcitabine; BAI, baicalein.

Figure 3.

Effects of BAI alone, GEM alone or combined treatment on the protein expression levels of Bax, Bcl-2, caspase-3, PARP and survivin in the human CFPAC-1 pancreatic cancer cell line. (A) Representative images of Bax, Bcl-2, caspase-3, PARP and survivin protein expression, generated using a western blot analysis. β-actin was used as a loading control. Quantification of the intensity of (B) Bax, (C) Bcl-2, (D) caspase-3, (E) PARP and (F) and survivin. **P<0.01 and ***P<0.001 with comparisons indicated by lines. GEM, gemcitabine; BAI, baicalein; Bax, Bcl-2 associated protein X; Bcl-2, B-cell lymphoma 2; PARP, poly ADP ribose polymerase.

Figure 4.

Inhibitory effect of combined treatment with BAI and GEM on the proliferation of the human CFPAC-1 pancreatic cancer cell line in vivo. Representative images of (A) mice and (B) tumors. (C) Tumor volume changes in the xenografts. Resected tumors revealed that combination therapy decreased the tumor volume significantly compared with either the control group or the BAI-alone treated group. The data are presented as the mean ± SEM. (D) Following 28 days of treatment, the tumor weights were significantly decreased in all 3 treatment groups compared with the control group. The data are presented as the mean ± SEM. Representative immunohistochemical staining images and quantification for (E) Bcl-2, (F) Bax, (G) caspase-3 and (H) survivin from all groups of mice following treatment with BAI alone, GEM alone or a combination of the 2 treatments. Images were captured using a light microscope at a magnification of ×20. **P<0.01, ***P<0.001 and ****P<0.0001 vs. the control group or with comparisons indicated by lines. GEM, gemcitabine; BAI, baicalein; Bax, Bcl-2 associated protein X; Bcl-2, B-cell lymphoma 2; SEM, standard error of the mean.