Icariin potentiates the antitumor activity of gemcitabine in gallbladder cancer by suppressing NF-κB

Abstract

Aim: Gemcitabine has been increasingly prescribed for the treatment of gallbladder cancer. However, the response rate is low. The aim of this study is to determine whether icariin, a flavonoid isolated from Epimedi herba, could potentiate the antitumor activity of gemcitabine in gallbladder cancer.

Methods: Human gallbladder carcinoma cell lines GBC-SD and SGC-996 were tested. Cell proliferation and apoptosis were analyzed using MTT assay and flow cytometry, respectively. The expression of apoptosis- and proliferation-related molecules was detected with Western blotting. Caspase-3 activity was analyzed using colorimetric assay, and NF-κB activity was measured with ELISA. A gallbladder cancer xenograft model was established in female BALB/c (nu/nu) mice. The mice were intraperitoneally administered gemcitabine (125 mg/kg) in combination with icariin (40 mg/kg) for 2 weeks.

Results: Icariin (40-160 μg/mL) dose-dependently suppressed cell proliferation and induced apoptosis in both GBC-SD and SGC-996 cells, with SGC-996 cells being less sensitive to the drug. Icariin (40 μg/mL) significantly enhanced the antitumor activity of gemcitabine (0.5 μmol/L) in both GBC-SD and SGC-996 cells. The mice bearing gallbladder cancer xenograft treated with gemcitabine in combination with icariin exhibited significantly smaller tumor size than the mice treated with either drug alone. In GBC-SD cells, icariin significantly inhibited both the constitutive and gemcitabine-induced NF-κB activity, enhanced caspase-3 activity, induced G(0)-G(1) phase arrest, and suppressed the expression of Bcl-2, Bcl-xL and surviving proteins.

Conclusion: Icariin, by suppressing NF-κB activity, exerts antitumor activity, and potentiates the antitumor activity of gemcitabine in gallbladder cancer. Combined administration of gemcitabine and icariin may offer a better therapeutic option for the patients with gallbladder cancer.

Figure 1

Cell growth inhibition and apoptosis induction by icariin in GBC-SD (A) and SGC-996 (B) cells, and the effect of icariin on normal mouse gallbladder cells (C). Cells were treated with the indicated concentration of Icariin for 24 h. Cell viability was analyzed by the MTT assay, and apoptosis was measured by flow cytometry. Mean±SD. n=3. ^bP<0.05, ^cP<0.01 vs 0 μg/mL icariin.

Figure 2

Downregulation of apoptosis-related proteins (A), increased caspase-3 activity (B) and decreased NF-κB activity (C) in GBC-SD cells following icariin treatment. Cells were treated with the indicated concentration of Icariin for 24 h. Expression of apoptosis-related proteins was analyzed by Western blotting; caspase-3 activity was measured by a colorimetric assay; NF-κB activity was determined using an ELISA. Mean±SD. ^bP<0.05, ^cP<0.01 vs 0 μg/mL icariin.

Figure 3

Enhanced anti-proliferative activity (A) and induction of apoptosis (B) of gemcitabine when administered in combination with icariin and representative cytometric apoptosis graphs (C). Cells were treated with both icariin (40 μg/mL) and gemcitabine (0.5 μmol/L) for 24 h. Cell viability was analyzed by the MTT assay, and apoptosis was measured by flow cytometry. Mean±SD. n=3. ^bP<0.05, ^cP<0.01 vs the vehicle.

Figure 4

Downregulation of apoptosis-related proteins (A), increased caspase-3 activity (B), decreased NF-κB activity (C), and enhanced G₀-G₁ cell cycle arrest (D) in GBC-SD cells treated with both gemcitabine and Icariin with representative cell cycle analysis graphs (E). GBC-SD cells were treated with both icariin (40 μg/mL) and gemcitabine (0.5 μmol/L) for 24 h. Expression of apoptosis-related proteins was analyzed by western blotting; caspase-3 activity was measured by a colorimetric assay; NF-κB activity was determined using an ELISA. Cell cycle distribution is presented as the mean. mean±SD. n=3. ^bP<0.05 vs the vehicle; ^eP<0.05, ^fP<0.01 vs the treatment with icariin or gemcitabine alone.

Figure 5

Measurements of gallbladder tumor volume at the indicated time points, depicting the in vivo therapeutic efficacy of icariin and gemcitabine (A) with a representative picture of a tumor at the end of the experiment (B). n=15. ^bP<0.05 vs animals treated with vehicle. ^eP<0.05 vs animals treated with icariin or gemcitabine alone.