Curcumin inhibits AP-2γ-induced apoptosis in the human malignant testicular germ cells in vitro

Abstract

Aim: To investigate the effects of curcumin on proliferation and apoptosis in testicular cancer cells in vitro and to investigate its molecular mechanisms of action.

Methods: NTera-2 human malignant testicular germ cell line and F9 mouse teratocarcinoma stem cell line were used. The anti-proliferative effect was examined using MTT and colony formation assays. Hoechst 33258 staining, TUNEL and Annexin V-FITC/PI staining assays were used to analyze cell apoptosis. Protein expression was examined with Western blot analysis and immunocytochemical staining.

Results: Curcumin (5, 10 and 15 μmol/L) inhibited the viability of NTera-2 cells in dose- and time-dependent manners. Curcumin significantly inhibited the colony formation in both NTera-2 and F9 cells. Curcumin dose-dependently induced apoptosis of NTera-2 cells by reducing FasL expression and Bcl-2-to-Bax ratio, and activating caspase-9, -8 and -3. Furthermore, curcumin dose-dependently reduced the expression of AP transcription factor AP-2γ in NTera-2 cells, whereas the pretreatment with the proteasome inhibitor MG132 blocked both the curcumin-induced reduction of AP-2γ and antiproliferative effect. Curcumin inhibited ErbB2 expression, and decreased the phosphorylation of Akt and ERK in NTera-2 cells.

Conclusion: Curcumin induces apoptosis and inhibits proliferation in NTera-2 cells via the inhibition of AP-2γ-mediated downstream cell survival signaling pathways.

Figure 1

Curcumin treatment inhibited the proliferation of testicular germ cell lines. (A) Curcumin inhibited the viability of NTera-2 cells at 8, 16, 24, and 32 h, in a time- and dose-dependent manner. ^bP<0.05, ^cP<0.01 vs control group. (B) Cell viability was determined using colony formation assays. Testicular cancer cells were incubated with curcumin (10 μmol/L) for 24 h, and then the cells were allowed to grow into colonies for 15 d. Incubation with curcumin inhibits colony formation. Data represent the mean±SD of three independent experiments. ^cP<0.01 vs control cells using the Student's t-test.

Figure 2

Effects of curcumin on apoptosis in NTera-2 cells. (A) The morphology of apoptotic nuclei was observed after Hoechst staining using a fluorescence microscope (magnification ×40). The control group was treated with DMSO. (B) A TUNEL Apoptosis Assay Kit was used to detect apoptosis in NTera-2 cells. The brown staining, indicative of apoptotic nuclei, significantly increased with increasing concentration of curcumin. (C) The apoptosis rate was analyzed by flow cytometry. Numbers in the respective quadrant profiles indicate the percentage of apoptotic cells. Each bar corresponds to the mean±SD of three independent experiments. ^bP<0.05, ^cP<0.01 vs control cells.

Figure 3

Curcumin modulates the expression of apoptotic genes in NTera- 2 cells. Cells were treated with different concentrations of curcumin for 24 h, as indicated. (A) Curcumin reduced the expression of the anti-apoptotic proteins, Bcl-2 and FasL, whereas it increased the expression of pro-apoptotic proteins compared to untreated cells. (B) Western blotting analyses showed a reduction of cytochrome c and the cleavage of caspase-9, -8, -3, and PARP in response to various concentrations of curcumin. β-actin was used as a control. One representative of three different experiments is shown.

Figure 4

The effect of siRNA-AP-2γ was consistent with that of curcumin treated in NTera-2 cells. (A) NTera-2 cells were treated with indicated dose of curcumin. RT-PCR and Western blot assays were used to monitor AP-2γ expression. Expression was normalized to GAPDH. (B) Western blot analysis of AP-2γ expression, cleaved PARP, caspase-8, caspase-9, caspase-3, and β-actin after 24 h treatment with siRNA-AP-2γ or siRNA-NC.

Figure 5

The proteasome inhibitor, MG132, blocks the curcumin-mediated reduction of AP-2γ. (A) Anti-AP-2γ Western blot of whole cell extracts incubated with the MG132 (100 μmol/L) for 5 h, prior to curcumin (10 μmol/L) treatment for 24 h. (B) Immunocytochemical staining of AP-2γ expression. (C) MG132 reversed the curcumin-mediated inhibition of cell proliferation, as determined by MTT assays. Data represent the mean±SD of three independent experiments. ^cP<0.01 vs curcumin.

Figure 6

Effect of curcumin on the expression of ErbB2 and the phosphorylation of ERK and AKT in NTera-2 cells. β-actin was used as an internal standard. Bands were analyzed using ImageJ software. The values represent the mean±SD of three assays. ^bP<0.05, ^cP<0.01 vs control cells.