Cell lines and cultures

The ovarian cancer cell line A2780 was purchased from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in RPMI 1640 medium (Gibco BRL, Grand Island, NY, USA) supplemented with 10% FBS (HyClone, Logan, UT, USA) at 37°C with 5% CO₂ in a humidified incubator.

Cell viability assay

The effects of AT-I on the viability of A2780 cells were detected by MTT assay. The cells (1×10⁵ per well) were seeded into 96-well plates and incubated for 24 h. After treatment with AT-I (0, 12.5, 25, 50, 100 and 200 μM) for 24, 48, and 72 h, the viability of the cancer cells was detected with MTT. We added 10 μl MTT solution (5 mg/ml) to each well, and the mixtures were incubated for 4 h at 37°C. Then, the MTT solution was removed and 150 μl of DMSO was added to the wells. The absorbance was measured using a Multiskan MK3 Reader (Thermo Fisher Scientific, Waltham, MA, USA) at 570 nm wavelength. Cell viability=(OD of experimental well/OD of control)×100%.

Colony formation assay

A2780 cells at densities of 2×10⁵ cells were seeded on 6-well plates with a top layer of 0.3% agar and a bottom layer of 0.6% agar. Treatment groups were administered with AT-I for 14 days in A2780 cells. The normal growth medium was applied on top of the cell layer and changed every 3 days. Colonies were counted manually and images were captured using a Panasonic Lumix DMC-FH3 camera (Panasonic, Osaka, Japan).

Cell cycle analysis

The A2780 cells were seeded in 6-well plates (1×10⁶ per well). After treatment with AT-I for 48 h, the cells were digested, centrifuged, collected, and washed with PBS, resuspended with precooled 75% ethanol, and fixed at 4°C overnight. Each sample was suspended in 450 μl PBS, and 50 μl PI (0.5 mg/ml) was added to the cell suspension, mixed, and incubated at 37°C for 30 min in the dark. The mixture was centrifuged to remove the supernatant, and cells were resuspended in PBS and assessed by flow cytometry (BD Biosciences, Franklin L, NJ, USA) to analyze the cell cycle distribution.

Apoptosis detection

For apoptosis analysis, Annexin V/PI staining (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) was performed by flow cytometry (BD Biosciences, Franklin L, NJ, USA). Briefly, after 48 h of AT-I treatment, the A2780 cells were washed with ice-cold PBS and incubated with Annexin V/PI in the dark at room temperature for 15 min. The fluorescence of the cells was detected by flow cytometry using a FITC signal detector (FL1) and a PI signal detector (FL2). Early-stage and late-stage apoptotic cells (Ann+/PI+) were examined by fluorescence-activated cell sorting (FACS) analysis performed with a FACScan flow cytometer using Kaluza analysis software (Beckman Coulter, Fullerton, CA, USA).

RNA isolation and qPCR

Total RNA was extracted from A2780 cells (1×10⁶ cells per well) using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. cDNA was synthesized from 1 μg of RNA using a reverse transcription system (Bio-Rad, Hercules, CA, USA), and qPCR was performed using the iQ5 Real-Time PCR system (Bio-Rad, Hercules, CA, USA). Reaction mixtures consisted of 5 μl SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA), 0.5 μl primer (10 mM), 1 μl cDNA, and 3 μl ddH₂O. Thermal cycling conditions consisted of an initial denaturation step of 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 5 s and annealing and extension at 55°C for 10 s. qPCR was performed using β-actin as an internal control to analyze cyclin B1 and CDK1 mRNA expression in A2780 cells and the primers listed in Table 1.

ELISA assay

A2780 cells were seeded in 6-well plates (1×10⁵ per well) in 1 ml of medium and treated with or without AT-I for 48 h. The supernatants were collected, and then the levels of cyclin B1 and CDK1 were measured using ELISA kits according to the manufacturer’s instructions.

Protein lysate preparation and Western blot analysis

Protein lysates were prepared using RIPA buffer (Beyotime Biotechnology, Jiangsu, China) supplemented with protein inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA). The cytosolic extracts were isolated using a cytoplasmic protein extraction kit (Beyotime Biotechnology, Jiangshu, China) due to determination of subcellular localization of cytochrome c and AIF. Protein samples were fractioned by 10% SDS-PAGE and transferred onto the PVDF membrane. After blocking with 5% BSA in TBST for 1 h, the cropped membranes were then incubated with a primary antibody against Bcl-2, Bax, AIF, cytochrome c, caspase-9, caspase-3 (1: 1000), Akt, p-Akt, PI3K, p-PI3K, mTOR, p-mTOR (1: 500), and β-actin (1: 2000) at 4°C overnight, followed by detection with horseradish peroxidase-conjugated secondary antibodies (Boster Biological Technology, Wuhan, China) and the ECL chemiluminescence kit (Bio-Rad, Hercules, CA, USA). The optical density of each band was determined using Image Lab 5.0 software (Bio-Rad, Hercules, CA, USA).

AT-I suppressed anchorage-independent growth of A2780 cells

To explore the effect of AT-I on the anchorage-independent growth of A2780 cells, the soft agar assay was used. A2780 cells were grown in soft agar containing AT-I (12.5–100 μM) for 14 days. As illustrated in Figure 2, colony formation of A2780 cells was significantly reduced by AT-I at 12.5, 25, 50, and 100 μM by 37.49%, 40.35%, 59.05%, and 76.45%, respectively. These results indicate that AT-I inhibits the anchorage-independent growth of A2780 cells in soft agar.

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Figure 2

AT-I inhibited anchorage-independent growth of A2780 ovarian cancer cells. (A) A2780 cells were grown in soft agar containing AT-I (0, 12.5, 25, 50, and 100 μM) for 14 days. (B) Quantitative results obtained using colony formation assay. The data are expressed as mean ±SEM (n=3). The experiments were performed in triplicate. * P<0.05 and ** P<0.01, compared with vehicle-treated control.

AT-I induced cell cycle arrest at G2/M phase

To investigate the effects of AT-I on cell cycle, PI staining and flow cytometry analyses were used to detect the cell cycle phases of A2780 cells treat with AT-I (12.5–100 μM) for 48 h. As demonstrated in Figure 3A, the percentage of A2780 cells in G2/M phase was significantly increased in the AT-I (50 and 100 μM) treatment group, while the proportion of cells in G0/G1 and S phase were decreased in the AT-I (100μM) treatment group, compared with the control group, suggesting that AT-I induced cell cycle arrest through affecting cell cycle progression to mitosis.

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Figure 3

AT-I induced cell cycle arrest at the G2/M phase. (A) PI staining and FACS analysis were used to investigate the induction of G2/M phase arrest in A2780 cells treated with AT-I at the indicated concentrations for 48 h. (B) AT-I regulated cell cycle related factors in A2780 cells. qPCR and ELISA analysis were used to detect expression of cell cycle-related factors in A2780 cells treated with AT-I at the indicated concentrations for 48 h. The data are expressed as mean±SEM (n=3). The experiments were performed in triplicate. * P<0.05; ** P<0.01 and *** P<0.001, compared with vehicle-treated controls.

To investigate the underlying mechanisms of AT-I-induced cell cycle arrest and its inhibitory effects on cell growth of A2780 cells, the expressions of cell cycle-related protein (cyclin B1 and CDK1) were examined using qPCR and ELISA. As shown in Figure 3B, AT-I dose-dependently reduced mRNA and protein levels of cyclin B1 and CDK1 in A2780 cells, confirming that AT-I induced cell cycle arrest through affecting cell cycle progression to mitosis. Altogether, our results suggest that AT-I prevented A2780 cells mitosis by down-regulation of cyclin B1 and CDK1 and cell cycle G2/M arrest, thus contributing to the inhibition of A2780 cell growth.

AT-I induced apoptosis in A2780 cells

To investigate whether AT-I induced apoptosis, we analyzed Annexin V/PI-stained A2780 cells by flow cytometry. As demonstrated in Figure 4, treatment with 0, 12.5, 25, 50, and 100 μM AT-I for 48 h dose-dependently increased early and late stages of apoptotic cells with the increase of Ann+/PI− and Ann+/PI+ cells in a dose-dependent manner.

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Figure 4

AT-I induced apoptosis in ovarian cancer cells. (A) AT-I induced apoptosis in A2780 cells in a dose-dependent manner. FACS analysis following Annexin V/PI staining was used to examine the percentage of early apoptotic cells (Ann+/PI−) and late-stage apoptotic cells (Ann+/PI+) in A2780 cells treated with AT-I at the indicated concentrations for 48 h. (B) Quantitative results obtained using Annexin V/PI staining. The data are expressed as mean ±SEM (n=3). The experiments were performed in triplicate. ** P<0.01 and *** P<0.001, compared with vehicle-treated control.

To investigate the underlying mechanisms by which AT-I induced apoptosis in A2780 cells, Western blot analysis was used to examine changes in the molecules of the apoptosis signaling pathways. As shown in Figure 5, the results revealed that AT-I induced up-regulation of proapoptotic protein Bax and down-regulation of anti-apoptotic protein Bcl-2, which could increase cytochrome C and AIF in the cytosol, and then activated the cytosolic caspase-9. Activated caspase 9 could further activate caspase 3, which then induced cell apoptosis. Taken together, these results suggest that AT-I can induce A2780 cells apoptosis through caspase-dependent and -independent mitochondrial pathways.

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Figure 5

AT-I activated mitochondria-mediated apoptosis pathways in A2780 cells. Western blot analysis was performed to detect expression of apoptosis related factors in A2780 cells treated with AT-I at the indicated concentrations for 48 h. (A) Representative Western blots of the main factors related to mitochondria-mediated apoptotic signaling pathways. (B–E) The quantification of the expression of apoptosis-related factors. The data are expressed as mean ±SEM (n=3). The experiments were performed in triplicate. * P<0.05; ** P<0.01 and *** P<0.001, compared with vehicle-treated controls.