Isoalantolactone was purchased from Tauto Biotech Company (Shanghai, China), purity >99% as determined by analytical HPLC. Propidium iodide (PI), dimethylsulfoxide (DMSO), acridine orange dye (AO), 3-meth-yladenine (3-MA), trypan blue dye, cell culture media (McCoy's 5A medium, RPMI-1640 medium), pentobarbital sodium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Sigma-Aldrich (Beijing, China). Cell-Light EdU imaging detection kit was purchased from RiboBio (Guangzhou, China). On-TARGETplus SMARTpool siRNA for PEA-15 kit was purchased from Dharmacon (Lafayette, CO, USA). Lipofectamine 2000 kit, TRIzol reagent and the Primescript™ reverse transcription reagent kit were purchased from Invitrogen (Beijing, China). SYBR Premix Ex Taq™ II was purchased from Takara (Dalian, China). Polyclonal anti-human Beclin1, PEA-15, ERK, pERK, LC3 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies specific to β-actin, α-tubulin and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The human ovarian cancer cell line SKOV₃ was purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were maintained in McCoy's 5A medium containing 10% FBS and 1% penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO₂. Cell detachments were achieved by rinsing with 0.05% trypsin/0.02% EDTA solution. The cells were treated with different concentrations of isoalantolactone dissolved in DMSO with a final concentration of 1% for 24 h. DMSO-treated cells were used as a control.

Eight-week-old, C57/BL6 mice, weighing 20 g were used. The mice were maintained in a specific pathogen-free grade animal facility on a 12-h light/dark cycles at 22±2°C. The mouse procedures were approved by the Experimental Animal Committee of Liaoning Medical University. Mice were anesthetized using pentobarbital sodium (65 mg/kg i.p.) and were perfused transcardially with PBS. Following midline abdominal incision spleen was removed and the freshly isolated splenocytes were cultured in RPMI-1640 medium supplemented with 20% heat-inactivated FBS and maintained at 37°C with 5% CO₂ in a humidified atmosphere.

For the in vitro cell viability experiments, a trypan blue exclusion assay was performed using Vi-CELL series cell viability analyzers (Beckman Coulter Inc., Brea, CA, USA). To determine the inhibition effect of isoalantolactone on SKOV₃ cell growth, cells were incubated with 35 or 75 µM isoalantolactone for 24 h. Subsequently, the cells were collected and washed with PBS and then incubated with 0.04% trypan blue for 5 min at room temperature. Trypan blue only stains dead cells. After washing, the cells were resuspended in PBS and the percentage of living and dead cells were counted. The living cell rate (%) = number of living cells/number of living cells + number of dead cells) ×100.

To analyze cell proliferation for isoalantolactone treatment, cells were treated as described above. Then cell proliferation was examined by the 5-ethynyl-2-deoxyuridine (EdU) incorporation assay using the Cell-Light EdU imaging detection kit according to manufacturer's instructions.

SKOV₃ cells were incubated with 35 and 75 µM isoalantolactone for 24 h. Then the cells were collected, fixed, stained with PI staining solution (3.8 mM sodium citrate, 50 µg/ml PI in PBS) and 20 g/ml RNase A in the dark for 30 min. Cell cycle distribution was assessed by flow cytometry (Beckman Coulter, Epics XL). For the flow cytometric analysis, at least 10,000 cells were used for each sample. The data were analyzed using Cell Quest software (Becton Dickinson, San Jose, CA, USA) to measure the DNA content of cells in the G0/G1, S and G2/M phases.

Cells (2×10⁶) were stained with AO according to a published procedure (23). Briefly, after the cells were treated with 35 and 75 µM isoalantolactone for 24 h, the cells were incubated with 1 mg/ml AO for 20 min in the dark at 37°C. Subsequently, the cells were washed twice with PBS. Images of the cells were obtained by fluorescence microscopy.

Cells were collected after treatment with 35 and 75 µM isoalantolactone in the presence or absence of the autophagy inhibitor 3-methyladenine (3-MA 5 mM) for 24 h. Next, the cells were incubated with 1 mg/ml AO for 20 min in the dark at 37°C. After washing, the cells were analyzed by flow cytometry and CellQuest software. Red fluorescence emissions from 10⁶ cells were analyzed, and quantified as percentage of cells containing AVOs.

In all, 5×10⁵ cells were seeded in 6-well plates. After 24 h, the cells were transfected with siRNA using Lipofectamine 2000. After 3 days, the transfected cells that were treated with isoalantolactone or DSMO were collected for the clonogenic survival assay and western blot analysis.

Cells were treated with isoalantolactone for 24 h, washed twice with PBS, and lysed on ice with WIP cell lysis reagent supplemented with 1% PMSF for 30 min. The insoluble protein lysate was removed by centrifugation at 12,000 rpm for 15 min at 4°C. The protein concentrations were determined using a NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Proteins (20 µg) were electrophoresed using 10% SDS-PAGE gels and transferred to a PVDF membrane. After blocking with 5% non-fat milk and washing with a Tris-buffered saline/Tween solution (TBST), the membranes were incubated overnight at 4°C with specific primary antibodies and then with anti-rabbit IgG or anti-mouse IgG secondary antibodies for 1 h at room temperature. Signals were detected using the ECL plus chemiluminescence kit and X-ray film (Millipore Billerica, MA, USA). All the bands obtained were quantified by densitometry using ImageJ software.

The results are expressed as the means ± SEM. Analyses were performed using GraphPad prism 5 software. One-way analysis of variance (ANOVA) was used to analyze significant differences between groups under different conditions. Student's t-test was used to determine significance when only two groups were compared and P<0.05 was considered to indicate a statistically significant difference.

The chemical structure of isoalantolactone is shown in Fig. 1A. SKOV₃ cells were treated with different doses of isoalantolactone (0, 35, 75, or 110 µM) for 24 and 48 h. As shown in Fig. 1B, isoalantolactone significantly inhibited the growth of SKOV₃ cells in a dose- and time-dependent manner. To further confirm the effect of isoalantolactone on growth inhibition, cellular morphology changes were observed using inverted phase contrast microscopy. The cells became rounder and shrunken, and they floated in the culture medium as the concentration of isoalantolactone increased (Fig. 1C), supporting the finding that isoalantolactone inhibited SKOV₃ cell growth in a dose-dependent manner. The cytotoxic effect of isoalantolactone was further evaluated by trypan blue staining on normal mouse splenocytes. The data showed that fewer inhibitory effects were observed in spleno-types treated with isoalantolactone at different doses (Fig. 1C), suggesting that SKOV₃ cells are more sensitive than normal cells to isoalantolactone. The effect of isoalantolactone at different concentrations on the viability of SKOV₃ cells was assessed by live/death cells staining. As Fig. 1D shows, isoalantolactone inhibited SKOV₃ cells growth in a dose-dependent manner. The data indicated that isoalantolactone was able to inhibit SKOV₃ cell growth selectively.

To further confirm that isoalantolactone inhibited SKOV₃ cell growth, we tested the cell proliferation using the EdU incorporation assay. Fig. 2A shows the EdU-positive cells on SKOV₃ cells treated with 35 or 75 µM isoalantolactone for 24 h. Compared to the control group (48.2%), the EdU-positive cells in the isoalantolactone-treated groups at 35 (27.4%, P<0.05) or 75 µM (16.6%, P<0.05) were significantly reduced in a dose-dependent manner (Fig. 2B). Thus, isoalantolactone shows promise as an antitumor drug for human ovarian cancer.

One of the major mechanisms underlying the anti-proliferative effect of anticancer drugs is the prevention of cell cycle progression. To explore the possible mechanism underlying the inhibitory effect of isoalantolactone on the growth of SKOV₃ cells, cell cycle distribution was examined by flow cytometry to analyze the DNA content of cell cycle phase. As shown in Fig. 3A, isoalantolactone induced a dose-dependent increase in the number of SKOV₃ cells in G2/M phase, the percentage of accumulation of cells was increased from 20.8% in the control group to 33.2% (P<0.05) and 46.3% (P<0.01) in the cells treated with 35 and 75 µM of isoalantolactone, respectively for 24 h, leading to a decrease in the proportion of cells in G0/G1 phase from 41.9% in the control group to 27.9% (P<0.05) and 20.6% (P<0.01) in the cells with the above-mentioned different concentration (Fig. 3B). We also investigated the expression of cyclin B1 and CDK1 of cell cycle regulators by western blot analysis. The results revealed that the expression of cyclin B1 and CDK1 were gradually decreased with increasing concentrations of isoalantolactone in a dose-dependent manner (Fig. 3C), indicating that cell cycle arrest at G2/M phase may be associated with the inhibition of SKOV₃ cell growth by isoalantolactone treatment.

Autophagy is closely associated with tumors and plays an important role in human tumor suppression. AVO formation (autophagosomes and autolysosomes) is a characteristic feature of autophagy (24). To investigate whether autophagy was involved in the inhibitory effects of isoalantolactone on SKOV₃ cell growth, the accumulation of AVOs was analyzed by AO staining and quantified by flow cytometry (25). AO has a weak base that freely passes across the plasma membrane in a neutral state distinguished by green fluorescence. After entrance into acidic compartments, AO changes into the protonated form which is distinguished by bright red fluorescence while control cells primarily showed green fluorescence. The intensity of the red fluorescence was proportional to the degree of acidity. Thus, the formation of AVOs could be quantified. As shown in Fig. 4A, after cell treatment with isoalantolactone, the intensity of the bright red fluorescence was markedly increased in a dose-dependent manner compared to the control. We further quantified AVOs by flow cytometry. Fig. 4B illustrates significant formation of AVOs in isoalantolactone-treated cells (26.5 or 39.2%, P<0.01) compared to the control cells (2.1%). Accordingly, we added the autophagy inhibitor 3-MA, which controls the autophagy pathway at various points (26). We found that isoalantolactone-induced AVO formation was suppressed when the cells were treated in combination with the specific autophagy inhibitor (28.8%, P<0.05) (Fig. 4C). These results show that isoalantolactone-induced autophagy is involved in inducing death of SKOV₃ cells.

To further confirm involvement of autophagy in isoalantolactone-induced SKOV₃ cell death, Western blot analysis was performed to evaluate the effects of isoalantolactone on the autophagy protein expression of Beclin1, which was the initiation factor of autophagosome formation, LC3-I and LC3-II (autophagosome marker). As shown in Fig. 5, isoalantolactone activated Beclin1 expression and increased the ratio of LC3-I and LC3-II protein expression in a dose-dependent manner, while markedly decreased in cells pre-treated with the combination 3-MA and 75 µM isoalantolactone compared to cells treated only with 75 µM isoalantolactone, confirming that autophagy was involved in isoalantolactone-induced cell death.

Autophagy is a complicated regulatory process, which involves a great number of upstream regulating signaling pathways (27,28). While ERK, the target of PEA-15, is relatively new regulator studied of autophagy (20,29,30). To elucidate the underlying mechanism of autophagy induced by isoalantolactone in SKOV₃ cells, we examined the expression of PEA-15, ERK and pERK by western blot analysis. As shown in Fig. 6A and B, isoalantolactone greatly increased PEA-15 expression and activated ERK phosphorylation in a dose-dependent manner compared to the control cells, whereas no great change on ERK total protein level was seen (Fig. 6C). These results suggest that isoalantolactone-induced upregulation of PEA-15 expression and activation of ERK signaling pathway are associated with autophagy in SKOV₃ cells.

Based on the results described above, we further confirm whether isoalantolactone induced autophagy on SKOV₃ cells through upregulation PEA-15 expression. The PEA-15 knockdown was performed by siRNA. Indeed, PEA-15 knockdown to ~50% of the original level significantly reduced AVO formation of SKOV₃ cells by isoalantolactone induction (Fig. 5B). Western blot analysis showed that the expression of Beclin1, LC3-I and LC3-II protein levels on SKOV₃ cells were greatly decreased by PEA-15 knockdown compared to cells treated with high-dose isoalantolactone (P<0.05 or P<0.01) (Fig. 5C). Thus, the PEA-15 levels correlated with isoalantolactone-induced autophagy on SKOV₃ cells.