The human endometrial cancer Ishikawa and HEC-1A cell lines were obtained from the Shanghai Institute of Cellular Biology of the Chinese Academy of Sciences (Shanghai, China) and cultivated in Dulbecco' modified Eagle's medium (DMEM) (Invitrogen; Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific Inc.), 50 µg/ml penicillin (Invitrogen; Thermo Fisher Scientific Inc.), 50 µg/ml streptomycin (Invitrogen; Thermo Fisher Scientific Inc.) and 2 mmol/l glutamine (Gibco; Thermo Fisher Scientific Inc.) at 37°C in 5% CO₂. All experiments were performed with cells in the logarithmic phase.

RAD001 (catalogue no. 07741), MTT and chloroquine (CQ) were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany). RAD001 was formulated in 1 mmol/l dimethyl sulfoxide (DMSO) diluent. Polyclonal rabbit anti-phospho-AKT (Ser473) antibody (#9271; 1:1,000), monoclonal rabbit anti-mTOR antibody (#2983; 1:1,000), monoclonal rabbit anti-phospho-mTOR (Ser2448) antibody (#5536; 1:1,000), monoclonal rabbit anti-p70S6K antibody (#2708; 1:1,000) and polyclonal rabbit anti-phospho-p70S6K (Thr421/Ser424) antibody (#9204; 1:1,000) were acquired from Cell Signaling Technology Inc. (Danvers, MA, USA). Polyclonal rabbit anti-LC3 antibody (#NB100-2220; 1:1,000) was obtained from Novus Biological LLC (Littleton, CO, USA). Goat anti-rabbit immunoglobulin G horseradish peroxidase-conjugated secondary antibody (#sc-2004; 1:2,000) was purchased from Santa Cruz Biotechnology Inc. (Dallas, TX, USA) and monoclonal mouse anti-GAPDH antibody (KC-5G4; 1:5,000) was purchased from Kangcheng Bio-tech Inc. (Shanghai, China).

Cells in the logarithmic phase were seeded in 96-well plates at a density of 8,000 cells/well in 195 µl DMEM and cultivated at 37°C overnight. The cells were then treated with CQ (10 mg/ml in DMSO), paclitaxel (1, 2, 4, 8 and 16 µM or the indicated concentrations in DMSO), 3-methyladenine (3-MA) (10 µM in H₂O, which was used as an autophagy inhibitor) and DAPI (1 µg/ml in H₂O) for the indicated time points at 37°C in 5% CO₂. Solvent was used as the control in each experiment. Experiments were terminated by adding 10 µl of 5 mg/ml MTT and incubated at 37°C for 4 h. Following complete removal of the medium, 100 µl of DMSO (Sigma-Aldrich; Merck Millipore) was added to each well to dissolve the purple formazan product. Absorbance values of the resultant purple solution were obtained with a test wavelength of 570 nm. The IC₅₀ values were calculated by the Bliss method: Inhibitory rate (%)= [1 - the average optical density (OD) value of the treatment group / the average OD value of the control group] × 100. Growth inhibition was calculated according to the results of the MTT assay, and the combination index was determined using CalcuSyn software version 2.0 (Biosoft, Cambridge, UK).

For the microscopic examination, 1 day prior to transfection, the cells were plated in 6-well plates with antibiotic-free RPMI 1640 growth medium at a density of 1.5×10⁵ cells/well. When the cells grew to a confluence of ~50% on the second day, green fluorescent protein microtubule-associated protein 1 light chain 3α (GFP-LC3)-expressing plasmids were transfected into the cells using Lipofectamine 2000 (Thermo Fisher Scientific Inc.), and cells stably transfected with the GFP-LC3 plasmid (kindly provided by Professor Beth Levine), which stably expressed GFP-LC3, were selected with growth medium containing G418 antibiotic. Subsequent to treatment with the experimental drugs, the cells were washed with 1X PBS three times and then evaluated under a confocal fluorescence microscope (LSM 710 Meta; Carl Zeiss AG, Oberkochen, Germany).

Prior to the drug treatment, the cells were plated in 6-well plates at a density of 4×10⁵ cells/well and incubated at 37°C in 5% CO₂ overnight. Following treatment, the cells were collected and washed with 1X PBS three times and lysed in 100 µl/well of lysis buffer. Cell lysates were centrifuged at 13,600 × g for 10 min at 4°C and the protein concentrations were determined by Bio-Rad protein assay (Bio-Rad Laboratories Inc., Hercules, CA, USA). SDS-PAG loading buffer was added to the cell lysate, which was then heated at 95°C for 10 min. Each sample containing 40 µg of protein was then loaded into each well of the SDS-PAGE gels and the resolved proteins were transferred to a polyvinylidene difluoride membrane electrophoretically. Subsequent to blocking with 5% skimmed milk, the membranes were probed sequentially with primary and secondary antibodies overnight at 4°C. Following washing three times with TBS plus Tween 20 [10 mmol/l Tris-HCl (pH 7.4), 150 mmol/l NaCl and 0.1% Tween 20] (TBST), the proteins were detected using enhanced chemiluminescence reagent (GE Healthcare Life Sciences, Chalfont, UK) and XAR film (Kodak, New York, NY, USA).

Pre-treated cells were collected and washed twice with 1X PBS. The cells were then re-suspended in 1X PBS at a density of 1×10⁶ cells/ml. Next, 10 µl of 10 mg/l propidium iodide was added to 1-ml cell suspension, which was then incubated in the dark for 10 min. The samples were placed on ice prior to being analyzed by flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Protein depletion through RNA-mediated interference was mediated using the pSUPER small hairpin (sh)RNA system. Retroviruses were generated by co-transfection of pSUPER-shRNA plasmids (#30519; Addgene, Inc., Cambridge, MA, USA) with retrovirus plasmid PIK (Ecopac: M. Finer Cell Genosys, Redwood City, CA, USA) into 293T cells by liposome. Retroviruses were collected in high-serum media at 48 and 72 h post-transfection. Ishikawa and HEC-1A cells were transduced with retroviruses and 8 µg/ml Polybrene (hexadimethrine bromide; Sigma-Aldrich; Merck Millipore) followed by incubation with virus at 37°C for 4–6 h. shRNA-transduced cells were selected for with 1 µg/ml puromycin for 72 h. To confirm the efficiency of Atg5 shRNA, puromycin-selected cells transfected with a specific shRNA targeting human Atg5 (5′-GCAACUCUGGAUGGGAUUG-3′) were cultured three-dimensionally in vitro. Cells were then subjected to western blot detection with anti-Atg5 polyclonal rabbit antibody (1:1,000; #2630; Cell Signaling Technology Inc.) and anti-GAPDH antibody (1:5,000; KC-5G4; Zhejiang Kangchen Biotech Co., Ltd., Shanghai, China) for 12 h at 4°C. Following three washes with TBST, the proteins were detected using an enhanced chemiluminescence reagent and BioMax XAR Film (Kodak, Rochester, NY, USA).

Data were presented as the mean ± standard deviation, and analyzed with a one-way analysis of variance and Student-Newman-Keuls-q test (22) by SPSS 16.0 statistical software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

The inhibitory effect of RAD001 on Ishikawa and HEC-1A cells was demonstrated using MTT assay. The soluble yellow compound of MTT was reduced to insoluble formazan, which produced a purple color in living cells. As the amount of formazan produced was proportional to the number of viable cells, after dissolving it in DMSO, the absorbance values of the resultant purple solution were used to calculate the inhibitory rate of cell proliferation and thus evaluate the cytotoxicity of RAD001. Following treatment with different concentrations (0, 5, 10, 20, 40 and 80 nM) of RAD001 for 72 h, the proliferation of the Ishikawa and HEC-1A cells was suppressed in a dose-dependent manner, and all results were significant compared with the control group (P<0.01) (Fig. 1). The group treated with 0 nM PAD001 was considered as the control group, and the decreased proliferation rate of this group was normalized to 0% (which coincides with the origin of coordinates in Fig. 1). The IC₅₀ values were 36.80±1.64 and 25.72±1.16 nM for the Ishikawa and HEC-1A cells, respectively. The results suggested that RAD001 alone could effectively inhibit the proliferation of the Ishikawa and HEC-1A cells.

Following treatment with RAD001 in combination with different concentrations of paclitaxel, the proliferation of the Ishikawa and HEC-1A cells was significantly inhibited in a dose-dependent manner. The IC₅₀ values for the Ishikawa and HEC-1A cells treated with paclitaxel alone were 7.91 and 9.27 µM, respectively. The corresponding combination index was <1 for the two cell lines, which was statistically significant (Fig. 2A). Apoptosis was observed in the Ishikawa cells treated with paclitaxel, as indicated by the presence of cleaved caspase3 and cleaved poly ADP ribose polymerase; when RAD001 was also added, the cleaved band became markedly more intense (Fig. 2B). According to flow cytometry, the apoptotic cell count increased from 15.2 to 45% (Fig. 2C). These results indicated that the combination treatment of RAD001 and paclitaxel is synergistic for suppressing the human endometrial cancer cell proliferation.

LC3 is an autophagic marker protein whose lipidated form, LC3II, is recruited to the autophagosomal membranes at a late stage of autophagy (23). As shown in Fig. 3A, upon treatment with RAD001, green puncta were detected in the cytosol of GFP-LC3-expressing Ishikawa and HEC-1A cells, denoting the formation of autophagosomes. Western blot analysis results further confirmed this, as the expression level of LC3II protein increased in a dose-dependent manner for the two cell lines following treatment with different concentrations of RAD001 for 24 h (Fig. 3B). This suggested that RAD001 induced autophagy in the Ishikawa and HEC-1A cells.

MTT results confirmed that the inhibitory effect of RAD001 on Ishikawa and HEC-1A cell proliferation was decreased when autophagy was suppressed by CQ, suggesting that RAD001 induced autophagic cell death in the Ishikawa and HEC-1A cells (Fig. 4A). Furthermore, inhibition of autophagy by shRNA knockdown of Atg5 also resulted in the reduced inhibition of cell death as induced by RAD001 (Fig. 4B). The levels of Atg5 were effectively reduced by Atg5 shRNA (data not shown). Together, these results showed that RAD001 induced autophagic cell death in the endometrial cancer Ishikawa and HEC-1A cells.

To further investigate whether the AKT/mTOR/p70S6K signaling pathway plays a role in RAD001-induced autophagy, the expression levels of the key proteins in this signaling pathway were examined in Ishikawa and HEC-1A cells by western blot analysis. The results indicated that RAD001 inhibited serine 473 phosphorylation and mTOR phosphorylation in a dose-dependently manner in the two cell lines, with no significant change in the expression level of mTOR (Fig. 5). Furthermore, it was observed that the phosphorylation of one of the essential substrates of mTORC1, p70S6 kinase, was also significantly suppressed. These results suggested that the AKT/mTOR/p70S6K signaling transduction pathway was suppressed upon treatment with RAD001 in the Ishikawa and HEC-1A cells.

To establish whether RAD001 sensitized endometrial cancer Ishikawa and HEC-1A cells to paclitaxel treatment via the induction of autophagy, Ishikawa cells were treated with 3-MA, the autophagy inhibitor, together with RAD001. Consistent with aforementioned results, RAD001 triggered autophagy, as shown by the presence of LC3II, and 3-MA inhibited this change (Fig. 6A). As shown in Fig. 6B, the effect of autophagy inhibition with 3-MA plus RAD001 was tested on cell death in the Ishikawa cells; it was found that RAD001-induced autophagic cell death was efficiently suppressed when autophagy was blocked. Also, the result from the flow cytometric apoptosis assay indicated that Ishikawa cell apoptosis was significantly enhanced when the cells were treated with RAD001 in combination with paclitaxel, and this effect was repressed upon inhibition of autophagy (Fig. 6C). These results suggested that RAD001 sensitized the Ishikawa cells to paclitaxel via the induction of autophagy.