The NSCLC A549 cells were obtained from the First Affiliated Hospital of Xi'an Jiaotong University as a gift. The curcumin, MTT, rapamycin, and LY294002 were purchased from Sigma (San Francisco, CA, USA). Roswell Park Memorial Institute culture medium (RPMI-1640) and penicillin-streptomycin were purchased from Hyclone Co. (Logan, USA). Fetal bovine serum (FBS; Biological Industries, Kibbutz Beit-Haemek, Israel). Dimethyl sulphoxide (DMSO) was purchased from Amresco (Houston, TX, USA). The Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit was purchased from 7Sea Pharmatech Co., Ltd. (Shanghai, China). TRIzol reagent was purchased from Life Technologies (Carlsbad, CA, USA). The Revert Aid First Strand cDNA Synthesis kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). FastStart Universal SYBR Green Master (Rox) was purchased from Roche (Basel, Switzerland). The Pierce bicinchoninic acid (BCA) protein assay kit was purchased from Merck (Darmstadt, Germany). The skim milk was purchased from Wandashan Dairy Co., Ltd. (Heilongjiang, China). Goat anti-rabbit IgG-HRP, Akt (60 kDa), p-Akt (60 kDa), mTOR (289 kDa), p-mTOR (289 kDa), GAPDH (38 kDa) antibodies were purchased from Abcam (Cambridge, MA, USA). The antibodies against Beclin1 (60 kDa), LC3 (LC3-II, 14 kDa and LC3-I, 16 kDa) and p62 (62 kDa) were purchased from Cell Signaling Technology Inc. (Beverly, MA, USA).

Curcumin, rapamycin, and LY294002 were dissolved into micro-DMSO stock solution and then RPMI-1640 culture medium was added to the desired concentration, waiting to be used. DMSO is a control for the entire study at a final concentration of <0.1%.

Methods

A549 cells were cultured in RPMI-1640 medium supplemented with 10% (v/v) FBS, 1% penicillin-streptomycin and specifically maintained in a 5% CO₂, 95% air humidified incubator at 37°C. Taking the logarithmic growth phase of the A549 cells for following tests and the cells were seeded and adhered on petri dishes for 24 h before starting the treatment. A549 cells were exposed to curcumin at the indicated concentrations and time periods in each experiment. The rapamycin and LY294002 were widely used as blockers of mTOR and PI3K/Akt, respectively. When co-cultured with curcumin the cells were pre-treated with rapamycin of 40 µM or LY294002 of 20 µM for 3 h based on a large number of references (6,18) and our pre-experimental results.

The cytotoxic activity of different stimulus in A549 cells was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A549 cells were seeded in 96-well plates at a density of 5×10⁴ cells/well for 24 h before challenged by different concentrations of curcumin for 24, 48, 72 and 96 h. Or the cells were pretreated with 40 µM rapamycin or 20 µM LY294002 for 3 h and subsequently with or without 40 µM curcumin for 48 h. Each 96-well plate was set up with control (cells-only) and zero-adjustment (medium only). Additionally, then washed once and incubated with 20 µl MTT (5 mg/ml) at 37°C for 4 h. Carefully aspirating the liquid, the purple formazan crystals were dissolved in 150 µl DMSO. Shaking 5 min, the absorbance of each well was read with the microplate reader (Infinire M200; Tecan Group Ltd., Mannedorf, Switzerland) at 570 nm. Assays were performed in triplicate on three independent experiments. Cell viability rate (%) = (experimental group OD-zero adjustment group OD)/(control group OD-zero adjustment group OD) ×100%.

The Annexin V-FITC/PI detection kit was used for the determination of cell apoptosis. A549 cells (4×10⁵ cells/well) were seeded in 6-well tissue culture plates followed by exposure with curcumin (0–40 µM) as well as co-incubation with curcumin (40 µM) and rapamycin (40 µM) or curcumin (40 µM) and LY294002 (20 µM) for 48 h at 37°C and then were collected, washed once with cold phosphate-buffered saline (PBS), then re-suspended in 400 µl binding buffer at a concentration of 1×10⁶ cells/ml. After 5 µl of Annexin V-FITC was added, the cells were incubated for 15 min at room temperature in the dark. Then 10 µl of PI (propidium iodide) was added and the cells were incubated for 5 min at 4°C in the dark. The rate of apoptosis was immediately analyzed by a flow cytometer (Guava^® easy Cyte HT; Merck Millipore, Billerica, MA, USA). Annexin V-FITC-positive cells were considered to be undergoing apoptosis and those negative for FITC were considered to be alive.

Formating and promoting the AVs is one of the characters of autophagy (19). To detect autophagy, MDC labeling was performed, which could infer the activation of autophagy from the changes in fluorescent particles. Collecting the logarithmic growth phase A549 cells seeded at 3×10⁴ cells per well in 24-well culture plates and treated with 0 µM curcumin (control), 40 µM curcumin, rapamycin+curcumin (40 µM), LY294002+curcumin (40 µM) respectively. MDC (50 µM) was added to living cells 48 h after different treatments. The cells were then incubated for 15 min at 37°C and 5% CO₂ in the dark, washed twice with PBS, and the anti-quencher was added. Visualizing and imaging quickly with UV excitation by an inverted fluorescence microscope (Nikon Eclipse Ti; Nikon, Tokyo, Japan). In order to quantify MDC staining, the relative MDC fluorescence intensity of the different treatment groups were measured by the Image-Pro Plus. The experiment was repeated three times.

The A549 cells (2×10⁶ cells/well) were seeded in 6-well tissue culture plates followed by exposure to curcumin (0–40 µM) or co-incubation with curcumin (40 µM) and rapamycin (40 µM) or curcumin (40 µM) and LY294002 (20 µM) for 48 h. Then total RNA was isolated with Trizol reagent from A549 cells and 2 µg of the total RNA was reverse-transcribed into cDNA with the RevertAid First Strand cDNA Synthesis kit according to the manufacturer's instructions. Quantitative real-time PCR (qPCR) was performed with a FastStart Universal SYBR Green Master (Rox) kit. Each sample was run in triplicate in final volume of 25 µl containing 2.5 µl first-strand cDNA, 1 µl (7.5 µM) of each primer (purchased from Oke Dingsheng Biological Technology Co., Ltd., Beijing, China), 12.5 µl of 2X Fast Start Universal SYBR Green Master (ROX) and 8 µl distilled water. Cycling parameters of the qPCR were as follows: 1 cycle at 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec, 60°C for 60 sec in the Step One Plus fluorescence quantitative PCR instrument (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). After the reaction, the results were obtained by ΔΔCT method. Primer sequences were: Akt forward, 5′-CAAGTCCTTGCTTTCAGGGC-3′ and reverse, 5′-ATACCTGGTGTCAGTCTCCGA-3′ (184-bp product); mTOR forward, 5′-AACCTCCTCCCCTCCAATGA-3′ and reverse, 5′-CTCACGGAGAACCAGGACAG-3′ (186-bp product); GAPDH forward, 5′-CAAGGTCATCCATGACAACTTTG-3′ and reverse, 5′-GTCCACCACCCTGTTGCTGTAG-3′ (496-bp product). Amplification of housekeeping gene GAPDH was taken as an endogenous control.

A549 cells were seeded in culture dish at a density of 2×10⁶ cells/well and then incubated with or without curcumin (0–40 µM, 48 h) in the presence or absence of various inhibitors (40 µM rapamycin or 20 µM LY294002). The cells were lysed in the radio-immunoprecipitation assay buffer (RIPA). Proteins were quantified using a BCA protein assay kit. Equal amount (20 µg) of protein samples were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and further transferred to polyvinylidene fluoride (PVDF) membranes which were soaked with methanol for 30 sec. After blocking with 5% bovine serum albumin for 1 h at room temperature and probed with the indicated antibodies. The membrane was washed thrice with TBST and incubated with goat anti-rabbit IgG conjugated to HRP. The membranes were visualized using the electrochemiluminescence and quantitated using the Image-Pro Plus6.0 software. Equal loading was assessed by GAPDH as internal control for western blotting.

SPSS18.0 statistical software was used for data analysis. The data are presented as mean ± SEM. The one-way ANOVA were used to analyze data. Comparisons between groups were done using the Dunnett's t-test (*P<0.05, **P<0.01 or ^#P<0.05, ^##P<0.01).

In the first place, the toxicity of curcumin treatment at concentration ranging from 0–40 µM for different time period (24, 48, 72 and 96 h) was assayed by using an MTT test on human lung cancer A549 cells. As is shown in Fig. 1 the growth inhibition of curcumin on the cells was manifested in a dose- and time-dependent manner. Of note, A549 cell viability could be obviously reduced with an incubation period for 48 h at 20 and 40 µM of curcumin consistent with our previous findings (17) selected as an effective condition for the subsequent studies to elucidate the underling molecular mechanisms of curcumin.

Next, Annexin V-FITC and PI staining were used to examine whether this inhibitory effect of curcumin on cell ability was related to the induction of apoptosis. Apoptotic or necrotic cells were detected after treated with curcumin of increasing concentrations for 48 h. The apoptosis phenomenon was observed in cells exposed to different doses of curcumin, whereas necrotic population was almost negligible. As shown in Fig. 2, the apoptotic (Annexin V-positive) cell populations (early and late apoptosis) were increased to 20.91 and 23.68% after treatment of A549 cells with 20 and 40 µM curcumin, respectively, with a 1.35- and 1.53-fold increase, respectively, compared with control cells. These data suggested that curcumin resulted in a dose-dependent increase in the apoptotic cell death in human lung cancer A549 cells when used for 48 h. Taken together, these results revealed its central role in regulating apoptosis mediated by curcumin.

Autophagy, one of the most studied fields in cancer biology, has been verified by using a variety of methods. MDC is a luminescent dye that can be absorbed by cells and displayed on AVs. The activation of autophagy was inferred from changes in fluorescent particles infected with MDC first. After curcumin treatment MDC staining showed that the relative fluorescence intensity and density of cells was significantly increased compared with control cells, indicating the occurrence of autophagy (Fig. 3A and B). This was the result expected and consistent with our previous study that curcumin enhanced autophagy in a dose-dependent manner in A549 cells (17).

Taking into account the non-specificity of MDC staining, the protein levels of autophagy markers Beclin1 (60 kDa), LC3-II (14 kDa), LC3-I (16 kDa), and p62 (62 kDa) were detected by western blotting. As shown in Fig. 3C, curcumin administration dramatically increased Beclin1 and LC3-II expression, decreasing p62 protein level. As expected, the ratio of LC3-II/LC3-I was enhanced (Fig. 3C and D) in a dose-dependent manner. All the results above provided real evidence for curcumin-induced autophagy of A549 cells.

Thus far our results revealed that curcumin could substantially induce both apoptosis and autophagy. Given the critical role of PI3K/Akt/mTOR pathway in controlling cell survival/death in cancer cells, including lung cancer cells (20–22), we investigated whether curcumin induced apoptosis and autophagy via the inhibition of PI3K/Akt/mTOR signaling pathway.

To determine the role of this way, two critical molecules, Akt and mTOR, were examined. The expression levels of Akt and mTOR mRNA was detected in A549 cells by qRT-PCR (Fig. 4A and B). As expected, compared with the control group (1.14±0.22), 40 µM of curcumin treated A549 cells 48 h, greatly reduced the transcription level of mTOR (0.36±0.05) (P<0.01), as indicated in Fig. 4A. As shown in Fig. 4B, the expression of Akt mRNA in curcumin group (0.44±0.09) was significantly lower than that in control (1.04±0.09) (P<0.01). In order to further ascertain and identify the role of mTOR and Akt in the curcumin-modulation PI3K/Akt/mTOR signaling in A549 cells, the effect of rapamycin and LY294002 were detected. The rapamycin and LY294002 were widely used as inhibitors of mTOR and PI3K/Akt, respectively (23,24). Similar to the effect of curcumin, there was a more significant inhibitory effect on mTOR and Akt mRNA levels when it combined with rapamycin or LY294002 compared with curcumin treatment alone (Fig. 4A and B).

Eukaryotic gene expression and regulation is complex, mRNA levels can not fully represent the expression of protein. Thus, the protein expression of important signaling molecules such as p-Akt, Akt, p-mTOR, and mTOR was determined by western blotting. As shown in Fig. 4C, dose-dependent inhition of Akt phosphorylation and mTOR phosphorylation were observed when A549 cells were treated with curcumin for 48 h. To be more intuitive, the inhibition of PI3K/Akt/mTOR pathway was judged by the ratio of p-Akt to total Akt as well as p-mTOR to total mTOR, which was consistent with the above results (Fig. 4D). These data together imply that the cytotoxicity of curcumin may be related to the PI3K/Akt/mTOR pathway inhibition.

Above observations prompted to infer that downregulation of PI3K/Akt/mTOR might have a crucial role in curcumin-induced cytotoxicity due to apoptosis and autophagy. Therefore, the cell viability was examined by MTT. To determine that the curcumin-induced apoptosis and autophagy occurred via the PI3K/Akt/mTOR signaling pathway, we employed mTOR blocker rapamycin and PI3K/Akt inhibitor LY294002. The MTT assay showed that inactivation of PI3K/Akt or inactivation of mTOR tremendously sensitized A549 cells toward cytotoxicity of curcumin (Fig. 5A). The level of curcumin-induced cell death was enhanced when the PI3K/Akt/mTOR signal transduction pathway was blocked, which may be related to the induction of PCDs.

As shown in Figs. 2A and 5B, the Annexin V-FITC/PI double stained assays revealed that the cell pre-treatment with rapamycin of 40 µM or LY294002 of 20 µM resulted in a significantly greater number of apoptotic (Annexin V-positive) cells than treatment with curcumin alone. MDC labeling analyses were done to detect the acidic vacuoles. Similarly, fluorescence microscopy shows significant increase in the number of MDC-labeled vacuoles (Fig. 3A and B) in cells exposed to rapamycin or LY294002 co-cultured with curcumin compared with curcumin treatment alone. Compared to curcumin alone, the ratio of LC3-II/LC3-I was significantly enhanced (Fig. 5C and D) in a dose-dependent manner when cells were pretreated with rapamycin, resulting in consistent results when curcumin was co-cultured with LY294002 (Fig. 5E and F), suggesting autophagy was clearly induced. The above results illustrated that combined treatment of PI3K/Akt or mTOR blockers significantly enhanced apoptosis and autophagy, accompanied by the inhibition of PI3K/Akt/mTOR pathway can be derived from the ratio of p-mTOR to total mTOR (Fig. 5C and D) as well as p-Akt to total Akt (Fig. 5E and F) downgrade. These data might support the idea that cytotoxicity derived from apoptosis and autophagy in A549 cells by curcumin proceeds through PI3K/Akt/mTOR inhibition. Thus, our findings unequivocally substantiated the fact that PI3K/Akt/mTOR is involved in the regulation of both apoptosis and autophagy induced by curcumin. Inhibition of mTOR promoted the development of both autophagy and apoptosis.

The present study collectively showed that curcumin has a similar function as the PI3K/Akt/mTOR pathway inhibitor resulting in varying degrees of reduction of the Akt phosphorylation and mTOR phosphorylation as well as mRNA expression. From the present results, our data indeed identified that curcumin exerts cytotoxic effect on A549 cells by inhibiting the PI3K/Akt/mTOR pathway to promote apoptosis and autophagy induced, indicating that PI3K/Akt/mTOR signal transduction pathway is a key pathway involved in the role of curcumin in lung cancer, and might be also the main target of curcumin.