Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), the TRIzol^® RNA purification kit and Fluo-4 probe were obtained from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). The ROS inhibitor (2R, 4R)-4-aminopyrrolidine-2,4-dicarboxylic acid (APDC) was supplied by Enzo Life Sciences, Inc. (Farmingdale, NY, USA). The dichlorodihydrofluorescein diacetate (H₂DCF-DA) probe was purchased from Thermo Fisher Scientific, Inc. MTT, Aloin and rhodamine 123 were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). The commercial kits for protein isolation and Bicinchoninic acid (BCA) protein quantification were products of Bio-Rad Laboratories, Inc. (Hercules, CA, USA). An annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit was obtained from MultiSciences (Lianke) Biotech Co., Ltd (Hangzhou, Zhejiang, China). A SYBR^® Green polymerase chain reaction (PCR) Master Mix was purchased from Applied Biosystems; Thermo Fisher Scientific, Inc. The phosphorylated (p)-p38 and p-c-Jun inhibitors, SB203580 and SP600125, were supplied by Beyotime Institute of Biotechnology (Haimen, China). The PrimeScript Real Time Reagent kit was obtained from Takara Biotechnology Co., Ltd. (Dalian, China).

Primary antibodies against caspase-3 (cat. no. sc-56053), caspase-9 (cat. no. sc-81589), caspase-8 (cat. no. sc-81661), caspase-10 (cat. no. sc-6186), CD95 (cat. no. sc-4276), p53 (cat. no. sc-126), p53-Ser15 (cat. no. sc-101762), p53-Thr18 (cat. no. sc-135631), p53-Ser20 (cat. no. sc-18,079-R), p53-Ser46 (cat. no. sc-101764), p53-Ser392 (cat. no. sc-7997), p-DNA-PK (cat. no. sc-101664), p-ATM (cat. no. sc-47739), p-ATR (cat. no. sc-109912), p-p38 (cat. no. sc-7973), p-c-Jun (cat. no. sc-822) and p-ERK (cat. no. sc-7976) were all purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The primary antibody against β-actin, and the IRDye-conjugated anti-rabbit (cat. no. 926-32211), anti-goat (cat. no. 926-32214) and anti-mouse (cat. no. 926-32280) secondary antibodies were purchased from LI-COR Biosciences (Lincoln, NE, USA).

A549 lung cancer cells were supplied by the Cell Bank of Type Culture Collection of the Chinese Academy of Science (Shanghai, China). The A549 cells were grown in DMEM medium supplemented with 10% (v/v) FBS and incubated at 37°C in a humidified (5% CO₂) incubator.

For experiments that included pretreatment with inhibitors of p-p38, p-c-Jun and ROS, A549 cells were pretreated with SB203580 (30 µM), SP600125 (10 µM) or APDC (25 µM) for 30 min at 37°C prior to Aloin (400 µM) treatment for 48 h at 37°C. Inhibitors were diluted in PBS and added to the cell culture supernatant. After 48 h Aloin treatment, the supernatant was immediately removed and experiments were performed.

A549 cells were plated into 96-well plates at 5×10³ cells per well. Following incubation for 24 h, the cells were exposed to 0 to 400 µM concentrations of Aloin. At 24, 48 and 72 h following exposure, the supernatant was removed and 100 µl (500 µg/ml) MTT solution was added to each well. Following 4 h of incubation at 37°C, the MTT solution from each well was replaced with 150 µl dimethyl sulfoxide, which was pipetted up and down several times to dissolve formazan crystals. The optical density was measured at a wavelength of 570 nm for each well using a microtiter plate reader (BioTek Instruments, Inc., Winooski, VT, USA). The results were expressed as the percentage of the control (0 µM Aloin).

The Annexin V-FITC and PI double staining method was used to analyze the percentage of cells undergoing apoptosis, according to the manufacturer's protocol. Briefly, A549 cells were seeded into 6-well plates at 2×10⁵ cells per well. Following incubation with or without the indicated concentrations of Aloin (0 to 400 µM) for 48 h, the cells were trypsinized, washed by PBS, extracted by centrifugation (1,500 × g for 10 min at 4°C), resuspended in binding buffer containing 1% (v:v) Annexin V-FITC and 2% (v:v) PI and incubated in the dark at room temperature for 20 min. Analysis was performed using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA). At least 1×10⁵ cells were collected, recorded on a dot plot and analyzed by ModFit v2.0.0 software (Verity Software House, Inc., Topsham, ME, USA).

Expression levels of the target proteins were analyzed by western blotting. Following incubation for 48 h, 1×10⁷ A549 cells treated in the presence or absence of Aloin (0 to 400 µM) were harvested and lysed with a radioimmunoprecipitation assay lysis buffer supplied by Beyotime Institute of Biotechnology (20 Mm Tris-HCl buffer, pH 7.5, containing 1 mM protease inhibitor mix) for 30 min on ice. Following this, the lysates were centrifuged at 1,500 × g for 10 min at 4°C and the supernatant was collected as a whole cell lysate for western blotting analysis. The whole cell lysate (~8 µg) was mixed with a loading buffer [125 mM Tris-HCl, 4% sodium dodecyl sulfate (SDS), 10% sucrose, 0.004% bromophenol blue (BPB) and 10% mercaptoethanol], boiled at 95°C for 5 min and then gel electrophoresis was performed with 8 to 12% SDS-PAGE gels. The protein bands were transferred to polyvinylidene fluoride (PVDF) membranes following manufacturer's protocol (EMD Millipore, Billerica, MA, USA). The PVDF membranes were then blocked with TBS containing 5% non-fat milk at 4°C for 1 h, rinsed with TBS with Tween 20 and incubated with specific primary antibodies, which were diluted in PBS (1:400), at 4°C overnight, followed by the corresponding IRDye-conjugated secondary antibody, which were diluted 1:2,000 in PBS, at room temperature for 1 h. Membranes were detected using an Odyssey Infrared Imaging System and Odyssey v1.3 software (LI-COR Biosciences). The relative optical densities of the target proteins were analyzed using Quantity One software v3.0 (Bio-Rad Laboratories, Inc.). Relative levels of the proteins were normalized to the control b-actin bands in each series and for protein loading. Each test was performed in at least triplicate.

The mRNA expression level of p53 was analyzed by RT-qPCR following incubation of the A549 cells with or without the indicated concentrations of Aloin (0 to 400 µM) for 48 h. Total RNA from ~5×10⁶ cells was isolated using a TRIzol reagent, and cDNA was synthesized using the PrimeScript Real Time Reagent kit according to the manufacturer's protocol. cDNA amplification was performed at 95°C for 10 sec and 56°C for 30 sec (45 cycles) on a 7500 Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.), using the QuantiTect SYBR^® Green PCR kit (Qiagen GmbH, Hilden, Germany) and 2 µl cDNA, according to the manufacturer's protocol. Specific primers for p53 and β-actin were custom-designed and synthesized by Takara Biotechnology Co., Ltd. The results from three independent experiments are expressed as the n-fold change of untreated (0 µM) cells and the relative mRNA level of each sample was normalized to β-actin using the 2^−∆∆Cq method (29).

MMP was examined by staining A549 cells with rhodamine 123. In healthy cells, rhodamine 123 accumulates and aggregates in the mitochondria; the loss of MMP leads to the release of rhodamine 123 from the mitochondria into the cytosol, increasing intracellular fluorescence (30). Following incubation for 48 h, 5×10⁵ cells A549 cells treated with or without the indicated concentrations of Aloin (0 to 400 µM) were harvested and resuspended in 200 µl PBS containing 10 µg/µl rhodamine 123 at 37°C for 30 min in the dark. Following staining, the A549 cells were rinsed three times with PBS and analyzed directly by flow cytometry using ModFit software v2.0.0 (Verity Software House, Inc.). The percentage of rhodamine 123 fluorescence positive cells was used to evaluate the extent of mitochondrial depolarization.

The level of Ca²⁺ released was measured by staining A549 cells with Fluo-4, which is a fluorescent dye for quantifying the levels of released mitochondrial Ca²⁺ (31). The A549 cells were incubated with or without the indicated concentrations of Aloin (0 to 400 µM) for 48 h. A total of 1×10⁶ cells were harvested and resuspended in 200 µl PBS containing 2.5 µM Fluo-4, prior to incubation at 37°C for 30 min in the dark. Following staining, the A549 cells were rinsed three times with PBS and analyzed directly by flow cytometry. The percentage of Fluo-4 fluorescence positive cells was used to evaluate the extent of mitochondrial depolarization.

The generation of ROS was measured by staining A549 cells with a H₂DCF-DA probe, which commonly reacts with a number of ROS molecules including hydrogen peroxide, hydroxyl radicals and peroxynitrite (32). Following incubation for 48 h, 1×10⁶ cells A549 cells treated with or without the indicated concentrations of Aloin (0 to 400 µM) were harvested, resuspended in 200 µl PBS containing 10 µM H₂DCF-DA, and incubated at 37°C for 30 min in the dark. Following staining, the A549 cells were rinsed three times with PBS and analyzed directly by flow cytometry using ModFit software v2.0.0 (Verity Software House, Inc.). The percentage of DCFH fluorescence positive cells were used to evaluate the production of ROS.

Data are presented as the mean ± standard deviation. Comparisons among groups were performed using one-way analysis of variance. If the variation was significant, significant differences between the means of control and of individual Aloin-treated cell groups were analyzed by Fisher's least significant difference test. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using SPSS 19.0 (IBM Corp., Armonk, NY, USA).

To evaluate the inhibitory effects of Aloin on the rate of A549 cell proliferation, cells were exposed to various concentrations (0, 100, 200, 300 and 400 µM) of Aloin for 24, 48 and 72 h. As presented in Fig. 1B, Aloin reduced A549 cell viability in a dose- and time-dependent manner. When compared with the untreated (0 µM) group, the viabilities of the 200, 300 and 400 µM Aloin treated groups were all significantly reduced at the 24, 48 and 72 h time points; however, no significant difference was observed in the cell viabilities at 24, 48 and 72 h for the 100 µM Aloin-treated group.

To determine whether the Aloin-induced inhibition of A549 cell proliferation was the result of apoptosis induction, cell apoptosis was assessed using flow cytometry. As presented in Fig. 1C and D, compared with the untreated group (0 µM), there was a significant increase in the number of apoptotic A549 cells in the 200, 300 and 400 µM Aloin-treated groups at 48 h. However, no significant difference was detected in the level of cell apoptosis in the 100 µM Aloin-treated group. Based on these findings, the 48-h time point was selected for further analyses.

To further elucidate the involvement of the apoptosis pathway initiated by Aloin in A549 cells, the protein expression levels of apoptosis-associated effectors including cleaved caspase-8 and −10, CD95, BAK, BAX, PUMA and NOXA were measured by western blotting. As presented in Fig. 2A, Aloin treatment failed to induce activation of the extrinsic apoptosis pathway effectors, cleaved-caspase-8 and −10, and CD95. However, compared with the untreated (0 µM) group, treatment with 200, 300 and 400 µM Aloin significantly increased the protein expression levels of the intrinsic apoptosis pathway effectors, BAK, BAX, PUMA and NOXA (Fig. 2B). In addition, as presented in Fig. 2C, treatment with 200, 300 and 400 µM Aloin also significantly increased the protein expression levels of cleaved caspase-3 and −9, which are downstream effectors of the intrinsic and extrinsic apoptosis pathways.

To further define the role of the mitochondria in Aloin-induced A549 cell apoptosis, the MMP and levels of released mitochondrial Ca²⁺ were determined using flow cytometry. When compared with the untreated (0 µM) group, treatment with 200, 300 and 400 µM Aloin induced a significant disruption in the MMP and release of mitochondrial Ca²⁺, as indicated by the observed increase in the percentage of rhodamine 123 or Fluo-4 positive A549 cells, respectively (Fig. 3).

It was hypothesized that Aloin-induced A549 cell apoptosis may be p53-dependent. To investigate this, the levels of p53 mRNA were measured using RT-qPCR. As presented in Fig. 4A, 100 to 400 µM Aloin did not exert any directly inductive or inhibitory effects on p53 mRNA expression following treatment for 48 h. Following this, the roles of Aloin in the protein expression of p53 and its antagonist MDM2, and p53 protein phosphorylation status, were investigated. As presented in Fig. 4B, compared with the untreated (0 µM) group, there were no detectable differences in the protein expression levels of p53, MDM2 or p53-Ser46. In contrast, treatment with 200, 300 and 400 µM Aloin resulted in strong activation of p53-Ser15, p53-Thr18, p53-Ser20 and p53-Ser392. These observations indicated that the signaling system responsible for p53 phosphorylation of Ser15, Thr18, Ser-20 and Ser-392 was involved in Aloin-induced apoptosis in A549 cells.

As DNA-PK and MAPKs are traditionally phosphokinases responsible for p53 phosphorylation, the extent of DNA-PK (p-ATM, p-ATR and p-DNA-PK) and MAPK (p-p38, p-c-Jun and p-ERK) activation in A549 cells following Aloin exposure were examined. As presented in Fig. 5A, when compared with the untreated (0 µM) group, treatment with 100 to 400 µM Aloin did not alter the levels of p-ATM, p-ATR and p-DNA-PK. However, when compared with untreated (0 µM) groups, treatment with 100 to 400 µM and 300 to 400 µM Aloin for 48 h significantly increased p-c-Jun and p-p38 levels, respectively. In addition, Aloin treatment did not alter the protein expression levels of p-ERK. These results suggest that c-Jun and/or p38-mediated cell signaling pathways may contribute to Aloin activities in p53 phosphorylation.

To determine whether Aloin-induced A549 cell death is attributable to the activation of p-p38 and/or p-c-Jun MAPKs, the specific inhibitors of p-p38 and p-c-Jun, SB203580 (30 µM) and SP600125 (10 µM), were used to pre-treat A549 cells for 30 min. The inhibiting efficiencies of SB203580 and SP600125 are shown in Fig. 6A. The levels of p-p38 and p-c-Jun in the SB203580 and SP600125 pretreated groups were significantly reduced when compared with the group treated with 400 µM Aloin only; the levels observed were nearly equal to those of the untreated group (0 µM Aloin). The impact of SB203580 and SP600125 on Aloin-induced A549 cell death was evaluated by MTT assay. As presented in Fig. 6B, SB203580 and SP600125 pretreatment, alone or in combination, significantly attenuated the Aloin-induced decrease in A549 cell viabilities. These results further support the hypothesis that Aloin-induced A549 cell apoptosis is associated with the c-Jun- and p38-mediated cell signaling pathways.

It has been widely reported that the generation of ROS induces the activation of MAPKs and subsequently, apoptosis (33,34). To investigate whether ROS generation is associated with Aloin-induced activation of c-Jun and p38, and apoptosis, the levels of ROS in Aloin-treated A549 cells were evaluated by flow cytometry following 48 h incubation and H₂DCF-DA probe labeling. As presented in Fig. 7A and B, when compared with the untreated (0 µM) group, treatment with 200, 300 and 400 µM Aloin induced a significant accumulation of ROS. To further define the role of ROS in the Aloin-induced decrease in cell viability, the ROS inhibitor, APDC (25 µM), was used for A549 cell pretreatment for 30 min. As presented in Fig. 7C and D, APDC pretreatment inhibited the 400 µM Aloin-induced generation of ROS in A549 cells. When compared with the untreated group, APDC pretreatment attenuated the 400 µM Aloin-induced inhibition of A549 cell viability (Fig. 7E). These results indicated that ROS generation may serve a crucial role in Aloin-induced apoptosis.