Human lung cancer epithelial H460 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were cultivated in Roswell Park Memorial Institute (RPMI)-1640 medium supplemented with 2 mM L-glutamine, 10% fetal bovine serum (FBS) and 100 U/ml of penicillin/streptomycin. Cell cultures were maintained in a 5% CO₂ environment at 37°C. Cells were routinely passaged at preconfluent density using a 0.25% trypsin solution with 0.53 mM EDTA. RPMI-1640 medium, FBS, L-glutamine, penicillin/streptomycin, phosphate-buffered saline (PBS), trypsin, and EDTA were purchased from Gibco (Grand Island, NY, USA). LA, cisplatin, catalase, Hoechst33342, N-acetylcysteine (NAC), propidium iodide (PI), dimethylsulfoxide (DMSO), absolute ethanol, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), 2,7-dichlorofluorescein diacetate (DCFH₂-DA), dihydroethidium (DHE) and hydroxyphenyl fluorescein (HPF) were obtained from Sigma Chemical, Inc. (St. Louis, MO, USA). LA was prepared by dissolving in absolute ethanol, and stock sample was further diluted in culture medium. The final concentration of absolute ethanol used in all of the experiments was 0.1%. The results from the treated cells were compared with the non-treated cell exposed to the 0.1% final concentration of absolute ethanol. Paclitaxel, etoposide, Mn (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP) were obtained from Calbiochem (San Diego, CA, USA). Agarose was obtained from Bio-Rad (Hercules, CA, USA). Antibodies for Bcl-2, Mcl-1, Bax, caspase-3, FAK, phosphorylated FAK (Y397), AKT, phosphorylated AKT (S473), integrin α5, integrin αv, integrin β1, integrin β3, β-actin and peroxidase-labeled specific secondary antibodies were obtained from Cell Signaling Technology, Inc. (Denver, MA, USA).

Cell viability was determined by MTT colorimetric assay. Briefly, after specific treatments, cells in 96-well plates were incubated with 0.4 mg/ml of MTT for 4 h at 37°C in the dark. The supernatant was then removed and DMSO was added to dissolve the formazan product. The intensity was measured at 570 nm using a microplate reader (Anthros, Durham, NC, USA). The optical density ratio of treated to non-treated control cells was calculated and presented in terms of relative cell viability.

Apoptotic and necrotic cell death were detected by Hoechst33342 and PI co-staining. After specific treatments, cells were stained with 10 μM of the Hoechst33342 and 5 μM PI dyes for 30 min at 37°C. The apoptotic cells having condensed chromatin and/or fragmented nuclei are presented as bright blue fluorescence of Hoechst33342, while PI stained cells indicate necrosis. Mode of cell death was visualized and scored under a fluorescent microscope (Olympus IX51 with DP70).

Human lung cancer cells were seeded at a density of 2×10³ cells/well in 96-well plates. Cell proliferation was determined by MTT assay at 0, 24, 48, and 72 h after exposure to LA at indicated concentrations (0–10 μM) for 48 h. The absorbance of formazan product which was dissolved by DMSO was measured by spectrophotometry at 570 nm using a microplate reader.

Anchorage-independent growth was analyzed by the colony formation assay in soft agar. Briefly, 250 μl of the bottom layer which was a mixture of 1% agarose and completed RPMI-1640 media at a ratio 1:1 was prepared in 24-well plates. A single cell suspension of treated-H460 cells with non-toxic concentration (0–10 μM) of LA for 48 h was used to prepare the upper layer. The top layer composed with 0.335% agarose containing the cells at 1×10³ in 250 μl. Then completed RPMI medium (250 μl/well) was added and refilled every 3 days. The colony formation was determined after 2 weeks using a phase-contrast microscope (Olympus IX51 with DP70).

After specific treatments, the cells were incubated in lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 150 mM sodium chloride, 10% glycerol, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 100 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN, USA) for 1 h on ice. The cell lysate was collected, and the protein content was determined using the BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). Equal amount of protein from each sample were denatured by heating at 95°C for 5 min and subsequently loaded onto 10% SDS-PAGE. After separation, proteins were transferred onto 0.45-μm nitrocellulose membranes (Bio-Rad). Transferred membranes were blocked for 1 h in 5% non-fat dry milk in TBST (25 mM Tris-HCl pH 7.5, 125 mM NaCl, and 0.05% Tween-20) and incubated overnight with specific primary antibodies at 4°C. Then, the membranes were washed three times with TBST and incubated with appropriate horseradish peroxidase-labeled secondary antibodies for 2 h at room temperature. The immune complexes were detected by SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology) and quantified using analyst/PC densitometry software (Bio-Rad).

Intracellular hydrogen peroxide (H₂O₂), superoxide anion (O₂^·−) and hydroxyl radical (OH^·) were determined by flow cytometry using DCFH₂-DA, DHE and HPF as fluorescent probes, respectively. Cells at 1.5×10⁵ cells/well were seeded overnight into 6-well plates. Before LA treatment, the cells were incubated either with 10 μM DCFH₂-DA, 10 μM DHE or 10 μM HPF for 30 min at 4°C, after which they were washed with PBS and treated with 10 μM of LA for 1–3 h. After the indicated time, cells were washed, resuspended in PBS, and immediately analyzed for fluorescence intensity using FACSCalibur (Beckton-Dickinson, Rutheford, NJ, USA) at the excitation and emission wavelengths of 488 and 538 nm, respectively, for detecting DCF fluorescence, at 488 and 610 nm for DHE and 490 and 515 nm for HPF. Mean fluorescence intensity was quantified by CellQuest software (Becton-Dickinson, Franklin Lakes, NJ, USA) analysis of the recorded histograms. Relative fluorescence was calculated as a ratio of the treated to the non-treated control fluorescence intensity.

All data were expressed as the means ± SD from three or more independent experiments. Multiple comparisons were examined for significant differences of multiple groups, using analysis of variance (ANOVA), followed by individual comparisons with the Scheffe’s post hoc test. Statistical significance was set at p<0.05.

The non-toxic concentrations of LA were first determined. Human lung cancer H460 cells were cultured in RPMI medium in the absence or presence of LA (0–100 μM) for 48 h, and cell viability was determined by MTT viability assay. The results indicated that treatment with 0–10 μM of LA caused no significant difference of % cell viability in H460 lung cancer cells compared with non-treated control cells (Fig. 1A). The cytotoxicity effect of LA was early observed in the presence of 50 μM LA with ~85% viable cells. To confirm the effect of LA on cell toxicity, mode of cell death was evaluated by Hoechst33342 and PI co-staining assay. Fig. 1B and C demonstrate that apoptotic cells containing condensed and/or fragmented nuclei were not detectable in response to LA treatment at the concentrations of 0–10 μM. Treatment doses of 50 and 100 μM caused a significant increase in cell apoptosis over the control, while necrosis was barely detected in any of the concentrations.

The ability to grow independently of cell adhesion of tumor cells has been shown to be an important hallmark of aggressive metastatic cells (4,14). Next, the effect of LA on anchorage-independent survival and growth was investigated by soft agar colony formation assay. The cells were pretreated with LA at non-toxic concentrations for 48 h prior to subject in the layer of agarose containing RPMI medium as described in Materials and methods. Fig. 2A, C and D show that LA treatment significantly inhibited survival and growth of the lung cancer cells in a dose-dependent manner. Both the number and size of colonies were significantly suppressed in LA-treated cells in comparison to those of non-treated control. Our results revealed that LA treatment sensitizes anoikis and inhibits growth of these cells in detached condition as indicated by the significant decrease in the number and size of colonies, respectively.

Additionally, anti-proliferative effect of LA at non-toxic concentrations (0–10 μM) was further evaluated in normal cell adherent condition. Fig. 2B indicates that 10 μM of LA obviously suppressed proliferation of human lung cancer cells. Taken together, our results have demonstrated the effects of LA in sensitization of anoikis, inhibition of tumor growth in both anchorage-dependent and -independence conditions.

As recent evidence has reported the role of certain integrins such as integrin α5, αv, β1 and β3 in potentiating lung cancer anoikis resistance, migration, and metastasis (4,27,28), the expression of integrins in response to LA treatment was analyzed. The lung cancer cells were exposure to non-toxic concentrations of LA as previously described and the levels of integrin α5, αv, β1 and β3 were examined by western blotting. Fig. 3A and B illustrate the dramatic reduction of integrin β1 and β3 in response to LA treatment. In terms of integrin α5 and αv, we found no alteration.

Integrins were shown to mediate anoikis resistance in various cancer cells by increasing cellular survival signals such as FAK and PI3K/AKT pathways (4,29). We further tested such downstream molecular targets of integrins and found that in correlation to integrin β1 and β3 depletion, p-FAK and p-AKT were strongly downregulated in the presence of LA at the concentrations of 0–10 μM, whereas, there was no difference in the level of total FAK and AKT (Fig. 3C and D). These data suggested the possible mechanism of LA in attenuation of anoikis resistance and growth via the suppression of integrins and their related downstream pro-survival pathway.

Substantial evidence has indicated the role of integrins in regulation of chemotherapy resistance (27,30–32). Therefore, we evaluated the effect of LA treatment on the susceptibility to current chemotherapeutic drugs used for lung cancer treatment. H460 lung cancer cells were cultured in completed RPMI medium with or without non-toxic concentrations (0–10 μM) of LA for 48 h. Cisplatin, etoposide or paclitaxel was then added into pretreated cells for 24 h, before viable cells were detected by MTT assay. As presented in Fig. 4A–C, the incubation of H460 cells with either 25 μM cisplatin, 25 μM etoposide, or 0.1 μM paclitaxel for 24 h significantly reduced cell viability to 88.89, 81.62 and 77.17%, respectively. Importantly, pretreatment of the cells with non-toxic concentration of LA (10 μM) for 48 h prior to drug treatment remarkably sensitized the lung cancer cells to drug-induced apoptosis (Fig. 4D–F). It is worth noting herein that the sensitization to cisplatin- and etoposide-induced apoptosis was also found in lung cancer cell pretreated with of LA at low dose (5 μM) (Fig. 4D and E). These findings add the information that the observed depletion of integrin β1 and β3 and their survival counterparts mediated by LA influence drug susceptibility in these lung cancer cells.

Furthermore, we evaluated the effect of LA on survival and apoptosis regulatory proteins namely p-FAK, FAK, p-AKT, AKT, Mcl-1, Bcl-2, Bax, and caspase-3. The cells were pretreated with 10 μM of LA for 48 h, treated with cisplatin, etoposide, or paclitaxel for 24 h, and the proteins were determined by western blotting. As expected treatment of the cells with either LA or drug alone significantly reduced p-FAK and p-AKT (Fig. 5). The combination of LA and drug caused further reduction of such survival proteins. Also, the downstream anti- and proapoptotic members of Bcl-2 family protein were evaluated. We found the key anti-apoptotic proteins Bcl-2 and Mcl-1 significantly depleted in response to LA and drug treatment, while the proapoptotic Bax significantly increased (Fig. 6). Also, the cleavage form of caspase-3 significantly increased in response to the combination treatment. Taken together, we provide supportive information that LA sensitizes the cell response to chemotherapeutic drugs via FAK and AKT-dependent mechanism.

Reactive oxygen species (ROS) are crucial signaling molecules in cell biology. They have been shown to regulate protein expression in many steps of protein processing including transcription, translation, and degradation (24,33–37). In order to clarify the underlying mechanism of LA in downregulation of integrins, we determined the change in cellular ROS in response to LA treatment. Flow cytometry was used to evaluate cellular ROS level with the specific ROS fluorescence dyes. Fluorescent intensity of DCFH₂-DA, DHE and HPF reflects the amounts of H₂O₂, O₂^·− and OH^·, respectively. Fig. 7B indicates that treatment of the cells with 10 μM LA significantly increased intracellular O₂^·− and H₂O₂. However, there was no alteration of OH^· level in the cell treated with LA (Fig. 7).

In order to link that such ROS-induced by LA regulate integrin alteration, broad and specific ROS scavengers were added prior to LA treatment, and the level of integrin β1 and β3 was determined by western blot analysis. Fig. 8A and B show that treatment of the cells with NAC, a broad ROS scavenger successfully restored the expression of integrin β1 and β3. Attractively, H₂O₂ scavenger, catalase specifically prevented the reduction of integrin β1 but not β3 in LA-treated H460 cells (Fig. 8C and D). Integrin β3 was preserved by the pretreatment with O₂^·− scavenger (MnTBAP), but such scavenger has no effect on integrin β1 (Fig. 8E and F). Thus, our results suggested that LA decreases integrin β1 via H₂O₂ induction, while reduces integrin β3 via O₂^·−.

To provide supportive information regarding the role of specific ROS-mediating effect of LA on integrin alteration, anoikis, and drug responses, the cells were pretreated with specific ROS scavengers, treated with LA, and subjected to anchorage-independent growth or drug sensitization assays as previously described. Results indicated that the pan ROS scavenger NAC and H₂O₂ scavenger, catalase abolished effects of LA on anoikis as well as drug-mediated apoptosis (Fig. 8G and H). Moreover, MnTBAP, O₂^·− scavenger, inhibited anoikis induction effect but had only slightly effect on chemosensitization (Fig. 8G and H). These results confirmed the mechanistic roles of specific ROS on LA sensitization of lung cancer cells to anoikis and chemotherapeutic agents.