The human non-small cell lung cancer (NSCLC) cells (A549 and H460) purchased from the American Type Culture Collection (Manassas, VA, USA) were maintained in DMEM supplemented with 10% FBS (Gibco, Gaithersburg, MD, USA) at 37°C in a humidified atmosphere with 5% CO₂. The heat-shock protein 70 (Hsp70) inhibitor PES, (pifithrin-μ), was purchased from Calbiochem (San Diego, CA, USA). Recombinant TRAIL was obtained from Invitrogen (Carlsbad, CA, USA). PES and recombinant TRAIL were dissolved in DMSO (Sigma, St. Louis, MO, USA) and PBS containing 0.1% (w/v) bovine serum albumin (BSA), respectively.

The plasmid pSG5-Hsp70 was kindly provided by Professor X. Sun (School of Basic Medical Sciences, Wuhan University, Wuhan, China). Hsp70 siRNA and control siRNA were obtained from GenePharma (GenePharma, Shanghai, China). A549 cells were transiently transfected with pSG5-Hsp70 using X-tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland) according to the manufacturer's instructions. A549 cells were transiently transfected with Hsp70 siRNA or control siRNA using Oligofectamine (Invitrogen) according to the manufacturer's instructions.

The cell viability was determined by the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) assay. Briefly, A549 and H460 cells were incubated in 96-well plates at a density of 5×10³ per 100 µl of culture medium overnight. After treated with indicated concentration of PES for 24 and 48 h, 10 µl of tetrazolium substrate were added to each well of the plate. After incubation at 37°C for 1 h, the absorbance was recorded at a wavelength of 450 nm using a microplate reader (EXL800; BioTek, Winooski, VT, USA). Each experiment was determined in triplicate and repeated at least three times.

A549 and H460 cells (5×10⁵) were placed and culture in 6-well plates overnight. A sterile 10-µl pipette tip was used to create wounds in 6-well plates. The cells were washed with PBS and cultured with fresh medium with or without PES. After incubation for further 48 h, an inverted microscope was used to determine the wounds.

Transwell migration assay was conducted in a Transwell chambers (Corning, New York, NY, USA) with a polycarbonate membrane (8-µm polyester membrane filter pores). Cells were starved for 24 h prior to the experiment, and then 1×10⁵ cells were seeded into the upper chamber with 100 µl serum-free medium. The bottom chamber contained 500 µl of medium containing 10% FBS to serve as a chemoattractant. Following incubation at 37°C for 48 h, the cells adhering to the lower surface of the membrane were fixed, stained, and captured.

Cell cycle arrest and induction of apoptosis by PES were assessed by flow cytometry (BD Biosciences). A549 and H460 cells (2×10⁵/well) were seeded in 6-well plates and treated with vehicle control or PES (20 µM) for 24 h, respectively. For cell cycle analysis, above cells were collected and fixed with 500 µl of 70% ethanol at −20°C overnight. The fixed cells were washed with cold PBS 3 times. Cell cycle distribution was determined by using Cell cycle Detection kit (Multisciences, Hangzhou, China) according to the manufacturer's instructions. For apoptosis analysis, PES or vehicle control treatment of A549 and H460 cells were collected. Annexin V-FITC/propidium iodide (PI) Apoptosis Detection kit (Multisciences) was used to detect cell apoptosis induced by PES according to the manufacturer's instructions.

Caspase-3 activity assay kits from Beyotime (Shanghai, China) were used to detect caspase-3 activity. In brief, human NSCLC cells were treated with PES and TRAIL, either alone or in combination for 24 h. The above cells were washed with cold PBS 3 times, followed by lysis buffer treatment on ice for 30 min. After centrifuged at 12,000 g for 10 min, cell lysate supernatant (10 µl), assay buffer (80 µl) and caspase-3 substrate (Ac-DEVD-pNA, 10 µl) were added to each well in a 96-well plate. The samples were further incubated at 37°C for 12 h, and their optical density (OD) was detected at a wavelength of 405 nm using a microplate reader (BioTek, EXL800; BioTek). Each experiment was determined in triplicate and repeated at least three times.

Cells were lysed in RIPA lysis buffer (Beyotime) supplemented with 0.5% cocktail protease inhibitor (Roche). The cell lysates were centrifuged at 12,000 g for 10 min at 4°C, and the supernatants were collected. Protein concentrations in the supernatants were measured according to the bicinchoninic acid method using bovine serum albumin as a standard. Equal amounts of proteins mixed with 5X loading buffer were subjected to 10% SDS-PAGE gels and transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). After blocking the membranes with 5% non-fat milk in TBST for 1 h at room temperature, the membranes were incubated with desired primary antibodies overnight at 4°C. After 3×5 min washes in TBST, the membranes were incubated with corresponding horseradish-peroxidase-conjugated secondary antibodies for 1 h at room temperature. ECL systems (Bio-Rad) were used to detect expression of antibody-bound proteins.

The primary antibodies used in this study were as follows: GAPDH (10494-1-AP) from Proteintech (Peking, China), Vimentin (5741), cleaved caspase-3 (9664), cleaved caspase-9 (7237), Hsp70 (4872), Akt (9272), p-Akt (4060), ERK (9102), and p-ERK (4370) from Cell Signaling Technology (Cambridge, UK), p21 (sc-397), cyclin A (sc-751), CDK2 (sc-163), MMP9 (sc-21733), E-cadherin (sc-8426) cleaved PARP (sc-56196), DR4 (sc-8411), and DR5 (sc-166624) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The secondary antibodies were horseradish-peroxidase-conjugated secondary anti-mouse IgG (Kerui Tech, Wuhan, China) or anti-rabbit IgG (Kerui Tech).

Athymic female nude mice (6 weeks of age) were purchased from Beijing HFK Bioscience Co. Ltd. (Beijing, China) and housed under pathogen-free conditions. Animal care and use were approved by the Medical Ethics Committee of Wuhan University.

A549 cells (1×10⁷) were suspended in Matrigel (BD Biosciences) and inoculated subcutaneously into the mice. Twelve mice bearing evident tumors were arbitrarily assigned to PBS control group and PES treatment groups (six mice per group). When tumors reached a size of ~5×5 mm², mice were treated with either a single of intraperitoneal injection of PES (20 mg/kg) or PBS every two days. After 3-week treatment, mice were euthanized with carbon dioxide. Tumor burdens were evaluated by measuring body weight, tumor weight, and tumor volume. Tumor volume was determined as 0.5 × length × width². Tumor samples were collected and fixed in 10% neutral buffered formalin. Hematoxylin and eosin staining and immunohistochemistry for histological analysis of tumor samples were performed as described previously (16).

Data were expressed as the mean ± standard deviation (mean ± SD). Statistical analysis was performed using SPSS software (version 19.0, SPSS, Chicago, IL, USA). The significance of the difference between two groups was determined using the Student's t-test. Values of P<0.05 were considered statistically significant.

The chemical structure of PES is shown in Fig. 1A. To determine the effect of PES on cell viability of human NSCLC cells, A549 and H460 cells were exposed to vehicle control or a series of different PES concentration, ranging from 2.5 to 40 µM, for 24 and 48 h, respectively. CCK-8 assays were used to detect cell viability. As shown in Fig. 1B, PES induced a time- and dose-dependent loss of viability in A549 cells, with 50% inhibitory concentration (IC₅₀) of 44.9 and 25.7 µM by a 24- and 48-h PES treatment. Similar results were also found in H460 cells treated by a series of different PES concentration, and the IC₅₀ values were calculated to be 40.1 and 24.3 µM by a 24- and 48-h PES treatment (Fig. 1C). These results suggested that PES efficiently inhibited cell proliferation of A549 and H460 cells.

Accumulating evidence has demonstrated that the metastasis activity of cancer cells is a critical mechanism for cancer mortality and invasion, which facilitated the development of cancer. To this end, the wound healing assay and Transwell migration assay were performed to study the effect of PES on migration of human NSCLC cells. When compared to the control group, PES suppressed the wound healing in A549 and H460 cells (Fig. 1D). To exclude the effect of cell proliferation on cell migration, Transwell migration assay was further used to evaluate effect of PES on cell migration. Similarly, the inhibitory effect of PES on cell migration was confirmed by the Transwell migration assay, in which the number of migrated A549 and H460 cells was significantly reduced after treatment with PES for 48 h (Fig. 1E). These results suggested that PES inhibited cell migration of human NSCLC cells.

To determine the mechanism by which PES inhibits human NSCLC cell proliferation, flow cytometry was used to analyze cell cycle contribution of A549 and H460 cells after PES (20 µM) treatment for 24 h. As shown in Fig. 2A, PES treatment markedly inhibited cell cycle progression in A549 and H460 cells, which was observed by a significant decrease in the percentage of cells in the S phase and concomitant increases in cells in the G0/G1 phase. When compared to the control, treatment of PES increased the fraction of G0/G1 from 32.8 to 54.1%, decreased the fraction of S phase from 55.4 to 32.8% in A549 cells (Fig. 2B). Similarly, when compared to the control, PES increased the fraction of G0/G1 from 34.2 to 48.4%, decreased the fraction of S phase from 44.6 to 33.5% in H460 cells (Fig. 2C). Notably, a marked change in percentage of cells in G2/M phase was not found in A549 and H460 cells (Fig. 2B and C).

Consistent with the observation by flow cytometry, western blotting results indicated that PES inhibited expression of cyclin A and CDK2, which are cell cycle progression markers (Fig. 2D). In contrast, expression of CDK inhibitor p21, a negative regulator of cell cycle progression, was increased by PES treatment (Fig. 2D). Since inhibition of cell migration may be partly caused by inhibition of cell proliferation, western blotting was used to further detect the expression of genes associated with cell invasion. Consistent with the results of wound healing assay, PES increased expression of the epithelial marker E-cadherin and reduced expression of vimentin and MMP9, suggesting that PES inhibited human NSCLC cell migration and invasion via regulation of epithelial to mesenchymal transition (EMT) (Fig. 2D).

To determine whether the inhibitory effect of PES on cell viability was associated with the induction of cell apoptosis, A549 and H460 cells were treated with or without PES (20 µM) for 24 h. Flow cytometry was used to analyze apoptosis induced by PES. As shown in Fig. 3A, the induction of apoptosis by PES was validated by increased percentage of Annexin V-positive cells. Quantitative analysis of Annexin V-positive cells induced by PES treatment is shown in Fig. 3B.

In addition to Annexin V-FITC assay, western blot analysis was used to evaluate the effect of PES on induction of apoptosis. A549 and H460 cells were treated with or without PES (10 or 20 µM) for 48 h. Whole-cell extracts were harvested and subjected to western blotting. As shown in Fig. 3C, PES treatment increased expression of cleaved caspase-3, cleaved caspase-9, and cleaved PARP in both A549 and H460 cells. Furthermore, activities of caspase-3 were dose-dependently increased by PES treatment in A549 and H460 cells (Fig. 3D). These results suggested that PES induced apoptosis, at least in part, through a caspase-dependent intrinsic mitochondrial pathway in human NSCLC cells.

AKT and MAPK signaling pathway is essential to the action of chemotherapeutic drugs in the regulation of cell cycle progression and apoptosis (17,18). Previous reports have indicated that AKT and ERK are key modulators of cell proliferation, and activation of AKT and ERK by phosphorylation is associated with tumor aggressiveness and prognosis in NSCLC (19,20). To determine the effect of PES on AKT and ERK signaling pathway, A549 and H460 cells were treated with vehicle control or PES (10 or 20 µM) for 48 h. Results shown in Fig. 4A indicate that PES dose-dependently decreased phosphorylated AKT and ERK in A549 and H460 cells. In addition, DR4 and DR5 were increased following PES treatment.

Previous results have indicated that Hsp70 is essential to NSCLC growth regulation, and compared to normal counterpart cell lines, Hsp70 was overexpressed in NSCLC cells (5). Since PES is an Hsp70 inhibitor, the effect of PES on Hsp70 was evaluated in human NSCLC cells. As shown in Fig. 4A, PES did not reduce expression of Hsp70 in A549 and H460 cells, suggesting that PES might inhibit activity of Hsp70 via specifically interacting with the ATPase binding domain.

To further validate if cell viability decrease by PES treatment is caused by acting on Hsp70, pSG5-Hsp70 or vehicle control (pSG5) were transiently transfected into A549 cells. At 4 h after transfection, cells were treated with or without PES (20 µM) for further 44 h. CCK-8 assay was used to detect cell viability after overexpression of Hsp70. As shown in Fig. 4B, Hsp70 was successfully overexpressed in A549 cells. Though overexpression of Hsp70 itself did not increase cell viability, it can efficiently attenuate the inhibitory efficiency of PES (Fig. 4C). To further characterize the role of Hsp70 in cell proliferation, Hsp70 was knocked down in A549 cells by siRNA. Results shown in Fig. 4D confirmed reduction of Hsp70 expression following by transfection with siHsp70. Moreover, Hsp70 knockdown by siHsp70 alone decreased cell viability by 58.8%. Most importantly, A549 cells transfected with siHsp70 were more sensitive to PES-induced cell proliferation inhibition (Fig. 4E). These results suggested that PES inhibited cell proliferation of human NSCLS cells through Hsp70-dependent mechanism.

TRAIL belongs to a member of the tumor necrosis factor family that can trigger apoptosis in a broad spectrum of tumor cells while not in most normal cells (21). TRAIL can selectively trigger apoptosis of tumor cells through recognizing and binding to DR4 and DR5 on cell surfaces (22). Based on the upregulation of DR4 and DR5 in response to PES, we sought to investigate whether PES combined with TRAIL synergistically inhibits cell proliferation and enhance induction of apoptosis in NSCLC cells. A549 and H460 cells were treated with or without PES (20 µM) in the absence or presence of TRAIL (40 ng/ml) for 24 h, cell were harvested and subjected to cell viability, apoptosis, and caspase-3 activation analysis, respectively.

As shown in Fig. 5A, PES alone and TRAIL alone reduced cell viability by 67.2 and 84.9%, while co-treatment resulted in cell viability inhibition by 28.5% in A549 cells. Similarly, PES alone and TRAIL alone reduced cell viability by 59.9 and 66.1%, while co-treatment resulted in cell viability inhibition by 19.7% in H460 cells (Fig. 5B). Flow cytometry results suggested that co-treatment with PES and TRAIL enhances cell apoptosis to a greater degree compared to PES treatment alone. Interestingly, TRAIL alone did not result in a marked induction of apoptosis in A549 and H460 cells. Quantitative analysis is shown in Fig. 5C and D. As expected, co-treatment of A549 and H460 cells with PES and TRAIL led to a marked increase in caspase-3 activation, when compared to PES or TRAIL treatment alone. Results indicated in Fig. 5E demonstrate that treatment of cells with TRAIL alone did not induce the cleavage of PARP protein, while co-treatment of cells with PES and TRAIL enhanced expression of cleaved PARP to a greater degree compared to PES treatment alone. Taken together, these results suggested that PES sensitized A549 and H460 cells to TRAIL-induced cell proliferation inhibition and apoptosis.

To evaluate the antitumor activity of PES in a xenograft model, A549 cells (1×10⁷) suspended in Matrigel were injected subcutaneously into flanks of female mice. After 7 days, mice bearing visible tumors (~5×5 mm²) were treated with PES (10 mg/kg) or PBS via intraperitoneal injection every two days. Three weeks later, the mice were euthanized, and tumors were measured and weighed. As shown in Fig. 6A and B, compared to PBS treatment, tumor volume and weight were reduced by PES treatment. Moreover, no significant mouse body weight loss was demonstrated in PES treatment groups (Fig. 5C).

Representative images of mice treated with either PBS or PES alone are shown in Fig. 6D. To further determine PES-associated toxicity in mice, major organs and tumors were dissected and subjected to hematoxylin and eosin staining and immunohistochemistry analysis. Results shown as Fig. 6E indicated that no notable differences between PES and PBS treatment group were observed among the organs involved, the liver, kidney, spleen, and lung. In addition, immunohistochemistry analysis results demonstrated that tumor tissues from the mice treated with PES also exhibited growth inhibition and increased apoptosis, as assessed by the staining for cleaved caspase-3, Ki-67, and TUNEL (Fig. 6F). Taken collectively, the above data suggested that PES exerts a potent in vivo inhibitory effect on lung carcinoma xenograft growth by inducing tumor cell apoptosis in mice. Moreover, PES used in this study made no marked signs of systemic toxicity, indicating that the dosage of PES in vivo in this study was safe.