Heat-shock protein (Hsp) 70, known as a pro-survival protein, is aberrantly expressed in several malignancies. The small molecule 2-phenylethyenesulfonamide (PES), also referred to as pifithrin-μ, is known as an HSP70 inhibitor, which exhibits antitumor activities in a variety of cancer cell lines. However, little is known about its effect on non-small cell lung cancer (NSCLC) cell lines. This study aimed to investigate the effect of PES on human NSCLC cell lines A549 and H460, and explore the possible underlying mechanism of action. Cell viability assay by using CCK-8 kits was performed to demonstrate that PES dose- and time-dependently inhibited proliferation of A549 and H460 cells. Wound healing assay and Transwell migration assay results indicated that PES inhibited cell migration of A549 and H460 cells. Flow cytometry results demonstrated that PES resulted in G0/G1 phase cell cycle arrest, and induced apoptosis via a caspase-dependent manner in A549 and H460 cells. Western blotting results suggested that phosphorylation of AKT and ERK was inhibited by PES treatment. In addition, death receptor 4 (DR4) and DR5 were increased by PES treatment. Overexpression of Hsp70 in A549 cells attenuated the growth inhibitory efficiency of PES. Knockdown of Hsp70 in A549 cells enhanced sensitivity of PES to cell growth inhibition, suggesting that the inhibitory effect of PES on cell proliferation is specifically through Hsp70-dependent mechanism. PES and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) exerts a potent synergistic effect on cell proliferation inhibition and induction of apoptosis in A549 and H460 cells. In a mouse xenograft model of lung cancer by A549 cells, PES treatment displayed significant inhibitory effects on tumor growth. All these findings suggest that PES shows antitumor activity against human NSCLC
Lung cancer is one of the most prevalent and fatal type of cancers in the world, accounting for ~20% of all cancer-related death (
Heat shock protein (Hsp) family is a group of conserved molecular chaperons that facilitate proper protein folding, modification, and transportation, and are known as inhibitors of apoptosis (
Previous results identified the small molecular 2-phenylethynesulfonamide (PES), also known as pifithrin-μ, as a specific inhibitor of stress-inducible Hsp70, which induced tumor cell death but markedly showed less toxic to non-transformed cells (
We investigated the ability of PES to inhibit proliferation of NSCLC cell lines
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 (
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
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 (
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
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 (
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
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
AKT and MAPK signaling pathway is essential to the action of chemotherapeutic drugs in the regulation of cell cycle progression and apoptosis (
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 (
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
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 (
As shown in
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
Representative images of mice treated with either PBS or PES alone are shown in
Over the past decades, lung cancer is one of the most commonly diagnosed cancers, and its morbidity and mortality have markedly increased in the world (
In this study, the biologic effects of a small molecular Hsp70 inhibitor, PES, on human NSCLC cells were determined
In addition to inhibitory effect of growth, PES can induce cell cycle arrest and apoptosis. Flow cytometry results suggested that PES disrupted cell cycle progression, which was shown as a markedly increased percentage of NSCLC cells in G0/G1 phase. Furthermore, cell cycle progression markers, CDK2 and Cyclin A, were decreased, while CDK inhibitor, p21 was increased by PES treatments. The answer for the cell death through apoptotic or non-apoptotic pathway induced by PES is controversial. PES resulted in cell death in primary effusion lymphoma through induction of lysosomal cathepsin D release, not through canonical apoptotic processes (
Multiple signaling pathways, such as ERK and AKT signal transduction pathways, play an important role in cell survival and regulation of apoptosis (
TRAIL, also referred as Apo-2L, is an attractive antitumor therapeutic, which can selectively induce tumor cell apoptosis through binding to DR4 and DR5 in the cell surface (
In addition to the effect of PES on human NSCLC cells
In conclusion, these results indicated the abilities of PES
This study was supported by the intramural funding from Jingzhou First People's Hospital of Hubei Province (to L.C.) and the research grant provided by JingZhou Hospital Affiliated to Huazhong University of Science and Technology.
PES inhibits cell proliferation and migration of human NSCLC cells
PES induces cell cycle arrest of human NSCLC cells. (A) Cell cycle analysis of vehicle control and PES treatment in human NSCLC cells. A549 and H460 cells were treated with vehicle control or PES (20 µM) for 24 h, respectively. After straining with PI, flow cytometry was used to detect contribution of the cell cycle. The cell cycle distributions in A549 (B) and H460 (C) treated by PES are quantified by the bar graphs. (D) A549 and H460 cells were treated with vehicle control or PES (10 or 20 µM) for 48 h, whole cell extracts were collected and immunoblotted with anti-MMP-9, Vimentin, E-cadherin, CDK2, Cyclin A, and p21. GAPDH was used for loading control.
PES induces apoptosis of human NSCLC cells. (A) The effects of PES on induction of apoptosis. A549 and H460 cells were treated with vehicle control or PES (20 µM) for 24 h. After straining with Annexin V-FITC/PI, flow cytometry was used to detect cell apoptosis. (B) Annexin V-positive cells treated by PES for 24 h are quantified by the bar graphs. (C) A549 and H460 cells were treated with vehicle control or PES (20 µM) for 48 h, whole cell extracts were collected and immunoblotted with anti-cleaved caspase-3, anti-cleaved caspase-9, and anti-cleaved PARP. GAPDH was used for loading control. (D) A549 and H460 cells were treated with vehicle control or PES (10 or 20 µM) for 24 h, followed by lysis buffer treatment. Caspase-3 activity assay kits were used to evaluate effect of PES on apoptosis.
PES inhibits activities of AKT, ERK, and Hsp70 in human NSCLC cells. Effect of PES on activities of AKT and ERK. (A) A549 and H460 cells were treated with vehicle control or PES (10 or 20 µM) for 48 h. Whole cell extracts were collected and immunoblotted with Hsp70, DR4, DR5, AKT, p-AKT, ERK, and p-ERK. GAPDH was used for loading control. (B and C) A549 cells transfected with or without pSG5-Hsp70 for 4 h were treated with or without PES for further 44 h. Whole cells were collected and subjected to western blotting or CCK-8 assay as in Materials and methods above, respectively. (D and E) A549 cells transfected with or without siHsp70 for 4 h were treated with or without PES for further 44 h. Whole cells were collected and subjected to western blotting or CCK-8 assay as in Materials and methods, respectively.
PES and TRAIL synergistically enhance cell proliferation inhibition and apoptosis in human NSCLC cells. (A) A549 and (B) H460 cells were incubated with or without PES in the absence and presence of TRAIL for 24 h. The cell viabilities (%) were measured by CCK-8 assay. Annexin V-positive cells for (C) A549 and (D) H460 cells treated as above are quantified by the bar graphs. (E) Caspase-3 activity for A549 and H460 cells treated as above using caspase-3 activity assay kits. (F) A549 and H460 cells were incubated with or without PES in the absence and presence of TRAIL for 48 h. Whole cell extracts were collected and subjected to western blotting for cleaved PARP and GAPDH.
PES inhibits human NSCLC cells proliferation