JS‑K induces G2/M phase cell cycle arrest and apoptosis in A549 and H460 cells via the p53/p21WAF1/CIP1 and p27KIP1 pathways

  • Authors:
    • Zeqing Song
    • Yongxin Yin
    • Siyuan Hao
    • Jianmao Wei
    • Bin Liu
    • Xiaojie Huang
    • Chenfeng Gao
    • Runzhi Zhu
    • Weiguo Liao
    • De Cai
  • View Affiliations

  • Published online on: April 9, 2019     https://doi.org/10.3892/or.2019.7104
  • Pages: 3475-3487
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Lung cancer is one of the most common malignancies worldwide, with high mortality and morbidity rates. O2‑​(2,4‑​dinitrophenyl)‑1‑​[(4‑ethoxycarbonyl)piperazin‑1‑yl]diazen‑1‑ium‑1,2‑diolate (JS‑K) is a potent anticancer agent that acts against a subset of human non‑small cell lung cancer (NSCLC) cell lines; however, the underlying mechanisms of JS‑K in NSCLC remain unclear. The present study aimed to evaluate the anticancer effect of JS‑K and investigate its underlying mechanisms in A549 and H460 cells. In the present study, A549 and H460 cells were treated with JS‑K, and then evaluated by cell viability assay, flow cytometry and western blot analysis. JS‑K markedly induced cell cycle arrest at the G2/M phase in a concentration and time‑dependent manner in both cell lines. This was associated with increased expression levels of p53, and the cell cycle inhibitors p21WAF1/CIP1 and p27KIP1, which, in turn, inhibited the expression of Cdc2, cyclin B1 and cyclin‑dependent kinase 2. In addition, JS‑K‑induced inhibition of proliferation was revealed to be partially modulated by the upregulation of p53 and p21WAF1, the ratio of Bax/Bcl‑2, and the activation of both the intrinsic and extrinsic apoptotic pathways in A549 and H460 cells. These results demonstrated that JS‑K could trigger cell cycle arrest at the G2/M phase and apoptosis in A549 and H460 cells, which was likely mediated via the p53/p21WAF1/CIP1 and p27KIP1 pathways. Overall, the results indicated that JS‑K may be used as an anticancer agent for the treatment of NSCLC.

Introduction

Lung cancer is the second most common type of cancer (1) and is the leading cause of cancer-associated mortality (2), with >1.38 million mortalities reported worldwide (3). At present, the majority of patients with lung cancer have a poor prognosis, with a 5-year relative survival rate of <17% (4). Lung cancer can be classified into small-cell lung cancer and non-small cell lung cancer (NSCLC), according to histological characteristics (5). More than 80% of lung cancer cases are NSCLC (6), which is composed of adenocarcinoma, squamous cell carcinoma and large cell carcinoma (7). Due to a lack of symptoms associated with early-stage lung cancer, the majority of lung cancer cases are diagnosed at an advanced stage when there are limited treatment options (810). The survival rate for patients with early-stage NSCLC is relatively higher following surgery (11). However, the majority of patients have a high risk of recurrence. Systemic chemotherapy is the most mainstream therapeutic option for the treatment of advanced NSCLC; however, the median survival time is only slightly >18 months from the time of diagnosis (12,13). Furthermore, it has been demonstrated that chemotherapy has notable resistance and insensitivity in the treatment of lung cancer (14). Therefore, the investigation of novel anticancer drugs with high efficiency and selectivity against lung cancer is required.

The well-known tumor suppressor gene p53 has been identified to be involved in DNA repair, transcription, genomic stability, cell cycle and apoptosis following cellular exposure to various types of stress (1517). Both cell cycle arrest and apoptosis are important tumor-suppressive pathways that involve p53 (18,19). Cell cycle arrest is regulated by the depletion of critical cell cycle proteins. Upon p53 activation, the cyclin-dependent kinase (CDK) inhibitor p21WAF1/CIP1 is upregulated, which leads to the inhibition of numerous cell cycle proteins, including CDK-4, CDK-6/cyclin D, CDK2/cyclin E and cyclin B1/Cdc2 (20). Cell apoptosis is associated with complex signaling pathways. p53 mediates cell apoptosis by upregulating BH3-only proteins, including Bax, and downregulating Bcl-2 family members, including Bcl-2 (21). As an intracellular suicide program, cell apoptosis is also mediated by the activation of caspases, including caspase-3, −8 and −9, and poly(ADP-ribose) polymerase (22,23). Therefore, triggering pathways involved with apoptosis and inhibiting cell cycle progression may present new strategies for the treatment of lung cancer.

Nitric oxide (NO) serves a crucial role in regulating the activity of cancer (24). As an arylated diazeniumdiolate-based anticancer agent, O2-(2,4-dinitrophenyl)-1-[(4- ethoxycarbonyl)piperazin- 1-yl]diazen-1-ium-1,2-diolate (JS-K; Fig. 1) exhibits cytotoxic and potent antitumor effects in several types of human cancer, including prostate cancer (25), hepatoma (26), acute myeloid leukemia (27), multiple myeloma (28) and bladder cancer (29). JS-K can also enhance cytotoxicity in doxorubicin-treated renal carcinoma cells by upregulating the expression of p53 (30). Notably, a number of studies have reported that JS-K was selectively cytotoxic to cancer cells and demonstrated no significant toxicity towards normal cells (25,28,31). A previous study demonstrated that a reactive oxygen species (ROS) activation mechanism contributed to JS-K-induced apoptosis in human NSCLC cells, including H1944 and H1703 cells (32). However, there is limited understanding regarding the mechanism of action of JS-K in other lung cancer cells, including A549 and H460 cells. The present study aimed to investigate the underlying antitumor mechanism of JS-K in A549 and H460 human NSCLC cells. To the best of our knowledge, the present study is the first to demonstrate that p53/p21WAF1/CIP1 and p27KIP1 proteins are involved in JS-K-induced apoptosis and cell cycle arrest.

Materials and methods

Chemicals

JS-K was acquired from Sigma-Aldrich; Merck KGA (Darmstadt, Germany; cat. no. J4137), and stored at −80°C at a concentration of 20 mM in 100% dimethyl sulfoxide (DMSO). The final density of DMSO used in the present study was <0.1%. Pifithrin-α (PFT-α) was purchased from MedChemExpress (New Jersey, USA; cat. no. HY-15484) and diluted to a final concentration of 30 µM with 100% DMSO. Antibodies against caspase-3 (cat. no. 9665S), caspase-9 (cat. no. 9502S), caspase-8 (cat. no. 4790S), Bcl-2 (cat. no. 4223S), Bax (cat. no. 2772S), p53 (cat. no. 9282S), p21Waf1/Cip1 (cat. no. 2947S), cyclin B1 (cat. no. 4138P), CDK2 (cat. no. 2546P), cyclin E1 (cat. no. 4129P), p27Kip1 (cat. no. 3686S), Cdc2 (cat. no. 9112S), p-Cdc2 (cat. no. 4539S) and GAPDH (cat. no. 2118L) were all obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Antibodies (caspase-3, caspase-9, caspase-8, Bcl-2, Bax, p53, p21Waf1/Cip1, cyclin B1, CDK2, cyclin E1, p27Kip1, Cdc2, p-Cdc2 and GAPDH) were diluted at the 1:1,000 with 5% bovine serum albumin (BSA). Goat anti-rabbit immunoglobulin G (IgG)-horseradish peroxidase antibody was used as the secondary antibody (Sino Biological, Inc., Beijing, China; cat. no. SSA004). The secondary antibody was diluted with 5% non-fat milk in proportion of 1:2,000. The Annexin V-FITC Apoptosis Detection kit was obtained from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan; cat. no. AD10). Cell Cycle Analysis kit (cat. no. C1052) and MTT (cat. no. ST316) were all obtained from Beyotime Institute of Biotechnology (Shanghai, China).

Cell culture and JS-K treatment

The human NSCLC cell lines A549 and H460 were obtained from the Cell Bank of the Institute of Biochemistry and Cell Biology at the China Academy of Sciences (Shanghai, China). In brief, A549 and H460 cells were respectively cultured in Dulbecco's modified Eagle's medium (DMEM; cat. no. C11995500BT) and RPMI-1640 medium (cat. no. C11875500BT) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), which were supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.; cat. no. 10099141), 100 U/ml penicillin and 100 U/ml streptomycin. The cells were maintained in a humidified incubator at 37°C with 5% CO2. Subsequently, the A549 cells were treated with various concentrations of JS-K (0, 1, 2 or 5 µM) for 48 h. In addition, the H460 cells were treated with different concentrations of JS-K (0, 5, 10 or 15 µM) for 24 h at which point the confluency was 60–70%. Another group of A549 cells were cultured with 5 µM JS-K for 0, 12, 24 or 48 h, and H460 cells were treated with 15 µM JS-K for 0, 6, 12 and 24 h. Moreover, A549 and H460 cells were pretreated with PFT-α (40 and 10 µM, respectively) for 1 h in the presence or absence of JS-K (5 and 15 µM, respectively).

MTT assay

An 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the cell toxicity and growth inhibition following JS-K treatment. Briefly, A549 and H460 cells were seeded in 96-well plates at a density of 5,000 cells/well overnight for attachment and recovery. Subsequently, the cells were pretreated with 0, 1, 2, 5, 10, 15 and 20 µM JS-K for 24 or 48 h. After the indicated time, 20 µl MTT solution (5 mg/ml) was added to each well and the cells were maintained in a humidified incubator at 37°C with 5% CO2 for 4 h. The supernatants were removed and 150 µl DMSO was added to each well to completely dissolve the formazan crystals. Subsequently, the plates were placed on an orbital shaker for 15 min and the absorbance was then assessed at 490 nm with an automated spectrophotometric plate reader (PerkinElmer, Inc., Waltham, MA, USA). All experiments were repeated independently in triplicate. The inhibition ratio was calculated using the following equation: Inhibition ratio (%) = (OD490 control-OD490 JS-K-treated)/OD490 control × 100%, where OD is the optical density.

Cell cycle analysis

Cells were quantified using a Cell Cycle Analysis kit (Beyotime Institute of Biotechnology, Haimen, China). A549 cells were pretreated with 0, 1, 2 or 5 µM JS-K for 48 h, and H460 cells were pretreated with 0, 5, 10 or 15 µM for 24 h. The cells were then washed with cold phosphate-buffered saline (PBS) and fixed with 70% ethanol overnight at 4°C. Subsequently, the ethanol was removed and the cells were incubated with propyl iodide organism dye for 30 min at 37°C. The dyed cells were detected with a BD Biosciences FACSCanto II flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and then analyzed using ModFit and CellQuest software 6.1 (BD Biosciences).

Analysis of cell apoptosis

To detect JS-K-induced apoptosis of the lung cancer cells, an Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining assay was performed with the Annexin V-FITC/PI kit (Sigma-Aldrich; Merck KGAA). Briefly, A549 cells were treated with 0, 1, 2 or 5 µM JS-K for 48 h, and H460 cells were treated with 0, 5, 10 or 15 µM JS-K for 24 h. The cells were then collected and resuspended in 1X binding buffer, which contained 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (pH 7.5), 2.5 mM CaCl2 and 140 nM NaCl. Cells were stained in the dark with Annexin V-FITC and PI for 15 min, according to the manufacturer's protocol, prior to flow cytometric analysis. Annexin V-positive cells were regarded to be in the early stage of apoptosis, whereas Annexin V and PI-positive cells were considered to be in the late phase of apoptosis.

Western blot analysis of JS-K-regulated apoptotic proteins and cell cycle proteins

The JS-K-treated cells were collected and lysed in lysis buffer, containing 1 ml RIPA buffer and 10 µl PMSF (Beyotime Institute of Biotechnology). The samples were then centrifuged for 20 min at 16,900 × g at 4°C to acquire the supernatants. The protein levels were quantified using a bicinchoninic acid assay (Beyotime Institute of Biotechnology) and equal amounts of protein were loaded to a 10% sodium dodecyl sulfate-polyacrylamide gel. The amount of p-Cdc2 loaded per lane was 40 µg, and for the other proteins 20 µg was loaded. Following electrophoresis, the separated proteins were transferred to polyvinylidene difluoride membranes, which were then blocked with 5% non-fat milk for 40 min at room temperature. The primary antibodies against cleaved-caspase-3, cleaved-caspase-9, cleaved-caspase-8, Bcl-2, Bax, p53, p21, p27, cyclin B1, CDK2, cyclin E1, Cdc2, cleaved-Cdc2 and GAPDH were incubated with the membranes at 4°C overnight. Subsequently, anti-rabbit IgG monoclonal antibody conjugated with horseradish peroxidase was added for 1 h at room temperature. Proteins were visualized with chemiluminescent reagent and a Tanon 5200 system (Tanon Science & Technology Co., Ltd., Shanghai, China). The data were analyzed using ImageJ (v1.8.0) analysis software (National Institutes of Health, Bethesda, MD, USA).

Statistical analysis

All data were acquired from a minimum of three independent repetitions. The results are presented as the mean ± standard deviation (SD) and the SD is indicated with an error bar in all figures. Statistical analysis was performed using SPSS software (version 17.0; IBM Corp., Armonk, NY, USA). Comparisons between groups were evaluated using one-way analysis of variance (ANOVA) (followed by Tukey's post hoc test). P<0.05 and P<0.01 were considered to indicate statistically significant differences.

Results

JS-K inhibits proliferation and induces apoptosis of A549 and H460 cells

The MTT assay with different concentrations of JS-K for 24 and 48 h demonstrated that JS-K exerted a significant inhibitory effect on the proliferation of A549 and H460 cells in a concentration- and time-dependent manner (Fig. 2). The IC50 value of JS-K at 48 h for A549 cells was 3.48±0.02 µM. The IC50 value of JS-K at 24 h for H460 cells was 11.17±0.03 µM. The Annexin V-FITC/PI cell apoptosis detection kit was also used to detect apoptosis of H460 and A549 cells (Fig. 3). The results revealed a significant increase in the apoptosis rates of these cells with increasing concentrations of JS-K. In addition, the Q2 and Q4 cell population in A549 (4.05–29.95%) and H460 (5.83–21.0%) cells was increased compared with the control group. As demonstrated in Figs. 2 and 3, JS-K inhibited proliferation and promoted apoptosis of H460 and A549 cells in a concentration-dependent manner.

JS-K promotes cell cycle arrest

To reveal whether JS-K exhibits an effect on cell cycle arrest in human lung carcinoma cells, A549 and H460 cells were treated with various concentrations of JS-K for 48 and 24 h, respectively, and were then subjected to flow cytometric analysis following DNA staining. The population of cells in the G2/M phase increased in A549 and H460 cells, coinciding with a reduction in cells in the G1 phase, when compared with the control group. The results revealed that the proportion of cells in the G2/M phase increased with increasing concentrations of JS-K following the indicated time-points (Fig. 4).

JS-K activates proteins associated with apoptosis and cell cycle arrest

The expression levels of apoptosis-associated proteins, including cleaved-caspase-9, cleaved-caspase-8, cleaved-caspase-3 and p53, and the ratio of Bax/Bcl-2 were identified to increase in a concentration- and time-dependent manner following treatment with JS-K compared with the control cells (Figs. 5 and 6). The amount of p-Cdc2 loaded per lane was 40 µg, and for the others 20 µg was loaded. In addition, the expression levels of proteins associated with cycle arrest, including cyclin E1, cyclin B1, p21, p27 and p-Cdc2, were revealed to increase in a concentration- and time-dependent manner following treatment with JS-K compared with the control cells. Conversely, the expression levels of CDK2, cyclin B1 and Cdc2 were revealed to decrease in a concentration- and time-dependent manner following treatment with JS-K (Figs. 7 and 8).

p53 serves a crucial role in JS-K-induced apoptosis and cell cycle arrest in A549 and H460 cells

As demonstrated in Figs. 9 and 10 the expression levels of proteins associated with apoptosis and cell cycle arrest were altered following JS-K treatment. However, it was identified that PFT-α could alleviate JS-K-induced cell apoptosis and cell cycle arrest. The results revealed that PFT-α significantly reduced the expression levels of p53, Bax, cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9, and upregulated the expression level of Bcl-2 in comparison with cells treated with JS-K alone. In addition, the expression levels of proteins associated with cell cycle arrest were altered following treatment with PFT-α. The results indicated that PFT-α significantly reduced the expression level of cyclin E1, p21, p27 and p-Cdc2. and upregulated the expression level of CDK2, cyclin B1 and Cdc2 in comparison with cells treated with JS-K alone. In summary, the results indicated a crucial role of p53 in JS-K-induced cellular apoptosis and cell cycle arrest in A549 and H460 cells.

Discussion

Despite advancements in therapeutic strategies and the development of new anticancer drugs, lung cancer is usually incurable. Therefore, investigations of novel therapeutic strategies and anticancer agents are urgently required. The antitumor effect of JS-K has attracted attention in recent years. JS-K is a diazeniumdiolate-based NO-donor prodrug, which has been widely studied due to its potential in increasing ROS levels, and the mechanisms of JS-K are largely understood. For example, JS-K has been reported to induce apoptosis of bladder cancer cells by increasing the levels of ROS (29). Although JS-K has been revealed to induce apoptosis and ROS accumulation in several types of human cancer cells, its effects on cell cycle signaling and apoptosis are poorly understood. In the present study, a potent antiproliferative effect of JS-K was observed in A549 and H460 cells. JS-K was identified to exert a marked effect on G2/M phase arrest and evidently triggered apoptosis in concentration- and time-dependent manners in both cell lines via the p53/p21WAF1/CIP1 and p27KIP1 pathways.

The proliferation of tumor cells is closely associated with cell cycle arrest, which can serve as a marker for the chemopreventative or antitumor activity of drugs (33). Three checkpoints in the G1, S and G2/M phases are mediated by various cell cycle regulatory proteins, including cyclins, CDKs and tumor suppressor proteins, including p53, p21WAF1/CIP1 and p27KIP1 (34). CDK-cyclin complexes are drivers of the cell cycle and are mediated by two families of CKIs, the CIP/KIP family, which includes p21WAF1/CIP1 and p27KIP1, and the INK4 family, which includes p15INK4b and p19INK4d (35). As the only CDK2-associated cyclin, cyclin E is a strong independent prognostic marker in patients with early-stage NSCLC (36). Results of the present study demonstrated that JS-K upregulated the expression level of cyclin E and downregulated the expression level of CDK2 in a concentration and time-dependent manner in A549 and H460 cells. Cdc2, also termed CDK-1, is associated with cyclin B1. The G2/M transition is often regulated by Cdc2 in mammalian cells (23). Cdc2 serves a crucial role in the induction of mitosis. Suppressing the activity of Cdc2 resulted in cell cycle arrest at the G2/M phase in several types of cell lines (37,38). In addition, it has been demonstrated that activation of the Cdc2/cyclin B1 complex was indispensable for the transition from the G2 to the M phase (39). In the present study, JS-K was identified to induce cell cycle arrest at the G2/M phase (Fig. 4). This effect was associated with a downregulation of cyclin B1 and Cdc2, an upregulation of p21WAF1/CIP1, and phosphorylation of Cdc2 in a concentration and time-dependent manner in A549 and H460 cells (Fig. 7). For the western blot analysis, the amount of p-Cdc2 loaded per lane was 40 µg, while for the other 20 µg was loaded. We consider that different amounts of samples may lead to some errors in the results. After adjusting the amounts of samples, we determined that the amount of sample of each analyzed lane was uniform by exposing the internal reference protein (GAPDH). The results revealed that the expression of the reference protein (GAPDH) and the target protein (p-Cdc2) was increased after adjusting the amounts of samples. Satisfactory observation results were obtained. The trend of GAPDH expression of each analyzed lane was consistent with that before samples adjustment. Although the amount of p-Cdc2 was different from that of Cdc2, the batches of samples on the analyzed lanes were the same. In addition, three batches of samples were repeated to obtain the means and relative errors. Then the relative errors were greatly reduced. In order to determine the phosphorylation, the following was assessed in the quantification section: ‘(p-Cdc2/control)/(Cdc2/control)’. Therefore, the errors caused by adjusting the amounts of samples had little effect on the results of the quantification analysis.

Following binding to cyclin-CDK complexes, members of the Cip/Kip family, including p21WAF1/CIP1 and p27KIP1, can prevent kinase activation and subsequently inhibit the progression of the cell cycle at the G2/M phase (40). p27KIP1 acts as a tumor suppressor by binding to complexes of cyclin E/CDK2 to prevent cell cycle progression from the G1 phase to the S phase (41). A previous study revealed that a low expression level of p27KIP1 was associated with tumor aggressiveness and poor patient survival (42). One of the functions of p21WAF1/CIP1 is to maintain cells at G2/M phases following DNA damage (18). In addition, p21WAF1/CIP1 can interact with cyclin B1, which prevents dephosphorylation of Cdc2 by Cdc25 and inhibit the activity of CDK-1 (43). Activated p53 has been revealed to serve a role in the regulation of cell cycle progression following DNA damage. The mechanism of p53 in G2/M phase cell cycle arrest involves the transactivation of the CDK inhibitor p21 (44). The results of the present study revealed that the expression levels of p53, p21WAF1/CIP1 and p27KIP1 increased in a concentration- and time-dependent manner in A549 and H460 cells following treatment with JS-K. These findings indicated that JS-K-induced G2/M phase cell cycle arrest may be mediated by the upregulation of p53, p21WAF1 and p27KIP1.

To maintain tissue homeostasis, apoptosis serves as a physiological pathway to eliminate damaged or infected cells (45). Apoptosis induction has been considered to be a useful strategy for cancer therapy. In the present study, the results of MTT assay suggested that JS-K acted as an anti-proliferation agent against A549 and H460 cells (Fig. 2). Therefore, in addition to cell cycle arrest, the present study investigated whether JS-K-induced cell growth arrest could be due to the induction of apoptosis. In the apoptosis assay, a significant increase in the apoptosis rates of A549 and H460 cells were revealed by flow cytometry following treatment with JS-K (Fig. 3).

Apoptosis can be activated via the extrinsic- or death receptor-associated pathways, as well the intrinsic- or organelle-mediated pathways (45). It is well known that p53 acts upstream of other pro-apoptotic proteins and mediates both the intrinsic and the extrinsic apoptotic pathways (46). Cysteine proteases, particularly caspases, can be classified as initiator caspases, including caspases-8 and −9, or as executioner caspases, including caspases-3, −6 and −7, based on their mechanism of action (47). Caspase-3, an executioner caspase, can be activated by the extrinsic pathway involving the activation of caspase-8 and the intrinsic pathway involving the activation of caspase-9 (48,49). The present study revealed that JS-K treatment could trigger both the mitochondria-initiated intrinsic pathway and extrinsic pathway in A549 and H460 cells. The results demonstrated that cleaved caspase-3, −8 and −9 were significantly upregulated following treatment with JS-K, which indicated that JS-K induced apoptosis in A549 and H460 cells via the mitochondria-initiated intrinsic pathway and extrinsic pathway by the upregulation of p53 (Figs. 5 and 6).

p53 is a critical transcription factor that regulates the transcription and expression of various target genes, including Bcl-2 and Bax, which can result in cell cycle arrest and apoptosis (50,51). The anti-apoptotic ability of Bcl-2 is suppressed following binding with Bax protein (52). Overall, p53 can positively regulate the expression of the proapoptotic proteins Bax, Bad and Bak, which prevents inhibition of Bcl-2 (49). Compared with the expression level of Bcl-2 alone, the ratio of Bcl-2 to Bax has been reported to a serve a larger role in cell survival or death following an apoptotic stimulus (53,54). The present study demonstrated that JS-K markedly upregulated the expression levels of cell cycle-associated proteins, including p21WAF1/CIP1 and p53, and also enhanced the ratio of Bax/Bcl-2 by increasing the expression level of Bax and reducing the expression level of the anti-apoptotic protein Bcl-2 in A549 and H460 cells (Fig. 5). These results indicated that p53 and Bax/Bcl-2 served an important role in JS-K-induced cell apoptosis. In addition, the results demonstrated that JS-K can be inhibited by PFT-α (Figs. 9 and 10). Following inhibition of JS-K with PFT-α, Bcl-2 suppression was markedly reversed, and p21WAF1/CIP1 and p27KIP1 were significantly downregulated with a decrease in the expression level of p53. Therefore, targeting the p53/p21WAF1/CIP1 and p27KIP1 signaling pathway may be considered as a potential strategy to inhibit the proliferation of A549 and H460 cells.

This research was aimed at evaluating the anticancer effect of JS-K and exploring its mechanism in A549 and H460 human NSCLC cells. It is unclear whether JS-K acts on other NSCLC cells via the p53/p21WAF1/CIP1 and p27KIP1 pathways. In addition, in vivo experiments were not carried out. In order to further elucidate the mechanism of JS-K, we will use JS-K to screen NSCLC cells comprehensively, and conduct in vivo studies in the future. In conclusion, the present study revealed that JS-K could induce cell cycle arrest and apoptosis in A549 and H460 cells via the p53/p21WAF1/CIP1 and p27KIP1 pathways. These findings promote understanding regarding the antitumor effect of JS-K in NSCLC and suggest that JS-K may be a novel candidate for lung cancer therapy.

Acknowledgements

Not applicable.

Funding

The present study was supported by the Yangfan Plan of Talents Recruitment Grant, Guangdong, China (Yue Ren Cai Ban [2016] 6); the Scientific Research Fund of Guangdong Medical College, China (M2016031, M2016032).

Availability of data and materials

The analyzed data sets generated during the study are available from the corresponding author on reasonable request.

Authors' contributions

DC, WL and RZ conceived and designed the study. DC and WL provided administrative support. ZS, RZ, XH and YY provided the materials of the study. BL, JW, ZS, CG and XH collected and assembled the data. SH, BL, JW, YY and WL performed the data analysis and interpretation. SH, CG and WL wrote the manuscript. All authors read and approved the manuscript and agree to be accountable for all aspects of the research in ensuring that the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References

1 

Siegel R, Naishadham D and Jemal A: Cancer statistics, 2012. CA Cancer J Clin. 62:10–29. 2012. View Article : Google Scholar : PubMed/NCBI

2 

Daniels MG, Bowman RV, Yang IA, Govindan R and Fong KM: An emerging place for lung cancer genomics in 2013. J Thorac Dis. 5 (Suppl 5):S491–S497. 2013.PubMed/NCBI

3 

Jemal A, Siegel R, Ward E, Hao Y, Xu J and Thun MJ: Cancer statistics, 2009. CA Cancer J Clin. 59:225–249. 2009. View Article : Google Scholar : PubMed/NCBI

4 

Askoxylakis V, Thieke C, Pleger ST, Most P, Tanner J, Lindel K, Katus HA, Debus J and Bischof M: Long-term survival of cancer patients compared to heart failure and stroke: A systematic review. BMC Cancer. 10:1052010. View Article : Google Scholar : PubMed/NCBI

5 

Saito S, Espinoza-Mercado F, Liu H, Sata N, Cui X and Soukiasian HJ: Current status of research and treatment for non-small cell lung cancer in never-smoking females. Cancer Biol Ther. 18:359–368. 2017. View Article : Google Scholar : PubMed/NCBI

6 

Zarogoulidis K, Zarogoulidis P, Darwiche K, Boutsikou E, Machairiotis N, Tsakiridis K, Katsikogiannis N, Kougioumtzi I, Karapantzos I, Huang H and Spyratos D: Treatment of non-small cell lung cancer (NSCLC). J Thorac Dis. 4 (Suppl 5):S389–S396. 2013.

7 

Farhat FS and Houhou W: Targeted therapies in non-small cell lung carcinoma: What have we achieved so far? Ther Adv Med Oncol. 5:249–270. 2013. View Article : Google Scholar : PubMed/NCBI

8 

Pirozynski M: 100 years of lung cancer. Respir Med. 100:2073–2084. 2006. View Article : Google Scholar : PubMed/NCBI

9 

Schwartz AG, Prysak GM, Bock CH and Cote ML: The molecular epidemiology of lung cancer. Carcinogenesis. 28:507–518. 2007. View Article : Google Scholar : PubMed/NCBI

10 

Spiro SG and Silvestri GA: One hundred years of lung cancer. Am J Respir Crit Care Med. 172:523–529. 2005. View Article : Google Scholar : PubMed/NCBI

11 

Blandin Knight S, Crosbie PA, Balata H, Chudziak J, Hussell T and Dive C: Progress and prospects of early detection in lung cancer. Open Boil. 7:1700702017. View Article : Google Scholar

12 

Vilmar AC and Sorensen JB: Customising chemotherapy in advanced nonsmall cell lung cancer: Daily practice and perspectives. Eur Respir Rev. 20:45–52. 2011. View Article : Google Scholar : PubMed/NCBI

13 

Wang T, Nelson RA, Bogardus A and Grannis FW Jr: Five-year lung cancer survival: Which advanced stage nonsmall cell lung cancer patients attain long-term survival? Cancer. 116:1518–1525. 2010. View Article : Google Scholar : PubMed/NCBI

14 

Zhang H, Feng QQ, Gong JH and Ma JP: Anticancer effects of isofraxidin against A549 human lung cancer cells via the EGFR signaling pathway. Mol Med Rep. 18:407–414. 2018.PubMed/NCBI

15 

Bailey SM, Meyne J, Chen DJ, Kurimasa A, Li GC, Lehnert BE and Goodwin EH: DNA double-strand break repair proteins are required to cap the ends of mammalian chromosomes. Proc Natl Acad Sci USA. 96:14899–14904. 1999. View Article : Google Scholar : PubMed/NCBI

16 

Vazquez A, Bond EE, Levine AJ and Bond GL: The genetics of the p53 pathway, apoptosis and cancer therapy. Nat Rev Drug Discov. 7:979–987. 2008. View Article : Google Scholar : PubMed/NCBI

17 

Choisy-Rossi C, Reisdorf P and Yonish-Rouach E: Mechanisms of p53-induced apoptosis: In search of genes which are regulated during p53-mediated cell death. Toxicol Lett. 102-103:491–496. 1998. View Article : Google Scholar : PubMed/NCBI

18 

Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP, Sedivy JM, Kinzler KW and Vogelstein B: Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science. 282:1497–1501. 1998. View Article : Google Scholar : PubMed/NCBI

19 

Vogelstein B, Lane D and Levine AJ: Surfing the p53 network. Nature. 408:307–310. 2000. View Article : Google Scholar : PubMed/NCBI

20 

Pietenpol JA and Stewart ZA: Cell cycle checkpoint signaling: Cell cycle arrest versus apoptosis. Toxicology. 181-182:475–481. 2002. View Article : Google Scholar : PubMed/NCBI

21 

Pflaum J, Schlosser S and Muller M: p53 family and cellular stress responses in cancer. Front Oncol. 4:2852014. View Article : Google Scholar : PubMed/NCBI

22 

Shi Y: Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell. 9:459–470. 2002. View Article : Google Scholar : PubMed/NCBI

23 

Yunlan L, Juan Z and Qingshan L: Antitumor activity of di-n- butyl-(2,6-difluorobenzohydroxamato)tin(IV) against human gastric carcinoma SGC-7901 cells via G2/M cell cycle arrest and cell apoptosis. PLoS One. 9:e907932014. View Article : Google Scholar : PubMed/NCBI

24 

Huang Z, Fu J and Zhang Y: Nitric oxide donor-based cancer therapy: Advances and prospects. J Med Chem. 60:7617–7635. 2017. View Article : Google Scholar : PubMed/NCBI

25 

Laschak M, Spindler KD, Schrader AJ, Hessenauer A, Streicher W, Schrader M and Cronauer MV: JS-K, a glutathione/glutathione S-transferase-activated nitric oxide releasing prodrug inhibits androgen receptor and WNT-signaling in prostate cancer cells. BMC Cancer. 12:1302012. View Article : Google Scholar : PubMed/NCBI

26 

Liu L, Huang Z, Chen J, Wang J and Wang S: Protein phosphatase 2A mediates JS-K-induced apoptosis by affecting Bcl-2 family proteins in human hepatocellular carcinoma HepG2 cells. J Cell Biochem. 119:6633–6643. 2018. View Article : Google Scholar : PubMed/NCBI

27 

Shami PJ, Saavedra JE, Wang LY, Bonifant CL, Diwan BA, Singh SV, Gu Y, Fox SD, Buzard GS, Citro ML, et al: JS-K, a glutathione/glutathione S-transferase-activated nitric oxide donor of the diazeniumdiolate class with potent antineoplastic activity. Mol Cancer Ther. 2:409–417. 2003.PubMed/NCBI

28 

Kiziltepe T, Hideshima T, Ishitsuka K, Ocio EM, Raje N, Catley L, Li CQ, Trudel LJ, Yasui H, Vallet S, et al: JS-K, a GST-activated nitric oxide generator, induces DNA double-strand breaks, activates DNA damage response pathways, and induces apoptosis in vitro and in vivo in human multiple myeloma cells. Blood. 110:709–718. 2007. View Article : Google Scholar : PubMed/NCBI

29 

Qiu M, Chen L, Tan G, Ke L, Zhang S, Chen H and Liu J: A reactive oxygen species activation mechanism contributes to JS-K-induced apoptosis in human bladder cancer cells. Sci Rep. 5:151042015. View Article : Google Scholar : PubMed/NCBI

30 

Qiu M, Ke L, Zhang S, Zeng X, Fang Z and Liu J: JS-K, a GST-activated nitric oxide donor prodrug, enhances chemo- sensitivity in renal carcinoma cells and prevents cardiac myocytes toxicity induced by Doxorubicin. Cancer Chemother Pharmacol. 80:275–286. 2017. View Article : Google Scholar : PubMed/NCBI

31 

Simeone AM, McMurtry V, Nieves-Alicea R, Saavedra JE, Keefer LK, Johnson MM and Tari AM: TIMP-2 mediates the anti-invasive effects of the nitric oxide-releasing prodrug JS-K in breast cancer cells. Breast Cancer Res. 10:R442008. View Article : Google Scholar : PubMed/NCBI

32 

Maciag AE, Chakrapani H, Saavedra JE, Morris NL, Holland RJ, Kosak KM, Shami PJ, Anderson LM and Keefer LK: The nitric oxide prodrug JS-K is effective against non-small-cell lung cancer cells in vitro and in vivo: Involvement of reactive oxygen species. J Pharmacol Exp Ther. 336:313–320. 2011. View Article : Google Scholar : PubMed/NCBI

33 

Ou X, Lu Y, Liao L, Li D, Liu L, Liu H and Xu H: Nitidine chloride induces apoptosis in human hepatocellular carcinoma cells through a pathway involving p53, p21, Bax and Bcl-2. Oncol Rep. 33:1264–1274. 2015. View Article : Google Scholar : PubMed/NCBI

34 

Shin SY, Yong Y, Kim CG, Lee YH and Lim Y: Deoxypodophyllotoxin induces G2/M cell cycle arrest and apoptosis in HeLa cells. Cancer Lett. 287:231–239. 2010. View Article : Google Scholar : PubMed/NCBI

35 

Hwang HJ, Kang YJ, Hossain MA, Kim DH, Jang JY, Lee SH, Yoon JH, Moon HR, Kim HS, Chung HY, et al: Novel dihydrobenzofuro[4,5-b][1,8]naphthyridin-6-one derivative, MHY-449, induces apoptosis and cell cycle arrest in HCT116 human colon cancer cells. Int J Oncol. 41:2057–2064. 2012. View Article : Google Scholar : PubMed/NCBI

36 

Muller-Tidow C, Metzger R, Kugler K, Diederichs S, Idos G, Thomas M, Dockhorn-Dworniczak B, Schneider PM, Koeffler HP, Berdel WE, et al: Cyclin E is the only cyclin-dependent kinase 2-associated cyclin that predicts metastasis and survival in early stage non-small cell lung cancer. Cancer Res. 61:647–653. 2001.PubMed/NCBI

37 

Bulavin DV, Higashimoto Y, Popoff IJ, Gaarde WA, Basrur V, Potapova O, Appella E and Fornace AJ Jr: Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase. Nature. 411:102–107. 2001. View Article : Google Scholar : PubMed/NCBI

38 

Chen YL, Lin SZ, Chang JY, Cheng YL, Tsai NM, Chen SP, Chang WL and Harn HJ: In vitro and in vivo studies of a novel potential anticancer agent of isochaihulactone on human lung cancer A549 cells. Biochem Pharmacol. 72:308–319. 2006. View Article : Google Scholar : PubMed/NCBI

39 

Lew DJ and Kornbluth S: Regulatory roles of cyclin dependent kinase phosphorylation in cell cycle control. Curr Opin Cell Biol. 8:795–804. 1996. View Article : Google Scholar : PubMed/NCBI

40 

Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K and Linn S: Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annu Rev Biochem. 73:39–85. 2004. View Article : Google Scholar : PubMed/NCBI

41 

Srivastava RK, Chen Q, Siddiqui I, Sarva K and Shankar S: Linkage of curcumin-induced cell cycle arrest and apoptosis by cyclin-dependent kinase inhibitor p21(/WAF1/CIP1). Cell Cycle. 6:2953–2961. 2007. View Article : Google Scholar : PubMed/NCBI

42 

Migita T, Oda Y, Naito S and Tsuneyoshi M: Low expression of p27Kip1 is associated with tumor size and poor prognosis in patients with renal cell carcinoma. Cancer. 94:973–979. 2002. View Article : Google Scholar : PubMed/NCBI

43 

O'Connell MJ, Walworth NC and Carr AM: The G2-phase DNA-damage checkpoint. Trends Cell Biol. 10:296–303. 2000. View Article : Google Scholar : PubMed/NCBI

44 

Chaudhary P, Sharma R, Sahu M, Vishwanatha JK, Awasthi S and Awasthi YC: 4-Hydroxynonenal induces G2/M phase cell cycle arrest by activation of the ataxia telangiectasia mutated and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) signaling pathway. J Biol Chem. 288:20532–20546. 2013. View Article : Google Scholar : PubMed/NCBI

45 

Guicciardi ME, Malhi H, Mott JL and Gores GJ: Apoptosis and necrosis in the liver. Compr Physiol. 3:977–1010. 2013.PubMed/NCBI

46 

Zhu X, Wang K, Zhang K, Zhang T, Yin Y and Xu F: Ziyuglycoside I inhibits the proliferation of MDA-MB-231 breast carcinoma cells through inducing p53-mediated G2/M cell cycle arrest and intrinsic/extrinsic apoptosis. Int J Mol Sci. 17:E19032016. View Article : Google Scholar : PubMed/NCBI

47 

McIlwain DR, Berger T and Mak TW: Caspase functions in cell death and disease. Cold Spring Harb Perspect Biol. 5:a0086562013. View Article : Google Scholar : PubMed/NCBI

48 

Ouyang L, Shi Z, Zhao S, Wang FT, Zhou TT, Liu B and Bao JK: Programmed cell death pathways in cancer: A review of apoptosis, autophagy and programmed necrosis. Cell Prolif. 45:487–498. 2012. View Article : Google Scholar : PubMed/NCBI

49 

Zhang Q, Ma S, Liu B, Liu J, Zhu R and Li M: Chrysin induces cell apoptosis via activation of the p53/Bcl-2/caspase-9 pathway in hepatocellular carcinoma cells. Exp Ther Med. 12:469–474. 2016. View Article : Google Scholar : PubMed/NCBI

50 

Brady CA, Jiang D, Mello SS, Johnson TM, Jarvis LA, Kozak MM, Kenzelmann Broz D, Basak S, Park EJ, McLaughlin ME, et al: Distinct p53 transcriptional programs dictate acute DNA-damage responses and tumor suppression. Cell. 145:571–583. 2011. View Article : Google Scholar : PubMed/NCBI

51 

Burmakin M, Shi Y, Hedstrom E, Kogner P and Selivanova G: Dual targeting of wild-type and mutant p53 by small molecule RITA results in the inhibition of N-Myc and key survival oncogenes and kills neuroblastoma cells in vivo and in vitro. Clin Cancer Res. 19:5092–5103. 2013. View Article : Google Scholar : PubMed/NCBI

52 

Zeng G, Liu J, Chen H, Liu B, Zhang Q, Li M and Zhu R: Dihydromyricetin induces cell cycle arrest and apoptosis in melanoma SK-MEL-28 cells. Oncol Rep. 31:2713–2719. 2014. View Article : Google Scholar : PubMed/NCBI

53 

Reed JC: Bcl-2 family proteins: Regulators of apoptosis and chemoresistance in hematologic malignancies. Semin Hematol 34 (4 Suppl 5). S9–S19. 1997.

54 

Pettersson F, Dalgleish AG, Bissonnette RP and Colston KW: Retinoids cause apoptosis in pancreatic cancer cells via activation of RAR-gamma and altered expression of Bcl-2/Bax. Br J Cancer. 87:555–561. 2002. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

June 2019
Volume 41 Issue 6

Print ISSN: 1021-335X
Online ISSN:1791-2431

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
APA
Song, Z., Yin, Y., Hao, S., Wei, J., Liu, B., Huang, X. ... Cai, D. (2019). JS‑K induces G2/M phase cell cycle arrest and apoptosis in A549 and H460 cells via the p53/p21WAF1/CIP1 and p27KIP1 pathways . Oncology Reports, 41, 3475-3487. https://doi.org/10.3892/or.2019.7104
MLA
Song, Z., Yin, Y., Hao, S., Wei, J., Liu, B., Huang, X., Gao, C., Zhu, R., Liao, W., Cai, D."JS‑K induces G2/M phase cell cycle arrest and apoptosis in A549 and H460 cells via the p53/p21WAF1/CIP1 and p27KIP1 pathways ". Oncology Reports 41.6 (2019): 3475-3487.
Chicago
Song, Z., Yin, Y., Hao, S., Wei, J., Liu, B., Huang, X., Gao, C., Zhu, R., Liao, W., Cai, D."JS‑K induces G2/M phase cell cycle arrest and apoptosis in A549 and H460 cells via the p53/p21WAF1/CIP1 and p27KIP1 pathways ". Oncology Reports 41, no. 6 (2019): 3475-3487. https://doi.org/10.3892/or.2019.7104