Effects of 3-Tetrazolyl Methyl-3-Hydroxy-Oxindole Hybrid (THOH) on Cell Proliferation, Apoptosis, and G2/M Cell Cycle Arrest Occurs by Targeting Platelet-Derived Growth Factor D (PDGF-D) and the MEK/ERK Signaling Pathway in Human Lung Cell Lines SK-LU-1, A549, and A-427.

BACKGROUND The aim of this study was to evaluate the effects of 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH) on cell proliferation, apoptosis, and the cell cycle in human lung cancer cell lines SK-LU-1, A549, and A-427, and the normal lung fibroblast cell line, MRC-5, in vitro. MATERIAL AND METHODS Human lung adenocarcinoma cells SK-LU-1, A549, and A-427, and the normal lung fibroblast cells, MRC-5 were cultured and treated with increasing concentrations of 10 mM of a stock solution of THOH in dimethyl sulfoxide (DMSO). An MTT cell proliferation assay was used. Cell apoptosis and the cell cycle were studied using fluorescence-activated cell sorting (FACs) with fluorescein isothiocyanate (FITC), Annexin-V, propidium iodide (PI), and nuclear staining with 4',6-diamidino-2-phenylindole (DAPI). DNA damage was measured using the comet (single-cell gel electrophoresis) assay. Cell migration was evaluated using a wound healing assay, and Western blotting was used to measure protein expression levels. RESULTS Treatment of SK-LU-1 cells with THOH inhibited cell migration. Treatment of lung cancer cells, SK-LU-1, A549, and A-427, with THOH inhibited cell proliferation, with the most marked inhibition found in the SK-LU-1 lung cancer cells (IC50, 12 µM). Treatment of lung cancer cells, SK-LU-1, A549, and A-427, with THOH increased cell apoptosis, resulted in G2/M cell cycle arrest, and inhibited both the platelet-derived growth factor D (PDGF-D) and MEK/ERK signaling pathways. CONCLUSIONS Treatment of adenocarcinoma cells, SK-LU-1, A549, and A-427, with THOH inhibited cell proliferation, apoptosis, and resulted in G2/M cell cycle arrest by targeting PDGF-D and the MEK/ERK signaling pathway.

Background

Lung cancer is one of the most commonly diagnosed cancers in developed countries and has high rates of morbidity and mortality due to the difficulty in detecting early-stage cancer [1,2]. Because there may be few clinical symptoms associated with early-stage lung cancer, patients commonly (approximately 80%) present with late-stage lung cancer [3]. Although there is continuing research being undertaken to understand the mechanisms involved in the prevention, etiology, and treatment of lung cancer, including studies on targeted therapy based on molecular research, the prognosis for patients who present with lung cancer remains poor [4-8].

The approach for the treatment of lung cancer includes surgical tumor resection, chemotherapy, and radiation therapy, but in most cases, recurrence of lung cancer occurs following treatment, and in advanced-stage cases, only palliative treatment is given [9-15]. Therefore, there is a need for continuing research to discover novel molecules, which might prevent the recurrence of lung cancer, or inhibit tumor progression. During the past few decades, several anti-cancer agents have been investigated in clinical trials, but the development of drug resistance and the development of drug toxicity to some currently used anti-cancer drugs drive the need for the discovery of new chemotherapeutic agents in lung cancer [16-18].

Currently, synthetic chemists are combining potent anti-cancer molecules with the aim of developing improved chemotherapeutic hybrid agents, such as 3-Substituted-3-hydroxy-2-oxindole as a synthetic scaffold for emerging anti-cancer drug development as well as its modified derivatives [19,20]. Some of the 3-Substituted-3-hydroxy-2-oxindoles have been shown to inhibit the proliferation of A549 lung adenocarcinoma cells in vitro [19]. Tetrazole-bearing compounds have also shown a broad spectrum of biological activities [21]. Therefore, it is possible that hybrid molecules formed from 3-Substituted-3-hydroxy-2-oxindoles and tetrazole-bearing compounds might have potential anti-cancer therapeutic properties.

The aim of this study was to evaluate the effects of 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH) on cell proliferation, apoptosis, and the cell cycle in human lung cancer cell lines SK-LU-1, A549, and A-427, and the normal lung fibroblast cell line, MRC-5, in vitro.

Material and Methods

Cell lines and culture conditions

The human lung cancer cell lines SK-LU-1, A549, A-427, and the normal lung fibroblast cell line, MRC-5, were obtained from the Shanghai Xiang Shi Biotechnology Co., Ltd. (Shanghai, China). Cell culture was performed in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin). The cells were incubated in an incubator containing 95% air and 5% CO2 at room temperature, 37°C. A 10 mM stock solution of 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH) was prepared in dimethyl sulfoxide (DMSO) medium and dilutions were performed in medium to the required concentration. The reagents for the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction colorimetric assay (MTT assay) and the DMSO were purchased from Sigma–Aldrich (St. Louis, MO, USA).

The MTT colorimetric assay to assess cell metabolic activity

The human lung cancer cells, SK-LU-1, A549, A-427, and the normal lung fibroblasts, MRC-5, were cultured in the 96-well plates containing DMEM medium at a density of 2×106 cells per well. The cells were then incubated for 48 hours with the conditioned medium containing THOH at 37°C. After incubation, the cells were treated with a 5 mg per ml solution of MTT for 4 hours. Extraction of the cells with acidic isopropyl alcohol was followed by absorbance measurement at 570 nm wavelength (A570) using an enzyme-labeling instrument, the BioTek ELx800 Absorbance Microplate Reader (BioTek Instruments, Inc., Winooski, VT, USA).

Apoptosis assay using fluorescence microscopy

The lung cancer SK-LU-1 cells were seeded into 6-well plates (2×105 cells per well) and treated with THOH at concentrations of 0, 6, 12, and 24 μM for 24 hours at 37○C. The cells were then stained with the nuclear stain, 4′,6-diamidino-2-phenylindole (DAPI) for 20–30 min at 25°C to detect the apoptosis by fluorescence microscopy, as previously described [22]. The percentage of apoptotic cells were detected using a fluorescein isothiocyanate (FITC), Annexin-V, propidium iodide (PI) apoptosis detection kit according to the manufacturer’s instructions. DNA damage was measured using the comet assay (single-cell gel electrophoresis assay), as previously described [23].

Cell cycle analysis using fluorescence-activated cell sorting (FACs)

To estimate the number of cells in each phase of the cell cycle, the THOH-treated SK-LU-1 lung cancer cells were harvested and washed with PBS. The cells were fixed with 70% ethanol for about an hour and then washed again with PBS. The cells were finally resuspended in a solution of PI (50 μl/ml) and RNase1 (250 μg/ml), followed by incubation for 30 min at room temperature. The analysis was performed using FACS cater-plus flow cytometry, with at least 10,000 cells counted per group.

Cell migration assay

The cell migration potential of THOH-treated SK-LU-1 lung cancer cells was investigated using a wound healing assay. Briefly, 5×104 cells/well were seeded in 96-well plates. The plates were incubated overnight at 37°C to allow the cells to adhere to the plates. A wound in the confluent cells was created by making a scratch using a sterile pipette tip after the cells reached confluence. The cells were then washed with PBS to clear the detached cells. The cells were monitored after a 20-hour interval and photographed.

Western blot analysis

The lung cancer cells treated with THOH or DMSO were collected and washed with a lysis buffer consisting of Tris-HCl, sodium-dodecyl sulfate (SDS), mercaptoethanol, and glycerol. The extracts were boiled for 10 min in the presence of loading buffer followed by separation of cell extracts using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel. The samples were then placed onto polyvinylidene fluoride (PVDF) membranes, blocked using 5% dried skimmed milk powder.

PVDF membranes were incubated overnight at 4°C with primary antibodies to MEK (Cat. No. sc-6250), and ERK (Cat. No. sc-92) obtained from Santa Cruz Biotechnology. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary biotinylated antibodies (Cat. No. sc-2372) at 1: 1,000 dilution, for 2 hours. The membranes were washed with PBS, followed by visualization of the immunoreactive bands using enhanced chemiluminescence (ECL) with the ECL-PLUS Kit, according to the manufacturer’s instructions (Pierce Biotechnology, Inc., Waltham, MA, USA). The bands were analyzed using the Gel Doc 2000 imaging system (Bio-Rad Laboratories GmbH, Munich, Germany).

Statistical analysis

All experiments were performed in triplicate, and the results were expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA), followed by Tukey’s t-test using GraphPad7 software was used for statistical analysis. A P-value of P<0.01 was considered as statistically significant.

Results

Treatment with 3-tetrazolylmethyl-3-hydroxy-oxindole hybrid (THOH) reduced cell proliferation in human lung cancer cells, SK-LU-1, A549, and A-427 in vitro

The antiproliferative effects of 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH) were evaluated for three lung cancer cell lines, SK-LU-1, A549, and A-427, and the normal lung fibroblast cell line, MRC-5, in vitro. Treatment with THOH inhibited the proliferation of all the lung cancer cell lines (Table 1), but there was little effect on the normal MRC-5 cells. The greatest anti-proliferative effect was observed for the THOH-treated SK-LU-1 cells, with an IC50 of 12 μM, the IC50 being the concentration of an inhibitor that results in a 50% reduced response (Figure 1). Therefore, the SK-LU-1 lung cancer cell line was carried forward for further studies.

Table 1

IC50 values of THOH against different lung cancer and normal cell line determined by MTT assay.

Cell line	IC50 (μM)
SK-LU-1	12
A549	25
A-427	25
MRC-5	65

Figure 1

The MTT assay and the effects of 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH) on the cell viability of lung cancer cells, SK-LU-1, and the normal MRC-5 cells. The experiments were carried out in triplicate, and the results are expressed as the mean ± standard deviation (SD) (* P<0.01).

THOH treatment and apoptosis in lung cancer cells in vitro

The treatment with THOH induced cell apoptosis in SK-LU-1 cells in a concentration-dependent manner (Figure 2). The percentage of apoptotic cells detected using a fluorescein isothiocyanate (FITC), Annexin-V, propidium iodide (PI) staining showed that the percentage of the apoptotic cells increased from 5.6% for the MRC-5 control cells to 61.3% for the SK-LU-1 cells treated with THOH at a concentration of 24 μM (Figure 3). DNA damage, measured using the comet (single-cell gel electrophoresis) assay, also showed that THOH was associated with apoptosis via induction of DNA damage (Figure 4)

Figure 2

Cell apoptosis in SK-LU-1 cancer cells following treatment with 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH) using fluorescence flow cytometry with Annexin V, propidium iodide (PI), and nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI). The experiments were carried out in triplicate, and the results are expressed as the mean ± standard deviation (SD) (* P<0.01).

Figure 3

Cell apoptosis using fluorescence flow cytometry with Annexin V, propidium iodide (PI), and nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI). The experiments were carried out in triplicate.

Figure 4

DNA damage, in the SK-LU-1 lung cancer cells, measured using the comet assay (single-cell gel electrophoresis assay), following treatment with 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH). The experiments were carried out in triplicate.

THOH treatment induced G2/M cell cycle arrest in lung cancer cells in vitro

The effect of THOH treatment was also determined for the distribution of different phases of the cell cycle of the SK-LU-1 lung cancer cells in vitro. THOH treatment induced the accumulation of SK-LU-1 cells in the G2 phase of the cell cycle and triggered cell cycle arrest (Figure 5). The effect of THOH treatment on the cell cycle of the SK-LU-1 lung cancer cells was also found to be concentration-dependent.

Figure 5

Induction of G2/M cell cycle arrest of SK-LU-1 lung cancer cells, evaluated by flow cytometry, following treatment with 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH). The experiments were carried out in triplicate.

THOH treatment inhibited cell migration of lung cancer cells in vitro

Because cell migration is an important aspect of cancer invasion and metastasis, the effect of THOH treatment on SK-LU-1 cancer cell migration was investigated. THOH treatment was shown to inhibit the migration of the SK-LU-1 lung cancer cells at the IC50 dose of 12 μM (Figure 6).

Figure 6

Inhibition of cell migration of SK-LU-1 lung cancer cells, evaluated using a wound healing assay, following treatment with 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH). The experiments were carried out in triplicate, and the results are expressed as the mean ± standard deviation (SD) (* P<0.01)

THOH treatment targeted the platelet-derived growth factor D (PDGF-D) and MEK/ERK signaling pathways

Treatment of lung cancer cells with THOH inhibited the expression of platelet-derived growth factor D (PDGF-D) in a concentration-dependent manner (Figure 7A). Treatment of lung cancer cells with THOH inhibited the expression of p-MEK and p-ERK in a concentration-dependent manner, but the expression of MEK and ERK was unchanged (Figure 7B).

Figure 7

Western blot analysis of the proteins expressed following treatment of cells with 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH). (A) Platelet-derived growth factor D (PDGF-D) signaling pathway proteins, detected by Western blot. (B) MEK/ERK signaling pathway proteins, detected by Western blot. The experiments were carried out in triplicate.

Discussion

Despite recent research on the molecular biology of lung cancer and drug development of targeted treatment, worldwide, lung cancer remains as one of the most prevalent types of cancer, and because it is often detected at an advanced stage, it remains difficult to treat [1,2]. Because of the need for continued studies on novel molecules for the treatment of lung cancer, in the present study, the anticancer effects of 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH) was evaluated against a panel of lung cancer cell lines, SK-LU-1, A549, and A-427, and the normal lung fibroblast cells, MRC-5.

The preliminary results of this study showed that treatment of lung cancer cell lines with THOH inhibited cell proliferation, which was less than that found in the normal lung cell line MRC-5. Also, out of all the cell lines, the anti-cancer effects of THOH were more pronounced in the SK-LU-1 lung cancer cells, and so this cell line was used for further studies on the effects of THOH treatment.

Because different anticancer agents exert their antiproliferative effects via different mechanisms [23], in this study, a preliminary investigation of the underlying mechanism for the anticancer activity of THOH was undertaken. The study findings showed that THOH treatment of lung cancer cells in vitro could trigger cell cycle arrest in SK-LU-1 lung cancer cells as shown by fluorescence-activated cell sorting (FACs) with fluorescein isothiocyanate (FITC), Annexin-V, propidium iodide (PI), and nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) and shown by DNA damage as measured using the comet (single-cell gel electrophoresis) assay. Apoptosis is an important mechanism by which many of the currently used anticancer agents exert their effects [24]. Also, induction of apoptosis by anticancer agents prevents the development of drug resistance in cancer cells [25].

In addition to apoptosis, cell cycle arrest is a further mechanism of action for anti-cancer drugs that inhibit the proliferation of cancer cells [26]. The cell cycle is controlled at two main checkpoints, the G1/S transition, and the G2/M transition. The G2/M checkpoint has an important role in the preservation of chromosomal integrity by permitting cells to repair DNA damage before entering mitosis [26]. The findings of this study showed that THOH induced G2/M cell cycle arrest, and could also inhibit the migration of the lung cancer cells, which indicate that THOH might have the potential to be developed as an anti-cancer drug, depending on future studies.

The expression of RAS genes are associated with oncogenesis and might be target candidates for drug development in cancer chemotherapy, and molecules that directly target the RAF gene, RAF kinases, or mitogen-activated protein (MAP) kinase or MEK in the MAP kinase pathway downstream of RAS have shown promising results in clinical trials [27]. Therefore, as part of this study, the effect of THOH on the expression of platelet-derived growth factor D (PDGF-D) and MEK/ERK signaling pathways were studied, and the findings showed that THOH could inhibit both of these signal transduction pathways.

Conclusions

The findings of this preliminary in vitro study showed that treatment of lung adenocarcinoma cells, SK-LU-1, A549, and A-427, with the compound 3-tetrazolyl methyl-3-hydroxy-oxindole hybrid (THOH), inhibited cell proliferation, apoptosis, and resulted in G2/M cell cycle arrest by targeting the platelet-derived growth factor D (PDGF-D) and MEK/ERK signaling pathways. Further studies are recommended to investigate the mechanism of action of THOH to determine the potential role of this compound, and similar compounds, in the treatment of human cancer, including non-small cell lung cancer (NSCLC).