MCF7 (human breast cancer), HepG2 (human hepatocellular carcinoma), A549 (human lung cancer), HeLa (human cervical cancer) cells and QSG7701 healthy human hepatocytes were procured from the American Type Culture Collection (Manassas, VA, USA). The following antibodies were obtained from Cell Signaling Technology, Inc., (Danvers, MA, USA): Mouse monoclonal anti-phosphorylated EGFR (pTyr1068-EGFR) (1:1,000; cat. no. 2236), rabbit polyclonal anti-STAT3 (1:1,000; cat. no. 9132), rabbit polyclonal anti-phosphorylated STAT3 (pTyr705-STAT3) (1:1,000; cat. no. 9131), rabbit monoclonal anti-AKT (1:1,000; cat. no. 4685), rabbit monoclonal anti-phosphorylated AKT (pSer473-AKT) (1:1,000; cat. no. 4060), rabbit monoclonal anti-Bax (1:1,000; cat. no. 5023), rabbit monoclonal Bcl-2 (1:1,000; cat. no. 2870) and rabbit monoclonal HK-2 (1:1,000; cat. no. 2867). Rabbit polyclonal anti-EGFR (1:1,000; cat. no. 18986-1-AP), rabbit polyclonal anti-β-actin (1:2,000; cat. no. 20536-1-AP) primary antibodies and peroxidase-conjugated Affinipure goat anti-rabbit/mouse secondary antibodies (1:10,000; cat. nos. SA00001-2 and SA00001-1) were obtained from Proteintech Group, Inc. (Chicago, IL, USA). EasyScript Two-Step RT-PCR SuperMix kit was obtained from TransGen Biotech (Beijing, China). Propidium iodide (PI), RNase A and Hoechst 33342 were obtained from Sigma-Aldrich (St. Louis, MO, USA). Triton X-100 was obtained from Amresco LLC (Solon, OH, USA). TRIzol was obtained from Biyuntian biotech (Shanghai, China). Agarose was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA).

HepG2 cells were transfected with STAT3 siRNA or treated with the HK-2 inhibitor, 3-BrPA for 6 h. Subsequently, different concentrations of QIC1 (0, 1, 2 and 4 μM) was added. After 48 h, an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed to detect the cell viability to evaluate whether QIC1 regulates cancer cell growth through the STAT3-HK2 pathway.

Cells were cultured in RPMI-1640 (Invitrogen Life Technologies) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in 5% CO₂ at 37°C. The quinolone-indolone conjugate, QIC1, was synthesized chemically by aldol condensation of the quinolone C-3 position, in order to introduce indolin-2-one. QIC1 was dissolved in DMSO (Sigma-Aldrich). An MTT assay was used to detect cell viability and cytotoxicity. MCF7, HepG2, HeLa, A549 and QSG7701 cells, cultured in 96-well plates, were treated with final concentrations of 0.5, 1, 2 and 4 μM of QIC1 for 48 h. Treated cells were then incubated in fresh medium containing MTT (0.5 mg/ml; Sigma-Aldrich) at 37°C for 4 h followed by replacement with 150 μl DMSO. Finally, the spectrophotometric absorbance of the samples in DMSO was determined using a microplate reader (Sunrise™, Tecan Group Ltd., Männedorf, Switzerland) at 570 nm.

Cell cycle analysis was performed using flow cytometry. HepG2 and A549 cells treated with QIC1 (0, 1, 2 or 4 μM) for 48 h were collected and washed twice with ice-cold PBS. Cells (1×10⁶) were fixed in 75% ethanol at 4°C for ≥4 h. Cells were then washed twice with ice-cold PBS, followed by incubation with DNA staining solution [(PI; 50 μg/ml), RNase (50 μg/ml) and Triton-x-100 (0.5%)] for 20 min. Cell cycle analysis was immediately performed using flow cytometry (FACSCalibur, BD Biosciences, Franklin Lakes, NJ, USA).

Cell apoptosis was analyzed using an ArrayScan VTI HCS 600-type high content live cell imaging system (Thermo Fisher Scientific, Waltham, MA, USA). MCF7, HepG2 and A549 cells were seeded in 96-well plates. After 12 h, the cells were treated with QIC1 (0, 0.5, 1, 2 and 4 μM) for 48 h, and then washed with ice-cold PBS. Cells were incubated with 5 mg/ml Hoechst 33342 for 30 min, and then incubated with 5 mg/ml PI for a further 1 h at 37°C. The cells were subsequently washed with ice-cold PBS. The cell apoptosis analysis was immediately performed using the ArrayScan VTI HCS 600-type high content live cell imaging system. The second channel overall average fluorescence intensity (mean fluorescence intensity; MFI) represents cells in late apoptosis.

Cells treated with QIC1 were harvested in RIPA buffer (50 mM Tris-HCl, pH 8.0; 150 mM sodium chloride; 1.0% NP-40; 0.5% sodium deoxycholate; and 0.1% SDS) with 10 μg/ml of the protease inhibitor, PMSF (Sigma-Aldrich). Total cellular proteins (40 μg) were separated using SDS-PAGE and subjected to western blotting using primary antibodies against EGFR, pTyr1068EGFR, STAT3, pTyr705-STAT3, AKt, pSer473-AKT, HK, Bcl-2, Bax and β-actin (Cell Signaling Technology, Inc. and Proteintech Group, Inc.). β-actin was used as loading control. Bands were detected using an ECL detection system (AlphaImager; Proteinsimple, San Jose, CA, USA).

Total mRNA was extracted from cultured cells using TRIzol. Reverse transcription and PCR reactions were performed using an Easy Script Two-Step RT-PCR SuperMix kit (TransGen Biotech company, Beijing, China). The primer pairs for RT-PCR were as follows: Forward: 5′-ATGGTCAAGTGCTGGATG-3′ and reverse: 5′-GAGGAAGGTGTCGTCTATG-3′ for EGFR, forward: 5′-CCAAGGAGGAGGCATTCG-3′ and reverse: 5′-ACATCGGCAGGTCAATGG-3′ for STAT3 and forward: 5′-GGTGCTGAGTATGTCATGGA-3′ and reverse: 5′-TTCAGCTCTGGGATGACCTT-3′ for GAPDH (both sets from Sangon Biotech Co., Ltd., Shanghai, China). The PCR conditions were as follows: 94°C for 5 min, followed by 38 cycles at 94°C for 30 sec, 56°C for 30 sec and 72°C for 1 min, an 72°C for 7 min using an Arktik PCR system (Thermo Scientific, Waltham, MA, USA). mRNA expression of GAPDH, EGFR and STAT3 was determined with density scanning (AlphaImager; Proteinsimple, San Jose, CA, USA). Subsequently the EGFR:GAPDH ratio and the STAT3:GAPDH (mRNAs were detected in the same RT sample) in densitometric units was calculated and analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA).

Each analysis was repeated three times. Relative cell viability is expressed as a percentage relative to the untreated control cells. Error bars represent standard deviation. Data were analyzed using analysis of variance for each two-group comparison test. Statistical analyses were conducted using SPSS 17.0. P<0.05 was considered to indicate a statistically significant difference.

The quinolone-indolone conjugate, QIC1, was synthesized to target EGFR. It was hypothesized that QIC1 may target human cancers via its effect on EGFR. The schematic structural diagram of QIC1 is shown in Fig. 1A. The antiproliferative/survival activity of QIC1 in liver, lung, cervix and breast cancer cells was investigated and compared with that of the tyrosine kinase inhibitor, sunitinib (schematic structural diagram shown in Fig. 1B), which was used as a positive control. The activity of QIC1 was examined in MCF7, HepG2, HeLa and A549 cells, and QSG7701 healthy human hepatocytes, using an MTT assay. Among these cell types, QIC1 induced a significant dose-dependent decrease in cancer cell survival (Fig. 1C). In MCF7, HepG2, HeLa and A549 cells, the dose of QIC1 required to achieve 50% cell viability (IC₅₀) was 1.467, 1.994, 2.513 and 2.708 μM respectively. Furthermore, QIC1 was shown to be a non-cytotoxic compound as QIC1 (0.5, 1 and 2 μM) did not inhibit cell growth in the healthy QSG7701 hepatocyte cell line (Fig. 1C). QIC1 exhibited a stronger anticancer activity than that of sunitinib (Fig. 1D).

The effects of QIC1 on cell proliferation were also examined using cell cycle analysis. There was a significant accumulation of the G2/M (4N-DNA) cell population in HepG2 cells treated with QIC1 (17.90, 25.33 and 38.25% following incubation with 1, 2 and 4 μM, respectively) compared with untreated cells (14.29%; Fig. 2). In A549 cells, the S phase cell population was decreased, which indicated that QIC1 may inhibit DNA synthesis in A549 (Fig. 2). These observations suggest that QIC1 may be a promising candidate with which to inhibit cancer cell survival and proliferation.

In subsequent experiments, the hypothesis that QIC1 results in cancer cell death by promoting apoptosis was investigated. Cell apoptosis was detected using an ArrayScan VTI HCS 600-type high content live cell imaging system. MCF7, HepG2 and A549 cells underwent apoptotic death in a dose-dependent manner when they were treated with QIC1 at doses of 1, 2 and 4 μM for 48 h (Fig. 3A and B). Furthermore, enhanced Bax expression and reduced Bcl-2 expression was observed in HepG2, MCF7 and A549 cells that were treated with QIC1 (1, 2 or 4 μM; Fig. 3C). These results supported the hypothesis of the induction of an apoptotic response in MCF7, HepG2 and A549 cancer cells upon treatment with QIC1.

Previous studies have indicated that downregulation of either the expression or the activity of EGFR contributes to the inhibition of cell proliferation and the induction of apoptosis in cancer cells (22,23). In the present study, the effect of QIC1 on the expression and activity of EGFR was measured in MCF7, HepG2 and A549 cell lines. Notably, decreased EGFR activity and expression was observed in all three cell lines exposed to QIC1, in a dose-dependent manner (Fig. 4A). It has also been reported that certain downstream components of the EGFR signaling pathway, such as activated AKT and STAT3, are involved in cell survival and apoptosis (24–26). Therefore, the expression and activation status of these downstream signaling molecules was also investigated. Reduced expression and activity of AKt and STAT3 was observed in the three cancer cells treated with QIC1 (Fig. 4B). Previous studies by this group, have indicated that STAT3 may regulate the expression of HK2 in HepG2 cells. HK2 catalyzes the first committed step of glycolysis, an important metabolic pathway in cancer cells, which provides essential energy for the growth and proliferation of cancer cells. It has been shown to be overexpressed and activated in a number of cancers. The downregulation of HK2 inhibits the growth and proliferation of cancer cells. Therefore, the present study examined the effect of QIC1 on the expression status of HK2 in MCF7, HepG2 and A549 cell lines. Notably, decreased HK2 expression was observed in the cell lines exposed to QIC1, and this decrease occurred in a dose-dependent manner (Fig. 4C). In order to further confirm whether QIC1 regulates cancer cell growth through the EGFR-STAT3-HK2 pathway, cell growth status was measured after cancer cells were pretreated with STAT3 siRNA: Sense: 5′-GCAACAGAUUGCCUGCAUUdTdT-3′ and antisense: 5′-AAUGCAGGCAAUCUGCAUU-3′), and the HK2 inhibitor, 3-bromine pyruvate (3-BrPA; Adamas-Beta, Shanghai, China). IT was observed that the anticancer activity of QIC1 was counteracted by the administration of STAT3 siRNA and 3-BrPA (Fig. 4D). Furthermore, STAT3 siRNA decreased the expression of HK2 in these cells (Fig. 4E). These results indicated that the downregulation of the EGFR-STAT3-HK2 pathway may be one if the mechanisms underlying the effect of QIC1.

The results suggested that QIC1 may downregulate the expression and activity of EGFR and STAT3. Furthermore, a previous study demonstrated that the overexpression of STAT3 induces multidrug resistant in cancer cells and consequently, that STAT3 may be an effective target with which to counter multidrug-resistant cancer (27). Therefore, the expression of STAT3 and its upstream gene, EGFR, was measured in doxorubicin-resistant human breast cancer MCF7/DOX cells, and the involvement of QIC1 in the MCF7/DOX cells was also investigated. It was found that STAT3 as well as its upstream gene, EGFR, were overexpressed in the MCF7/DOX cells (Fig. 5A–D). QIC1 may downregulate the level of the STAT3 and EGFR proteins (Fig. 5E) in MCF7/DOX cells. An MTT assay demonstrated that QIC1 inhibited MCF7/DOX cell proliferation in a dose-dependent manner, and that non-toxic (cell survival rate >90%) doses of QIC1 (1 μM, 2 μM) enhanced the anticancer activity of doxorubicin in the MCF7/DOX cells, consequently reversing the drug resistance of MCF7/DOX cells (Fig. 5F and G). Therefore, QIC1 may also reverse multi-drug resistance in tumor cells.