4-Methylphenylhydrazine hydrochloride, 4-methoxyphenylhydrazine hydrochloride and other chemical intermediates were purchased from Meryer Chemical Technology Co., Ltd.; 3-methyl-2-butanone and isoniazid were purchased from Aladdin Reagent Company. Other reagents were all synthesized in the laboratory. Primary antibodies against hypoxia-inducible factor-1α (HIF-1α, cat. no. 3716, dilution 1:1,000), matrix metallopeptidase 2 (MMP2, cat. no. 87809, dilution 1:1,000), vascular endothelial growth factor (VEGF, cat. no. 2463, dilution 1:1,000), phosphorylated AKT (p-AKT, cat. no. 4060, dilution 1:2,000), AKT (cat. no. 9272, dilution 1:1,000), p21 (cat. no. 2947, dilution 1:1,000) and β-actin (cat. no. 4970, dilution 1:2,000), were obtained from Cell Signaling Technology, Inc.

2,3,3,5-Tetramethyl-3H-indole (intermediate compound 1A; 5 g; 28.9 mmol) and ethyl 4-bromobutyrate (28 g; 144 mmol) were added to 200 ml CH₃CN and the reaction proceeded under reflux for 40 h. After purification with silica column chromatography, 4 g of a yellow oily liquid was obtained. Subsequently, the obtained intermediate was dissolved in 20 ml 5 mol/l HCl and heated to reflux for 14 h to obtain 3 g of a colorless liquid (intermediate compound 2A), with a yield of 81%. 5-Methoxy-2,3,3-trimethyl-3H-indole (5 g; 26.5 mmol) and 1-bromobutane (17.9 g; 132 mmol) were suspended in 200 ml CH₃CN, and the mixture was refluxed for 40 h. Colorless liquid (intermediate compound 2B; 3.5 g; yield, 41%) was obtained by purification with a silica gel column. Next, 1-phenylamino-5-phenylimino-1,3-pentadiene hydrochloride (1 g; 3.5 mmol), 2B (1.14 g; 3.5 mmol) together with 1 ml Et₃N were added to 30 ml acetic anhydride at 50°C for 1 h. After cooling to an ambient temperature, the reaction mixture was poured into 500 ml Et₂O and filtered to obtain 1.5 g of a brownish precipitate (intermediate compound 3; yield, 83%). Subsequently, intermediate 3 (1 g; 1.92 mmol) and intermediate 2A (649 mg; 1.92 mmol) were added to 30 ml pyridine and heated to 50°C with stirring for 1 h. The mixture was evaporated under reduced pressure and acidified to obtain intermediate compound 4 (300 mg; yield, 24%) that was purified with a silica gel column. Finally, PyBOP (241 mg; 0.46 mmol), N,N-diisopropylethylamine (DIPEA; 59 mg; 0.46 mmol) and intermediate 4 (300 mg; 0.46 mmol) were added to 100 ml dichloromethane of ice-salt bath, and isoniazid (63 mg; 0.46 mmol) was added. The resulting mixture was stirred for an additional 24 h at room temperature, filtered, and the solvent was removed by reduced pressure distillation and purified by silica gel to obtain G10 (90 mg; yield, 26%).

A549, NCI-H460 and PC9 human lung adenocarcinoma cell lines were purchased from American Type Culture Collection. Cells were cultured in RPMI-1640 medium supplemented with 10% FBS (both from Gibco; Thermo Fisher Scientific, Inc.), and maintained at 37°C in a humidified incubator with 5% CO₂. The chemoresistant cells were obtained after treatment with cisplatin, PTX and erlotinib (MedChemExpress) at concentrations between 0.5 and 5 µM for 3 months.

MAOA enzymatic activity was measured in A549, A549/CDDP, A549/PTX, H460, H460/CDDP, H460/PTX, PC9 and PC9/Er cells as described previously (24). Briefly, cells were plated in culture medium supplemented with 10% FBS in a 10-mm dish for 24 h. Through extracting the reaction products, MAOA activity was measured using a Cell MAOA assay kit (Shanghai Chengong Biotechnology) according to the manufacturer's protocol, and was based on substrate p-tyramine levels following treatment with the MAOB inhibitor pargyline.

An MTT assay was used to determine the effects of G10 on cell viability in vitro. Briefly, cells (1×10⁵ cells/ml) were seeded into 96-well culture plates and incubated overnight. Subsequently, the cells were treated with a range of concentrations (0.1–100 µM) of G10 for 72 h. MTT solution (10 µl, 2.5 mg/ml) was added to each well, and the plates were incubated for another 4 h at 37°C. After centrifugation (1,000 × g for 10 min at room temperature), the medium was replaced with 100 µl DMSO. The optical density of these samples was measured using a Spectra Max Paradigm Reader (Molecular Devices, LLC) at 570 nm.

Apoptosis analysis was performed using an Annexin V-FITC apoptosis detection kit (BioVision, Inc.). Cells (1×10⁶) were treated with G10 for 48 h [RNA interference cells were transfected with a specific MAOA siRNA or scramble siRNA (Santa Cruz Biotechnology, Inc.) for 24 h]. Subsequently, cells were collected by centrifugation (1,000 × g at 20°C for 5 min) and resuspended in 500 µl binding buffer. Annexin V and PI were added to the cells. After incubation at room temperature for 5 mins in the dark, cells were analyzed using fluorescence activated cell sorting using a flow cytometer (Becton, Dickinson and Company). For autophagy analysis, the cells were treated with G10 for 48 h and incubated with MDC dye, followed by flow cytometry.

Total RNA from untreated and G10-treated H460/PTX cells was isolated using an RNeasy Mini kit (Qiagen, Inc.) according to the manufacturer's protocol. Changes in gene expression were detected using an Affymetrix GeneChip PrimeView Human Gene Expression Arrays. In order to confirm the changes in specific gene expression, RT-qPCR was used. The sequences of the primers are listed in Table SI.

Transwell chambers (Corning, Inc.) with 6.5-mm-diameter polycarbonate filters (pore size, 8 µm) were used to determine the effect of G10 on A549/PTX and H460/PTX cell migration and invasion in vitro (25). Briefly, the upper chambers of the insert were coated with Matrigel and the lower chambers were filled with fresh M200 medium (1% FBS) containing 10 ng/ml VEGF. After trypsinization, the cells were resuspended at a concentration of 1×10⁶ cells/ml in M200 containing 1% FBS and treated with different concentrations of G10 for 30 min at room temperature before seeding. A total of 100 µl of the cell suspension per well was loaded into the upper wells, and the chamber was incubated at 37°C for 12 h. The cells were fixed and stained with Calcein-AM at room temperature for 30 min. Cells on the upper surface of the filter were removed by wiping with a cotton swab, and chemotaxis was quantified using the high-content drug screening system ImageXpressR Micro (Molecular Devices, LLC) by counting the cells that had migrated to the lower side of the filter.

A total of 1×10⁷ H460/PTX cells treated with G10 were collected. Western blotting was performed as previously described (26). Briefly, total proteins (50 µg) were extracted from the cells (determined by BCA method), resolved using 8–15% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk at room temperature for three times (10 min/per times). Primary and secondary antibodies (Thermo Fisher Scientific, Inc.; cat. nos. 31430 and 31460, dilution 1:5,000) were then used to visualize the resolved proteins. Finally, an enhanced chemiluminescence system (ECL Plus; Amersham; Cytiva) was used to develop the blots.

To evaluate the antitumor activity of G10 in vivo, a total of 20 7–8-week old male BALB/c nude mice (weight, 18–22 g) were anesthetized with 3% sodium pentobarbital (50 mg/kg, i.p.) and injected subcutaneously at the right flank with viable (confirmed using trypan blue staining) H460/PTX cells (5×10⁶/100 µl PBS per mouse). When the mean subcutaneous size of the tumor reached 100 mm³, the mice were randomly divided into four groups treated with different concentrations of G10 and a control group (4–6 mice per group). The G10 single treatment group and PTX single treatment group were administered G10 [19 mg/kg/2 days, intraperitoneally (i.p.); n=5] or PTX (3 mg/kg/2 days, i.p.; n=5), respectively. The combination group was treated with both G10 (19 mg/kg/2 days; i.p.; n=5) and PTX (3 mg/kg/2 days; i.p.; n=5). Tumor size was measured with a caliper every 3 days using the following formula: Tumor volume=shortest diameter² × longest diameter/2 (maximum tumor volume, 930 mm³). Body weight was also recorded every 3 days. After 21 days, the mice were sacrificed with cervical dislocation, and lack of breathing and heartbeat for 3 min were observed to confirm death. The study protocol complied with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Shenyang Pharmaceutical University and all animal experiments were approved by the Committee on the Ethics of Animal Experiments of the Shenyang Pharmaceutical University.

Differences between experimental groups were evaluated by one-way ANOVA followed by Turkey's post hoc test using the SPSS v.11.5 for Windows (SPSS, Inc.). P<0.05 (two-tailed test) was considered to indicate statistically significant differences.

The target compound (G10) was synthesized as shown in Fig. 1 (23). First, intermediates 2A and 2B were synthesized by S_N2 nucleophilic substitution of 1A and 1B, with ethyl 4-bromobutyrate and 4-bromobutane as alkylating agents, respectively. Subsequently, the asymmetric heptamethine cyanine dye intermediate 4 was obtained by two-step aldol reactions of 2A and 2B reacting with 1-phenylamino-5-phenylimino-1,3-pentadiene hydrochloride in acetic anhydride, with pyridine as a catalyst. Finally, target compound G10 was obtained by condensation of compound 4 with isoniazid in the presence of DIPEA and PyBOP in anhydrous dichloromethane.

In order to investigate the association between MAOA and drug resistance in lung cancer, the MAOA activity was examined in NSCLC cells resistant to chemotherapy (cisplatin and PTX), erlotinib-resistant NSCLC cells and their parental cells. As shown in Fig. 2A, the activity of MAOA was increased in chemotherapy-resistant NSCLC cells, but not in the TKI-resistant NSCLCs. In particular, PTX-resistant NSCLCs exhibited notably increased MAOA activity compared with the parental cells. Subsequently, cell viability was measured following treatment with the novel MAOA inhibitor G10. As shown in Fig. 2B, G10 exerted potent inhibitory effects on H460/PTX cells, and was more effective against the PTX-resistant cells compared with the parental cells (H460); in addition, the cytotoxicity of G10 was negatively correlated with MAOA activity, suggesting that inhibition of MAOA could reverse PTX resistance of NSCLC. To further confirm that G10 induced PTX-resistant cell death, cell apoptosis was measured by flow cytometry (Fig. 2C). The results demonstrated that G10 induced cell apoptosis at relatively higher concentration (10 µM). Of note, when MAOA gene expression was knocked down, the apoptosis induced by G10 was abrogated (supplementary Fig. S1), indicating that cell apoptosis induced by G10 was dependent on MAOA activity.

Acquisition of invasive ability by epithelial cells is required for metastasis (27). It has been previously demonstrated that MAOA promotes the proliferation, invasion and metastasis of PCa cells through inducing EMT and stabilizing HIF-1α (9). Therefore, the effect of G10 on the expression of ECM-related genes, changes in which are closely associated with the migration, invasion and drug resistance of tumor cells, were examined. Using Gene Ontology (GO) cellular component (CC) analysis, the changes in the gene expression profile of H460/PTX cells, found to be relatively sensitive to G10 in the cell viability assay, following treatment with G10 were evaluated (Fig. 3A). The differentially expressed genes are involved in the intrinsic component of the plasma membrane, ECM, proteinaceous ECM, nuclear pores, endoplasmic reticulum lumen, anchored component of the membrane, cell surface, voltage-gated calcium channel complexes, external side of the plasma membrane and keratin filaments. Among these genes, the distinct changes in the expression of genes associated with the ECM that are closely associated with tumor cell migration, invasion and drug resistance were further explored (28–30). As shown in Fig. 3B, heatmap analysis was used to further analyze these genes, and revealed that changes in the expression levels of several genes associated with tumor invasion and migration. As MMP2, MMP14 and COL6A have been shown to be involved in the pathophysiological processes underlying migration and invasion of several types of cancer cells (31–33), the mRNA levels of MMP2, MMP14 and COL6A in H460/PTX cells following G10 administration was further explored. Treatment with G10 reduced the expression of MMP2, MMP14 and COL6A in H460/PTX cells (Fig. 3C). Of note, in addition to ECM-related genes, GO CC analysis suggested that the expression of cell surface-related genes was also significantly altered following treatment with G10. This may be explained by the fact that cell surface proteins may transduce signals from the ECM into the cells, which in turn regulate a range of cellular functions, such as survival, proliferation, migration and differentiation (34). Taken together, these results suggest that G10 may suppress tumor invasion and migration by affecting the expression of genes associated with the ECM.

To determine whether G10 affects tumor cell migration and invasion, Transwell assays were performed. First, to avoid the direct cytotoxic effects of G10, Transwell assays were performed in cells treated with non-cytotoxic concentrations of G10 (cell viability >80%), which was determined using an MTT assay (data not shown). As shown in Fig. 4A, the number of migrating cells in both the A549/PTX and H460/PTX cell lines was decreased significantly following treatment with non-cytotoxic doses of G10. Similarly, G10 suppressed the invasive ability of A549/PTX and H460/PTX cells in a concentration-dependent manner (Fig. 4B). Taken together, these data suggest that G10 can inhibit the migration and invasion of PTX-resistant cells, consistent with the results of the GO CC analysis.

Upregulation of MAOA can activate HIF-1α and AKT signaling (9). The transcription factor HIF-1α mediates hypoxia by activating target genes associated with the mechanisms involved in tumor progression, such as increased tumor glycolysis, angiogenesis and metastasis (35). Activated HIF-1α regulates the expression of multiple genes in response to hypoxia, including VEGF (a key regulator of angiogenesis) and MMP2, which is an important enzyme promoting migration and invasion. Additionally, MMPs and VEGF are both involved in the metastatic process through modifying ECM components of neoplastic and stromal cells (36). HIF-1α, MMP2 and VEGF all participate in tumor invasion, migration and metastasis. The role of the MAOA-dependent HIF-1α/MMP2 and VEGF pathways on the inhibitory effects of G10 on cell invasion was examined using H460/PTX cells. As shown in Fig. 5A and B, G10 suppressed the expression levels of HIF-1α in a concentration-dependent manner and inhibited the expression of its target genes MMP2 and VEGF. Furthermore, the effects of G10 on the EMT biomarker E-cadherin were also assessed. The data revealed that G10 upregulated the expression of E-cadherin. These results suggest that G10 exerts its inhibitory effects on tumor cell migration and invasion through downregulating HIF-1α/MMP2 and VEGF under hypoxic conditions.

AKT, a serine/threonine kinase, requires phosphorylation to be activated to regulate cancer cell proliferation (37). G10 was shown to suppress AKT phosphorylation, thereby inhibiting AKT signaling (Fig. 5C and D). When AKT signaling is activated, the expression of p21, a tumor suppressor gene that is associated with tumor proliferation, is reduced. The results also confirmed that G10 treatment increased the expression of p21 (Fig. 5C and D), suggesting that inhibiting AKT signaling resulted in p21 activation. The mechanism underlying the suppressive effect of G10 on tumor progression may involve the regulation of the p-AKT/p21 signaling pathway. Due to the involvement of the AKT pathway in autophagy, cell autophagy was assessed using flow cytometry and was found to be induced by G10 in a concentration-dependent manner (Fig. 5E), suggesting that autophagy may also be involved in the antitumor effects of G10.

As G10 exerted prominent antitumor effects in vitro, further investigation of the efficacy of G10 on the growth of H460/PTX xenograft models was assessed. In view of the obvious action of G10 on H460/PTX cells, they were selected to establish xenograft models. The results revealed that treatment with PTX exerted no significant effect on tumor growth (Fig. 6A), confirming that H460/PTX cells were PTX-resistant. In addition, treatment with G10 alone was ineffective in inhibiting tumor progression. However, combined treatment with G10 and PTX significantly inhibited the growth of the H460/PTX xenografts within a 3-week treatment period (Fig. 6A). During this combined treatment, there were no notable differences in mouse body weight and visceral index (organ weight vs. body weight) between the control and treatment groups (Fig. 6B), suggesting that there were no gross toxic effects induced by the combination of G10 and PTX.