2.1. MHY4571 synthesis
A solution of cyclohexanone (0.16 mL, 1.55 mmol) and 4-fluoro-3-methoxybenzaldehyde (471 mg, 3.06 mmol) in a 1 M HCl solution (0.62 mL) was stirred at ambient temperature for 24 h. Cold water was added to the reaction mixture and the resultant solid was filtered and washed with diethyl ether, ethanol, and dichloromethane. The filter cake was further purified by silica gel column chromatography using a mixture of hexane and ethyl acetate (7:1) as an eluent to obtain MHY4571 (411 mg, 73%) as a solid. 1H NMR (400 MHz, CDCl3) showed peaks at d 7.68 (s, 2H, 2×vinylic H), 7.09 – 6.97 (m, 6H, 2′-H, 2′′-H, 5′-H, 5′′-H, 6′-H, 6′′-H), 3.87 (s, 6H, 2×OCH3), 2.87 (t, 4H, J = 5.6 Hz, 3-H2, 5-H2), 1.77 (quint, 2H, J = 5.6 Hz, 4-H2); 13C NMR (100 MHz, CDCl3) showed peaks at d 190.0, 152.7 (d, J = 248.8 Hz), 147.7 (d, J = 12.1 Hz), 136.3, 136.0, 132.6 (d, J = 3.7 Hz), 123.4 (d, J = 6.8 Hz), 116.2 (d, J = 18.6 Hz), 116.0, 56.5, 28.5, 23.1; LRMS (ESI+) showed peaks at 393 [M+Na]+, 425 [M+MeOH+Na]+. The simplified code name and structure of MHY4571 are shown in Fig. 1.
2.2. In silico docking simulation
To determine the possible molecular mechanism of MHY4571 and daphnetin, a known PKA inhibitor, for the inhibition of PKA catalytic subunit alpha, Autodock Vina and Autodock4.2 X-ray crystallographic structures were used to show quaternary ligand binding to aromatic residues in the ATP binding pocket of PKA catalytic subunit alpha (PDB ID: 4WB5) from the RCSB Protein Data Bank (http://www.rcsb.org/adb) [19]. The 3D structure of the two compounds and ATP were created using ChemDraw Ultra. LigandScout 4.1.5 was used to predict possible interactions between MHY4571 and PKA catalytic subunit alpha, and for the identification of pharmacophores [20].
2.3. Chemicals and reagents
All chemicals and reagents were of the purest grade available. Detailed information on the specific antibodies and reagents used in this study is presented in (see Supplementary Table S1 of Additional file 1).
2.4. Cell lines and cell culture
BEAS-2B human lung normal epithelial cells and NCI-H1703 human lung squamous carcinoma cells were obtained from American Type Culture Collection (Manassas, VA, USA). HCC95 human lung squamous carcinoma cells were purchased from the Korean Cell Line Bank (Seoul, Republic of Korea). BEAS-2B cells were cultured using the Bronchial Epithelial Cell Growth Medium Bullet Kit (BEGM; Lonza, Basel, Switzerland) with 10% fetal bovine serum (FBS), 2 mM glutamine, 100 U mL-1 penicillin, and 100 μg mL-1 streptomycin (WELGENE, Gyeongsan-si, Republic of Korea). Other NSCLC cell lines were grown in RPMI-1640 medium supplemented with 10% FBS, 2 mM glutamine, and penicillin/streptomycin at 37°C in a humidified atmosphere containing 5% CO2. All human cell lines used in this study were authenticated using short tandem repeat profiling (Cosmogenetech, Seoul, Republic of Korea).
2.5. Small interfering RNA transfection
PKA siRNA was purchased from Bioneer (Daedeok-gu, Daejeon, Republic of Korea), and CREB siRNA was purchased from IDT & MBiotech (Coralville, Iowa, USA). Transfection was performed using Lipofectamine RNAiMAX (Invitrogen, MA, USA). Corresponding pairs of scrambled control and siRNA were mixed with Lipofectamine RNAiMAX in Opti-MEM and allowed to react. Cells were treated with a mixture of siRNA and Lipofectamine RNAiMAX in serum-free media and incubated at 37°C for 48 h.
2.6. MTT assay
Cell viability was determined using the MTT assay. Cells were incubated in the dark with 0.5 mg mL-1 MTT at 37°C for 2 h. Formazan granules generated by live cells were dissolved in DMSO, and the absorbance was measured at 540 nm using a multi-well plate reader (Thermo Fisher Scientific, Waltham, MA, USA). Results are expressed as a percentage of control groups (untreated cells), with untreated cells considered as 100%.
2.7. Annexin V/propidium iodide (PI) double staining
We utilized the BD Pharmingen FITC Annexin V Apoptosis Detection Kit (BD Biosciences, San Diego, CA, USA) to quantitatively determine the percentage of apoptotic cells. Briefly, cells were stained with PI and Annexin V-fluorescein isothiocyanate (FITC) solution at room temperature for 15 min in the dark. The stained cells were then assessed using flow cytometry within 1 h. Flow cytometric analysis was performed using an Accuri C6 flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).
2.8 Colony-forming assay
Cells were seeded in six-well plates (500 cells well-1), incubated overnight, treated with or without cisplatin and/or MHY4571 at the indicated concentrations, and the medium was freshly replaced. Cells were further cultured for ten days and allowed to form clones; the medium was replaced every three days during this time. Subsequently, cells were fixed in 4% paraformaldehyde for 10 min and stained with 0.1% crystal violet for 15 min. Colonies in each treatment group were observed under a microscope and photographed.
2.9 Western blot analysis
Cells were harvested and lysed. Equal amounts of protein were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes for immunoblotting. The membranes were probed with the relevant primary antibodies overnight, incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Enzo Life Sciences, NY, USA), detected using an enhanced chemiluminescence system (Advansta Inc., San Jose, CA, USA), and then monitored using a FUSION Solo-X instrument (VILBER, Wembley, WA, Australia). Densitometric analysis was performed using the ImageJ software (National Institutes of Health, Bethesda, MD, USA).
2.10 Quantitative RT-PCR
RNA was isolated using TRIzol (Sigma, Missouri, USA), and cDNA was synthesized using the cDNA Synthesis Platinum Master Mix (GenDEPOT, Texas, USA). The mRNA was analyzed using the Rotor-Gene Q real-time PCR system. The relative abundance of mRNA was determined after normalization to GAPDH mRNA levels used as a control for experimental variations. The primer sequences used in this study are summarized in Supplementary Table S2 of Additional file 1.
2.11 Kinase profile assay
Kinase selectivity was assessed using the WildType+2Point-Profiler Assay (ProQinase GmbH, Breisgau, Germany), which included 410 kinases; 13 lipid kinases, as well as disease-relevant kinase mutants, were part of the kinase panel. The assay was performed at a single concentration (7.5 µM) using ATP Km for each kinase.
2.12 PKA kinase activity
PKA activity was evaluated using a PKA Kinase Activity Kit (Enzo Life Sciences, NY, USA). This assay is based on a solid-phase enzyme-linked immunosorbent assay (ELISA) that utilizes a specific synthetic peptide as a substrate for PKA and a polyclonal antibody that recognizes the phosphorylated form of the substrate. Samples (cell lysates) and diluted standard active PKA were added to the PKA substrate microtiter plate. After adding ATP to each well containing the sample, the plate was incubated at 30°C for 90 min. Each well was emptied, and the phosphor-specific substrate antibody was added and incubated for 60 min at room temperature. After washing each well four times, peroxidase-conjugated secondary antibodies were added and incubated for 30 min at room temperature. Subsequently, wells were washed four times, and tetramethylbenzidine substrate was added. Color development was stopped with an acid stop solution. The color (which is proportional to the PKA phosphotransferase activity) was measured in a microplate reader at 450 nm.
2.13.1 Xenograft mice model
All animal protocols conformed to guidelines for the care and maintenance of laboratory animals [21]. Female BALB/c nude mice were obtained from OrientBio (Kyunggi, Republic of Korea) and used for experiments after they were 5–6 weeks old. Animals were acclimated for seven days and weighed at the end of the acclimation period. NCI-H1703 cells were subcultured, stained with trypan blue, and counted to assess cell viability. These were then transplanted into the right dorsal flanks of mice (5 × 106 cells/animal; n = 24). Mice were assigned to each of the following groups: vehicle (n = 6) and three different MHY4571 concentration groups (5 mg kg-1 vs. 10 mg kg-1 vs. 20 mg kg-1) (n = 6 per group). The oral route of drug administration was used, as this administration route is used in clinical practice. MHY4571 was administered orally five times per week for three weeks. The mean body weight was measured for each group after the group assignment. Tumor volumes were measured with digital calipers and calculated according to the formula: 0.52×length×width2, and tumor masses were evaluated at 3-weeks post-injection. Mice were sacrificed at the end of the study by being placed in a CO2 chamber.
2.13.2 Orthotopic mouse model
Male BALB/c nude mice were obtained from ORIENT BIO, Inc. (Seongnam-si, Gyeonggi-do, Republic of Korea). NCI-H1703 cells were subcultured, stained with trypan blue, and counted to assess cell viability. These cells were then suspended in 30 μL HBSS and loaded into a 29G syringe for lung administration at 2 × 106 cells/animal. Subsequently, the growth of the tumor was assessed by bioluminescence imaging. Mice were assigned to each of the following groups: vehicle (n = 5), low-dose MHY4571 (10 mg kg-1) (n = 5), and high-dose MHY4571 (20 mg kg-1) (n = 5). The mean body weight was measured for each group after the group assignment. Mice were divided into groups of five per cage. To evaluate the drug’s therapeutic effect, bioluminescent imaging (using IVIS spectrum-CT equipment) was performed before administration (day 0) and on post-administration days 6, 13, and 20. All animals were euthanized with CO2 after the termination of the experiment. For the observation of lung cancer, the lungs were fixed with Bouin’s solution.
2.14 Tissue microarray construction
Tumor tissue microarray was constructed using two tissue cores (3-mm diameter) per tumor to obtain tissue from central and peripheral tumor areas after histologic examination of 236 NSCLC specimens in the Pathology Department of Pusan National University Yangsan Hospital. All tumors were histologically examined and classified using the 2015 World Health Organization International Classification of Lung Tumors.
2.15 Immunohistochemistry (IHC)
For immunohistochemical analysis, consecutive 3 µm-thick tissue sections were cut from formalin-fixed paraffin-embedded tissues. IHC staining was performed with the primary antibody (p-PKA, 1:200; p-CREB, 1:200) overnight at 4°C. Subsequently, 3,3'-diaminobenzidine staining and TUNEL assays were performed using an ApopTag Fluorescein Direct In Situ Apoptosis Detection Kit (Millipore, Billerica, MA, USA). Hematoxylin and eosin- and IHC-stained slides were digitally scanned with a Zeiss Axio Scan Z1 microscope using ZEN 2.3 software (Carl Zeiss Microscopy GmbH, Jena, Germany).
2.16 Kaplan-Meier plotter
The Kaplan-Meier Plotter was used to assess the prognostic significance of PKA catalytic subunit alpha in squamous cell lung carcinoma. PKA catalytic subunit alpha expression (GSE17710; high vs. low expression) and clinical data, such as overall survival (OS) and recurrence-free survival (RFS), were obtained from Gene Expression Omnibus (GEO) of the National Center for Biotechnology Information (https://ncbi.nlm.nih.gov/geo).
2.17 Combination index (CI) analysis
The CI method proposed by Chou-Talalay, based on the median-effect equation of the mass-action law, allows for the quantitative computerized simulation of synergy (CI < 1), additive effect (CI = 1), and antagonism (CI > 1) at all dose and effect levels in vitro or in vivo for various types of drugs, including radiation [22]. This analysis was applied using CompuSyn software, 2005 (www.combosyn.com).
2.18 Statistical analysis
Statistical analysis for in vivo experiments was performed using Prism 8 software (GraphPad Software Inc., CA, USA). For comparisons of more than two groups, one-way ANOVA was performed, followed by a posthoc test with Dunnett’s correction of pairwise group differences. All data are expressed as the mean ± standard deviation (SD) of three experiments and were analyzed using the student’s t-test. Means were considered significantly different at *P < 0.05, **P < 0.01, and ***P < 0.001.