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Introduction

The incidence of RAS gene mutations in all known human cancers is ~30% worldwide over the last 30 years (1). Although the mutation in the RAS gene is not the direct cause of all cancers, it has been reported to promote tumor occurrence, development and metastasis (2). Ras protein GTPase activity was reported to serve a key role in the process of human carcinogenesis more than 30 years ago (3,4). Furthermore, the RAS gene has been demonstrated to be one of the most mutated oncogenes in humans and to promote further development of tumors (5). A previous study demonstrated that mutant RAS gene stimulated cell proliferation and inhibited cell apoptosis, thereby promoting tumor occurrence, survival, migration, diffusion and angiogenesis (6). Therefore, Ras protein is used as a target for drug treatment; however, due to the complexity of the Ras signaling pathway and its unique structure, drugs cannot actively target Ras to bind directly of the protein (7).

In recent years, naphthoquinone derivatives have become a focus of interest due to their ability to effectively cause death in cancer cells, including those in lung cancer (8), breast cancer (9) and colon cancer (10). However, naphthoquinone reportedly exerts low cytotoxic effects on healthy cells compared with currently used, existing clinical drugs, including 5-fluorouracil, cisplatin and sorafenib (11). Naphtoquinone derivatives are primarily constructed through multiple additions and substitutions with shikonin as the primary substrate to obtain a variety of derivatives with different structures (12). The 5,8-dimethoxy-1,4-naphthoquinone (DMNQ) compound is commonly used to synthesize naphthoquinone derivatives. It has been reported that DMNQ derivatives can inhibit tumor activity in lung (13) and breast cancers (14). Since each derivative has different substituents, the resulting functions of each derivative are subsequently different (15). According to previous research, naphthoquinone derivatives primarily aim to target two different physiological processes to cause cell death: i) To induce cytotoxicity by increasing intracellular reactive oxygen species (ROS) levels, which causes damage to mitochondrial function in cells, endoplasmic reticulum stress and autophagy that lead to cell death (16); and ii) to directly cause cytotoxicity, DNA strand breakage and cell cycle arrest (17). In hepatocarcinoma cells, naphthoquinone derivatives were reported to suppress hepatocarcinoma liver cancer cell proliferation, inhibit angiogenesis and promote cellular apoptosis by inhibiting DNA synthesis (18,19). Research into drugs targeting Ras has made progress (20). Certain research demonstrated that 2,3-dichloro-1,4-naphthoquinone inhibited the growth of N-Ras-mutant melanoma (21), while 2-(2',4'-dihydroxyphenyl)-8-hydroxy-1,4-naphthoquinone and 5-hydroxy-2-(2,4-dihydroxyphenyl)naphthalene-1,4-dione inhibited Apc/K-Ras-mutant mouse organoid differentiation capacity (22). These results indicate that naphthoquinone derivatives have a certain inhibitory effect on the proliferation and differentiation of cancer cells and Ras signaling.

Therefore, the present study used shikonin as the main backbone to which to add different substituents and to design a variety of naphtoquinone derivatives, to select and investigate the anticancer mechanism of drugs with lower cytotoxicity for normal cells and higher lethality for cancer cells. Furthermore, the present study aimed to establish novel structural modification sites, while retaining the structure of derivatives for targeting Ras signaling to maximize the anticancer effect and reduce cytotoxicity, which may have practical significance for the development of targeted drugs.

Materials and methods

Materials

Solvents of reagent grade and were used without further purification. 1H NMR and 13C NMR spectra were recorded using a JNM-AL 400 (400 MHz) spectrometer, Chemical shift ppm (δ) were relative to internal standard tetramethylsilane. MS spectra were recorded with an AB SCIEX API 2000 LC/MS/MS (Applied Biosystems; Thermo Fisher Scientific, Inc.) and LCMS-IT-TOF (Shimadzu Corporation).

Synthesis of 5,8-dimethoxy-1,4-naphthoquinone (DMNQ) derivatives

Synthetic schemes for 12 DMNQ derivatives are summarized in Fig. 1.

Figure 1 Synthetic schemes and structures of the 12 different 5,8-dimethoxy-1,4-naphthoquinone derivatives. (A) Synthetic schemes of 5,8-dimethoxy-1,4-naphthoquinone derivatives. Compounds were synthesized by condensation, methylation, oxidation, and Michael addition reactions. (B) structures of 12 different 5,8-dimethoxy-1,4-naphthoquinone derivatives. OMe, methoxy; MeOH, Methanol; (CH2)5OH, hydroxy pentyl; (CH2)6CH3, heptyl; (CH2)9, nonyl;O, oxygen; AlCl3, aluminum chloride; NaCl, sodium chloride; OH, hydroxide; Me2SO4, dimethyl sulfate; Na2S2O4, sodium dithionite; CAN, Cerium (IV) diammonium nitrate; mCPBA, meta-Chloroperoxybenzoic acid; S, sulphur; HS, hydrosulfide; R, functional group; H2N, nitrogen hydride; NH, amine; ROS, reactive oxygen species.

Synthesis of 5,8-dihydoroxy-1,4-naphthoquinone

1,4-Dimethoxybenzene (1 equivalent) (Sigma-Aldrich; Merck KGaA) and maleic anhydride (2 equivalent) (Sigma-Aldrich; Merck KGaA) were added to the molten material of NaCl (4 equivalent) (Tianjin Damao Chemical Reagent Co., Ltd.) and aluminum chloride (8 equivalent, 171-177˚C) (Tianjin Damao Chemical Reagent Co., Ltd.) to give the condensed mixture. The derivatives were then treated with 1.2 liters H2O and 120 ml 95% HCl (Tianjin Damao Chemical Reagent Co., Ltd.) to generate DMNQ (Fig. 1A-a).

Synthesis of 1,4,5,8-tetramethoxynaphthalene

A solution of 25 ml methyl p-toluene sulfonate (Sigma-Aldrich; Merck KGaA), 10.6 g sodium dithionite (Tianjin Damao Chemical Reagent Co., Ltd.) in 40 ml 70% NaOH (Tianjin Damao Chemical Reagent Co., Ltd.) was added to a solution of 20.1 g (Fig. 1A-a) in 200 ml tetrahydrofuran (Tianjin Damao Chemical Reagent Co., Ltd.), 220 ml H2O and 2.2 g tetrabutylammonium bromide (Sigma-Aldrich; Merck KGaA). The mixture was stirred at 25˚C for 20 h until a red precipitate formed, which was filtered and recrystallized in petroleum ether (Tianjin Damao Chemical Reagent Co., Ltd.) (90-120˚C) to produce an intermediate 1,4,5,8-tetramethoxynaphthalene product (Fig. 1A-b).

Synthesis of DMNQ

An amount 5.5 g cerium (IV) diammonium nitrate (Tianjin Damao Chemical Reagent Co., Ltd.) was dissolved in 250 ml H2O and added drop-wise to a solution of 15 g 1,4,5,8-tetramethoxynaphthalene in 160 ml trichloromethane (Tianjin Damao Chemical Reagent Co., Ltd.) and 480 ml acetonitrile (Tianjin Damao Chemical Reagent Co., Ltd.). This mixture was stirred at 25˚C for 1.5 h. The key compound DMNQ (Fig. 1A-c) was obtained by this oxidation reaction.

Synthesis of the DMNQ derivatives #1

DMNQ solution (300 mg; 1.37 mmol) in 40 ml ethyl alcohol (EtOH, Tianjin Damao Chemical Reagent Co., Ltd.) was added to benzyl mercaptan (204.9 mg; 1.65 mmol; Sigma-Aldrich; Merck KGaA; Fig. 1B-#1) for derivative #1, 2-phenylethyl mercaptan (228.1 mg; 1.65 mmol; Sigma-Aldrich; Merck KGaA; Fig. 1B-#2) for derivative #2, 3,4-dimethoxybenzenethiol (280.9 mg; 1.65 mmol; Sigma-Aldrich; Merck KGaA; Fig. 1B-#3) for derivative #3, 4-tert-butylphenylmethanethiol (297.5 mg; 1.65 mmol; Sigma-Aldrich; Merck KGaA; Fig. 1B-#4) for derivative #4, phenyl amine (148.7 mg; 1.65 mmol; Sigma-Aldrich; Merck KGaA; Fig. 1B-#5) for derivative #5, cyclo-propyl amine (84.0 mg; 1.65 mmol; Sigma-Aldrich; Merck KGaA; Fig. 1B-#6) for derivative #6, 2-methylpropan-1-amine (120.6 mg; 1.65 mmol; Sigma-Aldrich; Merck KGaA; Fig. 1B-#7) for derivative #7, 2-mercaptoethanol (128.9 mg; 1.65 mmol; Sigma-Aldrich; Merck KGaA; Fig. 1B-#9) for derivative #9, 6-mercaptohexan-1-ol (221.5 mg; 1.65 mmol; Sigma-Aldrich; Merck KGaA; Fig. 1B-#10) for derivative #10, 4-mercaptophenol (208.2 mg; 1.65 mmol; Sigma-Aldrich; Merck KGaA; Fig. 1B-#11) for derivative #11 and 10-mercaptodecanoic acid (337.1 mg; 1.65 mmol; Sigma-Aldrich; Merck KGaA; Fig. 1B-#12) for derivative #12. The reaction mixture was stirred at room temperature for 4.5 h. The resulting solution was added dropwise to a solution of 0.24 mmol sodium dichromate (Tianjin Damao Chemical Reagent Co., Ltd.) and 0.77 mmol sulfuric acid (Tianjin Damao Chemical Reagent Co., Ltd.) in 10 ml H2O. The resulting mixture was stirred for a few minutes, extracted with methylene chloride (CH2Cl2; Tianjin Damao Chemical Reagent Co., Ltd.), concentrated and recrystallized in EtOH to generate the target compound #1 (Fig. 1B-#1).

Synthesis of DMNQ derivative #8

2-Heptyltho-DMNQ solution (300 mg, 1.37 mmol) in 40 ml dichloromethane was added to 1.65 mmol 3-chloroperoxybenzoic acid (Shanghai Aladdin Bio-Technology Co., Ltd.). The reaction mixture was stirred at room temperature for 2.5 h. The resulting solution was added to 40 ml saturated sodium chloride solution. The resulting mixture was stirred for a few minutes, extracted with CH2Cl2, concentrated and recrystallized in EtOH to produce the target compound #8 (Fig. 1B-#8).

The structures of compounds #1 were characterized by nuclear magnetic resonance (NMR) and mass spectroscopy.

Chemicals

The following chemicals were used in the current study: DMEM (Invitrogen; Thermo Fisher Scientific, Inc.), FBS (Cytiva), penicillin (100 U/ml) and streptomycin (100 mg/ml; Beijing Solarbio Science & Technology, Co., Ltd.), MTT (Sigma-Aldrich; Merck KGaA), an Annexin V-FITC and propidium iodide (PI) detection kit (Beijing Solarbio Science & Technology, Co., Ltd.), dihydroethidium (DHE; Beyotime Institute of Biotechnology), Hoechst 32258 (Thermo Fisher Scientific, Inc.), N-acetyl-L-cysteine (NAC; Beijing Solarbio Science & Technology, Co., Ltd.), horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Ig)G and anti-mouse IgG (Sangon Biotech, Co., Ltd.).

Cell culture

The following cell lines were used: NIH3T3 mouse embryonic fibroblasts (NC), H-RasG12V-transfected NIH3T3 mouse embryonic fibroblasts (NR), HepG2 human liver cancer (HC) cells and H-RasG12V-transfected HepG2 human liver cancer cells (HR). All cell lines were provided by Dr Dae-Yeul Yu (Aging Intervention Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea). Cells were all cultured in DMEM with 10% (v/v) FBS and maintained in a culture environment of 37˚C and 5% CO2. The culture medium was replaced once a day and cells were sub-cultured every 3 days. Cells were treated with 5 mM NAC for 30 min prior to treatment with 8 µM BZNQ.

Cytotoxicity analysis of the 12 different 5,8-dimethoxy-1,4-naphthoquinone derivatives

NC and NR cells were seeded on 96-well plates (10,000 cells/well) and cultured for 24 h at 37˚C in the presence of the 12 different 5,8-dimethoxy-1,4-naphthoquinone derivatives (0, 10, 25 and 50 µM). A total of 10 µl (0.5 mg/ml) MTT reagent was added and absorbance was measured at a wavelength of 490 nm using a UV MAX kinetic microplate reader (Molecular Devices, LLC.) to determine the accumulation of formazan. The purple formazan was subsequently dissolved using DMSO. The IC50 values were obtained using SigmaPlot Version 10.0 software (Systat Software Inc.).

Cell viability assay

MTT assays were used to analyze cell viability. NC, NR, HC and HR cells were seeded on 96-well plates (seeding density, 10,000 cells/well) and cultured for 24 h at 37˚C in the presence of 2-benzylthio-5,8-dimethoxynaphthalene-1,4-dione (BZNQ) at concentrations of 0, 2, 4, 6, 8 and 10 µM in NC and NR cells. For HC and HR cells, BZNQ was administered at 0, 2, 4, 6, 8, 10 and 20 µM. The 0 µM (untreated) group was used as the control. Following this, 10 µl (0.5 mg/ml) of MTT reagent was added and the absorbance was measured at the wavelength of 490 nm using a UV MAX kinetic microplate reader (Molecular Devices, LLC.) to determine the accumulation of formazan. The MTT reagent was dissolved with DMSO.

Early and late apoptosis detection using Annexin V-FITC/PI

NC, NR, HC and HR cells treated with BZNQ were harvested by trypsin and then suspended in PBS. Apoptosis was measured by staining with Annexin V-FITC/PI for 15 min at room temperature. The number and proportion of positively-labeled cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences). The results were analyzed using the WinMDI software (version 2.9; BD Biosciences).

Detection of ROS production

HC and HR cells were treated with BZNQ for 12 h. Cells were subsequently fixed with 4% paraformaldehyde for 10 min at room temperature. The changes in cellular ROS levels were determined using 5 µM DHE for 15 min at 37˚C. Nuclei were visualized using Hoechst 32258 staining. Nuclear staining was observed qualitatively under a fluorescence microscopy (EVOS®xl core cell culture microscope; Advanced Microscopy Group) following 20 min of incubation with the Hoechst dye at 37˚C.

Western blotting

For the isolation of proteins, cells was lysed in ice-cold cell extraction buffer [20 mM HEPES (pH 7.0), 50 mM NaCl, 10% Triton X-100, 10% Glycerol, 1 mM β-ME and a protease inhibitor cocktail tablet; Roche Diagnostics GmbH] for 30 min. Lysates were centrifugated at 13,200 x g and 4˚C for 20 min. Total protein (2 µg/µl) quantification was determined by a Bradford protein assay using an ultraviolet spectrophotometer (Aoyi Instruments Co., Ltd.). Denatured protein of cell lysates were separated using 12% SDS-PAGE and transferred to nitrocellulose membranes (EMD Millipore). The membranes were blocked with 5% skim milk (BD Biosciences) for 1 h at room temperature. The membranes were incubated with primary antibodies against H-Ras (cat. no bs-1071; BIOSS; 1:1,000), protein kinase B (AKT; cat. no. sc-8044; Santa Cruz Biotechnology, Inc.; 1:2,000), p-AKT (cat. no. sc-101629; Santa Cruz Biotechnology, Inc.; 1:2,000), extracellular regulated kinase (ERK; cat. no. sc-135900; Santa Cruz Biotechnology, Inc.; 1:2,000), p-ERK (cat. no. sc-7383; Santa Cruz Biotechnology, Inc.; 1:2,000), glycogen synthase kinase 3β (GSK3β; cat. no. AF1543; Beyotime Institute of Biotechnology, Inc.; 1:2,000). p-GSK3β (Ser9; cat. no. AF5830; Beyotime Institute of Biotechnology, Inc.; 1:2,000), β-catenin (cat. no. AF1189; Beyotime Institute of Biotechnology, Inc.; 1:2,000), B-cell lymphoma-2 (Bcl-2; cat. no. sc-7382; Santa Cruz Biotechnology, Inc.; 1:2,000), Bcl-2 associated agonist of cell death (Bad; cat. no. 8044; Santa Cruz Biotechnology, Inc.; 1:2,000), β-actin (cat. no. 3700; Cell Signaling Technology, Inc.; dilution, 1:2,000) at 4˚C overnight. The membranes were washed five times with TBST [10 mM Tris HCl (pH 7.5), 150 mM NaCl and 0.2% Tween-20] to remove excess antibodies and then incubated for 1 h at room temperature with horseradish peroxidase conjugated goat anti mouse and rabbit IgG secondary antibodies(cat. no. D110087 and D110058; Shanghai Sangon Biotech Co., Ltd.; 1:5,000). Bands of antibody-specific binding proteins were visualized by a chemiluminescence detection system (GE Healthcare) and densitometry was analyzed using ImageJ version 1.52a software (National institutes of Health).

Statistical analysis

Data are presented as the mean ± SEM from at least three independent repeated experiments. Two-way ANOVA was used to analyze the changes across time and differences between groups with the Tukey's honestly significant difference test used as the post-hoc test (α=0.05). P<0.05 was considered to indicate a statistically significant difference.

Results

Effect of BZNQ on cell viability

According to the synthetic methodology provided above, a total of 9 derivatives with a redox center S atom and 3 alkylamine derivatives that were able to clear ROS in cells were designed (Fig. 1A). The structure of each 5,8-dimethoxy-1,4-naphthoquinone derivative is provided in Fig. 1B. The results indicated that compounds #1-8 may be associated with ROS modulations, while compounds #9-12 may be involved in cell cytotoxicity (Fig. 1B). To examine the cytotoxicity of the synthesized compounds, the NC and NR NIH3T3 cells were treated with derived compounds #1-12 and the concentrations of IC50 for each compound were evaluated using MTT assays (Table I). The results demonstrated that at low concentrations, BZNQ exhibited higher cytotoxic activity in both NC and NR cells compared with the rest of the compounds. Compared with #11, #1 demonstrated lower dose targeting to HRAS. Additionally, the lower dosage produced fewer side effects. #1 was therefore selected for further study. Furthermore, compounds #2-4 and #8-10 demonstrated significant activities in NC and NR cells; however, there were no significant differences in cytotoxicity effects between NC and NR cells (data not shown). The results indicated that the different modulation of the substitution groups at the specified positions exhibited various inhibiting activities in cells. Therefore, the current study focused subsequent experiments on BZNQ to elucidate the possible mechanisms of action.

Table I IC50 values of the derived compounds on NC and NR cells.

Table I

IC50 values of the derived compounds on NC and NR cells.

Compound	NC	NR
#1 (BZNQ)	12.40	4.73a
#2	39.31	35.86
#3	<10.00	<10.00
#4	16.42	24.37
#5	>50.00	>50.00
#6	>50.00	>50.00
#7	>100.00	>100.00
#8	<1.00	<1.00
#9	<1.00	<1.00
#10	5.37	7.53
#11	>30.00	21.52a
#12	22.50	12.39

[i] ^aP<0.01. IC₅₀, half maximal inhibitory concentration (µM); NC, NIH3T3 mouse embryonic fibroblasts; NR, H-Ras^G12V-transfected NIH3T3 mouse embryonic fibroblasts; BZNQ, 2-benzylthio-5,8-dimethoxynaphthalene-1,4-dione.

BZNQ induces apoptosis in Ras-transfected NIH3T3 cells

To investigate the structure of BZNQ, NMR spectra were evaluated. According to the NMR spectra examination, the #1 compound as BZNQ: 1H-NMR (CDCl3, 400 MHz) δ7.40-7.28 [multiplet (m), 5 hydrogen (H)], 6.98-6.80 (m, 2H), 5.99 [singlet (s), 1H] 3.94 (s, 6H), 3.63 (s, 2H); 13C NMR (CDCl3, 100 MHz) δ 187.7 [C (carbon)-1], 180.7 (C-4), 154.3 (C-5), 154.1 (C-8), 153.7 (C-2), 135.2 (C-1'), 134.5 (C-3), 129.0 (C-3'), 129.0 (C-5'), 128.9 (C-2'), 128.9 (C-6'), 128.7 (C-4'), 128.1 (C-7), 127.5 (C-6), 121.1 (C-9), 119.6 (C-10), 56.9 (OCH3), 56.7 (OCH3), 35.4 (CH2)and m/z 362.63 [Molecule (M)+Na]+ (Fig. 2A).

Figure 2 Effect of BZNQ on cellular viability and apoptosis. (A) The structure of BZNQ as obtained from nuclear magnetic resonance analysis. (B) The effect of BZNQ on NC (black bars) and NR (white bars) cell viability was detected using MTT assays. The untreated NC group was used as the control group. (C) NC and NR cells were treated with BZNQ and cellular apoptosis was examined with Annexin V-FITC/PI kit using flow cytometry. (D) The level of apoptosis was presented as the mean ± standard deviation. Experiments were performed in triplicate. **P<0.01, ***P<0.001. BZNQ, 2-benzylthio-5,8-dimethoxynaphthalene-1,4-dione; NC, NIH3T3 mouse embryonic fibroblast cell line; NR, H-RasG12V-transfected NIH3T3 mouse embryonic fibroblast cell line; PI, propidium iodide; O, oxygen; S, sulphur; Con, control.

Following this, the effect of BZNQ on cellular apoptosis in NC and NR cells was examined. The results indicated that BZNQ induced NR cell apoptosis in a dose-dependent manner (Fig. 2B). Furthermore, flow cytometry analysis indicated that BZNQ treatment significantly increased NR cell apoptosis compared with NC cells (Fig. 2C and D).

BZNQ induces cellular apoptosis through the AKT, ERK and GSK signaling pathways

To elucidate the possible signaling pathways by which BZNQ induces NR cell apoptosis, H-Ras protein expression in cells treated with BZNQ for 24 h was evaluated. The results demonstrated that BZNQ treatment significantly downregulated H-Ras protein expression levels in NR cells (Fig. 3A). Due to these results, the known Ras-mediated downstream signaling pathways, including AKT, ERK and GSK, as well as β-catenin activation, were evaluated. The results revealed that BZNQ treatment significantly downregulated AKT, ERK and GSK3β protein phosphorylation in NR cells (white bar) compared with NC cells (black bar; Fig. 3B).

Figure 3 Effect of BZNQ on H-Ras protein expression and Ras-mediated downstream signaling. (A) NC and NR cells were treated with indicated concentrations of BZNQ for 24 h. H-Ras, p-AKT, AKT, p-ERK, ERK, p-GSK3β, GSK3β, β-catenin and β-actin protein expression levels were detected via western blotting. (B) Fold increase in protein expression levels. **P<0.01, ***P<0.001. Experiments were performed in triplicate. BZNQ, 2-benzylthio-5,8-dimethoxynaphthalene-1,4-dione; NC, NIH3T3 mouse embryonic fibroblast cell line; NR, H-RasG12V-transfected NIH3T3 mouse embryonic fibroblast cell line; AKT, protein kinase B; ERK, extracellular regulated kinase; GSK3β, glycogen synthase kinase 3β.

BZNQ increases cellular apoptosis and ROS accumulation in HepG2 liver cancer cells

Since BZNQ increased cell apoptosis by targeting Ras signaling proteins, the cytotoxic effects of BZNQ on HC and HR liver cancer cells was evaluated. BZNQ exhibited highly marked cytotoxicity in both HC and HR cells, as shown by MTT assays. After BZNQ treatment, fluorescence intensity was increased when compared with untreated cells, as observed by fluorescent microscopy (Fig. 4C) and flow cytometry analysis (Fig. 4D) by detecting DHE fluorescence intensity. The results demonstrated that BZNQ treatment elevated cellular ROS accumulations in HC and HR cells.

Figure 4 Effect of BZNQ on cancer cell viability, apoptosis and ROS levels. (A) HC and HR cells were treated with the indicated concentrations of BZNQ and cell viability was examined using MTT assays. (B) Cellular apoptosis was assayed using an Annexin V/propidium iodide staining kit and measured using flow cytometry. The increased fold of apoptosis was presented as mean ± SD. (C) Cellular ROS levels were detected using DHE staining and observed with fluorescent microcopy. Scale bar, 100 µM. (D) Fold increase of ROS levels was presented as mean ± SD. All experiments were performed in triplicate. *P<0.05, ***P<0.001. BZNQ, 2-Benzylthio-5,8-dimethoxynaphthalene-1,4-dione; ROS, reactive oxygen species; HC, HepG2 human liver cancer cell line; HR, H-RasG12V-transfected HepG2 human liver cancer cell line; DHE, dihydroethidium; SD, standard deviation; Con, control.

Scavenging ROS levels decreases BZNQ-induced cellular apoptosis by upregulating the protein expression of Bad

To understand the role of ROS on BZNQ-induced cellular apoptosis, HC and HR cells were pretreated with NAC, a ROS scavenger, for 30 min, followed with BZNQ treatment. NAC pre-treatment decreased cellular ROS levels and apoptosis in HC and HR cells (Fig. 5A-C). Furthermore, apoptosis-related protein expression levels, including Bcl-2 (anti-apoptotic) and Bad (pro-apoptotic), were evaluated following the BZNQ treatment. The results indicated that BZNQ treatment upregulated Bad protein expression and slightly downregulated the Bcl-2 expression in HC and HR cells (Fig. 5D).

Figure 5 ROS scavenging inhibited BZNQ-induced cellular apoptosis. (A) HC and HR cells were pre-treated with 5 mM NAC, an ROS scavenger, for 30 min, followed by BZNQ treatment. ROS levels were detected by DHE staining and observed with fluorescent microcopy. Scale bar, 100 µM. (B) Cellular ROS levels were assayed with DHE staining following NAC and BZNQ treatment and measured using flow cytometry. Fold increase in ROS levels was presented as mean ± SD. (C) Cellular apoptosis was assayed with Annexin V/propidium iodide (staining kit following NAC and BZNQ treatment and measured using flow cytometry assay. Fold increase in apoptosis was presented as mean ± SD. (D) The protein expression levels of Bcl-2 and Bad were examined following BZNQ treatment in HC and HR cells by western blotting. Experiments were performed in triplicate. ***P<0.001. ROS, reactive oxygen species; BZNQ, 2-benzylthio-5,8-dimethoxynaphthalene-1,4-dione; HC, HepG2 human liver cancer cell line; HR, H-RasG12V-transfected HepG2 human liver cancer cell line; NAC, N-acetyl-L-cysteine; DHE, dihydroethidium; SD, standard deviation; Bcl-2, B-cell lymphoma 2; Bad, Bcl-2 associated agonist of cell death; Con, control.

Discussion

Shikonin is a small-molecule compound extracted from the natural plant Arnebia euchroma and induces tumor cell apoptosis (23-25), inhibits DNA topoisomerase (26) and protein tyrosine kinase (26), represses human telomerase activity (27), suppresses tumor angiogenesis (28), alters tumor cell signal transduction and activates mitogen activated protein kinase (MAPK) through chemical protection and induces antimitotic effects (29). Furthermore, shikonin influences the metabolism, proliferation, differentiation, signal transmission, gene expression, cell growth, invasion and metastasis of tumor cells (30-32). However, due to the strong cytotoxicity and side effects of shikonin, including nausea, vomiting and loss of appetite, it has not been widely used in clinical practice (33). With further research into the mechanisms of action of shikonin, it has been reported that the toxic side effects of its derivatives are greatly reduced following the addition of the methylated 5' and 8' hydroxyl groups to methoxy groups (34). Moreover, naphthoquinone derivatives are more convenient for the latter modification and therefore, has more structural advantages in the synthesis of new compounds. Previous results have demonstrated that naphthoquinone derivatives specifically inhibit the p53-Snail binding in K-RasG12D mutant A549 cells to induce cell apoptosis (35), demonstrating that naphthoquinone derivatives have the potential to target the Ras signaling pathway.

1,4-Naphthoquinone is the main component of Arnebia purpurea and widely exists in nature as a small molecular compound with quinone moiety (36). Generally, when two kinds of substituents contain the same hydrocarbon group, the cytotoxicity of mercaptan derivatives is stronger compared with amino derivatives (37). While amino derivatives have anti-inflammatory effects (38), mercaptan derivatives have better anti-tumor (39,40) and anti-malaria activity (41,42). Bielitza et al (43) reported that benzyl carbon in naphthoquinone derivatives participates in the oxidation-reduction cycle of NADPH and that benzyl carbon can be oxidized to carbonyl by hydroxylation or peroxidation. If the para position of the benzene ring contains a tert-butyl group, it can reduce the toxicity of compounds (44). The substituent alkyl terminal containing hydroxyl or carboxyl group exhibits strong cytotoxicity (45). The current study synthesized and reported several groups of these kinds of compounds which exhibited significant anticancer property. In order to further investigate this series of DMNQ compounds, group compounds that contain a sulfur atom linked between naphthnoquinone core and linear substitutes were designed. The sulfur atom can be used as an oxidation-reduction center to eliminate ROS in cells (46). Several substitutes, including benzyl, butyl and cyclo propyl, which are normally selected to optimize the property of designed compounds, were used in the current study. The results demonstrated that benzyl substituted compound BZNQ demonstrated excellent effects, with increased cytotoxity in H-RasG12V mutated cells and a lower concentration.

In the current study, 12 DMNQ derivatives were designed, including BZNQ. Among the 12 kinds of derivatives, BZNQ had the highest cytotoxic effect in NR cells compared with NC cells compared with all other compounds from the IC50 value. Furthermore, compounds #2-4 and #8-10 demonstrated significant activities in NC and NR cells; however, there were no significant differences in cytotoxicity effects between NC and NR cells (data not shown), indicating that the different modulation of the substitution groups at the positions exhibited various inhibiting activities in cells. The possible molecular mechanisms that rely on structure-activity relationships should be studied further.

The intracellular signaling pathways that regulate apoptosis are diverse and complex. Generally, when Ras signaling has been activated, the downstream MAPK signaling pathway must also be activated (47). However, it has been reported that H-RasG12V preferentially activates the PI3K/AKT signaling pathway to promote cell survival following starvation and activated AKT inhibits s621 phosphorylation of RAF and downregulates ERK phosphorylation (48-50). Furthermore, NF-κB was revealed to be an important downstream signal molecule for AKT to regulate cell anti-apoptotic signaling and inhibiting NF-κB blocked the anti-apoptotic signals regulated by PDG/PI3K/AKT (51,52). Furthermore, BZNQ inhibited AKT and GSK3β S9 phosphorylation, which resulted in promotion of β-catenin in NIH3T3 cells These changes in the activity of signaling pathways promoted apoptosis-related protein expression levels (Bad upregulation and Bcl-2 downregulation), which led to cellular apoptosis. Additionally, Ras, a member of MAPK upstream signal, can cause the activation of downstream signals (53). It has also been reported that AKT phosphorylates Raf, which activates ERK phosphorylation (54) and that there have alternative active phenomenon between ERK and AKT, such as ERK upregulation and AKT downregulation (55). The current study did not result in direct evidence to clarify the differences in the activation of H-Ras and p-ERK expression levels. This should be further investigated in future studies.

Since BZNQ exhibited strong cytotoxic effects by targeting Ras signaling, the current study hypothesized that it may also have a toxic effect on cancer cells, particularly in Ras mutated cancer cells. Therefore, the effect of BZNQ in HC and HR cells was examined. The observed results demonstrated that there was a high toxic effect on BZNQ on HC and HR cells, there were significant differences in cellular apoptosis and ROS levels between HC and HR cells. This is closely associated with the 5,8-dimethoxynaphthalene-1,4-dione core and benzyl group of BZNQ as these subunits may serve an important role in the increase of intracellular ROS levels and cytotoxicity (56). The current study speculates that BZNQ promoted the apoptosis of Ras mutant cell lines by inhibiting the expression of Ras proteins and increasing the level of ROS in H-RasG12V transfected cells. The results indicated that the Ras signaling pathway may serve different roles in healthy cells and cancer cells. The possible mechanisms should be studied further.

In conclusion, based on the analysis of the structure-activity relationship of DMNQ derivatives, the current study designed, synthesize and screened compounds that specifically regulated the Ras mutation signal. The results confirmed the effects of the compounds on the apoptosis signaling pathway AKT/GSK3β/β-catenin, which is downstream of the Ras signaling pathway. Although there was no direct evidence of how BZNQ induced cell death in Ras-mutated cells, the results demonstrated that the apoptotic levels of NC, NR, HC and HR cells were significantly different following BZNQ treatment, resulting in significant changes in the downstream signal of Ras. This indicated that H-RasG12V transfected cells were more sensitive to BZNQ. The specific mechanism needs further study. These results indicated that the novel derivative synthesized based on DMNQ, BZNQ, may be a therapeutic drug for treating Ras-mediated tumorigenesis through the downregulation of H-Ras protein expression and Ras-mediated signaling pathways.

Acknowledgements

Not applicable.

Funding

The current study was supported by the Natural Science Foundation of Heilongjiang Province of China (grant no. QC2016012), the University Nursing Program for Young Scholars with Creative Talents in Heilongjiang Province (grant no. CXRC2017016) the Heilongjiang Undergraduate Training Programs for Innovation and Entrepreneurship (grant no. 201910223027), and the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1I1A2052417) and the grants from the Korean Research Institute of Bioscience and Biotechnology Research Initiative Program (KRIBB; KGM5162021, RBM0112011).

Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

GNS, JL and YHJ analyzed the data and wrote the manuscript. GNS, TK and YHH designed experiments. HNS, YYH, MHJ, RL, WLL and YQZ performed the experiments. JBY, NNY, WDW, LYY and JSK developed novel software and performed simulations. TK and YHH conceived and supervised the study. All authors read and approved the final manuscript.

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