The following antibodies were used: Ki-67 (Cat# Ab16667) from Abcam (Cambridge, Bratian); GAPDH (Cat# 10494-1-AP), phospho-PI3K (Cat# 310163), AKT (Cat# 10176-2-AP), Phospho-AKT (Cat# 66444-1-Ig), Bax (Cat# 50599-2-Ig), Bcl-2 (Cat# 12789-1-AP), and LC3 (Cat# 14600-1-AP) from Proteintech (USA); mTOR (Cat# 380411), phospho-mTOR (Cat# 385033), SQSTM1/P62 (Cat# 380612), PDK-1 (Cat# 220521), and PDH-E1α (Cat# 385512) from ZENBIO (China); caspase 9 (Cat# 9508), cleaved-caspase 9 (Cat# 52873), caspase 3 (Cat# 9662), cleaved-caspase 3 (Cat# 9664), PARP (Cat# 9532), and cleaved-PARP (Cat# 5625) from Cell Signaling Technology (USA); PI3K(Cat# AF6241) from Affinity (USA); phospho-PDHA1(Cat# Ap1022) from Abclonal (China). N-acetyl cysteine (NAC) (Cat# A7250) was purchased from Sigma (USA). As2O3 (ATO, Cat# H20080664) was purchased from Beijing SL Pharmaceutical Company (China).
Synthesis and characterization of Aa-Z2
The arsenic-containing compound was synthesized in two steps. First, the pro-drug 4-(1,3,2-dithiarsinan-2-yl) aniline (Z2) was described in detail. p-arsanilic acid (5 g, 18.45 mmol) and 70% ammonium thioglycolate (10 mL, 13 g, 120 mmol) were added into a 25 mL round flask containing a magnetic stir bar at 50 ℃. After 4 h, 1, 3-propanedithiol (4 mL, 23 mmol) was added dropwise and then stirred overnight to complete the reaction. Dichloromethane (DCM) and anhydrous Na2SO4 were used for extraction and drying respectively. The organic layer was purified by column chromatography on silica gel (PE/DCM = 1/4, v/v) to afford Z2 in 50% yield.
We further synthesized Aa-Z2 according to the following procedure: trimethylamine (0.25 mL, 2 mmol) was added to a solution of Z2 (0.274 g, 1 mmol) in dichloromethane (30 mL) under a N2 atmosphere. After half an hour, acryloyl chloride was added dropwise at 0 °C and stirred for 2 h. Then the reaction was quenched with H2O and extracted with CH2Cl2 (3 × 10 mL). The organic layer was washed with brine, dried over anhydrous Na2SO4, concentrated under reduced pressure and purified by column chromatography (PE/DCM = 1/50, v/v) to generate a white solid (C12H14AsNOS2, Aa-Z2) in 56% yield. In addition, Aa-Z2 was further characterized by full-scan mass spectrometry (MS) and 1H and 13C NMR spectroscopy (Additional file 1: Fig. S1).
Cell and cell culture
Human OS cell lines (143B, HOS, MG63, U2OS) were obtained from the American Type Culture Collection (Manassas, USA). UCMSCs were purchased from Yinfeng Dingcheng Biological Engineering (Wuhan, China), AML-12 cells were purchased from Procell Life Science and Technology (Wuhan, China), and 293 T and MC3T3-E1 cells were generously gifted to us from Professor Lin Cai (Wuhan University, China). 143B and HOS cells were cultured in MEM medium (HyClone, USA) with 10% fetal bovine serum (FBS) (Gibco, USA). U2OS cells were cultured in Mycco’5A medium (Procell, China) with 10% FBS. MG63, AML12, 293 T and MC3T3-E1 cells were cultured in high-glucose Dulbecco's modified Eagle's medium (HyClone, USA) with 10% FBS. UCMSCs were cultured in RPMI-1640 medium (HyClone, USA) with 20% FBS. All cells were cultured with 100 μg/ml penicillin and streptomycin at 37 °C under an atmosphere of 5% CO2.
Cell viability assay
All cells were plated in 96-well culture plates for 24 h at a density of 5 × 103 cells/well. Then, they were treated with different doses of Aa-Z2 (0–5 μM; dissolved in DMSO, diluted in MEM medium) or ATO (0–10 μM; dissolved in saline, diluted in MEM medium) for 24 h. Cell viability was measured using the Cell Counting Kit-8 assay (CCK8, Cat# MA0218-T, Meilunbio, China). Specifically, 100 μL of medium containing 10 μL of CCK-8 dye was added to each well, and the plate was placed in a 37 °C incubator. After 2 h, the absorbance values were read at 450 nm using a microplate reader (SpectraMax M2, Molecular Devices, USA). The half maximal inhibitory concentration (IC50) values of Aa-Z2 and ATO were calculated using GraphPad Prism 8. 0 software (GraphPad, USA).
Clone formation assay
143Band HOS cells were seeded in 6-well culture plates at a density of 1 × 106 cells/well and treated with Aa-Z2 (0, 0.4, 0.6, 0.8 μM) for 24 h. After being washed three times with PBS to remove dead cells, the remaining cells were recounted and seeded in 6-well plates at a density of 500 cells/well to grow for 10–14 days. When obvious colonies were observed under an inverted microscope (Olympus, Japan), the medium was discarded, and the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet for 15 min. After digital photos were taken of each well, the number of colonies (> 50 cells) in each image was counted by using ImageJ software (National Institutes of Health, USA).
Cellular ROS assay
143B and HOS cells were seeded in 6-well culture plates at a density of 1 × 106 cells/well and treated with different drug concentrations (Control, Aa-Z2 0.4 μM, Aa-Z2 0.6 μM, Aa-Z2 0.8 μM, NAC 5 mM + Aa-Z2 0.8 μM, NAC 5 mM) for 24 h. Subsequently, the cells were collected and incubated with the fluorescent probe 2, 7-dichlorodihydrofluorescindiacetate (DCFH-DA, Cat#BC01010, Bioss, China). Due to the presence of background fluorescence, cells transfected with the PDK-1 overexpression plasmid were incubated with fluorescent probe dihydroethidium (DHE, Cat#C1300-2, Applygen, China). After 30 min of incubation at 37 ℃ in the dark, the cells were washed three times in serum-free medium and detected by Cytoflex flow cytometry (Beckman, USA). According to the excitation/emission wavelengths of DCFH-DA and DHE probes in the manufacturer’s instructions (488 nm/522 nm, 488 nm/610 nm), appropriate channels were selected to measure fluorescence values, and the data were analysed using Flowjo V10 software (FlowJo LLC, USA). During flow detection, the acquisition voltage, rate and gating settings were consistent for each group of cells.
Cell apoptosis and cell cycle assays
Cell apoptosis assay was performed using an Annexin V-APC/7-AAD apoptosis kit (Cat# 70-AP105-100, Multi Sciences Biotech, China). The cell cycle was analysed by a Cell Cycle Staining Kit (Cat# CCS012, Multi Sciences Biotech, China). The cells (1 × 106/well) were seeded in 6-well plates overnight and treated with different drug concentrations (Control, Aa-Z2 0.4 μM, Aa-Z2 0.6 μM, Aa-Z2 0.8 μM NAC 5 mM + Aa-Z2 0.8 μM, NAC 5 mM) for 24 h. At the indicated time, cells were collected and washed by cooling PBS three times. For apoptosis detection, these cells were resuspended in binding buffer containing 5 μL Annexin V-APC and 10 μL 7-AAD (7-amino-actinomycin D7) and plated in the dark for 10 min at room temperature. For cell cycle analysis, 1 mL DNA staining solution and 10 μL propidium iodide (PI) were added to the above cells and incubated for 30 min at room temperature. All samples were measured by Cytoflex flow cytometry at a slow flow rate (10 μL/min) and the data were analysed using CytExpert 2. 4 software (Beckman, USA). During flow detection, the acquisition voltage, rate and gating settings were consistent for each group of cells.
Mitochondrial membrane potential assays
Mitochondrial membrane potential (MMP) was measured using an MMP assay kit with JC-1 probe (Beyotime, Cat# C2006, China). After being cultured with Aa-Z2 (0, 0.4, 0.6, 0.8 μM) for 24 h, the 143B and HOS cells were collected and stained with JC-1 dye for 20 min at 37 °C. According to the product instructions, JC-1 can aggregate in the matrix of mitochondria to form polymers with red fluorescence (488 nm/590 nm) at high mitochondrial membrane potential, while existing as monomers with green fluorescence (488 nm/529 nm). The changes in MMP were detected through Cytoflex flow cytometry and mitochondrial depolarization was measured by the percentages of JC-1 monomers. During flow detection, the acquisition voltage, rate and gating settings were consistent for each group of cells.
ATP, lactic acid and pyruvate measurements
ATP levels were quantified by using an Enhanced ATP Assay Kit (Cat# S0027, Beyotime, China). After 24 h treatment with different Aa-Z2 concentrations (0, 0.4, 0.6, 0.8 μM), 143B and HOS cells were disrupted by 200 μL cell lysis buffer, then cell supernatant was collected after centrifugation at 12,000 ×g for 5 min. 20 μL supernatant and 100 μL ATP working solution were mixed for 10 s at room temperature, and the relative light unit (RLU) values were measured timely by a SpectraMax M2 microplate reader. In addition, cell culture media was obtained by centrifugation (400 ×g, 5 min) and the supernatant of culture media was subjected to lactic acid and pyruvate analysis using commercial kits from Jiancheng Bioengineering Institution according to the instruction manuals. (Cat# A081, Cat# A019-2-1, Nanjing, China).
Transmission electron microscopy observations
The changes in cell ultrastructure caused by Aa-Z2 were visualized using transmission electron microscopy (TEM). 143B and HOS cells were prepared as described previously . Briefly, the cells were fixed with 2.5% glutaraldehyde overnight and postfixed in 1% osmium tetroxide for 2 h. After being dehydrated in different concentrations of alcohol, the cell pellets were embedded in epoxy resin. Representative areas were chosen for ultrathin sectioning and examined by TEM (HT7700, Hitachi, Japan).
Cell transfection, RNA extraction and quantitative Real-Time PCR
The negative control and PDK-1 overexpression plasmids were synthesized by Shanghai Jikai Company (China). 143B and HOS cells were transfected with plasmids using GP-Transfect-Mate (GenePharma, Suzhou, China) following the manufacturer’s protocol. After 48 h, total RNA was extracted with Trizol reagent (Vazyme, Cat# R401-01, China) based on the product, and then RNA was measured using a spectrometer (NanoDrop Technologies, USA). After cDNA synthesis, RT-qPCR was carried out with a CFX Connect Detector instrument (Bio-Rad, USA) and the relative mRNA expression levels were calculated by the 2−ΔΔCt method . The following primers were used:
PDK-1-F: 5′- GAGGAAGCAGGAAGGATCAGT-3′
PDK-1-R: 5′- GAACGGATGGTGTCCTGAGA-3′
Radioimmune precipitation assay (RIPA) buffer containing PMSF and protease inhibitor cocktail at a ratio of 100:1:1 was used to lyse cells for 30 min. Then the supernatant was collected after centrifugation at 12,000 ×g for 10 min. The protein concentration was measured by using BCA Protein Assay Kit (Beyotime, Cat# P0012, China). In total, 20 μg of protein was separated by SDS–PAGE, transferred to polyvinylidene difluoride (PVDF) membrane (Merck, Cat# 05317, USA), and then blocked with 5% fat-free milk for 2 h at room temperature. All primary antibodies were diluted at a ratio of 1:1500 and incubated with the membrane at 4 ℃ overnight. The next day, these blots were incubated with HRP-conjugated secondary antibodies (1:4000) for 1 h at room temperature. Immunoreactive proteins were detected by an enhanced chemiluminescence kit (Cat# abs920, Abisin, China) according to the manufacturer’s instructions. Quantifications were performed using ImageJ software.
Six- to eight-week-old male BALB/c nude mice (Charles River, China) were raised in a standard laboratory environment with food and water. An injection of a total of 2 × 106 143B cells suspended in 100 μl PBS was administered into the right axilla of mice (this day was marked as Day 0). At Day 9, these mice were randomly assigned to three groups: (1) control group: mice were injected intraperitoneally (i.p.) with saline vehicle; (2) Aa-Z2 (5 mg/kg) group: mice were injected i.p. with 5 mg/kg Aa-Z2 (dissolved in DMSO, diluted in saline, broken with ultrasonication) every 6 days; and (3) Aa-Z2 (10 mg/kg) group: mice were injected i.p. with 10 mg/kg Aa-Z2 every 6 days. The concentration of DMSO in mice was less than 1%. Animal weight and tumour size (volume = 0.5 × L × W2) were documented every 3 days. The mice were killed after four cycles of Aa-Z2 treatment. The tumours and vital organs were detached and fixed using 10% formalin for additional examination. All experiments were compliant with the National Institutes of Health Animal Use Guidelines and permitted by the Laboratory Animal Center of Zhongnan Hospital of Wuhan University (NO. ZN2021006).
All data were presented as the mean of three independent experiments, which were routinely performed in triplicate, and analysed using unpaired t test or one-way ANOVA by using GraphPad Prism 8. 0 software. P < 0.05 was regarded as statistically significant.