Materials

CYP11A1 cDNA, cloned in a pCMV-SPORT5 vector (Clone ID: hMU004796), was obtained from Korea Gene Bank. Plasmid Midi Kit was purchased from Qiagen (CA, USA). Dulbecco's modified Eagle’s medium–high glucose (DMEM) was purchased from GenDEPOT (TX, USA). Fetal bovine serum (FBS) and 1% penicillin/streptomycin solution were purchased from GIBCO (MA, USA). Lipofectamine 3000 reagent was procured from Invitrogen (MA, USA). RIPA buffer, protease, and phosphatase inhibitor cocktail were obtained from Cell Signaling (MA, USA) and Pierce™ BCA Protein Assay Kit and Pierce™ Protein A/G Agarose was purchased from Thermo Scientific (MA, USA).

Cholesterol, pregenenolone, mitomycin C, and aminoglutethimide standards were purchased from Selleck Chemicals LLC (TX, USA). The natural products set consisting of 1374 compounds in 5 μL volumes in 96-well polypropylene microtiter plates at concentrations ranging from 3.7 to 14.7 mM was kindly provided by the Korea Chemical Bank.

Primary antibodies directed against CYP11A1 (#14,217), Beclin1 (#3495), and LC3A/B (#12,741), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (#7074), and Ferroptosis Antibody Sampler Kit (#29,650) were purchased from Cell Signaling (MA, USA). Antibody against GAPDH was purchased from GeneTex (CA, USA).

Cell Culture and Transfection

Plasmid cDNA was isolated using the plasmid midi kit according to the manufacturer’s instructions. The concentration of CYP11A1 cDNA was measured using a NanoDrop spectrophotometer. Caki-1 cells were cultured in DMEM-high glucose containing 10% FBS and 1% penicillin/streptomycin solution. Culture plates were incubated at 37 °C with 5% CO2 in a humidified cell incubator. The cells were transiently transfected with CYP11A1 using Lipofectamine 3000 following the manufacturer’s protocol. All experiments were performed using cells in logarithmic growth phase.

For protein quantification, cells were collected in cold phosphate-buffered saline (PBS), washed twice with PBS, and then lysed in RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail. The cell lysates were incubated at 4 °C for 20 min and vortexed every 7 min. After centrifugation of the sample for 20 min at 14,000 × g at 4 °C, the supernatant was collected. Protein concentration in the lysate was determined using the Pierce™ BCA Protein Assay Kit according to the manufacturer’s protocol.

Wound-Healing Assay: 1st Screening

All 1374 compounds were screened at a final concentration of 10 μM with CYP11A1-overexpressing Caki-1 cells. Caki-1 cells (4 × 103) were cultured in 24-well plates at 37 °C with 5% CO2) until they reached 90% confluence. These cells were transfected with CYP11a1 plasmid DNA using Lipofectamine 3000 reagent for 24 h. The natural compounds were diluted to a final concentration at 10 μM and added to each well of the 24-well plate. Scratch wound-healing assay was performed by scratching across the confluent cell monolayer with a sterilized 200 μL pipette tip. Cell debris was removed by extensive washing with 1 × PBS and cells were allowed to migrate into the wound area for 24 h at 37 °C. Digital photographs were taken at 0 and 24 h after scratching. The ImageJ 1.53a software (National Institutes of Health, Bethesda, MD, USA) was used to measure the width of the wounds at three locations within each well. The percentage wound closure was quantified by dividing the width of healed wounds at 24 h with the initial width. Compounds that inhibited the migration of cancer cells by more than 75% were selected. The compounds that did not exhibit the wound-healing activity or killed 100% of cells were excluded from further analysis.

Cell Viability Assay for IC50 Determination: 2nd Screening

The viability of Caki-1 cells treated with 159 compounds was evaluated using an EZ-Cytox assay (DoGenBio, Seoul, South Korea). The cells were seeded into 96-well plates at a density of 1 × 104 cells/well and cultured for 24 h. They were subsequently treated with various concentrations of each compound (0, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100 μM) for 24 h. After treatment, EZ-Cytox solution (10 μL) was added to each well, and the cells were incubated for 1 h, protected from light. The absorbance was measured at 450 nm using a Bio-Rad microplate reader (CA, USA). Cell viability was calculated using the following formula: % cell viability = (OD of treatment − OD of blank)/(OD of control − OD of blank) × 100%. The assay was repeated three times. An IC50 lower than 50 μM was the selection criteria.

Assay of Enzymatic Activity: 3rd Screening

To test the effect of 38 selected compounds on CYP11A1 activity, we developed a quantitative analysis method for the steroid hormones, cholesterol and pregnenolone, using LC–MS/MS.

LC–MS/MS procedure

To determine cholesterol and pregnenolone concentrations, culture media (CM) samples were collected and processed using the liquid–liquid extraction method. Ethyl acetate and CM samples were mixed at a ratio of 5:1 (v/v) by vortexing for 1 min and then shaken in a rotator for 30 min. The mixture was then centrifuged at 1000 × g for 10 min at 4 °C, which allowed the solvent layer to be separated. Samples were frozen at − 80 °C and supernatant was transferred into a new clean tube. The liquid extraction was repeated two times for maximum recovery. The pooled supernatants were evaporated under a nitrogen stream. Samples were stored at − 20 °C until analysis.

Steroid analysis was conducted using a UHPLC-MS/MS system combined with an LTQ Orbitrap Velos Pro. A CORTECS C18 column (90 Å, 2.7 µm, 2.1 mm × 50 mm) (Waters Corporation, MA, USA) was used for separation. The column oven was maintained at 40 °C, and the injection volume was 10 μL. Isopropanol or 0.1% formic acid in water was used as a mobile phase at a flow rate of 0.4 mL/min. The gradient elution and MS operating conditions are presented in Table 1. The mass spectrometer was operated in positive electrospray ionization mode with total ion monitoring or multi-reaction monitoring. The spray voltage of the Orbitrap mass spectrometer was + 3.9 kV and the collision energy was 35 eV.

Table 1 High performance liquid chromatography gradient and mass spectrometry operating conditionMethod Validation

Calibration curves were constructed using 0.05–25 µg/mL cholesterol and pregnenolone standards, with finasteride as an internal standard (10 ng/mL). Six different concentrations of the standards were added to the cell culture medium and extracted using the method described above. The recovery was determined by calculating the mean percentage of the extracted sample area divided by the standard area. To calculate the limit of detection (LOD) and limit of quantification (LOQ) of steroid hormones, five replicates of low concentrations of target analytes were measured, and the concentrations that yielded a signal-to-noise ratio (S/N) of 3 for LOD and 10 for LOQ were selected. The accuracy of the method was determined as the % ratio of the measured quality control sample concentrations calculated using the calibration curve to the theoretical concentrations (1 and 10 µg/mL). Precision was calculated as the coefficient of variation (CV%) for triplicate measurements. The accuracy and precision of the method were assessed using intra- and inter-day variations from three repeated analyses.

Western Blot Analysis and Immunoprecipitation

Protein samples were separated using one-dimensional 12% Tris–glycine SDS-PAGE, and the proteins were transferred onto nitrocellulose membranes (Bio-Rad, CA, USA). The membrane was blocked by incubating in 5% skim milk in 1X TBS with 0.5% Tween 20 for 1 h and then washed with 1X TBST. The membranes were incubated overnight with primary antibodies diluted 1:1000 (v:v) in 5% BSA at 4 °C in the dark. After washing three times with TBST for 5 min each time, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (MA, USA) in 5% skim milk (1:5000 v:v diluted) at room temperature for 1 h. The blots were detected using a chemiluminescence SignalFire™ ECL Reagent from Cell Signaling Technology (MA, USA) with an Ez-Capture MG system ATTO (NY, USA). The relative intensities of the western blot bands were measured using the ImageJ software (MD, USA). The GAPDH antibody was used as a loading control.

Immunoprecipitation was performed using cell lysates with 500 µg of total protein and antibodies against CYP11A1. Cell lysate was incubated with 50 µL of Protein A/G agarose bead slurry for precleaning. The protein concentration was 1 mg/mL. The samples were mixed by rotation at 4 °C for 60 min, microcentrifuged for 10 min at 4 °C, and the supernatant was transferred to a fresh tube. The primary antibody (diluted 1:50, v:v) was added to 500 µg of precleaned cell lysates and the mixtures were incubated overnight with rotation at 4 °C. Thereafter, 50 µL of Protein A/G agarose bead slurry was added and the mixture was incubated with rotation at 4 °C for 1–3 h. The mixtures were microcentrifuged for 30 s at 4 °C. The pellet was washed five times with 500 µL of 1X cell lysis buffer (kept on ice between washes) and used for immunoblotting after SDS-PAGE.

Lipid Reactive Oxygen Species Assay

Cells, cultured to an appropriate density (5 × 107 cells), were collected and washed twice with ice-cold PBS. An EZ-Lipid Peroxidation (TBARS) Assay Kit (DoGenBio, Seoul, South Korea) was used to detect the malondialdehyde (MDA) levels, which reflect the level of lipid oxidation. The absorbance in this colorimetric assay was measured at 540 nm using a Bio-Rad microplate reader (CA, USA). The relative MDA levels were calculated according to the manufacturer’s protocol.

Statistical Analysis

All data are presented as mean ± standard deviation (SD) of triplicate results. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.0001, and ****p ≤ 0.0001 were considered to indicate significant differences. One-way ANOVA followed by Dunnett’s multiple comparison test was performed using GraphPad Prism version 8.0.0 for Windows (GraphPad Software, CA, USA).