Ciclopirox activates PERK-dependent endoplasmic reticulum stress to drive cell death in colorectal cancer

Cell lines and cell culture

Human CRC cell lines HCT-8, HCT-8/5-FU, and DLD-1 were purchased from the Cell Bank of Shanghai Institute of Cell Biology (Shanghai, China). HCT-8 and DLD-1 cells were maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, Life Technologies, Grand Island, NY, USA) and antibiotics (100 IU/mL penicillin and 100 μg/mL streptomycin) while HCT-8/5-FU cells were cultured in RPMI 1640 medium with 15 μM 5-FU. All cell lines were incubated at 37 °C with 5% CO2 in a humidified atmosphere unless otherwise noted. Cell lines were authenticated by short tandem repeats profiling before use and were also routinely tested for mycoplasma infection during this study.

Reagents and antibodies

Cell Counting Kit-8 (CCK-8), crystal violet, and NAC (N-acetyl-L-cysteine) were purchased from Beyotime Biotechnology (Shanghai, China). Cell-cycle analysis kit was purchased from KeyGEN BioTECH (Jiangsu, China). The annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit and PI/RNase staining buffer was purchased from BD Biosciences (San Jose, CA, USA). The bicinchoninic acid protein assay kit, Pierce ECL Western Blotting substrate, MitoSOXTM Red mitochondrial Superoxide Indicator, 2-NBDG and TRIzolTM Reagent, and Lipofectamine 3000 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Protease (Complete Mini) and phosphatase (PhosphoSTOPTM) inhibitor cocktail tablets were purchased from Roche Applied Science (Indianapolis, IN, USA). Matrigel and Transwell assays were purchased from Corning Incorporated (Corning, NY, USA). Seahorse XF96 V3 PS Cell Culture Microplates, Seahorse XFe96 FluxPak, Seahorse XF DMEM medium, Seahorse XF Calibrant Solution were purchased from Agilent Technologies Incorporated (Palo Alto, CA, USA). l-glutamine, glucose, sodium pyruvate, oligomycin, antimycin A, rotenone, carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP), 2-deoxyglucose, dimethyl sulfoxide (DMSO), and 5-FU were purchased from Sigma (St. Louis, MO, USA). CPX olamine was purchased from Dibai Biotechnology Co. (Shanghai, China). DNA extraction reagent was purchased from Solarbio Science & Technology Co., Ltd (Beijing, China). Enhanced chemiluminescence (ECL) kit was purchased from Perkin Elmer Life Science (Boston, MA, USA). The primary antibodies used in this study are provided in Table S1.

Cell viability and proliferation assay

For the cell viability assay, cells were seeded in triplicates into a 96-well plate at a density of 5 × 103 cells per well and cultured overnight. Cells were then treated with vehicle control (DMSO) or increasing concentrations of CPX (0, 5, 10, 20, 40, and 80 μM) for 48 h and cell viability was analyzed by CCK-8 assay according to manufacturer’s instructions. Briefly, CCK-8 reagent was added at a dilution of 1:10 to each well and incubated for 2–4 h. Optical density values were detected at a wavelength of 450 nm using a microplate reader (VarioskanTM LUX Multi-Plate Reader, Thermo Fisher Scientific, Waltham, MA, USA). For the cell proliferation assay, 2 × 103 cells were seeded into 96-well plates and cultured at overnight. The next day, cells were treated with different concentrations of CPX, as indicted above, or DMSO for 24, 48, and 72 h, and the relative cell number was measured by CCK-8 assay.

Colony formation assay

A total of 800 cells per well were plated into six-well plates and incubated at 37 °C with 5% CO2. The medium was changed every 2 days until several cell clusters are visible under the microscope, and then the medium was replaced with fresh RPMI 1640 medium containing different concentrations of CPX as indicated or vehicle control (DMSO). The cells were cultured until the macroscopic clone was visible and then stained with a 0.5% crystal violet solution as previous described27. Visible colonies were counted and representative views were photographed.

In vitro cell migration and invasion assays

For cell migration assay, HCT-8 (2 × 104), HCT-8/5-FU (4 × 104), and DLD-1 (6 × 104) cells were seeded in the upper chamber of 24-well Transwell plates in serum-free RPMI 160 medium, respectively. The lower chamber contained 650 μl of RPMI 160 medium with 15% FBS. After cellular attachment, the medium in the upper chamber was replenished with serum-free medium containing increasing concentrations of CPX or vehicle control (DMSO). After 48 h, the cells that migrated to the lower chamber were fixed for 30 min in methanol and then stained with 0.5% crystal violet. Five randomly selected fields from each Transwell were photographed and analyzed using ImageJ Plus software.

For cell invasion assay, Matrigel was diluted 1:10 with precooled F-12 medium after thawed and liquefied on ice. Then 40 μl of Matrigel mixture was added to the upper chamber and incubated overnight. Next day, warm F-12 medium (50 μl) was added the upper chamber and incubated for 30 min to rehydrate the Matrigel layer. Subsequent procedures were the same as cell migration assay.

Cell cycle and apoptosis assays by FACS

For cell-cycle analysis, HCT-8, HCT-8/5-FU, and DLD-1 cells were seeded in 6-cm dishes and cultured overnight, followed by treatment with vehicle control (DMSO) or a gradient concentration of CPX next day. After 24 h, cells were harvested and fixed in 70% ethanol and then stained with PI following RNase treatment. The stained cells were analyzed for cell-cycle distributions. For apoptosis assay, HCT-8, HCT-8/5-FU, and DLD-1 cells were incubated overnight, then treated with vehicle control (DMSO) or indicated concentration of CPX for 48 h. Cells were then collected and stained with Annexin V-FITC/PI for 20 min at room temperature.

Both cell-cycle distribution and cell apoptosis percentages were analyzed by flow cytometry on a BD AccuriTM C6 plus flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Mitochondrial and cellular reactive oxygen species (ROS) assays

To determine the effect of CPX on generation of ROS in CRC cells, cells (HCT-8, HCT-8/5-FU, and DLD-1 cells) were seeded in 6-cm cell culture dishes at a density of 4 × 105 in RPMI 1640 medium and cultured overnight. Cells were then replaced with medium containing vehicle control (DMSO) or a gradient concentration of CPX. After 48 h of incubation, cells were harvested and the intracellular ROS were determined using DCFH-DA fluorescent probe according to the manufacturer’s protocol. Stained cells were analyzed using a flow cytometer (BD AccuriTM C6 plus). Mitochondrial ROS were measured by staining cells with 5 μM MitoSOXTM Red Mitochondrial Superoxide Indicator and stained cells were analyzed using a flow cytometer (BD AccuriTM C6 plus).

Western blotting analysis

Total proteins (15 μg per well) from lysed cells were separated by SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Membranes were incubated with specific primary antibodies overnight at 4 °C and HRP-conjugated secondary antibody for 1 h at room temperature. β-actin was employed as a protein loading control. Protein expression was visualized with ECL and exposed to X-ray film (Carestream Health, Xiamen, China). Protein expression levels in cells were quantified by ImageJ software.

Mitochondrial respiration and glycolysis analysis

The Seahorse XF96 Extracellular Flux Analyzer (Agilent Technologies, Inc.) was used to measure the real-time oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of CRC cells according to the manufacturer’s instructions. Briefly, HCT-8, HCT-8/5-FU, and DLD-1 cells were seeded into 96-well cell plates, and incubated overnight. Meanwhile, the calibration plate was incubated at 37 °C, in a non-CO2 incubator overnight. Cells were pretreated with vehicle control (DMSO) or different concentration of CPX for 8 h, followed by replacing the medium with assay medium, and running the protocol until the calibration was completed, then OCR and ECAR were measured as previously described28.

Glucose uptake assay

HCT-8 (4 × 105), HCT-8/5-FU (5 × 105), and DLD-1 (5 × 105) cells were plated into 6-cm cell culture dishes and cultured overnight and then the medium, containing CPX or vehicle control (DMSO), was replenished. After 48 h, cells were harvested and glucose uptake was measured using 2-NBDG by flow cytometry.

Mitochondrial DNA copy numbers detection

The cells were split using cracking mixture (ratio of DNA lysate and proteinase K is 100:1) at 55 °C overnight. Total DNA was extracted following the manufacturer’s instructions. qRT-PCR analysis was performed to measure the mitochondrial DNA (mtDNA) copy numbers using SYBR Green kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol on the CFX ConnectTM real-time system (Bio-Rad, Hercules, CA, USA). 18S ribosomal DNA was used as an internal control for all samples. The primer sequences for mtDNA (Cyt b) and 18S ribosomal DNA are listed in Table S2.

In vivo subcutaneous xenograft models

Male nude mice were housed under specific pathogen-free conditions. HCT-8 (5 × 106), HCT-8/5-FU (5 × 106), and DLD-1 (5 × 106) cells were subcutaneously injected into the left flank of 5 weeks old Balb/c nude mice (n = 12 per cell line), respectively. When the tumors reached ~100 mm3, mice were randomly divided into two groups of six [physiological saline (0.9% NaCl) group and CPX-treated group]. The mice then received an intraperitoneal injection of CPX (20 mg/kg), dissolved in 0.9% NaCl, or 0.9% NaCl alone (saline control), once a day for 12 days. Tumor volume was evaluated at indicated time points using the formula: volume = 1/2 × L × W2. Gross weight of each mouse was assessed every 2 days. After 12 days, the mice were sacrificed and photographed, then tumors were dissected, weighed, fixed, and embedded. All animal experiments were carried out in accordance with the Institutional Animal Care and Use Committee, University Laboratory Animal Research of Wenzhou Medical University.

Immunohistochemistry

Immunohistochemistry (IHC) was performed as described previously27.

Statistical analysis

All experiments in this study were performed in triplicate and repeated at least three times, unless otherwise indicated. Student’s t test was used to compare the mean between two groups, and the graphs were created by GraphPad Prism 7.0 Plus software (GraphPad Software Inc., San Diego, CA, USA). Data were expressed as mean ± SD, and p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001; ns, no significant difference). Statistical analysis was carried out using SPSS software version 22.0 (SPSS Inc., Chicago, IL, USA).