Antibodies and reagents
The following antibodies were used: GADPH (#2118), anti-CHOP (#2895), anti-GRP78 (#3177), anti-cleaved caspase 9 (#7237), anti-cleaved caspase 3 (#9661), anti-cleaved PARP (#5625), anti-Bax (#5023), anti-Bcl-2 (#3498), anti-Cytochrome c (#4272), anti-PERK (#5683), anti-p44/42 MAPK (Erk1/2) (#9102), anti-phospho-p44/42 MAPK (Erk1/2) (#9101), anti-cyclin D1 (#2978), anti-phospho-Rb (#8516), anti-IRE1α (#3294), and anti-p21 (#2947), anti-mouse IgG-HRP (#7076), anti-rabbit IgG-HRP (#7074) were purchased from Cell Signaling (Beverly, MA, USA), and anti-Phospho-EIF2 alpha (AF3087) and anti-Phospho-PERK (Thr982) (DF7576) were purchased from Affinity Biosciences Ltd., anti-ATF4 (0835-1-AP) obtained from Proteintech (Wuhan Sanying) and anti-p53 (ab26) aquired from Abcam (Cambridge, UK).
Other reagents used in the present study were: 1 g (> 98% purity, HPLC) prepared in our laboratory. Cur and DMSO were obtained from Sigma-Aldrich (St. Louis, MO, USA). Cell Counting Kit-8 (CCK-8), N-acetylcysteine (NAC; ROS scavenger), and GSK2606414 were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Antibody diluent and Restore™ PLUS Western Blot Stripping Buffer were purchased from ThermoFisher Scientific (Shanghai, China).
The human colon cancer cell lines HCT116 and HT29 were obtained from the American Type Culture Collection (ATCC), SW480 and LOVO were obtained from the Cell Resource Center, Shanghai Institute of Life Sciences, Chinese Academy of Sciences. HCT116 and HT29 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM; BI, New York, NY, USA), and SW480 in Roswell Park Memorial Institute (RPMI)-1640 medium (BI). LOVO cells were maintained in DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12). The medium was supplemented with 10% fetal bovine serum (FBS; Biological Industries, Israel) and 1% penicillin G/streptomycin sulfate. Cells were maintained at 37 °C in a humidified incubator within 5% CO2.
Transcriptome gene sequencing
Untreated HCT116 cells and those treated with 1 g for 24 h (n = 3) underwent transcriptomic analysis by Gooal Gene Technologies Ltd (Wuhan, China). Briefly, total RNA was processed by mRNA enrichment and rRNA removal. The RNA was fragmented with interrupt buffer, and random N6 primers were reversed transcribed, from which double-stranded cDNA was synthesized. The resultant double-stranded DNA ends were flattened and phosphorylated at the 5'-ends. A “sticky” end was formed at the 3' end from which an 'A' protruded to which a bubblelike junction with a 'T' was connected. The ligands were amplified by PCR using specific primers. The PCR product was thermally denatured into a single strand, which was cycled with a bridge primer to obtain a single-stranded circular DNA library which was finally machine sequenced. The process is detailed in a flowchart in Additional file 1.
Cell proliferation assay
Cell proliferation was detected using the CCK-8 kit. Briefly, 1 g-treated cells were plated at 5 × 103 cells per well in 96-well culture plates and cultured for 12 h, 24 h, and 48 h. A 10 µl aliquot of CCK-8 reagent was added to each well then incubated at 37 °C for 3 hours. The absorbance at 450 nm of each well was recorded using a microplate reader (Tecan Austria GmbH 5082, Austria) (n = 5).
Flow cytometry analysis
Cell apoptosis analysis. Cells were seeded in 6-well plates (5 × 105 cells/well). After incubation with specified drug treatments, the cells were suspended in Annexin V binding buffer then incubated with Annexin V–FITC and PI in accordance with the manufacturer’s instructions (BD Biosciences, Bedford, MA), before flow cytometry analysis(n = 3).
Cell cycle analysis. Cells were treated with 1 g or Cur for 18 h, collected, fixed and permeated overnight in 75% ethanol overnight at -20 °C. All samples were washed twice with PBS, and then incubated with PI and RNase at room temperature for 30 min. The results were analyzed using Modifit software (n = 3).
ROS measurement. ROS levels were determined with a dichloro-dihydrofluorescein diacetate (DCF-DA) probe. After treatment, the cells were collected and incubated with 20 µM DCF-DA for 30 min. After washing twice in PBS, the fluorescence intensity of DCF was measured by flow cytometry. The results were analyzed using Flowjo v10 software (n = 3).
Mitochondrial membrane potential (ΔΨm) evaluation. Following treatment with 1 g or Cur for 24 h, the cells were incubated with JC-1 fluorescent probe (Elabscience, Wuhan, China) in a 5% CO2 humidified incubator at 37 °C for 20 min. The cells were then washed twice with a staining buffer and analyzed using a TMFC500 flow cytometer (Beckman Coulter, CA, USA) (n = 3).
Western blot analysis
Cells and tissues were collected in RIPA buffer (50 mM Tris, 10 mM EDTA, 1% v/v Triton-X100), and supplemented with PMSF protease inhibitor and phosphatase inhibitor. The cells and tissues were then sonicated (8 bursts of 12 s, 4 °C, 100 W, using a Labsonic sonicator, Hielscher, Teltow, Germany), and the protein concentration of the samples determined using a BCA protein assay. The same quantity of protein (35 µg) was added to an appropriate volume of 5x sample buffer (Beyotime Biotechnology, Shanghai, China), and then heated until boiling. The samples were then separated on a 10-12.5% SDS-polyacrylamide gel (PAGE) after which they were transferred to PVDF membranes using a Bio-Rad electro-transfer system (Bio-Rad Laboratories, Munich, Germany). Each membrane was then hybridized with a primary antibody overnight on a shaking table at 4℃, washed three times with TBST buffer (3 × 10 min), then incubated with the corresponding secondary antibody at room temperature for 1 h. Finally, Immobilon Western chemiluminescent HRP matrix (Millipore, Burlington, MA, USA) was added to develop the membrane. The positive bands were imprinted using the Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA), and the density of digital images was measured using ImageJ software (NIH, Bethesda, MD, ver.1.52a) and expressed as a ratio of the GAPDH loading control.
Quantitative real-time PCR analysis
qRT-PCR was used to measure the expression of GRP78 and CHOP. Total RNA was extracted using Trizol reagent (Invitrogen; Life Technologies Corporation, Grand Island, NY, USA) in accordance with the manufacturer’s instructions. A 1 µg quantity of RNA was reverse transcribed using a PrimeScript RT reagent kit (Takara, Tokyo, Japan). The cDNA was diluted (2 µl) and selectively amplified by real-time PCR using SYBR Green I (Takara) with specific primers. The sequences of human CHOP, GRP78, and GAPDH were obtained from previously published articles. The samples were amplified using a Roche LightCycler 480II (Switzerland). The relative amount of each mRNA was calculated by a comparative method (2−△△Ct) against a GAPDH endogenous control.
Colon cancer xenografts in nude mice
All animal husbandry and experimental procedures were approved by the Animal Research Ethics Committee of the Affiliated Hospital of Qingdao University in Shandong Province. Four six-week-old female Balb/c-nu/nu mice were from the Beijing Viton Lihua Experimental Animal Technology Co. Ltd. At an ambient temperature of 20–25 °C, all mice were maintained in specific pathogen-free ventilation chambers 45–50% relative humidity, and a 12 h light-dark cycle. All mice adapt to the environment for 7 days before the experiment, and get sterilized food and water free of charge. Suspensions of HCT116 cells were injected subcutaneously into each mouse (at a cell density of 5 × 106 in 150 µl PBS). When the tumor volume had reached approximately 150mm3, the mice were randomly divided into control and treatment groups (n = 6). The groups were: control group with vehicle; 1 g group (40 mg/kg); 1 g group (20 mg/kg), and Cur group (40 mg/kg). Each treatment group received an intraperitoneal injection once per day. The mice were monitored for 14 days during treatment. Bodyweight and tumor volume were measured every 2 days. The tumor volume was calculated using the formula: ([width]2×[length]/2). Moreover, weight was recorded throughout the experiment.
Transplanted tumor tissue biopsies were embedded in paraffin, sectioned, then processed in xylene Ⅰ for 20 min, xylene Ⅱ for 20 min, ethanol Ⅰ for 5 min, anhydrous ethanol Ⅱ for 5 min, and 75% alcohol for 5 min prior to washing in water. Sections were stained with hematoxylin stain for 3–5 min, rinsed with tap water, differentiated, rinsed with tap water, immersed in basic bluing reagent prior to rinsing in running water. Sections were then dehydrated through an 85%-95% gradient of alcohol for 5 min respectively, then stained in eosin solution for 5 min. The sections were placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol Ⅲ for 5 min, dimethyl Ⅰ for 5 min, xylene Ⅱ for 5 min for clearing then sealed in neutral rubber. Finally, the sections were examined by light microscopy and images acquired and analyzed.
GraphPad InStat 8.0 software (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. The results were expressed as means of arbitrary values ± SD. Differences were analyzed using a one-way analysis of variance followed by a Bonferroni's post-hoc test using GraphPad InStat 8.0 software. P-values < 0.05 were considered significant.