Curcumin (CAS Number 458-37-7, Sigma-Aldrich, Purity: ≧99.5%), demethoxycurcumin (CAS Number: 22608-11-3, Sigma-Aldrich, Purity: ≧98%), bisdemethoxycurcumin (CAS Number: 33171-05-0, Sigma-Aldrich, Purity: ≧98%), Thiazolyl Blue Tetrazolium Bromide (MTT) were purchased from Sigma–Aldrich (CAS Number: 298-93-1). β-actin (Cat No. GTX109639), p-p38 (Cat No. GTX24822), p-Akt (Cat No. GTX128414), p-Erk (Cat No. GTX24819), p-smad2 (Cat No. GTX133475), p-smad3 (Cat No. GTX129841), cleaved PARP (Cat No. GTX132329), cleaved caspase 3 (Cat No. GTX86952) antibodies were purchased from GeneTex. Annexin V Alexa Fluor 488 Ready Flow Conjugate (Lot number 2081235) and 7-aminoactinomycin D (7-AAD, Catalog number: A1310) both were purchased from Thermo Fisher Scientific.
Cell line and cell culture
The U2OS, HOS, A2058, MDA-MB-231 cell lines were obtained from Bioresource Collection and Research Center (BCRC, Hsinchu, Taiwan). Cells in culture were not more than 10 passages from the time of receipt. Cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with antibiotics (100 U/mL of penicillin A and 100 U/mL of streptomycin) and 10% fetal bovine serum (FBS), and maintained at 37 °C in 5% CO2 humidified air.
Cell viability assay
The effect of CUR, DMC and BDMC on cell viability in U2OS, HOS, A2058, and MDA-MB-231 cells were examined by MTT assay. Briefly, cells were seeded into 12-well plates with 10% FBS overnight. After attachment, the cells were put into 2% FBS media and then treated with varying concentrations of CUR, DMC and BDMC alone or in combination. After incubation for 24–48 h, 300 μL of MTT working solution (0.5 mg/mL) was added to each well and incubated for 3 h at 37 °C. The supernatant was aspirated, and the MTT-formazan crystals formed by metabolically viable cells were dissolved in DMSO. Finally, the absorbance was monitored by a 96-well microplate reader at a wavelength of 540 nm.
Flow cytometric analysis of apoptosis
HOS cells were cultured at a density of 1 × 106 cells in 10-cm dishes with 10% FBS overnight. After attachment, the cells were put into 2% FBS media and then treated with varying concentrations of CUR, DMC and BDMC alone or in combination. After treatment with varying concentrations of CUR, DMC and BDMC for 24 h, we collected the cell pellet by trypsinization and centrifugation at 2000 g for 5 min, and then discard the supernatant. Finally, 500 μL of 1 × annexin V-FITC Binding Buffer was added to resuspend the pellet, and then 5 μl of annexin V-FITC and 5 μL of 7-AAD were added to the cells. The samples were gently vortexed and incubated 15 min at 4 °C in the dark. The Flow cytometric analysis was performed by FAC-Scan cytometry (BD Biosciences, San Jose, CA).
Nuclear morphology assay and immunofluorescence assay
Nuclear morphology was detected by Hoechst staining. Immunofluorescence study was detected by apoptosis-inducing factor (AIF) primary antibodies. The HOS, U2OS, and A2058 cells were cultured with 10% FBS on coverslips placed in six-well plates for overnight. After attachment, the cells were changed into 2% FBS media and then treated with 10–20 μM of CUR, DMC and BDMC. After treatment with CUR, DMC and BDMC for 24 h, HOS, U2OS, and A2058 cells were washed with phosphate buffered saline (PBS), and then fixed with 4% formaldehyde in PBS for 30 min at room temperature. Finally, HOS cells were added apoptosis-inducing factor (AIF) primary antibodies at 4 °C overnight, followed with the fluorescein isothiocyanate-conjugated secondary antibodies for 1 h, and then were added 4 mg/mL Hoechst 33258 for 30 min, but U2OS, and A2058 cells were only added 4 mg/mL Hoechst 33258 for 30 min. The coverslips were mounted in Vectashield mounting medium and the nuclear morphology and AIF nuclear translocation were viewed under confocal laser scanning microscopy.
Western blot analysis
HOS cells with or without 10–20 μM CUR, DMC and BDMC treatment for 24 h were washed with PBS and lysed in the Golden lysis buffer containing protease inhibitors for 30 min at 4 °C. Protein content was determined against a standardized control, using the Bio-Rad protein assay kit (Bio-Rad Laboratories). The protein inputs in the western blot analyses were normalized by loading equal amounts of total protein lysates into the SDS-PAGE gel. Transferred onto polyvinylidene difluoride membranes, and then probed with primary antibody (β-actin, p-p38, p-Akt, p-Erk, p-Smad2, p-Smad3, cleaved PARP, or cleaved caspase 3) followed by secondary antibody conjugated with horseradish peroxidase. Reactive bands were visualized with enhanced chemiluminescence (ECL) reagents.
Colony formation assay (Clonogenic assay)
Cell proliferation was monitored using a colony formation assay. Briefly, HOS cells were seeded into 24-well plates with 10% FBS overnight. After attachment, cells were maintained in DMEM containing 10% FBS with or without various concentrations of CUR, DMC and BDMC for an additional 14 days. At last, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with crystal violet dye for 5 min.
All values were expressed as mean ± SD. Each value is the mean of at least three separate experiments in each group. Student’s t-test was used for statistical comparison. * indicates that the values are significantly different from the control (*, p < 0.05; **, p < 0.01; ***, p < 0.001).