Horseradish peroxidase-conjugated anti-mouse and anti-rabbit IgG, and rabbit polyclonal antibodies specific for β-actin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany; cat. no. SI-A5441; 1:10,000), voltage-dependent anion channel (GeneTex International Corporation, Hsinchu, Taiwan; cat. no. GTX-104745; 1:1,000), cytochrome c (GeneTex International Corporation; cat. no. GTX-108580; 1:1,000), B cell lymphoma (Bcl)-2 (GeneTex International Corporation; cat. no. GTX-100064; 1:1,000), Bcl-xL (GeneTex International Corporation; cat. no. GTX-105661; 1:1,000), Bcl-2-associated X (Bax; GeneTex International Corporation; cat. no. GTX-109683; 1:1,000), Bcl-2 homologous antagonist/killer (Bak; GeneTex International Corporation; cat. no. GTX-100063; 1:1,000), 78 kDa glucose-regulated protein (GRP78) (GeneTex International Corporation; cat. no. GTX113340; 1:1,000), GRP94 (GeneTex International Corporation; cat. no. GTX103203; 1:1,000), caspase-3 (Santa Cruz Biotechnology, Inc., Dallas, TX, USA; cat. no. SC-7272; 1:1,000), caspase-9 (Santa Cruz Biotechnology, Inc.; cat. no. SC-56073; 1:1,000) and poly (ADP-ribose) polymerase (PARP; GeneTex International Corporation; cat. no. GTX100573, 1:1,000) were used in the present study. Plumbagin from Plumbago indica and all other chemicals were obtained from Sigma-Aldrich; Merck KGaA.

The human osteosarcoma cell lines (MG-63, U2OS, HOS) and osteoblast cell line (hFOB 1.19) were purchased from American Type Culture Collection (Manassas, VA, USA). MG-63, U2OS, HOS and hFOB 1.19 cells were maintained in an α-modified Eagle's medium (MEM), McCoy's 5A, α-MEM and DMEM/Ham's F-12 medium, respectively (all Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). All cells were incubated at 37°C in an atmosphere of 5% CO₂ supplemented with 20 mM hydroxyethyl piperazineethanesulfonic acid (HEPES; Sigma-Aldrich; Merck KGaA), 10% heat-inactivated fetal calf serum (FBS), 2 mM of glutamine, 100 U/ml penicillin and 100 µg/ml of streptomycin (all Invitrogen; Thermo Fisher Scientific, Inc.). The media were changed every 48 h.

Cell viability was determined by MTT assay. Following treatment with indicated condition of plumbagin for 1 or 2 days, cultures were washed with PBS. All cells were incubated with MTT (0.5 mg/ml) for 2 h at 37°C. Following incubation, dimethylsulfoxide was added to dissolve formazan crystals and shaken at room temperature for 10 min. Finally, the cell viability was assessed by the absorbance of 570 nm using a microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

The cells were seeded in six-well plates (1×10³ cells/well) and incubated with indicated condition of plumbagin (1, 2.5, 5 and 10 µM) for 10 days. Culture media were changed every 2 days. At 10 days, colonies were washed with PBS, fixed for 15 min with 4% paraformaldehyde and stained with 0.1% crystal violet for 5 min. The colony forming cells were photographed. Following undergoing three washes with ddH₂O, acetic acid was added to a final concentration of 33% (v/v), which was achieved followed by measuring the absorbance at 550 nm. The colony formation assay was repeated three times with duplicate wells.

The cells were treated with indicated condition of plumbagin for 48 h, the cells were washed with PBS, fixed in a 3.7% formaldehyde solution for 10 min, and stained with DAPI (0.5 µg/ml) for 5 min. The cells were photographed using fluorescence microscopy.

The cells were treated with previously indicated conditions of plumbagin for 48 h. Total DNA were extracted and used to agarose electrophoresis (1.5% agarose gel). The agarose gel was stained with ethidium bromide to assess the DNA fragmentation. Ultraviolet spectroscopy at 302 nm was used to report results.

Total cell lysates were extracted as described previously (14). Proteins were resolved on a 10–15% SDS-PAGE and transferred to polyvinylidene membranes. The blots were blocked with 4% bovine serum albumin (cat. no. A9647; Sigma-Aldrich; Merck KGaA), for 1 h at room temperature and then probed with first antibodies (1:1,000) for 1 h at room temperature. Following three washes, the blots were subsequently incubated with peroxidase-conjugated secondary antibodies (1:1,000) for 1 h at room temperature. The blots were visualized by enhanced chemiluminescence using Kodak X-OMAT LS film (Kodak, Rochester, NY, USA). The bands were analyzed using ImageJ software (version 1.51k; https://imagej.nih.gov/ij/). Each experiment was repeated at least three times.

Apoptosis was assessed using Annexin V, a protein that binds to phosphatidylserine residues which are exposed on the cell surface of apoptotic cells, as previously described (15). Cells (2×10⁶ cells) were treated with indicated conditions of plumbagin. Following treatment, cells were washed twice with PBS, and resuspended in staining buffer containing 1 µg/ml PI and 0.025 µg/ml annexin V-fluorescein isothiocyanate (FITC). Double-labeling was performed at room temperature for 10 min in the dark prior to flow cytometric analysis. Cells were immediately analyzed using FACScan and the CellQuest pro software (version 5.1; BD Biosciences, Franklin Lakes, NJ, USA).

Quantitative assessment of apoptotic cells was also assessed by examining the cell cycle. Cells were collected by centrifugation (10 min at 950 × g and 4°C) and adjusted to 2×10⁶ cells/ml. Pre-chilled ethanol was added to 0.5 ml cell suspension and the mixture was incubated at 4°C for 30 min. Ethanol was then removed by centrifugation (15 min at 1,425 × g and 4°C), and cellular DNA was stained with 100 µg/ml PI (in PBS containing 0.1% Triton-X 100, and 1 mM EDTA) in the presence of an equal volume of DNase-free RNase A (200 µg/ml; Sigma-Aldrich; Merck KGaA). Following staining, cells were analyzed immediately with FACScan and CellQuest programs (version 5.1; BD Biosciences). The extent of apoptosis was determined by measuring the DNA content of cells below sub G1 peak (16).

Quantitative assessment of apoptotic cells was also assessed by TUNEL assay, which examines DNA-strand breaks during apoptosis by using the BD APO-DIRECT™ kit (BD Biosciences; cat. no. 556381). Briefly, cells (2×10⁶ cells) were incubated with plumbagin for the indicated times. The cells were trypsinized and collected by centrifugation (10 min at 950 × g and 4°C). The cells were fixed with 1% paraformaldehyde for 30 min on ice. Once the fixative was removed, cells were washed twice with PBS, ethanol was added to the 0.5 ml cell suspension and the mixture was incubated at −20°C for 2 h. Ethanol was then removed by centrifugation (15 min at 1,425 × g and 4°C) and cellular DNA was stained with TUNEL solution (3 ng/µl TdT enzyme and 0.04 nMol FITC dUTP) for 1 h at 37°C. Following incubation, 1 ml Rinse buffer was added and centrifuged again for 15 min at 1,425 × g and 4°C. The fluorescein labels incorporated in DNA strand breaks were detected and quantified immediately with FACScan and CellQuest programs (version 5.1; BD Biosciences) with a minimum of 10,000 counts/sample.

The mitochondrial membrane potential (ΔΨm) was assessed using the fluorometric probe Tetraethylbenzimidazolylcarbocyanine iodide (JC-1; EMD Millipore, Billerica, MA, USA), a positively charged mitochondria-specific fluorophore that indicates depolarization by a fluorescence emission shift from green (525 nm) to red (610 nm) (17). Briefly, cells (2×10⁶ cells) were plated in six-well culture dishes, grown to confluence, and treated with vehicle or plumbagin. Following incubation, cells were stained with JC-1 (5 µg/ml) for 15 min at 37°C and then analyzed by FACScan using an argon laser (488 nm). Mitochondrial depolarization, which is specifically indicated by a decrease in the red/green fluorescence intensity ratio, was analyzed by CellQuest program (version 5.1; BD Biosciences).

Levels of intracellular H₂O₂ were assessed spectrofluorimetrically by oxidation of specific probes: H₂DCFDA (Molecular Probes; Thermo Fisher Scientific, Inc.). Cells were plated at a density of 5×10⁵, allowed to attach overnight, and exposed to plumbagin for specified time intervals. The cells were stained with H₂DCFDA (10 µM) for 10 min at 37°C and the fluorescence intensity in cells was determined using the flow cytometry. The levels of ROS generation was stained with CellROX^® Deep Red reagent (Thermo Fisher Scientific, Inc.), and ROS generation was assessed by using the flow cytometry.

Cells (2×10⁵) seeded in 12-well plates were incubated with plumbagin for indicated times to detect changes in Ca²⁺ levels. Cells were harvested and washed twice, and re-suspension in Fluo 3/AM (3 µg/ml; Sigma-Aldrich; Merck KGaA) at 37°C for 30 min and analyzed by flow cytometry. For Ca²⁺ expression, the cells were stained with Calcium Assay kit (Cayman Chemical Company, Ann Arbor, MI, USA) and Ca²⁺ expression was assessed by using flow cytometry.

The assay is based on the ability of the active enzyme to cleave the chromophore from the enzyme substrate Ac-LEHD-pNA (for caspase-9; cat. no. APT173; EMD Millipore) and Ac-DEVD-pNA (for caspase-3; cat. no. APT131; EMD Millipore). The cell lysates were prepared and incubated with the specific anti-caspase-9 and caspase-3 substrate solution (final substrate concentration of 0.3 mg/ml) for 2 h at 37°C. The release of p-nitroaniline was monitored at 405 nm. Results are represented as the percentage change of the activity compared to the untreated control.

The upper chambers were coated with BD Matrigel Basement Membrane Matrix (50 µg/ml; BD Biosciences). Cells (1×10⁴) in serum-free medium were added to the upper chamber of a Transwell insert, and vehicle control or plumbagin (1, 2.5, 5 and 10 µM) in serum-free medium was applied to the lower chamber, followed by incubation. At 18 h, cells were fixed with 3.7% formaldehyde and stained with 0.05% crystal violet. Stained cells that migrated to the lower chamber were counted.

Data are presented as the mean ± standard error of the mean. Statistical comparisons between two groups were performed using the Student's t test. P<0.05 was considered to indicate a statistically significant difference.

The authors first examined whether plumbagin could trigger apoptosis in osteosarcoma cells. Osteosarcoma cells (MG63, U2OS, HOS and hFOB1.19) were incubated with plumbagin for 24 or 48 h, and the results indicated that plumbagin reduced cell viability in osteosarcoma cells but not normal bone cells (Fig. 1B). The IC₅₀ values at 48 h of plumbagin were 0.59, 0.78 and 1.24 µM for MG63, HOS and U2OS cells, respectively. The colony formation assay also consolidated the antitumor effect of plumbagin in osteosarcoma cells (Fig. 1C). Therefore, the authors investigated whether plumbagin induces apoptosis in osteosarcoma cells. The results indicated that plumbagin obviously increased DNA condensation (Fig. 1D). Degradation of chromatin was detected by a DNA ladder assay following plumbagin treatment (Fig. 2A). Cell apoptosis was also confirmed by cell cycle, Annexin V/PI double-labeling and TUNEL assay (Figs. 2B and C). To further verify the apoptosis induced by plumbagin, DNA fragmentation was conducted, as it is an important marker of apoptosis. It was observed that plumbagin induced cell apoptosis characterized by marked DNA fragmentation (Fig. 2D). These results support that plumbagin induces cell apoptosis in osteosarcoma cells.

ROS is known to induce mitochondrial dysfunction which promotes mitochondrial apoptotic pathway (18). Reactive oxygen species accumulation was detected after plumbagin treatment. The results demonstrated that H₂O₂ and ROS accumulation were increased following plumbagin treatment (Fig. 3A and B). Moreover, plumbagin-induced apoptosis was reversed by pretreating with ROS scavenger N-acetyl cysteine (NAC), NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) and H₂O₂ scavenging enzyme (catalase) (Fig. 3C and D). JC-1 is a cationic dye that accumulates in energized mitochondria. In healthy cells, JC-1 accumulated as aggregates in the mitochondrial membranes, presenting a red to orange fluorescence. In unhealthy cells, a monomer that yields green fluorescence was shown. Treatment of cells with plumbagin induced marked changes in mitochondrial membrane proteins, as demonstrated by the disappearance of orange-red fluorescence, or by the increase of green fluorescence (Fig. 3E). The increase of the proapoptotic/antiapoptotic Bcl-₂ ratio and release of cytochrome c confirmed that the mitochondrial apoptosis pathway was induced by plumbagin treatment (Fig. 3F-K). In summary, these results suggested that plumbagin induces cell apoptosis through the ROS-triggered mitochondrial apoptotic pathway in osteosarcoma cells.

A previous study indicated that ER stress-related events are correlated with ROS generation such as unfolded protein response (19). Since Ca²⁺ release is highly associated with ER stress-related mitochondria dysfunction (20), the authors therefore examined Ca²⁺ accumulation following plumbagin incubation. The results demonstrated that plumbagin treatment increased intracellular calcium concentration (Fig. 4A and B). The results also indicated that GRP78 and GRP94, two major components involved in ER stress, were upregulated by plumbagin incubation (Fig. 4C and D). The authors confirmed this suggestion by pretreatment cell with cell-permeant chelator of calcium (BAPTA-AM), and the data showed that plumbagin-induced apoptosis was inhibited by pretreatment with BAPTA-AM (Fig. 4E and F). Conclusively, these data indicated that plumbagin induces apoptosis through the ER-stress pathway.

Caspases are the critical components that regulate the apoptotic response. Finally, caspases activation was assessed following plumbagin incubation. Results indicated that caspase-3, caspase-9 and PARP (a downstream effector of caspase-3) were activated following plumbagin incubation (Fig. 5A and B). Plumbagin also increased caspase-3 and caspase-9 activity in osteosarcoma cells (Fig. 5C and D). Finally, pretreatment with caspase inhibitors abolished plumbagin induced cell apoptosis (Fig. 5E and F). These results suggested that caspase activation participates in plumbagin-induced cell apoptosis in osteosarcoma (Fig. 5G).