This article is mentioned in:

Introduction

Osteosarcoma is one of the common primary malignant bone tumor diagnosed in children and teenagers (1,2). Current chemotherapy regimens of osteosarcoma are based on a combination of doxorubicin, methotrexate (MTX) and cisplatin (3–6). Only 50–60% of tumors are chemosensitive, demonstrating the dismal outcome that occurs far too often in osteosarcoma (7–9). One potential strategy to overcome known chemotherapy agents in osteosarcoma is to seek out alternative anticancer agents, particularly those appearing from natural products or traditional Chinese medicine (TCM) (10–13).

Curcumin is from the plant Curcuma longa (tumeric) and has been used in traditional Chinese medicine for thousands of years (14–18). Many pharmacological effects have been reported including anti-amyloid, anti-bacterial, anti-depressant, anti-inflammatory, anti-oxidant and anticancer properties (19–24). Curcumin has also been proven to affect multiple signaling pathways such as inhibiting cancer cell proliferation, inducing apoptosis or autophagy (25–27), blocking cell invasion and migration (28–31) and suppressing inflammatory responses (19,32–34). Phase II and III clinical trials with curcumin have advocated its use for patients with colon and pancreatic cancers (35–41). The low water solubility contributed to the poor bioavailability is the primary limiting factor for the efficacy and safety of curcumin (42–46). To improve the oral bioavailability of curcumin, we designed and developed Cur-NPs (PLGA nanoparticles loaded with curcumin) (Fig. 1A) (42). The morphology of the Cur-NPs was examined by transmission electron microscopy. The produced Cur-NPs are spherical in shape with smooth surface (Fig. 1B). Our previous study showed that the Cur-NPs caused anti-proliferation effects on cisplatin-resistant human oral cancer CAR cells, but little cytotoxicity to the normal human gingival fibroblasts cells (HGF) and normal human oral keratinocyte cells (OK) (42). The aims of this study were to characterize the properties of Cur-NPs and to investigate the molecular mechanisms triggered by Cur-NPs in human osteosarcoma U2OS cells.

Figure 1. (A) The schematic diagram of Cur-NPs preparation. The curcumin was encapsulated in the PLGA by single emulsion. (B) Morphology observation of Cur-NPs by TEM. The Cur-NPs show spherical shape.

Materials and methods

Chemicals and reagents

Cisplatin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), poly(D,L-lactide-co-glycolide) (PLGA, copolymer ratio 75:25, molecular weight = 66,000–92,000), polyvinyl alcohol (PVA, average molecular weight = 30,000–70,000) and curcumin were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). Fetal bovine serum (FBS), L-glutamine, penicillin G and trypsin-EDTA were obtained from Life Technologies (Carlsbad, CA, USA). Caspase-3/-7 and caspase-9 activity assay kits were purchased from R&D Systems Inc. (Minneapolis, MN, USA). The primary antibodies against caspase-3, caspase-9, Apaf-1, cytochrome c, AKT, p-AKT and BAD were obtained from Cell Signaling Technology Inc. (Beverly, MA, USA). All other antibodies used in this study and horseradish peroxidase (HRP)-conjugated secondary antibodies against rabbit or mouse immunoglobulin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The enhanced chemiluminescence (ECL) detection kit (Immobilon Western Chemiluminescent HRP Substrate) was obtained from Merck Millipore (Billerica, MA, USA).

Cell culture

Human osteosarcoma U2OS cell line was obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). Cells were maintained at 37°C under a humidified 5% CO2 atmosphere in 90% McCoy’s 5a medium (Invitrogen Life Technologist, Grand Island, NY, USA) containing 2 mM L-glutamine, 10% fetal bovine serum (Life Technologies) and 1% penicillin-streptomycin (100 U/ml penicillin and 100 μg/ml streptomycin) (Life Technologies) (47–50).

Curcumin loaded nanoparticles

Curcumin-loaded PLGA nanoparticles (Cur-NPs) were prepared by using single emulsion solvent evaporation method. Cucurmin (1 mg) and PLGA (10 mg) were dissolved in dichloromethane. The curcumin and PLGA solution (1 ml) was added to 2 ml of 10% (w/v) PVA surfactant solution to form an oil-in-water emulsion by sonication. The emulsion was carried out by setting sonication at 55 W of energy output for 3 min over an ice bath. The formed emulsion was dispersed by dropping into the 0.5% (w/v) PVA solution and stirred for an additional 4 h at room temperature on a magnetic stir plate to allow evaporation of organic solvent. Nanoparticles were collected by centrifugation at 12,000 rpm for 30 min and washed twice with double distilled water to remove PVA and un-encapsulated curcumin. The prepared nanoparticles were collected and lyophilized (42).

Size, polydispersity index (PDI) and encapsulation efficiency

The size and the polydispersity of prepared nanoparticles (PLGA-NPs and Cur-NPs) were measured using DLS (Zetasizer Nano ZS, 3000HS, Malvern Instruments Ltd., Worcestershire, UK). To determine the encapsulation efficiency in Cur-NPs before freeze-drying, the amount of non-encapsulated curcumin was measured the absorption value of OD450 nm by ELISA reader. The encapsulation efficiency was calculated by [(Total amount of curcumin- non-encapsulated curcumin)/Total amount of curcumin] × 100% (51).

Transmission electron microscopy (TEM) observation

The morphology of test nanoparticles was examined by TEM (Jeol, Tokyo, Japan). A dilute suspension of nanoparticles (1/10 dilution) was prepared in double distilled water. One drop of this solution was placed on the TEM grid for 10 min, washed twice with double distilled water and allowed to dry overnight. The images were observed and captured at an accelerating voltage of 120 kV under a microscope (42).

NMR and mass spectra

NMR spectra were obtained on a Bruker 500 MHz FT-NMR (model: Avance III DPX-500) spectrometer in CDCl3. The following abbreviations are used: s, singlet; d, doublet; m, multiplet. Mass spectra were measured with a Finnigan/Thermo Quest MAT 95XL instrument (52,53).

Cell viability and apoptotic morphological features

The cell viability was assessed by the MTT assay. The U2OS cells were cultured in a 96-well plate at the density of 1×104 cells per well and were incubated with 0, 0.25, 0.5, 1 and 2 μg/ml of Cur-NPs for 24 and 48 h. Then, culture medium containing 500 μg/ml MTT was added to each well and incubated at 37°C for 4 h. After incubation, the supernatant was removed. The formed blue formazan crystals in viable U2OS cells were dissolved with isopropanol/0.04 N HCl, followed by measurement of the absorbance of each well at 570 nm with the ELISA reader. All experiments were performed in triplicate. The morphological examination in Cur-NPs-treated U2OS cells was determined under a phase-contrast microscope (50).

Internalization of curcumin

To track the internalization of Cur-NPs, U2OS cells (1×106) were seeded on 6-well plates and incubated overnight. Subsequently, cells were treated with 1 μg/ml of Cur-NPs for 24 h. Finally, cells were washed with PBS twice and the internalized curcumin particles were observed under a fluorescence microscope with the filter of 488-nm excitation wavelength and 520-nm emission (27).

DNA fragmentation assay

The U2OS cells (1×107) were exposed to 1 μg/ml Cur-NPs for 48 h. Cells were harvested, washed by PBS and then lysed in 500 μl lysis buffer at 4°C. The lysed cells were digested overnight with proteinase K (100 μg/ml) at 50°C followed by incubation with 50 μg/ml RNase A at 37°C for 1 h. DNA fragments were extracted twice with phenol/chloroform/isopropanol (24:25:1; v/v/v) and precipitated with 50% isopropanol with glycogen (20 μg/ml) before being re-suspended in 100 μl Tris-EDTA (TE) buffer (Amresco Inc., Solon, OH, USA). Samples were electrophoresed on 1.8% (w/v) agarose gel (Sigma-Aldrich Corp.) in 0.5X TBE buffer (Amresco Inc.) and DNA was stained with 1 μg/ml ethidium bromide (Life Technologies). The gel was observed and photographed under a UV lamp (54,55).

Western blot analysis

The U2OS cells (1×107) were treated with 0, 0.5, 1 and 2 μg/ml of Cur-NPs for 12 or 48 h. Cells were then harvested, lysed and the total proteins were collected by SDS sample buffer. Approximately 50 μg of proteins from each treatment were resolved on 10% SDS-polyacrylamide gel electrophoresis (PAGE) and electro-transferred to the Immobilon-P Transfer Membrane (Merck Millipore). The transferred membranes were blocked with 5% non-fat dry milk in 20 mM Tris-buffered saline/0.05% Tween-20 for 1 h at room temperature followed by incubation with appropriate primary antibodies at 4°C overnight. At the end of incubation, membranes were washed with Tris-buffered saline/Tween-20 and incubated with secondary antibodies conjugated with HRP. The blots were developed by the chemiluminescence kit and then exposed to X-ray film. Each membrane was stripped and reprobed with anti-β-actin antibody (Sigma-Aldrich Corp.) to ensure equal protein loading during the experiments (47,56,57).

Assays for caspase-3/-7 and caspase-9 activities

The U2OS cells (1×107) were exposed to 1 μg/ml of Cur-NPs for 0, 12, 24, 36 and 48 h. Subsequently, cells were harvested, and cell lysates were assessed in accordance with the manufacturer’s instructions provided in the caspase-3/-7 and caspase-9 colorimetric assay kits (R&D System Inc.). Cell lysate containing 50 μg proteins were then incubated for 1 h at 37°C with specific caspase-3/-7 substrate (DEVD-pNA) or caspase-9 substrate (LEHD-pNA) and determined by measuring OD405 of the released pNA (58–60).

Statistical analysis

All the statistical results are performed as the mean ± standard error of the mean (SEM) for the indicated numbers of independent experiments. Statistical analyses of data were done using one-way ANOVA followed by Student’s t-test and the levels of P<0.001 was considered significant between the treated and untreated group (61).

Results

Characterization of Cur-NPs

In order to improve the application of curcumin, curcumin was encapsulated by PLGA to form curcumin-loaded PLGA nanoparticles (Cur-NPs) by single emulsion method (Fig. 1A). The nanoparticle form of curcumin exhibited good water-solubility and formed a transparent solution while dissolving in water. TEM was used to examine the morphology of the Cur-NPs. As shown, the Cur-NPs showed spherical shape (Fig. 1B). The size distribution of Cur-NPs in aqueous solution was measured by DLS. As shown in Fig. 2A, the size of Cur-NPs is ∼250 nm, similar to the curcumin-empty PLGA-NPs. Both nanoparticles showed small polydispersity index (PDI) (Fig. 2A), indicating their homogeneous size distribution (Fig. 2B). The encapsulation efficiency of curcumin in Cur-NPs prepared by single emulsion was 90.5±3.0% (Fig. 2A).

Figure 2. (A) The size and polydispersity index of PLGA-NPs and Cur-NPs measured by DLS. The encapsulation efficiency of both nanoparticles was determined by measuring the value of OD450 nm. (B) The size distribution of PLGA-NPs and Cur-NPs was measured by DLS.

In order to confirm that curcumin was unaffected after nano-technologization, mass (MS) spectroscopy of curcumin and Cur-NPs were performed and we found the curcumin fragments on MS spectrum of Cur-NPs (Fig. 3). Furthermore, the proton nuclear magnetic resonance (1H-NMR) spectroscopy was used to obtain the 1H-NMR spectra of PLGA-NPs [poly(lactic-co-glycolic acid) nanoparticles], curcumin standard and Cur-NPs (Fig. 4). Comparing Fig. 4A and B, all the peaks of curcumin standard (Fig. 4A) could be found on the Cur-NPs spectrum (Fig. 4B) with identical chemical shifts and integration. Besides the peaks of curcumin, there were some other peaks, δ1.58–1.62 (354H, m), 4.66–4.93 (58H, m) and 5.17–5.27 (89H, m), on the Cur-NPs spectrum. The chemical shifts and integration ratio (∼4:0.6:1) of these additional peaks are identical with that of PLGA NPs, δ1.58–1.62 (4H, m), 4.63–4.93 (0.6H, m) and 5.17–5.27 (1H, m). From the careful inspections of spectral data described above, curcumin was constant and unaffected by nano-technologization.

Figure 3. Mass spectra of curcumin (A) and Cur-NPs (B). The mass spectra of curcumin and Cur-NPs were determined as described in Materials and methods.

Figure 4. 1H-NMR spectra of curcumin (A) and Cur-NPs (B). The 1H-NMR spectra of curcumin and Cur-NPs were determined as described in Materials and methods.

Cur-NPs reduce the viability of human osteosarcoma U2OS cells

The U2OS cells were treated with Cur-NPs (0, 0.25, 0.5, 1 and 2 μg/ml) for 24 and 48 h. The cells were collected and the cell viability was determined using MTT assay. Our results showed that the concentrations of 0.25, 0.5, 1 and 2 μg/ml Cur-NPs significantly decreased cell viability in U2OS cells concentration- and time-dependently (Fig. 5A). Cellular uptake of Cur-NPs was visualized by green fluorescence of curcumin using fluorescence microscopy (Fig. 5B). Intensified fluorescence was detected in the cytoplasm and nucleus of cells treated with Cur-NPs, suggesting the amount of curcumin internalized into the cells. Our results demonstrated that Cur-NPs display the anti-human osteosarcoma U2OS cells in vitro.

Figure 5. Effects of Cur-NPs on cell viability and cellular uptake in human osteosarcoma U2OS cells. (A) Effects of Cur-NPs on cell viability in U2OS cells for 24 and 48 h. Cells were incubated in the absence or presence of 0, 0.25, 0.5, 1 and 2 μg/ml of Cur-NPs for 24 and 48 h. Cell viability on Cur-NPs-treated cells were determined using the MTT assay as described in Materials and methods. Data are presented as the mean ± SEM in triplicate by comparing between the treated and untreated control cells. ***P<0.001 compared with the control value. (B) To track the internalization of Cur-NPs, U2OS cell were incubated without (control) or with Cur-NPs containing 2 μg/ml for 24 h. The internalized curcumin was observed under a fluorescence microscope with the filter of 488-nm excitation wavelength and 520-nm emission.

Cur-NPs induce apoptosis in human osteosarcoma U2OS cells

After treatment with 0.5, 1 and 2 μg/ml of Cur-NPs for 48 h, Fig. 6A revealed apoptotic bodies in Cur-NPs-treated U2OS cells and this effect is concentration-dependent. Further results are demonstrated in Fig. 6B, which indicated that Cur-NPs induced DNA fragmentation in Cur-NPs-treated U2OS cells.

Figure 6. Effects of Cur-NPs on cell morphology and DNA fragmentation in human osteosarcoma U2OS cells. (A) For morphological examination, U2OS cells were treated with 0, 0.5, 1 and 2 μg/ml of Cur-NPs for 48 h. The morphological examination was determined under a phase-contrast microscope. (B) For DNA fragmentation assay, U2OS cells were treated with 0 and 1 μg/ml of Cur-NPs for 48 h. Cells were harvested and then were measured for apoptosis by DNA gel electrophoresis as described in Materials and methods.

Cur-NPs trigger mitochondria-dependent apoptotic cell death in U2OS cells

To examine whether Cur-NPs induces apoptosis in U2OS cells, cells were treated with 1 μg/ml of Cur-NPs for 0, 12, 24, 36 and 48 h before subjected to caspase-3/-7 and caspase-9 activities. Cur-NPs stimulated caspase-9 (Fig. 7A) and caspase-3/-7 (Fig. 7B) activities in Cur-NPs-treated U2OS cells and this effect is time-dependent. Based on these findings, we provide evidence regarding the intrinsic caspase contributing to Cur-NPs-induced apoptosis in U2OS cells.

Figure 7. Effects of caspase-9 (A) and caspase-3/-7 (B) activities on Cur-NPs-treated human osteosarcoma U2OS cells. Cells were treated with 1 μg/ml of Cur-NPs and then incubated for 24 h. The whole-cell lysates were subjected to caspase activity assay as described in Materials and methods. Each result is shown as the mean ± SEM in triplicate by comparing between the treated and untreated control cells. ***P<0.001 compared with the control value.

Mitochondria-dependent and Akt-Bad signaling pathways were involved in Cur-NPs-induced apoptosis in U2OS cell apoptosis

We examined the effects of Cur-NPs on mitochondria-dependent and Akt-Bad signaling pathways in U2OS cells. The immunoblotting results showed that the protein level of p-Akt was decreased in Cur-NPs-treated U2OS cells (Fig. 8A). In contrast, the protein levels of cleaved caspase-3, cleaved caspase-9, cytochrome c, Apaf-1 and Bad were increased in Cur-NPs-treated U2OS cells (Fig. 8B). In conclusion, our data expand the current understanding of Cur-NPs treatment in U2OS cells on causing cell death through the mitochondrial-dependent caspase cascade and the Akt-Bad signaling in vitro.

Figure 8. Effects of Cur-NPs caused protein level change on mitochondria-dependent apoptosis and AKT-BAD signaling in human osteosarcoma U2OS cells. The U2OS cells were treated with 0, 0.5, 1 and 2 μg/ml of Cur-NPs for 0, 12 and 48 h then subjected to western blotting. The western blotting of (A) p-AKT, AKT and (B) BAD, cytochrome c, Apaf-1, caspase-3, caspase-9 protein levels in U2OS cells. The β-actin was detected for equivalent protein loading.

Discussion

The study published by Yin et al demonstrated that Cur-NPs are effective in inhibiting the growth of human lung cancer and exhibited little toxicity to normal tissues in an established A549 xenograft mouse model (42,62). Our previous study also showed that the Cur-NPs used in our study caused anti-proliferation effects on CAR cells in a dose- and time-dependent manner but little cytotoxicity to the normal human gingival fibroblasts cells (HGF) and normal human oral keratinocyte cells (OK) (29). This is the first study to investigate the anti-human osteosarcoma effects of Cur-NPs on human osteosarcoma U2OS cells. Our results showed that Cur-NPs inhibited U2OS cell proliferation (Fig. 5) and induced apoptotic cell death (Fig. 6) in a concentration- and time-dependent manner. The results in Fig. 7 show Cur-NPs enhanced cell apoptosis through the activation of caspase-9 and caspase-3/-7 in U2OS cells. Our results suggested that Cur-NPs-induced apoptosis might be through the mitochondria-dependent signaling pathway, which has a connection with the activation of caspase-9 and -3.

Previous research has shown that mitochondrial-mediated apoptosis is regulated by the Bcl-2 family proteins (63-65). The Bcl-2 family includes pro-apoptotic proteins (Bax and Bad) and anti-apoptotic proteins (Bcl-2 and Bcl-xL) (66-70). The ratio of pro-apoptotic and anti-apoptotic proteins is thought to determine, at least in part, the susceptibility of cells to a death signal (68,69,71). Previous studies have shown that the apoptotic stimuli can de-phosphorylate Bad and release Bad from the 14-3-3 protein (72-75). Thus, Bad will compete with Bcl-2/Bcl-xL in binding to Bax (76–79). Previously, it was shown that functional Akt phosphorylated Bad at Ser136 to promote the stabilization of the mitochondrial membrane system and cell survival (78,80). Our results demonstrated that the protein level of p-AKT was decreased (Fig. 8A), while the protein levels of cleaved caspase-3, cleaved caspase-9, cytochrome c, Apaf-1 and BAD were increased in Cur-NPs-treated U2OS cells (Fig. 8B). The results suggest that Cur-NPs enhance apoptotic cell death of human osteosarcoma U2OS cells through the Akt-Bad signaling pathway.

In conclusion, Cur-NPs induce cell apoptosis in human osteosarcoma U2OS cells. The findings suggest that the major pharmacologic action of Cur-NPs is to trigger apoptotic cell death through activation of caspase-9 and caspase-3/-7 connected to mitochondria-dependent and Akt-Bad signaling pathway in U2OS cells (Fig. 9). Cur-NPs could be one of the potential compounds to be developed as a novel medicine against human osteosarcoma.

Figure 9. Schematic diagram of Cur-NPs regulating the apoptotic signaling pathway in human osteosarcoma U2OS cells.

Acknowledgements

This study was supported by research grant from the China Medical University (CMU101-N2-07) to Dr Shu-Fen Peng.

References