MG63 human osteosarcoma cancer cells were procured from the Shanghai Institute of Cell Biology (Shanghai, China). The cancer cells were grown in Eagle’s minimal essential medium (Gibco-BRL, Carlsbad, CA, USA) supplemented with 5% (v/v) fetal bovine serum (FBS; Sigma, St. Louis, MO, USA), 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco-BRL, Carlsbad, CA, USA). The cells were allowed to attach for 2 h prior to the addition of sclareol. The cells were maintained at 37°C in a humidified atmosphere containing 5% (v/v) CO₂. Sclareol (Sigma) was dissolved in DMSO at 10 mg/ml as a stock solution and diluted to the necessary concentration with fresh medium prior to use. The final DMSO concentration in the cultures was <0.1% (v/v), which did not influence cell growth when compared with that of the vehicle-free controls. Cells were grown in the media containing an equivalent amount of DMSO without sclareol to serve as a negative control.

An MTT assay (Sigma) was used to assess cell viability. Briefly, cells were seeded in 96-well plates at a density of 1×10⁶ cells/well. Following a 12-h incubation, sclareol (10, 30, 50, 70 or 100 μM) was added to the cells, while DMSO served as a negative control. The cells were cultured for 12, 24 and 48 h, followed by incubation with MTT (0.5 mg/ml) for 3 h at 37°C. Water-insoluble formazan crystals formed during incubation, which were dissolved by the addition of 100 μl/well DMSO. The optical densities (OD) at 570 nm were measured using an immunosorbent assay plate reader (HR801; Shenzhen Highcreation Technology Co., Ltd. Guangdong, China). Wells that contained culture medium and MTT but no cells acted as blanks. The percentage cell viability was calculated with the following equation: Cell viability = (OD_drug − OD_blank/OD_control − OD_blank) × 100.

An LDH assay was performed using a CytoTox 96^® Non-Radioactive Cytotoxicity Assay kit from Promega (Madison, WI, USA). Cells were seeded at a density of 2×10⁵ cells per well into 24-well plates 12 h prior to the experiments. Following different treatments, media from each well was collected to quantify the amount of released LDH, whereas isolated wells exposed to lysis buffer (10% Triton X-100) and media were collected to measure the total amount of cellular LDH. Cellular LDH was measured using a Cytotoxicity Detection kit (Roche Pharmaceutical Co., Basal, Switzerland) and a Colorimetric microplate reader (Thermo Molecular Devices Co.). Lactate dehydrogenase activity was determined by change in absorbance at 490 nm. For the purpose of calculating percent cytotoxicity values, background LDH release from culture cells was considered as low control and triton-X 100 (0.01%) treated cells as high control. Leakage (%)=[A490 (sample)−A490 (low control)/A490 (high control) − A490 (low control)]×100%

Cells grown in 12-well plates on cover-slips were exposed to different concentrations (30, 50, 70 and 100 μM) of sclareol for 48 h and incubated with Hoechst 33258 (Hoechst Staining kit; Beyotime Biotechnology, Haimen, China) according to the manufacturer’s instructions. Fluorescence microscopy was used to monitor changes in cell shape; cells were washed once with PBS, and then observed under a RX50-RFL Biological fluorescence microscope (Nikon, Tokyo, Japan). The condensed DNA of apoptotic cells was identified by intense local staining in the nucleus, in contrast to diffused staining of DNA in normal cells. Cell morphology was evaluated from six random visual fields and minimum of 600 cells were counted; each experiment was performed in triplicate.

MG63 osteosarcoma cells (1×10⁶) in a 60-mm dish were exposed to varying concentrations (50, 70 and 100 μM) of sclareol for 48 h. The cells were collected by trypsinization and washed twice with phosphate-buffered saline (PBS; Guangzhou Geneshun Biotech, Ltd, Guangzhou, China). Cells were incubated in 60% ethanol at −20°C overnight and then treated with 40 μg/ml RNase A (Guangzhou Geneshun Biotech, Ltd) and stained with 20 μg/ml of propidium iodide (PI; Guangzhou Geneshun Biotech, Ltd). The stained cells were analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA) at a wavelength of 488 nm.

The ΛΨm in MG63 cells was measured with rhodamine 123 (Rh-123; Guangzhou Geneshun Biotech Ltd) dye. MG63 osteosarcoma cells (5×10⁵) were treated with different concentrations (50, 70 and 100 μM) of sclareol and the ΛΨm was measured by flow cytometry. Rh-123 (5 mM) was added 2 h prior to the termination of experiment. The cells were then washed with PBS and incubated with PI (20 μg/ml) for 30 min. The cells were analyzed using a FACScan flow cytometer.

The data are expressed as the mean ± standard deviation. Differences between two groups were analyzed using Student’s t-test and P<0.05 was considered to indicate a statistically significant difference.

The antiproliferative activity of sclareol was evaluated against cultured MG63 cells using an MTT assay following 12, 24 and 48 h incubation intervals. The osteosarcoma cancer cells showed susceptibility to treatment with increasing doses of sclareol. Sclareol displayed dose- and time-dependent inhibition of cancer cell growth as presented in Fig. 1. The IC₅₀ value of the drug was found to be 65.2 μM at 12 h of incubation. Following a 48-h incubation, growth inhibition was observed compared with the growth at 12- and 24-h incubation intervals.

To examine whether sclareol induces cell death in human MG63 osteosarcoma cancer cells, groups of cells were exposed to different concentrations (10, 30, 50, 70 and 100 μM) of sclareol. The extent of cell death was measured after a 48-h incubation period using an LDH release assay. Since the amount of LDH release from the dying cells into the culture medium is proportional to the amount of cell death, this assay provides an acceptable estimation of the cell death induced by sclareol. Fig. 2 shows the significant increase in the sclareol-induced LDH release in a dose-dependent manner compared with that of the control (P<0.05).

The apoptotic effects of sclareol were evaluated in MG63 osteosarcoma cells exposed to different concentrations (30, 50, 70 and 100 μM) of sclareol for 48 h. The osteosarcoma cells were stained and evaluated for nuclear morphological changes using an inverted fluorescence microscope. The untreated cells showed normal, evenly dispersed blue fluorescence within their nuclei, whereas sclareol-treated cells displayed chromatin condensation or dense staining fragmentation known as ‘apoptotic bodies’, which correspond to an early apoptotic event (Fig. 3) (9). Notably, at higher concentrations of sclareol, a more significant reduction in the total number of cancer cells was observed compared with that in the control cells, indicating that sclareol exerts potent apoptotic cell death effects at higher doses.

Apoptosis and cell cycle deregulation are closely related biochemical events, and any disruption in cell cycle progression may lead to apoptotic cell death. In order to have a mechanistic overview of the growth inhibitory effect exerted by sclareol in osteosarcoma cancer cells, flow cytometric analysis was performed to detect whether sclareol induces cell cycle arrest in the MG63 cell line. The results indicated that treatment with varying concentrations of sclareol for 48 h induced G₁-phase growth arrest in osteosarcoma cells (Fig. 4). Following a 48-h treatment with 50, 70 or 100 μM sclareol, the G₁-phase cell population was significantly increased from 56.02% in the DMSO control cells to 74.54, 79.12 and 88.21% in the 50, 70 and 100 μM sclareol-treated cells, respectively. This increase in the G₁-phase cell population was accompanied by corresponding decreases in the S and G₂ phases of the cell populations. In summary, the cancer growth inhibitory effects of sclareol can be explained on the basis that it induces strong G₁-phase arrest in osteosarcoma cells.

One important and indicative stage in the intrinsic apoptosis pathway is the depolarization of the mitochondrial membrane and the subsequent leakage of the outer membrane through pore formation. This is accompanied by the release of pro-apoptotic molecules and cytochrome c. The fluorescent dye Rh-123 is a specific probe for the detection of alterations in the ΛΨm in living cells (10). The results of the current study revealed that sclareol induced a progressive reduction in the number of cells with intact ΛΨm and increased the number of cells with low ΛΨm after 48 h incubation. The loss of ΛΨm in osteosarcoma cells increased in a dose-dependent manner following treatment with 50, 70 and 100 μM of sclareol for 48 h (Fig. 5). The percentage of cells with a reduced ΛΨm was 15.0% in the solvent-negative control and 23.9, 22.2 and 74.0% at 50, 70 and 100 μM sclareol, respectively. Thus, this experiment confirms that treatment of osteosarcoma cancer cells with sclareol induces a loss in ΛΨm significantly.