PA was obtained from Sigma-Aldrich, Inc. (St. Louis, MO). Antibodies against poly (ADP-ribose) polymerase (PARP), Bcl-2, Bax, pRB, and p21^Cip1 were purchased from a Santa Cruz Biotechnology (Santa Cruz, CA). Propidium iodide (PI) and RNase A were purchased from Sigma Chemical Corporation (St. Louis, MO).

The human osteosarcoma cell lines, HOS and U-2 OS, were obtained from the Korean Cell Line Bank (KCLB, Korea) and cultured in RPMI 1640 (HOS) or McCoy's 5A medium (U-2 OS) containing 10% heat-inactivated fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin (100 mg/ml) in a water-jacketed incubator with a humidified atmosphere (5% CO₂, 95% air) at 37℃. Experimental cultures were grown to 60~70% confluence. The cells were then treated with PA or ethanol alone by replacing the medium with fresh media containing 10% FBS and the test compound. The amount of ethanol added as the vehicle never exceeded 0.1% of the total volume.

Each cell line was seeded at 1×10⁶ cells/ml per 100 mm on a culture plate. The culture medium was replaced with fresh medium, and PA or ethanol was added after 24 h. After 24, 48, or 72 hours, the adherent cells were detached with trypsin/EDTA, and the cell number was determined using a hemocytometer. Cell viability was assessed by trypan blue exclusion assay.

A colony formation assay was used to determine the long-term survival of cells following a treatment with PA (5 mM) or ethanol alone. After a short period of time, exponentially growing cells were plated at a density of 4×10⁴ cells/ml onto 100 mm culture dishes, and the reagents were added after 24 hours. Two weeks later, the cells were fixed with methanol and stained with 3% crystal violet.

For the purposes of cell cycle analysis, the cells were plated at an initial density of 1×10⁶ cells/ml per 100 mm onto culture plates. The culture medium was replaced with fresh medium and PA or ethanol was added after 24 hours. Forty eight hours after treatment, the cells were trypsinized, washed once with phosphate buffered saline (PBS), and fixed in 70% ethanol for at least 1 hour at 4℃. After washing with PBS, they were incubated at room temperature with PI staining solution (0.005 mg/ml propidium iodide, 20 µg/ml RNase A) for 1 hour in the dark. Stained cells were analyzed by FACScan (Becton Dickinson, Mansfield, MA). The percentages of the cells in the G0/G1, S and G2/M phases were determined by cell Quest software (Becton Dickinson, Mansfield, MA).

The cell extracts were prepared as follows. Cells were collected by centrifugation and dissolved in a cold lysis buffer [1×PBS, 1% Triton X-100, 0.05% Sodium deoxycholate, 0.0l% SDS, 0.5 µg/ml leupeptin, l mM EDTA, 1 µg/ml pepstin and 0.2 mM PMSF] for 30 minutes on ice. The concentrations of total proteins in the extracts were determined using the Bradford protein assay. Bovine serum albumin (BSA) was used as a protein standard. Cell extracts (30 µg of protein/lane) were separated on 6~12% SDS-polyacrylamide gels with the Tri glycine buffer system, and the proteins were transferred to polyvinyliden difluoride (PVDF) membranes (Boehringer Mannheim, Germany) using a semi-dry transfer system as recommended by the manufacturer (Bio-Rad, Hercules, CA). After blocking with PBST (phosphate buffered saline pH 7.4, 0.1% Tween 20) containing 5% low fat milk, the membranes were incubated with the primary antibody in PBST for 1 hour. The membranes were then washed twice in PBST for 10 minutes. Next, the membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP) for 1 hour and developed with the enhanced chemiluminescence (ECL) detection system (Amersham Pharmacia Biotech, UK). Equal protein loading was controlled by a reprobing process with anti-actin antibody (1:1,000, Sigma-Aldrich, Inc. St. Louis, MO)

Growth inhibition studies were performed using osteosarcoma cell lines HOS and U-2 OS. These cells were tested with increasing nontoxic concentrations of PA at 2, 5, 7, 10 and 15 mM. As shown in Fig. 1, composite inhibitory effects exist at different concentrations of PA after 24, 48, and 72 hours of exposure. The antiproliferative effect of PA was time- and dose-dependent in both cell lines. The inhibitory effects of PA were not due to nonspecific cytotoxic action because cell viability, as assessed by trypan blue exclusion, was greater than 95%. This did not differ between control and treated cells. Both the HOS and U-2 OS cells, each treated with 5 mM of PA, failed to form colonies after 2 weeks on the colony survival assay (Fig. 2). The inhibition of cell proliferation could have been due to the induction of apoptosis elicited by PA. Therefore, further studies were performed to investigate whether PA could induce apoptosis in osteosarcoma cells.

Flow cytometric analysis revealed that PA treatment resulted in a higher proportion of cells in the G0/G1 phase relative to the control cells (HOS: 55% in control and 62.3% in treated cells, exclusive of apoptotic cells; U-2 OS: 49% in control and 63.3% in treated cells, exclusive of apoptotic cells). In contrast, PA treatment reduced the proportion of cells in the S and G2/M phase for both HOS and U-2 OS cells (Fig. 3). These results indicate that growth inhibition of osteosarcoma cells subsequent to PA treatment can be attributed to cell arrest in the G1 phase.

Using flow cytometric analysis, apoptosis was demonstrated in both the HOS and U-2 OS cells treated with 5 mM of PA. As shown in Fig. 3, 47% of HOS cells and 40% of U-2 OS cells are present in the sub-G0/G1 apoptotic phase after 48 hours of exposure to PA. The induction of apoptosis was first detected after 24 hours of treatment. This is directly correlated with the inhibition of cell proliferation. Western blot analysis revealed that after treatment with PA, the PARP protein (115 kDa) was degraded to an accumulation of the cleaved PARP (85 kDa) (Fig. 4). These two independent methods of measuring apoptosis have provided strong evidence that apoptosis was induced in both kinds of osteosarcoma cells treated with PA. In order to explore the mechanisms by which PA induces apoptosis, the alterations in the expression of Bcl-2 and Bax genes, known to be involved in the apoptotic processes, were investigated.

The effects of PA on the expression of Bcl-2 and Bax in osteosarcoma cells were studied by employing Western blot analysis. A significant decrease in Bcl-2 expression was observed in both HOS and U-2 OS cells after treatment with PA for 48 hours. However, the level of Bax expression in the osteosarcoma cells was mildly up-regulated (Fig. 5).

The phosphorylation pattern of pRb is an established indicator of the proliferative status of the cell. Hypophosphorylated pRb is found in G1 cells, whereas hyperphosphorylated pRb is characteristic of proliferating cells in the S-, G2- and M phases. As shown in Fig. 6, exposure of the cells to PA resulted in a marked decrease in the expression of phosphorylated pRb.

For negative cell cycle regulators, the expression of p21^Cip1 was investigated by employing Western blot analysis. Both of the osteosarcoma cell lines showed a significant increase in p21^Cip1 levels after 48 hours of exposure to 5 mM of PA (Fig. 7). The expression of p27 was not investigated.