The Effects of Ludartin on Cell Proliferation, Cell Migration, Cell Cycle Arrest and Apoptosis Are Associated with Upregulation of p21WAF1 in Saos-2 Osteosarcoma Cells In Vitro.

BACKGROUND The aim of this study was investigate the effects of the sesquiterpene lactone, ludartin, on cell proliferation, cell migration, apoptosis, and the cell cycle in osteosarcoma cell lines, compared with a normal osteoblast cell line. MATERIAL AND METHODS Osteosarcoma cell lines, MG-63 Saos-2 U-2OS, T1-73 143B, and HOS, and normal hFOB 1.19 osteoblasts, were cultured and treated with increasing doses of ludartin, The MTT colorimetric assay was used to measure cell metabolic activity and viability. Apoptosis was studied by fluorescence-activated cell sorting (FACS) using 4',6-diamidino-2-phenylindole (DAPI) nuclear staining and Annexin-V/propidium iodide (PI) staining. Cell cycle was studied using flow cytometry. Cell migration and invasion were studied using wound healing and Boyden chamber assays. Protein expression was measured by Western blotting. RESULTS Ludartin inhibited cell viability, cell migration, cell proliferation, and increased cell apoptosis, in all osteosarcoma cell lines, with an IC50 dose ranging from 15-30 µM. The greatest effects were on the Saso-2 osteosarcoma cells, with an IC50 of 15 µM. However, ludartin showed minor cytotoxic effects of the normal hFOB 1.19 osteoblasts (IC50 >100 µM). Ludartin exerted its anti-proliferative effects on Saos-2 cells via induction of apoptosis and cell cycle arrest at the G2/M checkpoint, associated with reduced expression of Cdc25c (Ser216), Cdc25c, pCdc2 (Tyr15), and Cdc2 and increased expression of p21WAF1. Ludartin inhibited cell migration and invasion of the Saos-2 cells. CONCLUSIONS The dose-dependent effects of ludartin on cell proliferation, migration, apoptosis, cell cycle arrest at the G2/M checkpoint involved p21WAFI in Saos-2 osteosarcoma cells.

Background

Osteosarcoma is the most common primary bone tumor in adolescents and children, with more than 70% of cases occurring in the age group of between 10–25 years [1]. In cases of childhood osteosarcoma, a 35% mortality rate has been reported, despite the use of available treatment strategies [2,3]. In adult patients with primary osteosarcoma, chemotherapeutic agents are often ineffective because of the development of chemoresistance. Although there has been increasing progress in the diagnosis and treatment of osteosarcoma, including surgery, chemotherapy, and radiation therapy, because of the development of early metastasis and resistance to chemotherapeutic agents, there is a need to discover novel diagnostic and prognostic biomarkers and supplement current treatments. Ongoing studies are being conducted to understand the development and progression of osteosarcoma and potential diagnostic and prognostic biomarkers [4-6]. However, the discovery of new molecules with the potential to inhibit osteosarcoma and the development of new treatment strategies are still needed.

In Chinese herbal medicine, natural or herbal derived medicines have shown promising results as chemotherapeutic agents for different types of cancers, and in China, more than half of the existing anticancer drugs are derived from natural products [7,8]. Ludartin is a sesquiterpene lactone that exhibits a wide range of pharmacological properties including, cytotoxic, anticancer, and bactericidal activities [9,10]. However, the anticancer activity of ludartin against the osteosarcoma has not been previously evaluated.

The aim of this in vitro study was to investigate the effects of the sesquiterpene lactone, ludartin, on cell proliferation, cell migration, apoptosis, and the cell cycle in osteosarcoma cell lines, compared with a normal osteoblast cell line.

Material and Methods

Cell culture

Osteosarcoma cell lines included MG-63 Saos-2 U-2OS, T1-73 143B, HOS, and normal osteoblast cells, hFOB 1.19 were purchased from the American Type Culture Collection (ATCC) (Rockville, MD, USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and antibiotics and maintained in a humidified atmosphere including 5% CO2 and maintained at a temperature of 37°C.

MTT assay

The proliferation rate of osteosarcoma and normal cells were analyzed by the MTT assay. The cells were cultured at a density of 3×106 cells per ml in a 96-well plate, and cultured for 24 h at 37°C. Incubation of the cells was performed for 48 h at a concentration of between 0–100 μM of ludartin in a humidified atmosphere of 5% CO2 at a temperature of 37°C. A volume of 150 μl of MTT solution (5 mg/ml) was added to each well of the 96-well plate and incubated for four more hours. The supernatant was decanted from each well. The formazan crystals that formed were dissolved by the addition of 150 μl of dimethyl sulfoxide (DMSO). The absorbance for each of the wells was recorded at 465 nm using a spectrophotometer.

Apoptosis analysis by flow cytometry

After 48 h of incubation, the cells were incubated with 0, 7.5, 15, and 30 μM concentrations of ludartin. The Saos-2 cells were selected, collected, and washed with phosphate buffered saline (PBS). The cells were then stained using 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining and apoptosis was detected by fluorescence microscopy, as previously reported [11]. For measurement of apoptotic cell populations, the ludartin-treated cells were then suspended in binding buffer at a density of 3×106 cells per ml followed by staining with 5 μl of Annexin-V fluorescein isothiocyanate (FITC) and 5 μl propidium iodide (PI). The cell suspension was incubated in the dark at room temperature for 25 min. Analysis of cell apoptosis was carried out using a BD FACSCalibur™ flow cytometer (BD Biosciences, NJ, USA).

Cell cycle analysis

To determine the distribution of cells in each phase of the cell cycle, the ludartin-treated Saso-2 osteosarcoma cells were collected and washed with PBS, fixed with ethanol (70%) for about an hour, and then washed again with PBS. The cells were resuspended in a solution of PI (50 μl/ml) and RNase1 (250 μg/ml), followed by incubation for 30 min at room temperature. Cell cycle was investigated using the fluorescence of Annexin-V and PI using FC500 fluorescence-activated cell sorting (FACS) cater-plus flow cytometry (Beckman Coulter, CA, USA) at an excitation wavelength of 488 nm and emission wavelengths of 525 and 625 nm, respectively using 10,000 cells/group.

Cell migration assay

The cell migration capacity of ludartin-treated osteosarcoma cells was examined using a wound healing assay. Briefly, 5×104 cells/well were cultured in 96-well plates and were incubated overnight at 37°C to allow the cells to adhere. A wound was then created with a scratch using a sterile pipette tip after the cells reached confluence. The cells were then washed with PBS to clear the detached cells. The cells were monitored after an interval of 20 h interval and photographed. The invasive properties of the ludartin-treated Saso-2 cells were determined using a Boyden Chamber assay as previously described [12]. Briefly, the Boyden Chamber assay used a plastic chamber containing a porous membrane, suspended over a larger well containing medium or chemoattractant and the cells that migrate through the pores to the other side of the membrane and were stained and counted.

Western blot analysis

The cells were incubated for 48 h with 25 nm concentration of ludartin, harvested, rinsed in cold PBS and lysed in cold lysis buffer for 45 min. The cell lysates were then centrifuged at 15,000×g for 15 min at a temperature of 4°C, and the supernatant was collected. A Bicinchoninic Acid Protein Assay kit (Pierce, Rockford, IL, USA) was used to measure the concentration of proteins according to the manufacturer’s instructions. Protein samples (50 μg) were isolated on 12% sodium dodecyl sulphate-polyacrylamide gels by electrophoresis. The proteins were transferred onto polyvinylidene difluoride (PVDF) membranes, blocked using 5% bovine serum albumin (BSA) and Tris-buffered saline with Tween® 20 (TBST) buffer using a semi-dry transfer system (Bio-Rad Laboratories, Hercules, CA, USA).

The membrane was then incubated overnight at 4°C using the dilution of 1: 1,000, with the following primary antibodies to: matrix metallopeptidase (MMP)-9 (Cat. No. sc-176046); MMP-2 (Cat. No. sc-176407); Cdc25c (Cat. No. sc-327); Chk2 (Cat. No. sc-56296), p-Cdc2 (Tyr15) (Cat. No. sc-12340); Cdc2 (Cat. No. sc-54); cyclin B1 (Cat. No. sc-70898); p27 (Cat. No. sc-1641); p21 (p21WAFI) (Cat. No. sc-122305); p53 (Cat. No. sc-47698); actin (Cat. No. sc-58673); GAPDH (Cat. No. sc-47724) (Santa Cruz Biotechnology Inc., Dallas TX, USA).

Following incubation with the primary antibodies, the membrane was washed with PBST and then incubated for 1 h with 5% dried skimmed milk powder, washed in PBST and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad Laboratories, Hercules, CA, USA). An enhanced chemiluminescence (ECL) detection kit (Amersham Pharmacia) was used for the analysis of the protein interactions.

Statistical analysis

The data were expressed as the mean ± standard deviation (SD). The Student’s t-test and one-way analysis of variance (ANOVA) were used for the comparison of the differences between the groups. For the purpose of relevance analysis of ordinal data, the cross chi-squared (χ2) test was used. The values were considered statistically significant at P<0.05.

Results

Inhibition of cell viability of the Saos-2 osteosarcoma cells treated with ludartin

The effect of increasing concentrations of ludartin (0–100 μM) on the viability of a panel of osteosarcoma cells (MG-63 Saos-2 U-2OS, T1-73 143B, and HOS) and normal osteoblast cells (hFOB 1.19) was investigated by the MTT assay. The results showed a dose-dependent inhibition in the viability of all the osteosarcoma cell lines treated with ludartin (Table 1). The anti-proliferative effects of ludartin treatment were found to be minimal for the normal osteoblast cell line (hFOB 1.19). Thee osteosarcoma cell line that showed the greatest anti-proliferative response following treatment with ludartin was Saos-2 osteosarcoma cell line, which showed an IC50 at a dose of 15 μM of ludartin (Figure 1A). Also, ludartin treatment induced distinctive morphological changes in the Saos-2 cells (Figure 1B)

Table 1

Antiproliferative effects of Ludartin on osteosarcoma and normal cell lines determined by MTT assay and expressed as IC50 (μM).

Cell lines	IC50 (μM)
MG-63	25
Saos-2	15
U-2 OS	25
T1–73	25
143B	30
HOS	30
hFOB 1.19	>100

Figure 1

Effect of ludartin on the viability of Saos-2 osteosarcoma cells. (A) The effect of ludartin on osteosarcoma Saos-2 cells in the MTT assay. (B) The effect of ludartin of the morphology of Saos-2 cells. The experiments were performed in triplicate and the values presented as mean ±SD (* P<0.05).

Induction of apoptosis in the Saos-2 osteosarcoma cells treated with ludartin

Analysis of the Saos-2 osteosarcoma cells following fluorescence-activated cell sorting (FACS) analysis using 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining and Annexin-V/propidium iodide (PI) staining showed that ludartin treatment was associated with the induction of cell apoptosis (Figure 2). Also, the apoptotic effects of ludartin on Saos-2 cells were found to be dose-dependent. The apoptotic cell populations were determined by Annexin-V/PI staining, which showed that the apoptotic cells increased from 2% in untreated cells to up to 77% with ludartin concentrations of 30 μM (Figure 3)

Figure 2

Ludartin triggered apoptosis in Saso-2 osteosarcoma cells. Apoptosis was studied by fluorescence-activated cell sorting (FACS) using 4′, 6-diamidino-2-phenylindole (DAPI) nuclear staining. The experiments were performed in triplicate and the values presented as mean ±SD (* P<0.05).

Figure 3

Estimation of apoptotic of Saos-2 osteosarcoma cell populations at indicated concentrations of ludartin. Annexin-V/propidium iodide (PI) staining. The experiments were performed in triplicate.

Induction of G2/M cell cycle arrest in of the Saos-2 osteosarcoma cells treated with ludartin

To further investigate other mechanisms that could cause ludartin-induced suppression of cell proliferation, the cell cycle was studied in Saos-2 cells after ludartin treatment, using flow cytometry. Ludartin treatment resulted in the accumulation of Saos-2 osteosarcoma cells in the G2-phase by 24 hours following ludartin treatment (Figure 4).

Figure 4

Ludartin treatment and G2/M cell cycle arrest in Saso-2 osteosarcoma cells as determined by flow cytometry. The experiments were performed in triplicate.

Further immunoblotting was performed to determine the effects of ludartin on the expression of G2/M cell cycle-associated proteins including cyclin B1, Cdc25c, and Cdc2 in Saos-2 osteosarcoma cells. As shown in Figure 5A, treatment of Saos-2 osteosarcoma cells with ludartin resulted in a dose-dependent reduction in the expression of pCdc25c (Ser216), Cdc25c, pCdc2 (Tyr15) and Cdc2 in Saos-2 osteosarcoma cells. Also, treatment of Saos-2 osteosarcoma cells with ludartin for 48 hours resulted in reduced expression of cyclin B1. Ludartin treatment increased the expression of cyclin-dependent kinase inhibitor p21 (WAF1) or p21WAF1 in Saso-2 osteosarcoma cells (Figure 5B). However, ludartin treatment did not alter the expression of either p27 or p53 tumor suppressor proteins (Figure 5B).

Figure 5

Western blots showing the effect of ludartin on cell cycle proteins in Saso-2 osteosarcoma cells. (A) Cdc25c, pCdc25c, Cdc2, pCdc2, p21WAFI, and cyclin B1, and (B) p21, p53, and p27. Ludartin treatment increased the expression of cyclin-dependent kinase inhibitor p21 (WAF1) or p21WAF1 in Saso-2 osteosarcoma cells but did not alter the expression of p27 or p53 tumor suppressor proteins. The experiments were performed in triplicate and the values presented as mean ±SD (* P<0.05).

Inhibition of cell migration and invasion of the Saos-2 osteosarcoma cells treated with ludartin

Treatment of Saso-2 osteosarcoma cells with ludartin inhibited cell migration and invasion (Figure 6A, 6B). Moreover, the inhibition of migration and invasion of Saos-2 were also associated with down-regulation of MMP-2 and MMP-9 expression (Figure 6C).

Figure 6

Effect of ludartin at the IC50 of the Saso-2 osteosarcoma cells. (A) Cell migration of Saso-2 osteosarcoma cells. (B) Cell invasion of Saso-2 osteosarcoma cells. (C) Expression of MMP-2 and 9 by Saso-2 osteosarcoma cells. The experiments were performed in triplicate and the values presented as mean ±SD (* P<0.05).

Discussion

Worldwide, osteosarcoma is a significant cause of morbidity and mortality, and the current methods of treatment may not be effective and are associated with side effects [13]. Therefore, there is a need to identify new chemotherapeutic agents to treat osteosarcoma. The natural products used in Chinese herbal medicine might provide promising treatments for malignant disease, including osteosarcoma [7,8]. Therefore, the aim of this study was to investigate the anticancer effects of ludartin, a natural product, in a panel of osteosarcoma cell lines (MG-63, Saos-2, U-2OS, T1-73 143B, and HOS), and a normal osteoblast cell line (hFOB 1.19) in vitro. The findings of this study showed that ludartin treatment resulted in an anti-proliferative effect on all the osteosarcoma cell lines studied cell lines. However, the anti-proliferative effects were more pronounced on the osteosarcoma Saos-2 cells (IC50 dose of 15 μM), and the least anti-proliferative effects were observed on the normal hFOB 1.19 osteoblast cells (IC50 dose >100 μM).

The findings of the present study are supported by those of previously published studies, which have shown that ludartin treatment inhibited the proliferation and metastasis of ovarian cancer cells [9]. Also, in the present study, the anti-proliferative effects ludartin treatment on the Saos-2 osteosarcoma cells studied in vitro were also associated with changes in cell morphology, which supports that ludartin selectively targeted osteosarcoma cells in this study. Given the initial findings in the Saos-2 osteosarcoma cells in the present study, further experiments were performed on these cells.

In this study, to evaluate the possible mechanism of the antiproliferative effects of ludartin treatment on Saos-2 osteosarcoma cells, the effects on cell apoptosis were studied by fluorescence-activated cell sorting (FACS) using 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining and Annexin-V/propidium iodide (PI) staining, which confirmed that ludartin induced apoptosis. Many anti-cancer drugs exert their therapeutic activity by triggering apoptosis of rapidly proliferating cancer cells, including the chemotherapeutic agent cisplatin [14,15]. Also, the resistance of the cancer cells to drugs is in part due to their capacity to escape apoptosis [16]. In the present study, the impact of ludartin on the phases of the cell cycle of the Saos-2 osteosarcoma cells were studied and the findings were that ludartin treatment induced G2/M cell cycle arrest, indicating that cells at the G2/M phase could not progress and undergo mitosis due to the inhibition of proteins such as cyclin B1, Cdc25C, and Cdc2. Also, p21WAF1 is an important inhibitor of cell cycle progression, and p21WAF1 expression was shown to be increased in Saos-2 osteosarcoma cells treated with ludartin in the present study [17]. There have been published findings that indicate that upregulation of p21WAF1 inhibits the expression of the Cdc2/cyclin B1 complex, which when activated triggers entry of the cell into mitosis [18].

Therefore, from the findings of this preliminary in vitro study, the treatment of Saos-2 osteosarcoma cells with ludartin inhibited the expression of cyclin B1, Cdc25C, and Cdc2 and increased the protein expression levels of p21WAF1, had no significant effect on the expression of p27 or p53. These findings might indicate that ludartin treatment induced the expression of p21WAF1, which in turn triggered G2/M-phase arrest. Cell migration and invasion is one of the important characteristic features cancer progression and metastasis [19]. Inhibition of cell migration and invasion prevents cancer metastasis and improves patient survival in vivo [19]. In the present study, treatment of Saos-2 osteosarcoma cells with ludartin inhibited cell migration and invasion in vitro. The findings of this preliminary study support the need for further studies to evaluate the effects of ludartin on other tumor cells lines in vitro together with an assessment of the effects of ludartin in vivo, to determine whether ludartin may prove to be a useful future chemotherapeutic agent.

Conclusions

The findings of this study showed that treatment of Saos-2 osteosarcoma cells in vitro with ludartin inhibited cell proliferation, cell migration, increased apoptosis of proliferating malignant cells, induced cell cycle arrest at the G2/M checkpoint, findings that were associated with increased expression of p21WAF1. Therefore, future in vivo studies are required to determine whether ludartin has a potential role as a lead molecule in the treatment of osteosarcoma and other types of malignancy.