Activity of Dobutamine on Human Osteosarcoma Cells

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The Antitumor Activity of Dobutamine on Human Osteosarcoma Cells

ABSTRACT

BACKGROUND: Dobutamine have been clinically used for the treatment of heart failure and cardiogenic shock. Osteosarcoma is the most common malignant bone tumor in children. Little information is known on the anticancer activity of dobutamine. The present study aimed to examine the effects of dobutamine on the cell proliferation, apoptosis, cycle and invasiveness of osteosarcoma cells. METHODS: Human osteosarcoma MG-63 cells were treated with dobutamine at various concentrations and incubation time. The inhibition of cell growth by doutamine was determined by MTT. Flow cytometry was used to evaluate the effect of doutamine on MG-63 cell apoptosis and cycling. Furthermore, the expression level of caspase-3 and caspase-9 with or without treatment was assessed by western-blot analysis. The influence of doutamine on cancer cell migration and invasion was investigated using wound-healing assay and Boyden Chamber migration method. RESULTS: Dobutamine significantly inhibited the growth of MG-63 cells at 10 µM or higher when incubated for 12 h or longer. Flow cytometry showed that dobutamine augmented cell apoptosis and arrested cell cycle in G2/M phase. Western-blot analysis revealed that dobutamine induces expression of caspase-3 and caspase-9. In addition, the invasiveness and migration of MG-63 cells were inhibited by dobutamine in a concentration-dependent manner. CONCLUSION: Taken together, our findings demonstrated that dobutamine inhibits the proliferation, induces apoptosis and inhibits invasiveness of osteosarcoma cells in vitro. It may thus have promise to become new a therapeutic agent against osteosarcoma.

Keywords: osteosarcoma; dobutamine; anticancer activity

Introduction

Ostosarcoma is the most common primary bone malignant cancer in children with high incidence and mortality rates (1). Since osteosarcoma is considered as a radioresistant tumor, chemotherapy is the main approach for the treatment of osteosarcoma. However, the chemotherapy regimens are not effective. The current chemotherapeutic agents such as ifosfamide, cisplatin, HDMTX (Methotrexate and Leucovorin) have a number of side-effects and can be acquired drug resistance by osteosarcoma (2). Moreover, the prognosis of osteosarcoma is very poor and >30% of patients might die of pulmonary metastases within 5 years (3). Therefore, there is an urgent need for the development of new effective therapeutic drugs for osteosarcoma.

Yes-associated protein (YAP), a transcriptional co-activator, is a key regulator of the Hippo pathway (4). When YAP is recruited to the nucleus, transcription of cell proliferation-promoting and anti-apoptotic genes is activated constantly (5, 6). Recently, high-expression of YAP has been found in many types of tumors, including hepatocellular, colorectal, gastric carcinoma, ovarian, breast, and lung cancers, and correlates with poor prognosis (7-10). These observations suggest that YAP might contribute to a malignant cellular phenotype and thus becomes an important target for anticancer drugs (11).

Dobutamine is a synthetic catecholamine developed by Eli Lilly and Company in 1970’s (12). It has been widely used as an inotropic drug for hemodynamic support in the treatment of congestive heart failure, cardiogenic and septic shock (13). A recent study showed that dobutamine is able to attenuate YAP-dependent transcription by inhibiting its nuclear translocation (14).

In the present study, we aimed to investigate the effect of dobutamine on the proliferation, apoptosis and invasiveness of human osteosarcoma cell line MG-63. Our results showed the usefulness of dobutamine for the treatment of osteosarcoma cancer.

Materials and methods

The human osteosarcoma cell line MG-63 was obtained from Shanghai cell bank of Chinese academy of sciences. Dulbecco's Modified Eagle's Medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone Corp (U.S.A.). Propidium iodide (PI) and dobutamine were purchased from Sigma-Aldrich Corp (St. Louis, MO, U.S.A.). Annexin V-FITC Kit was purchased from Beckman Coulter Inc (S.Kraemer Boulevard Brea, CA, U.S.A.). All other chemicals were of the highest grade commercially available in China.

Cell culture

MG-63 was grown in medium at 37°C in 5% CO2-95% air. As culture medium supplemented with 10% FBS, 100 U/mL penicillin, 100 μg/ mL streptomycin and DMEM were used for MG-63.

MTT assay

The influence of dobutamine on the cell viability was determined according to MTT assay. Cells were seeded on a 96-well plate overnight and stimulated with various concentrations of dobutamine (1 μM, 5 μM, 10 μM, 25 μM, 50 μM) for 12, 24, 48 and 72 h,after the indicated treatments, the cells were incubated with MTT (0.25 mg/ml in PBS) for 4 h, then the media were removed, and DMSO (1 ml of 100%) was added to solubilize the MTT-formazan product. The amount of violet crystals reflecting cellular growth and viability was determined by the absorbance at 490 nm. The inhibitory rate of cell growth was calculated as [1-treatment group/control group)] ×100%. The growth curve was drawn using time as abscissa and inhibition rate as ordinate. Each dose was done in triplicate, and the experiments were repeated at least twice.

Flow cytometry analysis

The rate of apoptosis and percentage of cells in G1, S and G2/M phase were measured by flow cytometry. Apoptosis analysis: after treated according to the experimental groups for 24 h, cells were harvested with trypsinase , washed twice with PBS, re-suspended in binding buffer, then stained with Annexin V-FITC and PI following the manufacturer’s instruction and processed by flow cytometry. The cell suspension was incubated with PI solution (50 μg/ml) and 50 units of RNase for 30 minfor cell cycle detection. Data acquisition and analysis were done on a BD (Becton Dickinson) FACSCaliber using CellQuest software (BD Biosciences).

Cell invasion analysis

The effect of dobutamine on the invasion of MG-63 cells was investigated using transwell chambers with polycarbonate filters (pore size of 8 μm). The cells were seeded on the upper chamber at a density of 1×105 cells/ml and incubated in 0.6 ml DMEM medium containing 10% FBS and various concentrations of drugs. The lower chamber was filled with 0.6 ml of DMEM medium containing 20% FBS. After 24 h, cells on the upper filter were removed by wiping, and then the filter was fixed in 4% paraformaldehyde for 1 h. Cells passing through the filter were stained with hematoxylin. Invasion cells were counted under a microscope.

Western blotting analysis

We examined the levels of protein expression of caspase-3 and caspase-9 in MG-63 cell before and after treatment with dobutamine. Cells treated as indicated were harvested in 5 mL of medium, pelleted by centrifugation (1000 × g for 5 min at 4 °C), then washed twice with ice-cold PBS and lysed in ice-cold HEPES buffer [HEPES (pH 7.5) 50 mmol/L, NaCl 10 mmol/L, MgCl2 5 mmol/L, EDTA 1 mmol/L, glycerol 110% (v/v), Triton X-100 1% (v/v), a cocktail of protease inhibitors, and 1 mg/L DOBUTAMINE on ice for 30 min. The lysates were clarified by centrifugation (15,000 × g for 10 min at 4 ℃) and the supernatants then either analyzed immediately or stored at -80℃. Equivalent amounts of protein (50 µg) from total cell lysates were resolved by SDS-PAGE using precast 12% Bis-Tris gradient gels and transferred onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked overnight at 4℃ in blocking buffer [nonfat dried milk 5% (v/v), NaCl 150 mmol/L, Tris (pH8.0) 10 mmol/L and 0.05% Tween 20 (v/v)]. Proteins were detected by incubation with primary antibodies at appropriate dilutions in blocking buffer overnight at 4℃. Unbound antibody was removed by washing with Tris-buffered saline (pH 7.2) containing 0.5% Tween 20 (TBS-T). The membrane was then incubated at room temperature with horseradish peroxidase-conjugated secondary antibody. After extensive washing with TBS-T, bands were visualized by enhanced chemiluminescence followed by exposure to autoradiography.

Statistical analysis

Statistical analyses were performed using the SPSS 15.0 software package (SPSS Inc.). Comparisons between two samples were employed by Student’s t-test. The test level was set to α=0.05.

Results and Discussion

Dobutamine inhibits the proliferation of ostosarcoma MG-63 cells

Our results showed that dobutamine significantly inhibited cell proliferation in time and concentration dependent manners compared with the control group. As shown on the proliferation inhibition curve (Figure 1), three (10, 25, 50 μM) had significant inhibitory effect on the survival of MG-63 cells out of the five tested concentrations of dobutamine (P<0.05).

Dobutamine augmented cell apoptosis and arrested cell cycle

Annexin V/PI staining was used to measure the dobutamine-induced apoptosis. Compared with control group, dobutamine induced a significant increase of apoptotic death, after pretreated with 5 μM, 10 μM, 25 μM and 50 μM for 24 h in MG-63 cells (P<0.05) (Table 1). The percentage of MG-63 cells in G2/M phases was significantly increased (P<0.01), at dobutamine concentration of 25 μM, 50 μM, and the percentage in S-phase was significantly decreased (P<0.05) (Figure 2).

Dobutamine reduces the cell migration and invasion of MG-63.

To check whether dobutamine has the effect on cell movement, we compared the migratory rate of the tumor cells in a wound-healing assay. Figure 3 showed that dobutamine significantly decreased cell migration from the edge of the wound (P<0.05). Similarly, invasion assay also showed a large number of cells passed through the filter in the control group, whereas the cells passing through the filter were markedly reduced after dobutamine treatment. Moreover, the dobutamine reduced the number of invasive cells in a concentration dependent manner (Figure 4). The number of invasive cells in combination group was significantly reduced than that in alone groups (P<0.05) (Figure 4).

Dobutamine induces expression of caspase-3 and caspase-9

Western blot analysis was used to examine the expression of caspase-3 and caspase-9 in MG-63 cells following dobutamine interventions. Protein expression analysis indicated that caspase-3 was increased after treated with dobutamine at the concentration of 10 and 50 μM for 72 h (P<0.05), and caspase-9 was increased after treated by dobutamine at the concentration of 50 μM for 72 h (P<0.05) (Figure 5).

Recent reports demonstrated that YAP protein is highly expressed in human osteosarcoma MG-63 cells (15). Our results indicate that the inhibitory effect of dobutamine might be associated with the inhibition of YAP protein translocation. Silencing of YAP gene with RNA interference led to the similar effect as dobutamine (16). In addition, dobutamine arrests cell cycle at G2/M transition and augmented cell apoptosis. Previous studies demonstrated that YAP activates cell apoptosis in response to DNA damage by interacting with p73 in several cancer cell lines (17).

Conclusion

In summary, our results showed that dobutamine can significantly inhibit osteosarcoma cell growth by inhibiting cell proliferation, inducing cell apoptosis and redistributing cell cycle. These data indicates dobutamine may become a new therapeutic agent for the treatment of osteosarcoma. However, further in vivo studies are required to confirm the effectiveness and safety of dobutamine in the treatment of osteosarcoma.

Figure legends

Figure 1 Effect of dobutamine on the proliferation of MG-63 cells. The cells were treated with different concentrations (1, 5, 10, 25 and 30 μM) of dobutamine for 12, 24, 48 and 72 h. The inhibition rate (%) of dobutamine was calculated as [1-treatment group/control group)] ×100%.

Figure 2 Cell cycle (a) and apoptosis distribution (b) of MG-63 cells in response to dobutamine treatment. A: control B: 10 μM. C: 25 μM. D: 50 μM. Data was mean± SD (n=6). * indicated the significant difference compared with the control group (P<0.05).

Figure 3 Effect of dobutamine treatment on the migration of MG-63 cells. Data was mean ± SD (n=6).* indicated the significant difference between control and the treatment (P<0.05).

Figure 4 The effect of dobutamine on MG-63 cell invasiveness. The cells were treated with different concentrations of dobutamine for 24 h. a: control b: 1 μM. c: 5 μM. d: 10 μM. e: 25 μM. f: 50 μM. The number of invasive cells of MG-63 after dobutamine treatment for 24 h. Data was mean ± SD (n=6). * indicated the significant difference between control and the treatment (P<0.05).

Figure 5 The effect of doubtamine on protein expression of caspase-3 and 9 in MG-63 cells. a: western blot analysis, b: protein expression of caspase-3 and 9. The cells were treated with various concentrations of dobutamine (0, 1, 10, and 50 µM) for 72 h. GAPDH was used as endogenous control gene. The expression level was calculated as the ratio of caspase-3 or caspase-9/GAPDH. Data was mean ± SD (n=6). *indicated the significant difference between control and the treatment.

Table 1 Cell cycle distribution and apoptosis of MG-63 cells

Groups

Cell cycle

Apoptosis%

G0/G1

S

G2/M

Control

54.12±6.53

25.60±5.33

20.28±1.79

2.2±0.4

DOBUTAMINE(1 μM)

55.19±3.16

25.37±3.66

19.44±5.01

2.5±1.1

DOBUTAMINE(5 μM)

53.96±4.58

23.18±6.34

22.86±4.27

2.9±1.7

DOBUTAMINE(10 μM)

49.82±2.99

26.78±5.92

23.40±6.52

7.0±2.5*

DOBUTAMINE(25 μM)

50.62±5.27

21.50±4.59

27.88±4.55*

11.6±4.7*

DOBUTAMINE(50 μM)

51.21±6.52

18.39±2.79

30.40±7.26*

13.2±1.8*

*Statistically significant difference from control group

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