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The Antitumor Activity of Dobutamine on Human Osteosarcoma Cells

Abstraction

Background: Dobutamine have been clinically used for the intervention of bosom failure and cardiogenic daze. Osteosarcoma is the most common malignant bone tumour in kids. Small information is known on the anticancer activity of dobutamine. The present survey aimed to analyze the effects of dobutamine on the cell proliferation, programmed cell death, rhythm and invasiveness of osteogenic sarcoma cells.Methods:Human osteosarcoma MG-63 cells were treated with dobutamine at assorted concentrations and incubation clip. The suppression of cell growing by doutamine was determined by MTT. Flow cytometry was used to measure the consequence of doutamine on MG-63 cell programmed cell death and cycling. Furthermore, the look degree of caspase-3 and caspase-9 with or without intervention was assessed by western-blot analysis. The influence of doutamine on malignant neoplastic disease cell migration and invasion was investigated utilizing wound-healing check and Boyden Chamber migration method.Consequence:Dobutamine significantly inhibited the growing of MG-63 cells at 10 µM or higher when incubated for 12 H or longer. Flow cytometry showed that dobutamine augmented cell programmed cell death and arrested cell rhythm in G2/M stage. Western-blot analysis revealed that dobutamine induces look of caspase-3 and caspase-9. In add-on, the invasiveness and migration of MG-63 cells were inhibited by dobutamine in a concentration-dependent mode.Decision:Taken together, our findings demonstrated that dobutamine inhibits the proliferation, induces programmed cell death and inhibits invasiveness of osteogenic sarcoma cells in vitro. It may therefore hold promise to go new a curative agent against osteogenic sarcoma.

Keywords: osteogenic sarcoma ; dobutamine ; anticancer activity

Introduction

Ostosarcoma is the most common primary bone malignant malignant neoplastic disease in kids with high incidence and mortality rates ( 1 ) . Since osteogenic sarcoma is considered as a radioresistant tumour, chemotherapy is the chief attack for the intervention of osteogenic sarcoma. However, the chemotherapy regimens are non effectual. The current chemotherapeutic agents such as ifosfamide, cisplatin, HDMTX ( Methotrexate and Leucovorin ) have a figure of side-effects and can be acquired drug opposition by osteogenic sarcoma ( 2 ) . Furthermore, the forecast of osteogenic sarcoma is really hapless and & A ; gt ; 30 % of patients might decease of pneumonic metastases within 5 old ages ( 3 ) . Therefore, there is an pressing demand for the development of new effectual curative drugs for osteogenic sarcoma.

Yes-associated protein ( YAP ) , a transcriptional co-activator, is a cardinal regulator of the Hippo tract ( 4 ) . When YAP is recruited to the karyon, written text of cell proliferation-promoting and anti-apoptotic cistrons is activated invariably ( 5, 6 ) . Recently, high-expression of YAP has been found in many types of tumours, including hepatocellular, colorectal, stomachic carcinoma, ovarian, chest, and lung malignant neoplastic diseases, and correlatives with hapless forecast ( 7-10 ) . These observations suggest that YAP might lend to a malignant cellular phenotype and therefore becomes an of import mark for anticancer drugs ( 11 ) .

Dobutamine is a man-made 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 intervention of congestive bosom failure, cardiogenic and infected daze ( 13 ) . A recent survey showed that dobutamine is able to rarefy YAP-dependent written text by suppressing its atomic translocation ( 14 ) .

In the present survey, we aimed to look into the consequence of dobutamine on the proliferation, programmed cell death and invasiveness of human osteogenic sarcoma cell line MG-63. Our consequences showed the utility of dobutamine for the intervention of osteogenic sarcoma malignant neoplastic disease.

Materials and methods

The human osteogenic sarcoma cell line MG-63 was obtained from Shanghai cell bank of Chinese academy of scientific disciplines. Dulbecco ‘s Modified Eagle ‘s Medium ( DMEM ) and foetal 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 class commercially available in China.

Cell civilization

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

MTT assay

The influence of dobutamine on the cell viability was determined harmonizing to MTT check. Cells were seeded on a 96-well home base overnight and stimulated with assorted concentrations of dobutamine ( 1 ?M, 5 ?M, 10 ?M, 25 ?M, 50 ?M ) for 12, 24, 48 and 72 hi??after the indicated interventions, the cells were incubated with MTT ( 0.25 mg/ml in PBS ) for 4 H, so the media were removed, and DMSO ( 1 milliliter of 100 % ) was added to solubilize the MTT-formazan merchandise. The sum of violet crystals reflecting cellular growing and viability was determined by the optical density at 490 nanometer. The repressive rate of cell growing was calculated as [ 1-treatment group/control group ) ] ?100 % . The growing curve was drawn utilizing clip as abscissa and suppression rate as ordinate. Each dosage was done in triplicate, and the experiments were repeated at least twice.

Flow cytometry analysis

The rate of programmed cell death and per centum of cells in G1, S and G2/M stage were measured by flow cytometry. Apoptosis analysis: after treated harmonizing to the experimental groups for 24 H, cells were harvested with trypsinase, washed twice with PBS, re-suspended in adhering buffer, so stained with Annexin V-FITC and PI following the manufacturer’s direction and processed by flow cytometry. The cell suspension was incubated with PI solution ( 50 ?g/ml ) and 50 units of RNase for 30 minfor cell rhythm sensing. Data acquisition and analysis were done on a BD ( Becton Dickinson ) FACSCaliber utilizing CellQuest package ( BD Biosciences ) .

Cell invasion analysis

The consequence of dobutamine on the invasion of MG-63 cells was investigated utilizing transwell Chamberss with polycarbonate filters ( pore size of 8 ?m ) . The cells were seeded on the upper chamber at a denseness of 1?105cells/ml and incubated in 0.6 milliliters DMEM medium incorporating 10 % FBS and assorted concentrations of drugs. The lower chamber was filled with 0.6 milliliters of DMEM medium incorporating 20 % FBS. After 24 H, cells on the upper filter were removed by pass overing, and so 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 degrees of protein look of caspase-3 and caspase-9 in MG-63 cell before and after intervention with dobutamine. Cells treated as indicated were harvested in 5 milliliter of medium, pelleted by centrifugation ( 1000 ? g for 5 min at 4 °C ) , so washed twice with ice-cold PBS and lysed in ice-cold HEPES buffer [ HEPES ( pH 7.5 ) 50 mmol/L, NaCl 10 mmol/L, MgCl25 mmol/L, EDTA 1 mmol/L, glycerol 110 % ( v/v ) , Triton X-100 1 % ( v/v ) , a cocktail of peptidase 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 a„? ) and the supernatants so either analyzed instantly or stored at -80a„? . Equivalent sums of protein ( 50 µg ) from entire cell lysates were resolved by SDS-PAGE utilizing precast 12 % Bis-Tris gradient gels and transferred onto polyvinylidene difluoride ( PVDF ) membranes. Membranes were blocked overnight at 4a„? in barricading 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 barricading buffer overnight at 4a„? . Unbound antibody was removed by rinsing with Tris-buffered saline ( pH 7.2 ) incorporating 0.5 % Tween 20 ( TBS-T ) . The membrane was so incubated at room temperature with horseradish peroxidase-conjugated secondary antibody. After extended rinsing with TBS-T, sets were visualized by enhanced chemiluminescence followed by exposure to autoradiography.

Statistical analysis

Statistical analyses were performed utilizing the SPSS 15.0 package bundle ( SPSS Inc. ) . Comparisons between two samples were employed by Student’s t-test. The trial degree was set to ?=0.05.

Consequences and Discussion

Dobutamine inhibits the proliferation ofostosarcomaMG-63 cells

Our consequences showed that dobutamine significantly inhibited cell proliferation in clip and concentration dependant manners compared with the control group. As shown on the proliferation suppression curve ( Figure 1 ) , three ( 10, 25, 50 ?M ) had important repressive consequence on the endurance of MG-63 cells out of the five tried concentrations of dobutamine (Phosphorus& A ; lt ; 0.05 ) .

Dobutamineaugmented cell programmed cell deathand arrested cell rhythm

Annexin V/PI staining was used to mensurate the dobutamine-induced programmed cell death. Compared with control group, dobutamine induced a important addition of apoptotic decease, after pretreated with 5 ?M, 10 ?M, 25 ?M and 50 ?M for 24 H in MG-63 cells (Phosphorus& A ; lt ; 0.05 ) ( Table 1 ) . The per centum of MG-63 cells in G2/M stages was significantly increased (Phosphorus& A ; lt ; 0.01 ) , at dobutamine concentration of 25 ?M, 50 ?M, and the per centum in S-phase was significantly decreased (Phosphorus& A ; lt ; 0.05 ) ( Figure 2 ) .

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

To look into whether dobutamine has the consequence on cell motion, we compared the migratory rate of the tumour cells in a wound-healing check. Figure 3 showed that dobutamine significantly decreased cell migration from the border of the lesion (Phosphorus& A ; lt ; 0.05 ) . Similarly, invasion check besides showed a big figure of cells passed through the filter in the control group, whereas the cells go throughing through the filter were markedly reduced after dobutamine intervention. Furthermore, the dobutamine reduced the figure of invasive cells in a concentration dependent mode ( Figure 4 ) . The figure of invasive cells in combination group was significantly reduced than that in entirely groups (Phosphorus& A ; lt ; 0.05 ) ( Figure 4 ) .

Dobutamine induces look of caspase-3 and caspase-9

Western smudge analysis was used to analyze the look of caspase-3 and caspase-9 in MG-63 cells following dobutamine intercessions. Protein look analysis indicated that caspase-3 was increased after treated with dobutamine at the concentration of 10 and 50 ?M for 72 H (Phosphorus& A ; lt ; 0.05 ) , and caspase-9 was increased after treated by dobutamine at the concentration of 50 ?M for 72 H (Phosphorus& A ; lt ; 0.05 ) ( Figure 5 ) .

Recent studies demonstrated that YAP protein is extremely expressed in human osteosarcoma MG-63 cells ( 15 ) . Our consequences indicate that the repressive consequence of dobutamine might be associated with the suppression of YAP protein translocation. Hushing of YAP cistron with RNA intervention led to the similar consequence as dobutamine ( 16 ) . In add-on, dobutamine apprehensions cell rhythm at G2/M passage and augmented cell programmed cell death. Previous surveies demonstrated that YAP activates cell programmed cell death in response to DNA harm by interacting with p73 in several malignant neoplastic disease cell lines ( 17 ) .

Decision

In drumhead, our consequences showed that dobutamine can significantly suppress osteosarcoma cell growing by suppressing cell proliferation, bring oning cell programmed cell death and redistributing cell rhythm. These informations indicates dobutamine may go a new curative agent for the intervention of osteogenic sarcoma. However, farther in vivo surveies are required to corroborate the effectivity and safety of dobutamine in the intervention of osteogenic sarcoma.

Figure fables

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

Figure 2 Cell rhythm ( a ) and apoptosis distribution ( B ) of MG-63 cells in response to dobutamine intervention. A: control B: 10 ?M. C: 25 ?M. Calciferol: 50 ?M. Data was mean± SD ( n=6 ) . * indicated the important difference compared with the control group (Phosphorus& A ; lt ; 0.05 ) .

Figure 3Consequence of dobutamine intervention on the migration of MG-63 cells. Data was average ± SD ( n=6 ) . * indicated the important difference between control and the intervention (Phosphorus& A ; lt ; 0.05 ) .

Figure 4The consequence of dobutamine on MG-63 cell invasiveness. The cells were treated with different concentrations of dobutamine for 24 h. a: control B: 1 ?M. degree Celsius: 5 ?M. vitamin D: 10 ?M. vitamin E: 25 ?M. degree Fahrenheit: 50 ?M. The figure of invasive cells of MG-63 after dobutamine intervention for 24 h. Data was average ± SD ( n=6 ) . * indicated the important difference between control and the intervention (Phosphorus& A ; lt ; 0.05 ) .

Figure 5The consequence of doubtamine on protein look of caspase-3 and 9 in MG-63 cells. a: western smudge analysis, B: protein look of caspase-3 and 9. The cells were treated with assorted concentrations of dobutamine ( 0, 1, 10, and 50 µM ) for 72 h. GAPDH was used as endogenous control cistron. The look degree was calculated as the ratio of caspase-3 or caspase-9/GAPDH. Data was average ± SD ( n=6 ) . *indicated the important difference between control and the intervention.

Table 1 Cell rhythm distribution and programmed cell death of MG-63 cells

Groups

Cell rhythm

Apoptosis %

G0/G1

Second

G2/M

Control

54.12±6.53

25.60±5.33

20.28±1.79

2.2±0.4

DOBUTAMINE ( 1 ?M )

55.19±3.16

25.37±3.66

19.44±5.01

2.5±1.1

DOBUTAMINE ( 5 ?M )

53.96±4.58

23.18±6.34

22.86±4.27

2.9±1.7

DOBUTAMINE ( 10 ?M )

49.82±2.99

26.78±5.92

23.40±6.52

7.0±2.5*

DOBUTAMINE ( 25 ?M )

50.62±5.27

21.50±4.59

27.88±4.55*

11.6±4.7*

DOBUTAMINE ( 50 ?M )

51.21±6.52

18.39±2.79

30.40±7.26*

13.2±1.8*

*Statistically important difference from control group

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