| Part 1 The effects of PARP-1 inhibitor on proliferation and apoptosis of human osteosarcoma cellsObjectThe effects of PARP-1 inhibitor on proliferation and apoptosis of human osteosarcoma cells were investigated.Methods1. The U2OS cells were cultured with McCoy's 5a Medium Modified containing 10% fetal bovine serum supplemented with penicillin (100 U/ml) and streptomycin (100μg/ml), and the MG-63 cells were cultured with DMEM containing 10% fetal bovine serum supplemented with penicillin (100 U/ml) and streptomycin (100μg/ml).2. The U2OS cells were seeded in three 96-well plates, respectively. The cells in the plate were divided into 6 groups:the blank group, the control group, the 5mM 3-AB group, the 10mM 3-AB group, the 15mM 3-AB group, the 20mM 3-AB group, each group have 4 wells. The three plates were treated with varying concentrations of 3-AB for 24, 48 and 72 h, the viability of the cells was assessed by the Cell count kit-8.3. The MG-63 cells were seeded in three 96-well plates, respectively. The cells in the plate were divided into 5 groups:the blank group, the control group, the 5mM 3-AB group, the lOmM 3-AB group, the 20mM 3-AB group, each group has 4 wells. The three plates were treated with varying concentrations of 3-AB for 24,48 and 72 h, the viability of the cells was assessed by the Cell count kit-8.4. The U2OS cells were seeded in 6 plates with a 6-cm diameter, respectively. The plates were divided into two groups:cells in one group were cultured with complete growth medium, while in the other group were cultured with complete growth medium plus 10mM 3-AB. Cells were harvested at 24,48 and 72 h after incubation, the cells were stained with annexinⅤ-FITC and propidium iodide, and then analyzed by flow cytometry.5. The MG-63 cells were seeded in 8 plates with a 6-cm diameter, respectively. The plates were divided into four groups:cells in one group were cultured with complete growth medium, while in the other group were cultured with complete growth medium plus 5mM 3-AB or 10mM 3-AB,20mM 3-AB. Cells were harvested at 24,48h after incubation, the cells were stained with annexinⅤ-FITC and propidium iodide, and then analyzed by flow cytometry.6. The U2OS cells were seeded in 4 plates, respectively. One plate was used as a control, the other three plate were treated with 10 mM 3-AB for 8,12 and 24 h, and then the cells were harvested, the caspase-3 activity was assayed by caspase-3 colorimetric assay kit.7. The U2OS cells were seeded in 4 plates, respectively. One plate was served as a control, the other three plate were treated with 10 mM 3-AB for 24,48 and 72 h, and then the cells were lysed, the cell extracts were collected, and the expression of Bcl-2 and Bax were analyzed with Western-blotting. Results1.3-AB suppressed the proliferation of U2OS cells in a dose and time dependent manner.2.3-AB suppressed the proliferation of MG-63 cells in a dose and time dependent manner.3.3-AB induced apoptosis of U2OS cells, and the apoptosis ratio increased with the extension of time4.3-AB induced apoptosis of MG-63 cells, and the apoptosis ratio increased with the increased concentration and the extension of time.5. The activity of caspase-3 in U2OS cells started to increase after 8 h treatment with 10 mM 3-AB and at 12 h the caspase-3 activity was significant higher than the control, at 24 h the activity decreased, approximately equal to the control group.6. Western blot analyses showed that in U2OS cells Bax gradually increased after treated with 3-AB for 24,48 and 72 h, but the Bcl-2 seemed to increased after 24 h and 48 h of treatment. At longer times of 3-AB treatment (72 h) Bcl-2 returned to initial levels.ConclusionPARP-1 inhibitor 3-AB suppresses proliferation and induced apoptosis of osteosarcoma cells. Part 2 The effects of PARP-1 inhibitor on invasion of human osteosarcoma cellsObjectThe effects of PARP-1 inhibitor on invasion of human osteosarcoma cells were investigated.Methods1. The U2OS cells were cultured as mentioned above.2. The U2OS cells were seeded in 6-well plates, three scratches were crossed with 200μl tips in each well, the wells were divided into 3 groups, one served as control, and the other two were treated with 5mM 3-AB or lOmM 3-AB, after 24h the migration distance was measured.3. The Transwell inserts coated with Matrigel Basement Membrane Matrix were placed over the bottom chamber, Serum containing medium (10%FBS) were used as chemo-attractant. Serum free medium containing 0.1% BSA served as negative control. U2OS cells were divided into two groups, one group served as a control, while the second group was pretreated with 10 mM 3-AB for 24 h,30000 cells were suspended in 100μl in serum free medium containing 0.1% BSA and added to the upper chamber. After 24 h of incubation at 37℃, the non-migrated cells on the upper surface of the filter were removed by gentle swabbing with cotton tipped applicators. The cells that had migrated to the lower side of the chamber were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Migrated cells were scored in 8 random fields using 10×objectives.Results1. The migration distance decreased after treated with 3-AB as compared with control group, and as the concentration increase, the distance decreased more obvious. 2.3-AB decreased the cell number cross through the membrane.ConclusionPARP-1 inhibitor 3-AB suppresses migration and invasion of osteosarcoma cells. Part 3 The effects of PARP-1 inhibitor co-treated with cisplatin on growth of human osteosarcoma cellsObjectThe anticancer effects of cisplatin co-treated with PARP-1 inhibitor 3-aminobenzamide (3-AB) on human osteosarcoma cells were investigated.Methods1. The U2OS cells were cultured as mentioned above.2. The U2OS cells were seeded in two 96-well plates, one plate were treated with different concentration of cisplatin and the other treated with different concentration cisplatin plus 3-AB, each plate was divided into 7 groups:the blank group, the control group, 0.5μg/ml cisplatin with or without 3-AB treated group, 1.0μg/ml cisplatin with or without 3-AB treated group,1.5μg/ml cisplatin with or without 3-AB treated group, 2.0μg/ml cisplatin with or without 3-AB treated group,2.5μg/ml cisplatin with or without 3-AB treated group, after 48h treatment, the viability of the cells was assessed by the Cell count kit-8.3. The U2OS cells were seeded in 12 plates with a 6-cm diameter, respectively. The plates were divided into four groups:control group,3-AB treated group, cisplatin treated group, cisplatin plus 3-AB treated group, each group have 3 plates, cells were harvested at 24,48 and 72 h after incubation, the cells were stained with propidium iodide (PI), and then analyzed by flow cytometry.Results1.3-AB sensitized U2OS to cisplatin by factor of 1.76.2. Compared with the control group, group treated with cisplatin or 3-AB alone for 24,48, 72 h had an increased apoptosis indice, and when cisplatin was combined with 3-AB, the apoptosis indices was higher then treated with cisplatin or 3-AB alone. 3. Compared to control, cell cycle kinetics was unaffected by 10 mM 3-AB alone, while 10μM cisplatin induced a S phase accumulation, and then convert to G2/M accumulation,10 mM 3-AB plus 10μmol cisplatin induced a more sustained S phase arrest.ConclusionPARP-1 inhibitor enhances the anti-proliferative and apoptosis-inducing effects of cisplatin in human osteosarcoma cells. |
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