Anti-osteosarcoma activity and underlying molecular mechanism of a novel small molecule spermine oxidase inhibitor SI-4650 / 药学学报

The purpose of this study is to further explore the effects of SI-4650, a newly discovered small molecule inhibitor of spermine oxidase (SMO) in our laboratory, on proliferation and migration of human osteosarcoma 143B cells and its underlying molecular mechanism. Chemiluminescence and high performance liquid chromatograph were used to analyze the effect of SI-4650 on SMO activity in 143B cells. DCFH-DA-staining/FCM was used to analyze the accumulation of cellular reactive oxygen species (ROS), whereas MTT and FCM were used to detect proliferation and cell cycle. Transwell culture and Western blot were used to analyze the expression levels of migration-related proteins. PI/FITC-Annexin V/FCM, fluorescence microscopy and Western blot were used to analyze apoptosis and autophagy. Our results showed that SI-4650 could significantly decrease SMO activity, inhibit cell proliferation or migration, and induce a S-phase cell cycle arrest in 143B human osteosarcoma cells. The mechanism may be related to interfering with polyamine metabolism, activating mitochondrial-mediated apoptosis and causing autophagic death. These results suggest that SI-4650 has the potential for clinical use in treatment of osteosarcoma.

Experimental research of Hedyotic diffusa injection on the inhibition of the proliferation of human osteosar- coma cell line MG-63 in vitro / 中国基层医药

Objective To investigate the inhibition of Hedyotic diffusa injection on the proliferation of hu-man osteosarcoma cell line MG -63 in vitro.Methods The human osteosarcoma cell line MG -63 was subcultivated aseptically in vitro.Different concentration of Hedyotic diffusa injection (50μL/mL,100μL/mL,200μL/mL,400μL/mL) successively acted on such cell .A total three time points were selected to determine the activity and numbers of cells by MTT assay including 12h,24h and 48h.Results The cellular proliferation inhibition rates of human osteosarcoma cell line MG-63 in drug groups of 50μL/mL concentration holes were (2.87 ±2.22)%,(13.42 ±2.14)% and (30.80 ±3.67)%after 12 h,24 h and 48 h.The rates of 100μL/mL concentration holes were (22.25 ±1.58)%, (43.34 ±2.84)%and (66.46 ±2.64)%,after 12 h,24 h and 48 h.The rates gradually increased and had statis-tical meaning,t12h =12.319,t24h =14.573,t48h =12.319,P<0.05;the cell in drug groups of 200μL/ml and 400μL/ml concentration holes was totally dead , and pathological findings showed that there were circular and floating cells in which nucleoplasmic ratio decreased and small fragments of cells increased .Conclusion Hedyotic diffusa extract has a certain inhibition in vitro on the proliferation of human osteosarcoma cell line MG -63.Moreover,the strength of its inhibition is relevant probably with drug concentration ,which deserves a further research .

Effect of Ginsenoside Rh2 on apoptosis of human osteosarcoma cell line MG-63 / 中国病理生理杂志

AIM:To investigate the effect of Ginsenoside Rh2 (Rh2) on the apoptosis of human osteosarcoma cell line MG-63.METHODS:The cell viability was determined by MTT assay .MG-63 cell apoptotic rate was examined by flow cytometry with Annexin V-PI double staining.The expression of Bcl-2, Bax, cytochrome C ( Cyt C) and cleaved caspase-3 were measured by Western blot .RESULTS: Rh2 enhanced the apoptosis of MG-63 cells in a dose-dependent manner.Furthermore, after treatment with Rh2, the release of mitochondrial Cyt C and Bax expression were increased , while Bcl-2 and the ratio of Bcl-2/Bax were decreased as compared with control group ( P<0.05 ) .The protein level of cleaved caspase-3 was also increased (P<0.05).CONCLUSION:Ginsenoside Rh2 accelerates the apoptosis of MG-63 cells through mitochondria-dependent pathway , suggesting that Rh2 is a novel approach for the treatment of osteosarcoma .

Knockdown of Notch-1 augments inhibitory effect of dihydroartemisinin on viability of human osteosarcoma cell line U-2OS / 中国病理生理杂志

AIM:To investigate the effect of Notch-1 knockdown on the growth of dihydroartemisinin-inhibited human osteosarcoma cell line U-2OS.METHODS:U-2OS cells treated with different concentrations of dihydroartemisinin (5, 10, 15 and 20μmol/L) were collected.The expression of Notch-1, MMP-2, MMP-9 and Hes-1 at mRNA and protein levels was measured by real-time PCR and Western blotting, respectively.U-2OS cells were transfected with Notch-1 siRNA for 24 h and incubated with dihydroartemisinin for another 24 h.The cell apoptotic rate , protein expression of MMP-2, MMP-9 and Hes-1, and the migration ability were measured by MTT assay , Western blotting and Transwell experiment , respectively.RESULTS:Dihydroartemisinin (5, 10, 15 and 20 μmol/L) decreased the expression of Notch-1, MMP-2, MMP-9 and Hes-1 at mRNA and protein levels in a dose-dependent manner .Down-regulation of Notch-1 significantly en-hanced the effect of dihydroartemisinin on the cell apoptosis , the protein expression of MMP-2, MMP-9 and Hes-1, and mi-gration ability ( P<0.05 ) .CONCLUSION: Notch-1 pathway is involved in the process of dihydroartemisinin-inhibited U-2OS cell growth.Knockdown of Notch-1 augments the inhibitory effect of dihydroartemisinin on U-2OS cell viability.

miR-30 a suppresses migration, invasion and vitality of human osteosarcoma cell line 143 B / 基础医学与临床

Objective To investigate the effect of miR-30a on human osteosarcoma cell 143B in migration,invasion andcellviability.Methods 143BcellswereinfectedortransfectedwithrecombinantadenovirusmiR-30a(Ad-miR30a) and miR-30a inhibitor respectively .Wound healing assay was performed to detect the cell healing ability ( P<0.05 ) .Cell migration and invasion ability were determined by Transwell assay ( P<0.05 ) .The cell viability was analyzed by MTT assay ( P<0.01 ) .Real-time quantitative PCR was performed to analyze the expression of RUNX2 mRNA level and confirmed the adenovirus miR-30a expressed in 143B cells.The expression of RUNX2 was analyzed by Western blot .miR-30a target to RUNX2 was verified by luciferase reported gene assay .Results The ability of migration and invasion was suppressed in osteosarcoma cell 143B by overexpression miR-30a,and the cell viability also decreased .After the endogenous miR-30 a being inhibited , the cell motility and invasion enhanced and the cell viability was promoted .The RUNX2 protein decreased after overexpression miR-30 a as compared with controlgroup.TheluciferaseactivityofRUNX2decreasedbyaddingmiR-30a.Conclusions 143Bcellmigration, invasion and viability were suppressed by miR-30a,and this process is potentially achieved via suppressing RUNX 2 protein expression .

The effect of exposure to 50 Hz magnetic fields on the proliferation of different cell densities of human MG-63 osteosarcoma cell line in vitro / 中华物理医学与康复杂志

Objective To study the effects of 50 Hz magnetic fields on the in vitro proliferation of human osteosareoma cell line MG-63 with different cell densities.Methods Four different magnetic intensities(1 mT, 2 mT,3 roT,4 mT)were used to stimulate the cells,and the experiment was repeated with different cell densities. The method of MTT was employed to evaluate the level of proliferation.Results Fifty Hz magnetic fields signifi- cantly affected the level of proliferation of human osteosareoma cell line MG-63,and the 2 mT intensity exerted the greatest influence on it.The effects of the magnetic field differed with different cell densities.Conclusion The effect of 50 Hz magnetic fields on the in vitro proliferation of human osteosarcoma cell line MG-63 was not only relat- ed to the magnetic intensity,but also the cell density,

The Expression of Insulin Receptor Substrate in Human Osteoblast / 中国医师杂志

Objective To determine the expression of insulin receptor substract(IRS) family in human osteosarcoma cell line MG-63 and cultured normal human osteoblast-like cells (HOB). Methods The mRNA and protein expression of IRS family was measured using semi-quantitative RT-PCR and western blot analysis, respectively. Results MG-63 cells and HOB had the mRNA and protein expressions of IRS-1,-2,-3, and -4. Among IRS family, the expressions levels of IRS-1 mRNA and protein were the highest, and those of the IRS-4 mRNA and protein were the lowest in MG-63 cell line and HOB. Conclusion MG-63 and HOB can express the mRNA and protein of IRS family members, and the expressional levels of IRS family members were different.