ABSTRACT
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.
ABSTRACT
Objective To evaluate the effect of spermine oxidase(SMO)inhibitor SI-4650 on the proliferation of a human malignant melanoma cell line A375, and to explore its molecular mechanism. Methods Some cultured A375 cells were divided into 6 groups to be treated with SI- 4650 at concentrations of 0, 10, 20, 40, 80 and 160 μmol/L respectively for 24, 48 and 72 hours, and methyl thiazolyl tetrazolium(MTT)assay was performed to evaluate changes in cellular proliferative activity. According to the cellular proliferative activity, 3 concentrations (0, 40, 80 μmol/L) were screened out. Some A375 cells were divided into 3 groups to be treated with 0(control group), 40 and 80μmol/L SI-4650 for 48 hours. Chemiluminescence assay was conducted to detect the SMO activity in A375 cells, high-performance liquid chromatography(HPLC)analysis to determine the polyamine content in A375 cells,flow cytometry to analyze the cell cycle and detect the apoptosis, and Western blot analysis to determine the protein expression of apoptotic marker proteins Bax and c-PARP, inhibitor of apoptosis protein Bcl-2, and autophagy marker proteins Beclin-1 and LC3-Ⅱ. Statistical analysis was carried out by using one-way analysis of variance for comparison of means among several groups, and by using Student-Newman-Keuls (SNK)-q test for multiple comparisons. Results MTT assay showed that there was a significant difference in the proliferative activity of A375 cells after the treatment with different concentrations of SI-4650 for different durations(F=977.23, 5.16 respectively, both P<0.001). Significant differences were observed in the SMO activity in A375 cells(F=242.58, P<0.001), spermine and the total polyamine content(F=338.02, 2931.07 respectively, both P < 0.001), proportion of S-phase cells (F = 31.66, P < 0.001), proportion of apoptotic cells(F=100.68, P<0.001), expression of apoptosis-related proteins Bax, c-PARP and Bcl-2(F = 35.51, 730.11, 27.54 respectively, all P < 0.001), and expression of autophagy marker proteins Beclin-1 and LC3-Ⅱ(F = 35.87, 425.04 respectively, P < 0.001)among the control group, 40-and 80-μmol/L SI-4650 groups. Compared with the control group, the 40-and 80-μmol/L SI-4650 groups showed significantly lower SMO activity(luminous intensity:61432.85 ± 2620.92, 43337.35 ± 1221.25 respectively, both P<0.05), lower spermine(1.97 ± 0.007, 1.88 ± 0.006 respectively, both P<0.05)and total polyamine content (3.18 ± 0.03, 2.81 ± 0.01 respectively, both P < 0.05), higher proportions of S-phase cells (27.61％ ± 2.05％, 31.58％ ± 1.45％ respectively, both P < 0.05) and apoptotic cells (27.61％ ± 2.05％, 31.58％ ± 1.45％ respectively, both P < 0.05), higher expression of apoptotic marker proteins Bax(0.83 ± 0.12, 1.18 ± 0.16 respectively, both P < 0.05)and c-PARP(0.32 ± 0.002, 0.79 ± 0.035 respectively, both P < 0.05)and autophagy marker proteins Beclin-1(1.00 ± 0.007, 1.14 ± 0.003 respectively, both P < 0.05)and LC3-Ⅱ(0.31 ± 0.001, 0.98 ± 0.003 respectively, both P < 0.05), and lower expression of inhibitor of apoptosis protein Bcl-2(0.65 ± 0.09, 0.12 ± 0.002 respectively, both P<0.05). Conclusion SI-4650 can inhibit the proliferation of A375 cells, likely by interfering with polyamine metabolism and inducing cell cycle arrest, apoptosis and autophagy.
ABSTRACT
Objective@#To evaluate the effect of spermine oxidase (SMO) inhibitor SI-4650 on the proliferation of a human malignant melanoma cell line A375, and to explore its molecular mechanism.@*Methods@#Some cultured A375 cells were divided into 6 groups to be treated with SI-4650 at concentrations of 0, 10, 20, 40, 80 and 160 μmol/L respectively for 24, 48 and 72 hours, and methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate changes in cellular proliferative activity. According to the cellular proliferative activity, 3 concentrations (0, 40, 80 μmol/L) were screened out. Some A375 cells were divided into 3 groups to be treated with 0 (control group) , 40 and 80 μmol/L SI-4650 for 48 hours. Chemiluminescence assay was conducted to detect the SMO activity in A375 cells, high-performance liquid chromatography (HPLC) analysis to determine the polyamine content in A375 cells, flow cytometry to analyze the cell cycle and detect the apoptosis, and Western blot analysis to determine the protein expression of apoptotic marker proteins Bax and c-PARP, inhibitor of apoptosis protein Bcl-2, and autophagy marker proteins Beclin-1 and LC3-Ⅱ. Statistical analysis was carried out by using one-way analysis of variance for comparison of means among several groups, and by using Student-Newman-Keuls (SNK) -q test for multiple comparisons.@*Results@#MTT assay showed that there was a significant difference in the proliferative activity of A375 cells after the treatment with different concentrations of SI-4650 for different durations (F = 977.23, 5.16 respectively, both P < 0.001) . Significant differences were observed in the SMO activity in A375 cells (F = 242.58, P < 0.001) , spermine and the total polyamine content (F = 338.02, 2 931.07 respectively, both P < 0.001) , proportion of S-phase cells (F = 31.66, P < 0.001) , proportion of apoptotic cells (F = 100.68, P < 0.001) , expression of apoptosis-related proteins Bax, c-PARP and Bcl-2 (F = 35.51, 730.11, 27.54 respectively, all P < 0.001) , and expression of autophagy marker proteins Beclin-1 and LC3-Ⅱ (F = 35.87, 425.04 respectively, P < 0.001) among the control group, 40- and 80-μmol/L SI-4650 groups. Compared with the control group, the 40- and 80-μmol/L SI-4650 groups showed significantly lower SMO activity (luminous intensity: 61 432.85 ± 2 620.92, 43 337.35 ± 1 221.25 respectively, both P < 0.05) , lower spermine (1.97 ± 0.007, 1.88 ± 0.006 respectively, both P < 0.05) and total polyamine content (3.18 ± 0.03, 2.81 ± 0.01 respectively, both P < 0.05) , higher proportions of S-phase cells (27.61% ± 2.05%, 31.58% ± 1.45% respectively, both P < 0.05) and apoptotic cells (27.61% ± 2.05%, 31.58% ± 1.45% respectively, both P < 0.05) , higher expression of apoptotic marker proteins Bax (0.83 ± 0.12, 1.18 ± 0.16 respectively, both P < 0.05) and c-PARP (0.32 ± 0.002, 0.79 ± 0.035 respectively, both P < 0.05) and autophagy marker proteins Beclin-1 (1.00 ± 0.007, 1.14 ± 0.003 respectively, both P < 0.05) and LC3-Ⅱ (0.31 ± 0.001, 0.98 ± 0.003 respectively, both P < 0.05) , and lower expression of inhibitor of apoptosis protein Bcl-2 (0.65 ± 0.09, 0.12 ± 0.002 respectively, both P < 0.05) .@*Conclusion@#SI-4650 can inhibit the proliferation of A375 cells, likely by interfering with polyamine metabolism and inducing cell cycle arrest, apoptosis and autophagy.
ABSTRACT
Aim To evaluate the effect of expression inhibition of spermine oxidase(SMO)on the actitumor activity of polyamine analogue CPENSpm (N~1-cyclopropylmethyl-N~(11)-ethylnorspermine).Methods siRNA technique was used to inhibit expression of SMO in human lung cancer line A549.QT-RT-PCR and enzyme activity assay was performed to determine the expression level of SMO.The cell proliferation was detected by MTT assay.The apoptosis of A549 cells were evaluated by DNA degradation and Sub-G_1/flow cytometry assay.Results The A549 cell line with silenced SMO expression was successfully obtained.Basic SMO mRNA and enzyme activity levels in the SMO-siRNA plasmid transfected cells were 0.53% and 14% lower than that in the control cells respectively. Treating A549 control cells by 10 μmol·L~(-1) CPENSpm for 24 hours resulted in a 10-folds up-regulation of SMO in mRNA level and 20-fold increase in enzyme activity,but this drug-induced SMO expression was obviously prevented in SMO-siRNA plasmid transfected cells.MTT assay demonstrated that SMO expression inhibition decreased the sensitivity of A549 cells to CPENSpm exposure(0～20 μmol·L~(-1)).DNA degradation and sub-G_1 assay proved a deceased ability of CPENSpm to induce apoptosis in SMO-siRNA plasmid transfected cells.Conclusion Up-regulation of SMO by CPENSpm is possibly one of the molecular basics for its antitumor activity.
ABSTRACT
AIM: To prepare recombinant human spermine oxidase (SMO) and polyclonal antibody against human SMO by gene recombination techniques. METHODS: Human SMO cDNA was amplified from total RNA of A549 cells through reverse transcription PCR. The cDNA was then cloned into pET-15b to construct SMO prokaryotic expression vector. After transforming, the vector was induced to express recombinant SMO by IPTG in E. coli BL21 (DE_3). Recombinant SMO was purified by Ni-NTA resin under denaturing condition and then was dialyzed to renature. The enzyme activity of recombinant SMO was analyzed by chemical fluorescent method. SMO polyclonal antibody was prepared by using recombinant human SMO protein purified by polyacrylamide gel electrophoresis as antigen to inoculate rabbit intradermally. The titer and specificity of anti-sera were determined by ELISA, Western blot and Immune Cell Chemistry. RESULTS: Purified and dialyzed recombinant human SMO has the specificicity of oxidizing the spermine. The polyclonal antibody has high titer and specificity against human SMO. CONCLUSION: This research established a method for prokaryotic expression, purification and polyclonal antibody preparation of human SMO. The method lays a foundation for the future functional research of SMO.