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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.
RESUMO
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.
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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.
