精胺氧化酶抑制剂SI-4650抑制人恶性黑素瘤A375细胞增殖的分子机制研究

Molecular mechanism underlying the inhibitory effect of spermine oxidase inhibitor SI-4650 on proliferation of a human malignant melanoma cell line A375

目的探讨精胺氧化酶抑制剂SI-4650对人恶性黑素瘤A375细胞增殖的影响及其机制。方法取0、10、20、40、80、160μmol/LSI-4650作用A375细胞24、48、72h,MTT法分析SI-4650对细胞增殖抑制率的影响。根据增殖活性,筛选出浓度分别为0(对照组)、40、80μmol/L的SI-4650处理A375细胞48h,化学发光法检测细胞内精胺氧化酶活性;高效液相色谱法(HPLC)检测细胞内多胺含量;流式细胞仪分析细胞周期及细胞凋亡;Western印迹分析凋亡标志蛋白Bax、c-PARP,凋亡抑制蛋白Bcl-2和自噬标志蛋白Beclin-1、LC3-Ⅱ的表达。多组均数比较采用单因素方差分析,组间比较采用SNK-q检验。结果不同浓度、不同处理时间的SI-4650对A375细胞增殖抑制率差异均有统计学意义(F=977.23、5.16,均P<0.001)。对照组、40μmol/L组、80μmol/L组A375细胞精胺氧化酶活性(F=242.58,P<0.001)、精脒和总多胺含量(F=338.02、2931.07,P<0.001)、S期细胞比例(F=31.66,P<0.001)、凋亡细胞比例(F=100.68,P<0.001)、凋亡相关蛋白Bax、c-PARP、Bcl-2水平(F=35.51、730.11、27.54,P<0.001)、自噬标志蛋白Beclin-1、LC3-Ⅱ水平(F=35.87、425.04,P<0.001)差异均有统计学意义;40μmol/L组、80μmol/L组精胺氧化酶活性(发光强度61432.85±2620.92、43337.35±1221.25)低于对照组,精脒(1.97±0.007、1.88±0.006)和总多胺(3.18±0.03、2.81±0.01)含量低于对照组,S期细胞比例(27.61%±2.05%、31.58%±1.45%)高于对照组,凋亡细胞比例(7.59%±0.63%、20.14%±2.27%)高于对照组,凋亡标志蛋白Bax(0.83±0.12、1.18±0.16)、c-PARP(0.32±0.002、0.79±0.035)和自噬标志蛋白Beclin-1(1.00±0.007、1.14±0.003)、LC3-Ⅱ(0.31±0.001、0.98±0.003)水平高于对照组,凋亡抑制蛋白Bcl-2水平(0.65±0.09、0.12±0.002)低于对照组(均P<0.05)。结论SI-4650能抑制A375细胞增殖,其机制可能与干扰多胺代谢和诱导细胞周期阻滞、细胞凋亡和自噬相关。

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 160μmol/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(0,40,80μmol/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=338.02,2 931.07 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.