灰黄霉素对人小细胞肺癌NCI-H446细胞的体内外作用及机制探讨
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摘要
目的:观察灰黄霉素在体内外对人小细胞肺癌细胞株NCI-H446抗肿瘤作用。
方法:①以不同浓度的灰黄霉素作用于NCI-H446细胞48小时,CCK-8法测定细胞的存活率;采用倒置显微镜、荧光显微镜、透射电镜观察细胞形态及超微结构的变化;采用流式细胞仪检测NCI-H446细胞周期的变化;AnnexinⅤ-FITC/PI双标记检测NCI-H446细胞的凋亡;比色法检测Caspase-3, Caspase-9活性变化;Western blotting检测Bax,Bcl-2,Caspase-3和细胞色素C的表达。②通过划痕实验、transwell小室及matrigel胶体外检测药物对NCI-H446细胞迁移、侵袭及粘附的影响。③用灰黄霉素在NCI-H446肺癌细胞荷瘤裸鼠的瘤体内进行注射治疗,观察肿瘤生长变化,两周后处死裸鼠,摘除瘤体称重量,通过免疫组化法检测Bcl-2、Bax、Caspase-3、p53和VEGF等细胞因子的表达。
结果:①灰黄霉素能显著抑制NCI-H446细胞的增殖,半数抑制率浓度为24.58±1.32μM;显微镜下可见有典型的细胞凋亡特征性形态学改变。G2/M期细胞数增多,S期和G0/G1期细胞数减少,同时伴有亚二倍体凋亡峰出现。灰黄霉素可活化NCI-H446细胞的caspase-3、caspase-9并上调Bax和细胞浆中细胞色素C的表达,下调Bcl-2的表达。②灰黄霉素可明显抑制NCI-H446细胞与matrigel的粘附及其对人工基底膜的侵袭力和迁移力,且抑制作用随剂量的升高而增强,划痕实验结果也显示灰黄霉素处理组细胞与对照组细胞相比迁移能力降低。③灰黄霉素瘤内注射小细胞肺癌细胞株NCI-H446裸鼠移植瘤后能显著抑制肿瘤生长,免疫组化结果显示灰黄霉素能明显上调细胞因子Bax、p53和Caspase-3的表达和下调Bcl-2与VEGF的表达。
结论:灰黄霉素能抑制肺癌NCI-H446细胞的生长,诱导NCI-H446细胞凋亡;灰黄霉素具有抑制NCI-H446细胞粘附、侵袭与转移的作用;灰黄霉素能抑制NCI-H446细胞的体内致瘤能力。
Objective: To study the anti-tumor effects of griseofulvin on human small cell lung cancer cell line NCI-H446 in vitro and in vivo.
Methods:①N CI-H446 cells were treated with different concentrations of griseofulvin for 48h.CCK-8 assay was used to determine the cell survival rate;The changes of morphology and ultrastructure were observed by inverted microscope,fluorescence microscope and transmission electron microscope;Cell cycle and apoptosis rate were analysed by flow cytometry(FCM);AnnexinⅤ-FITC apoptosis detection kit was used to detect the apoptosis of cells;The enzyme activity changes of Caspase-3 and Caspase-9 were detected by spectrophotometry;Western blotting was used to detect the changes of Bax,Bc1-2,and cytochrome C in cytosol of NCI-H446 cells in the protein level.②The migration,invasion and adhesion ability of NCI-H446 cells were detected by scratch test,transwell chamber assay and Transwell-Matrigel model respectively.The cell adhesion assay was carried out on 96-well plate precoated with matrigel.③For in vivo study, we first established the NCI-H446 tumor model by grafting NCI-H446 cells into the athymic nude mice,and then injected griseofulvin into the tumors.Two weeks after injection,we sacrificed the mice and removed the tumors,then weighed and calculated the ratios of tumor-suppression.We also detected the expressions of Bcl-2、Bax、Caspase-3、p53 and VEGF with immunohistochemistry.
Results:①CCK-8 assay results showed that griseofulvin could significantly inhibit the proliferation of NCI-H446 cells in vitro in a dose-dependant manner,and the IC50 of griseofulvin was 24.58±1.32μM.Typical morphological changes of NCI-H446 cells were observed after induced by griseofulvin. The cell cycle distribution of NCI-H446 were changed,the number of cell increased in the stage of G2/M and decreased in the stage of S and G0/G1, accompanied with the subdiploid apoptotic peak.After 48 hours treatment with griseofulvin,the rates of apoptosis of griseofulvin-treated NCI-H446 cells greatly increased in high concentration group but did not obviously change in low concentration group compared with control group.Colorimetric assay results showed that griseofulvin could activated caspase-3 and caspase-9;Western blotting results indicated that the expression of cytochrome C and Bax were increased but Bcl-2 was decreased by griseofulvin.②The adhesion,metastatic potential and invasiveness of NCI-H446 cells were obviously inhibited by griseofulvin in a dose-dependent manner.The result of scratch test also indicated that the migration ability of NCI-H446 cells in griseofulvin treated group was decreased compared with control group.③The in vivo data showed that griseofulvin inhibited the tumor growth conspicuously through down-regulating the expression of Bcl-2,VEGF and up-regulating the expression of Bax,caspase-3 and p53.The ratios of tumor weight-suppression of griseofulvin group was 42.07 % .
Conclusion: Griseofulvin can inhibit the growth and induce apoptosis of NCI-H446 cells;griseofulvin inhibits the adhesion, invasiveness and migration of NCI-H446 cells in vitro;griseofulvin inhibits the tumorigenicity of NCI-H446 cells in vivo.
方法:①以不同浓度的灰黄霉素作用于NCI-H446细胞48小时,CCK-8法测定细胞的存活率;采用倒置显微镜、荧光显微镜、透射电镜观察细胞形态及超微结构的变化;采用流式细胞仪检测NCI-H446细胞周期的变化;AnnexinⅤ-FITC/PI双标记检测NCI-H446细胞的凋亡;比色法检测Caspase-3, Caspase-9活性变化;Western blotting检测Bax,Bcl-2,Caspase-3和细胞色素C的表达。②通过划痕实验、transwell小室及matrigel胶体外检测药物对NCI-H446细胞迁移、侵袭及粘附的影响。③用灰黄霉素在NCI-H446肺癌细胞荷瘤裸鼠的瘤体内进行注射治疗,观察肿瘤生长变化,两周后处死裸鼠,摘除瘤体称重量,通过免疫组化法检测Bcl-2、Bax、Caspase-3、p53和VEGF等细胞因子的表达。
结果:①灰黄霉素能显著抑制NCI-H446细胞的增殖,半数抑制率浓度为24.58±1.32μM;显微镜下可见有典型的细胞凋亡特征性形态学改变。G2/M期细胞数增多,S期和G0/G1期细胞数减少,同时伴有亚二倍体凋亡峰出现。灰黄霉素可活化NCI-H446细胞的caspase-3、caspase-9并上调Bax和细胞浆中细胞色素C的表达,下调Bcl-2的表达。②灰黄霉素可明显抑制NCI-H446细胞与matrigel的粘附及其对人工基底膜的侵袭力和迁移力,且抑制作用随剂量的升高而增强,划痕实验结果也显示灰黄霉素处理组细胞与对照组细胞相比迁移能力降低。③灰黄霉素瘤内注射小细胞肺癌细胞株NCI-H446裸鼠移植瘤后能显著抑制肿瘤生长,免疫组化结果显示灰黄霉素能明显上调细胞因子Bax、p53和Caspase-3的表达和下调Bcl-2与VEGF的表达。
结论:灰黄霉素能抑制肺癌NCI-H446细胞的生长,诱导NCI-H446细胞凋亡;灰黄霉素具有抑制NCI-H446细胞粘附、侵袭与转移的作用;灰黄霉素能抑制NCI-H446细胞的体内致瘤能力。
Objective: To study the anti-tumor effects of griseofulvin on human small cell lung cancer cell line NCI-H446 in vitro and in vivo.
Methods:①N CI-H446 cells were treated with different concentrations of griseofulvin for 48h.CCK-8 assay was used to determine the cell survival rate;The changes of morphology and ultrastructure were observed by inverted microscope,fluorescence microscope and transmission electron microscope;Cell cycle and apoptosis rate were analysed by flow cytometry(FCM);AnnexinⅤ-FITC apoptosis detection kit was used to detect the apoptosis of cells;The enzyme activity changes of Caspase-3 and Caspase-9 were detected by spectrophotometry;Western blotting was used to detect the changes of Bax,Bc1-2,and cytochrome C in cytosol of NCI-H446 cells in the protein level.②The migration,invasion and adhesion ability of NCI-H446 cells were detected by scratch test,transwell chamber assay and Transwell-Matrigel model respectively.The cell adhesion assay was carried out on 96-well plate precoated with matrigel.③For in vivo study, we first established the NCI-H446 tumor model by grafting NCI-H446 cells into the athymic nude mice,and then injected griseofulvin into the tumors.Two weeks after injection,we sacrificed the mice and removed the tumors,then weighed and calculated the ratios of tumor-suppression.We also detected the expressions of Bcl-2、Bax、Caspase-3、p53 and VEGF with immunohistochemistry.
Results:①CCK-8 assay results showed that griseofulvin could significantly inhibit the proliferation of NCI-H446 cells in vitro in a dose-dependant manner,and the IC50 of griseofulvin was 24.58±1.32μM.Typical morphological changes of NCI-H446 cells were observed after induced by griseofulvin. The cell cycle distribution of NCI-H446 were changed,the number of cell increased in the stage of G2/M and decreased in the stage of S and G0/G1, accompanied with the subdiploid apoptotic peak.After 48 hours treatment with griseofulvin,the rates of apoptosis of griseofulvin-treated NCI-H446 cells greatly increased in high concentration group but did not obviously change in low concentration group compared with control group.Colorimetric assay results showed that griseofulvin could activated caspase-3 and caspase-9;Western blotting results indicated that the expression of cytochrome C and Bax were increased but Bcl-2 was decreased by griseofulvin.②The adhesion,metastatic potential and invasiveness of NCI-H446 cells were obviously inhibited by griseofulvin in a dose-dependent manner.The result of scratch test also indicated that the migration ability of NCI-H446 cells in griseofulvin treated group was decreased compared with control group.③The in vivo data showed that griseofulvin inhibited the tumor growth conspicuously through down-regulating the expression of Bcl-2,VEGF and up-regulating the expression of Bax,caspase-3 and p53.The ratios of tumor weight-suppression of griseofulvin group was 42.07 % .
Conclusion: Griseofulvin can inhibit the growth and induce apoptosis of NCI-H446 cells;griseofulvin inhibits the adhesion, invasiveness and migration of NCI-H446 cells in vitro;griseofulvin inhibits the tumorigenicity of NCI-H446 cells in vivo.
引文
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