| 赵亚萍,陈湘,傅利萍,等.野马追内酯O诱导三阴性乳腺癌BT-20细胞凋亡的作用机制研究[J].浙江中医药大学学报,2022,46(11):1181-1188. |
| 野马追内酯O诱导三阴性乳腺癌BT-20细胞凋亡的作用机制研究 |
| Study on the Mechanism of Eupalinolide O Inducing Apoptosis in Triple Negative Breast Cancer BT-20 Cells |
| DOI：10.16466/j.issn1005-5509.2022.11.002 |
| 中文关键词: 野马追内酯O 三阴性乳腺癌 细胞凋亡 细胞周期阻滞 半胱氨酸蛋白酶 BT-20 |
| 英文关键词: eupalinolide O triple negative breast cancer cell apoptosis cell cycle arrest caspase BT-20 |
| 基金项目:浙江省自然科学基金项目(LGF20H280002)；浙江中医药大学校级科研基金项目(2021ZZ04) |
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| 中文摘要: |
| [目的] 通过体内外实验探究野马追内酯O(eupalinolide O，EO)对人乳腺癌BT-20细胞的作用及机制。[方法] 采用噻唑蓝(methyl thiazolyl tetrazolium，MTT)法测定EO对BT-20细胞的抑制作用及半数抑制浓度，以确定EO浓度。将BT-20细胞分为空白对照组、EO 5 μmol·L-1组、EO 10 μmol·L-1组，后两组以EO处理24 h，采用乳酸脱氢酶(lactate dehydrogenase，LDH)法检测细胞毒性；流式细胞术检测细胞周期和凋亡情况。24只裸鼠通过乳腺脂肪垫注射BT-20细胞，建立乳腺癌模型，并随机分为对照组、阿霉素组(7 mg·kg-1)，EO低剂量组(15 mg·kg-1)和EO高剂量组(30 mg·kg-1)，每组6只。除对照组外，各组裸鼠连续腹腔注射对应药物治疗21 d，期间测量体表肿瘤体积；结束后称取肿瘤质量，并采用免疫组化染色检测肿瘤组织中增殖细胞核抗原(proliferating cell nuclear antigen，PCNA)和Ki-67表达。采用免疫印迹法检测细胞和裸鼠肿瘤组织中B细胞淋巴瘤-2(B-cell lymphoma-2，Bcl-2)、Bcl-2关联X(Bcl-2 associated X protein，Bax)、聚ADP核糖聚合酶(poly ADP-ribose polymerase，PARP)、活化的半胱氨酸蛋白酶-3(cleaved cysteinyl aspartate specific proteinase-3，cleaved caspase-3)和活化的半胱氨酸蛋白酶-9(cleaved cysteinyl aspartate specific proteinase-9，cleaved caspase-9)蛋白表达。 [结果] EO对BT-20细胞的抑制作用具有剂量-时间依赖性。与空白对照组比较，EO 5、10 μmol·L-1组细胞毒性和凋亡率增加(P＜0.05，P＜0.01)，且EO 10 μmol·L-1组的细胞周期明显阻滞于G2/M期(P＜0.01)。与对照组比较，阿霉素组、EO低剂量组、EO高剂量组的裸鼠肿瘤质量显著降低(P＜0.01，P＜0.05)，肿瘤组织内PCNA和Ki-67抗原表达显著降低(P＜0.01)，肿瘤体积随治疗进展显著缩小(P＜0.01，P＜0.05)。与空白对照组比较，各组细胞的Bcl-2蛋白表达降低(P＜0.01，P＜0.05)，Bax、PARP、cleaved caspase-3及cleaved caspase-9蛋白表达升高(P＜0.01，P＜0.05)；与对照组裸鼠比较，各给药组的Bcl-2蛋白表达降低(P＜0.01，P＜0.05)，Bax、PARP、cleaved caspase-3及cleaved caspase-9蛋白表达升高(P＜0.01，P＜0.05)。[结论] EO在体内外抑制BT-20细胞增殖并诱导细胞凋亡，作用机制可能与其抑制Bcl-2蛋白表达，促进Bax、PARP、cleaved caspase-3、cleaved caspase-9蛋白表达有关。 |
| 英文摘要: |
| [Objective] To investigate the effect of eupalinolide O(EO) on human breast cancer BT-20 cells and explore its mechanism through in vitro and vivo experiments. [Methods] Methyl thiazolyl tetrazolium(MTT) assay was used to detect the inhibitory effect of EO on BT-20 cells and its half inhibitory concentration， and to determine experimental concentration. BT-20 cells were divided into blank control group， EO 5 μmol·L-1 group and EO 10 μmol·L-1 group， the latter two groups were intervened with EO for 24 h， lactate dehydrogenase(LDH) release test was used to detect the cytotoxicity， and flow cytometry was used to detect the cell cycle and apoptosis. Twenty-four nude mice were injected BT-20 cells through mammary fat pad to establish breast cancer models， then the mice were randomly divided into control group， doxorubicin group(7 mg·kg-1)， EO low-dose group(15 mg·kg-1) and EO high-dose group(30 mg·kg-1)， with six mice in each group. Except for control group， other groups mice were administrated abdominal injection treatment for 21 d， tumor volume was measured during the treatment period. After treatment， tumor weight was measured and the expression of proliferating cell nuclear antigen(PCNA)， Ki-67 in tumor tissue were detected by immunohistochemical staining. Western blot was used to detect the B-cell lymphoma-2(Bcl-2)， Bcl-2 associated X protein(Bax)， poly ADP-ribose polymerase(PARP)， cleaved cysteinyl aspartate specific proteinase-3(cleaved caspase-3) and cleaved cysteinyl aspartate specific proteinase-9(cleaved caspase-9) protein expression in BT-20 cells and tumor tissue of BT-20 nude mice. [Results] Compared with blank control group， the EO 5， 10 μmol·L-1 group cell cytotoxicity and apoptosis increased significantly(P＜0.05， P＜0.01)， and the EO 10 μmol·L-1 group cell cycle was arrested in G2/M phase(P＜0.01). Compared with control group， doxorubicin， EO low-dose， EO high-dose groups tumor weight decreased markedly(P＜0.01， P＜0.05)， and the expression of PCNA and Ki-67 in tumor tissue also decreased remarkably(P＜0.01)， tumor volume decreased with the course of treatment(P＜0.01， P＜0.05). Compared with blank control group， the protein expression of Bcl-2 was decreased(P＜0.01， P＜0.05)， and the protein expression of Bax， PARP， cleaved caspase-3 and cleaved caspase-9 was increased(P＜0.01， P＜0.05) in each cell group. Compared with control group， the protein expression of Bcl-2 was decreased(P＜0.01， P＜0.05)， and the protein expression of Bax， PARP， cleaved caspase-3 and cleaved caspase-9 was increased in each treatment nude mice group(P＜0.01， P＜0.05). [Conclusion] EO can inhibit proliferation and induce apoptosis in BT-20 cells in vitro and in vivo， which may be related to inhibition of Bcl-2 protein expression and up-regulation of Bax， PARP， cleaved caspase-3， and cleaved caspase-9 protein expression. |
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