17-烯丙胺基-17-去甲氧基格尔德霉素对人绒毛膜癌JAR细胞系细胞周期和凋亡的影响及其相关机制

谷苗 巴一 崔丽军 刘百艺 安国庆 李玉红 许倩*

doi:10.16098/j.issn.0529-1356.2018.02.010

PDF(797 KB)

解剖学报 ›› 2018, Vol. 49 ›› Issue (2) : 198-203. DOI: 10.16098/j.issn.0529-1356.2018.02.010

肿瘤生物学

17-烯丙胺基-17-去甲氧基格尔德霉素对人绒毛膜癌JAR细胞系细胞周期和凋亡的影响及其相关机制

谷苗巴一崔丽军刘百艺安国庆李玉红许倩*

作者信息 +

Effect of 17-allylamino-17-demethoxygeldanamycin on cell cycle and apoptosis of JAR cells and the associated mechanisms

GU Miao BA Yi CUI Li-jun LIU Bai-yi AN Guo-qing LI Yu-hong XU Qian*

Author information +

文章历史 +

摘要

目的探讨热休克蛋白 90（HSP90）抑制剂17-烯丙胺基-17-去甲氧基格尔德霉素（17-AAG）对人绒毛膜癌JAR细胞系细胞周期和凋亡的影响及其相关机制。方法体外培养人绒毛膜癌JAR细胞系，经不同浓度的17-AAG作用24 h后，采用TUNEL细胞凋亡法及Annexin V-FITC/PI双标记流式细胞术检测细胞凋亡；流式细胞术检测细胞周期；Real-time PCR法和Western blotting法检测血管内皮生长因子（VEGF）、cyclinD1、cyclinA1和 Caspase-3在mRNA和蛋白水平的表达情况。结果 17-AAG对JAR细胞生长具有明显的抑制作用，并存在浓度依赖性。各组细胞发生G₂期阻滞及明显凋亡；5、10、20 mg/L 17-AAG 作用24 h 后，各组细胞增殖相关基因cyclinD1 mRNA及蛋白表达水平相对于0 mg/L 17-AAG处理组（对照组）均下调，VEGF蛋白表达水平相对于0 mg/L 17-AAG处理组（对照组）下调，且下调程度与处理浓度成正比，而凋亡相关基因Caspase-3的mRNA未出现明显上调，但其在蛋白表达水平上调。结论 17-AAG能够抑制JAR细胞增殖活力，诱导细胞凋亡，17-AAG 可能通过下调VEGF 的表达，产生抗肿瘤血管新生作用；可能通过下调cyclinD1使细胞周期发生G₂期阻滞；且可能通过上调Caspase-3诱导细胞凋亡。

Abstract

Objective To explore the effects of 17-AAG on cell cycle and apoptosis of Choriocarcinoma cells JAR and to clarify the related mechanisms． Methods Choriocarcinoma cells JAR were cultured in vitro and incubated with 17-allylamino-17-demethoxygeldanamycin (17-AAG) at different concentrations for 24 hours. The apoptosis of JAR cells with different concentrations of 17-AAG was measured by TUNEL analysis and flow cytometry (Annexin V-FITC/PI). Cell cycle was analyzed by flow cytometry. Real-time PCR and Western blotting analysis were used for the detection of vascular endothelial growth factor(VEGF), cyclinD1,cyclinA1 and Caspase-3 mRNA levels and protein levels. Results 17-AAG had obvious inhibitory effect on JAR cell growth with concentration dependent. When the 17-AAG concentration increased, G2 phase retardation and obvious apoptosis were found in every groups. Real-time PCR revealed an decreased expression level of cyclinD1. Western blotting analysis revealed an decreased expression level of VEGF, cyclinD1 and an increased expression level of Caspase-3. Conclusion 17-AAG can inhibit the JAR cell proliferation activity, induce cell apoptosis, and may exert the effect by down-regulation of VEGF, cyclinD1 and up-regulation of Caspase-3.

导出引用

谷苗巴一崔丽军刘百艺安国庆李玉红许倩. 17-烯丙胺基-17-去甲氧基格尔德霉素对人绒毛膜癌JAR细胞系细胞周期和凋亡的影响及其相关机制[J]. 解剖学报. 2018, 49(2): 198-203 https://doi.org/10.16098/j.issn.0529-1356.2018.02.010

GU Miao BA Yi CUI Li-jun LIU Bai-yi AN Guo-qing LI Yu-hong XU Qian. Effect of 17-allylamino-17-demethoxygeldanamycin on cell cycle and apoptosis of JAR cells and the associated mechanisms[J]. Acta Anatomica Sinica. 2018, 49(2): 198-203 https://doi.org/10.16098/j.issn.0529-1356.2018.02.010

参考文献

［1］ Noal S, Joly F, Leblanc E, et al. Management of gestational trophoblastic disease ［J］.Gynecol Obstet Fertil, 2010, 38(3):193-198．

［2］Seckl MJ, Sebire NJ, Berkowitz RS, et al. Gestational trophoblastic disease ［J］.Lancet, 2010, 376(9742):717-729.

［3］Oh YJ, Seo YH. A novel chalcone-based molecule, BDP inhibits MDA-MB-231 triplenegative breast cancer cell growth by suppressing Hsp90 function［J］. Oncol Rep, 2017, 38(4):2343-2350.

［4］Belalcazar A, Shaib WL, Farren MR, et al. Inhibiting heat shock protein 90 and the ubiquitin-proteasome pathway impairs metabolic homeostasis and leads to cell death in human pancreatic cancer cells ［J］. Cancer, 2017,123(24):4924-4933.

［5］Zhang H, Neely L, Lundgren K, et al. BIIB021, a synthetic Hsp90 inhibitor, has broad application against tumors with acquired multidrug resistance ［J］. Int J Cancer, 2010, 126(5):1226-1234.

［6］Soroka J, Wandinger SK, Mausbacher N, et al．Conformational switching of the molecular chaperone Hsp90 via regulated phosphorylation ［J］.Mol Cell, 2012, 45(4):517-528.

［7］Woyach JA, Shah MH. New therapeutic advances in the management of progressive thyroid cancer ［J］.Endocr Relat Cancer, 2009, 16(3):715-731.

［8］Jhaveri K, Taldone T, Modi S, et al. Advances in the clinical development of heat shock protein 90(Hsp 90) inhibitors in cancers［J］. Biochim Biophys Acta, 2012, 1823(3):742-755.

［9］Massimini M, Palmieri C, De Maria R, et al．17-AAG and apoptosis, autophagy, and mitophagy in canine osteosarcoma cell lines ［J］.Vet Patho, 2017, 54(3):405-412.

［10］Weber H, Valbuena JR, Barbhuiya MA, et al．Small molecule inhibitor screening identifified HSP90 inhibitor 17-AAG as potential therapeutic agent for gallbladder cancer ［J］.Oncotarget, 2017, 8(16):26169-26184.

［11］Woo JK, Jang JE, Kang JH, et al．Lectin, galactoside-binding soluble 3 binding protein promotes 17-N-allylamino-17-demethoxygeldanamycin resistance through PI3K/Akt pathway in lung cancer cell line ［J］.Mol Cancer Ther, 2017, 16(7):1355-1365.

［12］Zhang T, Li Y, Zhu Z, et al. MEK inhibition potentiates the activityof Hsp90 inhibitor 17-AAG against pancreatic cancer cells ［J］. Mol Pharm, 2010, 7(5):1576-1584.

［13］Wang G, Krishnamurthy K, Tangpisuthipongsa D. Protection of murine neural progenitor cells by the Hsp90 inhibitor17-allylamino-17-demethoxygeldanamycin in the low nanomolar concentration range ［J］. J Neurochem, 2011, 117(4):703-711.

［14］Wang X, Ju W, Renouard J, et al. 17-allylamino-17-demethoxygeldanamycin synergistically potentiates tumor necrosis factor induced lung cancer cell death by blocking the nuclear factor-κB pathway ［J］. Cancer Res, 2006, 66(2):1089-1095.

［15］Vlahakis NE, Young BA, Atakilit A,et al. Integrin alpha9beta1 directly binds to vascular endothelial growth factor (VEGF)-A and contributes to VEGF-A-induced angiogenesis ［J］.J Biol Chem, 2007, 282(20):15187-15196.

［16］Fang J, Zhou Q, Liu LZ, et al. Apigenin inhibits tumor angiogenesis through decreasing HIF-1alpha and VEGF expression ［J］. Carcinogenesis, 2007, 28 (4): 858-864.

［17］Hirakawa S, Brown LF, Kodama S, et al.VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites ［J］. Blood, 2007, 109 (3):1010-1017.