Wu Yaran,Ni Zhenhong,Yan Xiaojing,et al.Pirarubicin induces cytoprotective autophagy in HeLa cells through up-regulating autophapy related 4B[J].J Third Mil Med Univ,2016,38(02):129-135.

盐酸吡柔比星通过上调ATG4B增强HeLa细胞保护性自噬(/HTML )




Pirarubicin induces cytoprotective autophagy in HeLa cells through up-regulating autophapy related 4B
第三军医大学基础医学部生物化学与分子生物学教研室; 第三军医大学大坪医院野战外科研究所妇产科
Wu Yaran Ni Zhenhong Yan Xiaojing Zheng Yingru He Fengtian Lian Jiqin

Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Third Military Medical University, Chongqing, 400038; Department of Obstetrics and Gynecology, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Chongqing, 400042, China

pirarubicin autophagy autophapy related 4B uterine cervical neoplasm

目的       探讨盐酸吡柔比星增强宫颈癌HeLa细胞自噬水平的可能机制。       方法       采用CCK-8检测盐酸吡柔比星单独或联用自噬抑制剂氯喹对HeLa细胞增殖的影响;台盼蓝染色实验检测盐酸吡柔比星单独或联用自噬抑制剂氯喹对HeLa细胞死亡的影响;GFP-LC3 质粒转染HeLa细胞后观察盐酸吡柔比星对自噬小体形成的影响;Western blot检测细胞中自噬标志分子Ⅱ型微管相关蛋白轻链3(microtuble-associated protein light chain 3-Ⅱ, LC3-Ⅱ)、自噬相关分子自噬相关蛋白4B(autophapy related 4B,ATG4B)的表达情况。CCK-8实验及台盼蓝染色实验观察在有或者无自噬抑制剂氯喹作用下以及shRNA下调HeLa细胞中的ATG4B后盐酸吡柔比星对HeLa细胞的杀伤效应的影响。       结果       盐酸吡柔比星能剂量依赖地抑制HeLa细胞的增殖并促进其死亡(P<0.05);盐酸吡柔比星处理能显著增加HeLa 细胞中自噬小体的数量,并显著上调自噬标志分子LC3-II、自噬关键酶ATG4B的表达;自噬抑制剂氯喹可显著增强盐酸吡柔比星的杀细胞效应(P<0.05),shRNA干扰ATG4B可抑制盐酸吡柔比星对HeLa细胞中自噬的增强作用(P<0.05),并增强盐酸吡柔比星的杀细胞效应(P<0.05)。       结论       盐酸吡柔比星通过上调ATG4B而增强的自噬对HeLa细胞起保护作用。


Objective       To investigate the mechanism and significance of pirarubicin in enhancing the autophagy of HeLa cells.        Methods       Trypan blue exclusion assay and CCK-8 assay were used to detect cell mortality and proliferation in HeLa cells treated with different doses of pirarubicin alone or combined with chloroquine (autophagy inhibitor). The effect of pirarubicin on autophagosome formation in HeLa cells transfected with GFP-LC3 plasmid was observed with fluorescence microscopy. Western blotting was used to determine the protein levels of autophagy markers microtuble-associated protein light chain 3-Ⅱ (LC3-Ⅱ), and autophapy related 4B (ATG4B). After ATG4B was down-regulated with shRNA in HeLa cells, the effect of pirarubicin on killing HeLa cells in the presence of chloroquine was observed.        Results       Pirarubicin inhibited cell proliferation and induced cell death in a dose-dependent manner in HeLa cells (P<0.05). Pirarubicin induced the formation of LC3-GFP puncta in HeLa cells, and increased the expression of LC3Ⅱand ATG4B markedly. Chloroquine enhanced pirarubicin-triggered cell death in HeLa cells (P<0.05). Knockdown of ATG4B via shRNA suppressed pirarubicin-induced autophagy and increased pirarubicin-induced cell death in HeLa cells.        Conclusion       Pirarubicin induces cytoprotective autophagy in HeLa cells through up-regulating ATG4B.


[1]Ferlay J, Shin H R, Bray F, et al. Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008[J]. Int J Cancer, 2010, 127(12): 2893-2917. DOI:10.1002/ijc.25516
[2]Chauvergne J, Fumoleau P, Cappelaere P, et al. Phase II study of pirarubicin (THP) in patients with cervical, endometrial and ovarian cancer: study of the Clinical Screening Group of the European Organization for Research and Treatment of Cancer (EORTC)[J]. Eur J Cancer, 1993, 29A(3): 350-354.
[3]李席如, 王建东, 张艳君, 等. 多西他赛联合表柔比星/比柔比星新辅助治疗乳腺癌临床分析[J]. 中华医学杂志, 2009, 89(2): 87-90.
[4]Untch M, Sevin B U, Perras J P, et al. Chemosensitivity to the new anthracycline pirarubicin and other chemotherapeutic agents in primary and recurrent ovarian tumors in vitro[J]. Gynecol Oncol, 1992, 47(2): 172-178.
[5]Kato T, Nishimura H, Umezu J, et al. Phase II study of 4′-O-tetrahydropyranyl-adriamycin (THP-ADM) in patients with gynecological cancer[J]. Gan To Kagaku Ryoho, 1985, 12(10): 1962-1967.
[6]Guo X L, Li D, Hu F, et al. Targeting autophagy potentiates chemotherapy-induced apoptosis and proliferation inhibition in hepatocarcinoma cells[J]. Cancer Lett, 2012, 320(2): 171-179. DOI:10.1016/j.canlet.2012.03.002
[7]Xu Y, Yu H, Qin H, et al. Inhibition of autophagy enhances cisplatin cytotoxicity through endoplasmic reticulum stress in human cervical cancer cells[J]. Cancer Lett, 2012, 314(2): 232-243. DOI:10.1016/j.canlet.2011.09.034
[8]Li K, Chen X, Liu C, et al. Pirarubicin induces an autophagic cytoprotective response through suppression of the mammalian target of rapamycin signaling pathway in human bladder cancer cells[J]. Biochem Biophys Res Commun, 2015, 460(2): 380-385. DOI:10.1016/j.bbrc.2015.03.042
[9]Tsukagoshi S. Pirarubicin (THP-adriamycin)[J]. Gan To Kagaku Ryoho, 1988, 15(9): 2819-2827.
[10]Kaku S, Takahashi K, Murakami Y, et al. Neoadjuvant intraarterial chemotherapy for stage IIB-IIIB cervical cancer in Japanese women[J]. Exp Ther Med, 2010, 1(4): 651-655.
[11]Guo Y, Song L, Liu H. Cervical clear cell adenocarcinoma infected with human papillomavirus type 18 in an adolescent[J]. J Pediatr Adolesc Gynecol, 2014, 27(1): e1-e3. DOI:10.1016/j.jpag.2013.04.007
[12]Yang Z, Klionsky D J. Eaten alive: a history of macroautophagy[J]. Nat Cell Biol, 2010, 12(9): 814-822.
[13]Yen C Y, Chiang W F, Liu S Y, et al. Long-term stimulation of areca nut components results in increased chemoresistance through elevated autophagic activity[J]. J Oral Pathol Med, 2014, 43(2): 91-96. DOI:10.1111/jop.12102
[14]Liu S, Li X. Autophagy inhibition enhances sensitivity of endometrial carcinoma cells to paclitaxel[J]. Int J Oncol, 2015, 46(6): 2399-2408. DOI:10.3892/ijo.2015.2937
[15]范佳君, 曾贤, 李玉彬, 等. 细胞自噬在多柔比星诱导淋巴瘤细胞凋亡中的作用[J]. 第二军医大学学报, 2012, 33(6):595-598. DOI:10.3724/SP.J.1008.2012.00595
[16]Fernandes A, Azevedo M M, Pereira O, et al. Proteolytic systems and AMP-activated protein kinase are critical targets of acute myeloid leukemia therapeutic approaches[J]. Oncotarget, 2015, 6(31): 31428-31440. DOI:10.18632/oncotarget.2947
[17]Sun W L, Chen J, Wang Y P, et al. Autophagy protects breast cancer cells from epirubicin-induced apoptosis and facilitates epirubicin-resistance development[J]. Autophagy, 2011, 7(9): 1035-1044.
[18]Kong D, Ma S, Liang B, et al. The different regulatory effects of p53 status on multidrug resistance are determined by autophagy in ovarian cancer cells[J]. Biomed Pharmacother, 2012, 66(4): 271-278. DOI:10.1016/j.biopha.2011.12.002
[19]Cheng P, Ni Z, Dai X, et al. The novel BH-3 mimetic apogossypolone induces Beclin-1- and ROS-mediated autophagy in human hepatocellular carcinoma cells[J]. Cell Death Dis, 2013, 4:e489.
[20]Jiang X, Overholtzer M, Thompson C B. Autophagy in cellular metabolism and cancer[J]. J Clin Invest, 2015, 125(1): 47-54. DOI:10.1172/JCI73942 
[21]Mathew R, Karantza-Wadsworth V, White E. Role of autophagy in cancer[J]. Nat Rev Cancer, 2007, 7(12): 961-967.
[22]Dewaele M, Maes H, Agostinis P. ROS-mediated mechanisms of autophagy stimulation and their relevance in cancer therapy[J]. Autophagy, 2010, 6(7): 838-854.
[23]Ni Z, Wang B, Dai X, et al. HCC cells with high levels of Bcl-2 are resistant to ABT-737 via activation of the ROS-JNK-autophagy pathway[J]. Free Radic Biol Med, 2014, 70: 194-203. DOI:10.1016/j.freeradbiomed.2014.02.012
[24]Till A, Subramani S. A balancing act for autophagin[J]. J Clin Invest, 2010, 120(7): 2273-2276. DOI:10.1172/JCI43238 
[25]Guo L, Huang J X, Liu Y, et al. Transactivation of Atg4b by C/EBPβ promotes autophagy to facilitate adipogenesis[J]. Mol Cell Biol, 2013, 33(16): 3180-3190. DOI:10.1128/MCB.00193-13 
[26]Li Y, Luo Q, Yuan L, et al. JNK-dependent Atg4 upregulation mediates asperphenamate derivative BBP-induced autophagy in MCF-7 cells[J]. Toxicol Appl Pharmacol, 2012, 263(1): 21-31. DOI:10.1016/j.taap.2012.05.018
[27]Rothe K, Lin H, Lin K B, et al. The core autophagy protein ATG4B is a potential biomarker and therapeutic target in CML stem/progenitor cells[J]. Blood, 2014, 123(23): 3622-3634. DOI:10.1182/blood-2013-07-516807
[28]Akin D, Wang S K, Habibzadegah-Tari P, et al. A novel ATG4B antagonist inhibits autophagy and has a negative impact on osteosarcoma tumors[J]. Autophagy, 2014, 10(11): 2021-2035.
[29]Tran E, Chow A, Goda T, et al. Context-dependent role of ATG4B as target for autophagy inhibition in prostate cancer therapy[J]. Biochem Biophys Res Commun, 2013, 441(4): 726-731. DOI:10.1016/j.bbrc.2013.10.117


 Ding Wen,Ni Zhenhong,Cheng Panke,et al.Mechanism of (-)-gossypol down-regulating B lymphocyte stimulator expression in Namalwa cells via autophagy[J].J Third Mil Med Univ,2012,34(02):1613.
 Xu Mengxue,Qin Xinyue,Feng Jinzhou,et al.Anti-inflammatory effect of autophagy in experimental autoimmune encephalomyelitis mice[J].J Third Mil Med Univ,2015,37(02):1624.
 Ni Zhenhong,Wang Bin,Ding Wen,et al.(-)-gossypol induces autophagy in Daudi cells through ERK signal pathway[J].J Third Mil Med Univ,2012,34(02):2384.
 Yang Pengyu,Zhang Jiansheng,Li Qiang,et al.Rapamycin combined with nimuatine suppresses growth and proliferation of human U87MG glioma cells[J].J Third Mil Med Univ,2010,32(02):1529.
 YU Jian,CAO Hong-wei,LIANG Hua-ping,et al.Autophagy of peripheral monocytes following trauma: study on 51 cases[J].J Third Mil Med Univ,2008,30(02):1994.
 Zhang Zhimin,Yang Zhenzhou,He Hao,et al.APE1 acetylation regulates autophagy induced by ionizing radiation in HeLa cells[J].J Third Mil Med Univ,2016,38(02):380.
 Liu Yu,Wang Huanhuan,Shi Jiazhong,et al.Effect of deficiency of xeroderma pigmentosum group C on autophagy of bladder cancer cells[J].J Third Mil Med Univ,2016,38(02):1427.
 Yu Datang,Li Mingfang,Ni Bing,et al.Expression of autophagy related proteins LC3 and BNIP3 after acute spinal cord injury in rats[J].J Third Mil Med Univ,2013,35(02):841.
 Yang Ting,Ni Zhenhong,Gong Wei,et al.Anti-inflammation activity of sinomenine-induced autophagy in EA.hy926 cells[J].J Third Mil Med Univ,2013,35(02):1084.
 Xiao Yu,Long Chi,Zhang Minghui,et al.Expression of Beclin-1 in non-small cell lung cancer and its clinical significance[J].J Third Mil Med Univ,2013,35(02):1271.

更新日期/Last Update: 2016-01-14