Abstract
Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality globally. It accounts for the majority of primary liver cancer cases. Amyloid precursor protein (APP), a cell membrane protein, plays a vital role in the pathogenesis of Alzheimer’s disease, and has been found to be implicated in tumor growth and metastasis. Therefore, to understand the relationship between APP and 5-fluorouracil (5-FU) resistance in liver cancer, Cell Counting Kit-8, apoptosis and cell cycle assays, western blotting, and reverse transcription-quantitative polymerase chain reaction (qPCR) analysis were performed. The results demonstrated that APP expression in Bel7402-5-FU cells was significantly up-regulated, as compared with that in Bel7402 cells. Through successful construction of APP-silenced (siAPP) and overexpressed (OE) Bel7402 cell lines, data revealed that the Bel7402-APP751-OE cell line was insensitive, while the Bel7402-siAPP cell line was sensitive to 5-FU in comparison to the matched control group. Furthermore, APP overexpression decreased, while APP silencing increased 5-FU-induced apoptosis in Bel7402 cells. Mechanistically, APP overexpression and silencing can regulate the mitochondrial apoptotic pathway and the expression of apoptotic suppressor genes (B-cell lymphoma-2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl)). Taken together, these results preliminarily revealed that APP overexpression contributes to the resistance of liver cancer cells to 5-FU, providing a new perspective for drug resistance.
摘要
目 的
探讨淀粉样前体蛋白 (APP) 是否与肝癌细胞 5-氟尿嘧啶 (5-FU) 耐药的过程相关, 并探索其发挥作用的分子机制.
创新点
首次探索并初步证实 APP 通过影响线粒体凋亡通路信号的传递可以促进肝癌 5-FU 耐药.
方法
为探究肝癌中 APP 与 5-FU 耐药性之间的关系, 我们构建了 APP 沉默和过表达的 Bel7402 细胞系, 并进行了细胞活性检测, 细胞凋亡和细胞周期, 蛋白质印迹和荧光定量聚合酶链式反应 (qPCR) 等实验, 验证过表达或沉默 APP 时, 肝癌细胞的状态变化, 以及 APP 发挥作用的分子机制.
结论
与 Bel7402 细胞相比, 耐药细胞 Bel7402-5-FU 中 APP 的表达明显上调. 在 Bel7402 细胞中过表达 APP 降低了细胞的 5-FU 敏感性, 而沉默 Bel7402 细胞的 APP 表达提升了细胞对 5-FU 的敏感性. 从机制上讲, APP 的过表达和沉默可以调节线粒体的凋亡途径和凋亡抑制基因 (BAX、 BID、 Bcl-2 和 Bcl-xl) 的表达, 并进一步影响细胞凋亡的进程. 综上所述, 我们的结果初步表明 APP 在肝癌耐药过程中显著上调, APP 能够通过影响线粒体凋亡通路调节肝癌细胞的 5-FU 敏感性, 这为研究肝癌耐药机制提供了新的视角.
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References
Allemani C, Matsuda T, di Carlo V, et al., 2018. Global surveillance of trends in cancer survival 2000–14 (CONCORD-3): analysis of individual records for 37513025 patients diagnosed with one of 18 cancers from 322 population-based registries in 71 countries. Lancet, 391(10125):1023–1075. https://doi.org/10.1016/s0140-6736(17)33326-3
Belmokhtar CA, Hillion J, Ségal-Bendirdjian E, 2001. Staurosporine induces apoptosis through both caspase-dependent and caspase-independent mechanisms. Oncogene, 20(26):3354–3362. https://doi.org/10.1038/sj.onc.1204436
Bukhari H, Glotzbach A, Kolbe K, et al., 2017. Small things matter: implications of APP intracellular domain AICD nuclear signaling in the progression and pathogenesis of Alzheimer’s disease. Prog Neurobiol, 156:189–213. https://doi.org/10.1016/j.pneurobio.2017.05.005
Ceballos MP, Decàndido G, Quiroga AD, et al., 2018. Inhibition of sirtuins 1 and 2 impairs cell survival and migration and modulates the expression of P-glycoprotein and MRP3 in hepatocellular carcinoma cell lines. Toxicol Lett, 289:63–74. https://doi.org/10.1016/j.toxlet.2018.03.011
Dai W, Gao QG, Qiu JP, et al., 2016. Quercetin induces apoptosis and enhances 5-FU therapeutic efficacy in hepatocellular carcinoma. Tumor Biol, 37(5):6307–6313. https://doi.org/10.1007/s13277-015-4501-0
Gao SY, Lin J, Wang T, et al., 2019. Qingxin Kaiqiao Fang ameliorates memory impairment and inhibits apoptosis in APP/PS1 double transgenic mice through the MAPK pathway. Drug Des Devel Ther, 13:459–475. https://doi.org/10.2147/dddt.s188505
Gehrke A, Lee SS, Hilton K, et al., 2018. Development of the cancer survivor profile-breast cancer (CSPro-BC) app: patient and nurse perspectives on a new navigation tool. J Cancer Surviv, 12(3):291–305. https://doi.org/10.1007/s11764-017-0668-2
Gough M, Blanthorn-Hazell S, Delury C, et al., 2014. The E1 copper binding domain of full-length amyloid precursor protein mitigates copper-induced growth inhibition in brain metastatic prostate cancer DU145 cells. Biochem Biophys Res Commun, 453(4):741–747. https://doi.org/10.1016/j.bbrc.2014.10.004
Guo YK, Shi M, Qin YL, et al., 2017. Mechanism of apoptosis for resveratrol-mediated reversing the drug-resistance of AML HL-60/ADR cells. J Exp Hematol, 25(3):736–742 (in Chinese).
Hansel DE, Rahman A, Wehner S, et al., 2003. Increased expression and processing of the Alzheimer amyloid precursor protein in pancreatic cancer may influence cellular proliferation. Cancer Res, 63(21):7032–7037.
Harder H, Holroyd P, Burkinshaw L, et al., 2017. A user-centred approach to developing bWell, a mobile app for arm and shoulder exercises after breast cancer treatment. J Cancer Surviv, 11(6):732–742. https://doi.org/10.1007/s11764-017-0630-3
Hardy J, 2017. The discovery of Alzheimer-causing mutations in the APP gene and the formulation of the “amyloid cascade hypothesis”. FEBS J, 284(7):1040–1044. https://doi.org/10.1111/febs.14004
Hirata H, Sugimachi K, Takahashi Y, et al., 2015. Downregulation of PRRX1 confers cancer stem cell-like properties and predicts poor prognosis in hepatocellular carcinoma. Ann Surg Oncol, 22(S3):1402–1409. https://doi.org/10.1245/s10434-014-4242-0
Ikeda M, Morizane C, Ueno M, et al., 2018. Chemotherapy for hepatocellular carcinoma: current status and future perspectives. Jpn J Clin Oncol, 48(2):103–114. https://doi.org/10.1093/jjco/hyx180
Kallop DY, Meilandt WJ, Gogineni A, et al., 2014. A death receptor 6-amyloid precursor protein pathway regulates synapse density in the mature CNS but does not contribute to Alzheimer’s disease-related pathophysiology in murine models. J Neurosci, 34(19):6425–6437. https://doi.org/10.1523/jneurosci.4963-13.2014
Kovalenko PL, Kunovska L, Chen J, et al., 2012. Loss of MLK3 signaling impedes ulcer healing by modulating MAPK signaling in mouse intestinal mucosa. Am J Physiol Gastrointest Liver Physiol, 303(8):G951–G960. https://doi.org/10.1152/ajpgi.00158.2012
Li J, Duan BJ, Guo Y, et al., 2018. Baicalein sensitizes hepatocellular carcinoma cells to 5-FU and epirubicin by activating apoptosis and ameliorating P-glycoprotein activity. Biomed Pharmacother, 98:806–812. https://doi.org/10.1016/j.biopha.2018.01.002
Li JQ, Wu X, Gan L, et al., 2017. Hypoxia induces universal but differential drug resistance and impairs anticancer mechanisms of 5-fluorouracil in hepatoma cells. Acta Pharmacol Sin, 38(12):1642–1654. https://doi.org/10.1038/aps.2017.79
Liu JX, Cui XP, Qu LS, et al., 2016. Overexpression of DLX2 is associated with poor prognosis and sorafenib resistance in hepatocellular carcinoma. Exp Mol Pathol, 101(1):58–65. https://doi.org/10.1016/j.yexmp.2016.06.003
Ma ST, Tan WH, Du BT, et al., 2016. Oridonin effectively reverses cisplatin drug resistance in human ovarian cancer cells via induction of cell apoptosis and inhibition of matrix metalloproteinase expression. Mol Med Rep, 13(4):3342–3348. https://doi.org/10.3892/mmr.2016.4897
Meng JY, Kataoka H, Itoh H, et al., 2001. Amyloid β protein precursor is involved in the growth of human colon carcinoma cell in vitro and in vivo. Int J Cancer, 92(1):31–39. https://doi.org/10.1002/1097-0215(200102)9999:9999<::AID-IJC1155>3.0.CO;2-H
Miyazaki T, Ikeda K, Horie-Inoue K, et al., 2014. Amyloid precursor protein regulates migration and metalloproteinase gene expression in prostate cancer cells. Biochem Biophys Res Commun, 452(3):828–833. https://doi.org/10.1016/j.bbrc.2014.09.010
Nikolaev A, McLaughlin T, O’Leary DDM, et al., 2009. APP binds DR6 to trigger axon pruning and neuron death via distinct caspases. Nature, 457(7232):981–989. https://doi.org/10.1038/nature07767
Niu LL, Liu LP, Yang SL, et al., 2017. New insights into sorafenib resistance in hepatocellular carcinoma: responsible mechanisms and promising strategies. Biochim Biophys Acta Rev Cancer, 1868(2):564–570. https://doi.org/10.1016/j.bbcan.2017.10.002
Rodriguez-Ruiz M, Moreno E, Moreno-Delgado D, et al., 2017. Heteroreceptor complexes formed by dopamine D1, histamine H3, and N-methyl-d-aspartate glutamate receptors as targets to prevent neuronal death in Alzheimer’s disease. Mol Neurobiol, 54(6):4537–4550. https://doi.org/10.1007/s12035-016-9995-y
Shi YG, 2002. Mechanisms of caspase activation and inhibition during apoptosis. Mol Cell, 9(3):459–470. https://doi.org/10.1016/S1097-2765(02)00482-3
Siegel RL, Miller KD, Jemal A, 2018. Cancer statistics, 2018. CA Cancer J Clin, 68(1):7–30. https://doi.org/10.3322/caac.21442
Song MJ, 2015. Hepatic artery infusion chemotherapy for advanced hepatocellular carcinoma. World J Gastroenterol, 21(13):3843–3849. https://doi.org/10.3748/wjg.v21.i13.3843
Strilic B, Yang LD, Albarrán-Juárez J, et al., 2016. Tumour-cell-induced endothelial cell necroptosis via death receptor 6 promotes metastasis. Nature, 536(7615):215–218. https://doi.org/10.1038/nature19076
Sun T, Liu H, Ming L, 2017. Multiple roles of autophagy in the sorafenib resistance of hepatocellular carcinoma. Cell Physiol Biochem, 44(2):716–727. https://doi.org/10.1159/000485285
Takayama K, Tsutsumi S, Suzuki T, et al., 2009. Amyloid precursor protein is a primary androgen target gene that promotes prostate cancer growth. Cancer Res, 69(1):137–142. https://doi.org/10.1158/0008-5472.can-08-3633
Wang W, Xu B, Li QX, et al., 2018. Anti-cancer effects of a novel Pan-RAF inhibitor in a hepatocellular carcinoma cell line. Mol Med Rep, 17(4):6185–6193. https://doi.org/10.3892/mmr.2018.8615
Wang X, Lu XC, Zhu RL, et al., 2017. Betulinic acid induces apoptosis in differentiated PC12 cells via ROS-mediated mitochondrial pathway. Neurochem Res, 42(4):1130–1140. https://doi.org/10.1007/s11064-016-2147-y
Woods NK, Padmanabhan J, 2013. Inhibition of amyloid precursor protein processing enhances gemcitabine-mediated cytotoxicity in pancreatic cancer cells. J Biol Chem, 288(42):30114–30124. https://doi.org/10.1074/jbc.M113.459255
Zha Y, Gan P, Yao Q, et al., 2014. Downregulation of Rap1 promotes 5-fluorouracil-induced apoptosis in hepatocellular carcinoma cell line HepG2. Oncol Rep, 31(4):1691–1698. https://doi.org/10.3892/or.2014.3033
Zhang J, Luo N, Tian Y, et al., 2017. USP22 knockdown enhanced chemosensitivity of hepatocellular carcinoma cells to 5-FU by up-regulation of Smad4 and suppression of Akt. Oncotarget, 8(15):24728–24740. https://doi.org/10.18632/oncotarget.15798
Zhao YJ, Han HZ, Liang Y, et al., 2015. Alternative splicing of VEGFA, APP and NUMB genes in colorectal cancer. World J Gastroenterol, 21(21):6550–6560. https://doi.org/10.3748/wjg.v21.i21.6550
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Study concept and design: Zhong-quan ZHAO. Performing the experiments: Xiao-long WU, Ying CHEN, and Wen-cui KONG. Experimental technical guidance: Zhong-quan ZHAO. Data analysis and manuscript drafting: Xiao-long WU and Zhong-quan ZHAO. The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. All authors have read and approved the final manuscript. Therefore, all authors have full access to all the data in the study and take responsibility for the integrity and security of the data
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Xiao-long WU, Ying CHEN, Wen-cui KONG, and Zhongquan ZHAO declare that there are no conflicts of interest.
This article does not contain any studies with human or animal subjects performed by any of the authors.
Project supported by the International Science & Technology Cooperation Program of China (No. 2016G02)
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Wu, Xl., Chen, Y., Kong, Wc. et al. Amyloid precursor protein regulates 5-fluorouracil resistance in human hepatocellular carcinoma cells by inhibiting the mitochondrial apoptotic pathway. J. Zhejiang Univ. Sci. B 21, 234–245 (2020). https://doi.org/10.1631/jzus.B1900413
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DOI: https://doi.org/10.1631/jzus.B1900413
Key words
- Amyloid precursor protein
- 5-Fluorouracil resistance
- Mitochondrial apoptotic pathway
- Hepatocellular carcinoma