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. 2021 Jul 20;41(7):1056–1061. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2021.07.13

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Antitumor effects of AZD2014, a dual mTORC1/2 inhibitor, against human hepatocellular carcinoma xenograft in nude mice

Hui LIAO ¹, Yi WANG ¹, Xiaoping XU ¹, Chenjie ZHOU ¹, Jianmin ZHANG ¹, Kebo ZHONG ¹, Dinghua YANG ^2,^*

PMCID: PMC8329683 PMID: 34308856

Abstract

Objective

To investigate the antitumor effects of AZD2014 (a dual mTORC1/2 inhibitor) against human hepatocellular carcinoma (HCC) xenograft in mice.

Methods

HCCLM3 cells were injected subcutaneously in the right flank of nude mice, and when the tumors were macroscopic, the mice were randomized into 2 groups for daily intraperitoneal injection of AZD2014 (5 mg/kg, n=5) or vehicle (5 mL/kg, n=5) for 24 days. Tumor growth was assessed using calipers every 4 days and the tumor growth curve was drawn. After the final injection, the mice were euthanized and the tumors were dissected for measuring tumor weight and histopathological analysis with HE staining. Immunohistochemical staining was used to detect the expressions of Ki-67, cleaved caspase-3, CD31, and the epithelial-mesenchymal transition (EMT)-related proteins (Ecadherin, N-cadherin, and vimentin) in the tumor tissue.

Results

Daily treatment with AZD2014 significantly suppressed HCC growth as compared with the control group. HE staining showed significantly increased tumor necrosis in AZD2014-treated mice. AZD2014 treatment inhibited tumor cell proliferation, angiogenesis and EMT progression as shown by decreased expressions of Ki-67, CD31, N-cadherin, and vimentin and increased expression of E-cadherin in the tumor tissue, and significantly promoted tumor cell apoptosis as shown by an increased expression of cleaved caspase-3 in AZD2014-treated mice.

Conclusions

AZD2014 is a highly potent antitumor agent for HCC in nude mice bearing HCC xenografts. AZD2014 can effectively inhibit tumor proliferation, angiogenesis and EMT progression and induce tumor cell necrosis and apoptosis.

Keywords: hepatocellular carcinoma, mTOR inhibitor, molecular targeted therapy, AZD2014

肝细胞癌（HCC）发病率位居恶性肿瘤的第6位，起病隐匿，进展迅速^[1-2]。肝癌的治疗措施中有望获得治愈的方法有手术切除、肝移植和局部消融，但这些方法主要适用于早期患者^[2-3]。对于晚期和上述方法治疗失败的肝癌患者仍缺乏有效的治疗措施^[2-3]。近年来，随着对分子发病机制的深入研究，多条与HCC发生、发展相关的信号通路被阐明，促进了HCC分子靶向治疗的发展^[4-6]。另一方面，目前已有研究指出哺乳动物雷帕霉素靶蛋白（mTOR）与其他蛋白结合形成mTOR复合物1（mTORC1）和mTOR复合物2（mTORC2），这些蛋白复合物与多种肿瘤的发生发展有关^[7-9]。

研究表明HCC患者存在mTOR信号异常激活，是非常有前景的治疗肝细胞癌的分子靶点^[10]。目前，针对mTOR的靶向药物主要是传统mTORC1抑制剂雷帕鸣及其衍生物。从目前的临床数据来看，这些药物对HCC的治疗远远没有达到预期的临床效果，可能的原因主要为三点：不能完全地抑制mTORC1的活性；对mTORC2无效；反馈性地激活了AKT信号^[11-16]。因此，研究者们希望能够研发同时阻断mTORC1信号和mTORC2信号的新型双mTOR抑制剂。AZD2014属于APT竟争性的mTOR激酶抑制剂^[17]。研究表明它可以同时抑制mTORC1和mTORC2信号，且不激活AKT信号，具有显著的抗癌作用^[18-23]。前期我们在肝癌细胞株中也观察到AZD2014具有较好的抗癌作用^[24]。但AZD2014在动物体内对HCC是否具有抗癌作用尚不清楚。本研究拟在裸鼠皮下成瘤模型中，研究AZD2014在动物体内对肝细胞癌的抗癌作用及其机制，为HCC的防治提供更多思路。

1. 材料和方法

1.1. 肝癌细胞、裸鼠来源

人肝癌细胞株HCCLM3购买于中国科学院上海生命科学研究院细胞资源中心，雄性无特定病原体（SPF）Balb/c裸鼠16只购买于广东省医学动物实验中心，4~6周龄，体质量18~22 g，饲养于南方医科大学南方医院动物实验中心。AZD2014购买于美国Selleckchem公司。本实验通过南方医科大学动物实验伦理委员会批准。

1.2. 药品与试剂

PV-9000通用型二步法免疫组化检测试剂盒购买于北京中杉金桥公司），包括：E钙蛋白（E-cadherin）单克隆一抗（CST），神经性钙黏附素（N-cadherin）单克隆一抗（CST），波形蛋白（Vimentin）单克隆一抗（CST），CD31单克隆一抗（CST），Ki-67单克隆一抗（CST），Cleaved caspase-3单克隆一抗（CST）。

1.3. 裸鼠皮下成瘤实验和分组

HCCLM3用含10%胎牛血清的高糖DMEM培养基，在37 ℃、5% CO₂的培养箱中培养。HCCLM3细胞浓度培养至5×10⁷/mL时在每只裸鼠右侧腰腹部皮下接种100 μL细胞悬液。接种后观察裸鼠一般状态和肿瘤生长情况。待皮下成瘤块明显后（体积>20 mm³），将其随机分成对照组和AZD2014组，每组各5只。AZD2014组每天腹腔注射一次AZD2014药物，剂量为5 mg/kg；对照组每天腹腔注射药物溶剂（0.2% Tween80+2%聚乙二醇的PBS液）2.5 mL/kg。每4 d用游标卡尺测量裸鼠皮下瘤块的最长径和对应的正中垂直线，并计算肿瘤体积。连续给药24 d后处死裸鼠，剥离皮下瘤块并测量体积，并进行组织学检测。

1.4. HE染色和免疫组化染色

肿瘤组织经脱水、包埋后制成石蜡切片。切片经脱蜡、水化后置于苏木素溶液和伊红染液中染色，梯度酒精脱水，二甲苯透明，封片。

切片脱蜡、水化后采用高压修复组织抗原，3% H₂O₂溶液灭活内源性过氧化物酶。山羊血清封闭20 min。滴加一抗E-cadherin（1∶100）、N-cadherin（1∶125）、Vimentin（1∶200）、CD31（1∶100）、Ki-67（1∶400）、Cleaved caspase-3（1∶200）。按照试剂盒说明书滴加试剂，二氨基联苯胺显色，苏木素复染。组织切片梯度酒精中脱水，二甲苯透明及封片。

1.5. 统计学处理

根据预实验结果，采用每组5只实验动物已可达到统计学意义，为遵循动物伦理学相关要求，故选择每组5只实验动物进行实验。采用SPSS 13.0统计软件分析数据，正态分布计量资料用均数±标准差表示，两组比较采用t检验，两组多个时间点比较采用重复测量方差分析。P < 0.05为差异有统计意义。

2. 结果

2.1. AZD2014对裸鼠肿瘤生长的影响

对照组肿瘤明显大于AZD2014组（图 1）。不同时间点对照组和AZD2014组肿瘤的体积比较（图 2），AZD2014组肿瘤体积随时间延长，几乎未再增长，而与此相反的是，对照组中的肿瘤体积随着时间的延长而不断增大（F=43.021，P < 0.001）。

裸鼠及皮下肿瘤

Gross observatin of the tumor-bearing mice and the dissected tumors in control group (A) and AZD2014 group (B).

对照组和AZD2014组裸鼠皮下肿瘤增长情况

Growth curve of subcutaneous tumor in nude mice treated with AZD2014 or vehicle (n=5). *P < 0.05, **P < 0.01, ***P < 0.001.

2.2. AZD2014在体内对肝细胞癌组织学的影响

HE染色发现AZD2014组中发生坏死的细胞明显多于对照组（图 3）。我们对肿瘤组织中若干个细胞功能相关蛋白进行了检测。AZD2014给药组中Ki-67的表达量明显低于对照组（图 4）。在祼鼠体内研究中，发现AZD2014给药组中cleaved caspase-3的表达量明显高于对照组（图 5）。AZD2014给药组中CD31分子的表达量明显低于对照组（图 6）。在体内研究中，我们发现AZD2014给药组间质细胞表型蛋白N-cadherin和Vimentin的表达水平明显低于对照组，而上皮细胞表型蛋白E-cadherin的表达水平则明显高于对照组（图 7~9）。

肿瘤HE染色

HE staining of the tumor tissues (Original magnification: × 400). A: Control group. B: AZD2014 group.

免疫组化Ki-67的表达水平

Immunohistochemical staining for detecting Ki-67 in the tumor tissues (×400). A: Control group. B: AZD2014 group.

免疫组化Cleaved caspase-3的表达水平

Immunohistochemical staining for detecting cleaved caspase-3 in the tumor tissues (×400).A: Control group. B: AZD2014 group.

免疫组化CD31的表达水平

Immunohistochemical staining for detecting CD31 in the tumor tissues (×400). A: Control group. B: AZD2014 group.

免疫组化E-cadherin蛋白表达水平

Immunohistochemical staining for detecting E-cadherin in the tumor tissues (×400). A: Control group. B: AZD2014 group.

免疫组化N-cadherin蛋白表达水平

Immunohistochemical staining for detecting N-cadherin in the tumor tissues (×400). A: Control group. B: AZD2014 group.

免疫组化Vimentin蛋白表达水平

Immunohistochemical staining for detecting Vimentin in the tumor tissues (×400). A: Control group. B: AZD2014 group.

3. 讨论

HCC是一种普遍恶性肿瘤，其发病率逐年增加^[25]；其临床表现轻重主要与肿瘤部位、病理类型及是否发生转移有关，临床上大部分患者就诊时已是中晚期，不能或不适于进行手术治疗。晚期HCC患者多采用介入治疗、药物治疗或多种方案联合运用的综合措施进行治疗，然而治疗效果却并不理想，这主要与HCC生物学特性有关^[26]。

mTOR信号通路以mTORC1和mTORC2两种复合物的形式存在，mTORC1主要联合下游的4EBP1和S6K参与促进蛋白质合成，从而发挥促增殖作用^[27]；mTORC2则主要联合AKT通路发挥作用^[26]。约半数以上的HCC患者表现出mTOR信号通路的异常激活。临床上已将mTORC1的特异性抑制剂雷帕鸣用于HCC的治疗，然而，本课题组前期研究发现雷帕鸣诱导自噬作用有限^[24]，这在其他类型的肿瘤细胞中也有类似的报道^[28]。

AZD2014作为高度选择性的mTOR抑制剂，能够在完全抑制mTORC1通路的同时抑制mTORC2通路，可作为新的抗肿瘤药物应用于临床。目前其应用于其他类型肿瘤的报道较多，而在HCC中的应用研究极少。有研究在口腔癌细胞上证实AZD2014能够诱导G1/G2/M细胞周期阻滞^[19]，有研究应用永生化小鼠内皮细胞证实AZD2014通过内源性凋亡途径来诱导细胞凋亡^[22]。

本实验中，我们选用了一些指标对肿瘤的增殖能力及细胞凋亡进行了描述。Ki-67是一种细胞增殖相关抗原，其功能与细胞有丝分裂密切相关，在细胞增殖中发挥着不可或缺的作用^[29]。Caspase-3是细胞凋亡中承担重要功能的蛋白，已广泛用于描述肿瘤细胞凋亡及坏死情况^[30]。在实验中，我们发现注射AZD2014的实验组的肿瘤大小及增长速度均明显小于对照组，同时表达低Ki-67表达和高Caspase-3表达的特性，这在HCC上对AZD2014的细胞周期阻滞和诱导凋亡作用提供进一步佐证。另外，CD31作为一种广泛存在于血管内皮的细胞因子，常被用作描述微血管生成情况^[31]。我们的研究发现，实验组的CD31表达明显低于对照组，可以证明AZD2014对肿瘤微血管生成亦有抑制作用。因此，本实验证实了双mTORC1/2抑制剂AZD2014在体内可以显著地抑制HCC的生长。

目前认为上皮-间质转变在恶性肿瘤的转移和复发等过程中发挥着重要的作用。HCC具有较强的转移倾向和较高的复发率，这也正是近年来治疗预后未能得到明显改善的重要原因。若能抑制肿瘤细胞的EMT进程，或可以打破这一僵局。TGF-β信号在调控EMT进程中起着重要的作用^[32-33]。有研究发现，TGF-β可以通过PI3K信号通路激活mTORC1和mTORC2，增加细胞蛋白质的合成，增强细胞迁移和侵袭的能力，促进细胞从上皮表型转变为间质表型^[34-35]。我们前期的研究已证实AZD2014可以抑制体外肝癌细胞的EMT进程^[24]；本次研究中我们发现AZD2014处理后的裸鼠肿瘤中HCC间质表型蛋白N-cadherin和Vimentin的表达水平明显降低，而上皮表型蛋白E-cadherin的表达水平则明显升高，共同证明AZD2014在体内也能够有效地抑制HCC的EMT进程。

总之，我们证实了使用对mTORC1和mTORC2有联合抑制作用的AZD2014，能够更显著的抑制HCC的增殖、微血管形成及EMT进程，同时还能促进肝癌细胞的坏死及凋亡，共同作用发挥更理想的肝细胞癌抗癌效果。AZD2014有望能作为HCC分子靶向治疗新抑制剂在临床应用。

Biography

廖晖，博士，副主任医师，E-mail: jxliaohui@sina.com

Funding Statement

广东省自然科学基金（2018A030313214）

Contributor Information

廖晖 (Hui LIAO), Email: jxliaohui@sina.com.

杨定华 (Dinghua YANG), Email: dhyangyd@yahoo.com.

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