Blockade of Radiation-induced EGFR Nuclear Transport Enhances Radiosensitivity of Human Cervical Cancer
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摘要:目的
探讨抑制EGFR核转位是否会降低人宫颈癌细胞的放射抵抗。
方法Western blot测定pEGFR-T654多肽、pEGFR-T654对照多肽、西妥昔单抗或吉非替尼预处理后X线照射的人宫颈鳞癌CaSki和腺癌HeLa细胞pEGFR-T654和pDNA-PK-T2609的表达; 克隆形成法测定存活分数(SF2), 单击多靶模型拟合剂量-存活曲线, 计算放射增敏比(SER)。
结果4 Gy照射后, CaSki和HeLa细胞核EGFR表达呈时间依赖性增加; 与对照多肽比较, pEGFR-T654多肽显著降低了CaSki和HeLa细胞核内pEGFR-T654、DNA-PK和pDNA-PK-T2609的表达; 与单独放射比较, 西妥昔单抗联合放射明显降低了CaSki细胞核EGFR、pEGFR-T654和pDNA-PK-T2609的表达以及克隆形成率和存活分数(SF2=31.030), 增加CaSki细胞的放射敏感度(SER=2.34)。
结论放射诱导pEGFR-T654核转位介导pDNAPK-T2609的活化, 西妥昔单抗抑制pEGFR-T654核转运, 降低了DNAPK介导的宫颈鳞癌放射抵抗。
Abstract:ObjectiveTo investigate whether the inhibition of EGFR nuclear transport could reduce the radioresistance of human cervical cancer cells.
MethodsHuman cervical cancer CaSki and HeLa cells were exposed to radiation treated with or without Thr654 inhibitory peptide, cetuximab and gefitinib. The expression levels of pEGFR-T654 and pDNA-PK-T2609 were determined using Western blot. The survival fraction (SF2) was determined by colony formation and the dose-survival curve was fitted with a singlehit multi-target model to calculate the radiosensitization ratio (SER).
ResultsAfter 4 Gy irradiation, EGFR expression in the nuclear of CaSki and HeLa cells were up-regulated in a time-dependent manner. Compared with the control peptide, Thr654 inhibitory peptide significantly reduced the expression levels of pEGFR-T654, DNA-PK and pDNA-PK-T2609 in the nucleus of CaSki and HeLa cells; compared with irradiation alone, cetuximab combined with irradiation significantly reduced the expression levels of EGFR, pEGFR-T654 and DNA-PK-T2609 in the nucleus of CaSki cells, as well as clone formation rate and survival fraction (SF2=31.030), and increased the radiosensitivity (SER=2.34).
ConclusionRadiationinduced pEGFR-T654 nuclear translocation mediates the activation of pDNA-PK-T2609, and the inhibition of pEGFR-T654 nuclear transport by cetuximab reduces DNA-PK-mediated radiation resistance of cervical squamous cell carcinoma.
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Key words:
- Cervical cancer /
- EGFR /
- DNA-PK /
- Radiation therapy
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0 引言
目前，化学治疗仍是三阴性乳腺癌的主要治疗方法之一，但是肿瘤细胞对化疗药物的耐药性严重影响了治疗效果，化疗药物与肿瘤细胞的接触是诱导继发性耐药的主要原因[1]。由于阿霉素是乳腺癌化学方案的常用药物[2]，本研究观察阿霉素对三阴性乳腺癌耐药性的诱导作用并探究其机制。
ATP结合盒（ABC）转运蛋白在耐药的发展中起着至关重要的作用。ATP结合盒亚家族G成员2（ATP-binding cassette, sub-family G member 2, ABCG2）能排出大量异质化合物，导致耐药，引起治疗抵抗[3]。细胞耐药性的产生及耐药蛋白的表达受多种转录因子的调控。有研究报道cMyc能够调控包括ABCG2在内的ABC转运蛋白的表达[4]。cMyc是一个多功能的转录因子，参与调节细胞对阿霉素的敏感度[5]，而cMyc的表达受其上游基因Stat3的调控。Stat3在肿瘤组织中异常激活，引发其下游靶基因cMyc转录，从而使正常细胞转化为癌细胞，并增加肿瘤细胞的耐药性[6]。因此，本研究观察阿霉素对MDA-MB-468细胞耐药性的诱导作用并探讨Stat3-cMyc通路是否介导了耐药性的发生。
1 材料与方法
1.1 细胞株、试剂、仪器
人乳腺癌MDA-MB-468细胞株购自美国标准细胞库（American type culture collections, ATCC）。本研究实验剂和仪器包括：RPMI 1640培养基（Hyclone，美国）、青霉素/链霉素（索莱宝，北京，中国）、胎牛血清（四季青，杭州，中国）、阿霉素（索莱宝，北京，中国）、RIPA裂解液/苯甲基磺酰氟（索莱宝，北京，中国）、聚偏二氟乙烯膜（Millipore，Billerica，美国）、ABCG2抗体（Abcam，Cambridge，美国）、WP1066抑制剂（Selleckchem，上海，中国）和二甲基亚砜（索莱宝，北京，中国）等。
1.2 细胞培养
人乳腺癌MDA-MB-468细胞用含10%FBS和1%青霉素/链霉素的RPMI 1640在37℃、5%CO2培养箱中培养。以不同浓度的阿霉素（0、0.05、0.1和0.5 μmol/L）孵育细胞24 h，观察并筛选最适阿霉素浓度进行后续实验。
1.3 MTT法检测
细胞以3 000个/孔的密度接种至96孔板，然后分别加入终浓度为0、0.05、0.1和0.5 μmol/L的阿霉素。24 h后，每孔加入20 μl MTT溶液（5 mg/ml）继续培养4 h，吸弃培养液，每孔加150 μl DMSO溶液，振荡15 min后测定570 nm处的吸光度（OD570）。
1.4 细胞爬片及免疫荧光染色
将盖玻片置于24孔板孔底，分别将MDA-MB-468和MDA-MB-468/ADM细胞以1×104个/孔接种，待细胞爬满盖玻片后进行免疫荧光染色。用PBS轻轻冲洗后在4%多聚甲醛中固定15 min。PBS洗涤爬片3次，山羊血清封闭1 h。将细胞用ABCG2一抗在4℃冰箱中孵育、过夜。PBS洗涤后，用二抗于37℃温育1 h。PBS洗涤细胞，用DAPI染色10 min，再次洗涤3次后滴加荧光防淬灭剂，观察免疫荧光染色并拍照。
1.5 蛋白质印迹分析
抽提各组细胞的总蛋白，利用BCA法测定总蛋白浓度，以每个泳道20 μg浓度的蛋白样品上样，经SDS-PAGE电泳后，利用半干电转化法将蛋白转移至PVDF膜上，经过封闭、一抗（稀释倍数1:1 000）孵育、TBST洗脱、HRP标记的二抗（稀释倍数1:5 000）孵育、TBST再洗脱等步骤后，用增强化学发光法检测信号及X线片曝光，并且经定影显影处理，获得清晰条带。
1.6 统计学方法
运用SPSS13.0统计软件进行分析，所有结果采用（x±s）表示，组间均数的比较采用独立t检验（双侧），P < 0.05为差异有统计学意义。
2 结果
2.1 持续低剂量阿霉素刺激诱导MDA-MB-468细胞产生耐药性
不同浓度阿霉素作用于MDA-MB-468细胞24 h后可见0.05 μmol/L与0.1 μmol/L浓度的阿霉素未引起细胞明显的损伤，大部分细胞生长良好。当浓度增加到0.5 μmol/L时，几乎所有细胞都受损，可见大量坏死细胞；MTT法测得在0.05 μmol/L、0.1 μmol/L及0.5 μmol/L浓度下阿霉素对MDA-MB-468细胞的抑制率分别为0.14、0.20、0.38，而且阿霉素对MDA-MB-468细胞的半数最大效应浓度（concentration for 50% of maximal effect, EC50）为0.94 μmol/L（P=0.038）。综合以上结果，我们选用0.1 μmol/L的阿霉素继续进行后续研究。
用0.1 μmol/L的阿霉素持续刺激MDA-MB-468细胞4周后获得耐药细胞，命名为MDA-MB-468/ADM。MTT实验检测MDA-MB-468/ADM细胞对阿霉素敏感度，结果显示MDA-MB-468/ADM的EC50为5.2 μmol/L，较MDA-MB-468的EC50（0.94 μmol/L）显著升高（P=0.041），说明长期使用0.1 μmol/L的阿霉素后，MDA-MB-468细胞对阿霉素的敏感度显著下降，产生耐药，见图 1。
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图 1 MTT测定经24h处理后的MDA-MB-468细胞与MDA-MB-468/ADM细胞对阿霉素的敏感度(x±s, n=3)Figure 1 Sensitivity of MDA-MB-468 and MDA-MB-468/ADM cells to adriamycin after 24h treatment detected by MTT assay (x±s, n=3)2.2 MDA-MB-468/ADM细胞中高表达耐药蛋白ABCG2
与正常MDA-MB-468细胞相比，MDA-MB-468/ADM细胞中代表ABCG2表达水平的红色荧光明显增多增强，见图 2A。Western blot检测结果也表明了MDA-MB-468/ADM细胞中ABCG2的高表达，见图 2B。提示用0.1 μmol/L阿霉素持续刺激后，三阴性乳腺癌MDA-MB-468细胞对阿霉素产生耐药。
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图 2 MDA-MB-468/ADM细胞中耐药蛋白ABCG2的表达Figure 2 Expression of drug resistance protein ABCG2 in MDA-MB-468/ADM cellsA: Immunofluorescence staining results showed the increased expression of ABCG2 (red) in MDA-MB-468/ADM cells, staining with DAPI (blue); B: Western blot analysis results showed high expression of ABCG2 in MDA-MB-468/ADM cells (n=3, *: P < 0.05)2.3 MDA-MB-468/ADM细胞高表达p-Stat3与cMyc
为探究MDA-MB-468细胞对阿霉素产生耐药的机制，我们进一步检测了MDA-MB-468/ADM细胞中转录因子p-stat3与cMyc的表达水平，观察MDA-MB-468细胞对阿霉素耐药性的产生是否与Stat3-cMyc途径有关。Western blot结果显示，MDA-MB-468/ADM中p-Stat3与cMyc的表达均明显升高，而两组细胞中总的Stat3表达水平未见显著变化。这些结果表明Stat3的激活和cMyc表达的增多可能参与了MDA-MB-468细胞对阿霉素耐药性的产生，见图 3。
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图 3 Western blot检测在MDA-MB-468和MDA-MB-468/ADM细胞中p-Stat3、Stat3及cMyc的表达(n=3, *: P < 0.05, **: P < 0.01)Figure 3 p-Stat3, Stat3 and cMyc expression in MDA-MB-468 and MDA-MB-468/ADM cells analyzed by Western blot (n=3, *: P < 0.05, **: P < 0.01)2.4 抑制Stat3活化可下调cMyc及ABCG2的表达
为进一步证明Stat3-cMyc途径在阿霉素诱导三阴性乳腺癌MDA-MB-468细胞耐药性产生中的作用，我们用Stat3磷酸化的抑制剂WP1066抑制Stat3活化，观察转录因子cMyc的表达是否受到影响。结果显示WP1066（1.25 μmol/L）作用于MDA-MB-468/ADM细胞后，磷酸化的Stat3显著降低（P=0.014），同时cMyc表达水平明显下降（P=0.044）。另外WP1066处理后MDA-MB-468/ADM细胞耐药蛋白ABCG2的表达也显著减少（P=0.000）。这些结果进一步说明阿霉素通过Stat3-cMyc途径诱导了MDA-MB-468细胞耐药性的产生，而抑制Stat3的活化后，耐药蛋白表达减少，细胞的耐药性减弱，见图 4。
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图 4 Western blot检测MDA-MB-468、MDA-MB-468/ADM、MDA-MB-468/ADM/WP1066三组细胞中p-Stat3、Stat3、cMyc及ABCG2的表达Figure 4 Stat3, p-Stat3, cMyc and ABCG2 expression in MDA-MB-468, MDA-MB-468/ADM and MDA-MB-468/ADM/WP1066 cells analyzed by Western blot*: P < 0.05, **: P < 0.01 (x±s, n=3)2.5 抑制Stat3活化增强了MDA-MB-468/ADM细胞对阿霉素的敏感度
由于WP1066下调了耐药蛋白ABCG2的表达，因此我们进一步通过MTT法检测MDA-MB-468/ADM细胞对阿霉素敏感度的变化。结果显示，阿霉素对MDA-MB-468/ADM细胞的EC50为6.774 μmol/L，而在使用WP1066之后的EC50降低至1.29 μmol/L（P=0.000），这表明WP1066抑制Stat3的活化增强了MDA-MB-468/ADM细胞对阿霉素的敏感度，见图 5。
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图 5 MTT法检测经36 h处理后的MDA-MB-468、MDA-MB-468/ADM、MDA-MB-468/ADM/WP 1066三组细胞对阿霉素的敏感度(x±s, n=3)Figure 5 Sensitivity of MDA-MB-468, MDA-MB-468/ADM and MDA-MB-468/ADM/WP1066 cells to adriamycin after 36h treatment detected by MTT assay (x±s, n=3)3 讨论
目前肿瘤细胞的耐药性是临床治疗的难点与研究的热点，阐明肿瘤耐药的机制可以为肿瘤的治疗提供新的治疗方向和靶点。
本研究应用低剂量阿霉素持续诱导人三阴性乳腺癌MDA-MB-468细胞，导致细胞产生耐药性，对阿霉素的敏感度显著下降，耐药蛋白ABCG2表达增高。为探究MDA-MB-468细胞对阿霉素产生耐药的机制，本实验进一步检测了MDA-MB-468/ADM细胞中转录因子p-stat3与cMyc的表达水平，观察MDA-MB-468细胞对阿霉素耐药性的产生与Stat3-cMyc途径有关。为进一步证明Stat3-cMyc途径在阿霉素诱导三阴性乳腺癌MDA-MB-468细胞耐药性产生中的作用，实验用Stat3磷酸化的抑制剂WP1066抑制Stat3活化，发现转录因子cMyc的表达也受到影响。进一步的机制研究揭示了Stat3-cMyc通路在阿霉素诱导的耐药中具有重要作用。
文献报道，Stat3信号通路与肿瘤细胞对化疗的耐药性有关[7]。Stat3的激活可以帮助癌细胞逃避由药物引起的死亡，从而诱发耐药性。Yue等[8]证明了Stat3的过度活化可以促进顺铂耐药的卵巢癌进展，相反，如果抑制Stat3信号通路则会促进耐药性癌细胞的凋亡，增加癌细胞对各种药物的敏感度。Li等[9]研究也有相似的发现，抑制Stat3信号通路后人胃癌细胞的凋亡增强，耐药性减弱。那么Stat3在三阴性乳腺癌耐药性的产生中有何作用？文献报道，乳腺癌组织中Stat3的活化增强与乳腺癌的临床分期和侵袭转移密切相关[10]。多种致癌性细胞因子与细胞膜的相应受体结合后导致Stat3与酪氨酸磷酸化通道相偶联后被激活，激活后的Stat3可在核内与特异性DNA启动子相结合，调节cMyc、Oct4、Sox2等相关基因表达[11]。作为调节多种转录因子功能的重要枢纽，Stat3有望成为肿瘤基因治疗中的有效靶点。有研究表明，在肿瘤中cMyc的表达水平与耐药性有关[4, 12-13]，cMyc能够调控ABC转运蛋白的表达水平，而ABCG2与肿瘤细胞的耐药性直接相关，但Stat3/cMyc在三阴性乳腺癌产生耐药性方面的影响及机制却未见报道。
本研究发现低浓度（0.1 μmol/L）阿霉素持续刺激使MDA-MB-468细胞对阿霉素的敏感度明显降低，MDA-MB-468/ADM细胞中p-Stat3和cMyc的表达较MDA-MB-468细胞显著增加，这些发现与上述文献中对Stat3和cMyc在肿瘤耐药性中的作用相一致。另外，刘丽等[6]在喉鳞癌细胞的研究中也揭示了Stat3-cMyc通路的重要作用，与本研究的结果相吻合。由此推测，MDA-MB-468/ADM对阿霉素耐药的机制很可能与Stat3的激活和p-Stat3介导的cMyc表达的增多有关。为进一步证明Stat3-cMyc通路在阿霉素诱导的乳腺癌耐药性中的关键作用，本实验应用WP1066抑制MDA-MB-468/ADM中Stat3的活化，发现随着p-Stat3的降低，cMyc和ABCG2的表达也相应下降，这与Granato等[14]证实抑制Stat3信号可下调cMyc的表达一致。再次MTT检测发现WP1066作用后MDA-MB-468/ADM细胞对阿霉素的敏感度显著增强，这与Li等[9]研究结果一致。
总之，本实验结果表明阿霉素可以诱导Stat3活化，上调转录因子cMyc及耐药蛋白ABCG2的表达，促进了三阴性乳腺癌MDA-MB-468细胞对阿霉素耐药性的产生。因此，抑制Stat3的表达与活化可有效逆转乳腺癌对阿霉素的耐药性，特异性靶向Stat3-cMyc途径联合化疗药物治疗有望成为一种有效治疗乳腺癌的新措施，改善乳腺癌患者的预后。
作者贡献李璐：设计及实施实验、撰写论文黄建鸣：设计实验、撰写及审阅论文冯梅：设计实验、获取研究经费、审阅论文漆云翔、马士淇、谭明宇：设计及实施实验、采集数据郎锦义：设计实验、获取研究经费、指导论文 -
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图 1 CaSki和HeLa细胞照射后不同时间细胞质和细胞核EGFR表达变化
Figure 1 Relative expression of cEGFR and nEGFR in CaSki and HeLa cell lines at different time after radiation
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图 2 4Gy照射后不同时间CaSki和HeLa细胞核中pEGFR-T654、DNA-PK和pDNA-PK-T2609表达变化
Figure 2 Relative expression of pEGFR-T654, DNA-PK and pDNA-PK-T2609 in CaSki and HeLa cell lines at different time after 4 Gy radiation
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图 3 多肽和对照多肽不同时间预处理后CaSki和HeLa细胞核中pEGFR-T654、DNA-PK和pDNA-PK-T2609表达变化
Figure 3 Relative expression of pEGFR-T654, DNA-PK and pDNA-PK-T2609 in CaSki and HeLa cell lines with or without Thr654 inhibitory peptide
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图 4 放射前后采用吉非替尼(GFTN)和西妥昔单抗(C225)预处理后CaSki和HeLa细胞细胞核中pEGFR-T654、pDNA-PK-T2609和DNA-PK表达变化
Figure 4 Relative expression of pEGFR-T654, pDNA-PK-T2609 and DNA-PK in CaSki and HeLa cell lines treated with Gefitinib (GFTN) or Cetuximab (C225) before and after radiation
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图 5 单击多靶模型拟合细胞存活-曲线
Figure 5 Dose-survival curves of CaSki and HeLa cells fitted by a single-hit multi-target model
表 1 不同处理组CaSki和HeLa细胞生物学参数比较
Table 1 Biological parameters of CaSki and HeLa cells in different treatment groups
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