新型TK抑制剂达克替尼在肺腺癌细胞增殖和凋亡的影响
Effect of a Novel TK Inhibitor Dacomitinib on Proliferation and Apoptosis of Lung Adenocarcinoma Cells
DOI: 10.12677/HJMCe.2021.93014, PDF, HTML, XML, 下载: 458  浏览: 1,123 
作者: 郭李玲：甘肃医学院附属医院全科医学科，甘肃 平凉；毛 俊：甘肃医学院基础医学院生物化学与分子生物学，甘肃 平凉
关键词: 达克替尼；肺腺癌；增殖；凋亡；Dacomitinib； Lung Adenocarcinoma； Proliferation； Apoptosis
摘要: 目的：探究新型TK抑制剂达克替尼对人肺腺癌A549细胞增殖与侵袭作用及机制。方法：人肺腺癌A549细胞为研究对象，根据预实验结果，将人肺腺癌细胞系A549分成3组，即空白对照组，低剂量组和高剂量组。RT-PCR检测EFGR和凋亡蛋白Caspase-3和Caspase-9的表达，流式细胞仪检测细胞的分裂周期和细胞凋亡率，CCK-8检测细胞的增殖。结果：空白对照组，低剂量组和高剂量组的EFGR，Caspase-3和Caspase-9 mRNA相对表达量相对量比较具有统计学差异(P < 0.05)；空白对照组的EFGR mRNA相对表达量相对量要明显高于低剂量组和高剂量组(P < 0.05)，空白对照组的Caspase-3和Caspase-9 mRNA相对表达量要明显低于低剂量组和高剂量组(P < 0.05)，低剂量组和高剂量组的EFGR，Caspase-3和Caspase-9 mRNA相对表达量相对量比较无统计学差异(P > 0.05)。低剂量组和高剂量组的G0/G1期比例明显高于空白对照组(P < 0.05)；S期和G2期比例明显低于空白对照组(P < 0.05)。低剂量组和高剂量组的总凋亡率明显高于空白对照组(P < 0.05)；高剂量组晚期凋亡率明显高于低剂量组(P < 0.05)。药物处理2天后，空白对照组的细胞增值率要明显高于低剂量组和高剂量组(P < 0.05)；药物处理3天后，空白对照组的细胞增值率要明显高于低剂量组和高剂量组(P < 0.05)。结论：低剂量达克替尼可以抑制肺腺癌细胞的增殖，促进肺腺癌细胞的凋亡，并且可以抑制EFGR的表达，可以作为耐药性肺腺癌的治疗方案。
Abstract: Objective: The objective is to investigate the proliferation and invasion of human lung adenocar-cinoma A549 cells induced by a novel TK inhibitor, dacomitinib, and its mechanism. Methods: Human lung adenocarcinoma cell line A549 was divided into three groups according to the pre-liminary results, namely the blank control group, the low-dose group and the high-dose group. The expression of EFGR and Caspase-3 and Caspase-9 was detected by RT-PCR. The cycle of cell division and apoptotic rate of the cell were detected by flow cytometry. The cell proliferation was detected by CCK-8. Results: The relative expressions of EFGR, caspase-3 and caspase-9 mRNA in blank control group, low-dose group and high-dose group were statistically different (P < 0.05). The relative expression of EFGR mRNA in the blank control group was significantly higher than that in the low-dose group and the high-dose group (P < 0.05). The relative expression of Caspase-3 and caspase-9 mRNA in the blank control group was significantly lower than that in the low-dose group and high-dose group (P < 0.05). There was no significant difference in the relative expression of EFGR, caspase-3 and caspase-9 mRNA between the low-dose group and the high-dose group (P > 0.05). The proportions of G0/G1 phase in low-dose group and high-dose group were significantly higher than those in blank control group (P < 0.05), while the proportions of S phase and G2 phase were significantly lower than those in blank control group (P < 0.05). The total apoptotic rate of the low-dose group and the high-dose group significantly higher than that of the blank control group (P < 0.05), and the late apoptotic rate of the high-dose group was significantly higher than that of the low-dose group (P < 0.05). After 2 days of drug treatment, the cell increment rate of blank control group was significantly higher than that of low-dose group and high-dose group (P < 0.05); after 3 days of drug treatment, the cell increment rate of blank control group was significantly higher than that of low-dose group and high-dose group (P < 0.05). Conclusion: Low-dose of dacomitinib can inhibit the proliferation of lung adenocarcinoma cells, promote the apoptosis of lung adenocarcinoma cells, and inhibit the expression of EFGR. It can be used as a therapeutic regimen for drug-resistant lung adenocarcinoma.Objective: The objective is to investigate the proliferation and invasion of human lung adenocarci-noma A549 cells induced by a novel TK inhibitor, dacomitinib, and its mechanism. Methods: Human lung adenocarcinoma cell line A549 was divided into three groups according to the preliminary re-sults, namely the blank control group, the low-dose group and the high-dose group. The expression of EFGR and Caspase-3 and Caspase-9 was detected by RT-PCR. The cycle of cell division and apop-totic rate of the cell were detected by flow cytometry. The cell proliferation was detected by CCK-8. Results: The relative expressions of EFGR, caspase-3 and caspase-9 mRNA in blank control group, low-dose group and high-dose group were statistically different (P < 0.05). The relative expression of EFGR mRNA in the blank control group was significantly higher than that in the low-dose group and the high-dose group (P < 0.05). The relative expression of Caspase-3 and caspase-9 mRNA in the blank control group was significantly lower than that in the low-dose group and high-dose group (P < 0.05). There was no significant difference in the relative expression of EFGR, caspase-3 and caspa-se-9 mRNA between the low-dose group and the high-dose group (P > 0.05). The proportions of G0/G1 phase in low-dose group and high-dose group were significantly higher than those in blank control group (P < 0.05), while the proportions of S phase and G2 phase were significantly lower than those in blank control group (P < 0.05). The total apoptotic rate of the low-dose group and the high-dose group significantly higher than that of the blank control group (P < 0.05), and the late apoptotic rate of the high-dose group was significantly higher than that of the low-dose group (P < 0.05). After 2 days of drug treatment, the cell increment rate of blank control group was signifi-cantly higher than that of low-dose group and high-dose group (P < 0.05); after 3 days of drug treatment, the cell increment rate of blank control group was significantly higher than that of low-dose group and high-dose group (P < 0.05). Conclusion: Low-dose of dacomitinib can inhibit the proliferation of lung adenocarcinoma cells, promote the apoptosis of lung adenocarcinoma cells, and inhibit the expression of EFGR. It can be used as a therapeutic regimen for drug-resistant lung ade-nocarcinoma.
文章引用：郭李玲, 毛俊. 新型TK抑制剂达克替尼在肺腺癌细胞增殖和凋亡的影响[J]. 药物化学, 2021, 9(3): 112-118. https://doi.org/10.12677/HJMCe.2021.93014

1. 引言

肺癌是临床上的多发病，尤其是随着生活环境的改变，生活压力的激增，人们饮食习惯的改变，肺癌的发病率明显升高。据流行病学统计结果显示，全球每年新增的肺癌患者约几万例，多见于发展中国家 [1] [2] [3]。目前对于肺癌患者的治疗，化疗方案是较为理想的治疗方案。有研究表明，表皮生长因子受体酪氨酸激酶抑制剂(Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors, EGFR-TKIs)是比较有效的治疗方案之一，比如吉非替尼等 [4]，但是，该类治疗方案也存在一定的问题，比如药物耐药性的问题 [5]。并且与表皮生长因子(EFGR)的表达量相关，为了解决这个问题，第二代TKI应运而生，代表药物是达克替尼。有研究表明，达克替尼可以抑制EFGR的表达，从而达到抑制耐药性的作用 [6]。但是，关于达克替尼的细胞学实验较少，因此，本研究以人肺腺癌A549细胞为研究对象，验证达克替尼的生物学行为，以期为新型TK抑制剂达克替尼治疗人肺腺癌提供理论依据。

2. 材料与方法

2.1. 肺腺癌细胞系A549的培养

细胞系购自北京索莱宝生物有限公司，均经过str检测，证明细胞没有被污染。

2.2. 实验分组

根据预实验结果，将人肺腺癌细胞系A549分成3组，即空白对照组，低剂量组和高剂量组。低剂量组细胞给予1 μmol/L的达克替尼进行处理3 h；高剂量组细胞给予10 μmol/L的达克替尼进行处理3 h；空白对照组细胞给予等计量的PBS溶液进行对照处理。

2.3. 实时荧光定量PCR检测Akt1、Caspase-3和Caspase-9 mRNA的表达水平

收集上述3组细胞，加入1 mL Trizol试剂，充分混合后，提取细胞总RNA，室温条件下根据逆转录试剂盒说明书，将总RNA逆转录为cDNA，−20℃低温冰箱保存。然后根据荧光定量PCR试剂盒说明书，加入cDNA和USP-22基因和ERK5的引物模板(如表1所示)，置于Applied Biosystems PCR仪进行反应，设置条件按照说明书。统计并记录各样本CT值。

Table 1. The primer sequence of target gene

表1. 目的基因的引物序列

2.4. 细胞凋亡水平

利用流式细胞仪进行检测，加入AV-PI试剂盒的混合制剂，然后流式细胞仪上机测试，验证细胞的凋亡水平。

2.5. 细胞增殖能力

利用CCK-8试剂盒检测细胞增殖能力，加入CCK-8试剂盒的混合制剂，利用酶标仪进行测定，波长为450 nm。

2.6. 统计学方法

计数资料用 X ¯ ± s形式表示，SPSS 20.0软件进行统计学分析，3组的RT-PCR数据结果，细胞增值率和细胞凋亡率比较采用单因素方差分析，组间的比较采用q检验，认为P < 0.05有统计学意义。

3. 结果

3.1. EFGR，Caspase-3和Caspase-9的mRNA相对表达量

结果表明，空白对照组，低剂量组和高剂量组的EFGR，Caspase-3和Caspase-9的mRNA相对表达量比较具有统计学差异(F = 10.209, 9.892, 11.092; P < 0.05)；其中低剂量组和高剂量组的EFGR的mRNA相对表达量要明显低于空白对照组(P < 0.05)，Caspase-3和Caspase-9的mRNA相对表达量要明显高于空白对照组(P < 0.05) (见图1)。

Figure 1. The relative mRNA expression of EFGR, caspase-3 and caspase-9. Note: ** indicates statistical difference (P < 0.05); # indicates no statistical difference (P > 0.05)

图1. EFGR，Caspase-3和Caspase-9的mRNA相对表达量。注：**表示具有统计学差异(P < 0.05)；#表示没有统计学差异(P > 0.05)

3.2. 3组细胞周期比较结果

结果表明，低剂量组和高剂量组的G0/G1期比例明显高于空白对照组(P < 0.05)；S期和G2期比例明显低于空白对照组(P < 0.05) (见图2)。

3.3. 3组细胞凋亡率比较结果

结果表明，低剂量组和高剂量组的总凋亡率明显高于空白对照组(P < 0.05)；高剂量组晚期凋亡率明显高于低剂量组(P < 0.05) (见图3)。

3.4. 3组细胞增殖率比较结果

结果显示，药物处理2天后，空白对照组的细胞增值率要明显高于低剂量组和高剂量组(q = 2.293, 2.775; P < 0.05)；药物处理3天后，空白对照组的细胞增值率要明显高于低剂量组和高剂量组(q = 2.887, 2.461; P < 0.05) (见图4)。

(a) (b)(c)

Figure 2. The comparison of cell cycle among three groups. (a) The blank control group; (b) The low-dose group; (c) The high-dose group

图2. 3组细胞周期比较结果。(A) 空白对照组；(B) 低剂量组；(C) 高剂量组

(a) (b)(c)

Figure 3. The comparison of apoptosis rate among three groups. (a) The blank control group; (b) The low-dose group; (c) The high-dose group

图3. 3组细胞凋亡率比较结果。(a) 空白对照组；(b) 低剂量组；(c) 高剂量组

Figure 4. The comparison of proliferation rate among three groups. Note: * indicates statistical difference (P < 0.05)

图4. 3组细胞增值率比较结果。注：*表示具有统计学差异(P < 0.05)

4. 讨论

本研究结果表明，根据预实验结果给予1 μmol/L低剂量的达克替尼和10 μmol/L高剂量的达克替尼两种浓度进行处理，并且加入等量PBS溶液进行对比研究。RT-PCR检测EFGR和凋亡蛋白Caspase-3和Caspase-9的表达。流式细胞仪检测细胞的分裂周期和细胞凋亡率，CCK-8检测细胞的增殖。结果表明，达克替尼处理人肺腺癌A549细胞，可以明显降低EFGR mRNA相对表达量和蛋白表达量，增加Caspase-3和Caspase-9 mRNA相对表达量和蛋白表达量。并且，低剂量组和高剂量组的G0/G1期比例明显高于空白对照组(P < 0.05)；低剂量组和高剂量组的总凋亡率明显高于空白对照组(P < 0.05)；药物处理3天后，空白对照组的细胞增值率要明显高于低剂量组和高剂量组(P < 0.05)。说明，达克替尼可以抑制细胞增殖，促进细胞凋亡。可以作为EFGR阳性的肺腺癌A549细胞的治疗药物。

对于肺腺癌患者而言，尤其是晚期肺腺癌，表皮生长因子受体(Epidermal Growth Factor Receptor, EFGR)突变是其耐药性的主要特点 [7] [8] [9]。周玮玮等人 [10] 利用量子点(QDs)荧光探针检测人肺腺癌组织，结果表明，晚期肺腺癌组织可见EFGR的高表达。史张等人 [11] 对581例肺腺癌患者进行检测，可见132例EFGR的高突变，结果表明，EFGR的高突变与肺腺癌的增殖和侵袭有关。本研究在前人的基础上，利用第二代表皮生长因子受体酪氨酸激酶抑制剂(Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors, EGFR-TKIs)达克替尼进行治疗，结果表明，达克替尼可以抑制EFGR的mRNA相对表达水平和蛋白表达水平，说明达克替尼可以抑制EFGR的表达。

既往研究表明，Caspase-3和Caspase-9是与细胞凋亡密切相关，因此，也有学者称他们为“凋亡相关蛋白”，将Caspase-3和Caspase-9作为细胞凋亡的指示分子 [12] [13] [14]。本研究以Caspase-3作为凋亡的靶点，结果表明，达克替尼可以促进Caspase-3的mRNA相对表达水平和蛋白表达水平，说明达克替尼可以促进肺腺癌细胞的凋亡。另外，本研究利用CCK-8试剂盒检测细胞的增殖，结果表明，药物处理2天后，空白对照组的细胞增值率要明显高于低剂量组和高剂量组(P < 0.05)；药物处理3天后，空白对照组的细胞增值率要明显高于低剂量组和高剂量组(P < 0.05)。结果说明，达克替尼可以抑制肺腺癌细胞的增殖。并且与达克替尼的剂量无关。

综上所述，低剂量达克替尼可以抑制肺腺癌细胞的增殖，促进肺腺癌细胞的凋亡，并且可以抑制EFGR的表达，可以作为耐药性肺腺癌的治疗方案。

参考文献

参考文献

[1] Jordan, E.J., Kim, H.R., Arcila, M.E., et al. (2017) Prospective Comprehensive Molecular Characterization of Lung Adenocarcinomas for Efficient Patient Matching to Approved and Emerging Therapies. Cancer Discovery, 7, 15 p.
https://doi.org/10.1158/2159-8290.CD-16-1337
[2] Velcheti, V., Hida, T., Reckamp, K.L., et al. (2017) Phase 2 Study of Lenvatinib in Patients with RET Fusion-Positive Adenocarcinoma of the Lung. European Journal of Cancer, 72, S178.
https://doi.org/10.1016/S0959-8049(17)30651-2
[3] Feng, M., Zhu, J., Liang, L., et al. (2017) Diagnostic Value of Tumor Markers for Lung Adenocarcinoma-Associated Malignant Pleural Effusion: A Validation Study and Me-ta-Analysis. International Journal of Clinical Oncology, 22, 283-290.
https://doi.org/10.1007/s10147-016-1073-y
[4] Ono, A., Kenmotsu, H., Watanabe, M., et al. (2014) Mutant Al-lele Frequency Predicts the Efficacy of EGFR-TKIs in Lung Adenocarcinoma Harboring the L858R Mutation. Annals of Oncology, 25, 1948-1953.
https://doi.org/10.1093/annonc/mdu251
[5] Kim, I.A., Lee, J.S., Kim, H.J., et al. (2018) Cumulative Smoking Dose Affects the Clinical Outcomes of EGFR-Mutated Lung Adenocarcinoma Patients Treated with EGFR-TKIs: A Retrospective Study. BMC Cancer, 18, Article No. 768.
https://doi.org/10.1186/s12885-018-4691-0
[6] Zhou, F., Ma, W., Li, W., et al. (2018) Thick-Wall Cavity Predicts Worse Progression-Free Survival in Lung Adenocarcinoma Treated with First-Line EGFR-TKIs. BMC Cancer, 18, Article No. 1033.
https://doi.org/10.1186/s12885-018-4938-9
[7] Chen, H., Yang, X., Liu, H., et al. (2017) Correlation between Serum Tumor Markers and Efficacy of First-Line EGFR-TKIs in Patients with Advanced Lung Adenocarcinoma. Chi-nese Journal of Lung Cancer, 1, 76-87.
[8] Ma, X., Zhu, H., Guo, H., et al. (2016) Risk Factors of Brain Metastasis during the Course of EGFR-TKIs Therapy for Patients with EGFR-Mutated Advanced Lung Adenocarcinoma. Onco-target, 7, 81906-81917.
https://doi.org/10.18632/oncotarget.11918
[9] Ming-Szu, H., Jr-Hau, L., Lin, Y.C., et al. (2016) The Content of Mutant EGFR DNA Correlates with Response to EGFR-TKIs in Lung Adenocarcinoma Patients with Common EGFR Mutations. Medicine, 95, e3991.
https://doi.org/10.1097/MD.0000000000003991
[10] 周玮玮, 王磊, 苗玉, 等. 量子点荧光探针检测肺腺癌组织中EGFR的表达[J]. 标记免疫分析与临床, 2018, 25(5): 736-739.
[11] 史张, 宋长恩, 施睿峰, 等. 肺腺癌CT及临床特征与EGFR 19号外显子突变的相关性研究[J]. 临床放射学杂志, 2017, 36(4): 490-494.
[12] Bose, T.O., Pham, Q.M., Jellison, E.R., et al. (2013) CD11a Regulates Effector CD8 T Cell Differentiation and Central Memory Development in Response to Infection with Listeria Monocytogenes. Infection and Immunity, 81, 1140-1151.
https://doi.org/10.1128/IAI.00749-12
[13] Tai, W., Chen, Z. and Cheng, K. (2013) Expression Profile and Func-tional Activity of Peptide Transporters in Prostate Cancer Cells. Molecular Pharmaceutics, 10, 477-487.
https://doi.org/10.1021/mp300364k
[14] Ferrone, S. and Marincola, F.M. (1995) Loss of HLA Class I Antigens by Melanoma Cells: Molecular Mechanisms, Functional Significance and Clinical Relevance. Immunology Today, 16, 487-494.
https://doi.org/10.1016/0167-5699(95)80033-6