摘要
目的探讨肺金生方抗非小细胞肺癌(NSCLC)酪氨酸磷酸酶抑制剂(TKIs)靶向药物耐药的作用及分子机制。方法选用NSCLC细胞系HCC827,按0.01、0.05、0.10、0.50和1.0μM浓度梯度诱导构建吉非替尼耐药细胞株,采用CCK8法检测耐药株是否构建成功,采用荧光定量PCR(q-PCR)检测耐药株c-MET扩增水平。20只BALB/c裸鼠按随机数字表法分成空白对照组、吉非替尼组、肺金生方组和肺金生方+吉非替尼组,每组5只。按每只4×10~7/200μL将吉非替尼耐药细胞株注射于裸鼠后肢皮下,构建皮下移植瘤模型。四组均腹腔注射生理盐水,吉非替尼组给予吉非替尼50mg/kg灌胃;肺金生方组给予肺金生方剂量53.6g/kg灌胃;肺金生方+吉非替尼组给予吉非替尼50mg/kg+肺金生方剂量53.6g/kg联合灌胃给药。给药频次每2天1次,连续给药4周。CCK8法检测耐药HCC827 GR细胞增殖率,流式细胞技术检测耐药HCC827 GR细胞凋亡率,Western blot检测耐药HCC827 GR细胞p-EGFR、p-MET、p-AKT、p-ERK1/2表达水平。结果与空白对照组比较,吉非替尼组裸鼠皮下移植瘤瘤重无差异[(1.34±0.16)g比(1.41±0.13)g,P>0.05],肺金生方组裸鼠皮下移植瘤瘤重明显减轻[(0.77±0.28)g比(1.41±0.13)g,P<0.01],吉非替尼+肺金生方组裸鼠皮下移植瘤明显小于肺金生方组和吉非替尼组[(0.34±0.16)g比(0.77±0.28)g、(1.34±0.16)g,P均<0.01];肺金生方单用或与吉非替尼联用能够明显抑制耐药HCC827 GR细胞的增殖能力[(43.45±4.47)%、(24.25±5.21)%比(100.00±2.14)%,P均<0.01];肺金生方组、吉非替尼+肺金生方组细胞凋亡率显著高于空白对照组[(7.40±0.56)%、(12.80±0.72)%比(3.05±0.54)%,P均<0.01];肺金生方组、吉非替尼+肺金生方组c-MET和p-c-MET表达水平明显降低,并明显抑制下游AKT/ERK1/2磷酸化水平。结论肺金生方或和吉非替尼联用能有效抑制吉非替尼耐药皮下移植瘤的生长,抑制耐药细胞的增殖并促进凋亡,其作用机制可能与下调c-MET蛋白表达以及下游AKT/ERK1/2磷酸化水平有关。
Objective To investigate the effect and molecular mechanism of Feijinsheng recipe on drug resistance targeting tyrosine phosphatase inhibitor(TKI) in non-small cell lung cancer(NSCLC). Methods The NSCLC cell HCC827 was used to induce the construction of gefitinib resistant cell lines according to the concentration gradients of 0.01, 0.05, 0.10, 0.50 and 1.0 μM. CCK8 was used to detect whether the construction of drug resistant strains was successful, and fluorescent quantitative PCR(q-PCR) was performed to check the amplification level of c-MET of drug resistant strains. Twenty BALB/c nude mice were randomly divided into blank control group, gefitinib group, Feijinsheng recipe group and Feijinsheng recipe plus gefitinib group with 5 mice in each group. Gefitinib-resistant cells were injected subcutaneously into the hind limb of nude mouse at a dose of 4 ×10~7/200μL to construct a subcutaneous transplanted tumor model. The four groups were injected with saline intraperitoneally,while the gefitinib group was given 50 mg/kg gefitinib, Feijinsheng recipe group was given 53.6 g/kg Feijinsheng recipe by intragastric administration, Feijinsheng recipe plus gefitinib group was given 50 mg/kg gefitinib +53.6 g/kg Feijinsheng recipe. The dosing frequency was once every 2 days, and the administration was continued for 4 weeks. Proliferation rate of drug-resistant(GR) HCC827 cells was detected by CCK8, apoptosis rate was tested by flow cytometry, and expression levels of p-EGFR, p-MET, p-AKT and p-ERK1/2 was confirmed by Western blot.Results Compared to the blank control group, there was no difference in the tumor weight of subcutaneous transplantation in nude mice in gefitinib group[(1.34±0.16) vs(1.41±0.13)g]. The tumor weight in nude mice in Feijinsheng recipe group reduced significantly[(0.77±0.28) vs(1.41±0.13)g, P<0.01]. In gefitinib plus Feijinsheng recipe group, the tumor weight was significantly smaller than those of Feijinsheng recipe group and gefitinib group [(0.34±0.16) vs(0.77±0.28) and(1.34±0.16)g, P<0.01, respectively]. Feijinsheng recipe group or in combination with gefitinib significantly inhibited the proliferation of GR HCC827 cells [(43.45 ±4.47)% and(24.25 ±5.21)% vs(100.00±2.14)%, P<0.01, respectively]. Apoptosis rate in both Feijinsheng recipe group and Gefitinib+Feijinsheng recipe group was significantly higher than that of the blank control group [(7.40±0.56)% and(12.80±0.72)% vs(3.05±0.54)%, P<0.01, respectively]. Expression levels of c-MET and p-c-MET in both Feijinsheng recipe group and Gefitinib+Feijinsheng recipe group reduced significantly, and significantly inhibited the phosphorylation level of AKT/ERK1/2 in downstream. Conclusion Feijinsheng recipe or its combination with gefitinib could effectively inhibit the growth of gefitinib-resistant subcutaneously transplanted tumor, inhibit the proliferation of drug-resistant cells and promote apoptosis. The mechanism may be related to down-regulation of c-MET protein expression and phosphorylation level of downstream AKT/ERK1/2.
作者
姚成
高文仓
YAO Cheng;GAO Wencang(Department of Oncology,The Second Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou,Zhejiang province,310005,China)
出处
《浙江中西医结合杂志》
2020年第4期286-290,共5页
Zhejiang Journal of Integrated Traditional Chinese and Western Medicine
基金
浙江省中医药科技计划项目(No.2019ZB050)。