艾洛替尼耐药的卵巢癌SKOV3细胞系的建立及其耐药特性

Establishment of human ovarian cancer cell line SKOV3 resistant against erlotinib and its resistant characterization

目的通过体外建立艾洛替尼(Erl)耐药的人浆液性卵巢癌细胞系SKOV3/Erl,并探讨其耐药机制,为卵巢癌靶向性治疗的耐药性研究提供细胞模型及依据。方法 采用逐步递增联合大剂量冲击法诱导细胞,取对数生长期的SKOV3细胞,从Erl 1μmo.lL-1开始处理,以Erl 2,4,8,16和25μmo.lL-1反复换液传代,最终维持浓度为10μmo.lL-1,共诱导10个月。取SKOV3和SKOV3/Er1细胞,加入不同浓度艾洛替尼、紫杉醇、米托蒽醌、表柔比星、拓扑替康、长春新碱和甲氨蝶呤等药物,作用72 h,磺酰罗丹明B染色法检测耐药倍数。流式细胞术检测SKOV3细胞和SKOV3/Er1细胞的细胞周期。Western印迹法检测Erl刺激对敏感和耐药细胞中表皮生长因子受体(EGFR)相关信号通路产生的变化;流式细胞术检测SKOV3/Er1细胞表面ATP结合盒(ABC)转运蛋白中P糖蛋白(Pgp)、多药耐药相关蛋白1(MRP1)、乳腺癌耐药蛋白(BCRP)的表达以及Toll样受体4(TLR4)的表达;检测脂多糖(LPS)刺激对紫杉醇耐受的SKOV3/Erl细胞的敏感度。结果 Erl对SKOV3细胞的IC50为(9.54±1.04)μmol.L-1,而对SKOV3/Erl细胞的IC50为(21.63±1.05)μmo.l L-1,耐药倍数约为2.26,诱导成功的SKOV3/Erl对紫杉醇、长春新碱、米托蒽醌和甲氨蝶呤的耐药倍数均在3倍以上。与SKOV3细胞相比,SKOV3/Erl细胞G0/G1期比例上升,S期比例下降,G2/M期几乎无变化。Erl刺激后,与SKOV3细胞相比,SKOV3/Erl细胞中p-HER1,p-ERK与p-AKT水平上调。SKOV3/Erl细胞膜上Pgp,BCRP和MRP1表达有微弱上调,而TLR4显著上调;脂多糖可强烈刺激SKOV3/Erl细胞增殖,并且对紫杉醇杀伤起到保护作用,而亲本细胞则相对不敏感。结论 成功建立了对Erl耐受的人卵巢癌耐药细胞SKOV3/Erl。其表现出的多药耐药机制可能与TLR4蛋白表达上调有关。

OBJECTIVE To construct a drug-resistant human serous ovarian cancer cell model induced by erlotinb and explore its possible mechanism of resistance. METHODS The cell line SKOV3 was cultured by gradually increasing the concentration of erlotinib from 1,2, 4, 8, 16 to 25 μmoI·L-1 until 10 iJmoI-L-1 in vitro for 10 months to generate its resistance cell line SKOV3/Erl. In the induction process, the medium was treated with erlotinib 2, 4, 6 and 8 μmol·L-1 and changed step by step, accompanied by the passage of cells. The resistance index in SKOV3/Erl was tested by sulforhodamine B （SRB）, after being treated with a series of concentrations of erlotinib, paclitaxel, mitoxantrone, epirubicin, topotecan, vincristine and methotrexate. Cell cycle of SKOV3 cells and SKOV3/Erl cells was investigated by flow cytometry. The changes of signal transduction protein in sensitive and drug resistant cells treated with ero- Iotinib were detected by Western blotting. SKOV3 and SKOV3/Erl cells were stained with antibodies conju- gated with fluorescent dyes to determine the expression levels of cell surface P-glycoprotein （Pgp） , breast cancer drug resistance protein （BCRP） and multidrug resistance-related protein 1 （MRP1） and Toll-like receptor 4 （TLR4）. To evaluate the drug-resistance function of TLR4, the viability of cells was assayed af- ter stimulation by LPS. RESULTS IC5o Value of erlotinib to SKOV3 was （9.54 ±l.04）μmoI·L-1 while that of erlotinib to SKOV3 was （21.63 ± 1.05）μmol· L-1 with a resistant index of 2.26. The resistant indices of paclitaxel, vincristine, mitoxantrone and methotrexate all exceeded 3. Compared with SKOV3 cells, S-phase of SKOV3/Erl cells was reduced, and G0/G1 phase increased while the percentage of G2/M phase showed no significant change. The phosphorylated HER1 signal was upregulated in SKOV3/Erl cells, p-ERK and p-AKT levels in SKOV3/Erl were also higher than in SKOV3 cells. Major ABC transporter Pgp, BCRP and MRP1 expressions were slightly increased in SKOV3/Erl, while TLR4 showed intensive upregulation. LPS stimulated the proliferation of SKOV3/Erl, rescued the cytotoxicity from paclitaxel, but such function was not observed in SKOV3 cells. CONCLUSION The erolotinib-resistant human ovarian cancer cell line SKOV3/Erl has been established. The high TLR4 expression level in SKOV3/Erl cell line may be ascribed to its resistance to erlotinib.