Establishment Of PC9/ERlotinib Resistance Cell Line And Detect The Resistance Reversal Mechanism Of Octreotide On PC9/ERlotinib

Background and Objective:Lung cancer is the world’s highest mortality rates of malignant tumors. Human health is serious threatened.In recent years, molecular targeted therapy for lung cancer patients harbored epidermal growth factor receptor mutations have a good effect. Epidermal growth factor Tyrosine kinase inhibitors are the representative drugs. But all of the patients would get drug-resistance and tumor recurrence, therefore,mechanisms of acquired drug resistance become the hot spot.The mechanisms acquired drug resistance of EGFR TKIs have been studying,including EGFR second T790 M mutation, c-MET gene amplification, Insulin-like growth factor 1 receptor(IGF-1R) bypass pathway activation and so on. IGF-1R high expression is thought to activate PI3K/Akt pathway, contribute to tumor cell proliferation, inhibit apoptosis and result in EGFR TKIs resistance. We found that PC9/erlotinib cells high expression of IGF-1R, think acquired drug resistance of PC9/erlotinib may be related to high expression of IGF-1R. Octreotide(OCT) is a synthetic peptide, has not only suppress tumor proliferation and promote tumor cell apoptosis, can regulate the transcription of IGF-1 and inhibit secretion of IGF-1.So this experiment was to study whether OCT can reverse drug resistance, and to explore its possible mechanism.Methods:1. The establishment of a PC9/erlotinib(PC9/ER) resistance cell line and detection Resistance:Using low concentration erlotinib after 6 months cultivation, establish PC9/ER cell line, CCK 8 assay to detect PC9/ER resistance.2. Reversal function of octreotide on PC9/ER:CCK 8 assay for to detect reverse factor of erlotinib and erlotinib combine with OCT on PC9/ER cells,.Flow cytometry detection of PC9 / ER cell apoptosis.3. The mechanism of the reverse of OCT on erlotinib resistance of the PC9/ER cells: Western bolt assay to detect different treatment on PC9 / ER cells, IGF-1R、p-IGF-1R、Akt、p-Akt expression changes, qRT- PCR detection after OCT on PC9/ER IGF-1 and IGF-1R m RNA expression changes.ELISA assay detection OCT on PC9/ER the IGF-1 expression change.Results:1. 0.1μmol/L erlotinib induce PC9 cells after six months, CCK 8 assay to detect resistance index, IC50 of PC9/ER and PC9 were 4.809 μmol/L(PC9/ER) and 0.4608 μmol/L(PC9), resistance index was 10.43, successfully established resistant PC9/ER cells compared with the parental PC9 cells, IGF-1R、p-IGF-1R、Akt、p-Akt have higher expression in PC9/ER in protein levels.2. Erlotinib combine with OCT can improve PC9/ER resistance, CCK 8 assay to detect IC50 Erlotinib and Erlotinib combine with OCT were 4.809μmol/L and 0.4998μmol/L, and improve the ratio of 9.62 times. Erlotinib combine with OCT on PC9 / ER cells IGF-1R、p-IGF-1R、Akt、p-Akt protein expression were reduced.3. OCT can inhibit IGF-1R, IGF-1 mRNA expression.4. Erlotinib combine with OCT can promote cell apoptosis, and compared with OCT group, erlotinib group, erlotinib combine with OCT apoptosis rate increased.Conclusions:1. Successfully established on it for drug-resistant PC9/ER cells, and the PC9/ER can stably grow.2. The high expression of IGF-1R maybe PC9 / ER the Erlotinib mechanism of acquired resistance.3. Erlotinib combine with OCT on PC9 / ER can inhibit the IGF-1 secretion and IGF-1R activation and downstream Akt activation and inhibition of cell growth, promotes apoptosis.