A Study Of Acquired Resistance Mechanism Of HCC827-TR EGFR Mutant Cell And Afatinib Reversing The Resistance To EGFR-TKIs

Backgroud and Object:Lung cancer became one of the servere cancer in the world, Chemotherapy drugs because of side effects affecting the treatment of lung cancer. Most research found that the EGFR is overexpress in more than80%lung cancer.There are many EGFR target therapy drugs,among them the small molecular tyrosine kinase inhibitor(EGFR tyrosine kinase inhibitors,EGFR-TKI) is most impotant.In clinical research find that EGFR-TKIs are highly effective in lung cinitially ancer patients who harbor active mutations in the EGFR gene.Especilly the sensitive mutation including the5amino acid deletions occur ing from codon746to750in exon19or a point mutation in leucine(L)858to arginine(R)(L858R) in kinase domain of EGFR.However.patients who are sensitive to EGFR-TKIs eventually relapse within few years. To date,several major mechanisms of acquired resistance,such as secondary mutation of the EGFR gene,amplification of the MET gene, the two mechanisms possess70%.30-40%acquired resistant mechanisms are obscure,including loss of PTEN expression, IGF-1R high expression, HGF high expression et al.In order to study the EGFR-TKIs acquired resistant mechanisms,we use the different trays to detect the knowned acquired resistant mechanisms in the resistant cells HCC827-TR and the sensitive cell HC827-P.We know the most common mechanism of acquired resistance to EGFR exon20occurred outside a secondary T790M mutation, while the tumor is still dependent on the activity of the EGFR signaling pathway, so how to block the EGFR signaling pathway is a key to overcoming resistance, the main research including joint EGFR monoclonal antibody therapy, irreversible EGFR-TKIs inhibitors and inhibitors of the T790M mutation. The main topic of the irreversible inhibitor of EGFR-TKIs erlotinib A study issued. H1975and H1650cell lines because were point mutations in EGFR exons (L858R)21and exon deletion mutant (E746-A750del)19, where H1975also associated with a secondary mutation in exon (T790M)20, and H1975and H1650so use cell lines related experiment, explore A method to reverse EGFR-TKIs imatinib-resistant possibility of obtaining. Methods:1. Culting HCC827-P and HCC827-TR cells in vitro.Extracting total RNA and reverse transcribed into cDNA, Realtime PCR detects the PTEN expression level in HCC827-P and HCC827-TR.2. Western blot detect the expression level of PTEN in the HCC827-P and HCC827-TR cells,and detect the expression level of p-AKT in the HCC827-P and HCC827-TR cells with5u M Erlotinib2hours.3. MTS to detect toxic effects Afatinib and Erlotinib in H1650and H1975cell lineResults:1. Realtime-PCR analysis showed that PTEN mRNA expression levels inconsistent HCC827-TR and HCC827-P in both cell lines; which PTEN mRNA expression levels of genes HCC827-TR cell lines compared with HCC827-P cells is low.2. Western-blot analysis showed that expression of PTEN deletion HCC827-TR cells; give EGFR-TKI inhibitor Erlotinib5μM treatment2h, the downstream signaling protein p-AKT HCC827-P cells was significantly inhibited, HCC827-TR without Erlotinib treatment cell p-AKT levels slightly raised.3. The MTS results show that when the Erlotinib and Afatinib concentration is0.001μM, the cell growth inhibition rate of H1650cell growth inhibition rate were15%、6%, when the Erlotinib and Afatinib concentration is10μM, the cell growth inhibition rate of H1975were58%,14%.Conclution:1. HCC827-TR for Erlotinib mechanisms of acquired resistance may be associated with reduced PTEN protein expression caused sustained activation of p-AKT.2. Erlotinib can significantly inhibit the EGFR signaling protein(p-AKT) in HCC827-P cell not in HCC827-TR cell.3. Afatinib able to reverse drug resistance caused by the T790M mutation, the reduction of PTEN protein expression induced resistance reversal effect also has a certain degree.