Investigation Of Molecular Mechanism Of Gefitinib Acquired Resistance Induced By HMGB1 Regulating Autophagy And Its Follow-up Therapy Strategy In Non-small Cell Lung Cancer

ObjectiveTo clarify the relation between autophagy and resistance of Gefitinib in NSCLC, and the key role of HMGB1 in the resistance of Gefitinib mediated by autophagy, and illuminate the relationship of Bcl-2 and Beclin-1 pathway and the resistance of Gefitinib; and also to investigate the correlations among E-cadherin and vimentin expression, epithelial-mesenchymal transition factors, prognosis for non-small-cell lung cancer (NSCLC), and the sensitivity to platinum-based chemotherapy.Methods1. In gefitinib sensitive cell line PC9 (EGFR 19 exon deletion), establish the resistance cell line of gefitinib PC9-R by treating gefitinib for different time. Through AO, Western blot, the expression of LC3 Ⅰ/Ⅱ, p62 was detected, to clarify the relationship btween autohphagy and resistance of gefitinib.2. Through small molecular RNA interference technology/Liposome transfection technique, establish silence/over expression HMGB1 PC9; treat it with gefitinib in different concentration, and the anti-tumor activity was detected by MTT method, the expression of LC3 Ⅰ/Ⅱ, p62 were detected by Western blot, the distribution of LC3 Ⅰ/Ⅱ were detected by immunohistochemistry, to clarify the impact of HMGB1 and autophagy on the resistance of gefitinib.3. To investigate the pathway HMGB1/Beclin-1 and MAPK/Bc12/HMGB1, the expression of Beclin-1、Bcl2、pJNK、pERK and p38 were detected by Western blot, to clarify the interaction of HMGB1 and Beclin-1, Bcl2 in the resistance of gefitinib induced by autohphagy.Results1. In the process of gefitinib resistance, autophagy protein LC3 altered from I to II, with down-regulation of p62, indicating that autophagy existing in the resistance of gefitinib. Through autophagy inhibitor chloroquine and RNA interference blocking technology, it was found that inhibiting autophagy can increase the anti-tumor activity of gefitinib, proving that autophagy existing in the resistance of gefitinib.2. In the process of PC9-R establishment, the expression of HMGB1 up-regulate; By silence the expression of HMGB1, the anti-tumor activity of gefitinib significantly increased, indicating HMGB1 participate in the resistance of gefitinib. When HMGB1 expression was silenced, the autophagy protein LC3 decreased from I to II, p62 increased, indicating that HMGB1 mediated the autophagy in the resistance of gefitinib.3. In the resistance of gefitinib the expression of Beclin-1 and pBcl-2 increased, Bcl-2 decreased. By inhibiting Beclin-1 the anti-tumor activity of gefitinib significantly increased, indicating that Beclin-1 participate in the resistance of gefitinib. The combination of HMGB1 and Beclin-1 significantly increased in the autophagy, which promote the resistance of gefitinib.Conclusion Autophagy is the key of resistance of gefitinib. HMBG1 mediated the autophagy in of gefitinib resistance. Bcl2/Beclin-1 is important to HMGB1-promoted gefitinib resistance.