Reverse EGFR-TKI Resistance In NSCLC By Shikonin And Construct Shikonin Nanoparticles

Objective: EGFR-TKI resistance leads to treatment failure, even death in patients with EGFR sensitive mutations, have severe impact on human health. EGFR-TKI resistance mechanisms are complicated. How to overcome drug resistance has drawn much attentions in the field of NSCLC research. Our objects in this research are to observe the effect of shikonin and epidermal growth factor receptor tyrosine kinase inhibitor(EGFR-TKI) and explore its possible mechanisms; sequently, we construct single chain antibody mediated shikonin nanoparticles to targetedly deliver shikonin into NSCLC cells.Methods: 1. Shikonin effects on the proliferation, cell morphology, apoptosis and cell cycle of lung adenocarcinoma cell H1975 were evaluated by MTT, light microscope and flow cytometry assay for 24 h, 48 h and 72 h. Western blot was used to detect the expression of phosphorylatic level of EGFR signaling pathways, including EGFR, AKT and ERK. 2. MTT, cell cloning, flow cytometry and Western blot were used to analyze the cell proliferation, apoptosis and apoptosis-related proteins expression in low concentration(1.25 μM) of shikonin combined with gefitinib / icotinib. 3. Using the single emulsion solvent evaporation method to synthesize single-chain-antibody-coupled shikonin-loaded superparamagnetic iron oxide nanoparticles(SIONPs) and then we analyzed particle morphology, size, charge and cell internalization.Results: 1. The viability of H1975 cells decreased in the concentration and time dependent manner of shikonin. The inhibition rate of EGFR-TKI resistance cells was 70% in 10 μM of shikonin. Shikonin induced H1975 cell apoptosis with the apoptosis rate of 70%. Meanwhile, the cell cycles were arrested at S phase. The expression of EGFR downstream signaling molecules and apoptosis-related proteins were decreased. 2. Shikonin combined with gefitinib / icotinib significantly inhibited cell growth, induced cell death, which is associated with the down-regulation of phosphorylatic levels of EGFR, AKT and ERK compared with single drug group. 3. The shikonin nanoparticles(sc Fv-SHK-SIONPs) were observed by transmission electron microscopy and analyzed by laser particle size analyzer. The particles were spherical, the size was about 173.3 n M, and zeta charge was-28.55 m V. The uptake of targeted nanoparticles in NSCLC cells was showed by immunofluorescence microscope.Conclusion: 1. Shikonin can inhibit cell growth and induce apoptosis on EGFR-TKI resistant lung adenocarcinoma cell line H1975 by down-regulating EGFR signaling pathway and decreasing the expression of apoptosis-related protein Bcl-2, PARP. 2. Shikonin in low concentration combined with gefitinib / icotinib restores the sensitivity of EGFR-TKI resistant H1975 cell. 3. We successfully constructed sc Fv-SHK-SIONPs and the characters of these nanoparticels fulfilled the requirements of the experiment. They can targetedly deliver shikonin into EGFR-positive NSCLC cells.