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Tumor treatment has been a worldwide problem. As people of tumor cell signal transduction pathway knowledge increasing, the design of anticancer drugs targeting tumor-specific molecular targets and more attention. Plus crystal diffraction techniques, combinatorial chemistry, molecular modeling, high-throughput screening technology as well as the development of computational chemistry, rapid targeted drug offers a new strategy for the treatment of tumors. Structure and mechanism-based drug design has become the mainstream of the development of anticancer drugs. EGFR (epidermal growth factor receptor, EGFR) is one of the members of the ErbB family, and tyrosine kinases (tyrosine kinase, TK) activity, is an important transmembrane receptor. EGFR signaling pathways related to cell migration, adhesion, proliferation, differentiation, apoptosis, is closely related with tumor formation and progression. From the EGFR gene was cloned for the first time in 1984, more than 20 years of research, EGFR is a promising target molecule in cancer therapy. In recent years, the analysis of the biological activity of the compounds of the quinazoline visible, such compounds are a class of small molecule compounds with multiple pharmacological effects, especially to the inhibition of EGFR-TK activity. Erlotinib (Erlotinib) company Genentech, Roche and OSI jointly developed a small molecule EGFR-TK inhibitors (EGFR-TK inhibitor, EGFR-TKI), is a quinazoline compounds. Its main mechanism of action for competitive inhibition of adenosine triphosphate (adenosine-triphosphate, ATP) and EGFR intracellular catalytic sites combine to reduce the EGFR autophosphorylation role, resulting in cell growth arrest and undergo apoptosis. Another study found, Erlotinib induces cell cycle suppressor protein p27 KIP1 expression, cell cycle arrest at the G1 phase. Anticancer drugs targeting EGFR has achieved many milestone achievements, but there are still many problems to be solved: how to make these drugs only act on the intended target of the tumor cells, rather than acting on normal cells the same target; in combination therapy, how the efficacy of the additive or synergistic selection. These have hindered the clinical application of anticancer drugs targeting EGFR. Therefore, the mechanism of action and the scope of research has far-reaching implications on anticancer drugs targeting EGFR. The first part of this paper work and study its impact on TK activity in the anti-tumor effect of new compounds from the body level preliminary screening series quinoline oxazoline class; second part of Erlotinib as representatives to explore the new anti-tumor effect of quinazoline compounds mechanism aimed at the tumor's molecular targeted therapy to provide new candidate compounds and the quinazoline compounds accumulate the necessary academic and experimental basis for the development of more effective anticancer drugs. The first part quinazoline compounds screening purposes antitumor activity: the role of the screening series Quinazolines New anti-tumor compounds and study its impact on TK activity. METHODS: MTT assay tumor cell viability; enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) kit TK activity was measured. Results: Compared with Gefitinib, Erlotinib, G Ⅰ series of Compound No. 2 to 6, Ⅱ series G Compound No. 2 to 5, 2,6 G Ⅳ series compound, B Ⅰ series of 2 to 8 Compound No. B II series 2,5,7,8 Compound No. inhibit tumor cell proliferation, wherein B Ⅰ series of 2 to 8 Compound No. inhibitory effect was significantly stronger than the positive drug at the same concentration, the maximum inhibition rate exceeds 90%. Measured by ELISA 21 kinds of effective screening of new compounds of the TK activity of cells, the results show the, G Ⅰ series 5,6 compound, G Ⅱ series 4,5 Compound No. B Series 8 I Compound No. B Ⅱ series 5,7 Compound No. could significantly inhibit the TK activity. Conclusion: a total of 44 kinds of the seven series of new compounds, G Ⅰ series of 2 to 6 Compound No. Ⅱ series G Compound No. 2 to 5, G Ⅳ series compound 2,6 No. B Ⅰ series 2 to 8 Compound No. B Ⅱ series 2,5 7,8 compounds are able to significantly inhibit the proliferation of tumor cell lines in vitro showed good biological inhibitory effect, B Ⅰ family of 2 to 8 Compound No. significantly stronger than the same concentrations of gefitinib, Erlotinib is and the other new compounds; wherein G Ⅰ Series 5, Compound No. 6, G Ⅱ series 4,5 Compound No. B Ⅰ series of Compound No. 8, B Ⅱ series compound 5,7 inhibitory effect on cell TK activity was significantly (P <0.05), with good molecular targeting, is expected to become a tumor targeted therapy candidate compound. Objective: To study Erlotinib second part of Erlotinib antitumor mechanism of the new mechanism of the anti-tumor effect. Methods: hoechst 33342 staining Determination of apoptosis; by DCFH-DA fluorescent probe detection of intracellular reactive oxygen species (reactive oxygen species, ROS); extract A549 mitochondrial, oxygen electrode method for the determination of mitochondrial respiratory function; DHE fluorescent probes detect extracellular within superoxide anion (O 2 - ); RT-PCR assay changes in expression of the catalytic subunit of NADPH oxidase gp91; MTT assay cell viability; JC-1 fluorescent probe of mitochondrial membrane potential (mitochondrial membrane potential △ Ψ m ); western-blotting law. determination of cytochrome C and apoptosis-inducing factor (apoptosis-inducing factor, AIF), c-Jun NH 2 -terminal kinase (c-Jun NH 2 -terminal kinase, JNK), phosphorylated JNK (p-JNK) expression change. Results: 1) Erlotinib showed a concentration-dependent induction of apoptosis in A549 cells. 2) Erlotinib for 30 min in a concentration-dependent increase in the level of A549 cells intracellular ROS. 3) Erlotinib a dose-dependent increase in A549 cells, mitochondrial respiratory control ratio (respiratory control ratio, RCR), Erlotinib (10μM) reduces IV state breathing. 4) Erlotinib for 30 min in a concentration-dependent manner to promote the A549 cells O 2 - generation and increase the level of A549 cells NADPH oxidase catalytic subunit gp91 mRNA. 5) the role of antioxidant N-acetylcysteine ??(N-acetyl-L-cysteine, NAC) (1 mM) inhibit Erlotinib (10μM) decreased cell viability. 6) Erlotinib a dose-dependent reduction in A549 cells for 24 h △ Ψ m , and to promote the release of the A549 cell mitochondrial cytochrome C and AIF. 7) Erlotinib for 24 h in a concentration-dependent manner to promote phosphorylation of JNK in A549 cells. Conclusion: Erlotinib via increased ROS production, activation of JNK, activation of the mitochondrial apoptotic pathway and play an anti-tumor effect. This paper studies the the quinazoline compounds can form new compounds with higher activity after optimization design. They can not only inhibit tumor cell proliferation, and have a strong inhibition of TK activity. Erlotinib can promote the generation of ROS A549 cells, activation of JNK signal transduction induced mitochondrial pathway of apoptosis, the discovery of the new mechanism for the development of quinazoline compounds for more effective anticancer drugs have accumulated the necessary academic and experimental basis .
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