Screening For In Vitro Antitumor Activities Of Quinazoline Compounds And Study On The Novel Mechanism Involved In The Antitumor Action Of Erlotinib

Tumor therapy has been a worldwide problem. As our knowledge of signal transduction pathways in tumor cells increases, drug design against tumor-specific molecular targets attracts more and more attention. Furthermore, with the development of the crystal diffraction techniques, combinatorial chemistry, molecular model, high-throughput screening technology and computer chemistry, targeted agents change with each passing day, providing new stratigies for tumor therapy. Drug design based on the structure and mechanism has become the mainstream form of developing antitumor agents.Epidermal growth factor receptor (EGFR), one member of the ErbB family, is an important transmembrane receptor with tyrosine kinase (TK) activity. EGFR signaling passway is associated with cell migration, adhesion, proliferation, differentiation as well as apoptosis. Therefore, it is closely related to tumor initiation and deterioration. EGFR gene was first cloned in 1984. Studies over the past two decades have shown that EGFR is a promising molecular target for tumor therapy. In recent years, analyses of the bioactivities of quinazoline compounds suggest that they are a class of micromolecular compounds with mutiple pharmacological activities, particularly inhibiting EGFR-TK activity. Erlotinib is a micromolecular EGFR-TK inhibitor (EGFR-TKI) jointly developed by Genentech, Roche and OSI companies, belonging to the quinazoline compounds. It competitively inhibits the binding of ATP with the intracellular catalytic sites of EGFR, thus reducing the autophosphorylation of EGFR and resulting in cell growth arrest and apoptosis. Another study found that Erlotinib could induce the expression of cyclin dependent kinase inhibitor p27^KIP1 and suppress cell-cycle events involved in the G1/S transition.Although people have obtained many milestone achievements of the EGFR-targeted antitumor agents, yet a number of problems remain to be solved: how to make such agents only act on the assumed target of tumor cells, but not the same target of normal cells; in the therapeutic alliance, how to make the choice of combined or synergistic effects. These unsolved problems hamper the clinlical application of EGFR-targeted antitumor agents. Therefore, researches conercering the mechanisms and applications of EGFR-targeted antitumor agents are of great importance.In the present study, we first took in vitro screening for the antitumor activities of new quinazoline compounds and investigated their effects on TK activities. Then we explored the novel mechanism involved in the antitumor action of quinazoline compounds, taking erlotinib as a representative agent. The aim of this study is to provide candidate compounds for molecular target-based tumor therapy and accumulate the necessary academic and experimental bases for quinazoline compounds to become more efficient antitumor agents.Part I : Screening for the antitumor activities of quinazoline compoundsAIMS: To screen for the antitumor activities of new quinazoline compounds and investigate their effects on TK activities.METHODS: Tumor cell viability was measured by MTT method. ELISA kit was used to determine TK activity.RESULTS: Compared with gefitinib or erlotinib group, the proliferation of tumor cells treated with GI-2～GI-6 compounds, GII-2～GII-5 compounds, GIV-2,6 compounds, BI-2～BI-8 compounds, BII-2,5,7,8 compounds was markedly inhibited. The inhibitory effects of BI-2～BI-8 compounds were stronger than those of gefitinib or erlotinib at the same concentration; the highest inhibition ratio was over 90%. The results of ELISA showed that GI-5,6 compounds, GII-4,5 compounds, BI-8 compound, BII-5,7 compounds significantly suppressed TK activity.CONCULATIONS: Among the seven series, 44 kinds of new compounds, GI-2～GI-6 compounds, GII-2～GII-5 compounds, GIV-2,6 compounds, BI-2～BI-8 compounds, BII-2,5,7,8 compounds could markedly inhibit tumor cell proliferation, showing more effective biological antitumor activities in vitro. The inhibitory effects of BI-2～BI-8 compounds were stronger than those of gefitinib, erlotinib and other new compounds at the same concentration; GI-5,6 compounds, GII-4,5 compounds, BI-8 compound, BII-5,7 compounds significantly suppressed TK activity (P < 0.05). They were expected to become candidate compounds for tumor targeted therapy with a decent molecular target.Part II Study on the novel mechanism involved in the antitumor action of erlotinibAIMS: To investigate the novel mechanism involved in the antitumor action of erlotinib.METHODS: Cell apoptosis was determined by staining with Hoechst 33324. Intracellular production of ROS was measured by the fluorescent probe DCFH-DA. Mitochondrial preparations from A549 cells were extracted and mitochondrial respiration function was determined by oxygen electrode. Superoxide ions (O₂^-) were detected by the fluorescent probe DHE. RT-PCR was taken for the measurement of NADPH oxidase catalytic subunit gp91^phox. Cell viability was measured by MTT method. Mitochondrial membrane potential (ΔΨ_m) was assessed with the fluorescent probe JC-1. We introduced western blotting for the analyses of cytochrome C, apoptosis-inducing factor (AIF), c-Jun NH₂-terminal kinase (JNK), p-JNK.RESULTS: 1) Erlotinib induced apoptosis in A549 cells concentration-dependently. 2) Erlotinib incubation for 30 min promoted ROS generation in A549 cells in a concentration-dependent manner. 3) Erlotinib increased RCR dose-dependently in A549 cells. Erlotinib (10μM) inhibited state 4 respiration. 4) Erlotinib incubation for 30 min concentration-dependently induced O₂^- production and increased the mRNA levels of NADPH oxidase catalytic subunit gp91 in A549 cells. 5) Pretreatment with NAC (1 mM) inhibited erlotinib (10μM)-induced cell death. 6) Erlotinib incubation for 24 h caused loss ofΔΨ_m and induced the release of cytochrome C and AIF from mitochondria in a dose-dependent manner. 7) Erlotinib incubation for 24 h induced JNK phosphorylation in A549 cells in a concentration-dependent manner.CONCLUSIONS: Erlotinib exerts an antitumor activity by activating ROS-dependent, JNK mediated and mitochrondrial-initiated cell apoptosis.This study showed that quinazoline compounds could be developed into new agents with better antitumor activities after optimized design. They could not only prevent tumor cell proliferation but also suppress TK activity. Our study also demonstrated that erlotinib could exert an antitumor effect via increasing ROS production, activating JNK, and thereby initiating mitochondrial death pathways. These findings accumulated the necessary academic and experimental bases for quinazoline compounds to become more efficient antitumor agents.