EGFR is a cell-surface receptor activated in more than half of patients with NSCLC, and this activation can be the result of protein over-expression, increased gene copy number, or genetic mutations 
. Gefitinib's anti-lung cancer effects attribute to its binding to the adenosine triphosphate binding site in the tyrosine kinase domain of EGFR, thereby inhibiting EGFR kinase activity 
. Although many patients initially respond to gefitinib, resistance and tumor recurrence eventually develop. To overcome such drug resistance, novel mutant-selective EGFR kinase inhibitors against EGFR T790M were reported 
, and 4-pyrrylamino quinazolines as new gefitinib analogues were synthesized 
. In this study, we design, synthesize and evaluated a number of novel quinazoline derivatives by exchanging the positions of the C5 and C6 substituents and varying the C4-amino functionality of gefitinib, and report that compound V1801 having a trifluoromethyl group at the C5'-position and a bromine at the C2'-position of the aniline moiety substituted at the C4 position of quinazoline core overcomes gefitinib resistance via up-regulation of Noxa, suggesting a novel strategy to fight against NSCLC with EGFR T790M mutation.
The mitochondrial pathway of apoptosis constitutes one of the main safeguards against tumorigenesis, and the Bcl-2 family includes the central players of this pathway that regulate cell fate through the control of mitochondrial outer membrane permeabilization. Therefore, Bcl-2 family antagonists have been developed for cancer therapy 
. To inhibit Mcl-1 pro-survival function, occlusion of Mcl-1 hydrophobic groove by BH3-only proteins represents an effective strategy 
. Noxa belongs to the BH3-only proteins of the Bcl-2 family and is able to interact with Mcl-1, leading to initiation of apoptosis signals 
. Compared with the parental compound gefitinib, V1801 is a relatively weak EGFR inhibitor which bears a much more potent cytotoxicity in T790M EGFR-harboring NCI-H1975 cells (). These results suggest that V1801 may have “off-target” effects in gefitinib-resistance NSCLC cells. We carry out experiments to investigate the mechanisms of action of V1801 by measuring the expression of Bcl-2 family members including Noxa, Puma, Bim, Bcl-xl and Mcl-1, and found that in NCI-H1975 and A549 cells upon V1801, Noxa is up-regulated at an early stage (3 h) at both mRNA and protein levels (). In colon, gastric cancer cells and melanoma cells, V1801 also induces up-regulation of Noxa (), suggesting that this mechanism is valid in a spectrum of cancer cells. While the expression of Mcl-1 in NCI-H1975 cells is not affected by V1801 treatment, Mcl-1-Noxa interaction is enhanced (). Consequently, cytochrome C partially translocates from mitochondria to cytosol (). These results indicate that V1801 can induce Noxa expression and trigger intrinsic apoptotic pathway, leading to activation of effector caspases and cleavage of PARP (). Interestingly, V1801 can also activate caspase-8 (), demonstrating that V1801 also activates the extrinsic apoptotic pathway and the mechanisms of action of this compound in inducing apoptosis are complicated and warrant further investigation.
The expression of Noxa is regulated by transcription factors HIF-1α, E2F-1 and p53 
. Recent studies show that in a variety of tumor cell types, the induction of Noxa by proteasome inhibitor bortezomib is directly dependent on the oncogene c-Myc 
. Indeed, c-Myc not only regulates those genes capable of promoting cell growth and metabolism, but also activates apoptosis initiators 
. c-Myc amplifies mitochondrial apoptotic pathway by inhibiting anti-apoptotic Bcl-2 proteins and activating pro-apoptotic Bcl-2 family members such as BH3-only proteins 
. Here we report that c-Myc protein is up-regulated by V1801 treatment in NSCLC cells (). However, c-Myc silencing by siRNA only slightly but not significantly (p
0.14) attenuates V1801-caused programmed cell death of NSCLC cells (). Given that Erk pathway can mediate Noxa up-regulation 
and Erk itself has pro-apoptotic function 
, we test the effect of V1801 on Erk expression and report that this gefitinib analog increases p-Erk, but Erk inhibition only slightly (p
0.06) suppresses V1801-induced apoptosis of H1975 cells (). The results also suggest the complexity of the mechanisms of action of this compound in inducing apoptosis of lung cancer cells. In myeloma cells, bortezomib inactivates p-Erk and synergistically potentiates the apoptotic effects of a novel compound 6-O-Angeloylplenolin 
. Interestingly, we show that while bortezomib significantly enhances V1801-induced cytotoxicity () and up-regulation of Noxa () on NCI-H1975 cells, it antagonizes V1801-caused p-Erk activation (). These results indicate that bortezomib, as a multi-target agent, may increase Noxa expression through other unidentified mechanism which warrants further investigation, and combined use of V1801 and bortezomib may have therapeutic potential in gefinitib-resistance NSCLC cells.
The in vivo studies show that in nude mice inoculated with NCI-H1975 cells, treatment with V1801 at 30 mg/kg per day significantly inhibits tumor growth, while gefitinib at the same dosage does not show therapeutic benefit (). V1801 also up-regulates Noxa in vivo (), and V1801-treated mice have smaller tumors and higher Noxa expression compared to those treated with vehicle control or Gefitinib (). Therefore, our data indicates that gefinitib analogs with weak EGFR inhibitory activity can overcome TKI-resistance via activation of BH-3 only pro-apoptotic proteins, and V1801 represents a potential new therapeutic agent for patients with NSCLC.