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In non-small-cell lung carcinoma (NSCLC), aberrant activation of mammalian target of rapamycin (mTOR) contributes to tumorigenesis and cancer progression. PQR620 is a novel and highly-potent mTOR kinase inhibitor. We here tested its potential activity in NSCLC cells. In primary human NSCLC cells and established cell lines (A549 and NCI-H1944), PQR620 inhibited cell growth, proliferation, and cell cycle progression, as well as cell migration and invasion, while inducing significant apoptosis activation. PQR620 disrupted assembles of mTOR complex 1 (mTOR-Raptor) and mTOR complex 2 (mTOR-Rictor-Sin1), and blocked Akt, S6K1, and S6 phosphorylations in NSCLC cells. Restoring Akt-mTOR activation by a constitutively-active Akt1 (S473D) only partially inhibited PQR620-induced cytotoxicity in NSCLC cells. PQR620 was yet cytotoxic in Akt1/2-silenced NSCLC cells, supporting the existence of Akt-mTOR-independent mechanisms. Indeed, PQR620 induced sphingosine kinase 1 (SphK1) inhibition, ceramide production and oxidative stress in primary NSCLC cells. In vivo studies demonstrated that daily oral administration of a single dose of PQR620 potently inhibited primary NSCLC xenograft growth in severe combined immune deficient mice. In PQR620-treated xenograft tissues, Akt-mTOR inactivation, apoptosis induction, SphK1 inhibition and oxidative stress were detected. In conclusion, PQR620 exerted potent anti-NSCLC cell activity via mTOR-dependent and -independent mechanisms.

Objective: To investigate the effect of high expression of XAF1 in vivo or in vitro on lung cancer cell growth and apoptosis. Methods: 1. The A549 human lung cancer cell line was transfected with Ad5/F35 - XAF1, or Ad5/ F35 - Null at the same multiplicity of infection (MOI); (hereinafter referred to as transient transfected cell strain); XAF1 gene mRNA and protein expression was detected by reverse transcription polymerase chain reaction (RT- PCR) and Western blotting respectively. 2. Methyl thiazolyl tetrazolium (MTT) and annexin V-FITC/PI double staining were used to detect cell proliferation and apoptosis before and after infection of Ad5/F35 - XAF1 with Western blotting for apoptosis related proteins, caspase 3, caspase - 8 and PARP. 3. After the XAF1 gene was transfected into lung cancer A549 cells by lentiviral vectors, and selected by screening with Blasticidin, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were applied to detect mRNA and protein expression, to establish a line with a stable high expression of XAF1 (hereinafter referred to as stable expression cell strain). Twenty nude mice were randomly divided into groups A and B, 10 in each group: A549/ XAF1 stable expression cell strain was subcutaneously injected in group A, and A549/Ctrl stable cell line stable expression cell strain in group B (control group), to observe transplanted tumor growth in nude mice. Results: The mRNA and protein expression of XAF1 in A549 cells transfected by Ad5/F35 - XAF1 was significantly higher than in the control group. XAF1 mediated by adenovirus vector demonstrated a dose dependent inhibition of lung cancer cell proliferation and induction of apoptosis. This was accompanied by cleavage of caspase -3, -8, -9 and PARP, suggesting activation of intrinsic or extrinsic apoptotic pathways. A cell strain of lung cancer highly expressing XAF1 was established, and this demonstrated delayed tumor growth after transplantation in vivo. Conclusion: Adenovirus mediated XAF1 gene expression could inhibit proliferation and induce apoptosis in lung cancer cells in vitro; highly stable expression of XAF1 could also significantly inhibit the growth of transplanted tumors in nude mouse, with no obvious adverse reactions observed. Therefore, the XAF1 gene could become a new target for lung cancer treatment.

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations are responsive to EGFR-tyrosine kinase inhibitor (EGFR-TKI). However, NSCLC patients with secondary somatic EGFR mutations are resistant to EGFR-TKI treatment. In this study, we investigated the effect of TG101348 (a JAK2 inhibitor) on the tumor growth of erlotinib-resistant NSCLC cells. Cell proliferation, apoptosis, gene expression and tumor growth were evaluated by diphenyltetrazolium bromide (MTT) assay, flow cytometry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, Western Blot and a xenograft mouse model, respectively. Results showed that erlotinib had a stronger impact on the induction of apoptosis in erlotinib-sensitive PC-9 cells but had a weaker effect on erlotinib-resistant H1975 and H1650 cells than TG101348. TG101348 significantly enhanced the cytotoxicity of erlotinib to erlotinib-resistant NSCLC cells, stimulated erlotinib-induced apoptosis and downregulated the expressions of EGFR, p-EGFR, p-STAT3, Bcl-xL and survivin in erlotinib-resistant NSCLC cells. Moreover, the combined treatment of TG101348 and erlotinib induced apoptosis, inhibited the activation of p-EGFR and p-STAT3, and inhibited tumor growth of erlotinib-resistant NSCLC cells in vivo. Our results indicate that TG101348 is a potential adjuvant for NSCLC patients during erlotinib treatment.