Cap-independent protein synthesis is enhanced by betaine under hypertonic conditions.

Protein synthesis is one of the main cellular functions inhibited during hypertonic challenge. The subsequent accumulation of the compatible osmolyte betaine during the later adaptive response allows not only recovery of translation but also its stimulation. In this paper, we show that betaine modulates translation by enhancing the formation of cap-independent 48 S pre-initiation complexes, leaving cap-dependent 48 S pre-initiation complexes basically unchanged. In the presence of betaine, CrPV IRES- and sodium-dependent neutral amino acid transporter-2 (SNAT2) 5'-UTR-driven translation is 2- and 1.5-fold stimulated in MCF7 cells, respectively. Thus, betaine could provide an advantage in translation of messengers coding for proteins implicated in the response of cells to different stressors, which are often recognized by ribosomal 40 S subunit through simplified cap-independent mechanisms.

Effect of ABCG2/BCRP Expression on Efflux and Uptake of Gefitinib in NSCLC Cell Lines.

BACKGROUND: BCRP/ABCG2 emerged as an important multidrug resistance protein, because it confers resistance to several classes of cancer chemotherapeutic agents and to a number of novel molecularly-targeted therapeutics such as tyrosine kinase inhibitors. Gefitinib is an orally active, selective EGFR tyrosine kinase inhibitor used in the treatment of patients with advanced non small cell lung cancer (NSCLC) carrying activating EGFR mutations. Membrane transporters may affect the distribution and accumulation of gefitinib in tumour cells; in particular a reduced intracellular level of the drug may result from poor uptake, enhanced efflux or increased metabolism. AIM: The present study, performed in a panel of NSCLC cell lines expressing different ABCG2 plasma membrane levels, was designed to investigate the effect of the efflux transporter ABCG2 on intracellular gefitinib accumulation, by dissecting the contribution of uptake and efflux processes. METHODS AND RESULTS: Our findings indicate that gefitinib, in lung cancer cells, inhibits ABCG2 activity, as previously reported. In addition, we suggest that ABCG2 silencing or overexpression affects intracellular gefitinib content by modulating the uptake rather than the efflux. Similarly, overexpression of ABCG2 affected the expression of a number of drug transporters, altering the functional activities of nutrient and drug transport systems, in particular inhibiting MPP, glucose and glutamine uptake. CONCLUSIONS: Therefore, we conclude that gefitinib is an inhibitor but not a substrate for ABCG2 and that ABCG2 overexpression may modulate the expression and activity of other transporters involved in the uptake of different substrates into the cells.

Inhibition of PI3K Pathway Reduces Invasiveness and Epithelial-to-Mesenchymal Transition in Squamous Lung Cancer Cell Lines Harboring PIK3CA Gene Alterations.

A prominent role in the pathogenesis of squamous cell carcinoma of the lung (SQCLC) has been attributed to the aberrant activation of the PI3K signaling pathway, due to amplification or mutations of the p110α subunit of class I phosphatidylinositol 3-kinase (PIK3CA) gene. The aim of our study was to determine whether different genetic alterations of PIK3CA affect the biologic properties of SQCLC and to evaluate the response to specific targeting agents in vitro and in vivo. The effects of NVP-BEZ235, NVP-BKM120, and NVP-BYL719 on two-dimensional/three-dimensional (2D/3D) cellular growth, epithelial-to-mesenchymal transition, and invasiveness were evaluated in E545K or H1047R PIK3CA-mutated SQCLC cells and in newly generated clones carrying PIK3CA alterations, as well as in a xenograft model. PIK3CA mutated/amplified cells showed increased growth rate and enhanced migration and invasiveness, associated with an increased activity of RhoA family proteins and the acquisition of a mesenchymal phenotype. PI3K inhibitors reverted this aggressive phenotype by reducing metalloproteinase production, RhoA activity, and the expression of mesenchymal markers, with the specific PI3K inhibitors NVP-BKM120 and NVP-BYL719 being more effective than the dual PI3K/mTOR inhibitor NVP-BEZ235. A xenograft model of SQCLC confirmed that PIK3CA mutation promotes the acquisition of a mesenchymal phenotype in vivo and proved the efficacy of its specific targeting drug NVP-BYL719 in reducing the growth and the expression of mesenchymal markers in xenotransplanted tumors. These data indicate that PIK3CA mutation/amplification may represent a good predictive feature for the clinical application of specific PI3K inhibitors in SQCLC patients.

NF-κB drives acquired resistance to a novel mutant-selective EGFR inhibitor.

The clinical efficacy of EGFR tyrosine kinase inhibitors (TKIs) in non-small cell lung cancer (NSCLC) harbouring activating EGFR mutations is limited by the emergence of acquired resistance, mostly ascribed to the secondary EGFR-T790M mutation. Selective EGFR-T790M inhibitors have been proposed as a new, extremely relevant therapeutic approach. Here, we demonstrate that the novel irreversible EGFR-TKI CNX-2006, a structural analog of CO-1686, currently tested in a phase-1/2 trial, is active against in vitro and in vivo NSCLC models expressing mutant EGFR, with minimal effect on the wild-type receptor. By integration of genetic and functional analyses in isogenic cell pairs we provide evidence of the crucial role played by NF-κB1 in driving CNX-2006 acquired resistance and show that NF-κB activation may replace the oncogenic EGFR signaling in NSCLC when effective and persistent inhibition of the target is achieved in the presence of the T790M mutation. In this context, we demonstrate that the sole, either genetic or pharmacologic, inhibition of NF-κB is sufficient to reduce the viability of cells that adapted to EGFR-TKIs. Overall, our findings support the rational inhibition of members of the NF-κB pathway as a promising therapeutic option for patients who progress after treatment with novel mutant-selective EGFR-TKIs.

Trastuzumab emtansine is active on HER-2 overexpressing NSCLC cell lines and overcomes gefitinib resistance.

BACKGROUND: HER-2 represents a relatively new therapeutic target for non small cell lung cancer (NSCLC) patients. The incidence for reported HER-2 overexpression/amplification/mutations ranges from 2 to 20% in NSCLC. Moreover, HER-2 amplification is a potential mechanism of resistance to tyrosine kinase inhibitors of the epidermal growth factor receptor (EGFR-TKI) (about 10% of cases). T-DM1, trastuzumab emtansine is an antibody-drug conjugate composed by the monoclonal antibody trastuzumab and the microtubule polymerization inhibitor DM1. The activity of T-DM1 has been studied in breast cancer but the role of T-DM1 in lung cancer remains unexplored. METHODS: Antiproliferative and proapoptotic effects of T-DM1 have been investigated in different NSCLC cell lines by MTT, crystal violet staining, morphological study and Western blotting. HER-2 expression and cell cycle were evaluated by flow cytometry and Western blotting. Antibody dependent cell cytotoxicity (ADCC) was measured with a CytoTox assay. Xenografted mice model has been generated using a NSCLC cell line to evaluate the effect of T-DM1 on tumor growth. Moreover, a morphometric and immunohistochemical analysis of tumor xenografts was conducted. RESULTS: In this study we investigated the effect of T-DM1 in a panel of NSCLC cell lines with different HER-2 expression levels, in H1781 cell line carrying HER-2 mutation and in gefitinib resistant HER-2 overexpressing PC9/HER2cl1 cell clone. T-DM1 efficiently inhibited proliferation with arrest in G2-M phase and induced cell death by apoptosis in cells with a significant level of surface expression of HER-2. Antibody-dependent cytotoxicity assay documented that T-DM1 maintained the same activity of trastuzumab. Our data also suggest that targeting HER-2 with T-DM1 potentially overcomes gefitinib resistance. In addition a correlation between cell density/tumor size with both HER-2 expression and T-DM1 activity was established in vitro and in an in vivo xenograft model. CONCLUSIONS: Our results indicate that targeting HER-2 with T-DM1 may offer a new therapeutic approach in HER-2 over-expressing lung cancers including those resistant to EGFR TKIs.

Targeting PI3K/AKT/mTOR pathway in non small cell lung cancer.

While PI3K/AKT/mTOR pathway is altered in a variety of cancers including non small cell lung cancer, abnormalities in this pathway are more common in squamous cell lung carcinoma than in adenocarcinoma of the lung. Moreover, aberrant activation of PI3K/AKT/mTOR pathway is one of the mechanisms of acquired resistance to EGFR-TK inhibitors in patients with adenocarcinoma carrying EGFR activating mutations. Several inhibitors of the PI3K pathway are undergoing evaluation in preclinical and clinical studies. These include pan and selective inhibitors of PI3K, AKT inhibitors, rapamycin and rapalogs for mTOR inhibition, dual mTORC1-mTORC2 inhibitors and dual PI3K-mTOR inhibitors. This review focuses on recent preclinical and clinical data on the efficacy of PI3K pathway inhibitors in NSCLC either as monotherapy approach or in combination with chemotherapy or with drugs that target other signaling transduction pathways.

Predictive and prognostic value of early response assessment using 18FDG-PET in advanced non-small cell lung cancer patients treated with erlotinib.

BACKGROUND: [18F]fluorodeoxyglucose (FDG)-PET is being evaluated as a tool for the early detection of response to various targeted agents in solid tumors. The aim of this study was to evaluate the predictive value of PET response after 2 days of erlotinib in unselected pretreated patients with stage IV NSCLC. PATIENTS AND METHODS: FDG-PET/CT scans were conducted at baseline and after 2 days of erlotinib, with a CT evaluation performed at baseline and after 45-60 days of therapy. PET responses were evaluated by quantitative changes on SUVmax tumor/non-tumor ratio and classified according to EORTC criteria. PET responses were compared with RECIST responses and related to progression-free (PFS) and overall (OS) survival. Erlotinib effects on glucose uptake were also studied in a panel of NSCLC cell lines. RESULTS: Fifty-three patients were enrolled. At 2 days of erlotinib, 20 (38 %) patients showed partial metabolic response (PMR), 25 (47 %) had stable metabolic disease (SMD) and 8 (15 %) had progressive metabolic disease (PMD). All patients with PMD had confirmed RECIST progression at 45-60 days. Patients with early PMR and SMD had significantly longer PFS (p < 0.001 and p = 0.001, respectively) and OS (p = 0.001 for both) than PMD patients. CONCLUSIONS: FDG-PET assessment after 2 days of erlotinib could be useful to identify early resistant patients and to predict survival in unselected NSCLC pretreated population.

Gefitinib inhibits invasive phenotype and epithelial-mesenchymal transition in drug-resistant NSCLC cells with MET amplification.

Despite the initial response, all patients with epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer (NSCLC) eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). The EGFR-T790M secondary mutation is responsible for half of acquired resistance cases, while MET amplification has been associated with acquired resistance in about 5-15% of NSCLCs. Clinical findings indicate the retained addiction of resistant tumors on EGFR signaling. Therefore, we evaluated the molecular mechanisms supporting the therapeutic potential of gefitinib maintenance in the HCC827 GR5 NSCLC cell line harbouring MET amplification as acquired resistance mechanism. We demonstrated that resistant cells can proliferate and survive regardless of the presence of gefitinib, whereas the absence of the drug significantly enhanced cell migration and invasion. Moreover, the continuous exposure to gefitinib prevented the epithelial-mesenchymal transition (EMT) with increased E-cadherin expression and down-regulation of vimentin and N-cadherin. Importantly, the inhibition of cellular migration was correlated with the suppression of EGFR-dependent Src, STAT5 and p38 signaling as assessed by a specific kinase array, western blot analysis and silencing functional studies. On the contrary, the lack of effect of gefitinib on EGFR phosphorylation in the H1975 cells (EGFR-T790M) correlated with the absence of effects on cell migration and invasion. In conclusion, our findings suggest that certain EGFR-mutated patients may still benefit from a second-line therapy including gefitinib based on the specific mechanism underlying tumor cell resistance.

Effects of sorafenib on energy metabolism in breast cancer cells: role of AMPK-mTORC1 signaling.

In this study, we investigated the effects and the underlying molecular mechanisms of the multi-kinase inhibitor sorafenib in a panel of breast cancer cell lines. Sorafenib inhibited cell proliferation and induced apoptosis through the mitochondrial pathway. These effects were neither correlated with modulation of MAPK and AKT pathways nor dependent on the ERα status. Sorafenib promoted an early perturbation of mitochondrial function, inducing a deep depolarization of mitochondrial membrane, associated with drop of intracellular ATP levels and increase of ROS generation. As a response to this stress condition, the energy sensor AMPK was rapidly activated in all the cell lines analyzed. In MCF-7 and SKBR3 cells, AMPK enhanced glucose uptake by up-regulating the expression of GLUT-1 glucose transporter, as also demonstrated by AMPKα1 RNA interference, and stimulated aerobic glycolysis thus increasing lactate production. Moreover, the GLUT-1 inhibitor fasentin blocked sorafenib-induced glucose uptake and potentiated its cytotoxic activity in SKBR3 cells. Persistent activation of AMPK by sorafenib finally led to the impairment of glucose metabolism both in MCF-7 and SKBR3 cells as well as in the highly glycolytic MDA-MB-231 cells, resulting in cell death. This previously unrecognized long-term effect of sorafenib was mediated by AMPK-dependent inhibition of the mTORC1 pathway. Suppression of mTORC1 activity was sufficient for sorafenib to hinder glucose utilization in breast cancer cells, as demonstrated by the observation that the mTORC1 inhibitor rapamycin induced a comparable down-regulation of GLUT-1 expression and glucose uptake. The key role of AMPK-dependent inhibition of mTORC1 in sorafenib mechanisms of action was confirmed by AMPKα1 silencing, which restored mTORC1 activity conferring a significant protection from cell death. This study provides insights into the molecular mechanisms driving sorafenib anti-tumoral activity in breast cancer, and supports the need for going on with clinical trials aimed at proving the efficacy of sorafenib for breast cancer treatment.

Long-lasting inhibition of EGFR autophosphorylation in A549 tumor cells by intracellular accumulation of non-covalent inhibitors.

In the present study, a small set of reversible or irreversible 4-anilinoquinazoline EGFR inhibitors was tested in A549 cells at early (1h) and late (8h) time points after inhibitor removal from culture medium. A combination of assays was employed to explain the observed long-lasting inhibition of EGFR autophosphorylation. We found that EGFR inhibition at 8h can be due, besides to the covalent interaction of the inhibitor with Cys797, as for PD168393 (2) and its prodrug 4, to the intracellular accumulation of non-covalent inhibitors by means of an active cell uptake, as for 5 and 6. Compounds 5-6 showed similar potency and duration of inhibition of EGFR autophosphorylation as the covalent inhibitor 2, while being devoid of reactive groups forming covalent bonds with protein thiols.

Shiga toxin 1, as DNA repair inhibitor, synergistically potentiates the activity of the anticancer drug, mafosfamide, on raji cells.

Shiga toxin 1 (Stx1), produced by pathogenic Escherichia coli, targets a restricted subset of human cells, which possess the receptor globotriaosylceramide (Gb3Cer/CD77), causing hemolytic uremic syndrome. In spite of the high toxicity, Stx1 has been proposed in the treatment of Gb3Cer/CD77-expressing lymphoma. Here, we demonstrate in a Burkitt lymphoma cell model expressing this receptor, namely Raji cells, that Stx1, at quasi-non-toxic concentrations (0.05-0.1 pM), inhibits the repair of mafosfamide-induced DNA alkylating lesions, synergistically potentiating the cytotoxic activity of the anticancer drug. Conversely, human promyelocytic leukemia cells HL-60, which do not express Gb3Cer/CD77, were spared by the toxin as previously demonstrated for CD34+ human progenitor cells, and hence, in this cancer model, no additive nor synergistic effects were observed with the combined Stx1/mafosfamide treatment. Our findings suggest that Stx1 could be used to improve the mafosfamide-mediated purging of Gb3Cer/CD77+ tumor cells before autologous bone marrow transplantation.

5'-Untranslated region of heat shock protein 70 mRNA drives translation under hypertonic conditions.

In mammalian cells, adaptation to hypertonic conditions leads to the activation of an array of early (cell shrinkage, regulatory volume increase) and late (accumulation of compatible osmolytes) responses and increased level of HSPs (heat shock proteins). Protein synthesis is strongly inhibited few minutes after the hypertonic challenge as demonstrated in whole cells and as reproduced under controlled conditions in cell-free systems. Different mechanisms known to mediate the accumulation of HSP70, such as mRNA transcription and stabilization, require fully active protein synthesis. We show that the 5'-untranslated region of HSP70 messenger drives a hypertonicity-resistant translation (up to 0.425 osmol/kg of water), whereas cap-dependent protein synthesis is almost totally blocked under the same conditions. The results, obtained in cell-free systems and in whole cells, might help to explain why HSP70 is accumulated in cells when total protein synthesis is impaired. We also observed that translation initiated by viral IRES (from Cricket paralysis virus) is highly efficient in cells exposed to hyperosmolarity, suggesting that the resistance to hypertonic conditions is a more general feature of cap-independent translation. The described mechanism may also play a role in the control of translation of other messengers encoding for proteins involved in the adaptation to hypertonicity.

Combined use of anti-ErbB monoclonal antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type erlotinib-sensitive NSCLC cell lines.

BACKGROUND: The epidermal growth factor receptor (EGFR) is an established target for anti-cancer treatment in different tumour types. Two different strategies have been explored to inhibit this pivotal molecule in epithelial cancer development: small molecules TKIs and monoclonal antibodies. ErbB/HER-targeting by monoclonal antibodies such as cetuximab and trastuzumab or tyrosine-kinase inhibitors as gefitinib or erlotinib has been proven effective in the treatment of advanced NSCLC. RESULTS: In this study we explored the potential of combining either erlotinib with cetuximab or trastuzumab to improve the efficacy of EGFR targeted therapy in EGFR wild-type NSCLC cell lines. Erlotinib treatment was observed to increase EGFR and/or HER2 expression at the plasma membrane level only in NSCLC cell lines sensitive to the drug inducing protein stabilization. The combined treatment had marginal effect on cell proliferation but markedly increased antibody-dependent, NK mediated, cytotoxicity in vitro. Moreover, in the Calu-3 xenograft model, the combination significantly inhibited tumour growth when compared with erlotinib and cetuximab alone. CONCLUSION: Our results indicate that erlotinib increases surface expression of EGFR and/or HER2 only in EGFR-TKI sensitive NSCLC cell lines and, in turns, leads to increased susceptibility to ADCC both in vitro and in a xenograft models. The combination of erlotinib with monoclonal antibodies represents a potential strategy to improve the treatment of wild-type EGFR NSCLC patients sensitive to erlotinib.

Clinical perspectives for irreversible tyrosine kinase inhibitors in cancer.

Irreversible inhibitors provide potent and selective inhibition of tyrosine kinase enzymes. Use of such inhibitors has proved promising in overcoming the tumor resistance encountered with reversible tyrosine kinase inhibitors. Irreversible inhibitors inactivate their protein target through covalent interaction with a nucleophilic cysteine residue within the nucleotide binding pocket of the kinase domain. Different irreversible tyrosin kinase inhibitors directed against epidermal growth factor receptor (EGFR), Bruton's tyrosine kinase (BTK), vascular endothelial growth factor receptor (VEGFR) and fibroblast growth factor receptor tyrosine kinase (FGFR) have been developed and some of them have been employed clinically as anticancer agents. This review focuses on recent preclinical and clinical progress with currently available irreversible tyrosine kinase inhibitors. The chemical structures of the candidates, structure-activity relationships, biological activities and results of current clinical investigations are described.

Overcoming acquired resistance to letrozole by targeting the PI3K/AKT/mTOR pathway in breast cancer cell clones.

Development of resistance to endocrine therapy is a clinical issue in estrogen receptor (ER)-positive breast cancer. Here we show that persistent activation of AKT/mTOR signaling is crucial to the acquisition of letrozole resistance in cell clones generated from MCF-7/AROM-1 aromatase-expressing breast cancer cells after prolonged letrozole exposure. ERα plays a marginal role in this context. As a proof of concept, the association between PI3K/AKT/mTOR signaling and insensitivity to endocrine therapies was confirmed in breast cancer patients who developed early letrozole resistance in neoadjuvant setting. In addition our results suggest that, regardless of the mechanism mediating the activation of AKT/mTOR pathway, either RAD001 or NVP-BEZ235 treatment may represent a promising strategy to overcome acquired resistance to letrozole in breast cancers dependent on AKT/mTOR signaling.

Irreversible inhibition of epidermal growth factor receptor activity by 3-aminopropanamides.

Irreversible epidermal growth factor receptor (EGFR) inhibitors contain a reactive warhead which covalently interacts with a conserved cysteine residue in the kinase domain. The acrylamide fragment, a commonly employed warhead, effectively alkylates Cys797 of EGFR, but its reactivity can cause rapid metabolic deactivation or nonspecific reactions with off-targets. We describe here a new series of irreversible inhibitors containing a 3-aminopropanamide linked in position 6 to 4-anilinoquinazoline or 4-anilinoquinoline-3-carbonitrile driving portions. Some of these compounds proved to be as efficient as their acrylamide analogues in inhibiting EGFR-TK (TK = tyrosine kinase) autophosphorylation in A549 lung cancer cells. Moreover, several 3-aminopropanamides suppressed proliferation of gefitinib-resistant H1975 cells, harboring the T790M mutation in EGFR, at significantly lower concentrations than did gefitinib. A prototypical compound, N-(4-(3-bromoanilino)quinazolin-6-yl)-3-(dimethylamino)propanamide (5), did not show covalent binding to cell-free EGFR-TK in a fluorescence assay, while it underwent selective activation in the intracellular environment, releasing an acrylamide derivative which can react with thiol groups.

Metabolism of the EGFR tyrosin kinase inhibitor gefitinib by cytochrome P450 1A1 enzyme in EGFR-wild type non small cell lung cancer cell lines.

BACKGROUND: Gefitinib is a tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR) especially effective in tumors with activating EGFR gene mutations while EGFR wild-type non small cell lung cancer (NSCLC) patients at present do not benefit from this treatment.The primary site of gefitinib metabolism is the liver, nevertheless tumor cell metabolism can significantly affect treatment effectiveness. RESULTS: In this study, we investigated the intracellular metabolism of gefitinib in a panel of EGFR wild-type gefitinib-sensitive and -resistant NSCLC cell lines, assessing the role of cytochrome P450 1A1 (CYP1A1) inhibition on gefitinib efficacy. Our results indicate that there is a significant difference in drug metabolism between gefitinib-sensitive and -resistant cell lines. Unexpectedly, only sensitive cells metabolized gefitinib, producing metabolites which were detected both inside and outside the cells. As a consequence of gefitinib metabolism, the intracellular level of gefitinib was markedly reduced after 12-24 h of treatment. Consistent with this observation, RT-PCR analysis and EROD assay showed that mRNA and activity of CYP1A1 were present at significant levels and were induced by gefitinib only in sensitive cells. Gefitinib metabolism was elevated in crowded cells, stimulated by exposure to cigarette smoke extract and prevented by hypoxic condition. It is worth noting that the metabolism of gefitinib in the sensitive cells is a consequence and not the cause of drug responsiveness, indeed treatment with a CYP1A1 inhibitor increased the efficacy of the drug because it prevented the fall in intracellular gefitinib level and significantly enhanced the inhibition of EGFR autophosphorylation, MAPK and PI3K/AKT/mTOR signalling pathways and cell proliferation. CONCLUSION: Our findings suggest that gefitinib metabolism in lung cancer cells, elicited by CYP1A1 activity, might represent an early assessment of gefitinib responsiveness in NSCLC cells lacking activating mutations. On the other hand, in metabolizing cells, the inhibition of CYP1A1 might lead to increased local exposure to the active drug and thus increase gefitinib potency.

Synthesis and biological evaluation of tetracyclic fluoroquinolones as antibacterial and anticancer agents.

A simple and efficient synthesis of 6-fluoro-4-oxopyrido[2,3-a]carbazole-3-carboxylic acids (13a-e) and a structurally related 6-fluoro-4-oxothieno[2',3':4,5]pyrrolo[3,2-h]quinoline (13f) was achieved via Stille arylation of 7-chloro-6-fluoro-8-nitro-4-oxoquinoline-3-carboxylate and a subsequent microwave-assisted phosphite-mediated Cadogan reaction. The new compounds were tested for their in vitro antimicrobial and antiproliferative activity. The ability of 13a-f to inhibit the activity of DNA gyrase and topoisomerase IV was also investigated. The thieno isostere (13f) emerged as the most active antibacterial, while the 9-fluoro derivative (13e) was the most potent against multidrug-resistant staphylococci. Compounds 13a, 13c-f displayed growth inhibition against MCF-7 breast tumor and A549 non-small cell lung cancer cells coupled with an absence of cytotoxicity toward normal human-derm fibroblasts (HuDe). Compound 13e was the most active anticancer against MCF-7 cells, with greater potency than ellipticine (IC(50) 0.8 and 1.6muM, respectively). The most active compounds in this series show promise as dual acting anticancer and antibacterial chemotherapeutics.

Functional characterization of gefitinib uptake in non-small cell lung cancer cell lines.

Gefitinib, an inhibitor of epidermal growth factor receptor tyrosine kinase, has been developed and approved for treatment of advanced non-small cell lung cancer (NSCLC). In this study, we investigated the uptake of gefitinib in gefitinib-sensitive and -resistant NSCLC cell lines. The transport system was temperature-dependent, indicative of an active process and sodium- and potential-independent. Moreover, high cell densities and low extracellular pH significantly reduced the uptake of gefitinib. Inhibitors of the human organic cation transporter 1 (hOCT1) significantly decreased gefitinib uptake; however, gefitinib was not a substrate for hOCT1 or hOCT2 in overexpressing HEK293 cells. Interestingly, gefitinib significantly reduced uptake of the hOCT prototypical substrate MPP suggesting that gefitinib may exert an inhibitory effect on the intracellular accumulation of drugs transported by hOCT1 and hOCT2. After 15min of treatment at 1microM (the maximum plasma concentration of gefitinib obtained at the clinically relevant dose) gefitinib accumulated within the cell in resistant-cell lines at concentrations similar or even higher than in gefitinib-sensitive cells tending to rule out an alteration in drug uptake as a mechanism of resistance to gefitinib treatment. Moreover, our results suggest that the extrusion of lactate by crowded cells may contribute in decreasing the pH, which in turn can influence the uptake of gefinitib and as a result the inhibition of EGFR autophosphorylation.

Endothelial damage induced by Shiga toxins delivered by neutrophils during transmigration.

The endothelial damage induced by Stx represents the main pathogenic event in the HUS associated with STEC infections in humans. Stx, released in the gut by bacteria, enter the bloodstream and are targeted to renal endothelia. The role of PMN as a toxin carrier has been the object of controversy. In this paper, we confirm the binding of Stx1 to PMN, also showing its degranulating effects on full-loaded leukocytes, and support the carrier role of PMN by using a two-chamber transmigration device, in which PMN, loaded in vitro with different amounts of Stx1, transmigrated through confluent monolayers of endothelial cells, mimicking the toxin-induced renal endothelial injury. Stx1 was transferred during PMN transmigration, impairing protein synthesis and triggering production of proinflammatory cytokines in endothelial cells. PMN, carrying low toxin amounts, induced the release of high levels of cytokines in viable endothelial cells, whereas cytokine production was blocked in cells challenged with PMN fully loaded with Stx as a result of an almost total impairment of translation and of the activation of the apoptotic program. In agreement with previous unexplained observations in animal models, the results obtained with our experimental setting suggest that a self-amplifying circle triggered by low doses of toxin may lead to the production of proinflammatory mediators of renal damage in HUS.