Subcutaneous A549 Tumors Research Articles

Vitamin e succinate-glycol chitosan modified copper ferrite nanocomposites for lung cancer: Targeted oxidative stress regulation induces cuproptosis and ferroptosis

Although platinum-based drugs continue to be the mainstay of chemotherapy for non-small cell lung cancer (NSCLC), drug toxicity and resistance have caused considerable clinical problems. Copper species may overcome cisplatin-induced resistance, have a wider activity range, and be less toxic than platinum-based drugs. Despite the strong potential of copper-based nanomaterials to trigger cuproptosis and ferroptosis for the treatment of NSCLC, effectively targeting cancer cell death remains challenging. The copper ferrite nanocomposite (CF-GV-LBL) was designed to specifically induce tumor cell death by the responsiveness of vitamin E succinate-glycol chitosan (GV) polymers to cellular esterase levels, the protonation action of GV polymers and polylysine (PL) amino acids, and the active targeting of human serum albumin. Copper ferrite (CF) released iron and copper ions, increasing intracellular oxidative stress, depleting cellular glutathione, and initiating cuproptosis and ferroptosis. Because oxidative stress plays a crucial role in the processes of cuproptosis and ferroptosis, this work focuses on selectively regulating oxidative stress levels in intercellulars. In normal cells, GV degraded and produces the antioxidant vitamin E, which decreases cellular oxidative stress and subsequent damage. In cancer cells, GV generated reactive oxygen species, further promoting the occurrence of cuproptosis and ferroptosis. The CF-GV-LBL nanocomposite demonstrated greater anti-tumor efficacy and biocompatibility than carboplatin. Increased oxidative stress may activate the p53 signaling pathway, thus inhibiting tumor cell growth. Significantly, after 15 days of treatment, CF-GV-LBL efficiently suppressed the development of subcutaneous A549 tumors in the nude mice. Overall, this study focuses on providing insights for the treatment of NSCLC.

Inhibitory Effect of Iron Oxide Nanoparticle Liposome on the Growth of Non-Small Cell Lung Cancer In Vitro

In this study, we assessed the inhibitory potential of magnetic liposomes as gene vectors targeting human non-small cell lung cancer (NSCLC) growth In Vitro via hairpin RNA (shRNA) under the influence of an external magnetic field. Specifically, we engineered the plasmid pGFPshIGF-1R to co-express green fluorescent protein (GFP) and shRNA against insulin-like growth factor-1 receptor (IGF-1R), a gene associated with NSCLC progression. Utilizing superparamagnetic iron oxide nanoparticles (CombiMAG) and Lipofectamine2000,we constructed magnetic liposomes to deliver pGFPshIGF-1R into A549 NSCLC cells. Varying magnetic field strengths and exposure times were employed. The efficacy of transfection was gauged against liposome-only methods using Western blot analysis to measure IGF-1R protein reduction. In Vitro, the magnetic approach exhibited higher transfection efficiency (66.3%) compared to liposomes alone (22.1%), marking a threefold enhancement. In vivo, using a subcutaneous A549 tumor model in nude mice, shRNA-transfected A549 cells displayed substantial IGF-1R protein inhibition (interference rates: 56.1% and 85.1%). Importantly, magnetic transfection outperformed liposomal transfection in suppressing IGF-1R protein expression (P <0.05). Our findings suggest that magnetic liposome-mediated gene delivery, facilitated by an external magnetic field, holds promise as a targeted and safe therapeutic strategy for lung cancer.

In vivo evaluation of integrin αvβ6-targeting peptide in NSCLC and brain metastasis.

Integrin αvβ6, which is upregulated in malignancies and remains absent or weak in normal tissue, is a promising target in molecular imaging therapeutics. In vivo imaging of integrin αvβ6 could therefore be valuable for early tumor detection and intraoperative guidance. In this study, integrin αvβ6-targeting probe G2-SFLAP3 was labeled with near-infrared (NIR) dye Cy5.5 or radioisotope 68Ga. The resulting probes were evaluated in integrin αvβ6-positive A549 and αvβ6-negative H1703 xenograft mice models. The cellar uptake of G2-SFLAP3-Cy5.5 was consistent with the expression of integrin αvβ6. Both subcutaneous and brain metastatic A549 tumors could be clearly visualized by NIR fluorescent imaging of G2-SFLAP3-Cy5.5. A549 tumors demonstrated the highest G2-SFLAP3-Cy5.5 accumulation at 4h post-injection (p.i.) and remain detectable at 84h p.i. The fluorescent signal of G2-SFLAP3-Cy5.5 was significantly reduced in H1703 and A549-blocking groups. Consistently, small-animal PET imaging showed tumor-specific accumulation of 68Ga-DOTA-G2-SFLAP3. G2-SFLAP3 represents a promising agent for noninvasive imaging of non-small cell lung cancer (NSCLC) and brain metastases.

In Situ Silver-Based Electrochemical Oncolytic Bioreactor.

In this study, it is shown for the first time that a reduced graphene oxide (rGO) carrier has a 20-fold higher catalysis rate than graphene oxide in Ag+ reduction. Based on this, a tumor microenvironment-enabled in situ silver-based electrochemical oncolytic bioreactor (SEOB) which switched Ag+ prodrugs into in situ therapeutic silver nanoparticles with and above 95% transition rate is constructed to inhibit the growths of various tumors. In this SEOB-enabled intratumoral nanosynthetic medicine, intratumoral H2 O2 and rGO act as the reductant and the catalyst, respectively. Chelation of aptamers to the SEOB-unlocked prodrugs increases the production of silver nanoparticles in tumor cells, especially in the presence of Vitamin C, which is broken down in tumor cells to supply massive amounts of H2 O2 . Consequently, apoptosis and pyroptosis are induced to cooperatively contribute to the considerably-elevated anti-tumor effects on subcutaneous HepG2 and A549 tumors and orthotopic implanted HepG2 tumors in livers of nude mice. The specific aptamer targeting and intratumoral silver nanoparticle production guarantee excellent biosafety since it fails to elicit tissue damages in monkeys, which greatly increases the clinical translation potential of the SEOB system.

An Activatable Near‐Infrared Molecular Chemiluminescence Probe for Visualization of NQO1 Activity In Vivo†

Comprehensive SummaryChemiluminescence (CL) is a promising tool for diagnostics and sensing due to its inherent excitation light‐free. However, most CL probes emit exclusively visible light with limited penetration depth, hindering their broad application for in vivo deep tissue imaging. This work reports an activated unimolecular CL probe (CL‐P) that emits light in the near‐infrared (NIR) region allowing a superior real‐time visualization and detection of NAD(P)H quinone oxidoreductase 1 (NQO1) in vivo. The NQO1‐specific trimethyl‐locked quinone is a lock for masking the NQO1 trigger site, and it was conjugated on the NIR Schaap's dioxetane chemiluminophore, achieving not only a subtle signal‐to‐noise ratio even at a depth of 15 mm in the chicken breast tissues, but also sensitively detecting the positive expression of NQO1 in A549 cells. CL‐P has been successfully used for visualizing the aberrantly expressed NQO1 in subcutaneous xenograft A549 tumors. To our knowledge, this is the first work reporting the detection of NQO1 activity in the NIR region by unimolecular CL mode. Overall, the NQO1‐activated visualization platform provides a guideline to transform a unimolecular CL sensor into a licensed imaging agent for the in vivo detection of enzyme‐related diseases.

Radiotracer stereochemistry affects substrate affinity and kinetics for improved imaging of system xC- in tumors.

Amino acid utilization is perturbed in cancer cells, which rewire their metabolism to support cell survival and proliferation. This metabolic reprogramming can be exploited for diagnostic purposes through positron emission tomography imaging of fluorine-18 labeled amino acids. Despite its promise, little is known regarding transporter-recognition of non-natural amino acid stereoisomers or their utility for cancer imaging. We report here the synthesis and in vivo characterization of a radiolabeled amino acid (R)-4-(3-18F-fluoropropyl)-ʟ-glutamate ([18F]FRPG) and compared its tumor imaging properties to the 4S-isomer, [18F]FSPG.Methods: [18F]FRPG and [18F]FSPG uptake was assessed in H460 lung cancer cells, with efflux measured 30 min after removal of exogenous activity. Specificity of [18F]FRPG for system xC- was further examined following transporter inhibition and blocking studies with system xC- substrates. [18F]FRPG and [18F]FSPG pharmacokinetics was next quantified in mice bearing subcutaneous A549, H460, VCAP and PC3 tumors, with mice bearing A549 tumors imaged by PET/CT. To better-understand differential tumor retention, radiometabolite analysis was performed on tissue and blood samples after imaging. Next, [18F]FRPG and [18F]FSPG retention in lipopolysaccharide-treated lungs were compared to an orthotopic H460 lung cancer model. Finally, the sensitivity of [18F]FRPG to manipulation of the redox environment was examined in cell and in vivo models.Results: [18F]FRPG was specifically transported across the plasma membrane by the cystine/glutamate antiporter system xC- and retained at high levels in multiple tumor models. Conversely, [18F]FRPG was rapidly extracted from the blood and cleared from tissues with low system xC- expression. Due to its favorable imaging properties, tumor-to-blood ratios ≥10 were achieved with [18F]FRPG, which were either equal to or greater than [18F]FSPG. In addition, [18F]FRPG retention in orthotopic lung tumors with high system xC- expression was 2.5-fold higher than inflamed tissue, allowing for clear tumor visualization. In vivo, [18F]FRPG and [18F]FSPG were metabolized to a single species, with [18F]FRPG showing a higher percentage of parent radiotracer in tumors compared to [18F]FSPG. [18F]FRPG was sensitive to redox manipulations and tumor retention was reduced following treatment with liposomal doxorubicin in mice bearing ovarian tumors.Conclusions: Given the fast clearance and low background retention of [18F]FRPG throughout the body, this radiotracer holds promise for the imaging of system xC- activity and treatment response monitoring in tumors of the thorax, abdomen, and head and neck. [18F]FRPG PET imaging provides a sensitive noninvasive measure of system xC- and excellent properties for cancer imaging.

Codelivery of Paclitaxel and Parthenolide in Discoidal Bicelles for a Synergistic Anticancer Effect: Structure Matters

Nanotechnology has long been used to improve the tumor targeting of chemotherapeutics. The structure of these nanocarriers greatly affects their efficacy and toxicity. Herein, structurally stable nanodiscoidal phospholipid bicelles, which are capable of encapsulating anticancer drugs, paclitaxel (PTX) and parthenolide (PTL), are designed. This nanodiscoidal bicelle structure is shown previously to exhibit longer blood circulation, deeper tumor penetration, and increased tumor cell uptake compared with spherical particles of similar compositions. Structural characterization and loading capacity of the PTX‐ and PTL‐ loaded nanodiscs are conducted along with an investigation into the cytotoxicity of the bicelles based on the drug‐to‐lipid ratios of both drugs loaded separately and in combination. A significant synergistic effect between both loaded drugs is also observed in A549 lung cancer and drug‐resistant MDA‐MB‐231 cell lines. An in vivo study using mice bearing subcutaneous A549 tumors shows a long circulation time of the intravenously injected DiR‐loaded bicelles and significant antitumor efficacy of the PTX‐ and PTL‐loaded bicelles. This efficacious self‐assembled nanoparticle system displays great potential as a nanoplatform to codeliver multiple therapeutics or diagnostics for improving cancer therapy.

Sclareol ameliorated ERCC1-mediated cisplatin resistance in A549 human lung adenocarcinoma cells and a murine xenograft tumor model by suppressing AKT-GSK3β-AP1/Snail and JNK-AP1 pathways

Cisplatin-based chemotherapy is a common first-line regimen for treating non–small cell lung cancer (NSCLC). However, drug resistance is still a major problem. The purposes of this study were to evaluate whether sclareol can reverse cisplatin resistance and to investigate its possible mechanisms. A549 cells, the human NSCLC cells with inherent cisplatin resistance, were used to investigate synergistic effect of sclareol with cisplatin in cell proliferation and migration as well as its regulatory mechanisms in expression of excision repair cross-complementation group 1 (ERCC1), a cisplatin resistance-associated molecule. Nude mice bearing subcutaneous A549 tumors were applied to investigate synergistic activity of sclareol in anti-tumor. As comparing to the cisplatin alone group, the treatment of cisplatin combined with sclareol significantly suppressed survival rate and cell migration of A549 cells. Besides, sclareol also exhibited suppression in ERCC1 expression by inhibiting AKT-GSK3β-AP1/Snail and JNK-AP1 pathways. Furthermore, the experimental data from in vivo study also demonstrated that the combination group of cisplatin and sclareol showed the greatest anti-tumor activity, whose effect could be partially attributed to sclareol-mediated decrease in intratumoral level of ERCC1 protein. Accordingly, sclareol has potential as an adjuvant for the treatment in NSCLC patients with cisplatin resistance.

Targetability of hyaluronic acid nanogel to cancer cells: In vitro and in vivo studies

We have, in previous work developed, characterized and evaluated the biocompatibility of an engineered hyaluronic acid nanogel. Here we assess the targetability of a hyaluronic acid nanogel towards CD44 overexpressing cells, in vitro and in vivo. Results obtained by flow cytometry and confocal fluorescence microscopy shows that nanogel is greatly internalized by non-small cancer lung cells (A549 cells), that overexpress CD44 receptors. The biodistribution and tumor targetability of the nanogel labelled with a near-infrared (NIR) probe were performed, in mice, through a non-invasive imaging system. Results revealed nanogel high targetability towards an induced subcutaneous A549 tumor. Nanogels pharmacokinetics was evaluated also in healthy animals, and Alexa Fluor 680 labelled nanogel exhibited higher accumulation in liver, kidneys and skin. Also, a comparative biodistribution study was performed, using two NIR imaging probes, Cy5.5 and Alexa Fluor 680.

18F-EF5 PET Is Predictive of Response to Fractionated Radiotherapy in Preclinical Tumor Models.

We evaluated the relationship between pre-treatment positron emission tomography (PET) using the hypoxic tracer 18F-[2-(2-nitro-1-H-imidazol-1-yl)-N-(2,2,3,3,3- pentafluoropropyl) acetamide] (18F-EF5) and the response of preclinical tumor models to a range of fractionated radiotherapies. Subcutaneous HT29, A549 and RKO tumors grown in nude mice were imaged using 18F-EF5 positron emission tomography (PET) in order to characterize the extent and heterogeneity of hypoxia in these systems. Based on these results, 80 A549 tumors were subsequently grown and imaged using 18F-EF5 PET, and then treated with one, two, or four fraction radiation treatments to a total dose of 10–40 Gy. Response was monitored by serial caliper measurements of tumor volume. Longitudinal post-treatment 18F-EF5 PET imaging was performed on a subset of tumors. Terminal histologic analysis was performed to validate 18F-EF5 PET measures of hypoxia. EF5-positive tumors responded more poorly to low dose single fraction irradiation relative to EF5-negative tumors, however both groups responded similarly to larger single fraction doses. Irradiated tumors exhibited reduced 18F-EF5 uptake one month after treatment compared to control tumors. These findings indicate that pre- treatment 18F-EF5 PET can predict the response of tumors to single fraction radiation treatment. However, increasing the number of fractions delivered abrogates the difference in response between tumors with high and low EF5 uptake pre-treatment, in agreement with traditional radiobiology.

(68) Ga]-HP-DO3A-nitroimidazole: a promising agent for PET detection of tumor hypoxia.

The goal of this study is to evaluate a new (68) Ga-based imaging agent for detecting tumor hypoxia using positron emission tomography (PET). The new hypoxia targeting agent reported here, [(68) Ga]-HP-DO3A-nitroimidazole ([(68) Ga]-HP-DO3A-NI), was constructed by linking a nitroimidazole moiety with the macrocyclic ligand component of ProHance®, HP-DO3A. The hypoxia targeting capability of this agent was evaluated in A549 lung cancer cells in vitro and in SCID mice bearing subcutaneous A549 tumor xenografts. The cellular uptake assays showed that significantly more [(68) Ga]-HP-DO3A-NI accumulates in hypoxic tumor cells at 30, 60 and 120 min than in the same cells exposed to 21% O2 . The agent also accumulated in hypoxic tumors in vivo to give a tumor/muscle ratio (T/M) of 5.0 ± 1.2 (n = 3) as measured by PET at 2 h post-injection (p.i.). This was further confirmed by ex vivo biodistribution data. In addition, [(68) Ga]-HP-DO3A-NI displayed very favorable pharmacokinetic properties, as it was cleared largely through the kidneys with little to no accumulation in liver, heart or lung (%ID/g < 0.5%) at 2 h p.i. The specificity of the agent for hypoxic tissues was further validated in a comparative study with a control compound, [(68) Ga]-HP-DO3A, which lacks the nitroimidazole moiety, and by PET imaging of tumor-bearing mice breathing air versus 100% O2 . Given the commercial availability of cGMP (68) Ge/(68) Ga generators and the ease of (68) Ga labeling, the new agent could potentially be widely applied for imaging tumor hypoxia prior to radiation therapy.

Synergistic Antitumor Effect of a Combination of Paclitaxel and Carboplatin with Nobiletin from Citrus depressa on Non-Small-Cell Lung Cancer Cell Lines

Non-small-cell lung carcinomas do not sufficiently respond to cancer chemotherapeutic drugs. Combination effects of cancer chemotherapy drugs (paclitaxel and carboplatin) with nobiletin or powdered Shiikuwasha extract from Citrus depressa were examined by isobologram and combination index analyses. It was demonstrated that the combination generated a synergistic inhibitory effect against the proliferation of the human non-small-cell lung carcinoma cell lines A549 and H460 and that of the two chemotherapy drugs, paclitaxel was responsible for this synergistic effect. Furthermore, the percentage of apoptotic cells was decreased with increasing rates of nobiletin to paclitaxel and carboplatin. These findings were considered to be attributed to the ability of nobiletin to regulate cells in the G1 phase, which escaped cell death initiated by paclitaxel and carboplatin. An antitumor activity assay showed that this combination significantly suppressed the growth of subcutaneous A549 tumor xenografts in nude mice.

Multicompartment Micelles with Adjustable Poly(ethylene glycol) Shell for Efficient in Vivo Photodynamic Therapy

We describe the preparation of well-defined multicompartment micelles from polybutadiene-block-poly(1-methyl-2-vinyl pyridinium methyl sulfate)-block-poly(methacrylic acid) (BVqMAA) triblock terpolymers and their use as advanced drug delivery systems for photodynamic therapy (PDT). A porphyrazine derivative was incorporated into the hydrophobic core during self-assembly and served as a model drug and fluorescent probe at the same time. The initial micellar corona is formed by negatively charged PMAA and could be gradually changed to poly(ethylene glycol) (PEG) in a controlled fashion through interpolyelectrolyte complex formation of PMAA with positively charged poly(ethylene glycol)-block-poly(L-lysine) (PLL-b-PEG) diblock copolymers. At high degrees of PEGylation, a compartmentalized micellar corona was observed, with a stable bottlebrush-on-sphere morphology as demonstrated by cryo-TEM measurements. By in vitro cellular experiments, we confirmed that the porphyrazine-loaded micelles were PDT-active against A549 cells. The corona composition strongly influenced their in vitro PDT activity, which decreased with increasing PEGylation, correlating with the cellular uptake of the micelles. Also, a PEGylation-dependent influence on the in vivo blood circulation and tumor accumulation was found. Fully PEGylated micelles were detected for up to 24 h in the bloodstream and accumulated in solid subcutaneous A549 tumors, while non- or only partially PEGylated micelles were rapidly cleared and did not accumulate in tumor tissue. Efficient tumor growth suppression was shown for fully PEGylated micelles up to 20 days, demonstrating PDT efficacy in vivo.

Inhibition of class I histone deacetylases in non-small cell lung cancer by honokiol leads to suppression of cancer cell growth and induction of cell death in vitro and in vivo

Non-small-cell lung cancer (NSCLC) represents approximately 80% of all types of lung cancer. Here, we report the chemotherapeutic effect of honokiol, a phytochemical from Magnolia grandiflora, on NSCLC cells and the molecular mechanisms underlying these effects using in vitro and in vivo models. Treatment of NSCLC cells (A549, H1299, H460 and H226) with honokiol (20, 40 and 60 µM) inhibited histone deacetylase (HDAC) activity, reduced the levels of class I HDAC proteins and enhanced histone acetyltransferase activity in a dose-dependent manner. These effects of honokiol were associated with a significant reduction in the viability of NSCLC cells. Concomitant treatment of cells with a proteasome inhibitor, MG132, prevented honokiol-induced degradation of class I HDACs, suggesting that honokiol reduced the levels of HDACs in NSCLC cells through proteasomal degradation. Valproic acid, an inhibitor of HDACs, exhibited a similar pattern of reduced viability and induction of death of NSCLC cells. Treatment of A549 and H1299 cells with honokiol resulted in an increase in G1 phase arrest, and a decrease in the levels of cyclin D1, D2 and cyclin dependent kinases. Further, administration of honokiol by oral gavage significantly inhibited the growth of subcutaneous A549 and H1299 tumor xenografts in athymic nude mice, which was associated with the induction of apoptotic cell death and marked inhibition of class I HDACs proteins and HDAC activity in the tumor xenograft tissues. Together, our study provides new insights into the role of class I HDACs in the chemotherapeutic effects of honokiol on lung cancer cells.

Effective Treatment of Human Lung Cancer by Targeting Tissue Factor with a Factor VII-Targeted Photodynamic Therapy

Lung cancer is the leading cause of cancer death. Conventional photodynamic therapy (PDT) using a nontargeted photosensitizer (ntPDT) is a treatment option for central- and peripheral-type early-stage lung cancer. However, ntPDT can cause severe side effects for normal tissue due to its non-selective distribution. To improve the selectivity and effectiveness of ntPDT for human lung cancer, we hypothesized that tissue factor would be a common yet specific biomarker and a potential therapeutic target for both lung cancer cells and lung tumor vascular endothelial cells in factor VII-targeted PDT (fVII-tPDT), which uses the fVII-Sn(IV) chlorin e6 conjugate for the treatment of human lung cancer. We first identified that tissue factor is indeed expressed on the human non-small cell lung cancer (NSCLC) lines A549 and H460 as well as on tumor vascular endothelial cells of A549 tumor xenografts from nude mice, but it is not expressed by vascular endothelial cells in healthy mouse organs including the lungs. We then demonstrated that fVII-targeting in fVII-tPDT significantly enhanced (up to 25-fold) the in vitro effect of ntPDT on the destruction of A549 and H460 lung cancer cells via the rapid induction of apoptosis and necrosis. We further demonstrated that in vivo administration of fVIItPDT significantly inhibited or eliminated subcutaneous A549 and H460 tumor xenografts in an athymic nude (ATN) mouse model without any obvious side effects. We conclude that fVII-tPDT is effective and safe for the treatment of human lung cancer in preclinical studies and that this methodology holds therapeutic potential for lung cancer patients.

PM02734 (Elisidepsin) Induces Caspase-Independent Cell Death Associated with Features of Autophagy, Inhibition of the Akt/mTOR Signaling Pathway, and Activation of Death-Associated Protein Kinase

PM02734 (elisidepsin) is a synthetic marine-derived cyclic peptide of the kahalalide family currently in phase II clinical development. The mechanisms of cell death induced by PM02734 remain unknown. Human non-small-cell lung cancer (NSCLC) cell lines H322 and A549 were used to evaluate PM02734-induced cytotoxicity, apoptosis, and autophagy, as well as effects on cell death-related signaling pathways. PM02734 at clinically achievable concentrations (0.5-1 μmol/L) was cytotoxic to H322 and A549 cells but did not cause nuclear fragmentation, PARP cleavage, or caspase activation, suggesting that classical apoptosis is not its main mechanism of cell death. In contrast, PM02734-induced cell death was associated with several characteristics of autophagy, including an increase in acidic vesicular organelle content, levels of GFP-LC3-positive puncta, elevation of the levels of Atg-5/12 and LC3-II, and an associated compromise of the autophagic flux resulting in increased number of autophagosomes and/or autolysosomes. Cotreatment with 3-methyladenine (3-MA) and downregulation of Atg-5 gene expression by siRNA partially inhibited PM02734-induced cell death. PM02734 caused inhibition of Akt/mTOR signaling pathways and cotreatment with the Akt inhibitor wortmannin or with the mTOR inhibitor rapamycin led to a significant increase in PM02734-induced cell death. Furthermore, PM02734 caused the activation of death-associated protein kinase (DAPK) by dephosphorylation at Ser308, and downregulation of DAPK expression with siRNA caused also a partial but significant reduction of PM02734-induced cell death. In vivo, PM02734 significantly inhibited subcutaneous A549 tumor growth in nude mice (P < 0.05) in association with induction of autophagy. Our data indicate that PM02734 causes cell death by a complex mechanism that involves increased autophagosome content, due for the most part to impairment of autophagic flux, inhibition of the Akt/mTOR pathway, and activation of DAPK. This unique mechanism of action justifies the continued development of this agent for the treatment of NSCLC.

A fully replication-competent adenovirus vector with enhanced oncolytic properties

We have studied the oncolytic efficacy of two adenovirus vectors named KD3 and INGN 007, which differ from each other only in that whereas KD3 has two small deletions in its e1a gene that restrict its replication to rapidly cycling cells, INGN 007 has wild-type e1a gene. Both vectors overexpress the adenovirus death protein (ADP). Both KD3 and INGN 007 effectively suppressed the growth of subcutaneous human A549 and Hep3B tumors in nude mice upon intratumoral injection, and contained the growth of subcutaneous LNCaP tumors after intravenous injection, making some tumors shrink or disappear. However, in a more demanding model, intravenous injections of neither KD3 nor wild-type Ad5 were effective against subcutaneous A549 tumors, whereas INGN 007 increased the mean survival time by 35%. INGN 007 was also effective in suppressing tumor growth in a challenging A549 orthotopic lung cancer model. INGN 007 was superior to dl1520 (ONYX-015) in repressing subcutaneous A549 tumors. Our results suggest that vectors such as INGN 007 might provide better antitumor efficacy in the clinic as well.

Proliferation of human lung cancer in an orthotopic transplantation mouse model

The objective of this study was to clarify the growth and proliferation style of human lung cancer grown in an orthotopic transplantation model. The human lung squamous cell carcinoma SQ5 and adenocarcinoma A549 cell lines were used. Tumor cells suspended in serum-free medium were directly injected into the main bronchi of anesthetized female Balb/c athymic nude mice with simultaneous administration of 0.01 M ethylenediaminetetraacetic acid. Bromodeoxyuridine was injected into mice 20 min before sacrifice. Lung tissue with tumor nodules and subcutaneous tumors were fixed and confirmed by histological examinations. Bromodeoxyuridine-labeled cells in the tumor area were counted, and the proliferation index was calculated. Lung tumor colonies of various sizes were obtained in the SQ5- and A549-cell orthotopically transplanted mice. Orthotopic SQ5 tumors whose minor diameter was 40-700 μm and major diameter was 80-830 μm showed no definite necrosis. Orthotopic SQ5 tumors whose minor diameter was 540-5,200 μm and major diameter was 600-6,100 μm showed definite necrosis in the tumor center. Similar results were also found in the orthotopic A549 tumors. The proliferation index was 7.38 (3.03)/10.63 (3.10) in the orthotopic SQ5 tumors with/without necrosis and 6.99 (2.10) in the subcutaneous SQ5 tumors with necrosis, respectively. The proliferation index was 2.70 (0.88)/3.53 (1.70) in the orthotopic A549 tumors with/without necrosis and 3.91 (0.63) in the subcutaneous A549 tumors with necrosis, respectively. The data suggest that this orthotopic transplantation model may provide the proper organ microenvironment for lung cancer growth and may be suitable for the target therapy research of human lung cancer.

Hypoxia-inducible-factor 2 alpha mediates cisplatin resistance in the lung adenocarcinoma cell line A549

10573 Background: Hypoxia in solid tumors is associated with cancer progression, metastasis and resistance to chemotherapy. The hypoxia inducible factors (HIF-1a and HIF-2a) are key players in the response to hypoxia affecting angiogenesis, proliferation and apoptosis. Here we investigated the role of HIF-1a and HIF-2a in cellular resistance to cisplatin in non small lung cancer cells (A549). Results: Hypoxia reduced the efficiency of cisplatin treatment on A549 cells via enhancement of drug efflux activity. In addition, hypoxia induced the hypoxia-responsive-element (HRE) reporter gene assay, demonstrating the transcriptional activity of HIF-1a and HIF-2a. Administration of cisplatin further increased the reporter activity under both normoxic and hypoxic conditions. This correlated with the induction of HIF-2a protein expression upon cisplatin treatment in normoxia and hypoxia, whereas HIF-1a was suppressed. Additionally, cisplatin induced nuclear accumulation of HIF-2a. Suppression of HIF-2a by RNA interference potentiated the sensitivity to cisplatin treatment in hypoxic A549 cells. In vivo growth delay assay gave analogues results for subcutaneous A549 tumors treated with cisplatin and suppression of HIF-2a. In comparison, other cytostatic drugs such as doxorubicin and gemcitabine differed with respect to HIF signalling. Doxorubicin induced both HIF-1a and HIF- 2a. Cellular sensitivity to doxorubicin was increased via suppression of HIF-1a and HIF-2a. Gemcitabine appeared to act independent of HIF. The underlying mechanism of hypoxia-induced resistance was due to increased mRNA expression of drug resistance proteins such as MDR1, MRP1 and LRP. These were found to be HIF-2a but not HIF-1a dependent. Conclusion: This data identified HIF-2a as a key player in the hypoxia induced cisplatin resistance and show on the molecular level that HIF-2a is a credible target for therapeutic strategies. No significant financial relationships to disclose.

Mixed Metastatic Lung Cancer Lesions in Bone Are Inhibited by Noggin Overexpression and Rank:Fc Administration

Lung cancer metastases to bone produce a primarily mixed osteolytic/osteoblastic lesion. The purpose of this study was to determine if blockade of both pathways would inhibit the formation these lesions in bone. Inhibition of the osteoblastic lesion with noggin and the osteolytic lesion with RANK:Fc was a successful treatment strategy to inhibit progression of mixed lung cancer lesions in bone. Approximately 9-30% of patients with lung cancer develop bone metastases, leading to significant morbidity and mortality. A549 is a non-small-cell lung cancer (NSCLC) line that produces a mixed metastatic lesion in bone. We sought to determine if blockade of key components in both osteolytic and osteoblastic pathways would result in a reduction of a NSCLC tumor progression in a murine model of bony metastasis. The study used a retroviral vector overexpressing noggin (RN), a specific inhibitor of BMP, and RANK:Fc, a chimeric protein that inhibits the RANK-RANKL interaction. A549 cells were transduced with RN before implantation in SCID mice. Cells were implanted in a subcutaneous model and tibial injection model. RANK:Fc was administered twice weekly at 15 mg/kg. There were five treatment groups: A549; A549 + RN; A549 + RANK:Fc; A549 + empty vector; and A549 + RN + RANK:Fc (n = 10/group). In SCID mice who underwent subcutaneous A549 tumor cell injection, animals treated with A549 + RN had significantly smaller subcutaneous tumor size at 8 weeks. In an intratibial model of bony metastasis, animals injected with A549 cells developed a mixed lytic/blastic lesion with cortical destruction at 8 weeks. Treatment with RANK:Fc inhibited the formation of osteoclasts, led to a smaller tumor volume in bone, and inhibited the lytic component of the mixed lesion. Animals treated with A549 + RN had a decreased number of osteoblasts in bone lesions, smaller tumor volume, and inhibition of the blastic component of the mixed lesions. Combination treatment inhibited both the lytic and blastic components of the lesion. The NSCLC cell line A549 forms a mixed osteolytic/osteoblastic lesion in vivo. Noggin overexpression inhibited the formation of the osteoblastic aspect of the lesion in bone and the tumor growth in vivo. Treatment with RANK:Fc limited the formation of the lytic aspect of the mixed lesion and also inhibited the rate of in vivo tumor growth. Inhibition of both pathways is necessary to effectively inhibit the progression of mixed metastatic lesions in bone.