Human Non-small Lung Cancer Research Articles

The Correlation between CT Characteristics of Non-small Lung Cancer and the Performance of (18)F-FDG PET and the Expression of Glut1 Protein.

Glut1 is the main carrier which intervenes the glucose uptake of cell, and the expression of Glut1 was positively correlated to the uptake of FDG. The aim of this study is to select the CT characteristics and clinicopathological parameters which can reflect the levels of glucose metabolism of human non-small lung cancer (NSLC). Thirty-three cases of the NSLC (squamous carcinoma 11 cases and adenocarcinoma 22 cases) have undergone the scanning of FDG PET/CT before operation. The tumor pathological classification, degree of differentiation and lymph node metastasis were collected. The CT characteristics which included the diameter, deep lobulation, thin and short spiculation, spinous protuberance, cavity sign, vessel convergence sign and pleural retraction sign were described. Tracer uptake of suspected lesions was assessed by visual assessment and quantitatively by the maximum standardized uptake values (SUVmax). The expression of Glut1 which were examined by means of SP immunohistochemical technique was also analyzed. The positive rate of Glut1 was 66.67% (squamous carcinoma was 90.91% and adenocarcinoma was 54.55%). The positive rate of Glut1 and SUVmax of squamous carcinoma were significant higher than adenocarcinoma (Pbilateral=0.037, Punilateral=0.045). Through visual assessment and SUVmax, the uptake and the positive rate of Glut1 which size >=2 cm were higher than the size <2 cm (Pbilateral=0.002, Pbilateral=0.001, Punilateral=0.049). The uptake intense of FDG was related to the thin and short spiculation through visual assessment (Pbilateral=0.006). The study suggested that the levels of glucose metabolism of squamous carcinoma higher than adenocarcinoma, the diameter upper than 2 cm and the thin and short speculation were higher.

Metal‐based antitumor, cytotoxic and antimicrobial activity: pharmacological evaluation of Knoevenagel condensate β‐diketone Schiff base thiosemicarbazone Cu(II) and Zn(II) complexes

AbstractKnoevenagel condensate Schiff base ligands [L = 3‐cinnamalideneacetylacetone‐thiosemicarbazone (CAT)/3‐cinnama‐ lideneacetylacetoneethylthiosemicarbazone (CAET)/3‐cinnamalideneacetylacetonephenylthiosemicarbazone (CAPT)] and their copper/zinc complexes were synthesized. They were characterized by analytical and spectral techniques. From these data it was found that the ligands adopt square‐planar geometry on metalation with Cu2+ and Zn2+. To evaluate the antitumor and cytotoxic activity of the synthesized complexes in mice and human cancer cell lines, the antitumor activity of the complexes was evaluated against an Ehrlich ascites carcinoma (EAC) tumor model. The activity was assessed using survival time and short‐term in vitro cytotoxic activity. Oral administration of complexes (100 mg/kg) increased the survival time. The cytotoxic activity of complexes was evaluated using human breast cancer (MDA‐MB‐231), colon cancer (HCT‐116) and nonsmall lung cancer (NCI‐H‐23) cell lines. Both the complexes possessed significant antitumor and cytotoxic activity on EAC and human cancer cell lines. The in vitro antimicrobial screening effect of the investigated compounds was also tested against the various organisms by well diffusion method. Copyright © 2009 John Wiley &amp; Sons, Ltd.

Interaction between the p53 genotype of lung cancer and response to mTOR inhibitor combined with irradiation therapy

e22028 Background: Frequent activation of the PI3K/Akt/mTOR pathway and aberrations of tumor suppressor gene p53 are associated with therapeutic resistance in lung cancer. Nevertheless, the possibility of the variants of p53 genotype to affect response to mTOR inhibitor combined with irradiation therapy remains still unclear. Methods: Human non-small lung cancer cell line H1299 with p53 null genotype, was transfected with wild type or mutated p53 gene (H1299/wtp53 (WT), H1299/mp53 (MT)). Both cell survival and cell proliferation were estimated by colony formation assay to assess differences between WT and MT in sensitivity to rapamycin and ability of rapamycin to enhance radiation sensitivity. Cells were treated according to the individual study; DMSO (control), rapamycin (100 nM for 1 hour), irradiation (IR) (increasing doses), combination (RR) (rapamycin followed by irradiation). Changes in the cell cycle were also analyzed by flow cytometry. Results: Rapamycin decreased cell survival only in WT (P &lt; 0.01, vs. control). MT was resistant to rapamycin exhibiting slightly inhibited cell proliferation. Compared with IR, RR with no less than 6 Gy radiation enhanced inhibitory effects on both cell survival and proliferation independent of p53 genotype (P &lt; 0.01 in WT and P &lt; 0.01 in MT, respectively), that indicating additive interaction. Cell cycle analysis demonstrated rapamycin induced G1 cell cycle arrest in both types of cells compared with controls (P &lt; 0.01 in WT and P &lt; 0.05 in MT, respectively) at 24 hours after treatment. Enhancement of G1 arrest by RR was observed in both WT (P &lt; 0.01, vs. IR) and MT (P &lt; 0.01, vs. IR) at the same time point. In addition, RR caused a greater reduction of cells in S phase than that of IR regardless of p53 gene status (P &lt; 0.01 in WT and P &lt; 0.01 in MT, respectively). Conclusions: Rapamycin is an effective radiosensitizer in a p53 independent manner, in which increased G1 cell cycle arrest and decrease in S phase cells are responsible for increased growth inhibitory effect. It will enable us to provide more appropriate treatment to patients with mutated p53 gene. No significant financial relationships to disclose.

Synthesis and In‐Vitro Antitumor Activities of Some Mannich Bases of 9‐Alkyl‐1,2,3,4‐tetrahydrocarbazole‐1‐ones

A novel series of 2-substituted aminomethyl-9-alkyl-1,2,3,4-tetrahydrocarbazole-1-ones 5a-q was synthesized via aminomethylation of 9-alkyl-1,2,3,4-tetrahydrocarbazole-1-ones 4a-e with hydrochlorides of the respective amines 6a-m. The structures of these newly synthesized compounds were characterized by (1)H-NMR, MS, and elemental analysis. All the compounds were tested for their cytotoxic activity in vitro against four human tumor cell lines including human non-small lung cancer cells (A549), human gastric adenocarcinoma (SGC), human colon cancer cell (HCT116), human myeoloid leukemia cells (K562), and one multi-drug resistant subline (KB-VCR). Most compounds showed moderate to potent cytotoxic activity against the tested cell lines. Preliminary mechanism research indicated that the most promising compound, 2-diethylaminomethyl-9-methyl-1,2,3,4-tetrahydrocarbazole-1-one 5c, exhibited a potential inhibitory effect against microtubule.

Novel DNA-directed alkylating agents: Design, synthesis and potent antitumor effect of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker

A series of phenyl N-mustard-9-anilinoacridine conjugates via a carbamate or carbonate linker was synthesized for antitumor evaluation. The carbamate or carbonate linker is able to lower the reactivity of the phenyl N-mustard pharmacophore and thus, these conjugates are rather chemically stable. The in vitro studies revealed that these derivatives possessed significant cytotoxicity with IC 50 in sub-micromolar range in inhibiting human lymphoblastic leukemia (CCRF-CEM), breast carcinoma (MX-1), colon carcinoma (HCT-116) and human non-small cell lung cancer (H1299) cell growth in vitro. Compounds 10a, 10b, 10e, 10i, and 15a were selected for evaluating their antitumor activity in nude mice bearing MX-1 and HCT-116 xenografts. Remarkably, total tumor remission was achieved by these agents with only one cycle of treatment. Interestingly, no tumor relapse was found in mice treated with 10a over 129 days. This agent is capable of inducing DNA interstrand cross-linking in human non-small lung cancer H1299 cells in a dose dependent manner by modified comet assay and has a long half-life in rat plasma.

Binding to Protein by Isothiocyanates: A Potential Mechanism for Apoptosis Induction in Human Nonsmall Lung Cancer Cells

The upstream events by which isothiocyanates (ITCs) induce apoptosis have not been fully investigated. Numerous studies have reported that the apoptosis was induced by ITCs through generation of reactive oxygen species (ROS) as a result of conjugating with and, consequently, depleting cellular glutathione. As electrophiles, ITCs could potentially trigger apoptosis by binding to macromolecules including DNA and proteins. The results showed that DNA damage may not be an important early event for the apoptosis induction by ITCs. Phenethyl isothiocyanate (PEITC) is a more potent inducer of apoptosis than sulforaphane (SFN) in A549 cells, but SFN induces more ROS generation and oxidative damages than PEITC, suggesting that oxidative stress again is probably not a trigger for apoptosis in these cells. In contrast, we found that PEITC binds more to intracellular proteins than SFN. We identified tubulin as 1 of the protein targets of ITCs through proteomics approach. We showed that the relative tubulin binding affinity of ITCs correlates well with their potency of cell growth inhibition and apoptosis induction. These results collectively suggest that the covalent binding to protein targets, such as tubulin, by ITCs is an important chemical event in apoptosis induction by ITCs in human lung A549 cells.

Up‐regulation of CD40 with juxtacrine activity in human nonsmall lung cancer cells correlates with poor prognosis

CD40 and its ligand, CD154, play a regulatory role in several signaling pathways among lymphocytes. Recently, it was reported that CD40 is expressed in several malignant tumors. However, the clinical impact of CD40 expression in nonsmall cell lung cancer has not been studied widely. One hundred twenty-nine surgical specimens of nonsmall cell lung cancer were assessed immunohistochemically for CD40 and CD154 expression, and that expression was correlated with patients' clinicopathologic parameters and outcome. Subsequently, in vitro analysis of CD40-CD154 signaling was performed. Immunohistochemical staining of tumor cells confirmed that 67 patients (51.9%) were positive for CD40, and 76 patients (58.9%) were positive for CD154. The survival of patients who had tumors that were negative for CD40 was significantly better than the survival of patients who had tumors that were positive for CD40 (P = .0004). Multivariate analysis using a Cox regression model indicated that CD40 expression in cancer cells is an independent, unfavorable prognostic factor (risk ratio, 1.855; P = .0403). By using an in vitro juxtacrine growth factor assay, the growth of LK2 cells (CD40-positive/CD154-negative) was accelerated by CD154-positive cancer cells, such as PC10 cells (CD40-negative/CD154-positive), by a juxtacrine mechanism. The current results suggested that CD40 expression in tumors is associated with a poor prognosis and that the juxtacrine interaction of CD40-CD154 among cancer cells facilitates the development of malignant potential in nonsmall cell lung cancer.

Involvement of Rad51 in cytotoxicity induced by epidermal growth factor receptor inhibitor (gefitinib, IressaR) and chemotherapeutic agents in human lung cancer cells

Gefitinib (Iressa(R), ZD1839) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that blocks growth factor-mediated cell proliferation and extracellular signal-regulated kinases 1/2 (ERK1/2) signaling activation. Rad51 is an essential component of the homologous recombination repair pathway. High level of Rad51 expression has been reported in chemo- or radioresistant carcinomas. We hypothesized that gefitinib may enhance the effects of the alkylating agent cisplatin- or the antitumor antibiotic mitomycin C (MMC)-mediated cytotoxicity by decreasing ERK1/2 activation and Rad51 expression. Exposure of human non-small lung cancer cells to gefitinib decreased cisplatin- or MMC-elicited ERK1/2 activation and Rad51 protein induction. Neither cisplatin nor MMC treatment affected Rad51 messenger RNA (mRNA). However, gefitinib cotreatment with cisplatin or MMC significantly decreased Rad51 mRNA levels. In addition, gefitinib decreased cisplatin- or MMC-elicited Rad51 protein levels by increasing Rad51 protein instability. Enhancement of ERK1/2 signaling by constitutively active mitogen-activated protein kinase kinase 1/2 (MKK1/2-CA) increased Rad51 protein levels and protein stability in gefitinib and cisplatin or MMC cotreated cells. Moreover, the synergistic cytotoxic effects induced by gefitinib cotreatment with cisplatin or MMC were remarkably decreased by MKK1-CA-mediated enhancement of ERK1/2 activation. Depletion of endogenous Rad51 expression by si-Rad51 RNA transfection significantly enhanced lung cancer cell death upon treatment with cisplatin or MMC. We conclude that Rad51 protein protects lung cancer cells from synergistic cytotoxic effects induced by gefitinib and chemotherapeutic agents. Suppression of Rad51 expression may be a novel lung cancer therapeutic modality to overcome drug resistance to EGFR inhibitors and chemotherapeutic agents.

Synthesis, cytotoxic activity, and SAR analysis of the derivatives of taxchinin A and brevifoliol

Twenty-one derivatives of taxchinin A ( 1) and brevifoliol ( 2) were synthesized and evaluated for cytotoxicity against human non-small lung cancer (A549) cell line. Nine derivatives showed potent activity with IC 50 values from 0.48 to 6.22 μM. 5-Oxo-13-TBDMS-taxchinin A ( 11) and 5-oxo-13,15-epoxy-13- epi-taxchinin A ( 15) are the most potent derivatives, with IC 50 at 0.48 and 0.75 μM, respectively. The structure–activity relationship (SAR) of these compounds established that exocyclic unsaturated ketone at ring C is the key structural element for the activity, while the α,β-unsaturated ketone positioned at ring A has no effect for the activity. The significant cytotoxicity of derivatives 11 and 15 may be due to the conformational change in the taxane rings. The 3D-QSAR study was conducted on this series of compounds, which provided optimal predictive comparative molecular field (CoMFA) model with cross-validated r 2 ( q 2) value of 0.64.

Study on the structure–activity relationships of phoxalone and induction of apoptosis in H446 tumor cells

A novel macrolide with dioxa and double ring pentadecanone, named as phoxalone, isolated from the culture broth of a myxobacterium Sorangium cellulosum WXNXJ-C, was well investigated. The chemical structure of Phoxalone was elucidated by spectroscopic analysis including UV, IR, 1H NMR, MALDI-TOF-MS, and LC/MS spectra. The results of cytotoxic bioactivities on human non-small lung cancer H446, in vitro, showed that not only did phoxalone express higher antitumor bioactivity than many antitumor drugs, but it also displayed less cytotoxicity to normal human liver L02 cell lines (IC50, 286 μg mL−1). Further cytotoxic bioactivity study indicated that phoxalone could induce H446 cell line apoptosis in vitro. More investigation results of flow-cytometric analysis suggested that phoxalone arrested the mitosis of H446 cell line at G2/M phase. Hence, phoxalone and its derivatives or analogs would reveal huge research value and fascinating foreground.

Magnolol-lnduced H460 cells deathvia autophagy but not apoptosis

We have reported that the protective effect of Magnolol on TBHP-induced injury in human nonsmall lung cancer H460 cells is partially via a p53 dependent mechanism. In this study, we found that Magnolol displayed a stimulatory effect at low concentrations (< or = 20 microM) whilst inhibitory effect at high concentrations (> or = 40 microM) in H460 cells. To investigate the mechanism of inducing the biphasic effect in H460 cells with Magnolol, we showed that Magnolol stimulated DNA synthesis at low concentrations and displayed an inhibition effect at high concentrations in H460 cells. More importantly, the inhibition of DNA synthesis was accompanied by the S phase cell cycle arrest and the appearance of intense intracytoplasmic vacuoles. These vacuoles can be labeled by autophagic marker monodansylcadaverin (MDC), 3-methyladenine (3-MA), an inhibitor of autophagy, was able to inhibit the occurrence of autophagy. The results of the LDH activity assay and TUNEL assay also showed that Magnolol at high concentrations inhibiting H460 cell growth was not via apoptotic pathway. Furthermore, accompanied by the occurrence of autophagy, the expression of phospho-Akt was down-regulated but PTEN significantly was up-regulated. In conclusion, Magnolol induces H460 cells death by autophagy but not apoptotic pathway. Blockade of PI3K/PTEN/Akt pathway is maybe related to Magnolol-induced autophagy. Autophagic cells death induction by Magnolol underlines the potential utility of its induction as a new cancer treatment modality.

Pre-irradiation with low-dose 12C6+ beam significantly enhances the efficacy of AdCMV-p53 gene therapy in human non-small lung cancer

The combination of ionizing radiation and gene therapy has been investigated. However, there are very few reports about the combination of heavy-ion irradiation and gene therapy. To determine if the pre-exposure to low-dose heavy ion beam enhances the suppression of AdCMV-p53 on non-small lung cancer (NSLC), the cells pre-irradiated or non-irradiated were infected with 20, 40 MOI of AdCMV-p53. Survival fraction and the relative biology effect (RBE) were determined by clonogenic assay. The results showed that the proportions of p53 positive cells in 12C6+ beam induced AdCMV-p53 infected cells were more than 90%, which were significantly more than those in γ-ray induced AdCMV-p53 infected cells. The pre-exposure to low-dose 12C6+ beam significantly prevented the G0/G1 arrest and activated G2/M checkpoints. The pre-exposure to 12C6+ beam significantly improved cell to apoptosis. RBEs for the 12C6+ + AdCMV-p53 infection groups were 30%–60%, 20%–130% and 30%–70% more than those for the 12C6+-irradiated only, AdCMV-p53 infected only, and γ-irradiation induced AdCMVp53 infected groups, respectively. The data suggested that the pre-exposure to low-dose 12C6+ beam significantly promotes exogenous p53 expression in NSLC, and the suppression of AdCMV-p53 gene therapy on NSLC.

Symmetrical Derivatives of C2-Substituted Pyrrolo[2,3-f]quinolines:Synthesis, Cytotoxicity and Drug Delivery Studies

The synthesis, pharmacological properties and delivery characteristics in simulated aqueous gastric fluid of a series of symmetrical nitrogenated C2-substituted pyrrolo[2,3-f]quinolines are described. The cytotoxicity of the target molecules (5a-h) was evaluated in the human non-small lung cancer cell line NSCLCN16- L16 in vitro. One compound (5e) showed sufficient activity (IC50 = 26.4 μM) and satisfactory release characteristics from solid pharmaceutical formulations. Keywords: Symmetrical pyrrolo[2,3-f]quinolines, Synthesis, Cytotoxicity, Drug delivery

Reversal of inflammation‐associated dihydrodiol dehydrogenases (AKR1C1 and AKR1C2) overexpression and drug resistance in nonsmall cell lung cancer cells by wogonin and chrysin

Dihydrodiol dehydrogenase (DDH) is a member of the aldo-keto reductases superfamily (AKR1C1-AKR1C4), which plays central roles in the metabolism of steroid hormone, prostaglandin and xenobiotics. We have previously detected overexpression of DDH as an indicator of poor prognosis and chemoresistance in human non-small lung cancer (NSCLC). We also found DDH expression to be closely related to chronic inflammatory conditions. The aim of this study was to investigate the links between inflammation, DDH expression and drug resistance in NSCLC cells. We showed that pro-inflammatory mediators including interleukin-6 (IL-6) could induce AKR1C1/1C2 expression in NSCLC cells and increase cellular resistance to cisplatin and adriamycin. This effect was nullified by Safingol, a protein kinase C inhibitor. Moreover, the expression of AKR1C1/1C2 was inversely correlated to NBS1 and apoptosis-inducing factor (AIF). We also showed that IL-6-induced AKR1C1/1C2 expression and drug resistance were inhibited by wogonin and chrysin, which are major flavonoids in Scutellaria baicalensis, a widely used traditional Chinese and Japanese medicine. In conclusion, this study demonstrated novel links of pro-inflammatory signals, AKR1C1/1C2 expression and drug resistance in NSCLC. The protein kinase C pathway may play an important role in this process. Overexpression of AKR1C1/1C2 may serve as a marker of chemoresistance. Further studies are warranted to evaluate wogonin and chrysin as a potential adjuvant therapy for drug-resistant NSCLC, especially for those with AKR1C1/1C2 overexpression.

Biological Evaluation of Curcumin and Related Diarylheptanoids

Nine derivatives of three natural diarylheptanoids, curcumin, demethoxycurcumin and bisdemethoxycurcumin, were prepared. Their antioxidant, free radical scavenging, nitric oxide (NO) inhibitory and cytotoxic activities were evaluated and compared with those of the respective natural compounds. Curcumin (1), demethoxycurcumin (2), demethyldemethoxy-curcumin (C3), diacetyldemethoxycurcumin (AC2) and triacetyldemethylcurcumin (AC5) exhibited higher antioxidant activity than quercetin while products from demethylation of 1 and 2 exhibited higher free radical scavenging activity. Compounds AC2 and AC5 were found to be most active in inhibiting breast cancer cells (MCF-7) proliferation with IC50 values of 6.7 and 3.6 microM, respectively. The activity of AC2 is almost doubled and of AC5 almost tripled as compared to curcumin. Their selectivity towards different cell lines is also more noticeable. Compounds AC2 and AC5 also showed increased activity against a human prostate cancer cell line (DU-145) and non-small lung cancer cell line (NCI-H460) with IC50 values of 20.4, 16.3 and 18.3, 10.7 microM, respectively.

C5,C6-Disubstituted 1H-Indole-2-Carboxamides: Synthesis and Cytotoxic Activity in the Human Non-Small Lung Cancer Cell Line NSCLC-N16-L16

A facile synthesis of the disubstituted 1H-indole-2-carboxamides (6a-h) is described. Readily available 4-benzyloxy-3-methoxybenzaldehyde is converted to the parent acid (12) by nucleophilic attack of the azido-ester (9) and cyclization of the propenoic methyl ester (10). The target compounds (6a-d) were obtained by amidation of (12) with the appropriate primary amine. The new 6-hydroxy analogs (6e-h) were prepared by benzyl deprotection of (6a-d). The cytotoxicity of the new molecules was evaluated in the human non-small lung cancer cell line NSCLC-N16-L16 in vitro. One compound (6d) showed satisfactory activity (IC50 = 13.9 μM) worthy of further study. It is noteworthy that few agents are clinically effective against human non-small lung cancer, and thus there is a need for novel agents for use in this disease. Keywords: Disubstituted 1H-indole-2-carboxamides, synthesis, cytotoxicity

Expression of Hypoxia Inducible Factor 1α and Its Significance in Non-small Cell Lung Cancer

Objective: To explore the expression level and its clinical significance of hypoxia inducible factor 1α (HIF-1α) in non-small lung cancer. Methods: The expression of HIF-1α was detected in 68 human non-small lung cancer samples by immunohistochemistry. Results: (1) Thirty-nine (57.35%) out of the 68 human non-small lung cancer samples was positive for HIF-1α; (2) The positive rate of HIF-1α in adenocarcinoma and squamous carcinoma was 54.76% (23/42) and 61.54% (16/26) respectively. No significant difference was found between adenocarcinoma and squamous carcinoma of non-small lung cancer in the expression of HIF-1α (P>0.05). The positive rate of HIF-1α in middle-high differentiation was 74.28% (26/35), significantly higher than in low differentiation (39.39%, 13/33) (P<0.05); (3) The positive expression of HIF-1α was not correlated to the sexes, ages, tumor stage and lymph node status. Conclusion: The expression of HIF-1α is higher in non-small lung cancer and is correlated to differentiation.

Enhancement of radiosensitivity in H1299 cancer cells by actin-associated protein cofilin

Cofilin is an actin-associated protein that belongs to the actin depolymerization factor/cofilin family and is important for regulation of actin dynamics. Cofilin can import actin monomers into the nucleus under certain stress conditions, however the biological effects of nuclear transport are unclear. In this study, we found that over-expression of cofilin led to increased radiation sensitivity in human non-small lung cancer H1299 cells. Cell survival as determined by colony forming assay showed that cells over-expressing cofilin were more sensitive to ionizing radiation (IR) than normal cells. To determine whether the DNA repair capacity was altered in cofilin over-expressing cells, comet assays were performed on irradiated cells. Repair of DNA damage caused by ionizing radiation was detected in cofilin over-expressing cells after 24 h of recovery. Consistent with this observation, the key components for repair of DNA double-strand breaks, including Rad51, Rad52, and Ku70/Ku80, were down-regulated in cofilin over-expressing cells after IR exposure. These findings suggest that cofilin can influence radiosensitivity by altering DNA repair capacity.

Targeted therapy against VEGFR and EGFR signaling with ZD6474 enhances the therapeutic efficacy of irradiation in an orthotopic mouse model of human non-small cell lung cancer

Chemotherapy and radiation therapy have had limited therapeutic success in the treatment of advanced lung cancer. ZD6474 is a potent inhibitor of the VEGFR tyrosine kinase activity with additional activity against the EGFR tyrosine kinase. The purpose of the current study is to evaluate the feasibility of combining targeted therapy against VEGFR and EGFR (using ZD6474) with radiation therapy, as compared with chemoradiation, for human non-small lung cancer, in vitro and in vivo using an orthotopic nude mouse model that closely mimics patterns of growth observed in the clinic.

Targeted therapy against VEGFR and EGFR signaling with ZD6474 enhances the therapeutic efficacy of irradiation in an orthotopic mouse model of human non-small cell lung cancer

Chemotherapy and radiation therapy have had limited therapeutic success in the treatment of advanced lung cancer. ZD6474 is a potent inhibitor of the VEGFR tyrosine kinase activity with additional activity against the EGFR tyrosine kinase. The purpose of the current study is to evaluate the feasibility of combining targeted therapy against VEGFR and EGFR (using ZD6474) with radiation therapy, as compared with chemoradiation, for human non-small lung cancer, in vitro and in vivo using an orthotopic nude mouse model that closely mimics patterns of growth observed in the clinic. The human lung adenocarcinoma NCI-H441 cell line was used for all studies. After irradiation, cell proliferation with or without ZD6474 treatment was tested using an MTT assay. Surviving fractions of cells after irradiation and inhibition of sublethal damage repair by ZD6474 were determined using a clonogenic assay. For in vivo studies, 5 × 105 lung adenocarcinoma cells were injected into the left lung of nude mice. The mice were then randomized to treatment with vehicle (p.o. daily), ZD6474 (15mg/kg orally once daily), radiation (4 Gy fractions 3 times weekly to a total dose of 20Gy), ZD6474 and radiation, paclitaxel (200μg weekly i.p.), or paclitaxel and radiation. Therapy was initiated on day 8 and mice were sacrificed on day 24–26 after tumor injection, when control mice showed signs of disease-associated morbidity. Tumor burden was assessed by lung and tumor weight, pleural effusion volume, and total weight of disseminated thoracic lesions. Tumor and adjacent lung tissues were then subjected to immunohistochemical analyses. In the MTT assay, marked repopulation of the H441 cells was observed 72–96h after 4Gy irradiation. Cell proliferation was inhibited by 55 ± 4.7% (p = 0.007) of control by 2.5μM ZD6474 treatment for 4 h before and 72 h after irradiation. Radioresponse in the clonogenic assay was enhanced by a factor of 1.37. Sublethal damage repair was completely inhibited by incubation with 5μM ZD6474 for 4h split time. Recovery ratios between 4Gy single dose and 2 + 2Gy split dose were 1.34 in untreated control and 1.04 in ZD6474 treated group. In vivo, the suppression of both tumor growth and metastasis in the orthotopic lung model was greatest in mice treated with combined radiation and ZD6474 compared with radiation with paclitaxel or monotherapy with each modality. The lung weight of control, radiation, ZD6474, radiation with paclitaxel, and radiation with ZD6474 treated mice increased by 330%, 200%, 155%, and 147%, and 110%, respectively, as compared with normal lung weight and the total weight of disseminated lesions was 197 ± 38.1, 160 ± 49.7, 53.9 ± 14.5, 47.5 ± 16.6, and 17.2 ± 8.00, respectively. Radiation therapy had no effect on the formation of pleural effusion (565 ± 99.4μl) as compared with control (745 ± 112μl). Combined therapy with ZD6474 and radiation blocked pleural effusion (14.2 ± 4.84μl) more effectively than radiation and paclitaxel (246 ± 69.4μl). By immunohistochemical analysis, tumor cell apoptosis in the lung primary tumors was substantially increased in mice treated with combined radiation and ZD6474. Microvessel density of lung primary tumors was 55 ± 3.3, 52 ± 7.3, 13 ± 3.3, 42 ± 4.6, 31 ± 3.5, and 16 ± 1.9 in control, radiation, ZD6474, paclitaxel, radiation with paclitaxel, and radiation with ZD6474 treated groups, respectively. When radiation therapy is combined with ZD6474 as targeted therapy against VEGFR and EGFR a significant enhancement of antiangiogenic, anti-vascular and anti-tumor effects are seen in an orthotopic model of lung cancer. These data provide support for clinical trials combining biologically targeted therapies and conventional therapies in human lung cancer