Human Non-small Lung Research Articles

Isolation, identification, semi-synthesis of aziditaxel derivatives and their biological evaluation

Two new taxoids (5 and 6) were obtained by isolating impurities in aziditaxel, and their structures were characterized based on data analysis of 1H NMR, 13C NMR, HPLC-MS, and through comparison with literature. In order to test their cytotoxicities against human nonsmall lung cancer cell lines (A549), sufficient amounts of compounds 5 and 6 were obtained by semi-synthesis and both of them showed equipotent cytotoxiesty compared with taxol, docetaxel, and aziditaxel.

Growth inhibition of human non-small lung cancer cells h460 by green tea and ginger polyphenols.

Non small cell lung cancer is known to resist apoptotic stimuli of various antitumor agents and become progressively incurable. The present study was undertaken to evaluate the in vitro antineoplastic effect of polyphenols extracted from both green tea (GTPs) and ginger (GPs) on non-small cell lung cancer cells (NSCLC-NCI-H460). The direct antitumor effect of GTPs and GPs on H460 cells was assessed by investigating the proliferation rate, metabolic activity assay (MTT method) and the apoptotic effect (determined by an annexin V apoptosis assay). Also, the inhibition concentrations (IC50) of both extracts and the levels of P 53 and Bcl-2 proteins were determined. GTPs and GPs have inhibited the proliferation of H460 cells in a dose-dependent manner. At the end of treatment period (96 h) the cell population has decreased to 16% and 26% when treated with 80μg GTPs or GPs, respectively, compared to the untreated cells. The IC50 values of both extracts were 32.9 and 55.5 g/ml, respectively. GTPs was more effective in reduction of cell metabolic activity (measured by MTT assay), where cell count decreased to 22% compared to 64% in cells treated with similar concentration (80μg) of GPs. Lower concentration (20μg) of cisplatin induced 15% reduction in cell metabolic activity. Moreover, 80μg of GTPs and GPs extracts induced apoptosis by 71% and 39% of the living cells, respectively. The apoptotic effect of both extracts, especially GTPs, seems to be mediated by both P 53 and Bcl-2. The study reports the antiproliferative and apoptosis-mediated cytotoxic effects of green tea and ginger polyphenolic extracts on human H460 cell line, indicating their promising chemopreventive effect against lung cancer.

Effects of the inhibition of cytosolic phospholipase A2α in non-small cell lung cancer cells

The aim of this study was to investigate the expression of cPLA(2)α in non-small lung cancer cell lines and tissues, and we sought to determine the in vitro effects of the pyrrolidine-2 inhibitor on cPLA(2)α sensitivity in three different non-small lung cancer cell lines. The expression of cPLA(2)α was determined in lung cancer cells by Western blot. Cytotoxicity, cell growth and inhibition of cPLA(2)α activity were determined in relation to the concentration of pyrrolidine-2. Finally, this study investigated immunohistochemical expressions of cPLA(2)α in 23 species of human non-small lung cancer and 5 species of human normal lung to assess their clinicopathological relevance. cPLA(2)α is expressed in A549 and H460, however, no expression in H661 cells. Pyrrolidine-2 demonstrated a dose-dependent inhibitory effect on cell growth and its significantly inhibited BrdU incorporation of human non-small lung cancer cells. Inhibition with pyrrolidine-2 results in reduction in cPLA(2)α activity in A549 and H460 lung cancer cells by 50% when present at IC(50) concentration in arachidonoyl thio-PC assay. Immunohistochemistry of human lung tissue revealed that cPLA(2)α is increased in lung cancer tissues. Pyrrolidine-2 is a more potent and specific cPLA(2)α inhibitor than MAFP and AACOCF3 and represents an excellent pharmacological tool to investigate the biosynthesis and the biological roles of cancer. The present study suggests that pyrrolidine-2 could be a potential therapeutic agent for cancer therapy.

Antioxidant, antiproliferative, and α-glucosidase inhibitory activities of extracts from Impatiens textori Miq

Impatiens textori Miq is an annual plant used in traditional Chinese medicine to treat superficial infections and fingernail inflammation. In the present study, methanol extract and fractions of I. textori Miq were investigated for their antioxidant properties, α-glucosidase inhibitory activities, and antiproliferative activities against human gastric (AGS), human cervical (HeLa), human non-small lung (A549), and human colon adenocarcinoma (HT-29) cancer cell lines. Results showed that the ethyl-acetate fraction had high total phenolic and total flavonoid contents of 141.39 ± 1.54 mg GAE/g and 125.93 ± 7.60 mg QE/g, respectively, high 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity with an IC50 of 9.80 ± 0.08 μg/ml, and significant reducing power among all the extracts and fractions, suggesting that I. textori Miq is an exceptional source of natural antioxidants. In addition, the ethyl-acetate fraction possessed significant α-glucosidase inhibitory activity. The antiproliferative activity of the ethyl-acetate fraction was higher than that of the methanol extract and others fractions. These findings suggest the potential use of this plant as an antitumor agent. Key words: Impatiens textori Miq, antioxidant, 1,1-diphenyl-2-picrylhydrazyl (DPPH), reducing power, antitumor, α-glucosidase.

Expressions and Clinical Signficances of CAR and CD46 in Non-small Cell Lung Cancer

Objective To investigate the expressions of coxsackie and adenovirus receptor(CAR) and group B adenovirus receptor(CD46) in human non-small cell lung cancer(NSCLC) and its clinical significance.Methods The expressions of CAR and CD46 were detected by immunohistochemical technique in 144 cases of NSCLC and the relationships were analyzed between the expressions of CAR or CD46 and the transfection efficiency of adenovirus or development of NSCLC.Results In 144 samples from NSCLC patients,the expression rates of CAR in squmaous cell carcinoma and adenocarcinoma were 77.5%(31/40) and 36.8%(14/38) and CD46 were 58.3%(14/24) and 88.1%(37/42).There were significant differences between CAR and CD46 expression in the squmaous cell carcinoma and adenocarcinoma(P0.05).Meanwhile,CAR positive rate was 43.3%(13/30) in TNM stageⅠpatients and 66.7%(32/48) in stage Ⅱ~Ⅲ patients,(P0.05).The results showed that CAR expression was closely related to the clinical TNM staging in NSCLC.Conclusion The expression levels and pattern of CAR and CD46 were significantly different in various pathological types of NSCLC.Positive expression of CAR usually in squamous cell carcinoma,while CD46 is in adenocarcinoma,indicated that different receptor-dependent adenovirus might have transfection efficiency in various pathologic types of NSCLC.Expression of CAR protein is also correlated to the malignant degree of NSCLC.CAR might play an important roles in evalution of prognosis of NSCLC.

A Triterpenoid from Thalictrum fortunei Induces Apoptosis in BEL-7402 Cells Through the P53-Induced Apoptosis Pathway

Thalictrum fortunei S. Moore, a perennial plant distributed in the southeastern part of China, has been used in Traditional Chinese Medicine for thousands of years for its antitumor, antibacterial and immunoregulatory effects. In order to investigate the active components and the mechanism of the anti-tumor effects of Thalictrum fortunei, the growth inhibitory effects of eight triterpenoids isolated from the aerial parts of the plant on tumor cell lines were examined by 3-(4,5)-dimethylthiazoy1-3,5-diphenyltetrazolium bromide (MTT) assay. The MTT-assay results showed that the inhibitory activity of 3-O-β-d-glucopyranosyl-(1→4)-β-d-fucopyranosyl(22S,24Z)-cycloart-24-en-3β,22,26-triol 26-O-β-d-glucopyranoside (1) was stronger than that of the other seven tested triterpenoids on human hepatoma Bel-7402 cell line (Bel-7402), human colon lovo cells (LoVo), human non-small cells lung cancer NCIH-460 cells (NCIH-460) and human gastric carcinoma SGC-7901 cells (SGC-7901) after 48 h treatment in vitro, with the IC50 values of 66.4, 84.8, 73.5, 89.6 μM, respectively. Moreover, the antitumor mechanism of compound 1 on Bel-7402 cell was explored through nucleus dyeing, fluorescence assay, flow cytometry and western blot. The flow cytometric analysis results revealed that compound 1 caused apoptosis and mitochondrial membrane potential (MMP) loss in Bel-7402 cells. A fluorescence assay indicated that intracellular reactive oxygen species (ROS) were markedly provoked by compound 1 treatment compared to control cells. Immunoblot results showed that compound 1 significantly increased the expression levels of cleaved caspase-3, P53 and Bax protein, and decreased the expression level of Bcl-2 protein. These findings indicate that compound 1 inhibits the growth activity of tumor cells, probably through the P53 protein-induced apoptosis pathway.

Expression of granulocyte colony stimulating factor in patients with non-small cell lung cancer and its clinicopathological significance

To examine granulocyte colony stimulating factor (G-CSF) expression in human non-small cell lung cancer (NSCLC) as well as discuss its clinicopathological significance. Specimens were obtained from 114 cases (53 cases with granulocyte infiltration) diagnosed pathologically as NSCLC in General Hospital of PLA. Paraffin-embedded tissues from these 114 cases of NSCLC were examined for expression of G-CSF by immunohistochemical staining. Correlation between G-CSF expression and pathological features, clinical manifestation, prognosis of patients with NSCLC was analyzed statistically. All the patients were retrospectively followed-up. Fifty-five of the 114 NSCLC specimens expressed G-CSF, and among these 41 (41/54, 75.9%) were large cell carcinoma, nine (9/30, 30.0%) were adenocarcinoma and five (5/30, 16.7%) were squamous cell carcinoma. The expression was significantly correlated with infiltration of tumor mass by neutrophilic granulocytes, histological type, necrosis, differentiation, lymph node metastases, distant metastases, recurrence and survival period (P < 0.01). There was no significant correlation with primary tumor size (P > 0.05). Logistic multi-factor analysis revealed that necrosis, lymph nodes metastases and distant metastases RR (risk ratio) in G-CSF positive group was 5.57, 6.28 and 5.24 times higher than those of G-CSF negative group (P < 0.05). There were remarkable difference of 5-year survival rates (0 and 12.1% respectively) and survival period (42 and 62 months respectively) between positive and negative groups (P < 0.01). NSCLC with G-CSF excretion are mainly large cell lung cancer. The pathologic characteristics of these cases with G-CSF expression included poor differentiation, remarkable atypia, prominent necrosis and infiltration of tumor mass by neutrophils or emperipolesis. These tumors are usually more aggressive in biological behavior and have worse prognosis than those without G-CSF expression.

Curcumin enhances the mitomycin C-induced cytotoxicity via downregulation of MKK1/2–ERK1/2-mediated Rad51 expression in non-small cell lung cancer cells

Curcumin (diferuloylmethane), a major active component of turmeric ( Curcuma longa), has been reported to suppress the proliferation of a wide variety of tumor cells. Rad51 is a key protein in the homologous recombination (HR) pathway of DNA double-strand break repair, and HR represents a novel target for cancer therapy. A high expression of Rad51 has been reported in chemo- or radio-resistant carcinomas. Therefore, in the current study, we will examine whether curcumin could enhance the effects of mitomycin C (MMC), a DNA interstrand cross-linking agent, to induce cytotoxicity by decreasing Rad51 expression. Exposure of two human non-small lung cancer (NSCLC) cell lines (A549 and H1975) to curcumin could suppress MMC-induced MKK1/2–ERK1/2 signal activation and Rad51 protein expression. Enhancement of ERK1/2 activation by constitutively active MKK1/2 (MKK1/2-CA) increased Rad51 protein levels in curcumin and MMC co-treated human lung cells. Moreover, the synergistic cytotoxic effect induced by curcumin combined with MMC was decreased by MKK1-CA-mediated enhancement of ERK1/2 activation by a significant degree. In contrast, MKK1/2 inhibitor, U0126 was shown to augment the cytotoxicity of curcumin and MMC through downregulation of ERK1/2 activation and Rad51 expression. Depletion of endogenous Rad51 expression by siRad51 RNA transfection significantly enhanced MMC and/or curcumin induced cell death and cell growth inhibition. In contrast, an overexpression of Rad51 protected lung cancer cells from synergistic cytotoxic effects induced by curcumin and MMC. We concluded that Rad51 inhibition may be an additional action mechanism for enhancing the chemosensitization of MMC by curcumin in NSCLC.

Chitosan-coated triangular silver nanoparticles as a novel class of biocompatible, highly effective photothermal transducers for in vitro cancer cell therapy

One of the relevant directions that nanotechnology is taking nowadays is connected with nanomedicine and specifically related to the use of light and nanoparticles in early diagnosis and effective therapeutics of cancer. Noble-metal nanoparticles can act under laser irradiation as effective photothermal transducers for triggering localized hyperthermia of tumors. In this work we report the performance of newly synthesized chitosan-coated silver nanotriangles (Chit-AgNTs) with strong resonances in near-infrared (NIR) to operate as photothermal agents against a line of human non-small lung cancer cells (NCI-H460). The hyperthermia experiments were conducted by excitation of nanoparticles-loaded cells at 800 nm wavelength from a Ti:Sapphire laser. We found that the rate of cell mortality in the presence of Chit-AgNTs is higher than in the presence of thiolated poly(ethylene) glycol capped gold nanorods (PEG-AuNRs) – a common hyperthermia agent used as reference-, while no destructive effects were noticed on the control sample (cells without nanoparticles) under identical irradiation conditions. Additionally, we conducted cytotoxicity assays and found Chit-AgNTs to be efficiently uptaken by the cells while exhibiting good biocompatibility for healthy human embryonic cells (HEK), which is essential for any in vivo applications. Our results reveal a novel class of biocompatible plasmonic nanoparticles with high potential to be implemented as effective phototherapeutic agents in the battle against cancer.

Concurrent and sequential administration of sunitinib malate and docetaxel in human non-small cell lung cancer cells and xenografts

Optimal scheduling of chemotherapy with molecular-targeted agents is important to maximize clinical benefit. We compared the effects of concurrent and sequential administration of docetaxel and multi-target inhibitor sunitinib malate on tumor cells and xenografts and studied several mechanisms involved in drug interaction to provide experimental data in support of their clinical use in non-small cell lung cancer (NSCLC). Human umbilical vein endothelial cells (HUVECs), NCI-H460 human non-small lung carcinomas cells, and NCI-H460 xenograft were treated with docetaxel and Sunitinib malate, using concurrent and sequential treatment schedules. Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium (MTS) assay. Cell cycle analysis was conducted using flow cytometry. Extracellular signal-regulated kinases (ERK1/2) phosphorylation was evaluated by immunoblot analysis. Effects on xenografts were assessed by tumor growth delay. There were no significant difference in the cell proliferation and cell cycle distribution of NCI-H460 cells between concurrent treatment and the first sequential treatment. Both docetaxel and sunitinib malate had no effect on each other in inhibiting ERK1/2 phosphorylation in sequential treatments, while docetaxel eliminated inhibitory activity of sunitinib malate on ERK1/2 phosphorylation in concurrent treatment. For NCI-H460 xenografts, the first sequential treatment showed superior effect to concurrent treatment on inhibiting tumor growth. Combined treatment with sunitinib malate and docetaxel had a greater therapeutic effect than monotherapy, and the first sequential scheduling was more effective than concurrent scheduling, which partly due to the effect of docetaxel on receptor tyrosine kinase (RTK) signaling pathway.

Effect of inhibiting the expression of hSMG-1 on chemosensitivity of human non-small cell lung cancer H1299 cells

To investigate the effect of inhibiting the expression of human suppressor of morphogenesis in genitalia-1 (hSMG-1) on chemosensitivity in human lung cancer H1299 cells. Specialized small interference RNAs (siRNAs) of hSMG-1 were transfected into H1299 cells. The knockdown effect was evaluated by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence. After the administration of anti-cancer drugs, the cell viable rate was determined by cell counting kit (CCK-8) and apoptotic rate was measured by Annexin V-FITC PI double staining on flow cytometry. The apoptotic related activated caspase 3 and caspase 9 were determined by colorimetric assay. The expression of hSMG-1 mRNA was significantly inhibited by hSMG-1 siRNA. And the inhibition rate of (73.8 ± 10.3)% was obtained (P < 0.01). The knockdown effect was further confirmed by immunofluorescence. The inhibition of hSMG-1 enhanced the sensitivity of H1299 cells to gemcitabine and cisplatin. The survival rates significantly decreased when the hSMG-1 siRNA transfected cells were treated with 10.0 mg/L gemcitabine and 10.0 mg/L cisplatin for 48 h respectively (0.51 ± 0.02 vs 0.69 ± 0.01, P < 0.01 and 0.34 ± 0.03 vs 0.48 ± 0.01, P < 0.01;all compared with control siRNA group). Annexin V-FITC PI double staining showed that, under the treatment of anti-cancer drugs, the apoptotic rate of H1299 cells was significantly increased by hSMG-1 knockdown [gemcitabine, (20.9 ± 3.4)% vs (12.0 ± 2.7)%, P < 0.05; cisplatin, (10.2 ± 1.8)% vs (4.5 ± 2.0)%, P < 0.05; all compared with control siRNA group]. Further study showed the inhibition of hSMG-1 up-regulated the activated caspase 3 and caspase 9 in the treated H1299 cells (gemcitabine, 0.1 mg/L, 48 h, caspase 3: 14.4 ± 3.8 vs 2.3 ± 0.4, P < 0.01; caspase 9: 15.5 ± 2.4 vs 4.2 ± 0.9, P < 0.01; cisplatin, 1.0 mg/L, 48 h, caspase 3: 18.8 ± 3.0 vs 6.5 ± 1.5, P < 0.01; caspase 9: 20.3 ± 4.2 vs 8.1 ± 2.0, P < 0.05; all compared with control siRNA group). The inhibition of hSMG-1 significantly enhances the sensitivity of human lung cancer H1299 cells to gemcitabine and cisplatin through an induction of more apoptotic cells. And the activation of mitochondrial apoptotic pathway is involved.

Photodynamic therapy (PDT) – Initiation of apoptosis via activation of stress-activated p38 MAPK and JNK signal pathway in H460 cell lines

The purpose of this study was to investigate the photoefficacies of protoporphyrin IX (PpIX) generated by drug precursor 5-aminolevulinic acid (ALA) and its hexyl ester (H-ALA) on two human non-small lung carcinoma cell lines (H460/Bcl-2 and H460/neo). Drug uptake and the photoefficacies of PpIX were measured by flow cytometry and MTT assay; while the mode of cell death and alternation of signal transduction pathways were studied with 4',6-diamidino-2-phenylindole (DAPI) staining and Western blot analysis, respectively. The flow cytometric analysis of H-ALA (5μM) uptake revealed optimal fluorescent intensity at 8h incubation, while ALA (0.5mM) was still far from saturation. The LD(30) of H-ALA-PDT was 30μM, 2J/cm(2), while the LD(30) of ALA-PDT was 3mM, 2J/cm(2). The dark toxicities mediated by both pro-drug H-ALA and ALA were negligible. By DAPI staining, apoptotic cell death was observed. In addition, by Western blot analysis, H-ALA- and ALA-mediated PDT initiated apoptotic cell death via the up-regulation and activation of p38 mitogen activated protein kinase (MAPK), the stress-activated c-jun N-terminal kinases (JNK) and ERK. These results suggested that H-ALA and ALA mediated PDT displayed similar photocytotoxicities towards the two non-small lung cancer cells. Our present study also demonstrates H-ALA or ALA mediated PDT in H460 cells are closely related to the activation of p38 MAPK and JNK signalling pathway.

Proapoptotic Action of p53-Tom5 in p53-Resistant A549 Human Non-small Cell Lung Cancer Cells through Direct Mitochondrial Dysfunction

Transcription-dependent apoptosis triggered by p53 hardly occurs in alternative reading frame (ARF)-null cancer cells. Loss of ARF leads to hyperactivation of murine double minute 2 (MDM2), resulting in the degradation of p53. In the present study, A549 (ARF-null) human non-small lung cancer cells were transfected with a plasmid DNA encoding human wild-type p53 and the mitochondrial transmembrane domain of Tom5 (p53-Tom5) for delivering p53 to mitochondria. As a result, p53-Tom5 exclusively localized at mitochondria in A549 cells and suppressed the proliferation of them, whereas wild-type p53 did not. In addition, mitochondrial dysfunction and release of cytochrome c were induced by p53-Tom5 in A549 cells. These data suggest that p53-Tom5 suppressed the proliferation of A549 cells through direct mitochondrial dysfunction.

Effects of polyphyllin I on growth inhibition of human non-small lung cancer cells and in xenograft

Polyphyllin I (PPI), a small molecular monomer extracted from Rhizoma of Paris polyphyllin, shows strong anticancer effects in previous study. Human lung adenocarcinoma A549 cells, human lung squamous cell carcinoma SK-MES-1 cells, and human lung large cell carcinoma H460 cells were cultured and then treated with PPI. Cell proliferation and apoptosis were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, flow cytometry, western blot analysis, and DNA ladder. Athymic nude mice bearing tumors were injected with PPI, and tumor growth was recorded. Our results showed that PPI significantly inhibited the proliferation of three non-small cell lung cancer (NSCLC) cell lines, with the inhibitory concentrations (IC50) of 1.24, 2.40, and 2.33 μg/ml for A549, H460, and SK-MES-1 cells, respectively. After being treated with 2.5 µg/ml of PPI for 24 h, the apoptotic rate of A549 cells was 39.68%, which was remarkably higher than that of the control. Tumor growth was significantly inhibited in the PPI-treated group compared with the group treated with cisplatin (DDP) or PBS in the nude mice. PPI exhibits antitumor ability in NSCLC cells in vitro and in vivo, which might be related to the apoptosis induced by PPI.

Synthesis of new cytotoxic E-ring modified camptothecins

In an effort to decrease the toxicity and improve the stability of the E-ring of camptothecin, new analogues with an ‘inverted’ lactone ring were designed and synthesised. The compounds retained a good cytotoxic activity on human non-small lung cancer cells H-460.

Aqueous extract of ginger shows antiproliferative activity through disruption of microtubule network of cancer cells

Ginger has a long history of use as traditional medicine for varied human disease. Our present study has shown that the aqueous extract of ginger (GAE) interacts directly with cellular microtubules and disrupts its structure and induces apoptosis of cancer cells as well. The IC50 values of GAE, as determined from cell viability experiment on human non-small lung epithelium cancer (A549) cells and human cervical epithelial carcinoma (HeLa), were 239.4+7.4 and 253.4+8.9μg/ml, respectively. It has been found that the apoptosis of A549 cells by GAE is mediated by up regulation of tumor suppressor gene p53 and alteration of the normal Bax/Bcl-2 ratio followed by down regulation of cellular pro-caspase3. The morphological change of cells upon GAE treatment has also been demonstrated. Both the structural and functional properties of tubulin and microtubule were lost, as confirmed by both ex vivo and invitro experiments. The major component of GAE is poly-phenols (around 2.5%), which consist of ∼80% flavones and flavonols. Poly-phenolic compounds are well known to have anti-mitotic properties, and may be further screened for the development of a potential anti-cancer agent.

Abstract 4964: Transcriptional repression of MYCN is mediated by TAp63 in human neuroblastomas

Abstract p53 is one of the major tumor suppressors and promotes apoptotic cell death by transcriptional activation of pro-apoptotic target genes. In malignant human tumors, however, its tumor suppressive activity is frequently inactivated by somatic mutation and/or chromosomal deletion. To enhance the efficacy of chemotherapeutic agents in p53-deficient tumors, it is necessary to elucidate the p53-independent mechanisms of apoptotic cell death. Recently, we have shown that TATA-binding protein-like protein (TLP) transactivates one of p53 family members, TAp63, and induces apoptosis in a p53-independent manner. In response to DNA damage by etoposide, TLP is induced to translocate from cytoplasm to nucleus and enhance promoter activity of TAp63 through direct recruitment onto the promoter. TAp63 stimulated by TLP promotes apoptotic cell death in Hep3B cells null for the p53 alleles. The expression levels of TLP mRNA in human primary non-small lung cancers is low compared with corresponding normal tissues and the downregulation is significantly correlated with low levels of TAp63 and poor outcome of patients’ survival. These results suggest the potential for TLP-TAp63 pro-apoptotic pathway as a therapeutic target for malignant lung cancers. In this study, we analyzed expression of p53 family genes in 106 primary neuroblastomas using quantitative real time RT-PCR, and found that the levels of only TAp63 expression was significantly high in favorable neuroblastomas as compared with unfavorable tumors (p=0.00696) and that its expression was inversely correlated with MYCN expression (p=0.0350). MYCN oncogene is frequently amplified and overexpressed in unfavorable neuroblastoma, and in vitro studies have demonstrated that downregulation of MYCN is the critical steps for neuronal cell death induced by retinoic acids (RA) regardless of p53 status in tumors. However, the precise molecular mechanisms of the down-regulation of MYCN by RA have not been elusive. Intriguingly, TLP and TAp63 are induced in response to RA treatment and TAp63 directly represses MYCN transcription. In addition, RA treatment also inhibits positive auto-regulation of MYCN, leading to further down-regulation of MYCN mRNA levels. Collectively, our results suggest that TAp63 is highly expressed in favorable neuroblastomas and contributes to repression of MYCN by RA treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4964.

Abstract 1087: The expression and activity of actin-regulating protein cofilin are associated with the cell cycle progression

Abstract Cofilin is a member of actin depolymerizing factor (ADF)/cofilin family that can regulates actin dynamics for cancer growth and invasion/metastasis. Several lines of evidence have demonstrated that cofilin is associated with the cell cycle regulation, while the underlying mechanisms remain to be addressed. To better understand the involvement of cofilin in cell cycle progression, we dissected the cofilin expression and activity in different phases of the cell cycle. We synchronized human non-small lung A549 cancer cells in G1, S and G2/M phase by serum starvation, double thymidine blockage and nocodazole treatment, respectively. It was found that the expression of endogenous cofilin was weakest in G1 phase, while it was strongest in S phase and was modest in G2/M phase. This observation was further confirmed by detecting the down-regulation of highly expressed cofilin in S phase-synchronized cells entering mid-late S, G2/M and G1 phase of the cell cycle. Interestingly, it also showed that inactive form of cofilin (ser-3 phosphorylated) weakly expressed in S phase and gradually increased when cells progress through G2/M and G1 phases. These results suggest that cofilin expression and activity are coordinately regulated during the cell cycle progression. It is also consistent with our previous findings that enforced expression of cofilin results in G1 phase arrest but not S and G2/M phases. Moreover, the level of cofilin transcripts was not significantly altered in different phases of cell cycle. On the other hand, pretreatment of the proteasome inhibitor MG132 resulted in increase of cofilin in G1 phase, suggesting that the fluctuation of cofilin during cell cycle progression is associated with the protein stability. In addition, transduction of undegradable Y68F form of mutant cofilin potentially led to increase of the G1 percentage. Taken together, precisely regulation of cofilin expression and activity is important for cell cycle progression. Targeting on cofilin may provide alternative therapeutic strategy for cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1087.

Abstract 3517: Complete remission of xenograft tumor in nude mice induced by N-mustard-quinoline conjugates bearing hydrazinecarboxamide linker

Abstract Designing DNA-directed alkylating agents is a promising strategy to overcome the drawbacks of alkylating agents by enhancing DNA specific cross-linking and reducing the chemical reactivity of N-mustard. These compounds are generally synthesized by linking the alkylating warhead (such as alkyl or phenyl N-mustard pharmacophore) to the DNA-affinic carrier (such as DNA intercalating or groove binding agents). Recently, we reported that N-mustard-9-anilinoacridine conjugates via a urea, carbamate, or carbonate linker exhibited potent cytotoxicity and therapeutic efficacy against various human tumor xenografts indicating that these linkers are able to stabilize the reactive alkylating war head. Thus, these derivatives have a long half-life in plasma. Some of these conjugates are able to induce complete tumor remission (CR) in inhibiting human tumor xenograft model with low toxicity. To continue our research in finding potent DNA-directed alkylating agents, we have recently synthesized a series of N-mustard-quinoline conjugates bearing a hydrazinecarboxamide linker. These conjugates were prepared in good yield by the condensation of appropriate 4-hydrazinoquinolines with 4-bis(2-chloroethyl)- aminophenyl isocynate. The results showed that these conjugates possessed significant cytotoxicity against human lymphoblastic leukemia (CCRF-CEM) and various solid tumor xenografts (such as MX-1, HCT-116, H1299, CL 1-0, CL 1-5, PC3 and MCF-7) cell growth in vitro. Among these agents, BO-1392 and BO-1393 were able to induce CR against human breast carcinoma MX-1 xenograft and significantly suppress against human ovarian SK-OV3 xenograft in animal model with relative low toxicity. Interestingly, we found that BO-1393 (iv. injection) can significantly prolong the life span of nude mice bearing human glioma SK-NA-S xenograft (orthotropic implanted in brain). The experiment was compared with Temozolomide (oral administration). The modified comet assay and alkaline agarose gel shift assay showed that these conjugates are capable of inducing DNA interstrand cross-linking in human non-small lung cancer H1299 cells. In conclusion, the present studies demonstrated that the newly synthesized N-mustard-quinoline conjugates having a hydrazinecarboxamide linker exhibited a broad spectrum of antitumor activity and are chemically stable. The remarkable therapeutic efficacy of BO-1393 demonstrated that this agent has high potential for clinical development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3517.

Abstract B24: Establishment of human primary non-small cell lung cancer xenograft models to test anticancer drugs

Abstract Background: Human xenograft tumor models established by transplantation of human tumor cell lines into immunodifficient mice have been routinely used for preclinical test of anticancer agents. But such tumor models have a relatively low transplantability and limited correlation with clinical findings. Recently, we have developed human non-small lung cancer (NSCLC) xenograft models by transplanting human fresh lung cancer tissues into nude mice, which have been employed for test of clinically used chemotherapeutical drugs and are under way for new drug chemosensitivity in vivo screening. Methods: The fresh lung cancer samples were collected from local hospitals. The tumor fragments of 1–2 mm3 were subcutaneously implanted in the flanks of the Balb/c nude mice by trocar needle. Sixteen tumor fragments were grafted into four mice from one patient tumor tissue (passage 1). In the first passage, tumors derived from male patients were implanted into male mice, and tumors from women were inoculated into female mice. All therapeutic efficacy experiments used female mice. The drugs tested were clinically used drugs for NSCLC including cisplatin (4 mg/kg, q3d x 4), paclitaxel (15 mg/kg, q3d x 4), vinorelbine (2.5 mg/kg, q4d x 4), gemcitabine (120 mg/kg, q4d x 4), and the epidermal growth factor receptor (EGFR) inhibitor erlotinib (50 mg/kg/daily x 14, po). Results: A total of 42 NSCLC samples were implanted, and 13 tumor models were established (tumor taking rate 31%) for the first passage. The tumor taking rates were higher in the second and third passages (80–100%). Cisplatin, paclitaxel, vinorelbine, and gemcitatbine produced tumor growth inhibition rates of 48–55% regardless of the EGFR mutation status. While erlotinib demonstrated a significant antitumor activity only in the tumors bearing EGFR, which were consistent with their clinical findings. The tumor xenografts' architecture, the cell and histopathological morphology from the three generations mirrored the original patient cancers. Conclusions: These results suggest that human primary tumor xenograft models provide a unique renewable source of tumor material for test of novel anticancer agents. They may predict more relevant clinical response rates and higher correlation with clinical findings than use of xenograft models established from in vitro long-term cultured cancer cell lines, especially for test of target-oriented therapeutics in new drugs development programs. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B24