A549 Cell Xenograft Research Articles

TNF‑α inhibits xenograft tumor formation by A549 lung cancer cells in nude mice via the HIF‑1α/VASP signaling pathway.

Lung cancer is the leading cause of cancer‑associated mortality worldwide. Tumor necrosis factor α (TNF‑α) is an important cytokine in the tumor microenvironment that serves a function in the balance of cell survival and cell death pathways. Our previous studies indicated that hypoxia‑inducible factor 1α (HIF‑1α) acts downstream of TNF‑α in MCF‑7 luminal breast cancer cells. However, whether vasodilator‑stimulated phosphoprotein (VASP) is implicated in the direct regulation of HIF‑1α in response to TNF‑α in lung cancer remains unknown. In vitro studies were performed using A549 and H226 lung carcinoma cells and in vivo studies of tumor xenograft models were performed to investigate the effects of TNF‑α. The results demonstrated that TNF‑α decreased VASP expression by upregulating the expression of HIF‑1α to inhibit A549 cell proliferation and adhesion. Inhibition of transplanted tumor growth was associated with downregulation of VASP expression in nude mice. Bioinformatics analysis indicated that expression levels of VASP or HIF‑1α lead to differential outcomes of overall survival in lung carcinoma. These results suggest that the HIF‑1α/VASP signaling pathway serves an important function in the regulation of TNF‑α‑induced suppression of A549 cell proliferation and xenograft growth. This may improve our understanding of the antitumor effect of TNF‑α.

Free Adriamycin-Loaded pH/Reduction Dual-Responsive Hyaluronic Acid-Adriamycin Prodrug Micelles for Efficient Cancer Therapy.

Currently, tumor-targeted nanocarriers self-assembled from amphiphilic polymer-drug conjugates are of great demand. The appeal of these carriers arises mainly through their excellent loading efficiency of homologous drug molecules with microenvironment-triggered drug release. Herein, doxorubicin (DOX) was constructed to a hyaluronic acid (HA) backbone through hydrazone and disulfide linkages to construct pH and reduction coresponsive prodrug conjugates (HA-ss-DOX). During formulation, the amphipathic HA-ss-DOX spontaneously assembled into distinct core/shell micelles in aqueous media and showed conspicuous physical DOX loading capabilities (29.1%, DOX/HA-ss-DOX) based on homologous compatibility. DOX/HA-ss-DOX micelles were shown to be stable in normal physiological environments, while accomplishing selective, rapid DOX release at acidic pH and/or highly reducing conditions. The efficacy of DOX/HA-ss-DOX micelles was tested on A549 human lung cancer cells, wherein flow cytometry and confocal microscopy analysis revealed their HA receptor-mediated endocytosis mechanism. In comparison, DOX-loaded redox-insensitive micelles (DOX/HA-DOX) still demonstrated pH-dependent drug release. However, a more rapid intracellular DOX release profile was achieved in DOX/HA-ss-DOX micelles because of their sensitivity to both acidic and reducing environments. Resultantly, DOX/HA-ss-DOX exhibited the strongest cytotoxicity and apoptosis-inducing ability among all tested groups when tested on an A549 cell line and xenograft model.

Iodine‑125 interstitial brachytherapy reduces tumor growth via Warburg effect inhibition in non‑small cell lung cancer A549 xenografts

Iodine-125 interstitial brachytherapy (125I-IBT) is an alternative and effective treatment option for unresectable non-small cell lung cancer (NSCLC), and the Warburg effect is a determinant of tumor growth. The present study aimed to explore the influence of 125I-IBT on tumor growth and the Warburg effect, and the potential mechanisms underlying NSCLC progression. Mice with A549 cell xenografts were evenly divided into a control group without 125I-IBT, and three treatment groups receiving 125I-IBT with 20, 40 and 60 Gy. Tumor volume (TV), maximum standardized uptake value (SUVmax) determined by 18F-fluorodeoxyglucose (18F-FDG) micro-positron emission tomography/computed tomography and mean optical density (MOD) of mammalian target of rapamycin (mTOR), c-Myc, hypoxia inducible factor-1α (HIF-1α) and glucose transporter 1 (GLUT1) staining were compared among groups. Tumor inhibition rate (TIR), 18F-FDG uptake attenuation rate (FUAR) and expression suppression rate (ESR) were also calculated on day 14 and 28. The results demonstrated that the mean TV in the 60 and 40 Gy groups was smaller compared with the control TVs since days 14 and 16, respectively. The mean SUVmax value of the 60 Gy group at day 14, and all treatment group SUVmax values at day 28 were lower compared with the controls. In addition, the MOD of mTOR and GLUT1 was lower in the 60 Gy group, compared with the other groups, and c-Myc and HIF-1α values were lower in the 40 and 60 Gy groups, compared with the control and 20 Gy group (P<0.05). SUVmax positively correlated to TV (day 14, r=0.711; day 28, r=0.586) and the MOD of c-Myc and GLUT1 (r=0.621 and 0.546, respectively; P<0.01). Furthermore, dose dependent increases were observed for TIR, FUAR and ESR. In conclusion, 125I-IBT reduced tumor growth by inhibiting the Warburg effect, which may have resulted from downregulation of mTOR, c-Myc, HIF-1α and GLUT1 expression, particularly c-Myc and GLUT1, in NSCLC A549 ×enografts.

MicroRNA‐31‐5p enhances the Warburg effect via targeting FIH

The enhanced expression of miR-31 has been observed in many human malignancies including lung cancer, and this microRNA regulates several aspects of oncogenesis. However, the role of miR-31-5p in energy metabolism remains elusive. Here, we confirm that H1299 and A549 cells, 2 lung cancer cell lines, relay on aerobic glycolysis as main source of ATP. Inhibition of miR-31-5p leads to decreased glycolysis and ATP production, while miR-31-5p overexpression increases them. Hypoxia inducible factor 1 (HIF-1) up-regulates the expression of glycolytic enzymes, and the HIF-1α inhibitor (FIH) inhibits HIF-1 activity. Because FIH is a direct target of miR-31-5p, inhibition of miR-31-5p results in enhanced FIH expression and suppression of HIF-1 signaling, while overexpression of miR-31-5p has the opposite effects. Via this mechanism, miR-31-5p up-regulates aerobic glycolytic genes and maintains energy homeostasis. To further validate the mechanism of miR-31-5p in glycolysis regulation, we show that overexpression or knockdown of FIH rescued the effects of miR-31-5p or miR-31-5p inhibitor on HIF activation and its target gene expression, respectively. Finally, by means of an A549 cell xenograft mouse model, we demonstrate that the miR-31-5p promotes cell proliferation via enhancing glycolysis. In summary, this study reveals that miR-31-5p promotes the Warburg effect via direct targeting of FIH.-Zhu, B., Cao, X., Zhang, W., Pan, G., Yi, Q., Zhong, W., Yan, D. MicroRNA-31-5p enhances the Warburg effect via targeting FIH.

Wogonoside induces apoptosis in human non-small cell lung cancer A549 cells by promoting mitochondria dysfunction

Non-small cell lung cancer (NSCLC) is one of the most prevailing malignancies worldwide. It has been previously shown that wogonoside exerts anti-tumor activities in various kinds of human cancers. But its role in NSCLC remains elusive. In the present study, we determined the anti-tumor effect of wogonoside in human NSCLC A549 cells. We found that wogonoside effectively inhibits A549 cell viability through inducing cell cycle arrest and apoptosis. Moreover, administration of wogonoside by intraperitoneal injection inhibits the growth of A549 cell xenografts in athymic nude mice. Additionally, mitochondrial membrane potential was disrupted and cytochrome c was released to cytosol in the wogonoside-treated A549 cells. Finally, we found that AMPK/mTOR signaling might be implicated in the anti-NSCLC efficacy of wogonoside. Therefore, we may assume that wogonoside may be considered as a potential therapeutic agent for the treatment of NSCLC.

Effect of taurine on cell proliferation and apoptosis human lung cancer A549 cells.

To investigate the effects of taurine on cell proliferation and apoptosis, the human lung cancer A549 cell line and xenograft tumors in nude mice were used. The effects of taurine on cell proliferation and apoptosis were observed at time points of 24, 48 and 72 h after treatment using an MTT assay to detect the survival rate, and flow cytometry to detect the apoptotic rate. Western blot analysis was performed to examine the levels of p53 upregulated modulator of apoptosis (PUMA), BCL2, apoptosis regulator (Bcl-2) and BCL2-associated X, apoptosis regulator (Bax) in A549 cells. The level of PUMA, Bax and Bcl-2 proteins in the mouse xenograft tumors treated with taurine and/or exogenous PUMA were assessed by immunohistochemistry, with taurine suppressing the proliferation of the human lung cancer A549 cell line in a concentration-dependent manner, and it significantly enhanced the apoptosis rate at all concentrations. Taurine induced the significant upregulation of PUMA and Bax, but led to downregulation of Bcl-2. In comparison to the control group, taurine treatment markedly reduced the volume and weight of A549-derived xenograft tumors in nude mice. Expression of PUMA and Bax were upregulated in the xenograft tumors following taurine treatment, whereas Bcl-2 was downregulated. In addition, the inhibitory effect of taurine and exogenous PUMA on tumor growth was significantly higher than that of a single treatment of taurine or exogenous PUMA. It can therefore be concluded that taurine can inhibit cell proliferation of the human lung cancer A549 cell line and the growth of the xenograft tumors, whereas PUMA serves an important role in taurine-induced growth suppression.

Volatile oil from alpinia officinarum promotes lung cancer regression in vitro and in vivo.

The anti-lung cancer activity of volatile oil from Alpinia officinarum (VOAO) and the underlying mechanism has not been studied. Herein, VOAO was extracted by steam distillation and its components were identified by GC-MS analysis. Cells viability was measured by an MTT assay and the cell survival capacity was tested via a colony formation assay. Apoptosis cells were detected using the Annexin V-FITC/PI method. Hoechst 33342 cell unclear staining was employed to evaluate the nuclear morphology change. The mitochondrial membrane potential was detected by a JC-1 staining assay. Bcl-2, Mcl-1 and cleaved caspase-3 proteins were quantified by immune blotting. Furthermore, VOAO anti-cancer activity was evaluated on an A549 cell xenograft nude mice model. Our results have revealed that VOAO could inhibit the cell viability of lung carcinoma cells and the colony formation capacity. VOAO downregulates Bcl-2 and Mcl-1 and triggers dysfunction of the mitochondrial membrane potential. VOAO further activates caspase-3 cleavage and induces lung cancer cells apoptosis. In addition, VOAO administration significantly suppresses lung cancer growth in xenograft mice without obvious hepatotoxicity. We conclude that VOAO could be an effective, low cytotoxicity natural component for treatment of lung carcinoma.

A triazole-conjugated benzoxazone induces reactive oxygen species and promotes autophagic apoptosis in human lung cancer cells.

Numerous approaches suggested that compounds with conjugated triazole moieties or benzoxazone pharmacores are effective to antagonize proliferation of human tumors. The current study reported that a synthetic triazole-conjugated benzoxazone, 4-((5-benzyl-1H-1,2,3-triazol-3-yl)-methyl)-7-methoxy-2H-benzo[b][1,4]-oxazin-3(4H)-one (BTO), inhibited growth rates of human non-small cell lung cancer cells. The cytotoxicity can be enhanced with increasing drug concentrations. More evidence supported that the induced reactive oxygen species lead to ultimate apoptotic cell death by recruiting autophagy. The mechanistic pathway as elucidated involved tumor suppressor p53 activation and LC3-1 conversion followed by PARP and procaspase-3 cleavage. Autophagy inhibition reverted apoptotic death and restored cell viabilities. BTO suppressed the development of A549 cell xenograft tumors by activating autophagy and apoptosis simultaneously. As an efficient tumor growth inhibitor with relatively small molecular weight, BTO is a viable addition to the existing list of lung cancer treatment.

CCDC106 promotes non-small cell lung cancer cell proliferation.

Coiled-coil domain containing (CCDC) family members enhance tumor cell proliferation, and high CCDC protein levels correlate with unfavorable prognoses. Limited research demonstrated that CCDC106 may promote the degradation of p53/TP53 protein and inhibit its transactivity. The present study demonstrated that CCDC106 expression correlates with advanced TNM stage (P = 0.008), positive regional lymph node metastasis (P < 0.001), and poor overall survival (P < 0.001) in 183 non-small cell lung cancer cases. A549 and H1299 cells were selected as representative of CCDC106-low and CCDC106-high expressing cell lines, respectively. CCDC106 overexpression promoted A549 cell proliferation and xenograft tumor growth in nude mice, while siRNA-mediated CCDC106 knockdown inhibited H1299 cell proliferation. CCDC106 promoted AKT phosphorylation and upregulated the cell cycle-regulating proteins Cyclin A2 and Cyclin B1. Cell proliferation promoted by CCDC106 via Cyclin A2 and Cyclin B1 was rescued by treatment with the AKT inhibitor, LY294002. Our studies revealed that CCDC106 is associated with non-small cell lung cancer progression and unfavorable prognosis. CCDC106 enhanced Cyclin A2 and Cyclin B1 expression and promoted A549 and H1299 cell proliferation, which depended on AKT signaling. These results suggest that CCDC106 may be a novel target for lung cancer treatment.

Total flavonoid aglycones extract in Radix scutellariae inhibits lung carcinoma and lung metastasis by affecting cell cycle and DNA synthesis

Ethnopharmacological relevanceRadix Scutellariae (Scutellaria baicalensis Georgi, RS), a traditional herbal medicine commonly used to treat inflammation, hypertension, cardiovascular disease, bacterial and viral infections, is reported to treat lung cancer by supplements of modern medicine. The total flavonoid aglycones extract (TFAE) from RS is the most important composition for the pharmacodynamic effects. The present study was designed to evaluate the anti-lung tumor effect of TFAE on A549 cells and A549 cell nude mice xenografts. The aim of the study is to investigate the effect and mechanism of TFAE treating non-small cell lung cancer both in vitro and in vivo. Materials and methodsThe anti-tumor activity of TFAE in vitro was investigated using the MTT assay. The changes of cell invasion and migration were detected by Transwell assay and tube formation experiments were used to detect the anti-angiogenic effect. The anti-tumor effects of TFAE in vivo were evaluated in A549 cell nude mice xenografts. The mechanism of TFAE was detected by flow cytometry technology, western blot assay and immuno-histochemistry assay. ResultsIn vitro, TFAE inhibited the proliferation, invasion and migration of A549 cells in a dose- and time-dependent manner. In vivo, TFAE by oral administration at 100mg/kg for 30 days decreased the tumor volume and tumor weight in A549 cell xenograft by 25.5% with no statistical significance (P<0.05) compared to the cis-platinum positive control group (30.0%). The cell cycle and DNA synthesis experiment illustrated that TFAE could induce A549 cell cycle to arreste in S phase and DNA synthesis in A549 cells be inhibited, while TFAE had no influence on apoptosis of A549 cells. Western Blot assay demonstrated that the treatment of TFAE could make Cyclin D1 decrease and p53 increase both in vitro and in vivo. ConclusionTFAE displayed the inhibition effects of non-small cell lung cancer both in vitro and in vivo and the underlying mechanism might be related to the increased p53 protein expression and decreased Cyclin D1 expression, leading to cell cycle arrested in S phase and the decrease of DNA synthesis.

The targeted inhibitory effects of human amniotic fluid stem cells carrying CXCR4 promoter and DAL-1 on non-small cell lung carcinoma growth.

The differentially expressed in adenocarcinoma of the lung-1 (DAL-1) protein has been demonstrated to be suppressive to various types of tumors including lung cancer. This study aimed to determine the targeted effects of human amniotic fluid stem cells (hAFS cells) carrying CXCR4 promoter driven conditionally replicable adenovirus vector overexpressing DAL-1 (Ad-CXCR4-DAL-1) on non-small cell lung carcinoma (NSCLC) growth. The apoptotic effects of virus vectors were assessed using flow cytometry, and the cytotoxicity analyzed by CCK-8 assay. In vivo imaging system was used to determine the homing capability of hAFS cells. A549 cell xenograft mouse model was created to assess the in vivo effect of DAL-1 overexpression on NSCLC growth. We found that infection of Ad-CXCR4-DAL-1 increased the apoptosis of A549 NSCLC cells but not 16HBE normal human bronchial epithelial cells. Ad-CXCR4-DAL-1 administered via intratumoral injection led to significant reduced growth and greater necrosis of A549 xenograft tumors comparing to null vector treated animals. When infused via tail vein, hAFS cells carrying Ad-CXCR4-DAL-1 homed to lung cancer xenografts, caused virus replication and DAL-1 overexpression, and led to significant lower growth and greater necrosis of A549 cell xenografts comparing to non-treatment control. In conclusion, hAFS cells are capable of carrying Ad-CXCR4-DAL-1 vectors, specifically targeting to lung cancer, and causing oncolytic effects when administered in vivo.

Activation of endoplasmic reticulum stress is involved in the activity of icariin against human lung adenocarcinoma cells.

In this study, we investigated the anticancer activity of icariin (ICA) against human lung adenocarcinoma cells in vitro and in vivo and explored the role of endoplasmic reticulum (ER) stress (ERS) signaling in this process. ICA treatment resulted in a dose- and time-dependent decrease in the viability of human lung adenocarcinoma A549 cells. Additionally, ICA exhibited potent anticancer activity, as evidenced by reductions in A549 cell adhesion, migration and intracellular glutathione (GSH) levels and increases in the apoptotic index, Caspase 3 activity, and reactive oxygen species. Furthermore, ICA treatment increased the expression of ERS-related molecules (p-PERK, ATF6, GRP78, p-eIF2α, and CHOP), up-regulated the apoptosis-related protein PUMA and down-regulated the anti-apoptosis-related protein Bcl2. The down-regulation of ERS signaling using PERK siRNA desensitized lung adenocarcinoma cells to ICA treatment, whereas the up-regulation of ERS signaling using thapsigargin (THA) sensitized lung adenocarcinoma cells to ICA treatment. Additionally, ICA inhibited the growth of human lung adenocarcinoma A549 cell xenografts by increasing the expression of ERS-related molecules (p-PERK and CHOP), up-regulating PUMA, and down-regulating Bcl2. These data indicate that ICA is a potential inhibitor of lung adenocarcinoma cell growth by targeting ERS signaling and suggest that the activation of ERS signaling may represent a novel therapeutic intervention for lung adenocarcinoma.

Downregulation of peroxiredoxin-1 by β-elemene enhances the radiosensitivity of lung adenocarcinoma xenografts.

β-elemene, the active component of elemene (1-methyl-1-vinyl-2,4-diisopropenyl-cyclohexane), is a naturally occurring compound isolated from the traditional Chinese medicinal herb Curcuma wenyujin. Studies have confirmed that β-elemene enhances the radiosensitivity of lung cancer cell lines such as A549, by multiple pathways; however, their underlying mechanisms and pathways are yet to be elucidated. In the present study, two-dimensional differential in-gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry were used to profile the different proteins in A549 cell xenograft models of both treatment groups. The protein/mRNA expression was assessed by reverse transcription-polymerase chain reaction and western blotting techniques in tumor samples from all treatment groups. As a critical player in redox regulation of cancer cells, inhibition of peroxiredoxin-1 (Prx-1) may be an effective option for enhancing the tumor response to radiation. We further verified Prx-1 expression at the transcription and translation levels. β-elemene at a dose of 45 mg/kg had little effect on the Prx-1 protein expression, which was correlated with a moderate antitumor effect. However, a 45 mg/kg dose of β-elemene significantly inhibited the Prx-1 mRNA expression, thereby suggesting a possible influence on the transcriptional process, and radiation significantly increased the Prx-1 mRNA/protein expression compared to the control group (p<0.01). Notably, Prx-1 mRNA/protein expression was significantly lower in the β-elemene/radiation co-treatment group compared to the baseline levels in the control group (p<0.01). These results suggest that radiation-induced Prx-1 expression is directly or indirectly suppressed by β-elemene, thus suggesting a new pathway by which to reverse radioresistance.

Effects of artesunate on the invasion of lung adenocarcinoma A549 cells and expression of ICAM-1 and MMP-9

背景与目的近年来研究发现抗疟药青蒿琥酯对肺癌细胞具有抑制效应，但其作用机制尚未完全阐明。本研究旨在探讨青蒿琥酯对肺癌A549细胞侵袭能力的影响。方法MTT实验观察青蒿琥酯对体外培养的A549细胞增殖抑制作用；Transwell小室侵袭实验检测青蒿琥酯对体外A549细胞侵袭转移能力影响；构建A549细胞裸鼠皮下移植瘤模型，观察青蒿琥酯对A549细胞移植瘤的抑制作用，并采用Western blot检测A549细胞移植瘤ICAM-1和MMP-9蛋白表达的变化。结果MTT实验表明1.25 μg/L-5 μg/L的低浓度青蒿琥酯对A549细胞增殖无明显抑制作用。Transwell小室体外侵袭实验表明，与对照组（96.33±6.41）比较，1.25 μg/L青蒿琥酯即可抑制A549细胞侵袭转移（75.43±4.37, P < 0.05）。移植瘤实验表明10 mg/kg青蒿琥酯不能抑制裸鼠A549细胞移植瘤的生长（P > 0.05），但是可降低ICAM-1和MMP-9蛋白表达量（P < 0.05）。结论青蒿琥酯可抑制A549细胞侵袭潜能，其机制可能与降低ICAM-1和MMP-9表达有关。

Luciferase bioluminescence imaging monitoring gene therapeutic effect of apoptosis-inducing ligand for lung cancer A549 cells nude mice transplantation tumor in vivo

Objective To detect the expression and effect of human tumor necrosis factor related apoptosis-inducing ligand (hTRAIL) in vivo,by using a novel double expressing adenoviral vector encoding hTRAIL and firefly luciferase ( luc ) gene ( ad -luc-hTRAIL),in which luc was used as reporter gene.Methods Lung cancer A549 cell xenografts in 16 nude mice models were established in subcutaneous inoculation way,the adenovirus vectors ( ad-luc-hTRAIL,ad-hTRAIL,ad-luc) and phosphate buffer saline (PBS) (n =4) as control were injected into tumor respectively.The size of the tumor was measured at different time points (4,7,10,14,21,28 d)after injection.The activity of luciferase in surface of the tumor was detected in vivo by using high-sensitivity cooled-charged coupled device(CCD) camera.The expression of hTRAIL was demonstrated by immunohistochemistry staining after sacrificing the animals at different time points,and immunohistochemical scores (IHS) were measured. The apoptosis rate of tumor cells was detected by using TUNEL and calculated. Analysis of variance,the paired t test and linear correlation analysis was used for the statistics. Results The growing speed of tumour xenografts was more slowly in ad-luc-hTRAIL and ad-hTRAIL groups than PBS group (t =2.71,2.72,P ＜ 0.05 ).The tumor volumes of ad-luc-hTRAIL,ad-hTRAIL,ad-luc and PBS groups 28 days after injection were (208.4 ± 42.3 ),( 181.5 ±23.9),( 403.1 ± 54.0 ) and ( 427.0 ± 59.3 ) mm3, respectively. There was no significant difference between ad-luc group and PBS group(t =2.07,P ＞ 0.05).The expression of luciferase in ad-luc-hTRAILgroup reached its peak at 7th day ( 1.37 ± 1.04),and then decreased quickly.The IHS and apoptosis rate in ad-luc-hTRAIL and ad-hTRAIL groups reached their peaks at 7th day,the peak values of IHS were 6.25 ±2.06 and 6.5 ± 2.89,the peak values of apoptosis rate were (60.75 ± 8.06 ) ％ and ( 61.50 ± 8.47 ) ％,respectively.The amount of luciferase expression ( absolute number of photons detected by CCD camera)was linear positive correlated with IHS and apoptosis rate ( rphotons/IHs =0.942,rph /rate =0.842,rIHs/rate =0.887,P ＜ 0.05 ).Conclusion The target gene hTRAIL can be transfected into lung cancer A549 cell xenografts nude mice models efficiently with a high level expression,and the therapeutic effect of hTRAIL can be monitored by detecting the expression of luc. Key words: Luminescent proteins; Tumor necrosis factors; Fluorescein; Genes

Human positive coactivator 4 is a potential novel therapeutic target in non-small cell lung cancer

Transcriptional positive coactivator 4 (PC4) is a multifunctional nuclear protein that has important roles in DNA transcription, replication, repair and heterochromatinization. However, the role of PC4 in cancer remains to be clarified. Several studies propose that PC4 may act as a putative tumor suppressor. Here, we demonstrate for the first time that PC4 may represent a potential therapeutic target in non-small cell lung cancer (NSCLC). PC4 protein expression is significantly upregulated in NSCLC carcinoma tissues compared with their adjacent noncancerous counterparts as shown by immunohistochemical staining and western blotting in 104 pairs of formalin-fixed human NSCLC specimens and 6 fresh NSCLC samples. Knockdown of PC4 expression by sequence-specific small interfering RNA (siRNA) in human NSCLC cells (A549, H460 and H358) significantly inhibits the growth of cancer cells by the induction of cell cycle arrest and the increase of cell apoptosis in vitro. Interrupting the PC4 signaling pathway by injection of the PC4 siRNA liposome complex produced an effective regression of pre-established A549 cell xenografts in mice through growth inhibition and increased apoptosis. These results indicated that PC4 could be an attractive new therapeutic target for the treatment of NSCLC.

Controlled-release systemic delivery - a new concept in cancer chemoprevention

Many chemopreventive agents have encountered bioavailability issues in pre-clinical/clinical studies despite high oral doses. We report here a new concept utilizing polycaprolactone implants embedded with test compounds to obtain controlled systemic delivery, circumventing oral bioavailability issues and reducing the total administered dose. Compounds were released from the implants in vitro dose dependently and for long durations (months), which correlated with in vivo release. Polymeric implants of curcumin significantly inhibited tissue DNA adducts following the treatment of rats with benzo[a]pyrene, with the total administered dose being substantially lower than typical oral doses. A comparison of bioavailability of curcumin given by implants showed significantly higher levels of curcumin in the plasma, liver and brain 30 days after treatment compared with the dietary route. Withaferin A implants resulted in a nearly 60% inhibition of lung cancer A549 cell xenografts, but no inhibition occurred when the same total dose was administered intraperitoneally. More than 15 phytochemicals have been tested successfully by this formulation. Together, our data indicate that this novel implant-delivery system circumvents oral bioavailability issues, provides continuous delivery for long durations and lowers the total administered dose, eliciting both chemopreventive/chemotherapeutic activities. This would also allow the assessment of activity of minor constituents and synthetic metabolites, which otherwise remain uninvestigated in vivo.

Abstract 5030: Necitumumab (IMC-11F8), a recombinant human anti EGFR antibody, increases the antitumor effects of cisplatin/paclitaxel in human NSCLC xenograft models

Abstract Epidermal growth factor receptor (EGFR) targeted antibodies may have a role in the treatment of non-small cell lung cancer (NSCLC), based on preclinical and clinical data. Here we have demonstrated a significant benefit of adding necitumumab, a recombinant human IgG1 antibody targeting EGFR, to cisplatin+paclitaxel administered at its maximum tolerated dose, in subcutaneous xenograft tumor models established in nu/nu athymic mice with A549 or NCI-H1650 NSCLC cell lines. Necitumumab monotherapy or cisplatin+paclitaxel therapy inhibited xenograft tumor growth with a T/C% of 47 and 63, respectively in A549 tumor model. A similar anti tumor effect was seen in NCI-H1650 xenograft tumors treated with necitumumab monotherapy or cisplatin+paclitaxel therapy with a T/C% of 37 and 50, respectively. Furthermore, the combination of necitumumab with cisplatin+paclitaxel significantly inhibited tumor growth with a magnitude greater than any of the therapies alone in A549 xenograft tumors with a T/C% of 33 and in NCI-H1650 xenograft tumors with a T/C% of 18. Combination treatment resulted in partial regressions (3 out of 9 mice) in A549 and maximum tumor regressions (10 out of 12) in NCI-H1650. Chi-Squared test for all animals in the treatment groups showed p=0.02 in A549 and p=3.0E-08 in NCI-H1650 xenograft tumors. In vitro analysis on A549 and NCI-H1650 cell lines treated with cisplatin+paclitaxel, with/without necitumumab (5 μg/ml) demonstrated that this combination benefit was associated with the increased apoptotic index in both models. Cell cycle arrest during combination therapy was also observed as an increase in the percentage of cells in G2/M phase in A549 and S or G2/M phase in NCI-H1650 cell lines. We are further utilizing SABiosciences PCR arrays to demonstrate combination benefit related gene function in apoptosis, cell cycle signaling pathways and drug specific biomarkers in both A549 and NCI-H1650 cell lines. Thus our results demonstrate the potential utility of necitumumab for enhancing the efficacy of cytotoxic chemotherapy in both in A549 and NCI-H1650 cell lines and xenograft tumors models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5030. doi:10.1158/1538-7445.AM2011-5030

Abstract C196: Co-treatment with NVP-BEZ235 and heat shock protein (hsp) 90 or pan-deacetylase (DAC) inhibitor is synergistically active against non-small cell lung cancer (NSCLC) cells with mutant RAS and LKB1

Abstract Inactivating somatic mutation of LKB1 is found in approximately 30% of NSCLC and is often coincident with activating K-RAS mutation. In a mutant K-RAS-driven model of mouse lung cancer, strong cooperation and aggressive phenotype was seen with hemizygous inactivation of LKB1. Loss of LKB1 results in activation of mTOR pathway while somatic activating mutation in K-RAS results in activation of PI3K/AKT and RAS/RAF/ERK. Therefore therapeutic stratagies which target these pathways may have significant affect on the outcome of these cancers. NVP-BEZ235 is a dual PI3K and mTOR inhibitor and panobinostat (PS) (Novartis Pharmaceuticals Inc) is a pan-histone deacetylase (HDAC) inhibitor, which has been shown to induce proteasomal degradation and depletion of the levels of hsp90 client proteins, including AKT and c-RAF. Here, we determined the effects of BEZ235 and/or PS or AUY922 (an hsp90 inhibitor) on NSCLC A549 and H460 cells with LKB1 and K-RAS mutations (A549 and H460). Treatment with BEZ235 (200 to 1000 nM), PS (25 to 50 nM) or AUY922 (25 to 100 nM) dose-dependently increased % of cells in G2/M and decreased % of cells in S phase of cell cycle, as well as induced apoptosis of the NSCLC cells. Co-treatment with AUY922 or PS increased BEZ235 mediated depletion of p-AKT, p-4EBP1 and p-p70S6K levels, as well as synergistically induced apoptosis of A549 and H460 cells (combination indices &amp;lt; 1.0). Combined therapy with BEZ235 (25 mg/Kg) and PS (10 mg/Kg) or AUY922 (20 mg/Kg) resulted in significantly more tumor growth delay than treatment with each of the agents alone of A549 cell xenografts in nude mice (p &amp;lt; 05). We also determined the activity of BEZ235 and/or PS in the NSCLC H368 cells with mutant K-RAS and either wild-type (H358 cells) or stable knock down of LKB1 expression (H358/LKB1-KD cells), achieved by shRNA against LKB1, mimicking the presence of mutant LKB1. As compared to each agent alone, co-treatment with BEZ235 and PS exhibited a higher level of antitumor synergy against H358/LKB1-KD versus H358 cells. These pre-clinical in vitro and in vivo studies demonstrate that combined treatment with the dual PI3K/mTOR inhibitor BEZ235 and PS or AUY922 could potentially be a promising targeted therapy for NSCLC that harbors K-ras and LKB1 mutations. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C196.

Differential Radiation Effect in Tumor and Normal Tissue after Treatment with Ramipril, an Angiotensin-Converting Enzyme Inhibitor

The angiotensin-converting enzyme inhibitor, ramipril, has been shown to mitigate radiation injury in normal tissues. Using A549 cell xenografts grown in athymic mice, we measured the effect of ramipril on radiation damage to tumors. Ramipril did not alter tumor response to radiation despite different times of drug administration with respect to radiation delivery (drug started 2 weeks before or immediately after irradiation). In contrast, using the same dose, ramipril reduced normal tissue radiation injury (30 Gy x 2 or 6 Gy x 10) as assessed by a semi-quantitative scale of skin damage and relative leg contraction. The results indicate that ramipril could offer therapeutic gain due to its different effect on normal tissues and tumors.