Treatment Of Human Lung Cancer Research Articles

Thoc1 inhibits cell growth via induction of cell cycle arrest and apoptosis in lung cancer cells

THO complex 1 (Thoc1) is a human nuclear matrix protein that binds to the retinoblastoma tumor suppressor retinoblastoma protein (pRb). While some studies suggest that Thoc1 has characteristics of a tumor suppressor protein, whether Thoc1 can inhibit lung cancer cell growth is not clear. In the present study, we observed that Thoc1 is lowly expressed in the lung cancer cell lines SPC-A1 and NCI-H1975. Then, we investigated the potential effects of Thoc1 on lung cancer cell proliferation, cell cycle and apoptosis after stable transfection of these lines with a Thoc1 expression vector. We found that overexpression of Thoc1 can inhibit cell proliferation, induce G2/M cell cycle arrest and promote apoptosis. Further investigation indicated that overexpression of Thoc1 is involved in the inhibition of cell cycle-related proteins cyclin A1 and B1 and of pro-apoptotic factors Bax and caspase-3. In vivo experiments showed that tumors overexpressing Thoc1 display a slower growth rate than the control xenografts and show reduced expression of the protein Ki-67, which localized on the nuclear membrane. Taken together, our data show that in lung cancer cells, Thoc1 inhibits cell growth through induction of cell cycle arrest and apoptosis. These results indicate that Thoc1 may be used as a novel therapeutic target for human lung cancer treatment.

MiR-132 Suppresses the Migration and Invasion of Lung Cancer Cells via Targeting the EMT Regulator ZEB2

MicroRNAs (miRNAs) are small, non-coding RNAs which can function as oncogenes or tumor suppressor genes in human cancers. Emerging evidence reveals that deregulation of miRNAs contributes to the human non-small cell lung cancer (NSCLC). In the present study, we demonstrated that the expression levels of miR-132 were dramatically decreased in examined NSCLC cell lines and clinical NSCLC tissue samples. Then, we found that introduction of miR-132 significantly suppressed the migration and invasion of lung cancer cells in vitro, suggesting that miR-132 may be a novel tumor suppressor. Further studies indicated that the EMT-related transcription factor ZEB2 was one direct target genes of miR-132, evidenced by the direct binding of miR-132 with the 3′ untranslated region (3′ UTR) of ZEB2. Further, miR-132 could decrease the expression of ZEB2 at the levels of mRNA and protein. Notably, the EMT marker E-cadherin or vimentin, a downstream of ZEB2, was also down-regulated or up-regulated upon miR-132 treatment. Additionally, over-expressing or silencing ZEB2 was able to elevate or inhibit the migration and invasion of lung cancer cells, parallel to the effect of miR-132 on the lung cancer cells. Meanwhile, knockdown of ZEB2 reversed the enhanced migration and invasion mediated by anti-miR-132. These results indicate that miR-132 suppresses the migration and invasion of NSCLC cells through targeting ZEB2 involving the EMT process. Thus, our finding provides new insight into the mechanism of NSCLC progression. Therapeutically, miR-132 may serve as a potential target in the treatment of human lung cancer.

Flavonoids identified from Korean Citrus aurantium L. inhibit Non-Small Cell Lung Cancer growth in vivo and in vitro

Korean Citrus aurantium L. has long been used as a traditional medicine for the treatment of various cancers such as those of lung and prostat. C. aurantium L. has been reported to have anti-metastasis properties in in vitro experiments. However, the anti-metastasis effect of C. aurantium L. in vivo is not well understood. This study investigated the anti-metastatic effect of flavonoids isolated from Korean C. aurantium L. using high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) in NOD/SCID mice. PKH-26 staining and immunohistochemical analyses indicated that flavonoids prevented cancer cell infiltration and localization to the lung. Also, flavonoids induced cancer cell apoptosis through the regulation of the apoptosis related protein cleaved caspase-3 and p-p53. In vitro results also show that flavonoids inhibited A549 cells metastasis and induced apoptosis. Moreover, proteomic analysis indicated comparable down-regulation of the Ddx3x and ANP43B proteins with control group after treatment with flavonoids. These results provide scientific support for the use of flavonoids isolated from C. aurantium L. for the treatment of human lung cancer.

Combination Therapy of VEGF-Trap and Gemcitabine Results in Improved Anti-Tumor Efficacy in a Mouse Lung Cancer Model

BackgroundAngiogenesis is essential for the growth and metastasis of cancer. Although anti-angiogenic agents, particularly vascular endothelial growth factor (VEGF) inhibitors, have exhibited single-agent activity, there is considerable interest in combining these novel drugs with conventional chemotherapy reagents to achieve an optimal clinical efficacy. The objective of this study was to evaluate the benefits of the combination therapy of vascular endothelial growth factor trap (VEGF-Trap) with gemcitabine in a lung tumor model.MethodsA luciferase-expressing Lewis lung carcinoma (LLC) model was established in C57BL/6J mice and tumor-bearing mice were randomized into control, VEGF-Trap, gemcitabine and VEGF-Trap/gemcitabine combination groups. Tumor growth and animal survival were monitored. Tumor microvessel density and cell proliferation were evaluated by CD31 and Ki-67 immunohistochemical analysis. TUNEL assay was performed to detect apoptotic cells. The protein levels of Cyclin D1, Pro-Caspase-3, Bcl-2, MMP2 and MMP9 in tumor extracts were examined by western blot.ResultsVEGF-Trap in combination with gemcitabine showed significantly enhanced inhibition of tumor growth and prolonged mouse survival compared to the VEGF-Trap or gemcitabine monotherapy. The VEGF-Trap/gemcitabine combination therapy not only potently inhibited tumor angiogenesis and cell proliferation, but also increased cellular apoptosis within tumor tissues. In addition, the combination treatment markedly down-regulated the expression of proliferation, anti-apoptosis and invasion related proteins.ConclusionCombination therapy using VEGF-Trap and gemcitabine resulted in improved anti-tumor efficacy in a lung cancer model and VEGF-Trap/gemcitabine combination might represent a promising strategy in the treatment of human lung cancer.

CDK-associated Cullin 1 promotes cell proliferation with activation of ERK1/2 in human lung cancer A549 cells

Lung cancer is one of the most common causes of cancer-related death in the world, but the mechanisms remain unknown. In this study, we investigated the expression of CDK-associated Cullin 1 (CAC1) in lung cancer, the effect of CAC1 on the proliferation of human lung cancer A549 cells, and the activation of signaling pathways of mitogen-activated protein kinases (MAPKs). Results showed that CAC1 expression was higher levels in human lung carcinoma than normal lung tissue, and CAC1 siRNA reduced the proliferation of lung cancer A549 cells by decreasing cell activity and cell division in vitro. The proportion of cells treated with CAC1 siRNA increased in the G1 phase and decreased in the S and G2/M phase, indicative of G1 cell cycle arrest. Furthermore, the proportions of early/late apoptosis in lung cancer A549 cells were enhanced with CAC1 siRNA treatment. It was also found that activation of extracellular signal-regulated protein kinase (ERK) and p38 signaling pathways were involved in the proliferation of A549 cells. After CAC1 siRNA treatment, p-ERK1/2 levels decreased, and meanwhile p-p38 level increased, A549 cell proliferation increased when ERK1/2 signaling is activated by PMA. Our findings demonstrated that CAC1 promoted the proliferation of human lung cancer A549 cells with activation of ERK1/2 signaling pathways, suggesting a potential cure target for treatment of human lung cancer.

Effect of ARHI on lung cancer cell proliferation, apoptosis and invasion in vitro

The purposes of this study were to elucidate the effects of ARHI (aplysia ras homolog I) on several biological features of lung cancer cells, including growth, proliferation and invasion, to collect experimental evidence for the future biological treatment of human lung cancer. The eukaryotic expression vector, pcDNA3.1-ARHI, was constructed and transfected into the human lung cancer cell line SK-MES-1. The biological properties of the resulting ARHI-expressing lung cancer cell line were evaluated using methyl thiazolyl tetrazolium assay, flow cytometry, and a Transwell invasion assay. Additionally, the influence of ARHI on the gene expression levels of cyclin D1, p27(KIP1), death-associated protein kinase 1 (DAPK1), and matrix metalloproteinases1/2 (MMP-1/2) was determined. Compared to the non-transfected SK-MES-1 cells and the cells transfected with the empty pcDNA3.1 plasmid, the ARHI-transfected cells displayed significantly reduced growth rates and decreased viability (P < 0.05). The ARHI-transfected cells also displayed a significantly higher percentage of cells in G1 phase (P < 0.05) and a lower percentage of cells in S phase (P < 0.05); a higher percentage of apoptosis (P < 0.05); and finally, a notable reduction in the basement membrane-penetration rate in the Transwell invasion assay (P < 0.05). Furthermore, it was determined that ARHI is capable of inhibiting the expression of cyclin D1, MMP-1, and MMP-2; however, ARHI promotes the expression of both p27(KIP1) and DAPK1 in SK-MES-1 cells. In conclusion, overexpression of ARHI gene might be associated with the inhibition of lung cancer cell growth, proliferation and invasion, and the promotion of apoptosis.

PRR11 is a novel gene implicated in cell cycle progression and lung cancer

Identification and functional analysis of novel potential cancer-associated genes is of great importance for developing diagnostic, preventive and therapeutic strategies for cancer treatment and management. In the present study, we isolated and identified a novel gene, proline-rich protein 11 (PRR11), implicated in both cell cycle progression and lung cancer. Our results showed that PRR11 was periodically expressed in a cell cycle-dependent manner, and RNAi-mediated silencing of PRR11 caused significant S phase arrest as well as growth retardation in HeLa cells. Moreover, PRR11 was overexpressed at both mRNA and protein levels in lung cancer tissues as compared with normal lung tissues. Large scale in silico analysis of clinical microarray datasets also indicated that high expression of PRR11 was significantly associated with poor prognosis in lung cancer patients. RNAi-mediated silencing of PRR11 caused S phase arrest, suppressed cellular proliferation, colony formation ability in lung cancer cells and inhibited tumorigenic potential in nude mice. Knockdown of PRR11 also inhibited cell migration and invasion ability in lung cancer cells. Furthermore, microarray analysis revealed that PRR11 knockdown caused the dysregulation of multiple critical pathways and various important genes involved in cell cycle, tumorigenesis and metastasis (e.g. CCNA1, RRM1, MAP4K4 and EPB41L3). Taken together, our results strongly demonstrated that this newly identified gene, PRR11, had a critical role in both cell cycle progression and tumorigenesis, and might serve as a novel potential target in the diagnosis and/or treatment of human lung cancer.

Down-regulation of cellular FLICE-inhibitory protein (Long Form) contributes to apoptosis induced by Hsp90 inhibition in human lung cancer cells

BackgroundCellular FLICE-Inhibitory Protein (long form, c-FLIPL) is a critical negative regulator of death receptor-mediated apoptosis. Overexpression of c-FLIPL has been reported in many cancer cell lines and is associated with chemoresistance. In contrast, down-regulation of c-FLIP may drive cancer cells into cellular apoptosis. This study aims to demonstrate that inhibition of the heat shock protein 90 (Hsp90) either by inhibitors geldanamycin/17-N-Allylamino-17-demethoxygeldanamycin (GA/17-AAG) or siRNA technique in human lung cancer cells induces c-FLIPL degradation and cellular apoptosis through C-terminus of Hsp70-interacting protein (CHIP)-mediated mechanisms.MethodsCalu-1 and H157 cell lines (including H157-c-FLIPL overexpressing c-FLIPL and control cell H157-lacZ) were treated with 17-AAG and the cell lysates were prepared to detect the given proteins by Western Blot and the cell survival was assayed by SRB assay. CHIP and Hsp90 α/β proteins were knocked down by siRNA technique. CHIP and c-FLIPL plasmids were transfected into cells and immunoprecipitation experiments were performed to testify the interactions between c-FLIPL, CHIP and Hsp90.Resultsc-FLIPL down-regulation induced by 17-AAG can be reversed with the proteasome inhibitor MG132, which suggested that c-FLIPL degradation is mediated by a ubiquitin-proteasome system. Inhibition of Hsp90α/β reduced c-FLIPL level, whereas knocking down CHIP expression with siRNA technique inhibited c-FLIPL degradation. Furthermore, c-FLIPL and CHIP were co-precipitated in the IP complexes. In addition, overexpression of c-FLIPL can rescue cancer cells from apoptosis. When 17-AAG was combined with an anti-cancer agent celecoxib(CCB), c-FLIPL level declined further and there was a higher degree of caspase activation.ConclusionWe have elucidated c-FLIPL degradation contributes to apoptosis induced by Hsp90 inhibition, suggesting c-FLIP and Hsp90 may be the promising combined targets in human lung cancer treatment.

Mere15, a novel polypeptide from Meretrix meretrix, inhibits adhesion, migration and invasion of human lung cancer A549 cells via down-regulating MMPs

Context: Mere15 is a novel antitumor polypeptide purified from Meretrix meretrix Linn. (Veneridae). Previous studies have shown that the polypeptide induced cell death via intrinsic mitochondrial pathway.Objective: In the present study, the effects of Mere15 on cell adhesion, migration, invasion, as well as secretion and expression of matrix metalloproteinases (MMPs) were studied in human lung adenocarcinoma A549 cells.Materials and methods: The effect of Mere15 on cell adhesion, migration and invasion were studied by cell adhesion and transwell assay. The expression of MMPs was determined by gelatin zymography and RT-PCR analysis.Results: The ability of cell adhesion was decreased by 86.74% at the concentration of 12.0 μg/mL of Mere15. And the migration and invasion of A549 cells were decreased with the inhibition ratio of, 69.22 and 53.84% when treated with 15.0 μg/mL of Mere15. Further study also revealed that treatment of the cancer cells with Mere15 (15.0 μg/mL), the secretion of MMP-2 and MMP-9 were down-regulated with the inhibition ratio of 72.00 and 93.24% and the inhibition rate of mRNA expression of MMP-2 and MMP-9 was 57.54 and 91.22%, respectively.Discussion and conclusion: The study demonstrates that Mere15 inhibits tumor growth via both pro-apoptotic and antimetastasis pathways, and the polypeptide has potential to be developed as a multi-target therapeutic agent for the treatment of human lung cancer.

Inhibition of proliferation and induction of G1-phase cell-cycle arrest by dFMGEN, a novel genistein derivative, in lung carcinoma A549 cells

Genistein (GEN) is a molecule of great interest as a potent chemopreventive agent against atherosclerosis and cancer. However, the bioavailability of GEN is very low in vivo. Our previous study showed that a GEN derivative, 7-difluoromethyl-5,4′-dimethoxygenistein (dFMGEN) has a better bioavailability than GEN in vivo. In this study, we further evaluated the efficacy of dFMGEN as a candidate for cancer therapy. We demonstrated that dFMGEN treatment decreased the viability of A549 cells in a concentration- and time-dependent manner and induced cell-cycle arrest at the G1 phase. G1 phase arrest was correlated with a significant reduction of Cdk4 and cyclin D1 protein level. Further studies showed that cyclin-dependent kinase (Cdk)4 and cyclin D1 protein-level decrease was caused by Cdk inhibitors p15, p21, and p27 level increase, and decreased protein level directly suppressed Rb protein phosphorylation and E2F-1 expression, then cell-cycle progression was arrested. Finally, we also found that dFMGEN has a dosage effect in suppressing tumor growth in vivo, and that dFMGEN was well tolerated by animals. In summary, our results suggest that dFMGEN has therapeutic potential for the treatment of human lung cancer.

Knockdown of HMGN5 Expression by RNA Interference Induces Cell Cycle Arrest in Human Lung Cancer Cells

HMGN5 is a typical member of the HMGN (high mobility group nucleosome-binding protein) family which may function as a nucleosomal binding and transcriptional activating protein. Overexpression of HMGN5 has been observed in several human tumors but its role in tumorigenesis has not been fully clarified. To investigate its significance for human lung cancer progression, we successfully constructed a shRNA expression lentiviral vector in which sense and antisense sequences targeting the human HMGN5 were linked with a 9-nucleotide loop. Inhibitory effects of siRNA on endogenous HMGN5 gene expression and protein synthesis were demonstrated via real-time RT-PCR and western blotting. We found HMGN5 silencing to significantly inhibit A549 and H1299 cell proliferation assessed by MTT, BrdU incorporation and colony formation assays. Furthermore, flow cytometry analysis showed that specific knockdown of HMGN5 slowed down the cell cycle at the G0/G1 phase and decreased the populations of A549 and H1299 cells at the S and G2/M phases. Taken together, these results suggest that HMGN5 is directly involved in regulation cell proliferation in A549 and H1299 cells by influencing signaling pathways involved in cell cycle progression. Thus, our finding suggests that targeting HMGN5 may be an effective strategy for human lung cancer treatment.

Effects of SASH1 on lung cancer cell proliferation, apoptosis, and invasion in vitro

The purposes of this study were to investigate the effects of the SASH1 gene on the growth, proliferation, apoptosis, invasiveness, and metastatic potential of lung cancer cells and explore the potential use of SASH1 for the treatment of human lung cancer. The SASH1 gene was cloned into the pcDNA3.1 eukaryotic expression vector, and SASH1 shRNA were designed and constructed. The resulting constructs were transfected into A549 human lung cancer cells, and the changes in the relevant biological characteristics of the cells overexpressing SASH1 and cells with downregulated expression of SASH1 were analyzed using the MTT assay, transwell invasion assay, and flow cytometry. The effects of the SASH1 gene on the expression of cyclin D1, Bcl-2, and MMP-2/9 were also concurrently examined. In the A549 cells from the pcDNA3.1-SASH1 transfected group, cell viability, proliferation, and migration were significantly reduced compared to the control cells (p = 0.039, p = 0.013), and a cell cycle arrest in G1 was observed. The A549 cells transfected with the SASH1 shRNA demonstrated significantly higher cell viabilities, proliferation, and migration compared to the control cells (p = 0.012, p = 0.045). Additionally, the percentage of A549 cells undergoing apoptosis was significantly higher in the pcDNA3.1-SASH1 transfected cells and significantly lower in the SASH1 shRNA transfected cells compared to the control cells (p = 0.010, p = 0.000). The cyclin D1, Bcl-2, and MMP-9/2 protein expression levels were significantly lower in the pcDNA3.1-SASH1-transfected cells and were significantly higher in the SASH1 shRNA-transfected cells than that in the control cells. The SASH1 gene may inhibit A549 cell growth and proliferation as well as promote cellular apoptosis. The overexpression of the SASH1 gene may also be related to the decreased migration of A549 human lung cancer cells.

Effects of BTG2 on proliferation inhibition and anti-invasion in human lung cancer cells

The objective of the study was to investigate the impact of the B cell translocation gene 2 (BTG2) on lung cancer cell growth, proliferation, metastasis, and other biological characteristics and to provide experimental evidence for the biological treatment of human lung cancer. A pcDNA3.1-BTG2 eukaryotic expression vector was constructed and transfected into the human lung cancer cell line A549. The biological changes in the BTG2-expressing cells were analyzed using growth curves, the MTT (tetrazolium) assay, propidium iodide (PI) staining, and the Transwell invasion chamber. Additionally, Western blotting was used to determine the impact of BTG2 on the protein expression of cyclin D1, MMP-1, and MMP-2. Compared to the empty vector-transfected A549 cells or the mock-transfected A549 cells, the pcDNA3.1-BTG2-transfected A549 cells grew significantly slower. No significant differences were detected between the empty vector-transfected group and the mock-transfected A549 cells. The growth curve analysis and the PI staining showed that the pcDNA3.1-BTG2-transfected cells grew significantly slower than the empty vector-transfected A549 cells (P < 0.05). The cell invasion assay results suggested that the invasion rate of the pcDNA3.1-BTG2-transfected A549 cells was significantly slower than the invasion rate of the empty vector-transfected group and the mock-transfected group (P < 0.05). The overexpression of BTG2 may inhibit the protein expression of cyclin D1, MMP-1, and MMP-2 in A549 cells. The overexpression of BTG2 may inhibit the growth, proliferation, and invasiveness of the A549 human lung cancer cell line.

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

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

β-lapachone에 의한 A549 인체폐암세포의 apoptosis 유도와 cyclooxygenase-2 활성 저하

<TEX>${\beta}$</TEX>-lapachone은 남미에 자생하는 lapacho 나무(Tabeuia avellanedae)의 수액에 함유된 quinone계열의 일종으로 많은 인체암세포에서 apoptosis를 유발하는 것으로 알려져 있다. 본 연구는 A549 인체폐암세포를 대상으로 <TEX>${\beta}$</TEX>-lapachone에 의한 apoptosis 유발 과정에서 나타나는 또 다른 현상들을 조사하기 위하여 실시되었다. <TEX>${\beta}$</TEX>-lapachone이 처리된 A549 세포는 처리 농도의 증가에 따라 생존율이 감소되었으며, 이는 apoptosis 유발과 연관이 있음을 MTT assay와 flow cytometry 분석을 통하여 확인하였다. <TEX>${\beta}$</TEX>-lapachone에 의한 A549 세포의 증식억제는 종양억제유전자 p53과 cyclin-dependent kinase 저해제인 p21의 발현을 전사 및 번역 수준에서 증가시켰으며, p53 단백질의 인산화 증가와 연관성이 있었다. 또한 <TEX>${\beta}$</TEX>-lapachone은 caspase-3과 -9를 활성화시켰으며, 활성화된 caspase-3의 기질 단백질들[poly(ADP-ribose) polymerase, <TEX>${\beta}$</TEX>-catenin 및 phospholipase C-<TEX>$\gamma$</TEX>1]의 단편화를 유도하였다. 아울러 <TEX>${\beta}$</TEX>-lapachone은 cyclooxygenase (COX)-2의 mRNA 및 단백질의 발현을 억제하였으나 COX-1의 발현에는 영향을 미치지 않았으며, <TEX>${\beta}$</TEX>-lapachone에 의한 COX-2의 발현억제는 prostaglandin E2의 생성 저하에 관련이 있었다. 본 연구의 결과는 <TEX>${\beta}$</TEX>-lapachone의 항암활성 기전의 이해와 더불어 <TEX>${\beta}$</TEX>-lapachone이 폐암세포에서 강력한 항암활성이 있음을 보여 주는 것이다. <TEX>${\beta}$</TEX>-lapachone, a quinone of lapachol extracted from the bark of the lapacho tree, has been found to induce apoptosis in various human cancer cells. In the present study, we investigated further possible mechanisms by which <TEX>${\beta}$</TEX>-lapachone exerts its pro-apoptotic action in cultured human lung cancer A549 cells. <TEX>${\beta}$</TEX>-lapachone treatment resulted in inhibition of growth and induction of apoptosis in a concentration-dependent manner, as determined by MTT assay and flow cytometry analysis. The induction of apoptosis by <TEX>${\beta}$</TEX>-lapachone was associated with up-regulation of the expression of p53 and p21 in both transcriptional and translational levels, and the phosphorylation of p53. In addition, <TEX>${\beta}$</TEX>-lapachone activated caspase-3 and -9, and induced degradation of caspase-3 target proteins such as poly (ADP-ribose) polymerase (PARP) and <TEX>${\beta}$</TEX>-catenin. Furthermore, <TEX>${\beta}$</TEX>-lapachone treatment caused a progressive decrease in the expression levels of cyclooxygenase (COX)-2 without significant changes in the levels of COX-1, which was correlated with a decrease in prostaglandin E2 synthesis. Taken together, these results indicated that <TEX>${\beta}$</TEX>-lapachone may have therapeutic potential in human lung cancer treatment.

Abstract 1118: Role of the type I insulin-like growth factor receptor in lung tumorigenesis

Abstract Lung cancer is the leading cause of cancer related mortalities worldwide. The type I insulin-like growth factor receptor (IGF-IR) is frequently expressed at high levels in lung cancer. This receptor is involved in cell proliferation, apoptosis, migration and differentiation and is emerging as a potential target for molecular therapies. Furthermore, IGF-IR downregulation has been reported to sensitize cancer cells to agents that promote apoptosis. To investigate the importance of the IGF-IR in lung tumorigenesis, our lab generated doxycycline-inducible transgenic mice in which the IGF-IR was overexpressed in type II alveolar or Clara cells. IGF-IR overexpression was sufficient to induce lung tumor development in these mice indicating that this receptor has an important function in the initiation of lung tumorigenesis. To further investigate the role of IGF-IR in lung tumorigenesis, the levels and function of IGF-IR were evaluated in two human lung tumor cell lines (A549 and NCI-H358) and one murine lung tumor cell line (LA-4) as well as in human and murine normal bronchial epithelial cells. Western blotting revealed increased levels of IGF-IR in the lung cancer cell lines compared to the normal lung epithelial cells lines. The efficacy of the IGF-IR small-molecule inhibitor, BMS-754807, which is a drug currently in clinical trials, was also evaluated in the lung tumor cells. BMS-754807 inhibited A549, NCI-H358 and LA-4 cell survival in a dose-dependent manner with approximate IC50 concentrations of 1.70uM for A549, 1.25uM for NCI-H358 and 0.74uM for LA-4 cells. The effects of BMS-754807 on cell proliferation (Ki67 immunofluorescence), cell survival (JC-1 immunofluorescence) and migration (scratch wound and Boyden chamber assays) are currently being examined to determine which properties are affected by disruption of IGF-IR signaling. Similar studies will be performed following downregulation of IGF-IR expression using RNAi. To evaluate whether BMS-754807 could sensitize lung tumor cells to the cytotoxic effects of chemotherapeutic agents commonly employed in the treatment of human lung cancer, BMS-754807 was combined with cisplatin and with carboplatin. Neither the BMS-754807/cisplatin nor the BMS-754807/carboplatin combinations appeared to have a synergistic effect on cell survival. Combinations of BMS-754807 and lung cancer chemotherapeutic agents that do not target DNA are currently being evaluated. Additionally, key downstream molecules of the IGF-IR, including Akt and ERK1/2, are being studied as well as the transcription factor, KLF5, which is expressed at high levels in the lung tumors of the IGF-IR transgenic mice. Initial results support the importance of IGF-IR signaling in lung cancer. Further understanding of the function of IGF-IR in lung cancer will enable the development of more effective targeted therapies and improve existing therapeutic strategies. 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 1118. doi:10.1158/1538-7445.AM2011-1118

Osthole induces G2/M arrest and apoptosis in lung cancer A549 cells by modulating PI3K/Akt pathway

BackgroundTo explore the effects of Osthole on the proliferation, cell cycle and apoptosis of human lung cancer A549 cells.MethodsHuman lung cancer A549 cells were treated with Osthole at different concentrations. Cell proliferation was measured using the MTT assay. Cell cycle was evaluated using DNA flow cytometry analysis. Induction of apoptosis was determined by flow cytometry and fluorescent microscopy. The expressions of Cyclin B1, p-Cdc2, Bcl-2, Bax, t-Akt and p-Akt were evaluated by Western blotting.ResultsOsthole inhibited the growth of human lung cancer A549 cells by inducing G2/M arrest and apoptosis. Western blotting demonstrated that Osthole down-regulated the expressions of Cyclin B1, p-Cdc2 and Bcl-2 and up-regulated the expressions of Bax in A549 cells. Inhibition of PI3K/Akt signaling pathway was also observed after treating A549 cells with Osthole.ConclusionsOur findings suggest that Osthole may have a therapeutic application in the treatment of human lung cancer.

IL-24 gene transfer sensitizes melanoma cells to erlotinib through modulation of the Apaf-1 and Akt signaling pathways

Interleukin-24 (IL-24) is a novel tumor suppressor/cytokine gene expressed in normal human melanocytes but for which expression is nearly undetectable in metastatic melanoma. Overexpression of the IL-24 protein has been shown to inhibit tumor cell proliferation and induce apoptosis in many melanoma cell lines, and is now considered a tumor suppressor. Erlotinib, a small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has been widely studied for the treatment of human lung cancer and other solid tumors, but the erlotinib-targeted therapy has not been tested in melanoma. The objective of this study is to investigate the potency of erlotinib in suppressing the growth of human melanoma cells and whether IL-24 could enhance the antitumor activity of erlotinib. In cell viability and apoptosis assays, treatment with erlotinib dependently inhibited the growth of different melanoma cell lines and when combined with adenoviral vector-mediated IL-24 gene therapy, a significant increase in cell growth inhibition and apoptosis induction resulted (P<0.05). Immunoblot assay showed that the combination treatment of erlotinib and IL-24 considerably increased the cleavage of caspase-3 and caspase-9 and the expression of Apaf-1 protein in melanoma cells, inducing activation of the Apaf-1-dependent apoptotic pathways. Moreover, this combination treatment markedly inhibited phosphorylation of the EGFR, phosphatidylinositol-3 kinase, and Akt proteins, inactivating the Akt-dependent cell survival signaling pathway. These results show that a combination of IL-24-mediated molecular therapy and EGFR inhibitors such as erlotinib may be a promising treatment strategy for human melanoma and will serve as a basis for guiding the combination treatment designs in future preclinical and clinical trials.

Effect of a newly synthesized Zn sulfophthalocyanine derivative on cell morphology, viability, proliferation, and cytotoxicity in a human lung cancer cell line (A549)

Photodynamic therapy (PDT) is a photochemotherapeutic process that is used for the treatment of cancer. Photofrin is the most widely used photosensitizer, however, the chemical composition of Photofrin is unclear and it has a low absorption in the therapeutic wavelength (600-900nm). This factor has stimulated research in synthesis and testing of new photosensitizers. This in vitro study evaluated the effectiveness of a Zn sulfophthalocyanine (ZnPcS(mix)) as a potential photosensitizer in the treatment of human lung cancer. Lung cancer cells (A549) were divided into four groups: group 1 was control cells receiving neither light nor drug; group 2 was light control for cells exposed to laser irradiation at a fluence of 4.98J/cm(2); group 3 was drug control for cells incubated with 15.8μM photosensitizer and not exposed to laser irradiation, while group 4 was cells receiving the experimental treatment with 15.8μM photosensitizer and irradiation with 4.98J/cm(2). Laser irradiations were performed using a 636-nm diode laser with an output power of 110mW at 4.98J/cm(2). Changes in cellular responses were evaluated by cell morphology, viability, proliferation, and cytotoxicity. While control groups 1, 2, and 3 showed no changes in cell morphology, viability, proliferation, or cytotoxicity, group 4 receiving both photosensitizer and irradiation showed changes in cell morphology, a decrease in cell viability and proliferation, and an increase in cytotoxicity, cell death, and cell membrane damage. Irradiation or photosensitizer alone had no effect on the lung cancer cells since the cells remained viable and showed no evidence of damage. However, irradiation in the presence of a photosensitizer induced cell death.

Abstract A26: Direct interaction between ceramide and SET/I2PP2A restores nuclear PP2A activity leading to the degradation of cMyc and suppression of lung tumor growth

Abstract In our recent studies, (Mukhopdahyay A, Saddoughi, S. et al., FASEB J, 2009) ceramide, a bioactive sphingolipid, has been shown to exert anti-proliferative functions against lung cancers via degradation of the cMyc proto-oncogene through directly binding to the inhibitor 2 of PP2A, (I2PP2A) also known as the SET oncoprotein. Data demonstrated that the interaction between ceramide and SET/I2PP2A restores PP2A activity in vitro. In addition, studies in A549 human lung cancer cells revealed that ceramide mediates c-Myc degradation via its PP2A-dependent dephosphorylation of S62, and treatment with okadiac acid and expression of cMyc mutant with S62A conversion resulted in resistance to ceramide-mediated degradation. The role of I2PP2A was elucidated via the down-regulation of I2PP2A, which enhanced PP2A mediated cMyc degradation in response to ceramide, and the overexpression of I2PP2A prevented the growth inhibitory effects of ceramide both in anchorage independent growth assays and xenograft-driven tumors. Our unpublished data suggests that SET/I2PP2A preferentially binds endogenous ceramides with C18 and longer fatty acid chains in A549 cells. However, the subcellular localization of endogenous ceramides/SET(I2PP2A) interaction in the regulation of nuclear versus cytoplasmic anti-proliferative targets of PP2A in lung cancer cells remains unknown. SET/I2PP2A is mainly a nuclear protein, while endogenous ceramides are mainly generated in the ER by the de novo pathway. Thus, a SET/I2PP2A mutant (I2PP2A-ER) was generated to accumulate specifically in the ER. Although, I2PP2A-ER mutant was still able to bind ceramide in vitro, its association with ceramide was almost completely inhibited in A549 cells in-situ, suggesting the requirement of the nuclear localization of SET/I2PP2A for ceramide binding. Therefore, our ongoing research efforts are focused on understanding how ceramides reach the nucleus for SET/I2PP2A binding in A549 cells. Two enzymes, Ceramide Synthase (CerS) and Neutral Sphingomyelinase (nSMase) are known to be the main generators of ceramide in cells. The activity of these enzymes in the nucleus of A549 compared to non-cancerous WI38 lung epithelial fibroblasts are being explored to determine the mechanism of ceramide generation in the nucleus. To this end, the overexpression of CerS1, which generates C18-ceramide, leads to the degradation of cMyc, which is prevented by T58A mutation of cMyc. Interestingly, the downregulation of nSMase1 leads to an increase in the endogenous levels of cMyc, and this increase is prevented with SET/I2PP2A RNAi. Thus, these data support the role for endogenous ceramide in the regulation of SET/I2PP2A and PP2A-dependent regulation of cMyc in A549 cells. The clinical relevance of the ceramide-SET/I2PP2A interaction in lung cancer progression was explored using 10 patient lung adenocarcinoma tissues, with their paired pathologically non-cancerous adjacent lung tissues. In these samples, it was demonstrated that I2PP2A/SET is overexpressed in about 60% of these tumors and the mRNA expression of the ceramide generating enzymes, CerS1 and nSMase1 are decreased within the majority of tumors. In summary, this research highlights the structural and biological roles of ceramide-SET/I2PP2A binding in the regulation of PP2A/cMyc axis leading to the suppression of human lung cancers. These studies will lead to the development of mechanism-based therapeutic strategies, by targeting SET/I2PP2A through ceramide signaling for the treatment of human lung cancers. Citation Information: Clin Cancer Res 2010;16(7 Suppl):A26