Apoptosis In Non-small Cell Lung Carcinoma Cells Research Articles

ING2-WTAP is a potential therapeutic target in non-small cell lung cancer

Non-small-cell lung carcinoma (NSCLC) is the most common kind of lung cancer, which is also the largest cause of cancer-linked deaths globally. A lack of effective prognostic biomarkers contributes to the poor prognosis of NSCLC. For better patient outcomes, NSCLC diagnosing, prognostic, and predictive biomarkers are critically needed. A component of the chromatin-modulatory complex that dampens gene expression, ING2 (inhibitor of growth family member 2), has been linked to cellular mechanisms that enhance tumor repression. Nevertheless, its role in human NSCLC is unclear. Herein, we observed that ING2 downregulation in NSCLC tissues correlates with poor NSCLC prognosis. Functional analysis illustrated that ING2 dampened growth, infiltration along with metastasis and blocked apoptosis of NSCLC cells in vitro, as well as in vivo. The mechanism might be implicated in the EMT (epithelial-mesenchymal transition) process. Mechanistically, we found that ING2 silencing suppresses the expressions of Wilms tumor 1-associated protein (WTAP) and that WTAP expression negatively correlates with ING2 expression. Moreover, rescue experiments illustrated that WTAP overexpression partially counteracts the effect of ING2 silencing on proliferation. Finally, we found that elevated WTAP levels correlate with poor NSCLC prognosis. These results highlight the prognostic and therapeutic potential of ING2-WTAP in NSCLC.

Evodiamine suppresses the progression of non-small cell lung carcinoma via endoplasmic reticulum stress-mediated apoptosis pathway in vivo and in vitro.

BackgroundEvodiamine (EVO) is one of the major components isolated from Evodia rutaecarpa (Juss.). Recent studies have shown that EVO has an anti-cancer effect. However, the pharmacological mechanism by which EVO impacts cancer is still poorly understood.ObjectivesThis study focused on asking the anti-cancer effect of EVO in human non-small cell lung carcinoma (NSCLC), and in particular to investigate whether EVO acts via modulating the endoplasmic reticulum stress (ERS)-mediated apoptosis pathway.Materials and MethodsA Lewis lung carcinoma (LLC) tumor-bearing mouse model was treated with low-dose EVO (5 mg/kg) and high-dose EVO (10 mg/kg) intraperitoneally for 14 d. The effects of EVO on tumor growth, apoptosis, and ERS were assessed. In addition, NSCLC A549 and LLC cells were treated with EVO in vitro. The effects of EVO on cell proliferation, apoptosis, and ERS were investigated. Finally, 4-phenylbutyric acid (4-PBA), an ERS inhibitor, was used to validate whether EVO induced apoptosis of NSCLC cells by modulating ERS.ResultsEVO treatment significantly inhibited tumor growth in LLC tumor-bearing mice. H&E staining indicated that EVO treatment reduced the number of tumor cells and the nucleo-plasmic ratio. Immunostaining showed that EVO treatment significantly decreased the expression of Ki-67. TUNEL staining revealed that EVO induced apoptosis in the tumor. Likewise, EVO treatment up-regulated the expression of apoptosis-related genes and proteins and increased activation of the ERS pathway in the tumor. Additionally, EVO inhibited cell proliferation and increased cell apoptotic rates in A549 and LLC cells. EVO also increased the expression levels of genes and proteins associated with ERS-mediated apoptosis pathway in vitro. The effects of EVO on apoptosis were abolished by 4-PBA treatment.ConclusionsOur study demonstrated that EVO suppresses the progression of NSCLC by modulating the ERS-mediated apoptosis pathway.

Quercetin inhibits proliferation of and induces apoptosis in non-small-cell lung carcinoma via the lncRNA SNHG7/miR-34a-5p pathway

Objective To determine the role of quercetin in non-small cell lung carcinoma (NSCLC) and the biological outcomes using transfection experiments. Materials and methods Real-time reverse transcription-PCR and data collection were performed to determine lncRNA and miRNA levels. Transwell assay was performed to assess the invasion ability of cells. Apoptosis of cells digested with trypsin was determined using the Annexin V-FITC kit. Luciferase activity was determined using the luciferase reporter gene system. Cell viability was tested using the Cell Counting Kit-8 assay. A xenograft mouse model was established to investigate the effects of quercetin on tumor growth. Results The expression levels of the long non-coding RNA (lncRNA) small nucleolar RNA host gene 7 (SNHG7) were elevated in NSCLC cells, and the expression levels of the microRNA miR-34a-5p were decreased compared with those in normal cells. Further investigation revealed that quercetin decreased SNHG7 and elevated miR-34a-5p levels in NSCLC cells (p < .05). The luciferase reporter gene assay, RNA-binding protein immunoprecipitation assay, and transfection experiments revealed target-binding sequences between SNHG7 and miR-34a-5p. Overexpression of SNHG7 or miR-34a-5p inhibitor promoted NSCLC cell proliferation and accelerated tumor cell growth and metastasis. The therapeutic effect of quercetin on NSCLC cells was counteracted by co-transfection of SNHG7 mimic or miR-34a-5p inhibitor. Quercetin inhibited the survival, proliferation, migration, and invasion of NSCLC cells and enhanced their apoptosis. Using the mouse model, quercetin was shown to inhibit tumor growth. Conclusions Quercetin inhibits the proliferation and induces apoptosis of NSCLC cells by mediating signaling via the lncRNA SNHG7/miR-34a-5p pathway.

PGRN exacerbates the progression of non-small cell lung cancer via PI3K/AKT/Bcl-2 antiapoptotic signaling

Progranulin (PGRN) is a growth factor that is involved in the progression of multiple tumors. However, the effects and molecular mechanisms by which PGRN induces lung cancer remain unclear. The expression level of PGRN was analyzed by conducting immunohistochemistry of the histological sections of lung tissues from non-small-cell lung carcinoma (NSCLC) patients. The proliferation, apoptosis, migration, and invasion of NSCLC cells were assessed by the MTT assay, Western blot, degree of wound healing, and Transwell assays. A nude mouse xenograft model was used to validate the role of PGRN in vivo. The expression level of PGRN was higher in male patients with lung adenocarcinoma than in those with lung squamous cell carcinoma; by contrast, no difference was observed in female patients. The overexpression of PGRN promoted the proliferation and anti-apoptosis of H520 (derived from lung squamous cell carcinoma) cells, whereas knockdown of PGRN inhibited the proliferation and anti-apoptosis of A549 (derived from lung adenocarcinoma) cells. Copanlisib (targeting PI3K) inhibited the increase in the expression of cell anti-apoptosis marker Bcl-2 induced by rhPGRN protein; the PI3K agonist 740 Y–P partially reversed the decrease in Bcl-2 expression induced by PGRN deficiency in both A549 and H520 cells. PGRN increased the expression of Ki-67, PCNA, and Bcl-2 in vivo. PGRN inhibited cell apoptosis depending on the PI3K/Akt/Bcl-2 signaling axis; PGRN positivity correlated with lung adenocarcinoma. PGRN is a potential biomarker for the treatment and diagnosis of NSCLC, especially in lung adenocarcinoma.

Downregulation of circRNA_100876 Inhibited Progression of NSCLC In Vitro via Targeting miR-636.

Background:Non-small cell lung carcinoma (NSCLC) is a common malignant tumor with poor prognosis. CircRNA-100876 has been considered to be involved in NSCLC. However, the mechanism by which circRNA_100876 mediated the progression of NSCLC remains unclear.Methods:CCK8 assay and immunofluorescence were used to detect cell proliferation. Flow cytometry and transwell assay were performed to analyze cell apoptosis, migration and invasion, respectively. Verification of possible target for circRNA_100876 and related miR-636 were done using luciferase assay. In addition, western blot was performed to detect the protein expressions in NSCLC cells.Results:Silencing of circRNA_100876 notably inhibited the proliferation of NSCLC cells. Moreover, downregulation of circRNA_100876 significantly induce the apoptosis of NSCLC cells via mediation of apoptosis-related proteins. In addition, silencing of circRNA_100876 significantly inhibited migration and invasion of NSCLC cells. MiR-636 was the downstream target of circRNA_100876. Meanwhile, RET was the direct target of miR-636. Finally, circRNA_100876 shRNA2 notably suppressed the progression of NSCLC through PI3K/Akt signaling.Conclusion:CircRNA_100876 knockdown notably suppressed the progression of NSCLC through regulation of miR-636/RET axis, which may serve as a potential target for treatment of NSCLC.

TMEM106A inhibits cell proliferation, migration, and induces apoptosis of lung cancer cells.

Transmembrane protein 106A (TMEM106A) has been found to function as tumor suppressor in gastric and renal cancer. However, the role of TMEM106A in nonsmall-cell lung carcinoma (NSCLC) has not been investigated. In this study, we evaluated the expression profile of TMEM106A in NSCLC tissues and cell line, and explored the roles of TMEM106A in NSCLC cell lines. Our results showed that TMEM106A expression was significantly decreased in human NSCLC tissues. In vitro assays showed that TMEM106A expression in NSCLC cell lines was much lower than that in the bronchial epithelial cell line. Besides, overexpression of TMEM106A reduced cell proliferation, migration, and invasion, while induced cell apoptosis in NSCLC cells. TMEM106A overexpression repressed epithelial-mesenchymal transition (EMT), which was illustrated by increased E-cadherin expression and decreased the expressions of N-cadherin, and vimentin. In addition, TMEM106A overexpression suppressed the activation of phosphoinositide 3-kinase/protein kinase B/nuclear factor-κB (PI3K/Akt/NF-κB) signaling pathway in NSCLC cells. Our results indicated that TMEM106A acted as a tumor suppressor in NSCLC, and could be a therapeutic target for the management of NSCLC.

Phytochemical naphtho[1,2-b] furan-4,5‑dione induced topoisomerase II-mediated DNA damage response in human non-small-cell lung cancer

BackgroundPhytochemical naphtho[1,2-b] furan-4,5‑dione (NFD) presenting in Avicennia marina exert anti-cancer effects, but little is known regarding about DNA damage-mediated apoptosis in non-small-cell lung carcinoma (NSCLC). PurposeTo examine whether NFD-induced apoptosis of NSCLC cells is correlated with the induction of DNA damage, and to investigate its underlying mechanism. Study designThe anti-proliferative effects of NFD were assessed by MTS Assay Kit FACS assay, and in vivo nude mice xenograft assay. The DNA damage related proteins, the Bcl-2 family and pro-apoptotic factors were examined by immunofluorescence assay, q-PCR, and western blotting. The activity of NF-κB p65 in nuclear extracts was detected using a colorimetric DNA-binding ELISA assay. The inhibitory activity of topoisomerase II (TOPO II) was evaluated by molecular docking and TOPO II catalytic assay. ResultsNFD exerted selective cytotoxicity against NSCLC H1299, H1437 and A549 cells rather than normal lung-embryonated cells MRC-5. Remarkably, we found that NFD activated the hull marker and modulator of DNA damage repairs such as γ-H2AX, ATM, ATR, CHK1, and CHK2 probably caused by the accumulation of intracellular reactive oxygen species (ROS) and inhibition of TOPO II activity. Furthermore, the suppression of transcription factor NF-κB by NFD resulted in significantly decreased levels of pro-survival proteins including Bcl-2 family Bcl-2, Bcl-xL and Mcl-1 and the endogenous inhibitors of apoptosis XIAP and survivin in H1299 cells. Moreover, the nude mice xenograft assay further validated the suppression of H1299 growth by NFD, which is the first report for evaluating the anti-cancer effect of NFD in vivo. ConclusionThese findings provide a novel mechanism indicating the inhibition of TOPO II activity and NF-κB signaling by NFD, leading to DNA damage and apoptosis of NSCLC tumor cells.

TSPAN12 is overexpressed in NSCLC via p53 inhibition and promotes NSCLC cell growth in vitro and in vivo.

BackgroundTetraspanin 12 (TSPAN12), a member of the phylogenetically ancient tetraspanin family, is linked to impaired vascularization of the eye called familial exudative vitreoretinopathy, while the functional role of TSPAN12 in lung cancer has not been well characterized.ResultsIn our study, TSPAN12 is able to regulate the growth of non-small-cell lung carcinoma (NSCLC) cells both in vitro and in vivo. TSPAN12 mRNA level was significantly increased in human NSCLC samples compared with their corresponding paracancerous histologic normal tissues. In addition, TSPAN12 expression, which is frequently upregulated in NSCLC, is inversely correlated with p53 expression. Furthermore, the expression levels of TSPAN12 were also increased in three human NSCLC cell lines compared to human bronchial epithelial (16HBE) cells. Then, we studied the effects of TSPAN12 silencing by short hairpin ribonucleic acid on NSCLC cell growth in vitro and tumorigenesis in vivo, along with the effect on the p53 pathway. Knockdown of TSPAN12 in NSCLC cells inhibited cell proliferation and colony formation. In addition, knockdown of TSPAN12 increased apoptosis in NSCLC cells. Mechanistically, TSPAN12 could modulate the expression of p53, p21, and p27 in NSCLC cells. In a tumor xenograft model, TSPAN12 silencing inhibits the tumor growth of H1299 cells.ConclusionTaken together, our results reveal that TSPAN12 plays an important role in NSCLC and is a potential biomarker and a promising target in the treatment of NSCLC.

Overexpression of T-box Transcription Factor 5 (TBX5) Inhibits Proliferation and Invasion in Non-Small Cell Lung Carcinoma Cells

T-box transcription factor 5 (TBX5), a member of the conserved T-box transcription factor family that functions in organogenesis and embryogenesis, has recently been identified as a critical player in cancer development. The aim of this study was to determine the role of TBX5 in non-small cell lung carcinoma (NSCLC). Immunohistochemistry was used to detect the correlation between levels of TBX5 and clinicopathological features of NSCLC patients in tissue microarray. Expression of TBX5 in NSCLC tissues and cell lines was evaluated by quantitative PCR and Western blot. The role of TBX5 in regulating proliferation, colony formation, invasion, and apoptosis of NSCLC cells was evaluated in vitro. Finally, a tumorigenicity assay was performed to determine the effect of TBX5 on tumor growth in vivo. The levels of TBX5 in NSCLC tissues were significantly correlated with the TNM stage (p = 0.016), histopathologic type (p = 0.029), and lymph node status (p = 0.035) of NSCLC. TBX5 overexpression markedly suppressed in vitro NSCLC cell proliferation, colony formation, and invasion and induced apoptosis. In vivo tumor growth was significantly suppressed by TBX5. TBX5 has a tumor-suppressing effect in NSCLC and may serve as a therapeutic target for diagnoses and treatment of NSCLC.

MiR-124 Inhibits Growth and Enhances Radiation-Induced Apoptosis in Non-Small Cell Lung Cancer by Inhibiting STAT3

Background/Aims: A growing body of evidence indicates that the abnormal expression of microRNAs (miRNAs) play an important role in sensitizing the cellular response to ionizing radiation (IR). The aim of this study was to investigate whether the expression of miR-124 correlated with radiosensitivity in the context of non-small-cell lung carcinoma (NSCLC). Methods: Quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to quantify miR-124 expression in NSCLC tissues and cell lines. The role of miR-124 in NSCLC proliferation and radiosensitivity was analyzed using CCK-8 and flow cytometry apoptosis assays. Luciferase activity assays, RT-PCR, and Western blot assays were performed to confirm the target gene of miR-124. Results: In this study, we found that miR-124 was downregulated both in clinical NSCLC samples and in cell lines. miR-124 inhibited the proliferation of NSCLC cells and enhanced the apoptosis of NSCLC cells exposed to ionizing radiation. We identified signal transducer and activator of transcription 3 (STAT3) as a direct target of miR-124 by using target prediction algorithms and luciferase assays. Overexpression of STAT3 in A549 cell lines restored the enhanced radiosensitivity induced by miR-124. Conclusion: Taking these observations into consideration, we illustrated that miR-124 is a potential target for enhancing the radiosensitivity of NSCLC cells by targeting STAT3.

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.

Neuropilin 1 expression correlates with the Radio-resistance of human non-small-cell lung cancer cells.

The purpose of this study was to determine the correlation between over-expression of the neuropilin 1 (NRP1) gene and growth, survival, and radio-sensitivity of non-small cell lung carcinoma (NSCLC) cells. 3-[4,5-dimethylthylthiazol-2-yl]-2,5 diphenyltetrazolium broide (MTT) and colony assays were then performed to determine the effect of NRP1 inhibition on the in vitro growth of NSCLC cells. The Annexin V-Fluorescein Isothiocyanate (FITC) apoptosis detection assay was performed to analyse the effect of NRP1 enhancement on apoptosis of NSCLC cells. Transwell invasion and migration assays were employed to examine the metastatic ability of A549 cells post X-ray irradiation. In addition, Western blot assays were carried out to detect the protein level of VEGFR2, PI3K and NF-κB. Finally, to examine the effect of shNRP1 on proliferation and radio-sensitivity in vivo, a subcutaneous tumour formation assay in nude mice was performed. Microvessel density in tumour tissues was assessed by immunohistochemistry. The stable transfected cell line (shNRP1-A549) showed a significant reduction in colony-forming ability and proliferation not only in vitro, but also in vivo. Moreover, shRNA-mediated NRP1 inhibition also significantly enhanced the radio-sensitivity of NSCLC cells both in vitro and in vivo. The over-expression of NRP1 was correlated with growth, survival and radio-resistance of NSCLC cells via the VEGF-PI3K- NF-κB pathway, and NRP1 may be a molecular therapeutic target for gene therapy or radio-sensitization of NSCLC.

MCM2 is a therapeutic target of lovastatin in human non-small cell lung carcinomas.

Human non-small cell lung carcinoma (NSCLC) is one of the most common cancer worldwide. In previous studies, lovastatin, acting as an inhibitor of 3-hydroxy-3-methylglutaryl Co A (HMG-CoA) reductase, exhibited significant antitumor activity during tumorigenesis. However, whether or not this effect is mediated through changes in minichromosome maintenance (MCM) 2 expression remains unclear. The present study investigated whether lovastatin inhibits proliferation due to MCM2 in NSCLCs. We first assessed the effects of lovastatin on cell anti-proliferation, cell cycle progression and apoptosis in NSCLC cells. We found, by quantitative RT-PCR and western blot analysis, that lovastatin treatment markedly and consistently inhibited the expression of MCM2. Then, to further explore the anticancer mechanism of lovastatin involving MCM2, we silenced MCM2 by siRNA in two cell lines (A549 and GLC-82). Silencing of MCM2 triggered G1/S arrest. Following further examination of cell cycle-related factors, MCM2 knockdown inhibited protein retinoblastoma (Rb), cyclin D1 and CDK4 expression, but increased p21 and p53 expression, suggesting that siMCM2 indeed triggered cell cycle arrest. In addition, siMCM2 induced apoptosis. Finally, lovastatin treatment increased p-JNK, which is involved in the downregulation of MCM2. In conclusion, our data suggest that MCM2 may be a novel therapeutic target of lovastatin treatment in NSCLCs.

AURKA upregulation plays a role in fibroblast-reduced gefitinib sensitivity in the NSCLC cell line HCC827.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) have been used to treat non-small cell lung carcinoma (NSCLC) patients that have EGFR-activating mutations. EGFR-TKI monotherapy in most NSCLC patients with EGFR mutations who initially respond to EGFR-TKIs results in the development of acquired resistance. We investigated the role of fibroblasts in stromal cell-mediated resistance to gefitinib-induced apoptosis in EGFR-mutant NSCLC cells. While gefitinib induced apoptosis in EGFR-mutant NSCLC cells, apoptosis induction was diminished under stromal co-culture conditions. Protection appeared to be mediated in part by Aurora-A kinase (AURKA) upregulation. The protective effect of stromal cells was significantly reduced by pre-exposure to AURKA-shRNA. We suggest that combinations of AURKA antagonists and EGFR inhibitors may be effective in clinical trials targeting mutant EGFR NSCLCs.

Afatinib induces apoptosis in NSCLC without EGFR mutation through Elk-1-mediated suppression of CIP2A.

Afatinib has anti-tumor effect in non-small cell lung carcinoma (NSCLC) with epidermal growth factor receptor (EGFR) mutation. We found afatinib can also induce apoptosis in NSCLC cells without EGFR mutation through CIP2A pathway. Four NSCLC cell lines (H358 H441 H460 and A549) were treated with afatinib to determine their sensitivity to afatinib-induced cell death and apoptosis. The effects of CIP2A on afatinib-induced apoptosis were confirmed by overexpression and knockdown of CIP2A expression in the sensitive and resistant cells, respectively. Reduction of Elk-1 binding to the CIP2A promoter and suppression of CIP2A transcription were analyzed. In vivo efficacy of afatinib against H358 and H460 xenografts tumors were also determined in nude mice. Afatinib induced significant cell death and apoptosis in H358 and H441 cells, but not in H460 or A549 cells. The apoptotic effect of afatinib in sensitive cells was associated with downregulation of CIP2A, promotion of PP2A activity and decrease in AKT phosphorylation. Afatinib suppressed CIP2A at the gene transcription level by reducing the promoter binding activity of Elk-1. Clinical samples showed that higher CIP2A expression predicted a poor prognosis and Elk-1 and CIP2A expressions were highly correlated. In conclusion, afatinib induces apoptosis in NSCLC without EGFR mutations through Elk-1/CIP2A/PP2A/AKT pathway.

Cationic Lipid-Assisted Polymeric Nanoparticle Mediated GATA2 siRNA Delivery for Synthetic Lethal Therapy of KRAS Mutant Non-Small-Cell Lung Carcinoma

Synthetic lethal interaction provides a conceptual framework for the development of wiser cancer therapeutics. In this study, we exploited a therapeutic strategy based on the interaction between GATA binding protein 2 (GATA2) downregulation and the KRAS mutation status by delivering small interfering RNA targeting GATA2 (siGATA2) with cationic lipid-assisted polymeric nanoparticles for treatment of non-small-cell lung carcinoma (NSCLC) harboring oncogenic KRAS mutations. Nanoparticles carrying siGATA2 (NPsiGATA2) were effectively taken up by NSCLC cells and resulted in targeted gene suppression. NPsiGATA2 selectively inhibited cell proliferation and induced cell apoptosis in KRAS mutant NSCLC cells. However, this intervention was harmless to normal KRAS wild-type NSCLC cells and HL7702 hepatocytes, confirming the advantage of synthetic lethality-based therapy. Moreover, systemic delivery of NPsiGATA2 significantly inhibited tumor growth in the KRAS mutant A549 NSCLC xenograft murine model, suggesting the therapeutic promise of NPsiGATA2 delivery in KRAS mutant NSCLC therapy.

Chemical Constituents and Anticancer Activity of Curcuma zedoaria Roscoe Essential Oil against Non-Small Cell Lung Carcinoma Cells in Vitro and in Vivo

In this study, we report that the essential oil obtained from Curcuma zedoaria Roscoe, known as zedoary, possesses efficient cytotoxic effects on non-small cell lung carcinoma (NSCLC) cells and causes cell apoptosis. Zedoary essential oil increased the sub-G1 population and the level of annexin-V binding and induced cleavage and activation of caspase-3, -8, and -9 and poly(ADP ribose) polymerase. Decreases in the levels of Bcl-2 and Bcl-xL and an increase in the Bax/Bcl-2 ratio were also observed following zedoary essential oil treatment. Notably, zedoary essential oil led to the release of AIF, endonuclease G, and cytochrome c into the cytosol and increased levels of p53 in H1299 cells. Our results indicate that zedoary essential oil slightly inhibited the phosphorylation of ERK1/2 and enhanced the phosphorylation of JNK1/2 and p38. Zedoary essential oil also inhibited AKT/NF-κB signaling pathways in H1299 cells. Moreover, intraperitoneal administration of zedoary essential oil significantly suppressed the growth of H1299 cells in vivo. In addition, potential active compounds were detected using gas chromatography and mass spectrometry. 8,9-Dehydro-9-formyl-cycloisolongifolene, 6-ethenyl-4,5,6,7-tetrahydro-3,6-dimethyl-5-isopropenyl-trans-benzofuran, eucalyptol, and γ-elemene were found in zedoary essential oil. In summary, our findings provide insight into the molecular mechanisms underlying zedoary essential oil-induced apoptosis in NSCLC cells that are worthy of further study.

Matrine induction of reactive oxygen species activates p38 leading to caspase-dependent cell apoptosis in non-small cell lung cancer cells

Non-small cell lung carcinoma (NSCLC) is one of the most refractory cancers in the clinic; it is insensitive to chemotherapy and is usually excised. However, screening natural compounds from herbs is also considered a possible method for its therapy. In the present study, we investigated whether matrine, a natural compound isolated from Sophora flavescens Ait. and exerting an inhibitory effect on lung cancer cells, also indicates inhibition on NSCLC cells and elucidated its molecular mechanism. Firstly, it is confirmed that matrine induces apoptosis of human NSCLC cells with anti-apoptotic factors inhibited and dependent on caspase activity. In addition, we found that matrine increases the phosphorylation of p38 but not its total protein, and inhibition of the p38 pathway with SB202190 partially prevents matrine-induced apoptosis. Furthermore, matrine generates reactive oxygen species (ROS) in a dose- and time-dependent manner, which is reversed by pretreatment with N-acetyl-L-cysteine (NAC). Additionally, inhibition of cell proliferation and increase of phosphorylation of p38 was also partially reversed by NAC. Collectively, matrine activates p38 pathway leading to a caspase-dependent apoptosis by inducing generation of ROS in NSCLC cells and may be a potential chemical for NSCLC.

Abstract 1132: Oncogenic role of EAPII/TTRAP in lung cancer development and its activation of MAPK-ERK pathway

Abstract Cancer progression involves multiple complex and interdependent steps including progressive proliferation, angiogenesis and metastases. The complexity of these processes requires a comprehensive elucidation of the integrated signaling networks for better understanding. EAPII/TTRAP interacts with multiple cancer-related proteins, but its biological significance in cancer development remains unknown. In this report we identified the elevated level of EAPII/TTRAP protein in non-small cell lung carcinoma (NSCLC) patients and NSCLC cell lines in culture. The oncogenic role of EAPII in lung cancer development was demonstrated using NSCLC cells with genetic manipulations that influence EAPII expression: EAPII overexpression increases proliferation of NSCLC cells with an accelerated transition of cell cycle and facilitates xenograft tumor growth in vivo; EAPII knockdown results in apoptosis of NSCLC cells and reduces xenograft tumor formation. To further explore the mechanism of EAPII's oncogenic role in lung cancer development and to elucidate the potential signaling pathway(s) that EAPII may impact, we employed antibody array to investigate the alternation of the major signaling pathways in NSCLC cells with altered EAPII level. We found that EAPII overexpression significantly activated Raf-1 and ERK1/2, but not JNK and p38 pathways. Consistently, the protein and mRNA levels of MYC and cyclin D1, which are targets of the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK-ERK) pathway, are significantly increased by EAPII overexpression. Taken together, we demonstrated that EAPII is an oncogenic factor and the activation of MAPK-ERK signaling pathway by EAPII may contribute to lung cancer development. 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 1132. doi:10.1158/1538-7445.AM2011-1132

Abstract LB-134: C-terminal PKCδ1 protein cleavage product acts as a tumor suppressor in human non-small cell lung carcinoma cells

Abstract PKCδ1 is a member of family proteins of the lipid-activated serine/threonine kinases that have been implicated in a wide range of cellular functions, including apoptosis and cell proliferation. While the function of PKCδ1 in regulating apoptosis still remains controversial, it has been well documented that PKCδ1 can undergoe apoptosis-dependent protein cleavage. However, the function of its protein cleavage product has not been previously characterized. The PKCδ1 protein cleavage has also been frequently observed in our studies during apoptosis induced by various apoptosis stimuli, including proteasome inhibitors. To investigate the involvement of PKCδ1 protein cleavage product in apoptosis, in this study, we cloned 3′ portion of PKCδ1 cDNA, which encodes the predicted C-terminal PKCδ1 protein cleavage fragment starting from 318 asparagine residue to the stop codon of the protein, into pcDNA3.1Zeo(+) vector with Flag tag at its 5′end. The new vector was then named Flag-PKCδ1-Δ318. Subsequently, the Flag-PKCδ1-Δ318 demonstrated its ability to express a 35kd protein in COS7 cells following gene transfection. In human non-small cell lung carcinoma (NSCLC) NCI-H1703 and NCI-H358 cells, we observed that PKCδ1 underwent protein cleavage resulting in a 35kd cleavage product during proteasome inhibitor MG132-induced apoptosis. It was also observed that silencing of PKCδ1 expression by its siRNA mildly enhanced apoptosis induction in both NSCLC cell lines, suggesting that PKCδ1 can minimally inhibit MG132-induced apoptosis in NSCLC cells. However, unexpectedly, transfection with Flag-PKCδ1-Δ318, did not affect MG132-induced apoptosis. Instead, it caused a significant inhibition of cell proliferation as demonstrated by the slowed cell growth and reduced colony formation in both NSCLC cell lines. Moreover, it was found that expression of PKCδ1-Δ318 dramatically reduced in vitro migration and abolished in vivo tumor formation of NCI-H358 cells, strongly suggesting that PKCδ1-Δ318 acts as tumor suppressor. To further investigate the possible mechanisms underlying the tumor suppression function of PKCδ1-Δ318, we tested activity of MAPK, AKT and MLCK, the critical kinases associated with many oncogenic properties of tumor cells. It was then found that the activity of all these kinases were significantly reduced by PKCδ1-Δ318 in NCI-H358 cells, suggesting that the PKCδ1-Δ318 protein, different from its parental protein, exerts its tumor suppression function through inhibiting multiple oncogenic signaling pathways. Therefore, this study suggests that the apoptosis-dependent protein cleavage may represent a new mechanism in regulating tumorigenic activities and PKCδ1-Δ318 may be of great value of being a new cancer gene therapy agent. 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 LB-134. doi:10.1158/1538-7445.AM2011-LB-134