Lung Carcinoma Cell Growth Research Articles

Testing the Rebound Peer Review Concept

This invited editorial addresses the rescue of the article by Skrzypek et al. "Interplay between heme oxygenase-1 and miR-378 affects non-small cell lung carcinoma growth, vascularization, and metastasis." The work was rejected by the standard peer review system and subsequently rescued by the Rebound Peer Review (RPR) mechanism offered by Antioxidants and Redox Signaling (Antioxid Redox Signal 16: 293-296, 2012). The reviewers who openly rescued the article were James F. George, Justin C. Mason, Mahin D. Maines, and Yasufumi Sato. The initial article was a de novo resubmission of a previously rejected article, which was then reviewed by six reviewers. The reviewers raised substantial scientific concerns, including questions pertaining to the specificity of the findings, quality of the presentation, and other technical concerns; the editor returned a decision of reject. The authors voluntarily chose to exercise the option to rescue the article utilizing the RPR system, where the authors found qualified reviewers who were willing to advocate for acceptance with scientific reasoning. The open reviewers felt that the scientific and technical concerns raised by the reviewers were outweighed by the strengths and novelty of the findings to justify acceptance. The RPR, in this case, was a "success" in that it rescued a rejected article. Despite this assessment, we question the necessity of open peer review as a means to overturn a peer review decision, with concerns for the larger-than-usual peer review process, and the voluntary relinquishing of editorial privilege and disclosure of reviewer identity.

Interplay Between Heme Oxygenase-1 and miR-378 Affects Non-Small Cell Lung Carcinoma Growth, Vascularization, and Metastasis

Heme oxygenase-1 (HO-1, HMOX1) can prevent tumor initiation; while in various tumors, it has been demonstrated to promote growth, angiogenesis, and metastasis. Here, we investigated whether HMOX1 can modulate microRNAs (miRNAs) and regulate human non-small cell lung carcinoma (NSCLC) development. Stable HMOX1 overexpression in NSCLC NCI-H292 cells up-regulated tumor-suppressive miRNAs, whereas it significantly diminished the expression of oncomirs and angiomirs. The most potently down-regulated was miR-378. HMOX1 also up-regulated p53, down-regulated angiopoietin-1 (Ang-1) and mucin-5AC (MUC5AC), reduced proliferation, migration, and diminished angiogenic potential. Carbon monoxide was a mediator of HMOX1 effects on proliferation, migration, and miR-378 expression. In contrast, stable miR-378 overexpression decreased HMOX1 and p53; while enhanced expression of MUC5AC, vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), and Ang-1, and consequently increased proliferation, migration, and stimulation of endothelial cells. Adenoviral delivery of HMOX1 reversed miR-378 effect on the proliferation and migration of cancer cells. In vivo, HMOX1 overexpressing tumors were smaller, less vascularized and oxygenated, and less metastatic. Overexpression of miR-378 exerted opposite effects. Accordingly, in patients with NSCLC, HMOX1 expression was lower in metastases to lymph nodes than in primary tumors. In vitro and in vivo data indicate that the interplay between HMOX1 and miR-378 significantly modulates NSCLC progression and angiogenesis, suggesting miR-378 as a new therapeutic target. REBOUND TRACK: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16, 293-296, 2012) with the following serving as open reviewers: James F. George, Mahin D. Maines, Justin C. Mason, and Yasufumi Sato.

AAV-mediated gene transfer of human pigment epithelium-derived factor inhibits Lewis lung carcinoma growth in mice

Pigment epithelium-derived factor (PEDF) is the most potent inhibitor of angiogenesis in the mammalian eye, and mechanisms through which PEDF exerts its antitumour activity have recently been defined. The aim of our research was to evaluate the ability of adeno-associated virus (AAV) vector-mediated transfer of human PEDF to inhibit Lewis lung carcinoma (LCC) cell growth. Intratumoural injection of AAV-PEDF caused significant reduction of the tumour volume and prolonged the survival time of mice bearing LLC cells, which were associated with decreased microvessel density and increased apoptosis in the tumours. AAV vectors represent a very promising tool for cancer gene therapy. No noticeable toxicity concerning AAV was detected as inferred from monitoring changes in animal body weight as well as basic organ structure and histological morphology, and by analyzing mouse liver and kidney function. Our findings indicate that AAV-mediated PEDF gene expression may offer an active approach to inhibit LLC growth and that treatment with AAV-PEDF may provide a promising therapeutic strategy in lung cancer treatment.

Proteins kinase Cɛ is required for non-small cell lung carcinoma growth and regulates the expression of apoptotic genes

Protein kinase C (PKC)ɛ, a member of the novel PKC family, has key roles in mitogenesis and survival in normal and cancer cells. PKCɛ is frequently overexpressed in epithelial cancers, particularly in lung cancer. Using a short-hairpin RNA approach, here we established that PKCɛ is required for non-small cell lung carcinoma (NSCLC) growth in vitro as well as tumor growth when inoculated into athymic mice. Moreover, sustained delivery of a PKCɛ-selective inhibitor peptide, ɛV1-2, reduced xenograft growth in mice. Both RNA interference depletion and pharmacological inhibition of PKCɛ caused a marked elevation in the number of apoptotic cells in NSCLC tumors. PKCɛ-depleted NSCLC cells show elevated expression of pro-apoptotic proteins of the Bcl-2 family, caspase recruitment domain-containing proteins and tumor necrosis factor ligands/receptor superfamily members. Moreover, a Gene Set Enrichment Analysis revealed that a vast majority of the genes changed in PKCɛ-depleted cells were also deregulated in human NSCLC. Our results strongly suggest that PKCɛ is required for NSCLC cell survival and maintenance of NSCLC tumor growth. Therefore, PKCɛ may represent an attractive therapeutic target for NSCLC.

Targeted lung cancer therapy using ephrinA1-loaded albumin microspheres

EphrinA1, the ligand of EphA2 receptor tyrosine kinase, has been proven to suppress the growth of tumours. The aim of this study was to conjugate ephrinA1 on the surface of albumin microspheres and investigate the non-small cell lung carcinoma growth and migration in vitro. Bovine serum albumin microspheres were designed and synthesized using a natural polymer albumin by emulsification chemical cross-linking. EphrinA1 was then conjugated on the surface of microspheres by imine formation. The microspheres conjugated with ephrinA1 (ephrinA1-MS) were characterized for particle size, surface morphology, loading efficiency and stability in vitro. The ephrinA1-MS were labelled with fluorescein isothiocyanate to determine phagocytosis. In addition, the effects of ephrinA1-MS on A549 cell growth and migration were determined. Albumin microspheres exhibited low toxicity for A549 cells (above 90% cell viability). More than 80% of microspheres were phagocytosed within 2 h of incubation. EphrinA1-MS decreased the expression of focal adhesion kinase more effectively than recombinant ephrinA1 alone. Furthermore, ephrinA1-MS showed significant inhibition of non-small cell lung cancer migration when compared with resting cells. EphrinA1-MS attenuated the growth of tumour colonies in matrigels. The developed ephrinA1-MS may serve as potential carriers for targeted delivery of the tumour suppressive protein ephrinA1, with minimal cytotoxic effects and greater antitumour therapeutic efficacy against non-small cell lung cancer.

Hypoxia differentially affects proliferation and death of Lewis Lung Carcinoma cells containing or lacking PIN1

Protein Never in Mitosis Gene A Interacting-1 (PIN1) is a peptidyl-proline isomerase that specifically recognizes and isomerizes phosphorylated Ser/Thr-Pro motifs to modulate substrate protein function. We previously found that PIN1 depletion (KD) reduced EMT6 and Lewis Lung Carcinoma (LLC1) cell growth in culture, but increased tumor growth in immunocompetent mice. To explain this dichotomy, we hypothesized that LLC1 KD cells in culture would react differently than control cells to a hypoxic environment that is likely present in tumors. Hypoxia (1% oxygen) for 24h increased KD cell proliferation compared with normoxia, while control cells were unaffected. After 48h, hypoxia reduced control cell proliferation by nearly 30%, whereas proliferation remained elevated in KD cells. The level of proliferating cell nuclear antigen was also reduced by hypoxia in control cells, while its level in KD cells was unaffected. Though hypoxia reduced the amount of the apoptotic marker, cleaved caspase-3, its levels were lower in KD cells compared to controls. The results suggest that PIN1 regulates hypoxic responses such that depletion increases cell proliferation and reduces cell death in response to hypoxia in culture. These effects may explain the increased growth of tumors lacking PIN1, compared with control tumors, in mice.

ATP confers tumorigenic properties to dendritic cells by inducing amphiregulin secretion

AbstractATP, which has an important proinflam-matory action as danger signal, induces the semimaturation of dendritic cells (DCs) that can be associated with immune tolerance. We identified epidermal growth factor receptor ligands as target genes of ATPγS, a slowly hydrolyzed ATP derivative, by a gene profiling approach in DCs. Amphiregulin was the most highly up-regulated gene in response to ATPγS. Human monocyte–derived DCs and mouse bone marrow–derived DCs released amphiregulin (AREG) after purinergic receptor activation, with a contribution of P2Y11 and A2B receptor, respectively. Supernatants of LPS+ATPγS-stimulated DCs induced smooth muscle cell and Lewis Lung Carcinoma (LLC) cell growth in vitro. The coinjection of LPS+ATPγS-stimulated DCs or their supernatants with LLC cells increased tumor weight in mice compared with LPS-treated DCs. The preincubation of LPS+ATPγS-treated DC supernatants with an anti-AREG blocking antibody inhibited their positive effect on smooth muscle cell density and tumor growth. The present study demonstrates for the first time that DCs can be a source of AREG. ATP released from tumor cells might exert a tumorigenic action by stimulating the secretion of AREG from DCs. Antagonists of purinergic receptors expressed on DCs and anti-AREG blocking antibodies could have a therapeutic potential as antitumor agents.

Effect of fasudil on growth, adhesion, invasion, and migration of 95D lung carcinoma cells in vitro

The objective of the study was to investigate the effects of a Rho-kinase inhibitor on 95D lung carcinoma cell growth, adhesion, invasion, and migration and to explore the underlying molecular mechanisms involved in this process. After treatment of 95D lung carcinoma cells with fasudil, an inhibitor of Rho-kinase, cell biological behaviors such as growth, adhesion, invasion, and migration were observed. Matrix metalloproteinase (MMP) activity and Western blot assay were used to evaluate underlying molecular mechanisms. The IC50 of fasudil to 95D lung carcinoma cells was approximately 0.79mg/mL (95% confidence limits 0.58-1.11mg/mL). After treatment with 0.75mg/mL fasudil, the ability of 95D lung carcinoma cells for growth, adhesion, migration, and invasion was decreased significantly. Total active MMP2 was decreased approximately 22.7% (p< 0.05) and total MMP9 65.9% (p< 0.01). Myosin phosphatase target subunit 1 (MYPT1) was reduced by 29.4% (p< 0.05). We conclude that the Rho-kinase inhibitor prevents the growth, adhesion, invasion, and migration of 95D lung carcinoma cells by inhibiting the Rho/Rho-kinase pathway. Changes in MMP2, MMP9, and MYPT1 may be part of its molecular mechanisms.

Evaluation of Cisplatin in Combination with β‐Elemene as a Regimen for Prostate Cancer Chemotherapy

Cisplatin is one of the most potent chemotherapeutic agents for the treatment of many types of solid tumours. Nevertheless, it is not the first-line drug for prostate cancer chemotherapy, because prostate tumour cells exhibit intrinsic and acquired resistance to cisplatin. We have previously demonstrated that β-elemene, a novel plant-derived anti-neoplastic with low toxicity, inhibits lung and ovarian carcinoma cell growth in vitro. In the present study, we explored the therapeutically chemosensitizing effect of β-elemene on cisplatin anti-tumour efficacy in androgen-independent prostate cancer cells as well as the underlying mechanism. β-Elemene significantly increased cisplatin cytotoxicity in the androgen-independent prostate carcinoma cell lines DU145 and PC-3. In addition, β-elemene markedly promoted cisplatin-induced apoptotic cell death in both cell lines, as determined by three different apoptosis assays. β-Elemene augmented the cisplatin-induced activation of caspase-3/7/10 and caspase-9, cleavage of caspase-3 and -9, suppression of Bcl-2 and Bcl-X(L) expression, and release of cytochrome c from mitochondria in these cells. Thus, β-elemene enhancement of cisplatin-induced apoptosis via mitochondrial activation of the caspase-mediated apoptotic pathway may account for the augmented anti-cancer potency of cisplatin in prostate cancer. Cisplatin combined with β-elemene as a chemosensitizer or adjuvant warrants further study and may be potentially useful as a first-line treatment of androgen-independent prostate carcinomas.

Abstract 1223: Fibronectin induced lung cancer cell proliferation is dependent on induction of ILK through integrin B1-mediated activation of p38 MAPK and PI3-K

Abstract Fibronectin is a multi-functional matrix glycoprotein protein that has been linked to tumor progression. We previously demonstrated that cellular fibronectin (cFN) increased non-small cell lung carcinoma (NSCLC) cell growth through signals mediated by the integrin α5β1. Here, we explore the role of integrin-linked kinase (ILK), an integrin β1 subunit cytoplasmic domain interactor in cFN induced cell growth. We found that cFN increases ILK expression and kinase activity, promotes protein-protein interactions between ILK and integrin β1. Silencing of ILK attenuated the effect of cFN on cell proliferation. Knockdown of the integrin β1 subunit, but not the integrin α5, blocked the effect of cFN on ILK expression. In addition, the inhibitor of p38MAPK, SB239063, and the inhibitor of PI3-K, wortmannin, abrogated the effect of cFN on ILK expression, whereas the inhibitor of ERK, PD98059, had no effect. Taken together, our results demonstrate that fibronectin induces the expression of ILK through integrin β1-mediated signals that include p38 MAPK and PI3-K. This in turn, promotes lung carcinoma cell proliferation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1223.

Deuterium-depleted water inhibits human lung carcinoma cell growth by apoptosis

To investigate the in vivo and in vitro inhibitory effects of deuterium-depleted water (DDW) on human lung cancer and the possible mechanisms underlying these effects, we cultured and treated human lung carcinoma cell line A549 and human embryonic lung fibroblasts HLF-1 with various concentrations of DDW from 2 to 72 h. Cellular growth inhibition rates were determined using the 3-(4, 5-dimethyldiazol-2-yl)-2, 5-diphenyltetrazolium-bromide) (MTT) proliferation assay. A549 cells were treated with 50±5 ppm DDW, and the morphology and structure of cells were observed by scanning electron microscopy (SEM). We observed alterations in the cellular skeleton by transmission electron microscopy (TEM) and changes in cell cycle by flow cytometry. Our data showed that DDW significantly inhibited the proliferation of A549 cells at a specific time point, and cells demonstrated the characteristic morphological changes of apoptosis under SEM and TEM. The length of the S phase increased significantly in cells treated with 50 ppm DDW, whereas the G0 to G1 phase and G2 to M phase were decreased. We observed DDW-induced cellular apoptosis using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and DNA fragment analyses. In addition, we established a tumor transplantion model by injecting H460 tumor cells into subcutaneous tissue of BALB/c mice treated with DDW for 60 days. We determined the tumor inhibition rate of treated and control groups and found that the tumor weight was significantly decreased and the tumor inhibition rate was approximately 30% in the DDW group. We conclude that DDW is a promising new anticancer agent with potential for future clinical application.

Nicotine Stimulates PPARβ/δ Expression in Human Lung Carcinoma Cells through Activation of PI3K/mTOR and Suppression of AP-2α

We previously showed that nicotine stimulates non-small cell lung carcinoma (NSCLC) cell proliferation through nicotinic acetylcholine receptor (nAChR)-mediated signals. Activation of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) has also been shown to induce NSCLC cell growth. Here, we explore the potential link between nicotine and PPARbeta/delta and report that nicotine increases the expression of PPARbeta/delta protein; this effect was blocked by an alpha7 nAChR antagonist (alpha-bungarotoxin), by alpha7 nAChR short interfering RNA, and by inhibitors of phosphatidylinositol 3-kinase (PI3K; wortmannin and LY294002) and mammalian target of rapamycin (mTOR; rapamycin). In contrast, this effect was enhanced by PUN282987, an alpha7 nAChR agonist. Silencing of PPARbeta/delta attenuated the stimulatory effect of nicotine on cell growth, which was overcome by transfection of an exogenous PPARbeta/delta expression vector. Of note, nicotine induced complex formation between alpha7 nAChR and PPARbeta/delta protein and increased PPARbeta/delta gene promoter activity through inhibition of AP-2alpha as shown by reduced AP-2alpha binding using electrophoretic gel mobility shift and chromatin immunoprecipitation assays. In addition, silencing of Sp1 attenuated the effect of nicotine on PPARbeta/delta. Collectively, our results show that nicotine increases PPARbeta/delta gene expression through alpha7 nAChR-mediated activation of PI3K/mTOR signals that inhibit AP-2alpha protein expression and DNA binding activity to the PPARbeta/delta gene promoter. Sp1 seems to modulate this process. This study unveils a novel mechanism by which nicotine promotes human lung carcinoma cell growth.

Prostaglandin E2 Stimulates Human Lung Carcinoma Cell Growth through Induction of Integrin-Linked Kinase: The Involvement of EP4 and Sp1

Cyclooxygenase-2-derived prostaglandin E(2) (PGE(2)) stimulates tumor cell growth and progression. However, the mechanisms by which PGE(2) increases tumor growth remain incompletely understood. In studies performed in non-small cell lung carcinoma (NSCLC) cells, we found that PGE(2) stimulates the expression of integrin-linked kinase (ILK). ILK small interfering RNA (siRNA) inhibited the mitogenic effects of PGE(2). In view of its perceived importance, we turned our attention to the mechanisms involved in PGE(2)-induced ILK expression and found that this effect was blocked by an antagonist of the PGE(2) receptor subtype EP4 and by EP4 siRNA. Furthermore, we showed that PGE(2) induction of ILK was associated with phosphorylation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/Akt, which were abrogated by ILK siRNA. Transient transfection, gel mobility shift assays, and chromatin immunoprecipitation experiments showed that PGE(2) induced ILK promoter activity and increased Sp1, although it had no effect on nuclear factor-kappaB and AP-2 DNA-binding activity. Blockade of Sp1 abrogated the effect of PGE(2) on expression of ILK and promoter activity and on cell growth. In summary, our observations show that PGE(2) increases NSCLC cell growth through increased ILK expression, which is dependent on EP4 signaling and on induction of Sp1 protein and Sp1 DNA-binding activity in the ILK promoter. These studies suggest a novel molecular mechanism by which PGE(2) stimulates NSCLC cell growth and unveils a new molecular target for the development of therapies against NSCLC.

Relation between Cyclooxygenase-2 and Polo-like Kinase-1 in Non-Small Cell Lung Cancer

Background: Elevated expression of cyclooxygenase-2 (COX-2) and Polo-like kinase-1 (PLK-1) is observed in a wide variety of cancers. Augmented expression of COX-2 and enhanced production of prostaglandin E2 (PGE2) are associated with increased tumor cell survival and malignancy; COX-2 has been implicated in the control of human non-small cell lung carcinoma (NSCLC) cell growth. PLK-1 siRNA induced the cell death of lung cancer cells and the systemic administration of PLK-1 siRNA/atelocollagen complex inhibited the growth of lung cancer in a liver metastatic murine model. COX-2 and PLK-1 are involved in proliferation and in cell cycle regulation, and there is a significant correlation between their interaction in prostate carcinoma. Methods: In this study, we investigated the pattern of COX-2 and PLK-1 expression in NSCLC, after treatment with IL-1β, COX-2 inhibitor and PLK-1 siRNA. Results: Expression of PLK-1 was decreased in A549 COX-2 sense cells, and was increased in A549 COX-2 anti-sense cells. Knock out of PLK-1 expression by PLK-1 siRNA augmented COX-2 expression in A549 and NCl-H157 cells. When A549 and NCI-H157 cells were treated with COX-2 inhibitor on a dose-dependent basis, PLK-1 and COX-2 were reduced. However, when the expression of COX-2 was induced by IL-1β, the production of PLK-1 decreased. Conclusion: These results demonstrate that COX-2 and PLK-1 are regulated and inhibited by each other in NSCLC, and suggest that these proteins have a reverse relationship in NSCLC.

Fish Oil Inhibits Human Lung Carcinoma Cell Growth by Suppressing Integrin-Linked Kinase

We previously showed that synthetic peroxisome proliferator-activated receptor gamma (PPARgamma) ligands inhibit non-small cell lung carcinoma (NSCLC) cell growth through multiple signaling pathways. Here, we show that dietary compounds, such as fish oil (which contains certain kinds of fatty acids like omega3 and omega6 polyunsaturated fatty acids), also inhibit NSCLC cell growth by affecting PPARgamma and by inhibiting the expression of integrin-linked kinase (ILK). Exogenous expression of ILK overcame, whereas silencing ILK enhanced the inhibitory effect of fish oil on cell growth. The inhibitor of p38 mitogen-activated protein kinase, SB239023, abrogated the inhibitory effect of fish oil on ILK expression, whereas the inhibitor of extracellular signal-regulated kinase, PD98059, had no effect. Transient transfection experiments showed that fish oil reduced ILK promoter activity, and this effect was abolished by AP-2alpha small interfering RNA and SB239023 and by deletion of a specific portion of the ILK gene promoter. Western blot analysis and gel mobility shift assay showed that fish oil significantly induced AP-2alpha protein expression and AP-2 DNA-binding activity in the ILK gene promoter and that this was dependent on PPARgamma activation. Blockade of AP-2alpha abrogated the effect of fish oil on ILK expression and on cell growth, whereas exogenous expression of AP-2alpha enhanced cell growth in the setting of fish oil exposure. Taken together, these findings show that fish oil inhibits ILK expression through activation of PPARgamma-mediated and p38 mitogen-activated protein kinase-mediated induction of AP-2alpha. In turn, this leads to inhibition of NSCLC cell proliferation. This study unveils a novel mechanism by which fish oil inhibits human lung cancer cell growth.

Adenovirus-mediated ING4 expression suppresses lung carcinoma cell growth via induction of cell cycle alteration and apoptosis and inhibition of tumor invasion and angiogenesis

Previous studies demonstrated that ING4 as a novel member of ING (inhibitor of growth) family has potential effect on tumor inhibition via multiple pathways. However, adenovirus-mediated ING4 expression in inhibition of human tumors has not been reported. To explore its therapeutic effect on human lung carcinoma, we constructed a recombinant adenoviral vector Ad-ING4 expressing the humanized ING4 gene derived from murine ING4 with two amino acid modifications at residue 66 (Arg to Lys) and 156 (Ala to Thr) by site-directed mutagenesis. We demonstrated that Ad-ING4-mediated transfection of A549 human lung carcinoma cells induced cell apoptosis, altered cell cycle with S phase reduction and G2/M phase arrest, suppressed cell invasiveness, and down-regulated IL-6, IL-8, MMP-2, and MMP-9 expression of transfected tumor cells. In athymic mice bearing A549 lung tumors, intratumoral injections of Ad-ING4 suppressed the tumor growth and reduced the tumor microvessel formation. Therefore, Ad-ING4 may be useful in gene therapy of human lung carcinoma.

Prevention of local tumor growth with paclitaxel-loaded microspheres

Lung cancer is associated with a significant rate of locoregional recurrence after surgical resection, particularly when nonanatomic wedge resections are performed. The primary aim of this study was to assess the feasibility of a microsphere drug delivery system to locally deliver chemotherapy and prevent the establishment and growth of lung cancer cells and establish proof of concept for a potential future approach to target occult microscopic disease remaining at the surgical resection margin. Poly-(D,L-lactic-co-glycolic acid) (PLGA) microspheres loaded with the antineoplastic agent paclitaxel were prepared and tested for antitumor efficacy in an in vitro cell proliferation assay for tumor inhibition and induction of apoptosis. The in vivo prevention of Lewis lung carcinoma cell establishment and growth in subcutaneous tissues of mice was also assessed by comparing 4 treatment groups: Lewis lung carcinoma cells alone, Lewis lung carcinoma cells combined with 100 x 10(6) unloaded (carrier alone) PLGA microspheres, and Lewis lung carcinoma cells combined with 50 x 10(6) or 100 x 10(6) paclitaxel-loaded PLGA microspheres. After the coinjection of Lewis lung carcinoma cells with or without microspheres, in vivo tumor growth was monitored, and tumor weight was recorded on death. Paclitaxel-loaded PLGA microspheres were found to effectively prevent growth of tumor cells in culture through the induction of apoptosis. Similarly, paclitaxel-loaded PLGA microspheres significantly inhibited tumor growth in vivo at both the 50 x 10(6) and 100 x 10(6) microsphere dose (0.497 +/- 0.183 and 0.187 +/- 0.083 g total tumor weight, respectively) compared with 2.91 +/- 0.411 g for Lewis lung carcinoma cells with unloaded microspheres and 3.37 +/- 0.433 g for untreated tumor (P < .001). Toxicity was not clinically apparent in any animal treated with paclitaxel-loaded PLGA microspheres. Paclitaxel-loaded PLGA microspheres induce tumor apoptosis and inhibit the establishment and growth of lung cancer cells both in vitro and in vivo without obvious systemic toxicity. By using models consistent with localized microscopic tumor burdens, these results suggest that local delivery of paclitaxel through a microsphere system might lead to an effective future method of decreasing local tumor recurrence in non-small cell lung cancer when applied to the surgical margins at risk for microscopic tumor foci. Such an approach might be particularly efficacious after wedge resection in the setting of poor pulmonary reserve or significant comorbidity, where local recurrence rates are increased and acceptable alternative treatment options are limited.

PPARβ/δ agonist stimulates human lung carcinoma cell growth through inhibition of PTEN expression: the involvement of PI3K and NF-κB signals

Recent studies suggest that activation of peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) promotes cancer cell survival. We previously demonstrated that a selective PPARbeta/delta agonist, GW501516, stimulated human non-small cell lung carcinoma (NSCLC) cell growth. Here, we explore the mechanisms responsible for this effect. We show that GW501516 decreased phosphate and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor known to decrease cell growth and induce apoptosis. Activation of PPARbeta/delta and phosphatidylinositol 3-kinase (PI3K)/Akt signaling was associated with inhibition of PTEN. GW501516 increased NF-kappaB DNA binding activity and p65 protein expression through activation of PPARbeta/delta and PI3K/Akt signals and enhanced the physical interactions between PPARbeta/delta and p65 protein. Conversely, inhibition of PI3K and silencing of p65 by small RNA interference (siRNA) blocked the effect of GW501516 on PTEN expression and on NSCLC cell proliferation. GW501516 also inhibited IKBalpha protein expression. Silencing of IKBalpha enhanced the effect of GW501516 on PTEN protein expression and on cell proliferation. It also augmented the GW501516-induced complex formation of PPARbeta/delta and p65 proteins. Overexpression of PTEN suppressed NSCLC cell growth and eliminated the effect of GW501516 on phosphorylation of Akt. Together, our observations suggest that GW501516 induces the proliferation of NSCLC cells by inhibiting the expression of PTEN through activation of PPARbeta/delta, which stimulates PI3K/Akt and NF-kappaB signaling. Overexpression of PTEN overcomes this effect and unveils PPARbeta/delta and PTEN as potential therapeutic targets in NSCLC.

Stimulation of lung carcinoma cell growth by fibronectin–integrin signalling

Throughout many countries, lung cancer will kill more people this year than malignancies related to breast, prostate, colon, liver, kidney and melanoma combined. Despite recent advances in understanding the molecular biology of lung carcinoma and the introduction of multiple new chemotherapeutic agents for its treatment, its dismal five-year survival rate (<15%) has not changed substantially. The lack of advancement in this area reflects the limited knowledge available concerning the factors that promote oncogenic transformation and proliferation of carcinoma cells in the lung. Malignant transformation plays a key role in tumor growth and invasion; however, other factors such as the surrounding stroma, local growth factors, vascularity, and systemic hormones are important contributors as well. We believe that the composition of the lung extracellular matrix is also important due to its ability to affect malignant cell behavior in vitro. The matrix glycoprotein fibronectin, for example, is highly expressed in chronic lung disorders where most lung carcinomas are identified. This document reviews information that implicates fibronectin in the stimulation of lung carcinoma cell growth. Data available to date indicate that by binding to specific integrin receptors expressed on the surface of tumor cells, fibronectin stimulates intracellular signals implicated in the pathobiology of lung carcinogenesis and lung tumor chemoresistance including mitogen-activated protein kinases, GTPases, and the PI3-kinase/Akt/mTOR pathway. Thus, integrin-mediated signals triggered by fibronectin in tumor cells represent promising targets for the development of novel anti-cancer strategies.

M3 Muscarinic Receptor Antagonists Inhibit Small Cell Lung Carcinoma Growth and Mitogen-Activated Protein Kinase Phosphorylation Induced by Acetylcholine Secretion

The importance of acetylcholine as a neurotransmitter in the nervous system is well established, but little is yet known about its recently described role as an autocrine and paracrine hormone in a wide variety of nonneuronal cells. Consistent with the expression of acetylcholine in normal lung, small cell lung carcinoma (SCLC) synthesize and secrete acetylcholine, which acts as an autocrine growth factor through both nicotinic and muscarinic cholinergic mechanisms. The purpose of this study was to determine if interruption of autocrine muscarinic cholinergic signaling has potential to inhibit SCLC growth. Muscarinic receptor (mAChR) agonists caused concentration-dependent increases in intracellular calcium and mitogen-activated protein kinase (MAPK) and Akt phosphorylation in SCLC cell lines. The inhibitory potency of mAChR subtype-selective antagonists and small interfering RNAs (siRNAs) on acetylcholine-increased intracellular calcium and MAPK and Akt phosphorylation was consistent with mediation by M3 mAChR (M3R). Consistent with autocrine acetylcholine secretion stimulating MAPK and Akt phosphorylation, M3R antagonists and M3R siRNAs alone also caused a decrease in basal levels of MAPK and Akt phosphorylation in SCLC cell lines. Treatment of SCLC cells with M3R antagonists inhibited cell growth both in vitro and in vivo and also decreased MAPK phosphorylation in tumors in nude mice in vivo. Immunohistochemical staining of SCLC and additional cancer types showed frequent coexpression of acetylcholine and M3R. These findings suggest that M3R antagonists may be useful adjuvants for treatment of SCLC and, potentially, other cancers.