Cancer stem cell phenotype is supported by secretory phospholipase A2 in human lung cancer cells.

BACKGROUND: Lung cancer stem cells (CSCs) are a subpopulation of cells that drive growth, invasiveness, and resistance to therapy. Inflammatory eicosanoids are critical to maintain this malignant subpopulation. Secretory phospholipase A2 group IIa (sPLA2) is an important mediator of the growth and invasive potential of human lung cancer cells and regulates eicosanoid production. We hypothesized that sPLA2 plays a role in the maintenance of lung CSCs. METHODS: Cancer stem cells from lung adenocarcinoma cell lines H125 and A549 were isolated using aldehyde dehydrogenase activity and flow cytometry. Protein and mRNA levels for sPLA2 were compared between sorted cells using Western blotting and quantitative reverse transcriptase-polymerase chain reaction techniques. Chemical inhibition of sPLA2 and short-hairpin RNA knockdown of sPLA2 were used to evaluate effects on tumorsphere formation. RESULTS: Lung CSCs were isolated in 8.9%±4.1% (mean±SD) and 4.1%±1.6% of H125 and A549 cells respectively. Both sPLA2 protein and mRNA expression were significantly elevated in the CSC subpopulation of H125 (p=0.002) and A549 (p=0.005; n=4). Knockdown of sPLA2 significantly reduced tumorsphere formation in H125 (p=0.026) and A549 (p=0.001; n=3). Chemical inhibition of sPLA2 resulted in dose-dependent reduction in tumorsphere formation in H125 (p=0.003) and A549 (p=0.076; n=3). CONCLUSIONS: Lung CSCs express higher levels of sPLA2 than the non-stem cell population. Our findings that viral knockdown and chemical inhibition of sPLA2 reduce tumorsphere formation in lung cancer cells demonstrate for the first time that sPLA2 plays an important role in CSCs. These findings suggest that sPLA2 may be an important therapeutic target for human lung cancer.

Serum detection of thymidine kinase 1 as a means of early detection of lung cancer.

BACKGROUND: Thymidine kinase 1 (TK1) is a biomarker elevated in several malignancies, including lung cancer. Up-regulation of TK1 is an early event in carcinogenesis and therefore a target for early cancer detection. We have developed a novel Enzyme Linked Immunosorbent Assay (ELISA) to detect TK1 in serum. MATERIALS AND METHODS: Forty patients with pulmonary nodules and 18 healthy individuals had their serum collected prior to surgery. All samples were analyzed using a radioassay and ELISA. RESULTS: TK1 was significantly elevated in all lung cancer samples. Patients with stage I (n=16) and stage II (n=17) disease had significantly higher TK1 levels than controls. The area under the curve was 0.792, using 4.9 nM TK1 as cut-off, for early-stage lung cancer. The sensitivity and specificity were 75.0 and 83.3, respectively. TK1 concentration was a more sensitive and accurate indicator of lung cancer than TK1 activity. CONCLUSION: TK1 is significantly elevated in serum from patients with stage I and stage II lung cancer as measured using the established ELISA. This novel TK1 ELISA is both sensitive and specific for the detection of early-stage and advanced lung cancer, and therefore may be an important tool in the management of this disease.

Secretory phospholipase A2 mediates human esophageal adenocarcinoma cell growth and proliferation via ERK 1/2 pathway.

BACKGROUND/AIM: Secretory phospholipase-A2 (sPLA2) mediates growth and proliferation of human esophageal adenocarcinoma cells (HEAC). Two major molecular pathways of cancer growth regulation include extracellular signal-regulated kinase 1/2 (ERK 1/2) and protein kinase-B (AKT). Phospholipase enzymes have been demonstrated to significantly influence these two growth regulating pathways in other tumor cells. We hypothesize sPLA2 to mediate HEAC growth control through ERK 1/2 and AKT. MATERIALS AND METHODS: Verified HEAC (FLO-1), treated with either the mitogen-activated protein kinase kinase selective inhibitor (PD98059) or with group IIa sPLA2-specific inhibitor, were assessed for ERK1/2 phosphorylation and AKT activation after tumor necrosis factor-alpha stimulation, as well as for viability, proliferation, and apoptosis responses. RESULTS: PD98059 significantly inhibited ERK 1/2 activation, reduced cell viability, and reduced proliferation (p<0.05). sPLA2 inhibition attenuated ERK 1/2 activation (p<0.01) but had no effect on AKT activation or apoptosis. CONCLUSION: Specific inhibition of group IIa sPLA2 directly reduces HEAC viability and proliferation by attenuating ERK 1/2 activation with no effect on AKT activation or apoptosis. This may indicate that the mechanism of action of sPLA2 is mainly the induction of growth arrest.

Knockdown of secretory phospholipase A2 IIa reduces lung cancer growth in vitro and in vivo.

OBJECTIVE: Group IIa secretory phospholipase A2 (sPLA2 IIa) plays a role in the malignant potential of several epithelial cancers. Nuclear factor kappa B (NF-κB) regulates cancer cell growth and is modulated by phospholipase activity in many cancer cells. We hypothesized that knockdown of sPLA2 in lung cancer cells would reduce cell proliferation and NF-κB activity in vitro and attenuate tumor growth in vivo. METHODS: Two human non-small cell lung cancer cell lines (A549 and H358) were transduced with short hairpin RNA targeting sPLA2 group IIa. Quantitative reverse transcriptase-polymerase chain reaction and immunoblotting confirmed knockdown of sPLA2 IIa messenger RNA and protein, respectively. Cell proliferation was evaluated by the 5-bromo-2'-deoxyuridine DNA labeling assay. NF-κB phosphorylation was assayed by western blot. 1 × 10(6) of A549 or A549 sPLA2 knockdown cells were injected into the left flanks of nude mice (aged 6 to 8 weeks). Tumors were followed for 23 days, then removed and stained with hematoxylin and eosin, stained with Ki-67, and analyzed for sPLA2 IIa messenger RNA expression. RESULTS: sPLA2 knockdown reduced NF-κB phosphorylation and tumor growth in vivo. A549 wild-type tumors grew twice as fast as knockdown tumors. Ki-67 staining was more prominent throughout the wild-type tumors compared with knockdown tumors. Explanted knockdown tumors maintained lower sPLA2 levels compared with wild-type, confirmed by reverse transcriptase-polymerase chain reaction. CONCLUSIONS: Knockdown of sPLA2 IIa suppresses lung cancer growth in part by attenuating NF-κB activity. These findings justify further investigation into the cellular mechanisms of sPLA2 in lung cancer and its potential role as a therapeutic target.

Radiation induces osteogenesis in human aortic valve interstitial cells.

OBJECTIVE: Irradiation of the chest or chest wall has been shown to cause calcific aortic stenosis. However, the mechanisms are unknown. Aortic valve interstitial cells have been implicated in the pathogenesis of aortic stenosis; they have been shown to change from the phenotype of a myofibroblast to an osteoblastlike cell. We therefore hypothesized that irradiation of human aortic valve interstitial cells induces an osteogenic phenotype. In isolated human aortic valve interstitial cells, our purpose was to determine the effect of irradiation on the production of osteogenic factors: (1) bone morphogenetic protein 2, (2) osteopontin, (3) alkaline phosphatase, and (4) the transcription factor Runx2. METHODS: Human aortic valve interstitial cells were isolated from normal aortic valves obtained from explanted hearts of patients undergoing cardiac transplantation (n = 4) and were grown in culture. The cells were grown to confluence, irradiated with 10 Gy using a cesium-137 irradiator, and then lysed 24 hours after irradiation. Cell lysates were analyzed via immunoblot and densitometry for bone morphogenetic protein 2, osteopontin, alkaline phosphatase, and Runx2. Statistical analysis was performed using analysis of variance, with P < .05 indicating significance. RESULTS: Irradiation induced an osteogenic phenotype in human aortic valve interstitial cells. Irradiation induced a 2-fold increase in bone morphogenetic protein 2, a 7-fold increase in osteopontin, a 3-fold increase in alkaline phosphatase, and a 2-fold increase in Runx2. CONCLUSIONS: Radiation induces an osteogenic phenotype in human aortic valve interstitial cells. The irradiated cells had a significantly increased expression of the osteogenic factors bone morphogenetic protein 2, osteopontin, alkaline phosphatase, and Runx2. These data offer mechanistic insight into the pathogenesis of radiation-induced valvular heart disease.

Group IIa secretory phospholipase expression correlates with group IIa secretory phospholipase inhibition-mediated cell death in K-ras mutant lung cancer cells.

OBJECTIVE: There are currently no targeted therapies against lung tumors with oncogenic K-ras mutations that are found in 25% to -40% of lung cancers and are characterized by their resistance to epidermal growth factor receptor inhibitors. The isozyme group IIa secretory phospholipase A(2) (sPLA(2)IIa) is a potential biomarker and regulator of lung cancer cell invasion; however, the relationship between K-ras mutations and sPLA(2)IIa has yet to be investigated. We hypothesize that sPLA(2)IIa modulates lung cancer cell growth in K-ras mutant cells and that sPLA(2)IIa expression in human lung tumors is increased in K-ras mutant tumors. METHODS: Baseline sPLA(2)IIa expression in K-ras mutant lung cancer cell lines (A549, SW1573, H358, H2009) was assessed. Cells were treated with a specific sPLA(2)IIa inhibitor and evaluated for apoptosis and cell viability. Nuclear factor kappa-b (NF-κB) and extracellular signal-regulated kinase 1/2 activity were detected by Western blot. Human tumor samples were evaluated for sPLA(2)IIa mRNA expression by quantitative reverse-transcription polymerase chain reaction. RESULTS: Cytotoxicity of sPLA(2)IIa inhibition correlates with sPLA(2)IIa expression. Apoptosis in response to sPLA(2) inhibition parallels attenuation in NF-κB activity. In addition, sPLA(2)IIa expression in human tumors correlates with squamous cell pathology and increasing stage of K-ras mutant lung tumors. CONCLUSIONS: Baseline sPLA(2)IIa expression predicts response to sPLA(2)IIa inhibition in some K-ras mutant lung cancer cells. This finding is independent of p53 mutation status. Furthermore, squamous tumors and advanced-stage K-ras mutant tumors express more sPLA(2)IIa. These data support a role for sPLA(2)IIa as a potential global therapeutic target in the treatment of lung cancer.

The u and the sushi roll: a conceptual aid for lichtenstein hernia repair.

For surgical trainees, perfecting a systematic approach to open inguinal herniorrhaphy can be complicated by the difficulty of conceptualizing hernias in relationship to the relatively complex anatomy of the inguinal canal. Open inguinal hernia repair is a common general surgery operation and a precise understanding of the operation is essential for residents. We present a systematic approach to this operation that uses the U and sushi roll technique as a conceptual aid to understand inguinal anatomy and a method of hernia repair.

Group IIa sPLA2 inhibition attenuates NF-κB activity and promotes apoptosis of lung cancer cells.

BACKGROUND/AIM: Group IIa secretory phospholipase A2 (sPLA2 IIa) has been implicated in the regulation of metastasis of non-small cell lung cancer (NSCLC) and the present study investigates its contribution to lung cancer growth and progression. PLA2s initiate signaling in several pathways that mediate cell survival including phosphatidylinositol 3-kinase-AKT (PI3K-AKT), p38 mitogen-activated protein kinase (p38 MAPK), extracellular-signal-regulated kinase (ERK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). MATERIALS AND METHODS: Human NSCLC cell lines (A549 and NCI-H358) were treated with a specific sPLA2 IIa inhibitor. Cells were assayed for apoptosis, viability, proliferation and changes in cell morphology. Effects on AKT, p38 MAPK, ERK1/2 and NF-κB signaling pathways were investigated. RESULTS: sPLA2 IIa inhibition reduced proliferation and increased apoptosis. NF-κB activity was attenuated, whereas AKT, ERK1/2, p38 MAPK were variably affected by sPLA2 IIa inhibition. NF-κB inhibitor-associated apoptosis confirmed the dominant role of NF-κB. CONCLUSION: sPLA2 IIa attenuates growth and promotes apoptosis predominantly via its effects on NF-κB activity.

Biglycan induces the expression of osteogenic factors in human aortic valve interstitial cells via Toll-like receptor-2.

OBJECTIVE: Although biglycan (BGN) and oxidized low-density lipoprotein (oxLDL) accumulation has been observed in calcific, stenotic aortic valves, their role in the pathogenesis of calcific aortic valve disease is poorly understood. We hypothesized that soluble BGN induces the osteogenic response in human aortic valve interstitial cells via Toll-like receptor (TLR) 2 and TLR4 and mediates the proosteogenic effect of oxLDL. METHODS AND RESULTS: Aortic valve interstitial cells of stenotic valves express higher levels of BGN. Stimulation of cells from normal valves with BGN increased the expression of bone morphogenetic protein-2 (BMP-2) and alkaline phosphatase (ALP) among the chondrogenic/osteogenic markers examined and caused accumulation of calcium deposits. TLR2 silencing, but not TLR4 silencing, reduced BMP-2 and ALP levels after BGN stimulation although coimmunoprecipitation revealed that BGN interacts with both TLR2 and TLR4. BGN induced the phosphorylation of extracellular signal-regulated protein kinase-1/2, p38 mitogen-activated protein kinase and nuclear factor-κB. Inhibition of extracellular-regulated kinase-1/2 markedly reduced the upregulation of BMP-2 and ALP expression by BGN whereas inhibition of p38 mitogen-activated protein kinase or nuclear factor-κB had a moderate effect. Stimulation of aortic valve interstitial cells with oxLDL upregulated BGN expression and release. Knockdown and neutralization of BGN reduced the effect of oxLDL on BMP-2 and ALP expression. CONCLUSIONS: Extracellular soluble BGN induces the expression of BMP-2 and ALP in human aortic valve interstitial cells primarily via TLR2 and contributes to the proosteogenic effect of oxLDL. These findings highlight the potential role of soluble BGN and oxLDL in the development of calcific aortic valve disease.

Techniques of bronchial sleeve resection.

A bronchial sleeve resection can be considered for lesions arising from a lobar bronchus so as to preclude a standard lobectomy, yet without enough distal involvement as to warrant a pneumonectomy. Limited bronchial resection allows maximal conservation of pulmonary function in patients with benign or malignant disease, without compromising oncologic outcome. This article defines the indications and preoperative management of candidate patients and discusses key anesthetic considerations and surgical techniques for this complex airway reconstruction. The essential component of a successful operation is a tension-free bronchial anastomosis. Open communication and careful discussion of airway management between anesthesiologist and surgeon will help ensure a good outcome.

Surgical treatment of atypical mycobacterial infections.

Surgical evaluation in nontuberculous mycobacterial (NTM) infections plays an essential role as part of multidisciplinary management of this complex pulmonary process. Resection of damaged lung parenchyma combined with appropriate antimicrobial therapy may interrupt a cycle of disease progression and relapse in select patients. Relevant technical considerations for managing both minimally invasive and open anatomic resection in this unique population are discussed. Results of anatomic resection of NTM damaged lung in the modern era are also summarized.

Thoracoscopic lobectomy and segmentectomy for infectious lung disease.

BACKGROUND: The potential benefits of thoracoscopic lobectomy and segmentectomy for early stage non-small cell lung cancer have been well documented in the literature. However, little is known about the use of these techniques in patients requiring resection for infectious or inflammatory lung disease. METHODS: Using a prospectively collected database, we performed a retrospective review of consecutive operations from July 2004 to June 2010. All patients who underwent elective thoracoscopic lobectomy or segmentectomy for focal bronchiectasis or cavitary lung disease associated with active pulmonary infection were included. RESULTS: In all, 212 resections were performed in 171 patients. The average age was 59 years (range, 26 to 82 years). Patients were predominately white (93%) and female (93%). Indications for surgery included recurrent active infection, hemoptysis, or antibiotic intolerance associated with focal bronchiectasis (86%), cavitary disease (7%), or both (7%). Operations included 126 lobectomies, 73 segmentectomies, 10 lobe plus segmental resections, and 3 bilobectomies. Conversion to thoracotomy occurred in 10 patients. The operative mortality rate was zero. Complications occurred in 9%, consisting largely of prolonged air leak and atrial fibrillation. The mean hospital length of stay was 3.7 days. CONCLUSIONS: Thoracoscopic lobectomy and segmentectomy for individuals with infectious lung disease can be accomplished safely with minimal morbidity and mortality. These techniques may provide the optimal surgical approach for patients with focal bronchiectasis or cavitary lung disease requiring resection.

Assessment of the relationship between Contegra conduit size and early valvar insufficiency.

BACKGROUND: Contegra bovine jugular vein (BJV) conduit results vary widely, and little attention has been directed at assessment of early conduit insufficiency. Conduit insufficiency is graded subjectively, and criteria vary. Several studies have used branch pulmonary artery flow reversal (BPAFR) to define severe conduit insufficiency. BJV valves are larger than human pulmonary valves of similar diameter. We hypothesize that anatomic differences between BJV and human pulmonary valves limit the use of BPAFR in the evaluation of BJV competence. Our purposes were to (1) assess the prevalence of early and 6-month BJV conduit insufficiency in our patients, (2) determine if conduit size affects BJV competence, and (3) determine if BPAFR is a specific discriminator of severe conduit insufficiency. METHODS: We reviewed 135 BJV conduits. One cardiologist blinded to original reports reviewed postoperative and 6-month echocardiograms. Conduits were grouped by size: group 1, 12 to 14 mm (n=51), and group 2, 16 to 22 mm (n=84). Moderate or greater insufficiency was considered clinically significant. RESULTS: Early conduit insufficiency was common in group 1 (37%) and rare in group 2 (5%, p<0.0001). After excluding conduits with significant insufficiency, BPAFR occurred in 18% (group 1, 27%; group 2, 13%; p=0.02). At follow-up, insufficiency worsened in group 1 but was stable in group 2. CONCLUSIONS: Early conduit insufficiency is common and worsens with follow-up in small BJVs. Conduit insufficiency is limited in larger sizes and remains stable. BJV exhibits BPAFR commonly in the absence of significant conduit insufficiency. BPAFR should not be used as a primary criterion for grading insufficiency in BJV conduits.

Lung cancer cell invasion and expression of intercellular adhesion molecule-1 (ICAM-1) are attenuated by secretory phospholipase A2 inhibition.

OBJECTIVE: Invasive lung tumors are associated with intercellular adhesion molecule-1 (ICAM-1) expression. Secretory phospholipase A(2) (sPLA(2)) enzymes produce inflammatory mediators that stimulate ICAM-1 expression, and upregulation of PLA(2) activity can enhance metastasis. We hypothesize a link between sPLA(2) activity, ICAM-1 expression, and tumor cell invasion. We propose that inhibition of sPLA(2) modulates ICAM-1 expression in cancer cells and attenuates their invasiveness. METHODS: Human lung adenocarcinoma cells (A549) were treated with an ICAM-1 blocking antibody and assayed for invasion. Lung cancer cells (A549 and H358) were then treated with an sPLA(2) inhibitor and evaluated by immunoblotting for ICAM-1 expression. Next cells (A549) treated with sPLA(2) inhibitor were assayed for invasion. Finally, sPLA(2) messenger RNA and protein expression were evaluated by quantitative reverse-transcriptase polymerase chain reaction and immunofluorescence microscopy, respectively. Statistical analysis was performed by the Student t test or analysis of variance, as appropriate. RESULTS: Antibody blockade of ICAM-1 decreased lung cancer cell invasion. sPLA(2) inhibition significantly reduced ICAM-1 expression and invasion. sPLA(2) inhibition also significantly decreased sPLA(2) mRNA expression and immunofluorescent staining of sPLA(2). CONCLUSIONS: sPLA(2) plays a significant role in mediating the inflammatory signals that induce ICAM-1 expression in lung cancer cells. Inhibition of the enzyme can significantly decrease ICAM-1 expression and subsequent cancer cell invasion. This lays the groundwork for further investigation into the cellular mechanisms of sPLA(2) and its role in lung cancer.

Statin therapy attenuates growth and malignant potential of human esophageal adenocarcinoma cells.

OBJECTIVES: Esophageal adenocarcinoma is an aggressive malignancy generally diagnosed after metastatic spread and currently lacks effective medical therapy. Expression of intracellular adhesion molecule-1 (ICAM-1) is an adverse prognostic indicator in various human tumor cells and contributes significantly to their metastatic potential. Statin therapy reduces circulating ICAM-1 levels in patients with coronary heart disease and is associated with reduction in progression from Barrett esophagus to esophageal adenocarcinoma. We hypothesize that statin therapy may attenuate growth and malignant potential via ICAM-1 expression and nuclear factor-kappa beta activation in human esophageal adenocarcinoma cells. METHODS: Verified human esophageal adenocarcinoma cells (FLO-1) were treated with simvastatin, atorvastatin, or pravastatin (10-, 30-, and 50-µmol/L concentrations). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide viability, 5-bromo-2'-deoxyuridine proliferation, or annexin V apoptosis assays were performed, or cells were stimulated with tumor necrosis factor-alpha and collected for immunoblotting and flow cytometry. RESULTS: Simvastatin decreased cell viability and proliferation while increasing apoptosis in a dose-dependent manner (P < .05). Simvastatin attenuated total cellular and cell-surface ICAM-1 expression as well as nuclear factor-kappa beta activation (P < .05). Atorvastatin had mild effects and pravastatin had essentially no effect on growth and metastatic potential of these cells. CONCLUSIONS: We demonstrate that treatment of human esophageal adenocarcinoma cells with simvastatin attenuates growth, by decreasing cell viability, decreasing cell proliferation, and increasing apoptosis, and attenuates metastatic potential, by decreasing expression of key metastatic markers. These findings identify simvastatin as a potential therapeutic and chemopreventive modality to thwart the progression of esophageal adenocarcinoma.

Lady Windermere revisited: treatment with thoracoscopic lobectomy/segmentectomy for right middle lobe and lingular bronchiectasis associated with non-tuberculous mycobacterial disease.

OBJECTIVE: Lady Windermere syndrome is a well-known but poorly understood female predominant phenotype of isolated right middle lobe and lingular bronchiectasis associated with non-tuberculous mycobacterial (NTM) infection. Despite lengthy multidrug antibiotic treatment, the presence of damaged parenchymal tissue leads to symptomatic disease recurrence, often with resistant organisms. The use of surgical resection as an adjunct to medical therapy may alter this cycle, although little is known about the use of thoracoscopic lung resection in this patient population. METHODS: This is a retrospective review of a prospectively collected database of patients with pulmonary NTM disease from July 2004 to December 2009. All patients had focal bronchiectasis of the right middle lobe and lingula, treated with targeted antimicrobial therapy for several months prior to resection. RESULTS: A total of 134 patients underwent 172 operations, with 38 patients having staged bilateral resections. The cohort was predominately female (96%) and Caucasian (95%), with a mean age of 59 years (range 34-81 years). Using a thoracoscopic approach in all patients, 102 middle lobectomies and 70 lingulectomies were performed. Conversion to open thoracotomy occurred in five cases (3%). Secondary procedures were performed in 20 cases (12%). There was no operative mortality. Postoperative morbidity was noted following 12 operations (7%), primarily consisting of prolonged air leak. The mean length of stay was 3.3 days (range 1-15 days). CONCLUSIONS: Although medical therapy remains the primary treatment modality for patients with pulmonary NTM disease, the selective use of pulmonary resection may reduce the incidence of symptomatic disease recurrence. The addition of thoracoscopic resection to treatment regimens for patients with Lady Windermere syndrome can be accomplished with minimal morbidity and mortality.