Chronic Inhalation Exposure to Antimony Trioxide Exacerbates the MAPK Signaling in Alveolar Bronchiolar Carcinomas in B6C3F1/N Mice.

Antimony trioxide (AT) is used as a flame retardant in fabrics and plastics. Occupational exposure in miners and smelters is mainly through inhalation and dermal contact. Chronic inhalation exposure to AT particulates in B6C3F1/N mice and Wistar Han rats resulted in increased incidences and tumor multiplicities of alveolar/bronchiolar carcinomas (ABCs). In this study, we demonstrated Kras (43%) and Egfr (46%) hotspot mutations in mouse lung tumors (n = 80) and only Egfr (50%) mutations in rat lung tumors (n = 26). Interestingly, there were no differences in the incidences of these mutations in ABCs from rats and mice at exposure concentrations that did and did not exceed the pulmonary overload threshold. There was increased expression of p44/42 mitogen-activated protein kinase (MAPK) (Erk1/2) protein in ABCs harboring mutations in Kras and/or Egfr, confirming the activation of MAPK signaling. Transcriptomic analysis indicated significant alterations in MAPK signaling such as ephrin receptor signaling and signaling by Rho-family GTPases in AT-exposed ABCs. In addition, there was significant overlap between transcriptomic data from mouse ABCs due to AT exposure and human pulmonary adenocarcinoma data. Collectively, these data suggest chronic AT exposure exacerbates MAPK signaling in ABCs and, thus, may be translationally relevant to human lung cancers.

PolyI:C suppresses TGF-ß1-induced Akt phosphorylation and reduces the motility of A549 lung carcinoma cells.

BACKGROUNDS: Epithelial mesenchymal transition (EMT) is a critical process involved in the invasion and metastasis of cancer, including lung cancer (LC). Transforming growth factor (TGF)-ß is one of factors capable of inducing EMT. Polyinosinic-polycytidylic acid (polyI:C), a synthetic agonist for toll-like receptor (TLR) 3, can enhance immune responses and has been used as an adjuvant for cancer vaccines; however, it remains unclear whether it influences other process, such as EMT. In the present study, we examined the effects of polyI:C on TGF-ß-treated A549 human LC cells. METHODS AND RESULTS: By in vitro cell proliferation assay, polyI:C showed no effect on the growth of A549 cells treated with TGF-ß1 at the concentration range up to 10 µg/ml; however, it markedly suppressed the motility in a cell scratch and a cell invasion assay. By Western blotting, polyI:C dramatically decreased TGF-ß1-induced Ak strain transforming (Akt) phosphorylation and increased phosphatase and tensin homologue (PTEN) expression without affecting the Son of mothers against decapentaplegic (Smad) 3 phosphorylation or the expression level of E-cadherin, N-cadherin or Snail, indicating that polyI:C suppressed cell motility independently of the 'cadherin switching'. The Akt inhibitor perifosine inhibited TGF-ß1-induced cell invasion, and the PTEN-specific inhibitor VO-OHpic appeared to reverse the inhibitory effect of polyI:C. CONCLUSION: PolyI:C has a novel function to suppress the motility of LC cells undergoing EMT by targeting the phosphatidylinositol 3-kinase/Akt pathway partly via PTEN and may prevent or reduce the metastasis of LC cells.

Cobalt-induced oxidative stress contributes to alveolar/bronchiolar carcinogenesis in B6C3F1/N mice.

Rodent alveolar/bronchiolar carcinomas (ABC) that arise either spontaneously or due to chemical exposure are similar to a subtype of lung adenocarcinomas in humans. B6C3F1/N mice and F344/NTac rats exposed to cobalt metal dust (CMD) by inhalation developed ABCs in a dose dependent manner. In CMD-exposed mice, the incidence of Kras mutations in ABCs was 67% with 80% of those being G to T transversions on codon 12 suggesting a role of oxidative stress in the pathogenesis. In vitro studies, such as DMPO (5,5-dimethyl-1-pyrroline N-oxide) immune-spin trapping assay, and dihydroethidium (DHE) fluorescence assay on A549 and BEAS-2B cells demonstrated increased oxidative stress due to cobalt exposure. In addition, significantly increased 8-oxo-dG adducts were demonstrated by immunohistochemistry in lungs from mice exposed to CMD for 90 days. Furthermore, transcriptomic analysis on ABCs arising spontaneously or due to chronic CMD-exposure demonstrated significant alterations in canonical pathways related to MAPK signaling (IL-8, ErbB, Integrin, and PAK pathway) and oxidative stress (PI3K/AKT and Melatonin pathway) in ABCs from CMD-exposed mice. Oxidative stress can stimulate PI3K/AKT and MAPK signaling pathways. Nox4 was significantly upregulated only in CMD-exposed ABCs and NOX4 activation of PI3K/AKT can lead to increased ROS levels in human cancer cells. The gene encoding Ereg was markedly up-regulated in CMD-exposed mice. Oncogenic KRAS mutations have been shown to induce EREG overexpression. Collectively, all these data suggest that oxidative stress plays a significant role in CMD-induced pulmonary carcinogenesis in rodents and these findings may also be relevant in the context of human lung cancers.

Plant growth regulators and Axl and immune checkpoint inhibitors from the edible mushroom Leucopaxillus giganteus.

A novel compound, (R)-4-ethoxy-2-hydroxy-4-oxobutanoic acid (1), and six known compounds (2-7) were isolated from the fruiting bodies of the wild edible mushroom Leucopaxillus giganteus. The planar structure of 1 was determined by the interpretation of spectroscopic data analysis. The absolute configuration of 1 was determined by comparing specific rotation of the synthetic compounds. In the plant regulatory assay, the isolated compounds (1-7) and the chemically prepared compounds (8-10) were evaluated their biological activity against the lettuce (Lactuca sativa) growth. Compounds 1 and 3-10 showed the significant regulatory activity of lettuce growth. 1 showed the strongest inhibition activity among the all the compounds tested. In the lung cancer assay, all the compounds were assessed the mRNA expression of Axl and immune checkpoints (PD-L1, PD-L2) in the human A549 alveolar epithelial cell line by RT-PCR. Compounds 1-10 showed significant inhibition activity against Axl and/or immune checkpoint.

New Organometallic Tetraphenylethylene⋠Iridium(III) Complexes with Antineoplastic Activity.

Iridium(III) complexes have attracted more and more attention in the past few years because of their potential antineoplastic activity. In this study, four IrIII complexes of the types [(η5 -Cpx )Ir(N^N)Cl]PF6 (complexes 1 and 2) and [Ir(Phpy)2 (N^N)]PF6 (complexes 3 and 4) have been synthesized and characterized. They exhibit potential antineoplastic activity towards A549 cells, especially in the case of complex 1 [IC50 =(3.56±0.5) µm], which was nearly six times as effective as cisplatin [(21.31±1.7) µm]. Additionally, these complexes show some selectivity towards cancer cells over normal cells. They could be transported by serum albumin (binding constants were changed from 0.37×105 to 81.71×105 m-1 ). IrIII complexes 1 and 2 could catalyze the transformation of nicotinamide adenine dinucleotide reduced form (NADH) into NAD+ (turnover numbers 43.2, 11.9] and induce the accumulation of reactive oxygen species, thus confirming their antineoplastic mechanism of oxidation, whereas the cyclometalated complexes 3 and 4 were able to target the lysosome [Pearson co-localization coefficient (PCC)=0.73], cause lysosomal damage, and induce apoptosis. Understanding the mechanism of action would help further structure-activity optimization on these IrIII complexes as emerging cancer therapeutics.

A Nomogram for Predicting Cancer-Specific Survival of TNM 8th Edition Stage I Non-small-cell Lung Cancer.

BACKGROUND: Models for predicting the survival outcomes of stage I non-small-cell lung cancer (NSCLC) defined by the newly released 8th edition TNM staging system are scarce. This study aimed to develop a nomogram for predicting the cancer-specific survival (CSS) of these patients and identifying individuals with a higher risk for CSS. METHODS: A total of 30,475 NSCLC cases were extracted from the Surveillance, Epidemiology, and End Results (SEER) database. We identified and integrated the risk factors to build a nomogram. The model was subjected to bootstrap internal validation with the SEER database, and external validation with a multicenter cohort of 1133 patients from China. The difference in the impact of adjuvant chemotherapy on model-defined high- and low-risk patients was examined using the National Cancer Database (NCDB). RESULTS: Eight independent prognostic factors were identified and integrated into the model. The calibration curves showed good agreement. The concordance index (C-index) of the nomogram was higher than that of the staging system (IA1, IA2, IA3, and IB) (internal validation set 0.63 vs. 0.56; external validation set 0.66 vs. 0.55; both p < 0.01). Specifically, 21.7% of stage IB patients (7.5% of all stage I) were categorized into the high-risk group (score > 30). There was a significant interaction effect between the adjuvant chemotherapy and risk groups in the NCDB cohort (p = 0.003). CONCLUSIONS: We established a practical nomogram to predict CSS for 8th edition stage I NSCLC. A prospective study is warranted to determine its role in identifying adjuvant chemotherapy candidates.

Bronchioalveolar carcinoma in an adult alpaca (Vicugna pacos).

BACKGROUND: This report describes a case of a bronchiolar adenocarcinoma in a 6-year old alpaca mare. For the first time in an alpaca, neoplasia was classified by histopathology as a lepidic-predominant bronchiolar adenocarcinoma. CASE PRESENTATION: The mare was referred to the Clinic for Ruminants after a 6-week period of forced breathing and weight loss. The clinical examination included complete blood count, blood chemistry, ultrasound, radiographs and a CT-scan of the thorax. A bilateral pneumothorax and several, structures within the lung parenchyma were diagnosed. Differential diagnosis included neoplasia, tuberculosis and fungal granulomas. The owner requested euthanasia due to the mare's ongoing deterioration. At postmortem examination, the granulomatous changes in the lungs were histopathologically classified as lepidic dominant bronchiolar adenocarcinoma. CONCLUSIONS: Neoplastic diseases are more often seen in South American camelids compared to other farm animal species. The use of a CT scan was helpful in classifying the lung lesions and give a clear prognosis.

Lung cancer in Spanish women: The WORLD07 project.

The WORLD07 study was a female-specific database, to prospectively characterise the clinical, histological, molecular and treatment-related features in Spanish women with lung cancer. Data were collected from patients' medical records and patient interviews from October 2007 to December 2012. A total of 2,060 women were analysed: median age, 61.3 years; white, 98.6%; postmenopausal, 80.2%; and no smokers, 55% including never smokers and ex-smokers. A family history of cancer was found in 42.5% of patients, 12.0% of patients had had a previous history of cancer (breast cancer, 39.7%). Most patients (85.8%) were diagnosed of non-small-cell lung cancer (NSCLC), most commonly reported with adenocarcinoma (71.4%), which was stage IV at diagnosis in 57.6%. Median overall survival (OS) for the entire population was 24.0 months, with a 1- and 2-year survival rate of 70.7% and 50.0% respectively. Median OS in patients with small-cell lung cancer was 18.8 months versus 25.0 months in patients with NSCLC (p = 0.011). Lung cancer appears to be a biologically different disease in women. By collecting prospective information about characteristics of women with lung cancer attending university hospitals in Spain, we hope to highlight the need to develop strategies based on gender differences and influence future healthcare policy.

Lung transplantation for non-small cell lung cancer and multifocal bronchioalveolar cell carcinoma.

Lung transplantation for primary bronchogenic cancer could lead to increased survival and improved quality of life for patients who have malignant disease, for which other therapies might be inappropriate. This Review examines the development of experience and outcomes for this indication and explores the limitations that are inherent in lung transplantation for malignant disease. Bronchogenic malignancy is a rare indication for lung transplantation constituting only 0·13% of all lung transplants in the USA from 1987 to 2010 and is only indicated for early-stage disease when conventional surgical techniques are contraindicated by poor lung function in which an unacceptably high risk of short-term mortality is expected. Outcomes can be extrapolated from the experience of finding an unexpected malignancy in an explanted lung for which approximately 30% of recipients, dependent on stage, succumb from distant metastatic disease in the first few years after transplant, after which long-term survival is similar to transplantation for other conditions. Care must be taken for lung transplantation for multifocal bronchoalveolar cell carcinoma to ensure that the donor lung is not contaminated with residual bronchoalveolar cell carcinoma cells in the upper airways during surgical implantation. The rarity of lung transplantation for cancer, and the absence of head-to-head trials comparing lung transplantation with conventional cancer care, limit the conclusions that can be drawn about lung transplantation for this indication. Furthermore, the ethical balance of how to allocate a scarce resource, such as a donor lung, remains an unresolved dilemma given the uncertainties regarding long-term survival. Conversely, individual patients might have substantial increases in survival and quality of life equivalent or superior to conventional cancer treatment methods.

Comparative study of cyto- and genotoxic potential with mechanistic insights of tungsten oxide nano- and microparticles in lung carcinoma cells.

The exigency of semiconductor and super capacitor tungsten oxide nanoparticles (WO3 NPs) is increasing in various sectors. However, limited information on their toxicity and biological interactions are available. Hence, we explored the underlying mechanisms of toxicity induced by WO3 NPs and their microparticles (MPs) using different concentrations (0-300 µg ml-1 ) in human lung carcinoma (A549) cells. The mean size of WO3 NPs and MPs by transmission electron microscopy was 53.84 nm and 3.88 µm, respectively. WO3 NPs induced reduction in cell viability, membrane damage and the degree of induction was size- and dose-dependent. There was a significant increase in the percentage tail DNA and micronuclei formation at 200 and 300 µg ml-1 after 24 hours of exposure. The DNA damage induced by WO3 NPs could be attributed to increased oxidative stress and inflammation through reactive oxygen species generation, which correlated with the depletion of reduced glutathione content, catalase and an increase in malondialdehyde levels. Cellular uptake studies unveiled that both the particles were attached/surrounded to the cell membrane according to their size. In addition, NP inhibited the progression of the cell cycle in the G2 /M phase. Other studies such as caspase-9 and -3 and Annexin-V-fluorescein isothiocyanate revealed that NPs induced intrinsic apoptotic cell death at 200 and 300 µg ml-1 concentrations. However, in comparison to NPs, WO3 MPs did not incite any toxic effects at the tested concentrations. Under these experimental conditions, the no-observed-significant-effect level of WO3 NPs was determined to be ≤200 µg ml-1 in A549 cells.

Crystal Structures and Cytotoxicity of ent-Kaurane-Type Diterpenoids from Two Aspilia Species.

A phytochemical investigation of the roots of Aspilia plurisetaled to the isolation of ent-kaurane-type diterpenoids and additional phytochemicals (1⁻23). The structures of the isolated compounds were elucidated based on Nuclear Magnetic Resonance (NMR) spectroscopic and mass spectrometric analyses. The absolute configurations of seven of the ent-kaurane-type diterpenoids (3⁻6, 6b, 7 and 8) were determined by single crystal X-ray diffraction studies. Eleven of the compounds were also isolated from the roots and the aerial parts of Aspilia mossambicensis. The literature NMR assignments for compounds 1 and 5 were revised. In a cytotoxicity assay, 12α-methoxy-ent-kaur-9(11),16-dien-19-oic acid (1) (IC50 = 27.3 ± 1.9 µM) and 9ß-hydroxy-15α-angeloyloxy-ent-kaur-16-en-19-oic acid (3) (IC50 = 24.7 ± 2.8 µM) were the most cytotoxic against the hepatocellular carcinoma (Hep-G2) cell line, while 15α-angeloyloxy-16ß,17-epoxy-ent-kauran-19-oic acid (5) (IC50 = 30.7 ± 1.7 µM) was the most cytotoxic against adenocarcinomic human alveolar basal epithelial (A549) cells.

Jolkinolide A and Jolkinolide B Inhibit Proliferation of A549 Cells and Activity of Human Umbilical Vein Endothelial Cells.

BACKGROUND Jolkinolide A (JA) and Jolkinolide B (JB) are diterpenoids extracted from the roots of Euphorbia fischeriana Steud and have been shown to have anti-tumor activity. However, their effects on the ability of tumor cells to invade blood vessels and metastasize remain largely unknown. Investigations into the effects of JA and JB on the angiogenesis of tumor tissues may facilitate the identification of new natural drugs with anti-tumor growth and metastasis activities. MATERIAL AND METHODS We used different concentrations of JA and JB (20 µg/ml, 40 µg/ml, 60 µg/ml, 80 µg/ml, and 100 µg/ml) to stimulate A549 cells and then studied the effects on the growth and metastasis of lung cancers. In addition, we used conditional media from A549 cells (A549-CM) stimulated by either JA or JB in different concentrations to culture human umbilical vein endothelial cells (HUVECs). RESULTS We found that both JA and JB significantly inhibited the Akt-STAT3-mTOR signaling pathway and reduced the expression of VEGF in A549 cells, but JB exhibited more significant inhibitory effects than JA. The JB-stimulated A549 cell conditional media had a greater inhibitory effect on the proliferation and migration of HUVECs than did the conditional media of JA-stimulated A549 cells. This effect gradually increased with increasing concentrations of either type of Jolkinolide. CONCLUSIONS Our results suggest that JA and JB inhibited VEGF expression in A549 cells through the inhibition of the Akt-STAT3-mTOR signaling pathway, and directly inhibited the proliferation and migration of HUVECs. These findings are of great significance for the development of new plant-derived chemotherapy agents for the treatment of cancer.

Middle lobe syndrome: An exceptional presentation of concomitant lepidic adenocarcinoma and bronchial anthracofibrosis.

Lepidic adenocarcinoma previously known as bronchioloalveolar carcinoma (BAC) is a non-small cell lung cancer with an indolent presentation. Bronchial anthracofibrosis (BAF) is caused by long-standing exposure to biomass fuel smoke often in poorly ventilated kitchen. Middle lobe syndrome (MLS) due to BAF is not uncommon however, lepidic adenocarcinoma then known as BAC, presenting as MLS has been documented only once before in the Polish literature. A 68-year-old never-smoker female with biomass fuel smoke exposure presented with cough and breathlessness. Imaging revealed MLS. Fiberoptic bronchoscopy visualised bluish-black hyperpigmentation with narrowing and distortion of right middle lobe bronchus suggestive of BAF. Transbronchial biopsy confirmed presence of lepidic adenocarcinoma. To our knowledge, this is the first detailed description of lepidic adenocarcinoma and BAF presenting as MLS.

Downregulation of LRRC8A protects human ovarian and alveolar carcinoma cells against Cisplatin-induced expression of p53, MDM2, p21Waf1/Cip1, and Caspase-9/-3 activation.

The leucine-rich repeat containing 8A (LRRC8A) protein is an essential component of the volume-sensitive organic anion channel (VSOAC), and using pharmacological anion channel inhibitors (NS3728, DIDS) and LRRC8A siRNA we have investigated its role in development of Cisplatin resistance in human ovarian (A2780) and alveolar (A549) carcinoma cells. In Cisplatin-sensitive cells Cisplatin treatment increases p53-protein level as well as downstream signaling, e.g., expression of p21(Waf1/Cip1), Bax, Noxa, MDM2, and activation of Caspase-9/-3. In contrast, Cisplatin-resistant cells do not enter apoptosis, i.e., their p53 and downstream signaling are reduced and caspase activity unaltered following Cisplatin exposure. Reduced LRRC8A expression and VSOAC activity are previously shown to correlate with Cisplatin resistance, and here we demonstrate that pharmacological inhibition and transient knockdown of LRRC8A reduce the protein level of p53, MDM2, and p21(Waf1/Cip1) as well as Caspase-9/-3 activation in Cisplatin-sensitive cells. Cisplatin resistance is accompanied by reduction in total LRRC8A expression (A2780) or LRRC8A expression in the plasma membrane (A549). Activation of Caspase-3 dependent apoptosis by TNFα-exposure or hyperosmotic cell shrinkage is almost unaffected by pharmacological anion channel inhibition. Our data indicate 1) that expression/activity of LRRC8A is essential for Cisplatin-induced increase in p53 protein level and its downstream signaling, i.e., Caspase-9/-3 activation, expression of p21(Waf1/Cip1) and MDM2; and 2) that downregulation of LRRC8A-dependent osmolyte transporters contributes to acquirement of Cisplatin resistance in ovarian and lung carcinoma cells. Activation of LRRC8A-containing channels is upstream to apoptotic volume decrease as hypertonic cell shrinkage induces apoptosis independent of the presence of LRRC8A.

Morphine Suppresses Lung Cancer Cell Proliferation Through the Interaction with Opioid Growth Factor Receptor: An In Vitro and Human Lung Tissue Study.

BACKGROUND: There have been inconsistent reports on whether opioids promote or inhibit lung cancer growth. In this study, we suggest that opioid growth factor receptor (OGFR), a negative regulator of cell proliferation, is a binding site of morphine and is involved in subsequent morphine-induced lung cancer growth suppression. METHODS: The expression and distribution of OGFR in human lung cancer tissues and cell lines were assessed with immunohistochemistry and real-time reverse transcription polymerase chain reaction. The human lung cancer cell line, H1975 (adenocarcinoma), which overexpressed OGFR but not µ-opioid receptors, was selected for further analysis to verify the interaction between morphine and OGFR and the impact of morphine on cancer cell growth. RESULTS: OGFR was expressed in lung cancer tissues and all cancer cell lines tested. Adenocarcinoma showed a higher OGFR expression than squamous cell carcinoma (reverse transcription polymerase chain reaction relative quantitation value: median [interquartile range], 13.1 [9.3-20.0] vs 4.3 [2.2-6.6]; P = 0.003). OGFR expression showed an inverse correlation with cell proliferation (r = -0.92, P = 0.0001). Morphine treatment reduced the median H1975 cell number by approximately 23% (P = 0.03). Growth suppression by morphine was attenuated when OGFR was knocked down. A confocal experiment demonstrated binding of morphine to OGFR. Growth suppression by morphine occurred in the S phase of the cell cycle. CONCLUSIONS: Lung cancer tissues and cell lines express OGFR. Morphine interacts with OGFR and may suppress lung cancer progression.

Persistent effects of Libby amphibole and amosite asbestos following subchronic inhalation in rats.

BACKGROUND: Human exposure to Libby amphibole (LA) asbestos increases risk of lung cancer, mesothelioma, and non-malignant respiratory disease. This study evaluated potency and time-course effects of LA and positive control amosite (AM) asbestos fibers in male F344 rats following nose-only inhalation exposure. METHODS: Rats were exposed to air, LA (0.5, 3.5, or 25.0 mg/m(3) targets), or AM (3.5 mg/m(3) target) for 10 days and assessed for markers of lung inflammation, injury, and cell proliferation. Short-term results guided concentration levels for a stop-exposure study in which rats were exposed to air, LA (1.0, 3.3, or 10.0 mg/m(3)), or AM (3.3 mg/m(3)) 6 h/day, 5 days/week for 13 weeks, and assessed 1 day, 1, 3, and 18 months post-exposure. Fibers were relatively short; for 10 mg/m(3) LA, mean length of all structures was 3.7 µm and 1% were longer than 20 µm. RESULTS: Ten days exposure to 25.0 mg/m(3) LA resulted in significantly increased lung inflammation, fibrosis, bronchiolar epithelial cell proliferation and hyperplasia, and inflammatory cytokine gene expression compared to air. Exposure to 3.5 mg/m(3) LA resulted in modestly higher markers of acute lung injury and inflammation compared to AM. Following 13 weeks exposure, lung fiber burdens correlated with exposure mass concentrations, declining gradually over 18 months. LA (3.3 and 10.0 mg/m(3)) and AM produced significantly higher bronchoalveolar lavage markers of inflammation and lung tissue cytokines, Akt, and MAPK/ERK pathway components compared to air control from 1 day to 3 months post-exposure. Histopathology showed alveolar inflammation and interstitial fibrosis in all fiber-exposed groups up to 18 months post-exposure. Positive dose trends for incidence of alveolar epithelial hyperplasia and bronchiolar/alveolar adenoma or carcinoma were observed among LA groups. CONCLUSIONS: Inhalation of relatively short LA fibers produced inflammatory, fibrogenic, and tumorigenic effects in rats which replicate essential attributes of asbestos-related disease in exposed humans. Fiber burden, inflammation, and activation of growth factor pathways may persist and contribute to lung tumorigenesis long after initial LA exposure. Fiber burden data are being used to develop a dosimetry model for LA fibers, which may provide insights on mode of action for hazard assessment.

Use of high-throughput RT-qPCR to assess modulations of gene expression profiles related to genomic stability and interactions by cadmium.

Predictive test systems to assess the mode of action of chemical carcinogens are urgently required. Within the present study, we applied the Fluidigm dynamic array on the BioMark™ HD System for quantitative high-throughput RT-qPCR analysis of 95 genes and 96 samples in parallel, selecting genes crucial for maintaining genomic stability, including stress response as well as DNA repair, cell cycle control, apoptosis and mitotic signaling. The specificity of each individually designed sequence-specific primer pair and their respective target amplicons were evaluated via melting curve analysis as part of qPCR and size verification via agarose gel electrophoresis. For each gene, calibration curves displayed high efficiencies and correlation coefficients in the identified linear dynamic range as well as low intra-assay variations. Data were processed via Fluidigm real-time PCR analysis and GenEx software, and results were depicted as relative gene expression according to the ΔΔC q method. Subsequently, gene expression analyses were conducted in cadmium-treated adenocarcinoma A549 and epithelial bronchial BEAS-2B cells. They revealed distinct dose- and time-dependent and also cell-type-specific gene expression patterns, including the induction of genes coding for metallothioneins, the oxidative stress response, cell cycle control, mitotic signaling and apoptosis. Interestingly, while genes coding for the DNA damage response were induced, distinct DNA repair genes were down-regulated at the transcriptional level. Thus, this approach provided a comprehensive overview on the interaction by cadmium with distinct signaling pathways, also reflecting molecular modes of action in cadmium-induced carcinogenicity. Therefore, the test system appears to be a promising tool for toxicological risk assessment.

The synergistic effect of resveratrol in combination with cisplatin on apoptosis via modulating autophagy in A549 cells.

Several studies have shown that combination treatment with natural products and chemotherapy agents can improve the sensitivity and cytotoxicity of chemotherapy agents. Resveratrol, a natural product, has many biological effects including antitumor and antiviral activities, as well as vascular protective effect. The aim of this study is to investigate the synergistic anticancer effect of resveratrol in combination with cisplatin and the potential anticancer mechanisms involved in A549 cells. The results obtained from Cell Counting Kit-8 and isobolographic analysis demonstrated that combination of resveratrol and cisplatin resulted in synergistic cytotoxic effects in A549 cells. Results from Hoechst staining, flow cytometry and western blot analysis suggested that resveratrol enhanced cisplatin-mediated apoptosis. Meanwhile, the changes of LC3-II and P62 levels and formation of autophagosome suggested that resveratrol in combination with cisplatin triggered autophagy. More importantly, inhibiting autophagy by 3-methyladenine markedly attenuated the apoptosis caused by combination of resveratrol and cisplatin in A549 cells. Taken together, our study provides the first evidence that resveratrol combined with cisplatin synergistically induce apoptosis via modulating autophagic cell death in A549 cells. These findings also help us to understand the role of natural products in combination with chemotherapy agents in lung cancer.

Inhibitory effect of ursolic acid and oleanolic acid from Eriobotrya fragrans on A549 cell viability in vivo.

Loquat [Eriobotrya japonica (Lindl.)] is a traditional Chinese medicine, which has been used as an anti-inflammatory and for curing chronic bronchitis among other potential applications. Extracted ursolic acid (UA) and oleanolic acid (OA) from wild loquat were previously found capable of suppressing the proliferation of A549 cells in vitro. In the current study, nude mice were used to determine the inhibitory effect of UA and OA on tumor formation in vivo. The results demonstrate that UA and OA reduced the proliferation of A549 cells in nude mice, and increased the expression of Bid while decreasing the protein levels of MMP-2, Ki-67, and CD34. In this study, we identified potential antitumor activity in a wild loquat extract containing UA and OA, which demonstrates that traditional Chinese medicine may have a role in treating certain types of cancer.

Survival and treatment of non-small cell lung cancer stage I-II treated surgically or with stereotactic body radiotherapy: patient and tumor-specific factors affect the prognosis.

BACKGROUND: This study was designed to define clinical baseline parameters associated with impaired survival of patients with stage I or II non-small cell lung cancer (NSCLC) who underwent surgery or stereotactic body radiotherapy (SBRT). METHODS: From January 2001 to January 2011, 425 patients (216 surgery, 209 SBRT) were identified with clinical stage I or II NSCLC. Cox proportional-hazards regression analyses were used to investigate risk factors for mortality. RESULTS: Median age of patients in the surgery and SBRT groups was 65 and 74 years, respectively. A smaller proportion of the surgical group had Charlson Comorbidity Index (CCI) score ≥1 compared with the SBRT group: 52 and 72 % (p < 0.001), respectively. Overall survival in the surgical group at 2 and 4 years was 79 and 65 %, respectively. In the SBRT group, this was 65 % at 2 years and 44 % at 4 years. In the surgical group older age, CCI score = 4 and clinical stage = IIB were associated with long-term mortality. In the SBRT group, this was CCI score ≥5 and clinical stage >IA. The area under the curve was calculated for the model with clinical and tumor factors: 0.77 for the surgery and 0.85 for the SBRT group. CONCLUSIONS: Both patient characteristics and survival of NSCLC I-II patients undergoing surgical treatment or SBRT differ considerably. Long-term survival as a result of treatment strategy of NSCLC patients might be optimized by focusing on patient and tumor specific factors. In addition to TNM staging, the consideration of patient age and CCI can be useful for prognostication of NSCLC patients.