Purpose: CYP1B1and CYP1A1expression is up-regulated by activation of the aryl hydrocarbon receptor (AhR) through binding of ligands such as cigarette smoke components. We examined the association between AhR, CYP1B1, and CYP1A1expression in noninvasive bronchioloalveolar carcinomas (BAC) and lung adenocarcinomas and investigated the effects of AhR overexpression on cell physiology. Experimental Design: AhR, CYP1B1, and CYP1A1 expression was examined in 107 lung adenocarcinomas and 57 BAC by immunohistochemistry. AhR expression in lung adenocarcinoma H1355 cells was stably reduced by RNA interference (RNAi). AhR, CYP1B1, and CYP1A1 expression was examined using real-time reverse transcription-PCR. Cell physiology was evaluated by measuring anchorage-independent growth and intracellular reactive oxygen species. Results: Expression of AhR and CYP1A1 was associated in smoking adenocarcinoma patients, whereas expression of AhR and CYP1B1was associated regardless of smoking status. The level of CYP1B1, but not CYP1A1, was positively associated with AhR overexpression in BAC. 2,3,7,8-Tetrachlorobenzo-p-dioxin^induced CYP1A1/1B1 expression was reduced in AhR RNAi clones. In the absence of 2,3,7,8-tetrachlorobenzo-p-dioxin, CYP1B1 mRNA levels were reduced in AhR RNAi clones, whereas CYP1A1 mRNA levels were barely detectable. Furthermore, anchorageindependent growth and intracellular oxidative stress were significantly reduced in AhR RNAi cells. Conclusions: In the absence of exogenous AhR ligands (such as cigarette smoke components), AhR overexpression up-regulated the expression of CYP1B1in the early stage of lung adenocarcinoma. Elevated AhR expression in lung adenocarcinoma cells could increase intracellular oxidative stress and promote cell growth, implying that disrupting AhR expression might prevent the early development of lung adenocarcinomas.

This study investigated the effects of low intensity ultrasound on seeded Schwann cells within poly(DL-lactic acid-co-glycolic acid) (PLGA) conduits by in vitro and in vivo trials for peripheral nerve regeneration. The possible differences in the ultrasonic effects when using biodegradable and non-biodegradable materials as the conduits were also studied, using silicone rubber tubes as comparisons. In the in vitro study, seeded Schwann cells were cultured in serum deprivation culture medium that simulated the environment of mechanical trauma on injury nerve site. After 12, 24, and 48 h, only the PLGA conduit groups exposed to 0.05 W/cm(2), 3 min/treatment of ultrasound exhibited decreased LDH release and increased MTT values compared to the sham groups. Based on the results of the in vitro experiment in LDH and MTT testing, the silicone conduits with seeded Schwann cells group was ignored in the in vivo study. The PLGA nerve conduits seeded with Schwann cells (9 x 10(3) cells) were implanted to 15-mm right sciatic nerve defects in rats. Each conduit received 12 ultrasonic treatment sessions over 2 weeks after 1 day of rest. Ultrasound was applied as follows: frequency, 1MHz; intensity, 0.3 W/cm(2) (SATP); treatment, 5 min/day. Implanted graft specimens were harvested for histological analysis at 8 weeks following surgery. PLGA groups (with and without Schwann cells) treated with pulsed ultrasonic stimulation were found to have significantly greater number and area of regenerated axons at the mid-conduit of implanted grafts, as compared to the sham groups. Ultrasonic stimulation on silicone groups was found to induce a mass of fibrous tissues that covered the nerve conduits and retarded axon regeneration.

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Exposure to polycylic aromatic hydrocarbons (PAH) has been associated with increased risk of lung cancer. Aryl hydrocarbon receptor (AhR) is known to play an essential role in PAH-induced toxicity. The objectives of this study were to identify and evaluate AhR expression in normal human lung tissues and in lung carcinomas. AhR protein and mRNA levels in human lung cell lines were evaluated with immunoblot and quantitative real-time RT-PCR assays, respectively. AhR protein expression was high in cytosol homogenates of adenocarcinoma (AD) cell lines and AhR mRNA levels corresponded well with AhR protein levels in these cell lines. AhR expression in human lung tissues and carcinomas were examined by means of immunohistochemical staining method. In normal lung tissues, immunostaining was found in the cytosol of bronchiolar epithelial cells. AhR immunostaining was more intense in AD than in squamous cell carcinomas. When AhR expression was compared with noral bronchiolar epithelial cells and neoplastic cells in the same specimens, the neoplastic cells, especially those of AD, demonstrated an increased staining. The upregulation of AhR mRNA expression was also demonstrated among 2 of 4 paired tissues with the quantitative real-time RT-PCR assay. Our data indicated that AhR expression was upregulated in lung AD and suggested that AhR and its expression might play an important role in the development of lung AD.

Vitamin B-12 intake and mean plasma vitamin B-12 concentration were lower for vegetarian subjects than for nonvegetarian subjects, leading to an increase in plasma homocysteine concentration. Vitamin B-6 and folate had little effect on plasma homocysteine concentration when individuals had adequate vitamin B-6 and folate status.

The high outflow permeability of the nerve conduit used to emit the drained waste generated from the traumatized host nerve stump is critical in peripheral nerve regeneration. Our earlier studies have established that asymmetric conduits fulfill the basic requirements for use as nerve guide conduits. In this study, the inflow characteristics of optimal nerve conduits were further examined using in vivo and in vitro trials. Various asymmetric poly(DL-lactic acid-co-glycolic acid) (PLGA) conduits were controlled by modifying precipitation baths using 0, 20, and 95% isopropyl alcohol, with high-porosity (permeability), medium-porosity (high outflow and low inflow), and low-porosity (permeability), respectively. In the in vitro trial, the Schwann cells and fibroblasts were seeded on either side of the asymmetric PLGA films in a newly designed coculture system that simulated the repaired nerve conduit environment. The results of the directional permeable films indicated the statistically significant proliferation of Schwann cells and the inhibition of the division of fibroblasts in lactate dehydrogenase release and inhibition of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT) reduction, compared with the other films. In the in vivo trial, the PLGA conduits seeded with Schwann cells were implanted into 10 mm right sciatic nerve defects in rats. After 6 weeks, implanted conduits were harvested. Histological examination verified that directional permeable conduits had markedly more A-type and B-type myelin fibers in the midconduit and distal nerve. In this work, the directional transport characteristics were established as an extremely important factor to the design and development of optimal nerve guide conduits in peripheral nerve regeneration.

We had previously reported that aryl hydrocarbon receptors (AhRs) are overexpressed in lung adenocarcinomas. Benzo [a]pyrene (BaP), an AhR agonist, increased FGF-9 expression in human lung adenocarcinoma cells. Similarly, several AhR agonists increased FGF-9 mRNA levels, and BaP-induced FGF-9 expression was prevented by cotreatment with AhR antagonist in human lung adenocarcinoma cells. Furthermore, AhR agonists increased transcriptional activity of FGF-9 promoter. Modulation of AhR expression via RNA interference or transient overexpression respectively reduced or increased both constitutive and BaPinduced FGF-9 expression in human lung cells. These results suggested that AhR activation and overexpression increased FGF-9 expression in lung cells. FGF-9 increased growth of lung fibroblasts but not that of lung adenocarcinoma cells. However, conditioned media collected from FGF-9-treated fibroblasts increased cell growth of lung adenocarcinoma cells. Furthermore, lung adenocarcinoma cells expressed FGF receptor 2 and cotreatment with anti-FGF receptor 2 prevented the interaction between fibroblasts and tumor cells. It is likely that FGF-9-stimulated fibroblasts secreted unknown factors, which activated FGF receptor 2 and subsequently promoted growth of lung adenocarcinoma cells. We further compared AhR and FGF-9 expression in 146 nonsmall cell lung cancer (NSCLC) cases by immunohistochemistry. FGF-9 expression was more common in adenocarcinomas than in squamous cell carcinomas. Furthermore, FGF-9 and AhR expression were well correlated in lung adenocarcinomas. These results suggest that AhR expression correlated positively with FGF-9 expression in lung adenocarcinomas, which might promote tumor growth by modulating communication between tumor cells and fibroblasts. Preventing AhR overexpression or disturbing FGF-9 function may reduce the development of lung adenocarcinomas. ' 2009 UICC

Cigarette smoke (CS) is a leading cause of death worldwide. The aryl hydrocarbon receptor (AHR) is partially responsible for tobacco-induced carcinogenesis although the underlying mechanisms involving early effector genes have yet to be determined. Here, we report that adrenomedullin (ADM) significantly contributes to the carcinogenicity of tobacco activated AHR. CS and AHR activating ligands induced ADM in vitro and in vivo but not in AHR-deficient fibroblasts and mice. Ectopic transfection of AHR rescued ADM expression in AHR−/− fibroblasts while AHR blockage with siRNA in wild type cells significantly decreased ADM expression. AHR regulates ADM expression through two intronic xenobiotic response elements located close to the start codon in the ADM gene. Using tissue microarrays we showed that ADM and AHR were coupregulated in lung tumor biopsies from smoker patients. Microarray metaanalysis of 304 independent microarray experiments showed that ADM is elevated in smokers and smokers with cancer. Additionally, ADM coassociated with a subset of AHR responsive genes and efficiently differentiated patients with lung cancer from non-smokers. In a novel preclinical model of CS-induced tumor progression, host exposure to CS extracts significantly elevated tumor ADM while systemic treatment with the ADM antagonist NSC16311 efficiently blocked tobacco-induced tumor growth. In conclusion, ADM significantly contributes the carcinogenic effect of AHR and tobacco combustion products. We suggest that therapeutics targeting the AHR/ADM axis may be of clinical relevance in the treatment of tobacco-induced pulmonary malignancies.