Differential modulation of nicotine-induced gemcitabine resistance by GABA receptor agonists in pancreatic cancer cell xenografts and in vitro.

BACKGROUND: Pancreatic cancer is frequently resistant to cancer therapeutics. Smoking and alcoholism are risk factors and pancreatic cancer patients often undergo nicotine replacement therapy (NRT) and treatment for alcohol dependence. Based on our report that low dose nicotine within the range of NRT causes gemcitabine resistance in pancreatic cancer, our current study has tested the hypothesis that GABA or the selective GABA-B-R agonist baclofen used to treat alcohol dependence reverse nicotine-induced gemcitabine resistance in pancreatic cancer. METHODS: Using mouse xenografts from the gemcitabine--sensitive pancreatic cancer cell line BXPC-3, we tested the effects of GABA and baclofen on nicotine-induced gemcitabine resistance. The levels of cAMP, p-SRC, p-ERK, p-AKT, p-CREB and cleaved caspase-3 in xenograft tissues were determined by ELISA assays. Expression of the two GABA-B receptors, metalloproteinase-2 and 9 and EGR-1 in xenograft tissues was monitored by Western blotting. Mechanistic studies were conducted in vitro, using cell lines BXPC-3 and PANC-1 and included analyses of cAMP production by ELISA assay and Western blots to determine protein expression of GABA-B receptors, metalloproteinase-2 and 9 and EGR-1. RESULTS: Our data show that GABA was as effective as gemcitabine and significantly reversed gemcitabine resistance induced by low dose nicotine in xenografts whereas baclofen did not. These effects of GABA were accompanied by decreases in cAMP, p-CREB, p-AKT, p-Src, p-ERK metalloproteinases-9 and -2 and EGR-1 and increases in cleaved caspase-3 in xenografts whereas baclofen had the opposite effects. In vitro exposure of cells to single doses or seven days of nicotine induced the protein expression of MMP-2, MMP-9 and EGR-1 and these responses were blocked by GABA. Baclofen downregulated the protein expression of GABA-B-Rs in xenograft tissues and in cells exposed to baclofen for seven days in vitro. This response was accompanied by inversed baclofen effects from inhibition of cAMP formation after single dose exposures to stimulation of cAMP formation in cells pretreated for seven days. CONCLUSIONS: These findings suggest GABA as a promising single agent for the therapy of pancreatic cancer and to overcome nicotine-induced gemcitabine resistance whereas treatment with baclofen may increase gemcitabine resistance.

Computerized morphometric analysis of microvasculature in non-small cell lung carcinoma.

Interactions between tumor cells and microvasculature are particularly critical. This computerized morphometric study was designed to analyze the distance between cancer cells and blood vessels and microvasculature organization in non-small cell lung carcinoma (NSCLC) comparing squamous cell carcinoma (SCC) and adenocarcinoma (ADC). Seventy nine nests of tumor cells, located less than or more than 3 mm from the invading edge, with a similar surface area, and their surrounding stroma were analyzed. After immunolabeling with an antihuman CD34 monoclonal antibody, computerized morphometric analyses of microvascular density (MVD), distribution of microvessels within stroma, and fractions of carcinomatous cells over various distances from microvessels were performed. This analysis showed a significantly higher MVD score in ADC than in SCC, particularly close to the invading edge (382+/-57 in ADC <3 mm; 242+/-28 in SCC <3 mm, p=0.015). Moreover, a significantly higher proportion of cancer cells was situated more than 75 microm from microvessels in SCC than in ADC, regardless of their site in relation to the invading edge (for example, 25+/-5% in ADC <3 mm; 52+/-3% in SCC <3 mm, p=0.001).

Expression of erythropoietin and erythropoietin receptor in non-small cell lung carcinomas.

PURPOSE: Expression of erythropoietin (Epo) and its receptor (Epo-R) has been shown in various normal and neoplastic nonhematopoietic tissues. This study, in non-small cell lung carcinoma, was designed to investigate the previously unreported expression of Epo and Epo-R as well as hypoxia-inducible factor-1alpha (HIF-1alpha), which is known to control Epo expression. EXPERIMENTAL DESIGN: Samples from lung squamous cell carcinomas (n = 17) and adenocarcinomas (n = 12) were obtained from patients undergoing curative surgery. mRNA transcripts of Epo, Epo-R, soluble Epo-R (sEpo-R), HIF-1alpha, and factor inhibiting HIF-1 (FIH-1) were evaluated by reverse transcription-PCR, whereas localization of Epo, Epo-R, and HIF-1alpha was assessed by immunohistochemistry. RESULTS: Epo, Epo-R, sEpo-R, HIF-1alpha, and FIH-1 transcripts were detected by reverse transcription-PCR in all samples tested, but with heterogeneous levels of expression for Epo, Epo-R, and sEpo-R. Coordinated levels of mRNA were observed for HIF-1alpha and FIH-1.Epo was detected in carcinomatous cells by immunohistochemistry in 50% of samples and Epo-R was detected in 96% of samples. Co-expression of Epo and Epo-R was observed on contiguous sections from 50% of tumors. HIF-1alpha was immunolocalized in 80% of non-small cell lung carcinomas. CONCLUSION: Epo-R was expressed in almost all samples and Epo was expressed in one half of samples on immunohistochemistry and in 100% of samples by mRNA detection, suggesting a potential paracrine and/or autocrine role of endogenous Epo in non-small cell lung carcinoma. The detection of stabilized HIF-1alpha suggests a possible role in Epo expression. Moreover, in the light of these results, the potential interactions between therapeutic recombinant Epo and the putative neoplastic Epo/Epo-R signaling pathways must be considered.

Multidrug resistance-associated protein (MRP1) is overexpressed in DNA aneuploid carcinomatous cells in non-small cell lung cancer (NSCLC).

Resistance to chemotherapy is intrinsically present in most nonsmall-cell lung carcinomas (NSCLC). No parameter has yet been determined to predict the response to chemotherapy. However, MRP1 (multidrug resistance-associated protein) is suspected to play an important role in resistance to treatment. The genetic basis for this resistance is not clearly understood, but it could result from chromosome reassortments catalyzed by aneuploidy. The aim of this study was to investigate MRP1 expression concurrently to DNA ploidy analysis in order to evaluate the link between MRP1 expression and chromosome 16 (MRP1 gene location) aberrations in NSCLC before treatment. Eighty-four surgical tumor specimens, 18 selected samples containing more than 80% of carcinomatous cells and 11 samples from normal bronchial epithelium were studied. Samples were stained by MRP1 FITC indirect staining and propidium iodide and analyzed by Flow Cytometry. Fifty tumors contained at least 1 DNA-aneuploid clone and the percentage of MRP1-positive cells was higher in DNA-aneuploid cells (p = 0.0003). All tumors expressed MRP1, but the level of expression was 3-fold higher in DNA-aneuploid cells than in DNA-diploid cells (normal bronchial cells as well as carcinomatous cells) (p < 0.0001). FISH analysis of 24 tumor imprints using a chromosome 16 alpha-satellite centromere probe demonstrated significantly more frequent gain of chromosome 16 in DNA-aneuploid tumors. These results suggest that MRP1 overexpression in NSCLC could be a consequence of chromosome 16 reassortments catalyzed by aneuploidy and that DNA-aneuploid tumors could require different treatment modalities from those applied to DNA-diploid tumors.