André M. Cantin

Glutathione and inflammatory disorders of the lung.

Glutathione (GSH) is an essential tripeptide present in most eukaryotic cells. Because of its sulfhydryl group, GSH is a versatile molecule capable of protecting cells against oxidants and toxic xenobiotics. However, it also plays key roles in multiple metabolic pathways, such as the synthesis of certain leukotrienes, proteins, and DNA precursors as well as the activation of enzymes, the regulation of immune responses and others. Not only is GSH synthesized by cells for local use but it also participates in an elaborate intercellular exchange process regulated by theγ-glutamyl cycle. Extracellular GSH in plasma and in alveolar epithelial lining fluid is thus subject to variations according to the degree of expression ofγ-glutamyl cycle enzymes and the rate of consumption of GSH by electrophilic molecules. Bronchoalveolar lavage has allowed us to observe many of these variations of GSH within the extracellular environment of the normal and diseased human lung. Studies of lung GSH have lead to a better understanding of pathogenic processes and have stimulated investigations of novel therapeutic approaches in lung inflammatory disorders.

Activation of matrix metalloproteinase-2 and -9 by 2- and 4-hydroxyestradiol.

Breast cancer patients frequently develop metastases. This process requires the degradation of extracellular matrix proteins which act as a barrier to tumour cell passage. These proteins can be degraded by proteases, mainly the matrix metalloproteinases (MMPs). MMP-2 and -9 which are frequently detected in breast cancer tissues. ProMMPs are released from cancer cells, and their activation is considered to be a crucial step in metastases development. In breast cancer, estrogen metabolism is altered favouring the accumulation of 2- and 4-hydroxyestradiol (2- and 4-OHE(2)). These estradiol metabolites can generate free radicals. Since reactive species are known activators of proMMPs, this study was designed to determine if the free radicals generated by 2- and 4-OHE(2) can activate proMMP-2 and -9. Activation of MMPs by hydroxyestradiol was determined by monitoring the cleavage of a fluorogenic peptide and by zymography analysis. Both estradiol metabolites activated the MMP-2 and -9. 4-OHE(2) was a more potent activator than 2-OHE(2), which reflects its higher capacity to generate free radicals. ProMMPs activation was mainly mediated through O(2)*-, although the free radical HO* also activated the proMMPs but to a lesser extent. ProMMPs activation was not observed with estrogens that cannot generate free radicals, i.e. estradiol, estrone, 2- and 4-methoxyestradiol, and 16alpha-hydroxyestrone. These results demonstrate that 2- and 4-OHE(2) at a concentration as low as 10(-8)M can activate the proMMP-2 and -9 and might play an important role in the invasion of breast cancer cells.

Invasiveness of breast cancer cells MDA-MB-231 through extracellular matrix is increased by the estradiol metabolite 4-hydroxyestradiol.

In malignant breast cancer, estrogen metabolism is altered, favoring the accumulation of hydroxyestradiols, which can generate free radicals. These reactive species can activate matrix metalloproteinases (MMPs), which in turn can hydrolyze the proteins of the extracellular matrix (ECM) that act as a barrier to tumor cell passage. The aim of this study was to determine whether reactive oxygen species generated by 4‐hydroxyestradiol (4‐OHE2) can activate MMP‐2 and then enhance the invasiveness of breast cancer cells MDA‐MB‐231 in vitro. Enzymatic assay and gel zymography demonstrated that 4‐OHE2 at a concentration as low as 10−8 M led to the conversion of proMMP‐2 to active MMP‐2. Activation of proMMP‐2 by 4‐OHE2 was inhibited by the Cu,Zn‐SOD supporting the involvement of the free radical superoxide anion (O u20092·− ). Using invasion chambers coated with matrigel (artificial ECM), 4‐OHE2 (10−8 M) enhanced the invasiveness of MDA‐MB‐231 breast cancer cells by 3‐fold. The addition of Cu,Zn‐SOD reduced the invasiveness of MDA‐MB‐231 cells by more than 2‐fold, supporting the involvement of O u20092·− generated by 4‐OHE2. Addition of an MMP‐2 inhibitor completely inhibited the enhancement of invasiveness induced by 4‐OHE2, which demonstrates the importance of activating MMP‐2 by 4‐OHE2. On the other hand, estradiol, which does not have a catechol structure, did not generate free radicals, and it could not activate proMMP‐2 or enhance the invasiveness of beast cancer cells. Although these data need to be confirmed in an animal model, this study suggests that the accumulation of 4‐OHE2 in breast tumors could enhance the invasiveness of breast cancer cells.

Hyaluronan (hyaluronic acid) in lung lavage of asbestos-exposed humans and sheep.

The concentration of hyaluronan was measured in the bronchoalveolar lavage fluid (BALF) of 18 control subjects and 27 workers from the asbestos mills and mines of Québec, 9 without asbestosis and 18 with asbestosis. Hyaluronan was also measured in the BALF of 9 control sheep exposed to 100 ml phosphate-buffered saline (PBS) at 10 day intervals for 39 months, and 13 sheep exposed at the same intervals to 100 mg chrysotile in 100 ml PBS for 24 months. At month 24, the asbestos-exposed sheep were classified into 3 groups: (A) 4 sheep exposed to PBS alone, (B) 4 sheep exposed to 10 mg chrysotile asbestos every 10 days, and (C) 5 sheep exposed to 100 mg chrysotile asbestos every 10 days for 15 months. The BALF hyaluronan averaged 53.9 ± 7.4 ng/ml in human controls, 67.5 ± 10.3 ng/ml in asbestos-exposed workers without asbestosis, and 206 ± 83 ng/ml in workers with asbestosis (p < 0.05 vs. normal). In the control sheep, BALF hyaluronan was 34.7 ± 6.9 ng/ml, and it was 31.5 ± 17.8 ng/ml in the low-dosage asbestos-exposed group (A), 83.0 ± 27.7 ng/ml in the intermediate-dose group (B), and 248.0 ± 134.7 ng/ml in the high-dosage group (C) (p < 0.05 vs. controls). In contrast, the release of plasminogen activator, a protease that may play a role in limiting the fibrotic process, was increased in group A, but not in groups B and C. In conclusion, BALF hyaluronan constitutes an indicator of lung interstitial tissue changes that may reflect the activity of the fibrosing alveolitis associated with chronic asbestos exposure.

Ascorbate modulation of H2O2 and camptothecin-induced cell death in Jurkat cells

The effect of ascorbate on cell death was examined in Jurkat cells (human T-cell leukemia) by incubation with dehydroascorbate (DHA), which is rapidly taken up by cells and efficiently reduced to ascorbate. Apoptosis was evaluated by caspase-3 activity in cell extracts and flow cytometry of annexin V-labeled cells. In parallel, necrosis was estimated by the release of lactate dehydrogenase. Minor effects on cell death were observed when Jurkat cells were incubated with either DHA alone (100–1,000xa0μM) or a single dose of 10xa0μM H2O2. However, pre-incubation with DHA followed by exposure to H2O2 clearly stimulated both apoptosis and necrosis. In complete contrast, pre-incubation of cells with DHA significantly inhibited apoptosis, but did not affect necrosis, induced by the topoisomerase I inhibitor camptothecin. Our results indicate that intracellular ascorbate can modulate cell death in a manner which depends upon the nature of the apoptotic stimulus, which in turn has critical implications regarding the mechanism and potential application of ascorbate in cancer therapy.