Exposure to 9,10-phenanthrenequinone accelerates malignant progression of lung cancer cells through up-regulation of aldo-keto reductase 1B10.

Inhalation of 9,10-phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust, exerts fatal damage against a variety of cells involved in respiratory function. Here, we show that treatment with high concentrations of 9,10-PQ evokes apoptosis of lung cancer A549 cells through production of reactive oxygen species (ROS). In contrast, 9,10-PQ at its concentrations of 2 and 5 µM elevated the potentials for proliferation, invasion, metastasis and tumorigenesis, all of which were almost completely inhibited by addition of an antioxidant N-acetyl-l-cysteine, inferring a crucial role of ROS in the overgrowth and malignant progression of lung cancer cells. Comparison of mRNA expression levels of six aldo-keto reductases (AKRs) in the 9,10-PQ-treated cells advocated up-regulation of AKR1B10 as a major cause contributing to the lung cancer malignancy. In support of this, the elevation of invasive, metastatic and tumorigenic activities in the 9,10-PQ-treated cells was significantly abolished by the addition of a selective AKR1B10 inhibitor oleanolic acid. Intriguingly, zymographic and real-time PCR analyses revealed remarkable increases in secretion and expression, respectively, of matrix metalloproteinase 2 during the 9,10-PQ treatment, and suggested that the AKR1B10 up-regulation and resultant activation of mitogen-activated protein kinase cascade are predominant mechanisms underlying the metalloproteinase induction. In addition, HPLC analysis and cytochrome c reduction assay in in vitro 9,10-PQ reduction by AKR1B10 demonstrated that the enzyme catalyzes redox-cycling of this quinone, by which ROS are produced. Collectively, these results suggest that AKR1B10 is a key regulator involved in overgrowth and malignant progression of the lung cancer cells through ROS production due to 9,10-PQ redox-cycling.

9,10-Phenanthrenequinone promotes secretion of pulmonary aldo-keto reductases with surfactant.

9,10-Phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust particles, induces apoptosis via the generation of reactive oxygen species (ROS) because of 9,10-PQ redox cycling. We have found that intratracheal infusion of 9,10-PQ facilitates the secretion of surfactant into rat alveolus. In the cultured rat lung, treatment with 9,10-PQ results in an increase in a lower-density surfactant by ROS generation through redox cycling of the quinone. The surfactant contains aldo-keto reductase (AKR) 1C15, which reduces 9,10-PQ and the enzyme level in the surfactant increases on treatment with 9,10-PQ suggesting an involvement of AKR1C15 in the redox cycling of the quinone. In six human cell types (A549, MKN45, Caco2, Hela, Molt4 and U937) only type II epithelial A549 cells secrete three human AKR1C subfamily members (AKR1C1, AKR1C2 and AKR1C3) with the surfactant into the medium; this secretion is highly increased by 9,10-PQ treatment. Using in vitro enzyme inhibition analysis, we have identified AKR1C3 as the most abundantly secreted AKR1C member. The AKR1C enzymes in the medium efficiently reduce 9,10-PQ and initiate its redox cycling accompanied by ROS production. The exposure of A549 cells to 9,10-PQ provokes viability loss, which is significantly protected by the addition of the AKR1C3 inhibitor and antioxidant enzyme and by the removal of the surfactants from the culture medium. Thus, the AKR1C enzymes secreted in pulmonary surfactants probably participate in the toxic mechanism triggered by 9,10-PQ.

Aldo-keto reductase 1C15 as a quinone reductase in rat endothelial cell: its involvement in redox cycling of 9,10-phenanthrenequinone.

9,10-Phenanthrenequinone (9,10-PQ), a redox-active quinone in diesel exhausts, triggers cellular apoptosis via reactive oxygen species (ROS) generation in its redox cycling. This study found that induction of CCAAT/enhancer-binding protein-homologous protein (CHOP), a pro-apoptotic factor derived from endoplasmic reticulum stress, participates in the mechanism of rat endothelial cell damage. The 9,10-PQ-mediated CHOP induction was strengthened by a proteasome inhibitor (MG132) and the MG132-induced cell sensitization to the 9,10-PQ toxicity was abolished by a ROS inhibitor, suggesting that ROS generation and consequent proteasomal dysfunction are responsible for the CHOP up-regulation caused by 9,10-PQ. Aldo-keto reductase (AKR) 1C15 expressed in rat endothelial cells reduced 9,10-PQ into 9,10-dihydroxyphenanthrene concomitantly with superoxide anion formation, implying its participation in evoking the 9,10-PQ-redox cycling. The 9,10-PQ-induced damage was augmented by AKR1C15 over-expression. 9,10-PQ also provoked the AKR1C15 up-regulation, which sensitized against the quinone toxicity. These results suggest the presence of a negative feedback loop exacerbating the quinone toxicity in rat endothelial cells.

Nitric oxide mitigates apoptosis in human endothelial cells induced by 9,10-phenanthrenequinone: role of proteasomal function.

It has been widely recognized that nitric oxide (NO) suppresses oxidative damage of endothelial cell, but little is known about its pathophysiological role in apoptotic induction by 9,10-phenanthrenequinone (9,10-PQ), a major quinone component in diesel exhaust particles. Here, we have investigated the change in NO level in human aortic endothelial cells and the effect of NO in each step of apoptotic signaling initiated by 9,10-PQ. Treatment with 9,10-PQ evoked a bell-shaped production of NO, which was presumably due to increase in an active form of endothelial NO synthase. Pretreatment with exogenous NO decreased the susceptibility of the cells to 9,10-PQ, and retrieved from apoptotic signaling (reactive oxygen species generation, glutathione depletion and caspase activation) induced during exposure to high concentrations of 9,10-PQ. In addition, inhibition of endogenous NO production augmented the toxicity of 9,10-PQ. Interestingly, the 9,10-PQ treatment resulted in marked decreases in the proteasomal activities, which were partially abrogated by NO and a cell-permeable cGMP analog. These results indicate that proteasomal dysfunction by oxidative stress participates in the 9,10-PQ-induced apoptotic signaling and is ameliorated by NO via a cGMP-dependent pathway, thereby suggesting the protective role of NO in vascular damage caused by 9,10-PQ.