Pemetrexed, a multitarget antifolate used to treat malignant mesothelioma and non-small cell lung cancer (NSCLC), has been shown to stimulate autophagy. In this study, we determined whether autophagy could be induced by pemetrexed and simvastatin cotreatment in malignant mesothelioma and NSCLC cells. Furthermore, we determined whether inhibition of autophagy drives apoptosis in malignant mesothelioma and NSCLC cells. Malignant mesothelioma MSTO-211H and A549 NSCLC cells were treated with pemetrexed and simvastatin alone and in combination to evaluate their effect on autophagy and apoptosis. Cotreatment with pemetrexed and simvastatin induced greater caspase-dependent apoptosis and autophagy than either drug alone in malignant mesothelioma and NSCLC cells. 3-Methyladenine (3-MA), ATG5 siRNA, bafilomycin A, and E64D/pepstatin A enhanced the apoptotic potential of pemetrexed and simvastatin, whereas rapamycin and LY294002 attenuated their induction of caspase-dependent apoptosis. Our data indicate that pemetrexed and simvastatin cotreatment augmented apoptosis and autophagy in malignant mesothelioma and NSCLC cells. Inhibition of pemetrexed and simvastatin-induced autophagy was shown to enhance apoptosis, suggesting that this could be a novel therapeutic strategy against malignant mesothelioma and NSCLC.

The non-steroidal anti-inflammatory drugs (NSAIDs) celecoxib and sulindac have been reported to suppress lung cancer migration and invasion. The class III deacetylase sirtuin 1 (SIRT1) possesses both pro- and anticarcinogenic properties. However, its role in inhibition of lung cancer cell epithelial-mesenchymal transition (EMT) by NSAIDs is not clearly known. We attempted to investigate the potential use of NSAIDs as inhibitors of TGF-β1-induced EMT in A549 cells, and the underlying mechanisms of suppression of lung cancer migration and invasion by celecoxib and sulindac. We demonstrated that celecoxib and sulindac were effective in preventing TGF-β1-induced EMT, as indicated by upregulation of the epithelial marker, E-cadherin, and downregulation of mesenchymal markers and transcription factors. Moreover, celecoxib and sulindac could inhibit TGF-β1-enhanced migration and invasion of A549 cells. SIRT1 downregulation enhanced the reversal of TGF-β1-induced EMT by celecoxib or sulindac. In contrast, SIRT1 upregulation promoted TGF-β1-induced EMT. Taken together, these results indicate that celecoxib and sulindac can inhibit TGF-β1-induced EMT and suppress lung cancer cell migration and invasion via downregulation of SIRT1. Our findings implicate overexpressed SIRT1 as a potential therapeutic target to reverse TGF-β1-induced EMT and to prevent lung cancer cell migration and invasion.

Pemetrexed is a multitargeted antifolate used for the treatment of malignant mesothelioma and non-small cell lung cancer (NSCLC). However, the mechanism by which pemetrexed induces apoptosis remains unclear. In the present study, we investigated the involvement of reactive oxygen species (ROS) and sirtuin 1 (SIRT1) in pemetrexed-induced apoptosis in MSTO-211 malignant mesothelioma cells and A549 NSCLC cells. Pemetrexed enhanced caspase-dependent apoptosis, induced intracellular ROS generation, and downregulated SIRT1 in the MSTO-211 and A549 cells. Pemetrexed-induced apoptosis, which was prevented by pretreatment with N-acetyl-cysteine (NAC), was mediated by effects on the mitochondria, including mitochondrial membrane potential transition (MPT) and cytosolic release of cytochrome c, and also involved regulation of SIRT1 expression. Interference with SIRT1 expression using siRNA enhanced pemetrexed-induced apoptosis through mitochondrial dysfunction and ROS generation, whereas resveratrol, an activator of SIRT1, protected against pemetrexed-induced apoptosis. These results show that pemetrexed induces apoptosis in MSTO-211 mesothelioma cells and A549 NSCLC cells through mitochondrial dysfunction mediated by ROS accumulation and SIRT1 downregulation.

Abstract. Pemetrexed is a multitarget antifolate currently used for the treatment of malignant mesothelioma and non-small cell lung cancer (NSCLC). Statins, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors used primarily for hyperlidpidemia, have been studied for their antiproliferative and pro-apoptotic effects. However, the effects of simvastatin on pemetrexed-induced apoptosis have not been investigated. In this study, we investigated whether combination treatment with pemetrexed and simvastatin potentiates the apoptotic activity above that is seen with either drug alone in malignant mesothelioma and NSCLC cells. We found that the combination of pemetrexed and simvastatin induced more extensive caspase-dependent apoptosis than either drug alone in malignant mesothelioma cells (MSTO-211) or NSCLC cells (A549). In addition, reactive oxygen species (ROS) generation in cells treated with both pemetrexed and simvastatin was markedly increased compared to cells treated with either pemetrexed or simvastatin alone. Combination treatment also increased the loss of mitochondrial membrane potential, increased cytosolic release of cytochrome c, and altered expression of inhibitor of apoptosis proteins (IAP) and B-cell lymphoma-2 (Bcl-2) families of apoptosis related proteins. On the other hand, pretreatment with N-acetylcysteine (NAC) prevented apoptosis and mitochondrial dysfunction by pemetrexed and simvastatin. In addition, Bim siRNA conferred protection against apoptosis induced by pemetrexed and simvastatin. These results suggest that combination of pemetrexed and simvastatin potentiates their apoptotic activity beyond that of either drug alone in malignant mesothelioma and lung cancer cells. This activity is mediated through ROS-dependent mitochondrial dysfunction and Bim induction.

Residual pleural thickening (RPT) is the most frequent complication associated with pleural tuberculosis, and may occur even after successful anti-tuberculosis medications. Matrix metalloproteinases (MMPs) are zinc-dependent proteinases capable of degrading all components of the extracellular matrix. The proteolytic action of MMPs may be involved in the pathogenesis of tuberculosis. MMP-9, secreted by monocytes and lymphocyte, may lead to long-term fibrosis. The aim of the present study was to determine whether MMP-2 and/or MMP-9 and their specific inhibitors, tissue inhibitors of metalloproteinase 1 (TIMP-1) and TIMP-2, could be used to predict RPT. This retrospective study enrolled 52 patients diagnosed with pleural tuberculosis. Levels of MMP-2, MMP-9, TIMP-1, and TIM-2 were determined in the pleural fluid by ELISA. The RPT was measured on chest X-ray at the completion of treatment and the final follow-up. The average periods of anti-tuberculosis medication and the follow-up after completion of treatment were 6.7 and 7.6 months, respectively. MMP-2 or MMP-9 levels had no significant correlation to RPT. The patients with RPT > 2 mm at the completion of anti-tuberculosis medication and the final follow-up had higher TIMP-1 levels (p = 0.00 and p = 0.001, respectively). However, patients with RPT > 2 mm at the completion of anti-tuberculosis medication had lower TIMP-2 levels (p = 0.005). In a logistic regression model, elevated TIMP-1 levels at the completion of anti-tuberculosis medications were associated with RPT. In conclusion, higher TIMP-1 levels are responsible for the development of RPT and may be helpful for predicting RPT in pleural tuberculosis.