[The role of subtypes of voltage-gated K+ channels in pulmonary vasoconstriction induced by 15-hydroeicosatetraenoic acid].

AIM: To observe the effect of subtypes of Kv channels in rat pulmonary artery smooth muscle cells (PASMCs) on the process of pulmonary vasoconstriction induced by 15-HETE. METHODS: In the present study, ring of rabbit PA with specific Kv channel blockers were employed to functionally identify certain channel subtypes that took part in the process of 15-HETE induced pulmonary vasoconstriction; RT-PCR and Western blotting analysis were also used to measure the expression of subtypes of Kv in PASMCs exposed to 15-HETE,chronic hypoxia. RESULTS: Blocking of Kv1. 1, Kv1. 2, Kv1. 3 and Kv1. 6 channels did not affect 15-HETE induced vasoconstriction in normoxic rats; 15-HETE did not affect expression of Kv1. 1 and Kv1. 2 channels; 15-HETE significantly downregulated the expression of mRNA and protein of Kv1. 5 and Kv2. 1 in rat PASMCs. CONCLUSION: The results suggested that hypoxia may block Kv1. 5 and Kv2. 1 channels via 15-HETE mediated mechanism, leading to decrease numbers of functional Kv1. 5 and Kv2. 1 channels in PASMCs, leading to PA vasoconstriction.

Schedule-dependent cytotoxicity of sunitinib and TRAIL in human non-small cell lung cancer cells with or without EGFR and KRAS mutations.

BACKGROUND: Non-small cell lung cancer (NSCLC) patients who do initially respond to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) may eventually develop resistance, which may at least partly be due to the acquisition of a secondary EGFR mutation (T790M). Additionally, it has been found that KRAS mutations may serve as poor prognostic biomarkers. Here, we aimed at establishing a suitable treatment regimen for the multi-target TKI sunitinib and the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) in NSCLC-derived cells with or without EGFR and KRAS mutations. METHODS: Four NSCLC-derived cell lines with or without EGFR and KRAS mutations were exposed to different sunitinib and TRAIL treatment regimens. Alterations in cell viability, cell cycle distribution, apoptosis, phosphorylation of AKT and expression of the death receptors DR4 and DR5 were evaluated using CCK8, flow cytometry and Western blotting assays, respectively. RESULTS: A synergistic cytotoxic effect was observed in all four cell lines treated with sunitinib (1 nM) followed by TRAIL (100 ng/ml), as well as after simultaneous treatment with both agents. We found that sunitinib enhances TRAIL-induced G0/G1-phase cell cycle arrest and blocks TRAIL-triggered activation of AKT as the underlying mechanism. In contrast, we observed antagonistic effects when sunitinib was administered after TRAIL to the cell lines tested. A decreased DR4 and DR5 expression was found to be correlated with this antagonism. CONCLUSION: From our data we conclude that administration of sunitinib followed by TRAIL, as well as a simultaneous administration of both agents, serve as favorable treatment regimens for NSCLC-derived cells, irrespective of their EGFR and/or KRAS mutation status.

ERK1/2 signaling pathway is involved in 15-hydroxyeicosatetraenoic acid-induced hypoxic pulmonary vasoconstriction.

Hypoxia-induced 15-hydroxyeicosatetraenoic acid (15-HETE) is an essential mediator to constrict pulmonary arteries (PA). The signaling pathway involved in 15-HETE-induced PA vasoconstriction remains obscure. The aim of the present study was to test the hypothesis that hypoxic PA constriction induced by 15-HETE was possibly regulated by the extracellular signal-regulated kinase-1/2 (ERK1/2) pathway. PA ring tension measurement, Western blot and immunocytochemistry were used in the study to determine the possible role of ERK1/2 in 15-HETE-induced PA vasoconstriction. The organ bath for PA rings tension study was employed. Adult male Wistar rats were raised in hypoxic environment with fractional inspired oxygen (FIO2, 0.12) for 9 d. PA 1~1.5 mm in diameter were dissected and cut into 3 mm long rings for tension study. ERK1/2 up-stream kinase (MEK) inhibitor PD98059, which blocks the activation of ERK1/2, was used. The results showed that pretreatment of PD98059 significantly blunted 15-HETE-induced PA vasoconstrictions in the rings from hypoxic rat. Moreover, in endothelium-denuded rings, PD98059 also significantly attenuated 15-HETE-induced vasoconstriction. Phosphorylation of ERK1/2 in pulmonary arterial smooth muscle cells (PASMCs) of rat was enhanced evidently when stimulated by 15-HETE. Thus, the data suggest that ERK1/2 signaling pathway is involved in 15-HETE-induced hypoxic pulmonary vasoconstriction.

15-hydroxyeicosatetraenoic acid depressed endothelial nitric oxide synthase activity in pulmonary artery.

15-hydroxyeicosatetraenoic acid (15-HETE) plays an important role in hypoxia-induced pulmonary vasoconstriction. Release of nitric oxide (NO) is apparently decreased and activity of endothelial nitric oxide synthase (eNOS) is impaired in chronic hypoxia. However, little is known whether 15-HETE contributes to eNOS/NO pathway in the constriction induced by 15-HETE. We examined the response of rat pulmonary artery (PA) rings to 15-HETE, the production of NO, total eNOS expression and the phosphorylation of eNOS in bovine pulmonary artery endothelial cells (BPAECs) stimulated by 15-HETE. Rat PA rings were divided into three groups: endothelium intact group, endothelium denuded group, and nitro-L-arginine methyl ester (L-NAME, 0.1 mmol/L, an inhibitor of eNOS) group. Constrictions to 15-HETE were significantly enhanced in endothelium denuded group and L-NAME group (both P< 0.05 vs endothelium intact group, n= 9); BPAECs were incubated in different conditions to test nitrite production by Greiss method. Nitrite production was significantly reduced by 1 mumol/L 15-HETE (P<0.05), and increased by the lipoxygenase inhibitors, 10 mumol/L cinnamyl 3,4- dihydroxy-[alpha] -cyanocinnamate (CDC, P< 0.05) and 0.1 mmol/L nordihydroguiairetic acid (NDGA, P< 0.01 ); Western blot analysis of extracts from BPAECs incubated with 15-HETE in different time was carried out to test total eNOS expression, and the expression was changed unobviously. Immunoprecipitation (IP) and Western blot analysis of cell extracts from BPAECs treated with 2 mumol/L 15-HETE in different length of time were accomplished, using phospo-eNOS-threonine 495 (Thr495, an inhibitory site) antibody for IP, and eNOS or 15-lipoxygenase (15-LO) antibodies for Western blot. 15-HETE depressed eNOS activity by increasing the levels of phospho-eNOS-Thr 495. The data suggest that eNOS/NO pathway is involved in PA constrictions induced by 15-HETE and that 15-HETE depresses eNOS activity by phosphorylation in Thr495 site. The protein interaction between phospho-eNOS (Thr495) and 15-LO is discovered for the first time.

Mutations of EGFR or KRAS and expression of chemotherapy-related genes based on small biopsy samples in stage IIIB and IV inoperable non-small cell lung cancer.

PURPOSES: Epidermal growth factor receptor (EGFR) and KRAS mutations may predict the outcome of targeted drug therapy and also may be associated with the efficacy of chemotherapy in patients with non-small cell lung cancer (NSCLC). This report investigated the relation of EGFR or KRAS mutation and expression of chemotherapy-related genes, including excision repair cross-complementing 1 (ERCC1), thymidylate synthetase (TYMS), ribonucleotide reductase subunit M1 (RRM1) and class III ß-tubulin (TUBB3), as a potential explanation for these observations. METHODS: A total of 143 patients with stage IIIB and IV NSCLC from bronchoscopy or percutaneous lung biopsy obtained tumor samples were analyzed concurrently for EGFR or KRAS mutations, and mRNA expression of ERCC1, TYMS, RRM1 and TUBB3. EGFR or KRAS mutations were detected with xTAG liquidchip technology (xTAG-LCT), and mRNA expression levels of four genes were detected by branched DNA-liquidchip technology (bDNA-LCT). RESULTS: Of 143 patients, 63 tumors were positive for EGFR-activating mutations, and 16 tumors were positive for KRAS mutations. EGFR-activating mutations are more frequent in females, adenocarcinoma and non-smokers patients, and KRAS mutations are more frequent in smoking patients. ERCC1 mRNA levels were significantly associated with histological type and tumor differentiation, whereas TYMS levels were significantly associated with age. NSCLC specimens that harboring EGFR-activating mutations are more likely to express low ERCC1 and high TUBB3 mRNA levels, whereas tumors from patients with NSCLC harboring KRAS mutation are more likely to express high ERCC1 mRNA levels. CONCLUSIONS: Mutations and expression of chemotherapy-related genes may provide a basis for the selection of suitable molecular markers for individual treatment in a population with stage IIIB and IV NSCLC.