sincalide and diacetyldichlorofluorescein

Epidermal growth factor receptor (EGFR) gene mutations are the most common driver mutations in non-small cell lung cancer (NSCLC). To prolong the survival of the patients, EGFR tyrosine kinase inhibitors (TKIs) resistance in NSCLC is a major challenge that needs to be addressed urgently, and this study focuses on investigating the mechanism of cigarette smoke (CS) induced Gefitinib resistance in NSCLC.. PC-9 and A549 cells were cultured in vitro and treated with 1 µmol/L Gefitinib for 4 h and 10% cigarette smoke extract (CSE) for 48 h. Western blot was used to detect Sirtuin 3 (Sirt3) and superoxide dismutase 2 (SOD2) protein expressions; DCFH-DA probe was used to detect intracellular reactive oxygen species (ROS); CCK-8 kit was used to detect cell activity, and EdU was used to detect cell proliferation ability. Sirt3 overexpression plasmid (OV-Sirt3) was transfected in PC-9 and A549 cells and treated with 1 µmol/L Gefitinib for 4 h and 10% CSE for 48 h after N-acetylcysteine (NAC) action. The expressions of Sirt3 and SOD2 were detected by Western blot; the ROS level in the cells was detected by DCFH-DA probe, and the cell activity was detected by CCK-8.. CSE induced an increase in the 50% inhibitory concentration (IC50) of both PC-9 and A549 cells to Gefitinib (P<0.01) and enhanced the proliferation of PC-9 and A549 cells, suggesting that CS induced Gefitinib resistance in NSCLC. ROS was involved in CSE-induced Gefitinib resistance (P<0.05). CSE induced low expressions of Sirt3 and SOD2 (P<0.01), and Sirt3/SOD2 was associated with poor prognosis in lung cancer patients (P<0.05). OV-Sirt3 in PC-9 and A549 cells reversed CSE-induced Gefitinib resistance (P<0.05) and significantly reduced ROS production. NAC reversed CSE-induced Gefitinib resistance in PC-9 and A549 cells (P<0.05).. The ROS/Sirt3/SOD2 pathway is involved in CS-induced Gefitinib resistance in NSCLC.. 【中文题目：烟草烟雾通过ROS/Sirt3/SOD2通路
诱导NSCLC细胞吉非替尼耐药】 【中文摘要：背景与目的 表皮生长因子受体（epidermal growth factor receptor, EGFR）基因突变是非小细胞肺癌（non-small cell lung cancer, NSCLC）最常见的驱动基因突变。为延长患者生存时间，NSCLC EGFR酪氨酸激酶抑制剂（tyrosine kinase inhibitors, TKIs）耐药是目前急需解决的重大难题。本研究主要探究烟草烟雾（cigarette smoke, CS）诱导NSCLC发生吉非替尼耐药的机制。方法 体外培养PC-9、A549细胞，分别经1 µmol/L吉非替尼处理4 h、10%CS萃取物（CS extract, CSE）处理48 h。Western blot检测沉默蛋白3（Sirtuin 3, Sirt3）、超氧化物歧化酶2（superoxide dismutase 2, SOD2）蛋白表达，使用DCFH-DA探针检测细胞内活性氧（reactive oxygen species, ROS）水平，CCK-8试剂盒检测细胞活性，EdU检测细胞增殖能力。Sirt3过表达质粒（OV-Sirt3）转染于PC-9和A549细胞中、N-乙酰半胱氨酸乙酯（N-acetylcysteine, NAC）作用于细胞后分别经1 µmol/L吉非替尼处理4 h和10%CSE处理48 h。Western blot检测Sirt3、SOD2蛋白表达，DCFH-DA探针检测细胞中的ROS水平，CCK-8检测细胞活性。结果 CSE均可促使PC-9、A549细胞对吉非替尼的半数抑制浓度（50% inhibitory concentration, IC50）提高（P<0.01），并且增强PC-9和A549细胞的增殖能力，提示CS可诱导NSCLC吉非替尼耐药；ROS参与CSE诱导的吉非替尼耐药（P<0.05）；CSE诱导Sirt3、SOD2低表达（P<0.01），且Sirt3/SOD2与肺癌患者不良预后有关（P<0.05）；OV-Sirt3的PC-9、A549细胞可逆转CSE诱导的吉非替尼耐药（P<0.05）且显著降低ROS生成；NAC可逆转CSE诱导的PC-9、A549细胞吉非替尼耐药（P<0.05）。结论 ROS/Sirt3/SOD2通路参与了CS诱导的NSCLC吉非替尼耐药。
】 【中文关键词：肺肿瘤；烟草烟雾；ROS；Sirt3/SOD2；吉非替尼耐药】.

Topics: Antineoplastic Agents; Carcinoma, Non-Small-Cell Lung; Cell Line, Tumor; Cigarette Smoking; Drug Resistance, Neoplasm; ErbB Receptors; Gefitinib; Humans; Lung Neoplasms; Reactive Oxygen Species; Sincalide; Sirtuin 3

We have employed confocal laser scanning microscopy to investigate how intracellular free calcium concentration ([Ca2+]i) is influenced by hydrogen peroxide (H2O2) in collagenase-dispersed mouse pancreatic acinar cells. In the absence of extracellular calcium, treatment of cells with increasing concentrations of H2O2 resulted in an increase in [Ca2+]i, indicating the release of calcium from intracellular stores. Micromolar concentrations of H2O2 induced an oscillatory pattern, whereas 1 mmol H2O2/L caused a slow and sustained increase in [Ca2+]i. H2O2 abolished the typical calcium release stimulated by thapsigargin or by the physiological agonist cholecystokinin octapeptide (CCK-8). Depletion of either agonist-sensitive or mitochondrial calcium pools was unable to prevent calcium release induced by 1 mmol H2O2/L, but depletion of both stores abolished it. Additionally, lower H2O2 concentrations were able to release calcium only after depletion of mitochondrial calcium stores. Treatment with either the phospholipase C inhibitor U-73122 or the inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor xestospongin C did not modify calcium release from the agonist-sensitive pool induced by 100 micromol H2O2/L, suggesting the involvement of a mechanism independent of IP3 generation. In addition, H2O2 reduced amylase release stimulated by CCK-8. Finally, either the H2O2-induced calcium mobilization or the inhibitory effect of H2O2 on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulphydryl reducing agent. We conclude that H2O2 at micromolar concentrations induces calcium release from agonist-sensitive stores, and at millimolar concentrations H2O2 can also evoke calcium release from the mitochondria. The action of H2O2 is mediated by oxidation of sulphydryl groups of calcium ATPases independently of IP3 generation.

Topics: Animals; Calcium; Calcium Channels; Calcium Signaling; Dose-Response Relationship, Drug; Fluoresceins; Hydrogen Peroxide; Inositol 1,4,5-Trisphosphate Receptors; Male; Mice; Mitochondria; Pancreas, Exocrine; Reactive Oxygen Species; Receptors, Cytoplasmic and Nuclear; Sincalide; Thapsigargin; Time Factors; Type C Phospholipases