Influence of SiRNA targeting survivin on chemosensitivity of H460/cDDP lung cancer cells

We investigated the difference in survivin expression between a multidrug-resistant lung cancer cell line (H460/cDDP) and its parental counterpart (H460) and the influence of siRNA targeting survivin on the chemosensitivity of H460/cDDP. SiRNA targeting survivin was transfected into H460/cDDP cells using a liposome approach. Survivin mRNA and protein expression were significantly higher in H460/cDDP than H460 cells. The median inhibitory concentrations (IC(50)s) for cisplatin and paclitaxol in vitro against H460/cDDP cells were significantly lower in cells treated with survivin-specific siRNA than in control cells. Apoptosis and cleaved caspase-3 expression were analysed using annexin V and Western blotting, respectively, and showed a significant increase in apoptosis after treatment with the chemotherapeutic agents plus specific siRNA. Specific siRNA sensitized H460/cDDP cells to both cisplatin and paclitaxol. Thus, survivin appears to participate in the multidrug resistance mechanism of H460/cDDP cells and siRNA targeting survivin has the potential to increase the sensitivity of drug-resistant cancer cells to anticancer drugs.     

Shikonin circumvents cancer drug resistance by induction of a necroptotic death

Defect in apoptotic signaling and up-regulation of drug transporters in cancer cells significantly limits the effectiveness of cancer chemotherapy. We propose that an agent inducing non-apoptotic cell death may overcome cancer drug resistance and showed that shikonin, a naturally occurring naphthoquinone, induced a cell death in MCF-7 and HEK293 distinct from apoptosis and characterized with (a) a morphology of necrotic cell death; (b) loss of plasma membrane integrity; (c) loss of mitochondrial membrane potentials; (d) activation of autophagy as a downstream consequence of cell death, but not a contributing factor; (e) elevation of reactive oxygen species with no critical roles contributing to cell death; and (f) that the cell death was prevented by a small molecule, necrostatin-1, that specifically prevents cells from necroptosis. The characteristics fully comply with those of necroptosis, a basic cell-death pathway recently identified by Degterev et al. with potential relevance to human pathology. Furthermore, we proved that shikonin showed a similar potency toward drug-sensitive cancer cell lines (MCF-7 and HEK293) and their drug-resistant lines overexpressing P-glycoprotein, Bcl-2, or Bcl-x(L), which account for most of the clinical cancer drug resistance. To our best knowledge, this is the first report to document the induction of necroptosis by a small molecular compound to circumvent cancer drug resistance.     

Antiproliferative and Apoptotic Activities of Ketonucleosides and Keto-C-Glycosides against Non-Small-Cell Lung Cancer Cells with Intrinsic Drug Resistance

We compared the biological activity of a new group of keto-C-glycosides to that of a narrow spectrum of unsaturated ketonucleosides in a panel of non-small-cell lung cancer (NSCLC) cells with various levels of intrinsic resistance to standard chemotherapy drugs. Unlike cisplatin, etoposide, adriamycin, or taxol, for which a significant difference in the cytotoxic effect was observed between sensitive cell lines (H460, H125, and MGH4) and drug-resistant cell lines (H661, MGH7, and FADU), nucleoside analogs were equally cytotoxic in NSCLC cell lines, with compound 92 being 10-fold more active than compound 43, 44, 81, or 161, while compound 3 was the least active. Apoptotic measurements with flow cytometric analysis of terminal uridine deoxynucleotide nick end-labeled cells revealed that the cytotoxic activity of these nucleosides correlated with their potency to induce apoptosis. Compound 92 triggered death in cells with wild-type p53, mutated p53, or p53 gene deletion. Our findings suggest that keto-C-glycosides may be promising alternative anticancer agents which merit further studies in in vivo cancer models refractory to standard chemotherapy drugs.     

TAK1 inhibition by natural cyclopeptide RA-V promotes apoptosis and inhibits protective autophagy in Kras-dependent non-small-cell lung carcinoma cells

TAK1 kinase is required for the survival of Kras-dependent non-small-cell lung carcinoma (NSCLC) cells. Here, we report that the inhibition of TAK1 by a small natural cyclopeptide (RA-V) can promote apoptosis and inhibit protective autophagy in Kras-dependent NSCLC cells. Using short hairpin RNAs to deplete K-Ras, we identified H441 and H358 cells as Kras-dependent NSCLC cells which require protective basal autophagy for cell viability. We found that RA-V could selectively kill and induce apoptosis in H441 and H358 cells but had little effect on A549 and H460 (Kras-independent) cells. Furthermore, RA-V could inhibit basal autophagy in H441 and H358 cells. Mechanistic studies further showed that RA-V inhibits the level of TAK1 phosphorylation by binding directly to TAK1, resulting in the inhibition of the autophagy-related TAK1–AMPK–mTOR pathway. In addition, we found that RA-V could inhibit TAK1–P70S6K interaction, which may also inhibit basal autophagy. Our study shows that RA-V acts as an inducer of apoptosis and inhibitor of autophagy via the inhibition of TAK1 and provides the first example of TAK1 inhibition as a potential therapeutic strategy to promote apoptosis and inhibit protective autophagy in Kras-dependent NSCLC.

TAK1 kinase is required for the survival of Kras-dependent non-small-cell lung carcinoma (NSCLC) cells.     

Silencing of X-linked inhibitor of apoptosis decreases resistance to cisplatin and paclitaxel but not gemcitabine in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a highly lethal malignancy that often becomes resistant to chemotherapy. The effect of silencing the X-linked inhibitor of apoptosis gene (XIAP) on resistance to cisplatin, paclitaxel and gemcitabine was studied in the NSCLC cell lines A549 and H460. Transfection of these cells with small interfering RNA (siRNA) for XIAP blocked overexpression of the gene, suppressed cell proliferation, increased apoptosis and increased the cells' sensitivity to cisplatin and paclitaxel by preventing the binding of XIAP to caspase3 and increasing the activity of this enzyme. There was no significant difference in resistance to gemcitabine between XIAP-silenced cells and non-transfected cells. Changes in chemoresistance were independent of the activity of caspase-9. Silencing XIAP with siRNA can decrease chemoresistance in NSCLC and may have a potential role in the treatment of this disease.     

Retracted Article: FOXC1 silencing promotes A549 cell apoptosis through inhibiting the PI3K/AKT/hedgehog/Gli2 signaling pathway

Lung cancer begins in the lung and is a leading cause of premature death. Forkhead box C1 (FOXC1) has been reported to play an important role in different types of cancer, and evidence suggests that FOXC1 is highly expressed in non-small cell lung cancer (NSCLC) patients. However, the function and molecular mechanism of FOXC1 in the NSCLC cell line A549 is still unclear. In the present study, we indicate that FOXC1 is expressed in the NSCLC cell lines A549, H460, and SK-MES-1 at a high level compared with control human bronchial epithelial (HBE) cells. FOXC1 silencing promotes A549 cell apoptosis, whereas it inhibits cell survival. The levels of anti-apoptosis protein Bcl-2 decreased and the expression of pro-apoptosis protein Bax increased in FOXC1 silenced cells. Further studies show that FOXC1 knockdown inhibits the PI3K/AKT/hedgehog/Gli2 pathway. Overexpressed AKT or Gli2 reversed the effects of FOXC1 silencing on A549 cell survival and apoptosis. Taken together, our results conclude that FOXC1 silencing reduced the survival of cancer cells and promoted their apoptosis, and that the PI3K/AKT/hedgehog/Gli2 pathway plays an important role in the functioning of FOXC1 silencing.

Lung cancer begins in the lung and is a leading cause of premature death.     

Bortezomib, but not cisplatin, induces mitochondria-dependent apoptosis accompanied by up-regulation of noxa in the non-small cell lung cancer cell line NCI-H460

Defects in the apoptotic machinery may contribute to chemoresistance of non-small cell lung cancer (NSCLC) cells. We have previously showed a deficiency in mitochondria-dependent caspase-9 activation in NSCLC H460 cells after exposure to cisplatin, a drug widely used to treat NSCLC. Here we show that, unlike cisplatin, the novel anticancer agent bortezomib efficiently induces caspase-9 activation and apoptosis in H460 cells. A comparative analysis of molecular events underlying cell death in bortezomib-treated versus cisplatin-treated H460 cells revealed that bortezomib, but not cisplatin, caused a rapid and abundant release of cytochrome c and Smac/DIABLO from mitochondria. This was associated with a marked increase in levels of the BH3-only proapoptotic protein Noxa and the antiapoptotic protein Mcl-1. Taken together, our data show that bortezomib, by promoting a proapoptotic shift in the levels of proteins involved in mitochondrial outer-membrane permeabilization, is a potent activator of the mitochondrial pathway of apoptosis in NSCLC cells. Our preclinical results support further investigation of bortezomib-based therapies as a possible new treatment modality for NSCLC.     

Amphiregulin Promotes Resistance to Gefitinib in NonSmall Cell Lung Cancer Cells by Regulating Ku70 Acetylation

Multiple molecular resistance mechanisms reduce the efficiency of receptor tyrosine kinase inhibitors such as gefitinib in non-small cell lung cancer (NSCLC). We previously demonstrated that amphiregulin (Areg) inhibits gefitinib-induced apoptosis in NSCLC cells by inactivating the proapoptotic protein BAX. In this part of the investigation, we studied the molecular mechanisms leading to BAX inactivation. We show that Areg prevents gefitinib-mediated acetylation of Ku70. This augments the BAX-Ku70 interaction and therefore prevents BAX-mediated apoptosis. Accordingly, Areg or Ku70 knock down restore BAX activation and apoptosis in gefitinib-treated H358 cells in vitro. In addition, overexpression of the histone acetyltransferase (HAT) CREB-binding protein (CBP) or treatments with histone deacetylase (HDAC) inhibitors sensitize H358 cells to gefitinib. Moreover, a treatment with vorinostat, a HDAC inhibitor strongly sensitized tumors to gefitinib in vivo. These findings suggest new prospects in combining both HDAC and epidermal growth factor receptor inhibitors for the treatment of NSCLC.     

Tumor-suppressive effects by adenovirus-mediated mda-7 gene transfer in non-small cell lung cancer cell in vitro

The melanoma differentiation-associated gene-7 (mda-7), cloned from a human melanoma cell line H0-1, is known to induce tumor cell-selective growth inhibition in breast cancer cells in vitro and loss of tumorigenicity ex vivo. Yet, the mechanisms underlying these effects are still unknown. Therefore, we investigated these mechanisms on the molecular level in human non-small cell lung carcinoma (NSCLC) cells in vitro. Overexpression of mda-7 protein by Ad-mda-7 significantly suppressed proliferation and induced G2/M cell cycle arrest in wild-type p53 (A549, H460), and p53-null (H1299) non-small cell lung cancer cell lines, but not in normal human lung fibroblast (NHLF) cells. p53, Bax, and Bak protein expression was up-regulated in wild-type p53 tumor cell lines, but not in p53-null cells, suggesting that an intact p53 pathway was required for Bax and Bak induction. However, in all three cancer cell lines tested, activation of the caspase cascade and cleavage of poly(ADP-ribose) polymerase (PARP) appeared to be independent of the p53 mutational status. Together, these results suggest that apoptosis may be induced via multiple pathways by Ad-mda-7 in lung cancer cells and that Ad-mda-7 has the potential to become a novel therapeutic for clinical cancer gene therapy. Gene Therapy (2000) 7, 2051-2057.     

Amphiregulin Promotes BAX Inhibition and Resistance to Gefitinib in Non-small-cell Lung Cancers

Molecular resistance mechanisms affecting the efficiency of receptor tyrosine kinase inhibitors such as gefitinib in non-small-cell lung cancer (NSCLC) cells are not fully understood. Amphiregulin (Areg) overexpression has been proposed to predict NSCLC resistance to gefitinib and we have established that Areg-overexpressing H358 NSCLC cells resist apoptosis. Here, we demonstrate that Areg prevents gefitinib-induced apoptosis in NSCLC cells. We show that H358 cells are resistant to gefitinib in contrast to H322 cells, which do not overexpress Areg. Inhibition of Areg expression by small-interfering RNAs (siRNAs) restores gefitinib sensitivity in H358 cells, whereas addition of recombinant Areg confers resistance in H322 cells. Areg knockdown overcomes resistance to gefitinib and induced apoptosis in NSCLC H358 cells in vitro and in vivo. Under gefitinib treatment, Areg decreases the expression of the proapoptotic protein BAX, inhibits its conformational change and its mitochondrial translocation. Thus, in the presence of Areg, gefitinib-mediated apoptosis is reduced because BAX is sequestered in the cytoplasm. This suggests that treatments using epidermal growth factor receptor (EGFR) inhibitors may be poorly efficient in patients with elevated levels of Areg. These findings indicate the need for inhibition of Areg to enhance the efficiency of the EGFR inhibitors in patients suffering NSCLC.     

Tigecycline targets nonsmall cell lung cancer through inhibition of mitochondrial function

Nonsmall cell lung cancer (NSCLC) is the most common type of lung cancer with a high mortality rate and still remains therapeutically a challenge. A strategy to target NSCLC is to identify agents that are effective against NSCLC cells while sparing normal cells. We show that tigecycline, an FDA‐approved antibiotic drug, preferentially targets NSCLC cells. Tigecycline is effective in inhibiting proliferation and inducing apoptosis of multiple cell lines derived from two common NSCLC subtypes: adenocarcinoma and squamous cell carcinoma. Tigecycline also dose‐dependently inhibits colony formation of NSCLC subpopulation of cells with highly proliferative and invasive properties. Compared to NSCLC cells, tigecycline affects proliferation and survival of normal fibroblast cells significantly to a less extent. More importantly, tigecycline significantly inhibits NSCLC tumor growth through decreasing proliferation and increasing apoptosis of tumor cells in vivo. Tigecycline significantly inhibits mitochondrial respiration, mitochondrial membrane potential, and ATP levels and increases reactive oxygen species (ROS), suggesting that tigecycline impairs mitochondrial functions. Our study suggests that tigecycline may be a useful therapeutic agent, and inhibiting mitochondrial functions may represent a new targeted therapy for NSCLC.     

灵芝三萜类化合物的体外抗肿瘤活性研究

目的研究灵芝三萜类化合物在不同肿瘤细胞上的抗肿瘤活性。方法四甲基偶氮唑盐(MTT)法检测3种灵芝三萜化合物对5种肿瘤细胞的抑制能力,绘制抑制曲线,并计算IC50,反映化合物的细胞毒活性。结果体外实验显示,3种灵芝三萜类化合物单体对5种肿瘤细胞表现出不同程度的抑制作用,其中灵芝酸Y的抑制作用比较强,对肺癌细胞H460的IC50为22.4μmol/L,其次是7-oxo-ganoderic acid Z2,IC50为43.1μmol/L。3种化合物灵芝酸Y、7-oxo-ganodericacid Z2和ganoderon B对其他被测细胞株未显示活性或活性很弱。结论灵芝酸Y对肺癌细胞H460显示了较强的抑制活性,7-oxo-ganoderic Z2对肺癌细胞H460显示了一定的抑制活性,且抑制作用具有剂量依赖性。灵芝酸Y和7-oxo-ganoderic Z2的抗肺癌作用有待于进一步深入研究。     

Rosiglitazone suppresses human lung carcinoma cell growth through PPARgamma-dependent and PPARgamma-independent signal pathways

Peroxisome proliferator-activated receptors gamma (PPARgamma) exert diverse effects on cancer cells. Recent studies showed that rosiglitazone, a synthetic ligand for PPARgamma, inhibits cell growth. However, the exact mechanisms underlying this effect are still being explored, and the relevance of these findings to lung cancer remains unclear. Here, we report that rosiglitazone reduced the phosphorylation of Akt and increased phosphatase and tensin homologue (PTEN) protein expression in non-small cell lung carcinoma (NSCLC) cells (H1792 and H1838), and this was associated with inhibition of NSCLC cell proliferation. These effects were blocked or diminished by GW9662, a specific PPARgamma antagonist. However, transfection with a CMX-PPARgamma2 overexpression vector restored the effects of rosiglitazone on Akt, PTEN, and cell growth in the presence of GW9662. In addition, rosiglitazone increased the phosphorylation of AMP-activated protein kinase alpha (AMPKalpha), a downstream kinase target for LKB1, whereas it decreased phosphorylation of p70 ribosomal protein S6 kinase (p70S6K), a downstream target of mammalian target of rapamycin (mTOR). Of note, GW9662 did not affect the phosphorylation of AMPKalpha and p70S6K protein. The inhibitory effect of rosiglitazone on NSCLC cell growth was enhanced by the mTOR inhibitor rapamycin; however, it was blocked, in part, by the AMPKalpha small interfering RNA. Taken together, these findings show that rosiglitazone, via up-regulation of the PTEN/AMPK and down-regulation of the Akt/mTOR/p70S6K signal cascades, inhibits NSCLC cell proliferation through PPARgamma-dependent and PPARgamma-independent signals.     

A novel anti-lung cancer agent inhibits proliferation and epithelial–mesenchymal transition

ObjectiveTo synthesize a novel chalcone-1,3,4-thiadiazole hybrid and investigate its anticancer effects against NCI-H460 cells.Methods(E)-3-(4-bromophenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one, 1,3-dibromopropane and 1,3,4-thiadiazole-2-thiol were used as chemical materials to synthesize compound ZW97. The NCI-H460 lung cancer cell line was selected to explore the antitumor effects of compound ZW97 in vitro and in vivo.ResultsCompound ZW97 selectively inhibited cell proliferation against lung cancer cell lines NCI-H460, HCC-44 and NCI-H3122 with IC₅₀ values of 0.15 μM, 2.06 μM and 1.17 μM, respectively. ZW97 suppressed migration and the epithelial–mesenchymal transition process in NCI-H460 cells in a concentration-dependent manner. Based on the kinase activity results and docking analysis, compound ZW97 is a novel tyrosine-protein kinase Met (c-Met kinase) inhibitor. It also inhibited NCI-H460 cell growth in xenograft models without obvious toxicity to normal tissues.ConclusionsCompound ZW97 is a potential c-Met inhibitor that might be a promising agent to treat lung cancer by inhibiting the epithelial–mesenchymal transition process.     

m6A甲基转移酶METTL3介导miR-127调控非小细胞肺癌细胞系自噬的机制研究

目的　分析N6 甲基腺苷（m6A）甲基转移酶 3（methyltransferase-like 3, METTL3）和miR-127 在非小细胞肺癌（non small cell lung cancer cells, NSCLC）细胞系中的表达及其相关性,并探究METTL3 介导miR-127 调控非小细胞肺癌自噬的作用机制。方法　采用qRT-PCR 法检测正常肺上皮细胞BEAS-2B 与非小细胞肺癌细胞HCC827,A549 和H460 中METTL3 和miR-127 的表达水平;通过Linked Domics 数据库筛选出肺癌中与miR-127 共表达的基因,并分析METTL3 和miR-127 之间的相关性;选择H460 细胞传代培养至对数生长期后,将浓度接近的细胞随机分为三组,分别转染METTL3-siR,NC-siR 及Control,验证转染后H460 细胞中METTL3 和miR-127 表达;通过吖啶橙染色,Lyso-Tracker Red 染色观察METTL3 对细胞自噬的影响;利用Western blot 检测PTEN,AKT,mTOR,ULK1,Beclin-1等自噬相关蛋白的表达。结果　非小细胞肺癌细胞HCC827,A549,H460 中METTL3 相对表达分别为1.35±0.17,1.54±0.11 和1.78±0.21,明显高于正常肺上皮细胞BEAS-2B 中表达水平（0.91±0.11）,差异有统计学意义(F=34.037,P=0.002)。非小细胞肺癌细胞HCC827,A549,H460 中miR-127 相对表达分别为1.56±0.21,1.85±0.19 和2.11±0.25,较正常肺上皮细胞BEAS-2B 中表达（1.02±0.20）亦显著升高,差异有统计学意义（F=28.152,P=0.005）。肺癌中METTL3 与miR-127 共同表达呈正相关性（r=0.452,P ＜ 0.001）。METTL3-siR 组细胞中METTL3 表达水平（0.61±0.15）较Control 组（1.71±0.28） 和NC-siR 组（1.65±0.19） 显著降低, 差异有统计学意义（F=78.357,P ＜ 0.001）。METTL3-siR 组细胞中miR-127 表达水平（0.48±0.15）较Control 组（2.02±0.33）和NC-siR 组（1.97±0.25）亦显著下降,差异有统计学意义（F=105.216,P ＜ 0.001）;吖啶橙和Lyso-Tracker Red 染色分别观察到METTL3-siR 组细胞酸性自噬小泡增多,自噬溶酶体数量也明显增加。与Control 组和NC-siR 组相比,METTL3-siR 组细胞中PTEN,ULK1,Beclin1 蛋白表达水平显著升高,差异均有统计学意义（F=62.420~175.615,均P<0.001）;p-AKT 和p-mTOR 表达水平显著下降,差异均有统计学意义（F=148.781,87.147,均P<0.001）。结论　METTL3 和miR-127 在非小细胞肺癌细胞系中均呈高表达,且它们之间呈正相关性,沉默METTL3 基因可以抑制miR-127 表达,促进非小细胞肺癌H460 细胞发生自噬,其调控机制可能与PTEN/AKT/mTOR 通路有关。     

The effects of cetuximab alone and in combination with endostatin on vascular endothelial growth factor and interleukin-8 expression in human lung adenocarcinoma cells

Background: Angiogenesis, the growth of new blood vessels, plays an important role in tumor growth and metastasis. Both cetuximab and endostatin have been found to reduce the expression of endothelial-stimulating growth factors such as vascular endothelial growth factor (VEGF) and interleukin (IL)-8. However, the effects of cetuximab alone or in combination with endostatin on human lung adenocarcinoma cell growth remain unclear.Objective: The aim of this study was to evaluate the cellular and molecular effects of cetuximab alone and in combination with endostatin on human lung adenocarcinoma cell lines HI 299, SPC-A1, and H460 in vitro.Methods The epidermal growth factor receptor (EGFR) status of a panel of human lung adenocarcinoma cell lines was characterized using Western blot analysis. We used a modified tetrazolium salt assay to evaluate the growth-inhibitory effects of cetuximab and endostatin alone and in combination on the cell lines. We also determined the effects of these 2 drugs on VEGF and IL-8 expression using enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Cells were treated for 4 days with cetuximab 12.5 μ/mL, endostatin 25 μ/mL, or cetuximab 12.5 μg/mL + endostatin 25 μg/mL. Untreated cells cultured for 4 days served as controls.Results: EGFR expression in the H1299 cells was higher than in the SPC-A1 and H460 cells. Varying concentrations of cetuximab alone were associated with a significant growth-inhibitory effect on all 3 cell lines in a dose-dependent manner after 4 days of exposure compared with controls (all, P < 0.05). Compared with controls, varying concentrations of endostatin alone were not associated with significant inhibition of cell growth in any of the 3 cell lines. The inhibitory ratio of cetuximab + endostatin at varying concentrations was significantly greater than that of cetuximab alone (all, P < 0.05). On ELISA, either drug alone was associated with significant reductions in secreted VEGF and IL-8 in the HI 299, SPC-A1, and H460 cell lines (all, P < 0.05), with the exception of IL-8 concentration in the H460 cells. Mean (SD) VEGF expression with combination treatment in the H1299 and SPC-A1 cell lines (687 [21] and 629 [23] pg/mL, respectively) was significantly lower than with cetuxi-mab alone (878 [31] and 708 [20] pg/mL; both, P < 0.001); in the H460 cell line, combination treatment was not associated with a significant further reduction in VEGF expression. IL-8 concentrations with cetuximab in the H1299, SPC-A1, and H460 cell lines were 628 (20), 484 (29), and 532 (28) pg/mL, respectively, while the IL-8 concentrations with the combination treatment were 516 (20), 480 (18), and 467 (30) pg/mL. An enhanced effect of endostatin on IL-8 was observed in the H1299 and H460 cell lines (P < 0.001 and P = 0.018, respectively); however, no enhanced effect in the SPC-A1 line was observed. Similar results for VEGF and IL-8 expression were found using Western blot analysis.Conclusions: The results from this in vitro study suggest that cetuximab treatment might both inhibit human lung adenocarcinoma cell line growth and reduce the expression of VEGF and IL-8, which are the biomarkers of angiogenesis. Endostatin was not associated with inhibition of human lung adenocarcinoma cell line growth directly. Findings with the combination of cetuximab + endostatin suggest that endostatin might enhance the antiangiogenic and antitumor activity of cetuximab through an apparent effect on VEGF expression and, to a lesser degree, on IL-8 expression.     

Retracted Article: MiR-148a agomir based targeting of c-Met and Her-3 is able to attenuate EGFR-T790M mutation driven gefitinib and erlotinib resistance in non-small cell lung cancer cells

MiR-148a inhibits NSCLC progression. Whether miR-148a would reduce EGFR tyrosine kinase inhibitor (TKI) resistance of NSCLC cells remains underexplored. In this study, 5 NSCLC patients received surgery and gefitinib treatment but developed pleural metastasis. Patients'' NSCLC adopted EGFR T790M mutation. 5 naïve and 5 gefitinib-resisting NSCLC cell lines were derived from patients primary and metastatic tumor tissues, and the 5 gefitinib-resisting NSCLC cell lines were trained with erlotinib to establish the erlotinib-resisting cell lines. MiR-148a levels in cells were analyzed by qRT-PCR. miR-148a overexpression was mimicked by agomir treatment. NSCLC cell malignancy was evaluated by cell proliferation, apoptosis, colony formation and transwell invasion assays. Protein levels of c-Met, Her-3 and IGF-1R were assessed by western blotting. miRNA-mRNA interaction was investigated by luciferase reporter assay and AGO2-RIP. Transient overexpression of MET, ERBB3 or IGF1R gene was achieved by plasmid transfection. Results showed that the MiR-148a level was decreased with the development of gefitinib and erlotinib resistance and that there was an increase in malignancy in NSCLC cells in vitro. Treatment with miR-148a agomir significantly enhanced the cytotoxicity of gefitinib and erlotinib to naïve, gefitinib-resisting and erlotinib-resisting NSCLC cells in vitro while reducing their protein levels of c-Met, Her-3 and IGF-1R, the mRNAs of which were verified as direct targets of miR-148a in NSCLC cells. Restoring c-Met or Her-3 protein levels partially reduced the gefitinib and erlotinib sensitizing effect of miR-148a agomir treatment on NSCLC cells. We concluded that MiR-148a attenuated gefitinib and erlotinib resistance in non-small cell lung cancer cells with EGFR T790M mutation by targeting c-Met and Her-3 expression.

MiR-148a inhibits NSCLC progression.     

miR-124 and miR-142 enhance cisplatin sensitivity of non-small cell lung cancer cells through repressing autophagy via directly targeting SIRT1

Background: Drug resistance is a major obstacle in the treatment of non-small cell lung cancer (NSCLC). Recently, miRNAs are reported to be involved in the drug resistance of NSCLC. The roles of miR-124 and miR-142 in the multidrug resistance of NSCLC cells have been reported. However, the underlying mechanism by which miR-124 and miR-142 regulate resistance to cisplatin (CDDP) remains unknown. Methods: The expressions of miR-124, miR-142 and sirtuin 1 (SIRT1) in CDDP-sensitive and CDDP-resistant NSCLC tissues and cells were detected by qRT-PCR and western blot. IC50 value and cell proliferation were determined by MTT assay. Apoptosis was assessed by flow cytometry analysis. Autophagy was evaluated by western blot analysis of the protein levels of LC3-I, LC3-II and p62, and FITC-LC3 punctate formation assay. The interaction between miR-124 or miR-142 and SIRT1 was determined by luciferase reporter, RNA immunoprecipitation (RIP) and western blot assays. A tumor xenograft was performed to further validate the role of miR-124 and miR-142 in the sensitivity of CDDP-resistant NSCLC to cisplatin. Results: miR-124 and miR-142 were downregulated, while SIRT1 was upregulated in CDDP-resistant NSCLC tissues and cells compared to CDDP-sensitive groups. Functionally, overexpression of miR-124 and miR-142 or SIRT1 silencing enhanced the CDDP sensitivity of H1299/CDDP cells via suppressing autophagy, as evidenced by the reduced LC3-II/LC3-I radio, elevated p62 protein, and suppressed FITC-LC3 punctate formation in H1299/CDDP cells. miR-124 and miR-142 were demonstrated to co-target SIRT1. Re-expression of SIRT1 overturned miR-124 and miR-142-mediated chemosensitivity in H1299/CDDP cells via triggering autophagy. Conclusion: miR-124 and miR-142 enhance the cytotoxic effect of CDDP through repressing autophagy via targeting SIRT1 in CDDP-resistant NSCLC cells.

Drug resistance is a major obstacle in the treatment of non-small cell lung cancer (NSCLC).     

Murine double minute 2 as a therapeutic target for radiation sensitization of lung cancer

Murine double minute 2 (MDM2) inhibits p53-mediated functions, which are essential for therapies using DNA-damaging agents. The purpose of this study was to determine whether MDM2 inhibition enhances the radiosensitivity of a lung cancer model. The effects of MDM2 inhibition on tumor vasculature were also studied. Transient transfection of H460 lung cancer cells and human umbilical vascular endothelial cells (HUVEC) with antisense oligonucleotides (ASODN) against MDM2 resulted in a reduced level of MDM2 and increased levels of p21 and p53. Clonogenic assays showed that inhibition of MDM2 greatly decreased cell survival following irradiation. Quantification of apoptotic cells by 7-aminoactinomycin D staining and of senescent cells by X-gal staining showed that both processes were significantly increased in H460 cells treated with MDM2-specific ASODN and radiation. H460 xenografts that were treated with MDM2 ASODN plus radiotherapy also showed significant growth delay (P < 0.001) and increased apoptosis by terminal deoxynucleotidyl transferase-mediated nick end labeling staining. HUVECs transfected with MDM2-specific ASODN showed impaired viability and migration with decreased tube formation. Doppler studies showed that tumor blood flow was compromised when H460 xenografts were treated with MDM2-specific ASODN and radiation. A combination of radiotherapy and inhibition of MDM2 through the antisense approach results in improved tumor control in the H460 lung cancer model. This implies that a similar strategy should be investigated among patients with locally advanced lung cancer, receiving thoracic radiotherapy.