The effect of PLK1 inhibitor in osimertinib resistant non-small cell lung carcinoma cells. / Poloæ ·æ¿€é ¶1抑制剂在奥希替尼耐药的非小细胞肺癌细胞中的作用.

OBJECTIVES: To investigate the effects of PLK1 inhibitors on osimertinib-resistant non-small cell lung carcinoma (NSCLC) cells and the anti-tumor effect combined with osimertinib. METHODS: An osimertinib resistant NCI-H1975 cell line was induced by exposure to gradually increasing drug concentrations. Osimertinib-resistant cells were co-treated with compounds from classical tumor pathway inhibitor library and osimertinib to screen for compounds with synergistic effects with osimertinib. The Gene Set Enrichment Analysis (GSEA) was used to investigate the activated signaling pathways in osimertinib-resistant cells; sulforhodamine B (SRB) staining was used to investigate the effect of PLK1 inhibitors on osimertinib-resistant cells and the synergistic effect of PLK1 inhibitors combined with osimertinib. RESULTS: Osimertinib-resistance in NCI-H1975 cell (resistance index=43.45) was successfully established. The PLK1 inhibitors GSK 461364 and BI 2536 had synergistic effect with osimertinib. Compared with osimertinib-sensitive cells, PLK1 regulatory pathway and cell cycle pathway were significantly activated in osimertinib-resistant cells. In NSCLC patients with epidermal growth factor receptor mutations treated with osimertinib, PLK1 mRNA levels were negatively correlated with progression free survival of patients (R=－0.62, P<0.05), indicating that excessive activation of PLK1 in NSCLC cells may cause cell resistant to osimertinib. Further in vitro experiments showed that IC50 of PLK1 inhibitors BI 6727 and GSK 461364 in osimertinib-resistant cells were lower than those in sensitive ones. Compared with the mono treatment of osimertinib, PLK1 inhibitors combined with osimertinib behaved significantly stronger effect on the proliferation of osimertinib-resistant cells. CONCLUSIONS: PLK1 inhibitors have a synergistic effect with osimertinib on osimertinib-resistant NSCLC cells which indicates that they may have potential clinical value in the treatment of NSCLC patients with osimertinib resistance.

In vivo efficacy studies of novel quinazoline derivatives as irreversible dual EGFR/HER2 inhibitors, in lung cancer xenografts (NCI-H1975) mice models.

Lung cancer is the most common cancer and leading cause of cancer-related deaths worldwide. The first-generation reversible, ATP-competitive inhibitors gefetinib and elotinib showed good clinical responses in lung adenocarcinoma tumors (NSCLC). But almost all patients developed resistance to these inhibitors over time. Such resistance of EGFR inhibitors was frequently linked to the acquired L858R and T790M point mutations in the kinase domain of EGFR. To overcome these resistance problems, the second and the third generation inhibitors have been discovered. FDA approved afatinib, the second generation irreversible inhibitor and osimitinib, the third generation irreversible EGFR inhibitors for the treatments of NSCLC. We identified new covalent quinazoline inhibitors (E)-N-(4-(3-chloro-4-fluorophenylamino)-7-(2-ethoxyethoxy)quinazolin-6-yl)-4-(dimethylamino)but-2-enamide (6d) and (E)-N-(4-(3-chloro-4-(pyridin-2-ylmethoxy)phenylamino)-7-(2-ethoxyethoxy)quinazolin-6-yl)-4-(dimethyl-amino)but-2-enamide (6h) that exhibited potent EGFR kinase inhibitory activities on L858R and T790M mutations. The compound 6 h showed selectivity similar to AZD9291 (osimertinib) in mutated and wild type tumor cell lines. In vitro cell assay 6d and 6h were better than afatinib and osimertinib. In vivo antitumor efficacy studies of these compounds were done in NCI-H1975 mice xenografts.

Synergistic effects of Bcl-2 inhibitors with AZD9291 on overcoming the acquired resistance of AZD9291 in H1975 cells.

Non-small cell lung cancer (NSCLC) patients with epithermal growth factor receptor (EGFR) mutations can be treated with EGFR-tyrosine kinase inhibitors (EGFR-TKIs), however, development of acquired resistance could significantly limit curative effects of EGFR-TKIs. Different mechanisms of acquired resistance to first-generation and second-generation EGFR TKIs have been widely reported, but there were few reports on the resistant mechanism of third-generation EGFR-TKI such as osimertinib (AZD9291). In the present study, significant upregulation of Bcl-2 was found in AZD9291-resistant H1975 cells (H1975AR) compared with H1975, which may constitute an important resistant mechanism of acquired resistance to AZD9291. More importantly, our study showed that synergism between AZD9291 and Bcl-2 inhibitor ABT263 (0.25 µM) or ABT199 (1 µM) could effectively overcome the acquired resistance of AZD9291 in H1975AR in vitro. Flow cytometry analyses demonstrated that AZD9291 + ABT263/ABT199 caused a significantly different cell cycle distribution and produced significantly more apoptosis compared with either AZD9291 or ABT263/ABT199 treatment alone. Further multiscreen/Western blot analyses revealed that NF-κB was significantly downregulated in AZD9291 + ABT263/ABT199 treatment groups compared with AZD9291 or ABT263/ABT199 treatment alone, with a more significant reduction of NF-κB in AZD9291 + ABT199 compared with AZD9291 + ABT263. It is also noticeable that AZD9291 + ABT263 specifically caused a significantly reduced expression of p21 compared with AZD9291 or ABT263 treatment alone while AZD9291 + ABT199 specifically caused significantly reduced expressions of SQSTM1 and survivin, but increased expression of autophagosome marker LC3-II compared with AZD9291 or ABT199 treatment alone. Furthermore, cytotoxicity of AZD9291 + ABT199 could be partially reversed by autophagy inhibitor chloroquine. These results suggest that ABT263 and ABT199 may work through different signaling pathways to achieve synergistic cytotoxicity with AZD9291 in H1975AR. These findings suggest that Bcl-2 inhibitor may provide an effective option in combination therapy with EGFR-TKIs to treat NSCLC with EGFR-TKI acquired resistance.

The apoptotic effect of GSK-3 inhibitors: BIO and CHIR 98014 on H1975 lung cancer cells through ROS generation and mitochondrial dysfunction.

OBJECTIVE: GSK-3 has been reported to be upregulated in malignant diseases, including lung cancers, thus suggesting it to be a valid target for cancer treatment. The study elucidates the possible mechanism involved in the ability of GSK-3 inhibitors: BIO and CHIR 98014 to regulate proteins involved in cell death of H1975 lung cancer cells. RESULTS: BIO and CHIR 98014 successfully induced apoptosis at lower concentrations in H1975 cells but not in H460 lung cancer cells. Moreover, increased ROS generation and depolarization of mitochondrial membrane potential were observed in both treatments. Cleavage of caspase-3 was observed in both BIO and CHIR 98014-treated cells after 72 h with monolayer and tumorsphere cell culture models. CONCLUSIONS: The use of GSK-3 inhibitors shows promising apoptotic abilities in clinical cancer treatments, particularly for lung cancer cells. This study is the first report to describe the significant apoptotic effects of BIO and CHIR 98014 through multiple mechanisms of H1975 NSCLC that are linked to their proliferative and migratory capacities.

Arrayed microfluidic chip for detection of circulating tumor cells and evaluation of drug potency.

Circulating tumor cells (CTCs) from peripheral blood of cancer patients are considered as one of the most promising pharmacodynamic (PD) biomarkers due to its non-invasive property in disease diagnosis and prognosis. However, the detection of extremely low number of CTCs in patient blood requires methods with high sensitivity and accuracy. We fabricated an arrayed geometrically enhanced mixing (GEM) chip with a "dislocation herringbone" layout based on cell immunoaffinity. By optimizing the injection and rinsing flow rate, an average cell capture rate of 87.02% and an average capture purity of 99.58% were achieved using the human lung adenocarcinoma cell lines H1975. In addition, we determined the specificity, precision, accuracy, and detection limit of our chip. The results demonstrated the chip was stable, accurate and reliable for the "liquid biopsy" of lung cancer cells using the peripheral blood of patients. Our chip can also be used to evaluate the potency of different drugs against tumor cells in parallel due to the presence of four independent microchannels.

T59, a New Compound Reconstructed from Curcumin, Induces Cell Apoptosis through Reactive Oxygen Species Activation in Human Lung Cancer Cells.

Curcumin is acknowledged for its antioxidant, anti-inflammatory, anti-cancer, and wound-healing properties. However, the biological activity and the molecular mechanisms of T59, which is a new derivative of curcumin, are not fully understood. The present study was aimed to determine the cytoxicity role of T59 in human lung cancer and the molecular mechanisms. Cytotoxicity and cell apoptosis effects induced by T59 were determined by MTT, AO staining, Annexin V, and JC-1. Compared with curcumin, T59 exerted more effective cytotoxicity and cell apoptosis effects in A549 and H1975. With the decreasing level of the mitochondrion membrane potential, the generation of reactive oxygen species (ROS) was increased and induced by T59. Furthermore, the expressions of cleaved-caspase-3 and Bax were increased, which were reversed by NAC mainly through the PI3K/AKT signaling pathway. Our results suggested that T59 has the potential for further investigation and study to act as an anti-cancer therapeutic against human lung cancer.

Cordycepin Induces Apoptosis and Inhibits Proliferation of Human Lung Cancer Cell Line H1975 via Inhibiting the Phosphorylation of EGFR.

Cordycepin is an active component of the traditional Chinese medicine Cordyceps sinensis and Cordyceps militaris with notable anticancer activity. Though the prominent inhibitory activity was reported in different kinds of cancer cell lines, the concrete mechanisms remain elusive. It was reported that cordycepin could be converted into tri-phosphates in vivo to confuse a number of enzymes and interfere the normal cell function. For the inhibitory mechanism of EGFR inhibitors and the structure similarity of ATP and tri-phosphated cordycepin, human lung cancer cell line H1975 was employed to investigate the inhibitory effect of cordycepin. The results showed that cordycepin could inhibit cell proliferation and induce apoptosis in a dose-dependent manner. Cell cycle analysis revealed that H1975 cells could be arrested at the G0/G1 phase after cordycepin treatment. The expression levels of apoptosis-related protein Caspase-3 and Bcl-2 and phosphorylated expression levels of EGFR, AKT and ERK1/2 were all decreased compared with the control group stimulated with EGF. However, the protein expression levels of proapoptotic protein Bax and cleaved caspase-3 were increased. These results implied that cordycepin could inhibit cell proliferation and induce apoptosis via the EGFR signaling pathway. Our results indicated that there was potential to seek a novel EGFR inhibitor from cordycepin and its chemical derivatives.

Disclosing Potential Therapeutic Targets Associated With Osimertinib Resistance in the Non-small Cell Lung Cancer Cell Line H1975.

BACKGROUND/AIM: Osimertinib, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor, is a highly effective and valuable treatment option for advanced non-small cell lung cancer (NSCLC) patients with EGFR mutations, such as T790M. However, acquired resistance ultimately limits its clinical application. In this study, we aimed to identify potential targets for overcoming osimertinib resistance. MATERIALS AND METHODS: The H1975/OSI cell line was induced in vitro through intermittent induction. Cell activity was measured using a cell counting kit-8 assay. Uni-omics and multi-omics analyses were conducted on the transcriptomic and proteomic (4D label-free) expression profiles, which involved differential expression analysis, GO functional annotation and KEGG pathway enrichment analysis, as well as correlation analysis of transcription factors and PPI network. RESULTS: H1975/OSI cells showed resistance towards osimertinib with IC50 values approximately 5.25-fold higher than H1975 cells. A total of 2519 genes were found to be differentially expressed genes (DEGs) and 1533 proteins were found to be differentially abundant proteins (DAPs). Furthermore, 147 genes that were differentially expressed at both the transcription and protein levels (TPGs) were identified as being differentially expressed in both the transcriptome and proteome. It was revealed that many pathways related to the structure and function of ribosomes, as well as metabolites, were altered. The highest connectivity genes of 147 TPGs included NOP56, DDX21, PDCD11, CCNB1, and TOP2A. The hub genes of the transcriptional regulatory network included DDX21, KPNA2, DDX5, BRCA1, LMNB1, and HIF1A. CONCLUSION: Collectively, our high-throughput analysis uncovered functional properties that interacted with gene signatures of H1975/OSI cells, and highlighted certain pathways and eleven hub genes that may be the potential targets for improving clinical osimertinib resistance.

Inhibitory effects of total triterpenoids isolated from the Hedyotis diffusa willd on H1975 cells.

It is estimated that non-small-cell lung cancer (NSCLC) is responsible for 80% of human deaths related to lung cancer worldwide. Currently, it has been discovered that two transcription factors. Nuclear factor-κB (NF-κB) and Signal transducer and activator of transcription 3 (STAT3) were the main factors affecting inflammation and cancer, and their activation promoted lung cancer cell proliferation. Hedyotis diffusa Willd. (H. diffusa) is an herbal Chinese medicine, which has always been used for the treatment of malignant tumors in clinical. Previous research found that H. diffusa could inhibit the proliferation of H1975 cells, but the specific mechanisms remain elusive. We investigated the effects of total triterpenes extracted from H. diffusa (TTH) on the migration, proliferation and apoptosis of H1975 cells. Cell-cycle and immunofluorescence analysis showed that TTH could block H1975 cells at G0/G1 phase and induce apoptosis of experimental cells. The protein levels of Bcl-2 were decreased, while the levels of pro-apoptotic Bax were increased. In addition, TTH could also inhibit the migration of H1975 cells through downregulated MMP-2 and MMP-9 and upregulated TIMP-2. Further research found that the level of phospho-STAT3 was significantly decreased after administration of TTH. And protein expression level of NF-κB in nucleus was decreased after TTH treatment, while NF-κB in cytoplasm increased. These results suggested that TTH could inhibit the proliferation and migration of H1975 cells, and also could induce cell apoptosis. These effects were closely connected to the activation of NF-κB and the phosphorylation of STAT3.

The Synthesis and Antitumor Activity of 1,8-Naphthalimide Derivatives Linked 1,2,3-Triazole.

In this study, acenaphthylene was used as the raw material, and a series of novel 1,8-naphthalimide-1,2,3-triazole derivatives was obtained through oxidation, acylation, alkylation, and click reactions, and subsequently, their anti-tumor activities were tested. After screening, we found that Compound 5e showed good activity against H1975 lung cancer cells, with the half maximal inhibitory concentration (IC50) reaching 16.56 µM.

Extracellular Vesicles (EVs) from Lung Adenocarcinoma Cells Promote Human Umbilical Vein Endothelial Cell (HUVEC) Angiogenesis through Yes Kinase-associated Protein (YAP) Transport.

Yes kinase-associated protein (YAP) plays an important role in angiogenesis and can promote the occurrence and development of many tumor types. However, whether YAP affects tumor angiogenesis in lung cancer, and its potential mechanism in lung cancer, are unknown. In this study, we explored the role of YAP in the angiogenesis of lung adenocarcinoma, and further illustrated its possible mechanism. The expression levels of YAP and the vascular endothelial marker protein CD31 were examined by immunohistochemistry and immunofluorescence in human lung adenocarcinoma tissues, revealing a possible positive correlation between YAP and CD31 in lung adenocarcinoma. The results of the western blotting (WB) of Human Umbilical Vein Endothelial Cells (HUVECs) after coculture with lung adenocarcinoma H1975 cells, H1975 cell-supernatants and H1975-derived EVs showed that YAP derived from H1975 cells can enter HUVECs via EVs. These results were confirmed by immunofluorescence. Finally, we generated H1975 low-YAP expression cells by transfecting the cells with a shYAP lentivirus, and confirmed that the low expression of YAP in H1975 cells inhibits HUVEC angiogenesis by reducing the amount of YAP that enters HUVECs. We found, for the first time, that YAP promotes angiogenesis in lung adenocarcinoma via EVs, at least partially. Our work may provide a promising method for lung cancer treatment by targeting angiogenesis in the future.

Inhibitory Effects of HangAmDan-B1 (HAD-B1) Combined With Afatinib on H1975 Lung Cancer Cell-Bearing Mice.

Epidermal growth factor receptor mutation-positive non-small cell lung cancer is cared for mainly by target therapeutics in the clinical treatment at present. We investigated the antitumor effect of HangAmDan-B1 (HAD-B1) combined with afatinib on H1975 (L858R/T790M double mutation) lung cancer cells. The combined treatment of HAD-B1 with afatinib inhibited the proliferation of H1975 cells in a dose-dependent manner compared with the treatment of afatinib or HAD-B1 alone. The combined treatment group significantly induced early apoptosis and cell cycle arrest of the cells compared with afatinib- or HAD-B1-treated control group. Profile analysis of cell cycle proteins in H1975 cells treated with the combination of HAD-B1 and afatinib using InnoPharmaScreen antibody microarray showed downregulation of pERK1/2 and upregulation of p16 in the cells. In vivo tumor growth assay in xenograft animal model of human H1975 lung cancer cells revealed that the mean tumor volume in the group treated with the combination of HAD-B1 and afatinib showed a significant reduction compared with the control groups.

The effect of PLK1 inhibitor in osimertinib resistant non-small cell lung carcinoma cells / 浙江大学学报·医学版

OBJECTIVES@#To investigate the effects of PLK1 inhibitors on osimertinib-resistant non-small cell lung carcinoma (NSCLC) cells and the anti-tumor effect combined with osimertinib.@*METHODS@#An osimertinib resistant NCI-H1975 cell line was induced by exposure to gradually increasing drug concentrations. Osimertinib-resistant cells were co-treated with compounds from classical tumor pathway inhibitor library and osimertinib to screen for compounds with synergistic effects with osimertinib. The Gene Set Enrichment Analysis (GSEA) was used to investigate the activated signaling pathways in osimertinib-resistant cells; sulforhodamine B (SRB) staining was used to investigate the effect of PLK1 inhibitors on osimertinib-resistant cells and the synergistic effect of PLK1 inhibitors combined with osimertinib.@*RESULTS@#Osimertinib-resistance in NCI-H1975 cell (resistance index=43.45) was successfully established. The PLK1 inhibitors GSK 461364 and BI 2536 had synergistic effect with osimertinib. Compared with osimertinib-sensitive cells, PLK1 regulatory pathway and cell cycle pathway were significantly activated in osimertinib-resistant cells. In NSCLC patients with epidermal growth factor receptor mutations treated with osimertinib, PLK1 mRNA levels were negatively correlated with progression free survival of patients (R=－0.62, P<0.05), indicating that excessive activation of PLK1 in NSCLC cells may cause cell resistant to osimertinib. Further in vitro experiments showed that IC50 of PLK1 inhibitors BI 6727 and GSK 461364 in osimertinib-resistant cells were lower than those in sensitive ones. Compared with the mono treatment of osimertinib, PLK1 inhibitors combined with osimertinib behaved significantly stronger effect on the proliferation of osimertinib-resistant cells.@*CONCLUSIONS@#PLK1 inhibitors have a synergistic effect with osimertinib on osimertinib-resistant NSCLC cells which indicates that they may have potential clinical value in the treatment of NSCLC patients with osimertinib resistance.

Regulation and mechanism of Myosin X on radiosensitivity of non-small cell lung cancer cell line H1975 in vitro / 中华放射肿瘤学杂志

Objective:To investigate the effect and mechanism of Myosin X on the radiosensitivity of non-small cell lung cancer (NSCLC) cell line H1975 in vitro. Methods:Western blot was applied to detect the expression level of Myosin X expression. The H1975 cell line with stable knockout of Myosin X (KO group) and infected with control virus (NC group) were constucted by using CRISPR/Cas9 technique. The knockout efficiency was validated. The radiosensitivity of two cell lines was measured by colony formation assay and single-hit multi-target model. γ-H 2AX focus formation test and RNA sequencing (RNAseq) analysis were employed to identify the regulatory mechanism of the radiosensitivity of lung cancer cell lines mediated by Myosin X. Results:The expression level of Myosin X in the H1975 cells was significantly up-regulated than those in other NSCLC cell lines (all P<0.01). The lentiviral vector of Myosin X sgRNA-Lenti-CRISPR v2 was successfully constructed. After the puromycin screening, H1975 cell lines with complete knockout of Myosin X and control cell lines (NC group) were obtained. Colony formation assay demonstrated that compared with the NC group, the radiosensitivity in the KO group was significantly higher (The D 0 value was decreased from 1.28 Gy to 1.03 Gy, SF 2 decreased from 0.29 to 0.21, and the sensitization ratio was 1.24). The γ-H 2AX focus formation test showed that the number of damage focus formed at 1 h and 6 h after irradiation in the KO group was significantly larger than that in the NC group ( P<0.05. RNAseq analysis indicated that the expression level of ISLR in the KO group was significantly down-regulated than that IN the NC group ( P<0.05). Conclusion:Knockout of Myosin X can increase the radiosensitivity of H1975 cells probably by interfering the repair of DNA double-strand damage and down-regulating the expression level of ISLR.

The afatinib resistance of in vivo generated H1975 lung cancer cell clones is mediated by SRC/ERBB3/c-KIT/c-MET compensatory survival signaling.

We generated afatinib resistant clones of H1975 lung cancer cells by transient exposure of established tumors to the drug and collected the re-grown tumors. Afatinib resistant H1975 clones did not exhibit any additional mutations in proto-oncogenes when compared to control clones. Afatinib resistant H1975 tumor clones expressed less PTEN than control clones and in afatinib resistant clones this correlated with increased basal SRC Y416, ERBB3 Y1289, AKT T308 and mTOR S2448 phosphorylation, decreased expression of ERBB1, ERBB2 and ERBB3 and increased total expression of c-MET, c-KIT and PDGFRß. Afatinib resistant clones were selectively killed by knock down of [ERBB3 + c-MET + c-KIT] but not by the individual or doublet knock down combinations. The combination of the ERBB1/2/4 inhibitor afatinib with the SRC family inhibitor dasatinib killed afatinib resistant H1975 cells in a greater than additive fashion; other drugs used in combination with dasatinib such as sunitinib, crizotinib and amufatinib were less effective. [Afatinib + dasatinib] treatment profoundly inactivated ERBB3, AKT and mTOR in the H1975 afatinib resistant clones and increased ATG13 S318 phosphorylation. Knock down of ATG13, Beclin1 or eIF2α strong suppressed killing by [ERBB3 + c-MET + c-KIT] knock down, but were only modestly protective against [afatinib + dasatinib] lethality. Thus afatinib resistant H1975 NSCLC cells rely on ERBB1- and SRC-dependent hyper-activation of residual ERBB3 and elevated signaling, due to elevated protein expression, from wild type c-MET and c-KIT to remain alive. Inhibition of ERBB3 signaling via both blockade of SRC and ERBB1 results in tumor cell death.

Marsdenia tenacissima extract enhances gefitinib efficacy in non-small cell lung cancer xenografts.

PURPOSE: The stem of Marsdenia tenacissima (Roxb.) Wight et Arn. has long been used as a medicine to treat cancer in China. Our previous in vitro results showed that Marsdenia tenacissima extract (MTE) overcomes gefitinib resistance in non-small cell lung cancer (NSCLC) cells. However, it is unknown whether MTE could enhance gefitinib efficacy in vivo. The present study was intended to investigate the in vivo anti-tumour activity of MTE combined with gefitinib. METHODS: Human NSCLC H460 (K-ras mutation) or H1975 cells (EGFR T790M mutation) were subcutaneously inoculated into nude mice. Tumour volume and body weight were measured regularly. Resected tumours were weighed after the animals were sacrificed. Immunoblotting or immunohistochemistry was used to assess the cellular proliferation and apoptosis in xenograft tumour tissue. Expression of the EGFR downstream pathways and c-Met were measured with western blot analysis to explore possible mechanisms. RESULTS: MTE (5, 10, 20 g/kg) dose-dependently reduced tumour growth and induced cell apoptosis. MTE suppressed EGFR related signals, and 20 g/kg was the most effective dose. Low-dose MTE (5 g/kg) significantly enhanced gefitinib efficacy in resistant H460 and H1975 xenografts. The combination inhibited tumour proliferation and induced cell apoptosis in both resistant NSCLC xenografts. Constitutive activation of the PI3K/Akt and MEK/ERK pathways is related to EGFR-TKI resistance. Accordingly, phosphorylation of PI3K/Akt/mTOR and ERK1/2 was suppressed after combination treatment. Simultaneously, cross-talked c-Met and EGFR were also prominently lowered in the presence of MTE combined with gefitinib. CONCLUSION: The present results suggest that the combination of MTE and gefitinib may be a promising therapeutic approach to enhance gefitinib efficacy in resistant NSCLC.

Structure-activity study of quinazoline derivatives leading to the discovery of potent EGFR-T790M inhibitors.

We have developed a series of 6, 7-disubstituted-4-(arylamino) quinazoline derivatives that functioned as irreversible EGFR inhibitors, and these compounds exhibited excellent enzyme inhibition potency. As compared with afatinib, some of them showed significantly enhanced activities towards H1975 cells (EGFR-T790M). Furthermore, the optimized compounds 7q and 8f also demonstrated good pharmacokinetic profiles, oral bioavailability as well as excellent in vivo efficacy in H1975 and HCC827 xenografts at a non-toxic dose. Based on the improved safety and efficacy against EGFR-T790M resistance, 7q and 8f are promising candidates for further studies.

SOX2 expression is an early event in a murine model of EGFR mutant lung cancer and promotes proliferation of a subset of EGFR mutant lung adenocarcinoma cell lines.

OBJECTIVES: Primary and acquired resistance to EGFR TKIs in EGFR mutant lung cancer occurs primarily through secondary mutations in EGFR or Met amplification. Drug resistance can also be mediated by expression of pluripotency transcription factors, such as OCT4, SOX2 and NANOG that decrease terminal differentiation. In this study, we investigated the expression and role of SOX2 in model systems of EGFR mutant tumors. MATERIALS AND METHODS: Immunoblotting or immunohistochemistry was used to assess expression of pluripotency transcription factors in lungs of transgenic mice or in human NSCLC cell lines. Expression of SOX2 was reduced by shRNA knockdown, and response to erlotinib and cellular proliferation were assessed. RESULTS AND CONCLUSION: Induction of mutant EGFR in transgenic CCSP-rtTA/TetO-EGFR(L858R/T790M) mice correlated with increased OCT4 and SOX2 expression in lung tissue prior to tumor development. Established lung tumors retained SOX2 expression. To assess a role for SOX2 in tumorigenesis, a panel of NSCLC cell lines with activating EGFR mutations was assessed for SOX2 expression. Two of six cell lines with mutant EGFR showed detectable SOX2 levels, suggesting SOX2 expression did not correlate with EGFR mutation status. To assess the role of SOX2 in these cell lines, HCC827 and H1975 cells were infected with lentivirus containing SOX2 shRNA. Knockdown of SOX2 decreased proliferation in both cell lines and increased sensitivity to erlotinib in HCC827 cells. Because constitutive activation of the PI3K/Akt pathway is associated with EGFR TKI resistance, cells were treated with PI3K/AKT inhibitors and expression of SOX2 was examined. PI3K/Akt inhibitors decreased SOX2 expression in a time-dependent manner. These data suggest targeting SOX2 may provide therapeutic benefit in the subset of EGFR-mutant tumors with high constitutive levels of SOX2, and that until more direct means of inhibiting SOX2 are developed, PI3K/Akt inhibitors might be useful to inhibit SOX2 in EGFR TKI resistant tumors.

The effect of apoptosis-2 ligand on irradiation-induced apoptosis in lung adenocarcinima H1975 cells resistant to EGFR-TKI / 中华放射医学与防护杂志

Objective To investigate whether the apoptosis-2 ligand (Apo-2L),known as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),could enhance irradiation-induced apoptosis in lung adenocarcinima H1975 cells that are resistant to the epithelial growth factor receptor (EGFR)-TKI.Methods Adenocarcinima H1975 cells were randomly divided into four groups:the control group,Apo2L group,irradiation group,and both Apo-2L and irradiation group.H1975 cells were pretreated with Apo2L under different concentrations of 200 and 228 ng/ml at 24 h before irradiation with doses of 1,1.5,2,2.5,3,3.5 and 4 Gy.The apoptosis rates of all groups were analyzed by flow cytometry 24 h post-irradiation.The inhibition rates of cell proliferation were measured by the MTT assay.Results MTT assay showed that the Apo-2L treatment significantly inhibited cell proliferation(x2 =136.17,P ＜ 0.05).The apoptosis rates of the four groups were different significantly,and the apoptosis rate of radiation combined with drug group was significantly higher than the other three groups(x2 =78.02,P ＜0.05).Conclusions The Apo-2L could not only inhibit the proliferation but also promote radiation-induced apoptosis of adenocarcinoma H1975 cells.

NGX6 gene combined with cisplatin in treatment of lung cancer in vitro and in vivo / 中国生化药物杂志

Objective To evaluate the effect of NGX6 combined with cisplatin on the inhibition rate of A549 cells and NCI-H1975 cells and antitumor effects in vivo.Methods The NGX6 was loaded in the LPD, and prepare Liposome protamine DNA complexes.A549 cells and NCI-H1975 cells were seperately divided into NGX6 group ( 30 μg/mL NGX6 concentration ) , cisplatin group, NGX6 +cisplatin group, PBS as negative control group.The effect of cytotoxicity of four group on A549 cells and NCI-H1975 cell were evaluated by MTT assay.the clone forming rate and the inhibition rate were determined by Cell colony count.lung transplantation tumor model were successfully established, then nude mice were divided into four groups as abeve, each group of 10, tumor size and survival period were determined tumor cell apoptosis were observed.Results The cell viability of A549 cells and NCI-H1975 cells of (NGX6 +cisplatin) were lower than that of NGX6 group, cisplatin group and saline group, respectively(P<0.01).The cloning efficiency of A549 cells and NCI-H1975 cells of ( NGX6 +cisplatin) were lower than that of NGX6 group, cisplatin group and saline group, respectively(P<0.01).The tumor inhibitory rate was in vivo for (NGX6 +cisplatin) was higher than other group(P<0.01).The median survival of nude mice in (NGX6 +cisplatin), NGX6, cisplatin and saline group were 43,31,29 and 15 days.Conclusion NGX6 combination with cisplatin can inhibit the cell proliferate of lung cancer cells and inhibit the tumor growth and the combination of NGX6 and cisplatin may be a potentially effective treatment for lung cancer.