Targeting Polo-like kinase 1 and TRAIL enhances apoptosis in non-small cell lung cancer

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can selectively induce apoptosis in cancer cells without causing damage to normal cells. However, some tumors are resistant to TRAIL monotherapy and clinical studies assessing targeted agents towards the TRAIL receptor have failed to show robust therapeutic activity. Evidence has shown that standard anti-mitotic drugs can induce synergistic apoptosis upon combination with TRAIL via cell cycle arrest. Polo like kinase-1 (PLK1) plays a critical role in different stages of cell cycle progression and mitosis. A number of investigations have demonstrated that PLK1 inhibition causes cell cycle arrest and mitotic catastrophe in non-small cell lung cancer (NSCLC), and we thus postulated that PLK1 inhibition could enhance TRAIL-induced apoptosis. We demonstrate that the combination of a TRAIL receptor agonist and a PLK1 inhibitor synergistically reduces cell viability, and strongly increases apoptosis in NSCLC cellular models. Consistent with our in vitro observations, this drug combination also significantly reduces tumor growth in vivo. Our data additionally reveal that G2/M cell cycle arrest and downregulation of Mcl-1 and signal transducer and activator of transcription 3 (STAT3) activity following PLK1 inhibition may contribute to the sensitization of TRAIL-induced apoptosis in NSCLC. Together, these data support the further exploration of combined TRAIL and PLK1 inhibition in the treatment of NSCLC

Pengaruh Sumpah Pemuda terhadap keturunan Arab di Indonesia

Adapun fokus penelitian yang dibahas dalam skripsi ini adalah : (1) Apa pengaruh Sumpah Pemuda 1928 terhadap Keturunan Arab di Indonesia ?. (2) Apa kontribusi Sumpah Pemuda Indonesia Keturunan Arab untuk Indonesia ?. (3) Bagaimana pengaruh nasionalisme etnis keturunan Arab kepada etnis keturunan lain di Indonesia ?. Penulisan skripsi ini disusun dengan menggunakan metode penelitian sejarah yaitu heuristik (pengumpulan data), verifikasi (kritik terhadap data), interpretasi (penafsiran) dan historiografi (penulisan sejarah). Pendekatan yang digunakan adalah pendekatan historis kritis (mendeskripsikan peristiwa yang terjadi pada masa lampau). Disamping itu, penulis juga menggunakan pendekatan politik dan sosiologi. Sedangkan teori yang digunakan adalah Teori Challenge and Response (Tantangan dan Jawaban) oleh Arnold Joseph Toynbee. Dari hasil penelitian yang dilakukan, dapat disimpulkan bahwa : (1) Sumpah Pemuda 1928 memberikan pengaruh yang besar khususnya bagi keturunan Arab yaitu dengan tercetusnya Sumpah Pemuda Indonesia Keturunan Arab (SPIKA) 1934 (2) Kontribusi atas dicetuskannya SPIKA untuk Indonesia adalah berdirinya Partai Arab Indonesia (PAI), banyaknya tokoh-tokoh Arab yang turut serta memperjuangkan kemerdekaan, dan pasca kemerdekaan pun peran etnis peranakan Arab sangat besar untuk Indonesia. (3) Nasionalisme dalam diri etnis peranakan Arab ternyata memberikan pengaruh terhadap etnis-etnis peranakan lain di Indonesia dalam hal Nasionalisme dan cinta tanah air Indonesia

Analisis penggunaan leverage, kualitas audit, dan employee diff dalam mendeteksi kecurangan laporan keuangan: Studi empiris perusahaan manufaktur yang terdaftar di Bursa Efek Indonesia tahun 2007-2011

Tujuan penelitian ini adalah untuk mengetahui apakah leverage, kualitas audit dan employee diff (selisih ukuran keuangan dan nonkeuangan) dapat digunakan untuk mendeteksi kecurangan laporan keuangan, studi empiris pada perusahaan sektor manufaktur yang terdaftar di Bursa Efek Indonesia tahun 2007- 2011. Sampel pada penelitian ini dipilih berdasarkan metode purposive sampling dengan jumlah populasi sebanyak 134 perusahaan dan sampel sebanyak 23 perusahaan. Metode analisis yang digunakan adalah analisis regresi berganda dengan program SPSS versi 20. Teknik analisis yang digunakan pada penelitian ini adalah analisis statistik deskriptif, uji asumsi klasik, uji hipotesis F-statistik untuk menguji pengaruh secara bersama-sama dengan tingkat kepercayaan 5% serta menggunakan t-statistik untuk menguji koefisien regresi parsial. Hasil penelitian ini menunjukkan bahwa secara simultan leverage, kualitas audit, dan employee diff berpengaruh secara signifikan terhadap kecurangan laporan keuangan dengan proksi discretionary accruals. Sementara secara parsial kualitas audit dan employee diff berpengaruh signifikan terhadap kecurangan laporan keuangan, leverage tidak berpengaruh signifikan terhadap kecurangan laporan keuanga

Combined targeting of EGFR/HER promotes anti-tumor efficacy in subsets of KRAS mutant lung cancer resistant to single EGFR blockade.

peer reviewedKRAS is a frequently mutated oncogene in lung cancer and among the most refractory to EGFR targeted therapy. Recently, preclinical evidence in pancreatic cancer has demonstrated that mutant KRAS can be regulated by EGFR. However, the distinct correlation between the EGFR/HER family members and mutant KRAS has not been investigated. Here, we show that non-small cell lung cancer cell lines harboring differing isoforms of mutant KRAS, can be broadly divided into EGFR/HER dependent and EGFR/HER independent groups. Combined therapeutic targeting of EGFR, HER2 and HER3 in isoforms regulated by extracellular growth signals promotes in vitro and in vivo efficacy. We also provide evidence that depletion of EGFR via RNA interference specifically abolishes the EGFR/KRAS interaction in the dependent subset. Taken together, these findings suggest that upstream inhibition of the EGFR/HER receptors may be effective in treating a subset of KRAS mutant lung cancers

Synergistic effect of afatinib with su11274 in non-small cell lung cancer cells resistant to gefitinib or erlotinib.

Epidermal growth factor receptor (EGFR) and c-MET receptors are expressed on many non-small cell lung cancer (NSCLC) cells. Current single agent therapeutic targeting of a mutant EGFR has a high efficacy in the clinic, but is not curative. Here, we investigated the combination of targeting EGFR and c-MET pathways in NSCLC cells resistant to receptor tyrosine kinase inhibitors (TKIs), using RNA interference and inhibition by TKIs. Different NSCLC cell lines with various genomic characteristics (H358, H1650 and H1975) were transfected with EGFR-specific-siRNA, T790M-specific-siRNA, c-MET siRNA or the combination. Subsequently EGFR TKIs (gefitinib, erlotinib or afatinib) or monoclonal antibody cetuximab were combined respectively with the c-MET-specific TKI su11274 in NSCLC cell lines. The cell proliferation, viability, caspase-3/7 activity and apoptotic morphology were monitored by spectrophotometry, fluorimetry and fluorescence microscopy. The combined effect of EGFR TKIs, or cetuximab and su11274, was evaluated using a combination index. The results showed that the cell lines that were relatively resistant to EGFR TKIs, especially the H1975 cell line containing the resistance T790M mutation, were found to be more sensitive to EGFR-specific-siRNA. The combination of EGFR siRNA plus c-MET siRNA enhanced cell growth inhibition, apoptosis induction and inhibition of downstream signaling in EGFR TKI resistant H358, H1650 and H1975 cells, despite the absence of activity of the c-MET siRNA alone. EGFR TKIs or cetuximab plus su11274 were also consistently superior to either agent alone. The strongest biological effect was observed when afatinib, an irreversible pan-HER blocker was combined with su11274, which achieved a synergistic effect in the T790M mutant H1975 cells. In a conclusion, our findings offer preclinical proof of principle for combined inhibition as a promising treatment strategy for NSCLC, especially for patients in whom current EGFR-targeted treatments fail due to the presence of the T790M-EGFR-mutation or high c-MET expression

Combined inhibition of rho-associated protein kinase and EGFR suppresses the invasive phenotype in EGFR-dependent lung cancer cells.

INTRODUCTION: Lung cancer remains the leading cause of cancer-related mortality worldwide, with metastatic disease frequently a prominent feature at the time of diagnosis. The role of NSCLC-derived EGFR mutations in cancer cell proliferation and survival has been widely reported, but little is known about the function of these mutations in invasive growth and metastasis. In this study, we sought to evaluate the intrinsic invasive properties of NSCLC cells with differing EGFR status and examine possible therapeutic targets that can abrogate invasive growth. MATERIALS AND METHODS: Collagen-based assays and 3D cell cultures were used to assess morphological features, actin cytoskeleton dynamics and the invasive capacity of NSCLC cell lines with differing EGFR status. The role of the RhoA/ROCK/MYPT1 and EGFR/HER pathways in NSCLC-related invasion was investigated by pharmacological inhibition and RNA interference techniques. RESULTS: We demonstrate a positive correlation between EGFR mutational/amplification status and invasive capacity. Knockdown of wild-type and mutant EGFR leads to depletion of active and total MYPT1 levels. Combined pharmacological inhibition or genetic ablation of ROCK/EGFR suppresses the hallmarks of cancer cells and abrogates the invasive phenotype in EGFR-dependent NSCLC cells. CONCLUSIONS: These observations suggest that combined targeting of the ROCK and EGFR/HER pathways may be a potential therapeutic approach in limiting invasive growth in NSCLC

Production of a mono-biotinylated EGFR nanobody in the E. coli periplasm using the pET22b vector

Abstract Objective Our aim was to produce a mono-biotinylated single domain antibody (‘nanobody’) specific for the epidermal growth factor receptor (EGFR), which is overexpressed in many cancer cells. The binding of the nanobody and its function are tested in cancer cells. The construct could be used to carry variable therapeutic or diagnostic load using biotin-streptavidin bridging. Results The EGFR-specific 7D12 nanobody was genetically fused to an IgA hinge linker and to a C-terminal biotin ligase acceptor sequence, allowing mono-biotinylation in E. coli. Expression was in strain BL21-DE3 from a T7 RNA polymerase driven pET22b vector. The biotinylated nanobody, isolated from the periplasm, was purified using streptavidin-mutein affinity chromatography. Final yields were up to 5 mg/l of cell culture. We showed that the construct could bind to EGFR expressing A431 epidermoid carcinoma cells, and to transiently transformed EGFR overexpressing HEK293T cells and not to EGFR negative control cells. The specificity for the EGFR was further demonstrated by immunoprecipitation. To test the functionality, PC9 non-small cell lung cancer cells were treated with mono-biotinylated nanobody or with streptavidin-coupled tetravalent nanobodies. Both were able to block mutant EGFR phosphorylation and slow down growth of PC9 cells. Tetravalent nanobodies were able to downregulate AKT phosphorylation

Functional effect of combination of EGFR-specific-siRNA with c-MET siRNAs.

<p><b>Panel A</b>: Cell proliferation following transfection with different siRNAs. Cell lines H358 (white), H1650 (grey) and H1975 (black) were transfected with EGFR-specific-siRNA 1247, T790M specific siRNA, a c-MET siRNA pool, or combinations of these, and the proliferation was assayed 72 h post transfection using a colorimetric MTS assay. <b>Panel B</b>: Caspase−3/7 activity. * <i>P</i><0.05 and ** <i>P</i><0.01 compared to both single treatment.</p

Effect of su11274 in lung cancer cells containing TKI resistance mutations.

<p>H358, H1650 and H1975 cells were incubated with a concentration range of su11274, and the cells were incubated for 72 h. <b>Panel A:</b> proliferation. <b>Panel B:</b> caspase−3/7 activity. <b>Panel C:</b> apoptosis. <b>Panel D:</b> dose-effect curves.</p

siRNAs used in the study.

<p>NA: not applicable.</p