casein-kinase-ii and Adenocarcinoma-of-Lung

The abnormal activation of the downstream signaling pathways of epidermal growth factor receptor (EGFR) that are independent of EGFR, contribute to the acquisition of EGFR‑tyrosine kinase inhibitor (TKI) resistance in non‑small cell lung cancer (NSCLC). The serine/threonine protein kinase casein kinase II (CK2) phosphorylates and modulates several members of the EGFR downstream signaling pathways. Thus, the purpose of the current study was to investigate the effects of the addition of quinalizarin (a specific CK2 inhibitor) to icotinib (an EGFR‑TKI) on the proliferation and apoptosis of four NSCLC cell lines and its underlying mechanisms. The human lung adenocarcinoma cell lines HCC827, A549, H1650 and H1975 were employed to represent the EGFR‑TKI‑sensitive EGFR (EGFR‑sensitive) mutation, wild‑type EGFR and the EGFR‑TKI‑resistant EGFR (EGFR‑resistant) mutations. The cell viability was determined by the MTT assay. Cell apoptosis was detected by flow cytometry using the Annexin V‑enhanced green fluorescent protein Apoptosis Detection kit. The level of proteins in the EGFR downstream pathway was observed using a western blot assay. The results showed that the cells with the EGFR‑sensitive mutation (HCC827, EGFR E716‑A750del) were more sensitive to icotinib compared with those possessing the EGFR wild‑type (A549) and the EGFR‑resistant mutations (H1650, EGFR E716‑A750del and PTEN lost; H1975, EGFR L858R+T790M). Quinalizarin inhibited proliferation and promoted apoptosis in the cells with the EGFR wild‑type and resistant mutations, and the addition of quinalizarin to icotinib partially restored their sensitivity to icotinib. Quinalizarin and/or icotinib increased the apoptotic rates in the EGFR‑TKI resistant cells, and the combination of these reduced the level of protein downstream of EGFR, including phosphorylated (p‑AKT) and p‑(ERK). In conclusion, quinalizarin may partially sensitize cells to icotinib by inhibiting proliferation and promoting apoptosis mediated by AKT and ERK in EGFR‑TKI resistant NSCLC cell lines.

Topics: Adenocarcinoma of Lung; Anthraquinones; Antineoplastic Agents; Apoptosis; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Crown Ethers; Drug Resistance, Neoplasm; Humans; Lung Neoplasms; Protein Kinase Inhibitors; Quinazolines

Lung cancer is the leading cancer-related death worldwide. Patients with lung cancer mainly died of tumor metastasis and invasion. Protein kinase CK2 is an ubiquitous serine/threonine protein kinase and is frequently upregulated in various human tumors. This study aims to explore the effect and molecular mechanism of the invasion and migration of lung adenocarcinoma A549 cells after knock-down of CK2α expression.. The pSilencerTM 4.1-siCK2α-eGFP of lentiviral-mediated shRNA was constructed. The expression of CK2α was knock-downed, and a stable A549 cell line was established. The invasion and migration of A549 cell line was detected through Transwell and Boyden chamber assays. The protein expression of the PI3K/Akt signaling pathway and mesenchymal-to-epithelial transition (EMT) was evaluated using Western blot analysis.. The invasion and migration of A549 cells were significantly inhibited after the knockdown of CK2α expression compared with that in the control group. p-PTEN, Akt, p-Akt473, p-Akt308, p-PDK1, p-c-Raf, and p-GSK-3β were significantly downregulated, whereas PTEN was upregulated. Moreover, vimentin, β-catenin, Snail, MMP2, and MMP9 were significantly downregulated after reducing the CK2α expression.. CK2α might regulate the invasion and migration of A549 cells through the PI3K/Akt/GSK-3β/Snail signaling pathway, which controls EMT in lung adenocarcinoma.
.. 背景与目的 肺癌已成为全球癌症死亡的首要原因，而侵袭和转移是导致肿瘤死亡的主要原因之一，蛋白激酶CK2是一种高度保守信使非依赖性丝氨酸苏氨酸蛋白激酶，其在各种肿瘤中高表达。本研究旨在探讨下调CK2α基因表达对肺腺癌A549细胞侵袭迁移的影响以及可能的机制。方法 构建pSilencerTM 4.1-shCK2α-eGFP慢病毒表达载体，建立稳定干扰CK2α表达的A549细胞株。利用Transwell和Boyden小室实验检测干扰CK2α表达前后A549细胞的侵袭及迁移的能力。Western blot检测PI3K/Akt信号通路和上皮-间充质转化（mesenchymal-to-epithelial transition, EMT）相关蛋白的表达。结果 与对照组相比，干扰CK2α表达后肺腺癌A549细胞的侵袭及迁移能力明显下降，p-PTEN、Akt、p-Akt473、p-Akt308、 p-PDK1、p-c-Raf、p-GSK-3β蛋白明显下调，PTEN蛋白表达水平显著上调。上皮-间充质转化的相关蛋白E-cadherin蛋白表达水平显著上调，而Vimentin、β-catenin、Snail蛋白表达水平显著下调，与侵袭转移相关蛋白的MMP2、MMP9表达水平显著下调。结论 CK2α可能通过PI3K/Akt/GSK-3β/Snail信号通路来调控上皮-间充质转化参与肺腺癌A549细胞的侵袭及迁移。.

Topics: A549 Cells; Adenocarcinoma; Adenocarcinoma of Lung; Casein Kinase II; Cell Movement; Glycogen Synthase Kinase 3 beta; Humans; Lung Neoplasms; Neoplasm Metastasis; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Signal Transduction

To explore whether quninalizarin, an specific inhibitor of protein kinase CK2, could sensitize icotinib in EGFR-TKIs (epithelial growth factor receptor-tyrosine kinase inhibitor)-resistant cell lines and uncover the underlying mechanisms.. MTT assay was performed to evaluate the inhibitory effect of quninalizarin, icotinib or the combination of both on cell proliferation in several lung adenocarcinoma cell lines. Western blot assay was used to assess if combined inhibition of EGFR and protein kinase CK2 by icotinib and quninalizarin, exerts effect on the expression and phosphorylation of major proteins of EGFR signaling pathways.. The IC50 of HCC827, H1650, H1975 and A549 cells for icotinib were (8.07±2.00)μmol/L, (66.01±6.64)μmol/L, (265.60±9.47)μmol/L and (87.88±6.8)μmol/L, respectively, indicating that HCC827 cells are sensitive to icotinib, and the H1650, H1975 and A549 cells are relatively resistant to icotinib. When treated with both quninalizarin and icotinib in the concentration of 50 μmol/L, the viability of H1650, H1975 and A549 cells was (40.64±3.73)%, (65.74±3.27)% and (44.96±0.48)%, respectively, significantly lower than that of H1650, H1975 and A549 cells treated with 50 μmol/L icotinib alone (55.05±1.22)%, (71.98±1.60)% and (61.74±6.18)%, respectively (P<0.01 for all). When treated with both 100 μmol/L quninalizarin and 100 μmol/L icotinib, the viability of H1650, H1975 and A549 ells were (23.35±0.81)%, (55.70±1.03)%, (33.42±1.33)%, respectively, significantly lower than the viability of H1650, H1975 and A549 cells treated with 100 μmol/L icotinib alone (40.57±2.65)%, (62.40±2.05)% and (44.97±8.20)%, respectively, (P<0.01 for all). The two-way ANOVA analysis showed that compared with the viability of EGFR-TKIs-resistant cells (H1650, H1975, A549) treated with 50 μmol/L and 100 μmol/L icotinib alone, the viability of cells treated with icotinib and quinalizarin were significantly suppressed, and the differences were statistically significant (P<0.01). In addition, the phosphorylation form of Akt and ERK (namely p-Akt and p-ERK) were significantly down-regulated by treating with quninalizarin and icotinib together in the H1650 cells while the expression of Akt and ERK changed little.. Quinalizarin, as a specific CK2 inhibitor, may overcome icotinib resistance by inhibiting proliferation mediated by Akt and ERK in human lung adenocarcinoma cell lines, and enhances the suppressive effect of icotinib on the proliferation of EGFR-TKIs-resistant human lung adenocarcinoma cells.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Analysis of Variance; Anthraquinones; Apoptosis; Casein Kinase II; Cell Line, Tumor; Cell Proliferation; Cell Survival; Crown Ethers; Down-Regulation; Drug Combinations; Drug Resistance, Neoplasm; Drug Synergism; ErbB Receptors; Humans; Lung Neoplasms; Phosphorylation; Protein-Tyrosine Kinases; Quinazolines; Signal Transduction

The epithelial-to-mesenchymal transition (EMT) is a major phenotype of cancer metastasis and invasion. As a druggable cancer target, the inhibition of protein kinase CK2 (formally named to casein kinase 2) has been suggested as a promising therapeutic strategy to treat EMT-controlled cancer metastasis. This study aimed to evaluate the effect of the CK2 inhibitor CX-4945 on the processes of cancer migration and invasion during the EMT in A549 human lung adenocarcinoma cells.. The effect of CX-4945 on TGF-β1-induced EMT was evaluated in A549 cells treated with TGF-β1 (5 ng/ml) and CX-4945. The effect of CX-4945 on TGF-β1-induced cadherin switch and activation of key signaling molecules involved in Smad, non-Smad, Wnt and focal adhesion signaling pathways were investigated by Western blot analysis, immunocytochemistry and reporter assay. Additionally, the effect of CX-4945 on TGF-β1-induced migration and invasion was investigated by wound healing assay, Boyden chamber assay, gelatin zymography, and the quantitative real-time PCR.. CX-4945 inhibits the TGF-β1-induced cadherin switch and the activation of key signaling molecules involved in Smad (Smad2/3, Twist and Snail), non-Smad (Akt and Erk), Wnt (β-catenin) and focal adhesion signaling pathways (FAK, Src and paxillin) that cooperatively regulate the overall process of EMT. As a result, CX-4945 inhibits the migration and invasion of A549 cells accompanied with the downregulation of MMP-2 and 9.. Clinical evaluation of CX-4945 in humans as a single agent in solid tumors and multiple myeloma has established its promising pharmacokinetic, pharmacodynamic, and safety profiles. Beyond regression of tumor mass, CX-4945 may be advanced as a new therapy for cancer metastasis and EMT-related disorders.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Cadherins; Casein Kinase II; Cell Line, Tumor; Cell Movement; Epithelial-Mesenchymal Transition; Focal Adhesion Kinase 1; Humans; Lung Neoplasms; Naphthyridines; Neoplasm Invasiveness; Paxillin; Phenazines; Protein Kinase Inhibitors; Signal Transduction; Smad Proteins; Transforming Growth Factor beta1; Wnt Proteins

Cell surface nucleolin (NCL) plays fundamental roles in tumor angiogenesis. However, the mechanism underlying its surface translocation remains obscure. The present study discovered that heat shock cognate 70 (Hsc70) is essential in both the surface translocation and the angiogenic function of NCL.. We identified that Hsc70 interacted with NCL in endothelial cells via the peptide-binding domain of Hsc70 and the RNA-binding domain of NCL. Functional knockdown of Hsc70 remarkably inhibited the expression of surface NCL, which was rescued by wild-type Hsc70 rather than its truncations. Phosphorylation of NCL by either protein kinase C-ξ or casein kinase 2 mediated its interaction with Hsc70 and the surface expression. Hsc70 regulated NCL translocation via stabilizing NCL and enhancing its interaction with nonmuscle myosin heavy chain 9. Moreover, Hsc70 was associated with NCL-induced endothelial cell migration and tubule formation in vitro and angiogenesis in both matrigel plugs and xenograft tumors. Tissue array analysis revealed that the expression levels of NCL and Hsc70 were intimately correlated in human lung adenocarcinomas.. Our study demonstrates that Hsc70 is a prerequisite for the surface translocation and angiogenic function of NCL, which suggests strategies to target both Hsc70 and NCL for more effective antiangiogenic therapies.

Topics: Adenocarcinoma; Adenocarcinoma of Lung; Animals; Benzhydryl Compounds; Binding Sites; Carcinoma, Non-Small-Cell Lung; Casein Kinase II; Cell Line, Tumor; Cell Movement; HSC70 Heat-Shock Proteins; Human Umbilical Vein Endothelial Cells; Humans; Lung Neoplasms; Mice; Mice, Nude; Myosin Heavy Chains; Neovascularization, Pathologic; Neovascularization, Physiologic; Nucleolin; Phosphoproteins; Phosphorylation; Protein Interaction Domains and Motifs; Protein Interaction Mapping; Protein Kinase C-epsilon; Protein Stability; Protein Transport; Pyrrolidinones; RNA Interference; RNA-Binding Proteins; Tissue Array Analysis; Transfection; Xenograft Model Antitumor Assays