Decrease in paracellular permeability and chemosensitivity to doxorubicin by claudin-1 in spheroid culture models of human lung adenocarcinoma A549 cells

Chemotherapy resistance is a major problem in the treatment of cancer, but the underlying mechanisms are not fully understood. We found that the expression levels of claudin-1 (CLDN1) and 3, tight junctional proteins, are upregulated in cisplatin (CDDP)-resistant human lung adenocarcinoma A549 (A549R) cells. A549R cells showed cross-resistance to doxorubicin (DXR). Here, the expression mechanism and function of CLDN1 and 3 were examined. CLDN1 and 3 were mainly localized at tight junctions concomitant with zonula occludens (ZO)-1, a scaffolding protein, in A549 and A549R cells. The phosphorylation levels of Src, MEK, ERK, c-Fos, and Akt in A549R cells were higher than those in A549 cells. The expression levels of CLDN1 and 3 were decreased by LY-294002, a phosphoinositide 3-kinase (PI3K) inhibitor, and BAY 11-7082, an NF-κB inhibitor. The overexpression of CLDN1 and 3 decreased the paracellular permeability of DXR in A549 cells. Hypoxia levels in A549R and CLDN1-overexpressing cells (CLDN1/A549) were greater than those in A549, mock/A549, and CLDN3/A549 cells in a spheroid culture model. In contrast, accumulation in the region inside the spheroids and the toxicity of DXR in A549R and CLDN1/A549 cells were lower than those in other cells. Furthermore, the accumulation and toxicity of DXR were rescued by CLDN1 siRNA in A549R cells. We suggest that CLDN1 is upregulated by CDDP resistance through activation of a PI3K/Akt/NF-κB pathway, resulting in the inhibition of penetration of anticancer drugs into the inner area of spheroids.     

Epidermal growth factor increases clathrin-dependent endocytosis and degradation of claudin-2 protein in MDCK II cells

Epidermal growth factor (EGF) decreases the mRNA and protein levels of claudin-2 (CLDN2) in Madin-Darby canine kidney (MDCK) II cells. Here we examined whether EGF affects the stability and intracellular distribution of CLDN2 protein. EGF decreased surface and total levels of CLDN2, which was inhibited by U0126, a MEK inhibitor. CLDN2 was co-localized at the tight junctions (TJs) with ZO-1, a scaffolding protein. The fluorescence signal for CLDN2 disappeared on treatment with EGF, which was inhibited by U0126. EGF accelerated the decrease in CLDN2 in the presence of cycloheximide, a translation inhibitor, indicating that EGF reduces the stability of the protein. Chloroquine, a lysosomal protease inhibitor, blocked the EGF-induced decrease in CLDN2 protein and caused the co-localization of CLDN2 with Lamp-1, a marker of lysosome. Monodancylcadaverine, an inhibitor of endocytosis, and clathrin siRNA blocked the EGF-induced decrease in CLDN2 and the translocation of CLDN2 from the TJs to the lysosome. EGF increased the association of CLDN2 with clathrin and adaptin α which was inhibited by U0126. These results suggest that EGF accelerates clathrin-dependent endocytosis and lysosomal degradation of CLDN2 protein mediated by the activation of a MEK/ERK pathway.     

Inhibitory effects of norcantharidin against human lung cancer cell growth and migration

The effects of norcantharidin (NCTD), an anticancer drug in China, on the growth and migration in human lung cancer cells were investigated by in vitro and ex vivo assays. NCTD significantly inhibited the in vitro and ex vivo growth of human non-small cell lung cancer A549 cells in dose- and time-dependent manners. Western blot analysis indicated that NCTD dose-dependently down-regulated the expression of anti-apoptotic protein Bcl-2 and up-regulated the level of pro-apoptotic protein Bax, eventually leading the reduction of ratio of Bcl-2/Bax proteins in A549 cells. Moreover, NCTD significantly suppressed the A549 cell migration in the case of without reducing the cell viability. More importantly, NCTD significantly enhanced the anticancer activity of anticancer agents such as trichostatin A (the histone deacetylase inhibitor), celecoxib (the inhibitor of cyclooxygenase-2) and lovastatin (the inhibitor of HMG-CoA reductase) by strongly reducing the viability and/or the ratio of Bcl-2/Bax protein in A549 cells. Our findings suggest that NCTD may have the wide therapeutic and/or adjuvant therapeutic application in the treatment of human lung cancer.     

Expression and targeting of the tight junction protein CLDN1 in CLDN1-negative human breast tumor cells

Claudins and occludin constitute the major transmembrane proteins of tight junctions (TJs). We have previously identified the human homologue of the murine Cldn1, CLDN1 (SEMP1) that is expressed in normal, mammary gland-derived epithelial cells but is absent in most human breast cancer cell lines. To investigate the potential functions of CLDN1 protein in tumor and normal epithelial cells, we developed an I-NGFR retroviral vector and monoclonal anti-CLDN1 antibody. In subconfluent and confluent breast cancer cells, MDA-MB-435 and MDA-MB-361, endogenous CLDN1 expression was not detected by an anti-CLDN1 monoclonal antibody by Western blot analysis or quantitative RT-PCR. When CLDN1-negative breast cancer cell lines were transduced with a CLDN1 retrovirus the cells express CLDN1 mRNA constitutively as shown by quantitative RT-PCR. Immunofluorescence analyses of the CLDN1 retroviral transduced breast tumor cells using monoclonal antibodies against CLDN1 reveals a subcellular distribution at cell-cell contact sites similar to the CLDN1 homing pattern in T47-D cells, which express endogenous CLDN1. This cell-cell contact co-localization of CLDN1 was evident in CLDN1-transduced breast tumor cells which fail to express occludin protein (MDA-MB-361 and MDA-MB-435) and express relatively little ZO-1 protein (MDA-MB-435), suggesting that other proteins may be responsible for targeting of CLDN1 to cell-cell contact sites. The re-expression of CLDN1 decreases the paracellular flux of 3 and 40 kDa dextran despite the absence of occludin in the MDA-MB-361 tumor cells. Our findings indicate that in CLDN1-negative breast tumor cells, the basal protein partner requirements for physiological homing of the CLDN1 protein are intact, and that CLDN1 gene transfer and protein expression itself might be sufficient to exert a TJ-mediate gate function in metastatic tumor cells even in the absence of other TJ-associated proteins, such as occludin.     

靶向紧密连接蛋白6的药物在癌症治疗中的研究进展

紧密连接蛋白6(Claudin6,CLDN6)是紧密连接蛋白(Claudins,CLDNs)家族的一员,在卵巢癌、睾丸癌、子宫颈内膜癌、肝癌和肺腺癌等多种癌症中特异性高表达,而在成人正常组织中几乎不表达。其能够激活多条通路参与肿瘤发生的多个过程,包括促进肿瘤生长、迁移和侵袭,且促进肿瘤化疗耐药。近年来,CLDN6作为癌症治疗的新靶点引起了研究人员的广泛关注,针对CLDN6靶点开发了多种类型的抗癌药物,包括抗体偶联药物(antibody-drug conjugate,ADC)、单克隆抗体、双特异性抗体和嵌合抗原受体T细胞免疫疗法(chimeric antigen receptor T-cell immunotherapy,CAR-T)。本文简要概述了CLDN6的蛋白结构、表达分布以及在肿瘤中的功能,并对其作为药靶开发的抗癌药物研发现状和研发思路进行了综述。     

Suppression of human lung cancer cell growth and migration by berbamine

The purpose of this study is to investigate the effects of berbamine (BER), a naturally occurring small-molecule compound from Traditional Chinese Medicine (TCM) Berberis amurensis, on the growth and migration of human lung cancer A549 cell line. This cell line is the non–small cell lung cancer (NSCLC) which constitutes 80% of lung cancer cases and remains an aggressive lung cancer associated with a poor patient survival. Our present results have shown that BER significantly suppressed the in vitro and ex vivo growth of A549 cells in dose- and time-dependent manners. Furthermore, Western blot analysis confirmed that BER dose-dependently down-regulated the expression of anti-apoptotic protein Bcl-2 and up-regulated the level of pro-apoptotic protein Bax, eventually leading the reduction of Bcl-2/Bax protein ratio in A549 cells. In addition, BER significantly inhibited the A549 cell migration at the low concentrations without restraining the cell growth. More importantly, BER significantly enhanced the anticancer activity of anticancer agents such as trichostatin A (the histone deacetylase inhibitor) and celecoxib (the inhibitor of cyclooxygenase-2) by strongly reducing the viability and/or the Bcl-2/Bax protein ratio in A549 cells. Our findings suggest that BER may have the wide therapeutic and/or adjuvant therapeutic application in the treatment of human NSCLC.     

Virtual screening of National Cancer Institute database for claudin-4 inhibitors: Synthesis,biological evaluation,and molecular dynamics studies

Claudin-4 (CLDN4) is a vital member of tight-junction proteins that is often overexpressed in cancer and other malignancies. The three-dimensional structure of human CLDN4 was constructed based on homology modeling approach. A total of 265 242 molecules from the National Cancer Institute (NCI) database has been utilized as a dataset for this study. In the present work, structure-based virtual screening is performed with the NCI database using Glide. By molecular docking, 10 candidate molecules with high scoring functions, which binds to the active site of CLDN4 were identified. Subsequently, molecular dynamics simulations of membrane protein were used for optimization of the top-three lead compounds (NCI110039, NCI344682, and NCI661251) with CLDN4 in a dynamic system. The lead molecule from NCI database NCI11039 (purpurogallin carboxylic acid) was synthesized and cytotoxic properties were evaluated with A549, MCF7 cell lines. Our docking and dynamics simulations predicted that ARG31, ASN142, ASP146, and ARG158 as critically important residues involved in the CLDN4 activity. Finally, three lead candidates from the NCI database were identified as potent CLDN4 inhibitors. Cytotoxicity assays had proved that purpurogallin carboxylic acid had an inhibitory effect towards breast (MCF7) and lung (A549) cancer cell lines. Computational insights and in vitro (cytotoxicity) studies reported in this study are expected to be helpful for the development of novel anticancer agents.     

Tight junction protein 1 is regulated by transforming growth factor-β and contributes to cell motility in NSCLC cells

Tight junction protein 1 (TJP1), a component of tight junction, has been reported to play a role in protein networks as an adaptor protein, and TJP1 expression is altered during tumor development. Here, we found that TJP1 expression was increased at the RNA and protein levels in TGF-β-stimulated lung cancer cells, A549. SB431542, a type-I TGF-β receptor inhibitor, as well as SB203580, a p38 kinase inhibitor, significantly abrogated the effect of TGF-β on TJP1 expression. Diphenyleneiodonium, an NADPH oxidase inhibitor, also attenuated TJP1 expression in response to TGF-β in lung cancer cells. When TJP1 expression was reduced by shRNA lentiviral particles in A549 cells (A549-sh TJP1), wound healing was much lower than in cells infected with control viral particles. Taken together, these data suggest that TGF-β enhances TJP1 expression, which may play a role beyond structural support in tight junctions during cancer development. [BMB Reports 2015; 48(2): 115-120]     

Effects of theanine on growth of human lung cancer and leukemia cells as well as migration and invasion of human lung cancer cells

The aim of this study is to investigate the effects of theanine, a tea characteristic amino acid, on human lung cancer and leukemia cells. In the present study, we have demonstrated that theanine suppressed the in vitro and ex vivo growth of human non-small cell lung cancer A549 and leukemia K562 cell lines in dose- and time-dependant manners. In addition, theanine displayed the inhibitory effect on the migration of A549 cells. More importantly, theanine enhanced the anticancer activity of anticancer agents such as trichostatin A (the histone deacetylase inhibitor), berbamine and norcantharidin (the anticancer drugs in China) by strongly reducing the viability and/or migration rate in A549 cells. In addition, theanine significantly suppressed A549 cell invasion. Suppression of A549 cell migration may be one of the important mechanisms of action of theanine against the A549 cell invasion. Our present results suggest that theanine may have the wide therapeutic and/or adjuvant therapeutic application in the treatment of human lung cancer and leukemia.     

Effects of matrine against the growth of human lung cancer and hepatoma cells as well as lung cancer cell migration

The purpose of this study is to investigate in vitro and ex vivo effects of matrine on the growth of human lung cancer and hepatoma cells and the cancer cell migration as well as the expressions of related proteins in the cancer cells. Matrine significantly inhibited the in vitro and ex vivo growth of human non-small cell lung cancer A549 and hepatoma SMMC-7721 cells. Matrine induced the apoptosis in A549 and SMMC-7721 cells. Western blot analysis indicated that matrine dose-dependently down-regulated the expression of anti-apoptotic protein Bcl-2 and up-regulated the level of pro-apoptotic protein bax, eventually leading the reduction of ratios of Bcl-2/Bax proteins in A549 and SMMC-7721 cells. Furthermore, matrine significantly suppressed the A549 cell migration without reducing the cell viability. In addition, matrine dramatically reduced the secretion of vascular endothelial growth factor A in A549 cells. More importantly, matrine markedly enhanced the anticancer activity of anticancer agent trichostatin A (the histone deacetylase inhibitor) by strongly reducing the viability and/or the ratio of Bcl-2/Bax protein in A549 cells. Our findings suggest that matrine may have the broad therapeutic and/or adjuvant therapeutic application in the treatment of human non-small cell lung cancer and hepatoma.     

Mimulone-Induced Autophagy through p53-Mediated AMPK/mTOR Pathway Increases Caspase-Mediated Apoptotic Cell Death in A549 Human Lung Cancer Cells

Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy.     

Smac调控Caspase-3对耐药肺腺癌细胞株生物活性及相关信号的研究

摘要 目的：探讨Smac基因调控Caspase-3表达对紫杉醇耐药肺腺癌细胞株生物活性及经典凋亡信号通路的作用机制。方法：取构建好的耐药A549细胞,将其分为A549细胞（LC）组、A549细胞+Smac-NC（SN）组、A549细胞+Smac抑制剂（SI）组、A549细胞+Smac激动剂（SM）组、A549细胞+Caspase-3-NC（CN）组、A549细胞+Caspase-3抑制剂（CI）组、A549细胞+Caspase-3激动剂（CM）组、A549细胞+Smac激动剂+Caspase-3激动剂（MM）组;Real-time PCR法检测正常肺上皮细胞及4种肺腺癌细胞系中Smac、Caspase-3表达水平,将阴性对照、Smac、Caspase-3类似物转染至紫杉醇耐药肺腺癌细胞株,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,免疫印迹法检测经典凋亡信号通路表达,并分析Smac与Caspase-3的相关性。结果：肺腺癌细胞系中的Smac、Caspase-3 mRNA表达量显著低于正常肺上皮细胞系BEAS-2B（P＜0.05）,其中A549的Smac、Caspase-3 mRNA值最小（P＜0.05）,因此选取其作为此次实验细胞;LC组与SN组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异（P＞0.05）,与SN组相比,SI组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显降低（P＜0.05）,增殖率、Bcl-2表达明显升高（P＜0.05）,与SI组相比,SM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高（P＜0.05）,增殖率、Bcl-2表达明显降低（P＜0.05）;LC组与CN组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异（P＞0.05）,与CN组相比,CI组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显降低（P＜0.05）,增殖率、Bcl-2表达明显升高（P＜0.05）,与CI组相比,CM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高（P＜0.05）,增殖率、Bcl-2表达明显降低（P＜0.05）;SM组与CM组相比,细胞增殖率、凋亡率及Caspase-3、Bcl-2、Bax、Cyto-C蛋白表达基本无差异（P＞0.05）,与CM组相比,MM组细胞凋亡率及Caspase-3、Bax、Cyto-C蛋白表达明显升高（P＜0.05）,增殖率、Bcl-2表达明显降低（P＜0.05）;Smac与Caspase-3呈现正相关（r=0.470,P=0.002）,组间具有显著差异。结论：Smac基因可显著改善紫杉醇耐药肺腺癌细胞株细胞生物活性,并激活经典凋亡信号通路,其作用机制可能与调控Caspase-3表达有关。     

Podosome formation impairs endothelial barrier function by sequestering zonula occludens proteins

Podosomes and tight junctions (TJs) are subcellular compartments that both exist in endothelial cells and localize at cell surfaces. In contrast to the well-characterized role of TJs in maintaining cerebrovascular integrity, the specific function of endothelial podosomes remains unknown. Intriguingly, we discovered cross-talk between podosomes and TJs in human brain endothelial cells. Tight junction scaffold proteins ZO-1 and ZO-2 localize at podosomes in response to phorbol-12-myristate-13-acetate treatment. We found that both ZO proteins are essential for podosome formation and function. Rather than being derived from new protein synthesis, podosomal ZO-1 and ZO-2 are relocated from a pre-existing pool found at the peripheral plasma membrane with enhanced physical interaction with cortactin, a known protein marker for podosomes. Sequestration of ZO proteins in podosomes weakens tight junction complex formation, leading to increased endothelial cell permeability. This effect can be further attenuated by podosome inhibitor PP2. Altogether, our data revealed a novel cellular function of podosomes, specifically, their ability to negatively regulate tight junction and endothelial barrier integrity, which have been linked to a variety of cerebrovascular diseases.     

Haishengsu, a Protein from Shellfish Tegillarca L. granosa, Inhibits the Growth and the Activity of Matrix Metalloproteinases-2 and -9 in Human Lung Carcinoma

Haishengsu (HSS) is a seashell protein extracted from Tegillarca L. granosa, a type of Malaysian shellfish. Previous in vitro studies showed that HSS might possess biological anticancer activity. In this combined in vitro and in vivo study, we investigated the inhibitory effects of HSS on tumor growth, invasion, and metastasis using human lung carcinoma cell lines A549 and NCI-H292, both intensely positive for matrix metalloproteinases-2 (MMP-2) and MMP-9. HSS significantly inhibited the proliferation of A549 and NCI-H292 as estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The transwell chamber assay showed that HSS effectively blocked the invasion and migration of the carcinoma cells through the reconstituted extracellular matrix (Matrigel). Gelatin zymography analysis revealed that the secretion and activity of MMP-2 and MMP-9 in the supernatants of the cultured cells A549 and NCI-H292 were decreased after treatment with HSS. The levels of MMP-2 and MMP-9 in these cancer cells were further examined by Western blot assay in which a significant decrease of MMP-2 and MMP-9 was observed in A549 and NCI-H292 cells after 24 h of exposure to HSS. The anticancer activity of HSS was verified in a mouse model in which HSS delayed the growth of A549 xenografts after 3 weeks of oral administration. Inhibition of MMP-2 and MMP-9 expression was also demonstrated in the A549 xenografts as determined by Western blot analysis. These results suggest that HSS is a novel seashell protein that cannot only inhibit tumor growth but also prevent tumor invasion and metastasis through suppressing the activity of MMP-2 and MMP-9.     

Emodin enhances gefitinib-induced cytotoxicity via Rad51 downregulation and ERK1/2 inactivation

Emodin, a tyrosine kinase inhibitor, is a natural anthraquinone derivative found in the roots and rhizomes of numerous plants. It reportedly exhibits an anticancer effect on lung cancer. Gefitinib (Iressa) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor for human non-small cell lung cancer (NSCLC). However, the molecular mechanism of how emodin combined with gefitinib decreases NSCLC cell viability is unclear. The recombinase protein Rad51 is essential for homologous recombination repair, and Rad51 overexpression is resistant to DNA double-strand break-inducing cancer therapies. In this study, we found that emodin enhanced the cytotoxicity induced by gefitinib in two NSCLC cells lines, A549 and H1650. Emodin at low doses of 2-10 μM did not affect ERK1/2 activation, mRNA, and Rad51 protein levels; however, it enhanced a gefitinib-induced decrease in phospho-ERK1/2 and Rad51 protein levels by enhancing Rad51 protein instability. Expression of constitutively active MKK1/2 vectors (MKK1/2-CA) significantly rescued the reduced phospho-ERK1/2 and Rad51 protein levels as well as cell viability on gefitinib and emodin cotreatment. Blocking of ERK1/2 activation by U0126 (an MKK1/2 inhibitor) lowered Rad51 protein levels and cell viability in emodin-treated H1650 and A549 cells. Knockdown of Rad51 expression by transfection with si-Rad51 RNA enhanced emodin cytotoxicity. In contrast, Rad51 overexpression protected the cells from the cytotoxic effects induced by emodin and gefitinib. Consequently, emodin-gefitinib cotreatment may serve as the basis for a novel and better therapeutic modality in the management of advanced lung cancer.