Claudin-6通过P38 MAPK信号通路抑制肺腺癌细胞H1299恶性表型的作用
|
摘要
肺癌目前已成为世界上发病率及死亡率最高的肿瘤疾病。肺癌分为小细胞肺癌（small cell lung cancer, SCLC）及非小细胞肺癌（non-small cell lung cancer,NSCLC）。非小细胞肺癌分为鳞癌、腺癌和大细胞癌等。肺癌的主要确切治疗手段有手术治疗、化疗、放疗和分子靶向治疗。多数肺癌患者在疾病发现时已失去手术机会。
肺腺癌倍增时间较短，易转移，对于放化疗均不敏感，且有一定比例的肺腺癌在肿瘤直径<1cm时已发生远端转移。近来，Gefitinib作为分子靶向治疗药物为肺腺癌提供了有效的治疗方法，尽管如此，仍有很大比例的腺癌患者对于EGFR-TKI的治疗效果不佳，因此探寻新的分子靶向治疗方法对于肺腺癌的治疗十分必要。
根据目前的研究及报道显示，CLDN家族目前已经拥有27个成员，CLDN6是这个家族的成员之一。之前有研究从胚胎干细胞中采用ddPCR方法筛选了一些交迭的cDNA(20M24)，对20M24的DNA测序结果显示其结构是一段编码219个氨基酸的开放阅读框架，能够指导合成一种分子量大小为23kDa的蛋白，这种蛋白与之前研究中报道的CLDNs的家族成员在结构上具有很高的同源性，因此将其命名为CLDN6。CLDN6位于人类染色体的16p13.3位点，其在维持细胞间TJ结构的完整性及功能中起到了十分关键的作用。有研究发现在乳腺癌细胞MCF-7中稳定过表达CLDN6基因可以抑制乳腺癌细胞的增殖、侵袭和及迁移能力。
目前认为CLDNs蛋白在肺癌组织及肺癌细胞中均具有表达特异性。CLDNs家族成员既有部分在肺癌组织及肺癌细胞株中表达上调，又有部分在肺癌组织及肺癌细胞株中表达下调。但CLDNs蛋白在肺癌组织及肺癌细胞中无论表达情况如何，其最终结果总是引起紧密连接的正常结构破坏而导致细胞间分子异常侧向扩散、细胞极性消失等一系列变化。
TJ的其他家族成员通过一些特定的信号通路可以影响细胞的生物学行为。有研究显示在乳腺癌MCF-7细胞中过表达CLND6可以激活ASK1/P38通路，从而诱导细胞凋亡。目前有关CLDN6作为TJ的构成蛋白对肺腺癌细胞生物学行为影响的研究尚未见报道，相关机制还不清楚。本研究将首次证实CLDN6作为TJ的构成蛋白对肺腺癌H1299细胞生物学行为的影响及其相关作用机制，不仅对揭示肺腺癌的发生机理有重要意义，并且为肺腺癌的诊断方法及分子靶向治疗药物的研制带来广泛前景。
方法：
为探讨CLDN6在与肺腺癌的相关性以及CLDN6过表达对肺腺癌细胞H1299生物学表型的影响，本研究采用如下方法：
1.鉴定人正常支气管上皮细胞株16Hbe及人肺腺癌细胞株A549、A2和H1299中CLDN6的表达；
2.构建CLDN6真核表达载体并转染H1299细胞；
3.筛选并鉴定稳定过表达CLDN6的H1299细胞；
4.检测CLDN6过表达对H1299细胞增殖能力的影响；
5.检测CLDN6过表达对H1299细胞凋亡的影响；
6.检测CLDN6过表达对H1299细胞迁移、侵袭和粘附能力的影响；
7.检测CLDN6过表达对H1299细胞紧密连接结构和功能的影响；
8.分析CLDN6对H1299细胞影响可能的作用机制。
结果：
1. CLDN6在人正常支气管上皮细胞株16Hbe中有表达，在人肺腺癌细胞株A549、A2和H1299中低表达。
2.构建并鉴定了表达绿色荧光蛋白的pIRES2-EGFP-CLDN6质粒。
3. CLDN6转染H1299细胞稳定克隆的筛选和鉴定：经荧光显微镜观察、RT-PCR和Western blot方法筛选出3株稳定过表达CLDN6的H1299单克隆，分别为6、10和11号克隆。细胞免疫荧光染色结果发现，CLDN6表达于细胞膜及核膜上，而空载组细胞中无CLDN6的表达。
4. CLDN6过表达对H1299细胞增殖的影响：CCK8实验结果显示，过表达组H1299细胞的增殖速度较空载组细胞显著减慢，二维克隆形成实验和三维克隆形成实验结果显示过表达组细胞的克隆形成能力较空载组细胞显著减弱。流式细胞术细胞周期检测结果提示过表达组处于增殖期的细胞比例显著低于空载组。说明转染CLDN6后H1299细胞的增殖速度降低，二维和三维克隆形成能力均显著下降，同时细胞周期也发生了改变。
5. CLDN6过表达对H1299细胞凋亡的影响：DAPI染色结果显示，过表达组细胞凋亡率显著高于空载组细胞。Hoechst33342/PI双染法结果发现，过表达组细胞早期和晚期细胞凋亡率均显著高于空载组。流式细胞术进一步证实了上述结果，即与空载组细胞相比，过表达组细胞存在显著增加的凋亡率。说明转染CLDN6诱导了H1299细胞的凋亡。
6. CLDN6过表达对H1299细胞转移能力的影响：细胞划痕实验发现，过表达组细胞迁移速度显著低于空载组细胞。Transwell小室法检测发现，空载组细胞的侵袭能力显著大于过表达组细胞，说明CLDN6具有降低H1299细胞体外迁移和侵袭能力的作用。体外粘附能力检测发现，空载组与过表达组细胞间体外粘附能力无显著差别。
7. CLDN6基因过表达对H1299细胞紧密连接功能的影响：通过检测单层培养细胞的跨上皮电阻，我们发现，转染CLDN6细胞的跨上皮电阻显著高于空载组细胞，表明转染CLDN6可加强细胞间紧密连接的结构和功能。
8. CLDN6基因过表达影响肺腺癌细胞生物学行为的可能机制：Western blot检测发现，CLDN6过表达细胞中磷酸化ASK1和ASK1、磷酸化P38、磷酸化STAT3和STAT3的表达水平显著高于空载组细胞，表明过表达CLDN6可促进P38MAPK途径的活化。应用P38抑制剂SB203580处理过表达组H1299细胞后，CLDN6和ASK1的表达并未受到影响，而STAT3表达显著下降。说明P38位于CLDN6/ASK1信号通路的下游，位于STAT3信号通路的上游。应用P38抑制剂SB203580处理后可以显著抑制上述通路的活化，并逆转过表达CLDN6所致的抑制增殖、促凋亡、抑制迁移和侵袭的能力。因此可以认为CLDN6对细胞增殖、凋亡、迁移和侵袭能力的影响是通过P38信号通路实现的。
结论：
1. CLDN6在人正常支气管上皮细胞株16Hbe中有表达，在A549、A2和H1299三种人肺腺癌细胞株中低表达。
2. CLDN6抑制H1299细胞增殖、迁移和侵袭能力，诱导细胞凋亡。
3. CLDN6通过增强细胞间紧密连接功能，抑制H1299细胞的转移能力。
4.在肺腺癌细胞H1299中过表达CLDN6可以上调P38的表达。
5. CLDN6对H1299细胞生物学表型的作用与P38MAPK信号通路的激活有关。
Lung cancer has been a tumor disease of highest incidence and mortality in theworld. Lung cancer consists small cell lung cancer（SCLC） and non-small cell lungcancer（NSCLC） which includes squamous carcinoma, adenocarcinoma, large cellcarcinoma and so on. Surgry, chemotherapy, radiotherapy and molecular targetedtherapy are the main effective therapies to lung cancer. However, majority of thepatients lose the chance for surgery when the disease is diagnosed.
Lung adenocarcinoma usually resists to chemotherapy and radiotherapy, some ofwhich take place distant metastasis when the tumor is less the1cm. Recently，gefitinibplays an effective role to lung adenocarcinoma as molecular targeted therapymedicine. On the other side, it remains a majority of lung adenocarcinoma that cannot been controlled by EGFR-TKI. Thus, it makes much sense to explore new ways ofmolecular targeted therapy to lung adenocarcinoma.
CLDN6is a member of CLDN family which has27proteins. Previousreaserches identify many folded clones(20M24) in embryonic stem cells thoughddPCR. DNA sequencings for20M24revealed it an open reading frame with219amino acids guiding synthesis of a protein of23kDa called CLDN6which hashomology to TJ family. CLDN6localized in chromosome16p13.3which plays animportant role in maintaining TJ’ structure and function as a member of CLDN family.Previous research suggested that over expression of CLDN6could suppress theabilities of proliferation, migration and invasion in breast cancer cell MCF-7.
We consider that CLDNs in lung cancer tissues and cells express specifically.Members of CLDN family in lung cancer tissues and cells show both up-regulated and down-regulated. However, whatever the situation that CLDNs express in lungcancer tissues and cells, it will inevitably give rise to the destruction of TJ’s normalstructure which leads to changes of abnormal lateral molecular diffusion betweencells, dispearance of cell polarity and so on.
It is reported that some members of CLDNs influenced the biologicalbehaviours of some particular cancers by activating or inhibiting some signalingpathway. Some researches also revealed that over expression of CLDN6could inducecells apoptosis though activating ASK1/P38signaling pathway in breast cancerMCF-7. Few reports cover the effects and mechanism of CLDN6as a member of TJprotein on the biological phenotypes in lung adenocarcinoma cell line. For the firsttime，this research will identify the effects and mechanism of CLDN6on thebiological phenotype of lung adenocarcinoma cell H1299. This will contribute to notonly understand the mechanism on occurance of lung adenocarcinoma, but alsoexplore new methods to diagnosis and new medicines of molecular targetingtreatment for lung adenocarcinoma.
Metheds：
To explore the correlation between CLND6and some lung adenocarcinoma celllines and the effects of up-regulation of CLDN6on lung adenocarcinoma cell lineH1299, we employed the following metheds:
1. We evaluated the expression of CLDN6in human bronchial epithelial cell16Hbe and lung adenocarcinoma cell lines A549, A2, and H1299.
2. We designed and structured pIRES2-EGFP-CLDN6plasmid which was thentransfected into H1299cells.
3. We screened and indentified H1299cells with stable over expression ofCLDN6.
4. We explored the effects of CLDN6on cell growth ability.
5. We explored the effects of CLDN6on cell proliferation ability.
6. We explored the effects of CLDN6on cell invasive and migration ability.
7. We explored the effects of CLDN6on cell structure and function of tightjunction.
8. We analysed the mechanism of effects that CLDN6worked on H1299cells.
Results:
1. CLDN6had a normal expression in human bronchial epithelial cell16Hbe andlow or absent expressions in lung adenocarcinoma cell lines A549, A2and H1299.
2. A pIRES2-EGFP-CLDN6plasmid was structured and identified.
3. Screened and identified the clones expressing CLDN6steadily by transfection:evaluated by fluorescence microscope, RT-PCR and Western blot, three clones namedNO.6,10and11with stable expression of CLDN6were confirmed. Thecytoimmunity stain results showed that CLDN6localized on the cell membranes andnuclear membranes in stable expression clones and revealed absent in vector groups.
4. CCK8showed the over expression groups have a lower proliferation speedthan the vector groups. Two-dimensional clone forming assay and three-dimensionalclone forming assay revealed that comparing to the vector group cells, overexpression group cells have fewer clone formation. Flow cytometry cell cycle assaydiscovered that over expression group cells have a lower rate in S phase comparing tovector groups which suggested that over expression group have fewer cells inproliferation stage. Above all, over expression of CLDN6may decrease cellproliferation speed, two and three-dimensional clone forming abilities and change cellcycle stages.
5. The effects of CLDN6on H1299cell apoptosis: DAPI stain proved more cellapoptosis in over expression groups than vector group. Hoechst33342/PI double stainshowed that the rate of early and late apoptosis cells in over expression groups werehigher than vector group. Flow cytometry cell cycle assay further proved the aboveresults. Above all, CLDN6may induce cell apoptosis.
6. The effects of CLDN6on H1299cell metastatic ability: Wound healing assayfound that cell migration ability was lower in CLDN6expression cells than vector group. Transwell assay showed that invasive ability was lower in CLDN6expressioncells. Above all, CLDN6may decrease the cell migration and invasive abilities. Celladhesion assay in vitro showed there was no difference in all groups.
7. Transepithelial electric resistance of monolayer culture revealed thatcomparing to the vector group cells, over expression group cells showed a higherresistance which means that over expression of CLDN6may strengthen the structureand enhance the function of tight junction between monolayer cultured cells.
8. The mechanisms of CLDN6’s on H1299cell biological behaviour: Westernblot assay showed that the level of phospho-ASK1and ASK1, phospho-P38,phospho-STAT3and STAT3was higher in CLDN6expression groups than vectorgroup which meaned that over expression of CLDN6could activate P38MAPKsignaling pathway. After treating by P38inhibitor named SB203580, the expressionsof CLDN6and ASK1showed no difference and that of STAT3decreased significantlywhich suggested P38localized at downstream of CLDN6/ASK1signaling pathwayand upstream of STAT3signaling pathway. P38inhibitor could not only obviouslyinhibit the activation of P38pathway, but also partly suppress the effects of CLDN6expression on cell proliferation apoptosis, migration and invasive abilities, yet with noeffect on CLDN6expression. Above all, we believe that the effects of CLDN6on cellproliferation apoptosis, migration and invasive ability might work though P38signaling pathway.
Conclusion:
1. CLDN6has a normal expression in human bronchial epithelial cell16Hbe andlower expressions in lung adenocarcinoma cell lines A549, A2and H1299.
2. CLDN6can inhibit proliferation, migration and invasive abilities, induce cellapoptosis in H1299cell.
3. CLDN6can inhibit cell metastatic ability by strengthening the tight junction.
4. Over expression of CLDN6is able to up-rgeulate P38in human lungadenocarcinoma cell line H1299.
5. The effects of CLDN6on biological phenotype of H1299are related with theactivation of P38MAPK signal pathway.
肺腺癌倍增时间较短，易转移，对于放化疗均不敏感，且有一定比例的肺腺癌在肿瘤直径<1cm时已发生远端转移。近来，Gefitinib作为分子靶向治疗药物为肺腺癌提供了有效的治疗方法，尽管如此，仍有很大比例的腺癌患者对于EGFR-TKI的治疗效果不佳，因此探寻新的分子靶向治疗方法对于肺腺癌的治疗十分必要。
根据目前的研究及报道显示，CLDN家族目前已经拥有27个成员，CLDN6是这个家族的成员之一。之前有研究从胚胎干细胞中采用ddPCR方法筛选了一些交迭的cDNA(20M24)，对20M24的DNA测序结果显示其结构是一段编码219个氨基酸的开放阅读框架，能够指导合成一种分子量大小为23kDa的蛋白，这种蛋白与之前研究中报道的CLDNs的家族成员在结构上具有很高的同源性，因此将其命名为CLDN6。CLDN6位于人类染色体的16p13.3位点，其在维持细胞间TJ结构的完整性及功能中起到了十分关键的作用。有研究发现在乳腺癌细胞MCF-7中稳定过表达CLDN6基因可以抑制乳腺癌细胞的增殖、侵袭和及迁移能力。
目前认为CLDNs蛋白在肺癌组织及肺癌细胞中均具有表达特异性。CLDNs家族成员既有部分在肺癌组织及肺癌细胞株中表达上调，又有部分在肺癌组织及肺癌细胞株中表达下调。但CLDNs蛋白在肺癌组织及肺癌细胞中无论表达情况如何，其最终结果总是引起紧密连接的正常结构破坏而导致细胞间分子异常侧向扩散、细胞极性消失等一系列变化。
TJ的其他家族成员通过一些特定的信号通路可以影响细胞的生物学行为。有研究显示在乳腺癌MCF-7细胞中过表达CLND6可以激活ASK1/P38通路，从而诱导细胞凋亡。目前有关CLDN6作为TJ的构成蛋白对肺腺癌细胞生物学行为影响的研究尚未见报道，相关机制还不清楚。本研究将首次证实CLDN6作为TJ的构成蛋白对肺腺癌H1299细胞生物学行为的影响及其相关作用机制，不仅对揭示肺腺癌的发生机理有重要意义，并且为肺腺癌的诊断方法及分子靶向治疗药物的研制带来广泛前景。
方法：
为探讨CLDN6在与肺腺癌的相关性以及CLDN6过表达对肺腺癌细胞H1299生物学表型的影响，本研究采用如下方法：
1.鉴定人正常支气管上皮细胞株16Hbe及人肺腺癌细胞株A549、A2和H1299中CLDN6的表达；
2.构建CLDN6真核表达载体并转染H1299细胞；
3.筛选并鉴定稳定过表达CLDN6的H1299细胞；
4.检测CLDN6过表达对H1299细胞增殖能力的影响；
5.检测CLDN6过表达对H1299细胞凋亡的影响；
6.检测CLDN6过表达对H1299细胞迁移、侵袭和粘附能力的影响；
7.检测CLDN6过表达对H1299细胞紧密连接结构和功能的影响；
8.分析CLDN6对H1299细胞影响可能的作用机制。
结果：
1. CLDN6在人正常支气管上皮细胞株16Hbe中有表达，在人肺腺癌细胞株A549、A2和H1299中低表达。
2.构建并鉴定了表达绿色荧光蛋白的pIRES2-EGFP-CLDN6质粒。
3. CLDN6转染H1299细胞稳定克隆的筛选和鉴定：经荧光显微镜观察、RT-PCR和Western blot方法筛选出3株稳定过表达CLDN6的H1299单克隆，分别为6、10和11号克隆。细胞免疫荧光染色结果发现，CLDN6表达于细胞膜及核膜上，而空载组细胞中无CLDN6的表达。
4. CLDN6过表达对H1299细胞增殖的影响：CCK8实验结果显示，过表达组H1299细胞的增殖速度较空载组细胞显著减慢，二维克隆形成实验和三维克隆形成实验结果显示过表达组细胞的克隆形成能力较空载组细胞显著减弱。流式细胞术细胞周期检测结果提示过表达组处于增殖期的细胞比例显著低于空载组。说明转染CLDN6后H1299细胞的增殖速度降低，二维和三维克隆形成能力均显著下降，同时细胞周期也发生了改变。
5. CLDN6过表达对H1299细胞凋亡的影响：DAPI染色结果显示，过表达组细胞凋亡率显著高于空载组细胞。Hoechst33342/PI双染法结果发现，过表达组细胞早期和晚期细胞凋亡率均显著高于空载组。流式细胞术进一步证实了上述结果，即与空载组细胞相比，过表达组细胞存在显著增加的凋亡率。说明转染CLDN6诱导了H1299细胞的凋亡。
6. CLDN6过表达对H1299细胞转移能力的影响：细胞划痕实验发现，过表达组细胞迁移速度显著低于空载组细胞。Transwell小室法检测发现，空载组细胞的侵袭能力显著大于过表达组细胞，说明CLDN6具有降低H1299细胞体外迁移和侵袭能力的作用。体外粘附能力检测发现，空载组与过表达组细胞间体外粘附能力无显著差别。
7. CLDN6基因过表达对H1299细胞紧密连接功能的影响：通过检测单层培养细胞的跨上皮电阻，我们发现，转染CLDN6细胞的跨上皮电阻显著高于空载组细胞，表明转染CLDN6可加强细胞间紧密连接的结构和功能。
8. CLDN6基因过表达影响肺腺癌细胞生物学行为的可能机制：Western blot检测发现，CLDN6过表达细胞中磷酸化ASK1和ASK1、磷酸化P38、磷酸化STAT3和STAT3的表达水平显著高于空载组细胞，表明过表达CLDN6可促进P38MAPK途径的活化。应用P38抑制剂SB203580处理过表达组H1299细胞后，CLDN6和ASK1的表达并未受到影响，而STAT3表达显著下降。说明P38位于CLDN6/ASK1信号通路的下游，位于STAT3信号通路的上游。应用P38抑制剂SB203580处理后可以显著抑制上述通路的活化，并逆转过表达CLDN6所致的抑制增殖、促凋亡、抑制迁移和侵袭的能力。因此可以认为CLDN6对细胞增殖、凋亡、迁移和侵袭能力的影响是通过P38信号通路实现的。
结论：
1. CLDN6在人正常支气管上皮细胞株16Hbe中有表达，在A549、A2和H1299三种人肺腺癌细胞株中低表达。
2. CLDN6抑制H1299细胞增殖、迁移和侵袭能力，诱导细胞凋亡。
3. CLDN6通过增强细胞间紧密连接功能，抑制H1299细胞的转移能力。
4.在肺腺癌细胞H1299中过表达CLDN6可以上调P38的表达。
5. CLDN6对H1299细胞生物学表型的作用与P38MAPK信号通路的激活有关。
Lung cancer has been a tumor disease of highest incidence and mortality in theworld. Lung cancer consists small cell lung cancer（SCLC） and non-small cell lungcancer（NSCLC） which includes squamous carcinoma, adenocarcinoma, large cellcarcinoma and so on. Surgry, chemotherapy, radiotherapy and molecular targetedtherapy are the main effective therapies to lung cancer. However, majority of thepatients lose the chance for surgery when the disease is diagnosed.
Lung adenocarcinoma usually resists to chemotherapy and radiotherapy, some ofwhich take place distant metastasis when the tumor is less the1cm. Recently，gefitinibplays an effective role to lung adenocarcinoma as molecular targeted therapymedicine. On the other side, it remains a majority of lung adenocarcinoma that cannot been controlled by EGFR-TKI. Thus, it makes much sense to explore new ways ofmolecular targeted therapy to lung adenocarcinoma.
CLDN6is a member of CLDN family which has27proteins. Previousreaserches identify many folded clones(20M24) in embryonic stem cells thoughddPCR. DNA sequencings for20M24revealed it an open reading frame with219amino acids guiding synthesis of a protein of23kDa called CLDN6which hashomology to TJ family. CLDN6localized in chromosome16p13.3which plays animportant role in maintaining TJ’ structure and function as a member of CLDN family.Previous research suggested that over expression of CLDN6could suppress theabilities of proliferation, migration and invasion in breast cancer cell MCF-7.
We consider that CLDNs in lung cancer tissues and cells express specifically.Members of CLDN family in lung cancer tissues and cells show both up-regulated and down-regulated. However, whatever the situation that CLDNs express in lungcancer tissues and cells, it will inevitably give rise to the destruction of TJ’s normalstructure which leads to changes of abnormal lateral molecular diffusion betweencells, dispearance of cell polarity and so on.
It is reported that some members of CLDNs influenced the biologicalbehaviours of some particular cancers by activating or inhibiting some signalingpathway. Some researches also revealed that over expression of CLDN6could inducecells apoptosis though activating ASK1/P38signaling pathway in breast cancerMCF-7. Few reports cover the effects and mechanism of CLDN6as a member of TJprotein on the biological phenotypes in lung adenocarcinoma cell line. For the firsttime，this research will identify the effects and mechanism of CLDN6on thebiological phenotype of lung adenocarcinoma cell H1299. This will contribute to notonly understand the mechanism on occurance of lung adenocarcinoma, but alsoexplore new methods to diagnosis and new medicines of molecular targetingtreatment for lung adenocarcinoma.
Metheds：
To explore the correlation between CLND6and some lung adenocarcinoma celllines and the effects of up-regulation of CLDN6on lung adenocarcinoma cell lineH1299, we employed the following metheds:
1. We evaluated the expression of CLDN6in human bronchial epithelial cell16Hbe and lung adenocarcinoma cell lines A549, A2, and H1299.
2. We designed and structured pIRES2-EGFP-CLDN6plasmid which was thentransfected into H1299cells.
3. We screened and indentified H1299cells with stable over expression ofCLDN6.
4. We explored the effects of CLDN6on cell growth ability.
5. We explored the effects of CLDN6on cell proliferation ability.
6. We explored the effects of CLDN6on cell invasive and migration ability.
7. We explored the effects of CLDN6on cell structure and function of tightjunction.
8. We analysed the mechanism of effects that CLDN6worked on H1299cells.
Results:
1. CLDN6had a normal expression in human bronchial epithelial cell16Hbe andlow or absent expressions in lung adenocarcinoma cell lines A549, A2and H1299.
2. A pIRES2-EGFP-CLDN6plasmid was structured and identified.
3. Screened and identified the clones expressing CLDN6steadily by transfection:evaluated by fluorescence microscope, RT-PCR and Western blot, three clones namedNO.6,10and11with stable expression of CLDN6were confirmed. Thecytoimmunity stain results showed that CLDN6localized on the cell membranes andnuclear membranes in stable expression clones and revealed absent in vector groups.
4. CCK8showed the over expression groups have a lower proliferation speedthan the vector groups. Two-dimensional clone forming assay and three-dimensionalclone forming assay revealed that comparing to the vector group cells, overexpression group cells have fewer clone formation. Flow cytometry cell cycle assaydiscovered that over expression group cells have a lower rate in S phase comparing tovector groups which suggested that over expression group have fewer cells inproliferation stage. Above all, over expression of CLDN6may decrease cellproliferation speed, two and three-dimensional clone forming abilities and change cellcycle stages.
5. The effects of CLDN6on H1299cell apoptosis: DAPI stain proved more cellapoptosis in over expression groups than vector group. Hoechst33342/PI double stainshowed that the rate of early and late apoptosis cells in over expression groups werehigher than vector group. Flow cytometry cell cycle assay further proved the aboveresults. Above all, CLDN6may induce cell apoptosis.
6. The effects of CLDN6on H1299cell metastatic ability: Wound healing assayfound that cell migration ability was lower in CLDN6expression cells than vector group. Transwell assay showed that invasive ability was lower in CLDN6expressioncells. Above all, CLDN6may decrease the cell migration and invasive abilities. Celladhesion assay in vitro showed there was no difference in all groups.
7. Transepithelial electric resistance of monolayer culture revealed thatcomparing to the vector group cells, over expression group cells showed a higherresistance which means that over expression of CLDN6may strengthen the structureand enhance the function of tight junction between monolayer cultured cells.
8. The mechanisms of CLDN6’s on H1299cell biological behaviour: Westernblot assay showed that the level of phospho-ASK1and ASK1, phospho-P38,phospho-STAT3and STAT3was higher in CLDN6expression groups than vectorgroup which meaned that over expression of CLDN6could activate P38MAPKsignaling pathway. After treating by P38inhibitor named SB203580, the expressionsof CLDN6and ASK1showed no difference and that of STAT3decreased significantlywhich suggested P38localized at downstream of CLDN6/ASK1signaling pathwayand upstream of STAT3signaling pathway. P38inhibitor could not only obviouslyinhibit the activation of P38pathway, but also partly suppress the effects of CLDN6expression on cell proliferation apoptosis, migration and invasive abilities, yet with noeffect on CLDN6expression. Above all, we believe that the effects of CLDN6on cellproliferation apoptosis, migration and invasive ability might work though P38signaling pathway.
Conclusion:
1. CLDN6has a normal expression in human bronchial epithelial cell16Hbe andlower expressions in lung adenocarcinoma cell lines A549, A2and H1299.
2. CLDN6can inhibit proliferation, migration and invasive abilities, induce cellapoptosis in H1299cell.
3. CLDN6can inhibit cell metastatic ability by strengthening the tight junction.
4. Over expression of CLDN6is able to up-rgeulate P38in human lungadenocarcinoma cell line H1299.
5. The effects of CLDN6on biological phenotype of H1299are related with theactivation of P38MAPK signal pathway.
引文
[1] Furuse M, Hirase T, Itoh M, et al. Occludin: a novel integral membraneprotein localizing at tight junctions. J Cell Biol.1993Dec;123(6Pt2):1777-88.
[2] Gonzalez-Mariscal L, Betanzos A, Nava P, et al. Tight junction proteins[J].Prog Biophys Mol Biol,2003,81(1):1-44.
[3] Staehelin LA. Further observations on the fine structure of freezecleaved tightjunctions. J Cell Sci,1973,13:763–786.
[4] Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions. NatRev Mol Cell Biol,2001,2(4):285–293.
[5] Ebnet K, Aurrand2LionsM, Kuhn A, et al. The junctional adhesion molecule(JAM) family members JAM-2and JAM-3associate with the cell polarityprotein PAR-3: a possible role for JAMs in endothelial cell polarity. Journal ofCell Science,2003,116(2):3879-3891.
[6] Mitic LL, Anderson JM. Molecular architecture of tight junctions. Annu RevPhysiol,1998,60(10):121–142.。
[7] Furuse M, Sasaki H, Tsukita S. Manner of interaction of heterogeneous CLDNspecies within and between tight junction strands. J Cell Biol,1999,147:891-903.
[8] Liebner S, Fischmann A, Rascher G, et a1. CLDN-1and CLDN-5expressionand tight junction morphology are altered in blood vessels of humanglioblastoma multiforme. Acta Neuropathol(Berl),2000,100:323-331.
[9] Morita K, Sasaki H, Furuse M, Tsukita S. Endothelial CLDN: CLDN-5/TMVCF constitutes tight junction strands in endothelial cells. J Cell Biol.1999Oct4;147(1):185-94.
[10] Liebner S, Kniesel U, Kalbacher H, et al. Correlation of tight junctionmorphology with the expression of tight junction proteins in blood-brainbarrier endothelial cells. Eur J Cell Biol.2000Oct;79(10):707-717.
[11] Turksen K，Troy TC. Barriers built on CLDNs[J].Cell Biol，2004，117：2435-2447.
[12] Anderson J M, CereijidoM. Tight Junctions. Introduction: evolution of ideason the tight junction[M]. Boca Raton: CRC Press,2001:1-18.
[13] Morita K，Furuse M，Fujimoto K，et al. CLDN multigene family encodingfour-transmembrane domain protein components of tight junction strands[J].Cell Biol，1999，2：511-516.
[14] Eveline E. Schneeberger, Robert D. Lynch. The tight junction: amultifunctional complex. Am J Physiol Cell Physiol,2004,286(6):1213-1228.
[15] Miyamoto T., Morita K., Takemoto D., et al. Tight junctions in Schwann cellsof peripheral myelinated axons: a lesson from CLDN19-deficient mice[J].Cell Biol,2005,169:527-538.
[16] Hawkins BT, Davis TP. The blood-brain barrier/neurovascular unit in healthand disease. Pharmacol Rev,2005,57(2):173-185.
[17] Furuse M, Sasaki H, Tsukita S. Manner of interaction of heterogeneous CLDNspecies within and between tight junction strands. J Cell Biol,1999,147(4):891-903.
[18] Mikio F, Hiroyuki S, Shoichiro T. Manner of Interaction of HeterogeneousCLDN Species Within and Between Tight Junction Strands. J Cell Biol,1999,147(4):8912-90.
[19] Furuse M, Furuse K, Sasaki H, et al. Conversion of zonulae occludentes fromtight to leaky strand type by introducing CLDN-2into Madin-Darby caninekidney I cells. J Cell Bio,.2001,153:263–272.
[20] Oscar RC, Christina VI, Christoph R, et al. CLDN extracellular domainsdetermine paracellular charge selectivity and resistance but not tight junctionfibril architecture. Am J Physiol Cell Physiol,2003,284(6):1346-1354.
[21] Van IC, Rahner C, and Anderson JM. Regulated expression of CLDN4decreases paracellular conductance through a selective decrease in sodiumpermeability. J Clin Invest,2001,107:1319–1327.
[22] Colegio OR, Van Itallie CM, McCrea HJ, et al. CLDNs create charge-selective channels in the paracellular pathway between epithelial cells. Am JPhysiol Cell Physiol,2002,283: C142–C147.
[23] Simon D B, Lu Y, Choate K A, et al. Paracellin-1, a renal tight junctionprotein required for paracellular Mg2+resorption. Science,1999,285:103-106.
[24] Weber S, Schneider L, Peters M, et al. Novel paracellin-1mutations in25families with familial hypomagnesaemia with hypercalciuria andnephrocalcinosis. J Am Soc Nephrol,2001,12:1872-1881.
[25] Hirano T, Kobayashi N, Itoh T, et al. Null mutation of PCLN-1/CLDN-16results in bovine chronic interstitial nephritis. Genome Res,2000,10:659-663.
[26] Furuse M, Sasaki H, Tsukita S．Manner of interaction of heterogeneous CLDNspecies within and between tight junction strands．J Cell Biol,1999，147(4)：891-903.
[27] Nitta T., Hata M., Gotoh S., et al. Size-selective loosening of the blood-brainbarrier in CLDN5-deficient mice[J]. Cell Biol,2003,161:653-660.
[28] McLaughlin J., Padfield P.J., Burt J.P.,et al. Ochratoxin A increasespermeability through tight junctions by removal of specific CLDN isoforms[J].Am J Physiol Cell Physiol,2004,287:C1412-1417.
[29] Kyle JH, Rachana A，Patrice JM．The CLDN gene family：expression innormal and neoplastic tissues．BMC Canccr,2006,6:186.
[30] Borlak J,Meier T, Halter R,Spanel R,et al. Epidermal growth factor-inducedhepatocellular carcinoma: gene expression profiles in precursor lesions, earlystage and solitary tumours[J]. Oncogene,2005,24(11):1809-1819.
[31] Al Moustafa AE, Alaoui-Jamali MA, Batist G,et al. Identification of genesassociated with head and neck carcinogenesis by cDNA microarraycomparison between matched primary normal epithelial and squamouscarcinoma cells[J]. Oncogene,2002,21(17):2634-2640.
[32] Chao YC,Pan SH, Yang SC, et al. CLDN-1is a metastasis suppressor andcorrelates with clinical outcome in lung adenocarcinoma[J]. Am J Respir CritCare Med,2009,179(2):123-133.
[33] Paschoud S, Bongiovanni M,Pache JC,et al. CLDN-1and CLDN-5expressionpatterns differentiate lung squamous cell carcinomas from adenocarcinomas[J]. Mod Pathol,2007,20(9):947-954.
[34] Coyne CB, Gambling TM, Boucher RC, et al. Role of CLDN interactions inairway tight junctional permeability[J]. Am J Physiol Lung Cell Mol Physiol,2003,285(5):L1166-1178.
[35] Nunbhakdi-Craig V,Machleidt T, Ogris E, et al. Protein phosphatase2Aassociates with and regulates atypical PKC and the epithelial tight junctioncomplex[J]. Cell Biol,2002,158(5):967-978.
[36] Itoh M, Furuse M, Morita K, et a1. Direct binding of three tightjunction-associated MAGUKs, ZO-l, ZO-2, and ZO-3with the COOH terminiof CLDNs[J]. Cell Bio1,1999, l47:1351-1363.
[37] Ebnet K, Schulz C U, Meyer Zu Brickwedde M K, et a1. Junctional adhesionmolecule interacts with the PDZ domain-containing proteins AF-6and ZO-1.J Biol Chem,2000,275:27979-27988.
[38] Broadwell R D, Charlton H M, Ebert P, et a1. Angiogenesis and theblood-brain barrier in solid and dissociated cell grafts with in the CNS. ProgBrain Res,1990,82:95-101.
[39] Haskins J, Gu L, Wittchen E S, et a1. ZO-3, a novel member of the MAGUKprotein family found at the tight junction, interacts with ZO-1and occludin. JCell Bio1,1998,141:199-208.
[40] Simon D B, Lu Y, Choate K A, et al. Paracellin-1, a renal tight junctionprotein required for paracellular Mg2+resorption. Science,1999,285(5424):103–106.
[41] Konrad M, Schaller A, Seelow D,et al. Mutations in the tight-junction geneCLDN19(CLDN19) are associated with renal magnesium wasting, renalfailure, and severe ocular involvement[J]. Am J Hum Genet,2006,79(5):949–957.
[42] Wolburg H, Wolburg-Buchholz K, Liebner S, et a1. CLDN-l, CLDN-2andCLDN-11are present in tight junction of choroid plexus epithelium of themouse. Neurosci Lett,2001,307:77-80.
[43] Stewart P A, Wiley M J. Developing nervous tissue induces formation ofblood-brain barrier characteristics in invading endothelial cells: a study usingquail-chick transplantation chimeras. Dev Biol,1981,84:183-192.
[44] Janzer R C, Raft M C. Astrocytes induce blood-brain barrier properties inendothelial cells. Nature,1987,325:253-257.
[45] Wilcox E R, Burton Q L, Naz S,et al. Mutations in the gene encoding tightjunction CLDN-14cause autosomal recessive deafness DFNB29[J].Cell,2001,104(1):165–172.
[46] Hadj-Rabia S, Baala L, Vabres P, et al. CLDN-1gene mutations in neonatalsclerosing cholangitis associated with ichthyosis: a tight junction disease[J].Gastroenterology,2004,127(5):1386-1390.
[47] Lu Z, Ding L, Hong H, Hoggard J, Lu Q, Chen YH. CLDN-7inhibits humanlung cancer cell migration and invasion through ERK/MAPK signalingpathway. Exp Cell Res.2011Aug1;317(13):1935-46. doi:10.1016/j.yexcr.2011.05.019.
[48] Moldvay J., Jackel M., Paska C., et al. Distinct CLDN expression profile inhistologic subtypes of lung cancer[J]. Lung Cancer,2007,57:159-167.
[49] Bello I.O., Vilen S.T., Niinimaa A., et al. Expression of CLDNs1,4,5, and7and occludin, and relationship with prognosis in squamous cell carcinoma ofthe tongue[J]. Hum Pathol,2008,39:1212-1220.
[50] Moldvay J., Jackel M., Paska C., et al. Distinct CLDN expression profile inhistologic subtypes of lung cancer[J]. Lung Cancer,2007,57:159-167.
[51] Miyamoto K., Kusumi T., Sato F., et al. Decreased expression of CLDN1iscorrelated with recurrence status in esophageal squamous cell carcinoma[J].Biomed Res,2008,29:71-76.
[52] Takala H., Saarnio J., Wiik H.,et al. CLDNs1,3,4,5and7in esophagealcancer: loss of CLDN3and4expression is associated with metastaticbehavior [J]. APMIS,2007,115:838-847.
[53] Soini Y., Tommola S., Helin H., et al. CLDNs1,3,4and5in gastriccarcinoma, loss of CLDN expression associates with the diffuse subtype[J].Virchows Arch,2006,448:52-58.
[54] Wu Q., Liu Y., Ren Y., Xu X., Yu L., et al. Tight junction protein, CLDN6,downregulates the malignant phenotype of breast carcinoma [J]. Eur J CancerPrev,2010,19(3):186-194.
[55] Osanai M., Murata M., Chiba H., Kojima T., Sawada N. Epigenetic silencingof CLDN6promotes anchorage-independent growth of breast carcinoma cells[J]. Cancer Sci,2007,98(10):1557-1562.
[56] Kominsky SL, Argani P, Korz D et al. Loss of the tight junction proteinCLDN-7correlates with histological grade in both ductal carcinoma in situand invasive ductal carcinoma of the breast. Oncogene.2003,22(13):2021-33.
[57] Park D, Karesen R, Axcrona U et al. Expression pattern of adhesionmolecules (E-cadherin, alpha-, beta-，gamma-catenin and CLDN-7), theirinfluence on survival in primary breast carcinoma, and their correspondingaxillary Lymph node metastasis. APMIS.2007,115(1):52-65.
[58] Sauer T, Pedersen MK, Ebeltoft K et al. Reduced expression of CLDN-7infine needle aspirates from breast carcinomas correlate with grading andmetastatic disease. Cytopathology.2005,16(4):193-198.
[59] TokesAM, KulkaJ, PakuS et al. CLDN-l,-3and-4proteins and mRNAexpression in benign and malignan tbreast lesions: a researeh study. BreastCancerRes.2005,7(2):R296-305.
[60] Soini Y. CLDNs2,3,4, and5inPaget’s disease and breast carcinoma. HumPathol.2004,35(12):1531-6.
[61] Hoevel T, Macek R, Swisshelm K et al. Reexpression of the TJ ProteinCLDN1induces apoptosis in breast tumors Phereoids. Int J Cancer.2004,108(3):374-383.
[62] Rangel LB, Agarwal R, D'Souza T et al. Tight junction proteins CLDN-3andCLDN-4are frequently overexpressed in ovarian cancer but not in ovariancystadenomas. Clin Cancer Res.2003,9(7):2567-2575.
[63] Zhu Y, Brannstrom M, Janson PO et al. Differences in expression patterns ofthe tight junction proteins,CLDN1,3,4and5, in human ovarian surfaceepithelium as compared to epithelia in inclusion cysts and epithelial ovariantumours. Int J Cancer.2006,118(8):1884-1891.
[64] Kreusel KM, Fromm M, Schulzke JD, Hegel U Cl-secretion in epithelialmonolayers of mucus forming human colon cells(HT-29/B6).[J] Am J Physiol,1991，261:C574--C582.
[65] Rahner C, Mitic LL, Anderson JM. Heterogeneity in expression andsubcellular localization of CLDNs2,3,4, and5in the rat liver, pancreas, andgut.[J] Gastroenterology120:411-422.
[66] Amasheh, S; Schmidt,T,Mahn, M, et al. Contribution of CLDN-5to barrierproperties in tight junctions of epithelial cells [J] Cell Tissue Res2005:89-96.
[67] Dhawan P, Singh AB, Deane NG, et al. CLDN-1regulates cellulartransformation and metastatic behavior in colon cancer. J Clin Invest,2005,115(71):1765-1776.
[68] Miwa N, Furuse M, Tsukita S, et al. Involvement of CLDN-1in thebeta-catenin/Tcf signaling parhway and its frequent upregulation in humancolorectal cancers[J]Oncol Res,2001,469-476.
[69] de Oliveira SS, de Oliveira IM, De Souza W, et al. CLDNs upregulation inhuman colorectal cancer[J]. Febs Lett2005,6179-6185.
[70] Kinugasa T, Selective up-regulation of CLDN-l and CLDN-2in colorectalcancer [J].Gastroenterology2008,A446-A447.
[71] Grone J, Weber B, Staub E, et al. Differential expression of genes encodingtight junction proteins in colorectal cancer: frequent dysregulation ofCLDN-1,-8and-12. Int J Colorectal Dis2007,651-659.
[72] Borlak J., Meier T., Halter R., et al. Epidermal growth factor-inducedhepatocellular carcinoma: gene expression profiles in precursor lesions, earlystage and solitary tumours[J]. Oncogene,2005,24:1809-1819.
[73] Christopher D.CLDN-7Immunohistochemistry in Renal Tumors: A CandidateMarker for Chromophobe Renal Cell Carcinoma Identified by GeneExpression Profiling[J].Arch Pathol Lab Med,2007,131:1541-1546.
[74] Yoo Duk Choi, Ki Seung Kim.CLDN-7is Highly Expressed in ChromophobeRenal Cell Carcinoma and Renal Oncocytoma[J]. Korean Med Sci2007,22:305-310.
[75] Lin Li. Expression of CLDN-7in Benign Kidney and Kidney Tumors. Int JClin Exp Pathol,2008,1:57-64.
[76] Moldvay J., Jackel M., Paska C., et al. Distinct CLDN expression profile inhistologic subtypes of lung cancer[J]. Lung Cancer,2007,57:159-167.
[77] Paschoud S., Bongiovanni M., Pache J.C.,et al. CLDN-1and CLDN-5expression patterns differentiate lung squamous cell carcinomas fromadenocarcinomas[J].Mod Pathol,2007,20:947-954.
[78] Schuetz A.N., Rubin B.P., Goldblum J.R.,et al. Intercellular junctions inEwing sarcoma/primitive neuroectodermal tumor: additional evidence ofepithelial differentiation[J]. Mod Pathol,2005,18:1403-1410.
[79] Cohn M.L., Goncharuk V.N., Diwan A.H.,et al. Loss of CLDN1expression intumor-associated vessels correlates with acquisition of metastatic phenotype inmelanocytic neoplasms. J Cutan Pathol,2005,32:533-536.
[80] Soini Y. Expression of CLDNs1,2,3,4,5and7in various types of tumours.[J]Histopathology,2005,46:551-560.
[81] Tokes AM, Kulka J, Paku S et al. CLDN-l,-3and-4proteins and mRNAexpression in benign and malignant breast lesions: a research study[J].BreastCancerRes,2005,7(2):R296-305.
[82] A1Moustafa AE,Alaoui-Jamali MA, Batiste, et al. Identification of genesassociated with head and neck carcinogenesis by cDNAmicroarray comparison betweenmatched primary normal epithelialand squamous carcinoma cells[J].Oncogene,2002,21(17):2634-2640.
[83] Sauer T, Pedersen MK, EbeltoftK, et al. Reduced expression of CLDN-7infine needle aspirates from breast carcinomas correlate with grading andmetastatic disease[J]. Cytopathology,2005,16(4):193-198.
[84] Taddei, A; Giampietro, C; Conti, A, et al. Endothelial adherens junctionscontrol tight junctions by VE-cadherin-mediated upregulation of CLDN-5.[J]Nat Cell Biol2008,923-934.
[85] Michl P,Gress TM.Bacteria and bacterial toxins as therapeutic agents forsolid tumors. Curr Cancer Drug Targets,2004,4:689-702.
[86] Michl P，Buchholz M, Rolke M, et al.CLDN-4:a new target for pancreaticcancer treatment using clostridium perfringens enterotoxin. Gastroenterology，2001，121:678-684.
[87] Hoevel T,Macek R,Swisshelm K, et al induces apoptosis in breast tumorspheroids. Reexpression of the TJ protein CLDN1Int J Cancer2004,108:374-383.
[88] Yamauchi K, Rai T, Kobayashi K,et al. Disease causing mutant WNK4increases paracellular chloride permeability and phosphorylates CLDNs[J].Proc Natl Acad Sci USA,2004,101(13):4690-4694.
[89] Soini Y.CLDNs2,3,4, and5in Paget'sdisease and breastcarcinoma[J]. HumPatho,l2004,35(12):1531-1536.
[90] Rangel LB, Agarwal R, D'Souza T, et al. Tight junction proteins CLDN-3andCLDN-4are frequently overexpressed in ovarian cancer but not in ovariancystadenomas.[J] Clin Cancer Res,2003,9:2567-2575.
[91] Nichols LS, Ashfaq R, Iacobuzio-Donahue CA, et a1. CLDN4proteinexpression in primary andmetastatic pancreatic cancer: sup-port foruse as atherapeutic target[J].Am J Clin Pathol,2004,121(2):226-230.
[92] Tsukahara M, NagaiH, KamiakitoT, et a.1Distinct expression patterns CLDN-1and CLDN-4in intraductal papillary-mucinous tumors of pancreas[J].Pathol Int,2005,55(2):63-69.
[93] Ip, YC; Cheung, ST; Lee, YT, et al. Inhibition of hepatocellular carcinomainvasion by suppression of CLDN-10in HLE cells[J] Molecular CancerTherapeutics2007,6:2858-2867.
[94] IP, YC; Cheung, ST; Lee, YT, et al. CLDN-10enhanced metastatic potentialin hepatocellular carcinoma with MMP activation and modified expressionprofile of other CLDN family members[J] Ejc Supplements2007,5(4):85-86.
[95] Yamauchi K, Rai T, Kobayashi K, et al. Disease-causing mutant WNK4increases paracellular chloride permeability and phosphory-lates CLDNs. ProcNatl Acad Sci USA,2004,101:4690-3694.
[96] Oshima T., Miwa H., Joh T. Aspirin induces gastric epithelial barrierdysfunction by activating P38MAPK via CLDN-7[J]. American Journal ofPhysiology-Cell Physiology,2008,295(3): C800-C806.
[97] Carrozzino F., Pugnale P., Féraille E., Montesano R. Inhibition of basal P38orJNK activity enhances epithelial barrier function through differentialmodulation of CLDN expression [J]. American Journal of Physiology-CellPhysiology,2009,297(3): C775-C787.
[98] Atsumi T., Kato K., Uno K., Iijima K., Koike T., et al. Pathophysiological roleof the activation of P38mitogen-activated protein kinases in poorlydifferentiated gastric cancer [J]. Pathol Int,2007,57(10):635-644.
[99] Oshima T, Miwa H, Joh T, et al. Aspirin induces gastric epithelial barrierdysfunction by activating P38MAPK via CLDN-7[J]. Am J Physiol CellPhysiol,2008,295:800-806.
[100] Zhe Lu, Lei Ding. CLDN-7inhibits human lung cancer cell migration andinvasion through ERK/MAPK signaling pathway[J]. Experimental CellResearch,2011,317:1935-1946.
[101] Chang-Hwan Yoon, Min-Jung Kim, Mei-Hua Yang, et al.CLDN-1Actsthrough c-Abl-Protein Kinase Cδ(PKCδ)Signaling and Has a Causal Role inthe Acquisition of Invasive Capacity in Human Liver Cells. J Biol Chem,2010,285:226-233.
[102] Klover PJ, Muller WJ, Rfeiffer RM, et al. Loss of STAT1from mousemammary epithelium results in an increased Neu-induced tumor burden.Neoplasia,2010,12:899-905.
[103] G R Stark, I M Kerr, et al. How cells respond to interferons[J]. Annu RevBiochem,1998,67:227–264.
[104] G P Dunn, C M Koebel. Interferons,immunity and cancer immunoediting[J].Nat Rev Immunol,2006,6:836–848.
[105] Sultan AS, Xie J, LeBaron, et al. Stat5promotes homotypic adhesion andinhibits invasive characteristics of human breast cancer cells[J]. Oncogene,2005,24:746-760
[106] Chen HH,Chou CY, Chen D, et al. Constitutive STAT5activation correlateswith better survival in cervical cancer patients treated with radiation therapy.Int J Radiat Oncol Biol Phys,2012,82:658-66.
[107] Kataoka TR, Nishizawa Y. Stat4suppresses the proliferation of connectivetissue type mast cells[J]. Lab Invest,2008,88:856-864.
[108] SoiniY.Expression of CLDNs l,2,3,4,5and7in various types of tumours[J]HistoPathology.2005May:46(5):551-560.
[109] Resnick MB，Gavilanez M, Newton E，et al.CLDN expression in gastricadenocarcinomas:a tissue microarray study with prognostic correlation. HumPathol,2005,36:886-892.
[110] Loth C,Szabo E,Holczbauer A,et al.CLDN-4differentiates biliary tractcancers from hepatocellular carcinomas. Mod Pathol,2006,19:460-469.
[111] ResnickMB, GavilanezM, Newton E, et al. CLDN expression in gastricadenocarcinomas: a tissue microarray Hum Patho with prognosticcorrelation[J].2005,36(8):886-892.
[112] Cheung ST,Leung KL，Ip YC,et al.CLDN-10expression level is as-sociated with recurrence of primary hepatocellular carcinoma. Clin CancerRes,2005,11:551-556.
[113] Aldred MA,Huang Y,Liyanarachchi S,et al. Papillary carcinomas showdistinctly different microarray expression and follicular thyroid proliles andcan be distinguished by a minimum of five genes. J Clin Oncol2004,22:3531-3539.
[114] Hoevel T,Macek R, Swisshelm K,et al. Reexpression of the TJ protein CLDN1induces apoptosis in breast tumor spheroids. Int J Cancer.2004Jan20;108(3):374-83.
[115] Offner S,Hekele A,Teichmann U,et al.Epithelial tight junction proteins aspotential antibody targets for pancarcinoma therapy. Cancer ImmunolImmunother2005,54:431-445.
[116] PAEZ JG, JANNE PA, LEE JC, et al. EGFR mutations in lung cancer:correlation with clinical response to gefitinib therapy. Science (New York, NY,2004,304:1497-1500.
[117] CHAN SK, GULLICK WJ, HILL ME. Mutations of the epidermal growthfactor receptor in non-small cell lung cancer-search and destroy[J]. Eur JCancer,2006,42:17-23.
[118] TARON M, ICHINOSE Y, ROSELL R, et al. Activating mutations in thetyrosine kinase domain of the epidermal growth factor receptor are associatedwith improved survival in gefitinib-treated chemorefractory lungadenocarcinomas[J]. Clin Cancer Res,2005,11:5878-5885.
[119] Wu Q, Liu YF, Ren Y, et al. Effects of stable up-regulation of tight junctionprotein CLDN6upon biological phenotypes of breast cancer cell MCF-7.Zhonghua Yi Xue Za Zhi.2010Feb9;90(6):407-12.
[120] Wu Q, Liu Y, Ren Y, et al. Tight junction protein, CLDN6, downregulates themalignant phenotype of breast carcinoma. Eur J Cancer Prev.2010May;19(3):186-94.
[121] Singh A B, Harris R C. Epidermal growth factor receptor activationdifferentially regulates CLDN expression and enhances transepithelialresistance in Madin-Darby canine kidney cells. J Biol Chem,2004,279(5):3543-3552.
[122] Ouyang W, Ma Q, Li J, et al. Cyclin Dl induction tlwough IkappaB kinasebeta/nuclear factor-kappaB pathway is responsible forarsenite-induced increased cell cycle G1-S phase transition in humankeratinocytes. Cancer Res.2005.65(20):9287-9293.
[123] JD Taylor, D Briley, Q Nguyen. Flow cytometric platform for high-throughputsingle nucleotide polymorphism analysis Biotechniques.2001Mar;30(3):661-6,668-9
[124] Sgambato A, Han EK, Zhang YJ, Moon RC, Santella Weinstein IBDeregulated expression of cyclin D1and other cell cycle-related genes incarcinogen-induced rat mammary tumors. Carcinogenesis.1995.16(9):2193-2198.
[125] Lee CC, Yamamoto S, Wanibuchi H, et al. Cyclin D1overexpression in rattwo-stage bladder carcinogenesis and its relationship with oncogenes, tumorsuppressor genes, and cell proliferation. Cancer Res.1997.57(21):4765-4776.
[126] Sanmartin C., Plano D., Sharma A. K., Palop J. A. Selenium compounds,apoptosis and other types of cell death: an overview for cancer therapy. Int JMol Sci,2012,13(8):9649-9672.
[127] Evan G. I., Vousden K. H. Proliferation, cell cycle and apoptosis in cancer.Nature,2001,411(6835):342-348.
[128] Woodhouse E, Chuaqui R, Liotta L. General mechanisms of metastasis.Cancer1997;80:1529-37.
[129] Van Itallie C.M., Anderson J.M.. CLDNs and epithelial paracellulartransport[J]. Annu Rev Physiol,2006,68:403-429.
[130] Furuse M, Sasaki H, Fujimoto K,et.al.A single gene Produet, clandin-1or2,reeonstitutes tight junction strands and recruits occludinin fibroblasts. J CellBiol,1998,143(2):391-401
[131] Furuse M., Sasaki H., Tsukita S. Manner of interaction of heterogeneousCLDN species within and between tight junction strands. J Cell Biol,1999,147:891-903.
[132] McCarthy K.M., Francis S.A., McCormack J.M.,et al. Inducible expression ofCLDN1-myc but not occludin-VSV-G results in aberrant tight junction strandformation in MDCK cells. J Cell Sci,2000,113Pt19:3387-3398.
[133] Van Itallie C., Rahner C., Anderson J.M. Regulated expression of CLDN4decreases paracellular conductance through a selective decrease in sodiumpermeability. J Clin Invest,2001,107:1319-1327.
[134] Wen H., Watry D.D., Marcondes M.C, et al. Selective decrease in paracellularconductance of tight junctions: role of the first extracellular domain ofCLDN5[J]. Mol Cell Biol,2004,24:8408-8417.
[135] Alexandre M.D., Lu Q., Chen Y.H. Overexpression of CLDN7decreases theparacellular Cl-conductance and increases the paracellular Na+conductance inLLC-PK1cells. J Cell Sci,2005,118:2683-2693.
[136] Yu A.S., Enck A.H., Lencer W.I.,et al. CLDN8expression in Madin-Darbycanine kidney cells augments the paracellular barrier to cation permeation. JBiol Chem,2003,278:17350-17359.
[137] Peter Y., Goodenough D. CLDNs [J]. Curr Biol,2004,14(8): R293-294.
[138] Lu Z., Ding L., Hong H., Hoggard J., Lu Q., et al. CLDN-7inhibits humanlung cancer cell migration and invasion through ERK/MAPK signalingpathway [J]. Exp Cell Res,2011,317(13):1935-1946.
[139] Ikari A., Sato T., Watanabe R., Yamazaki Y., Sugatani J. Increase in CLDN-2expression by an EGFR/MEK/ERK/c-Fos pathway in lung adenocarcinomaA549cells [J]. Biochim Biophys Acta,2012,1823(6):1110-1118.
[140] Gong Y., Renigunta V., Himmerkus N., Zhang J., Renigunta A., et al.CLDN-14regulates renal Ca++ transport in response to CaSRsignalling via a novel microRNA pathway [J]. EMBO J.2012Feb28;31(8):1999-2012.
[141] Chiba K., Kondo Y., Yamaguchi K., Miyake H., Fujisawa M. Inhibition ofCLDN-11and Occludin Expression in Rat Sertoli Cells byMono-(2-Ethylhexyl) Phthalate Through p44/42Mitogen-Activated ProteinKinase Pathway. J Androl,2012,33(3):368-374.
[142] Oshima T., Miwa H., Joh T. Aspirin induces gastric epithelial barrierdysfunction by activating P38MAPK via CLDN-7[J]. American Journal ofPhysiology-Cell Physiology,2008,295(3): C800-C806.
[143] Bode JG, Ehlting C, H ussinger D. The macrophage response towards LPS andits control through the P38(MAPK)-STAT3axis. Cell Signal.2012Jun;24(6):1185-94.
[144] Riebe C, Pries R, Schroeder KN, Wollenberg B. Phosphorylation of STAT3inhead and neck cancer requires P38MAPKinase, whereas phosphorylation ofSTAT1occurs via a different signaling pathway. Anticancer Res.2011Nov;31(11):3819-25.
[145] Zhang X, Shan P, Alam J, Fu XY, Lee PJ. Carbon monoxide differentiallymodulates STAT1and STAT3and inhibits apoptosis via a phosphatidylinositol3-kinase/Akt and P38kinase-dependent STAT3pathway duringanoxia-reoxygenation injury. J Biol Chem.2005Mar11;280(10):8714-21.Epub2004Dec7.
[146] BruceE J, PasiA J. Rationale for a dimerization inhibitor, in patients withphase II non-small trial of Pertuzumab, a HER-2cell lung cancer.[J]. ClinCancer Res,2006,12(14Supp1):4436-4440.
[147] Gargova G;Derijard ZB;Wu IH An osmosening signal transduction pathway inmammalian cells. Science.1994Aug5;265(5173):806-8.
[148] Morazzani M;de Carvalbo DD;Kovacic H Monolayer versus aggregate balancein survival process for EGF-induced apoptosis in A431carcinoma cells:Implication of ROS-P38MAPK-integrin alpha2betalpathway.Int-J-Cancer,2004,110(06):788-799.
[149] Uzgare AR, Kaplan PJ, Greenberg NM. Differential expression and/oractivation of P38MAPK, erkl/2, and jnk during the initiation and progressionof prostate cancer[J].Prostate,2003,55(2):128-139.
[150] Kim BJ, Ryu SW, Song B J, et al. JNK-and P38kinase-mediatedphosphorrylation of Bax leads to its activation and mitochond rial translocationand to apoptosis of hum an hepatoma HepG2cells. J Biol Chem,2006,81(30):21256-21265.
[151] Kim MS, Lee EJ, Kim HR. H-Ras-induced cell motility and P38kinase is akey signaling molecule for invasive phenotype in human breast epithelialcells [J]. Cancer Res.2003,63(17):5454-5461.
[152] Bradham C, McClay DR. P38MAPK in development and cancer [J]. CellCycle,2006,5(8):824-828.
[2] Gonzalez-Mariscal L, Betanzos A, Nava P, et al. Tight junction proteins[J].Prog Biophys Mol Biol,2003,81(1):1-44.
[3] Staehelin LA. Further observations on the fine structure of freezecleaved tightjunctions. J Cell Sci,1973,13:763–786.
[4] Tsukita S, Furuse M, Itoh M. Multifunctional strands in tight junctions. NatRev Mol Cell Biol,2001,2(4):285–293.
[5] Ebnet K, Aurrand2LionsM, Kuhn A, et al. The junctional adhesion molecule(JAM) family members JAM-2and JAM-3associate with the cell polarityprotein PAR-3: a possible role for JAMs in endothelial cell polarity. Journal ofCell Science,2003,116(2):3879-3891.
[6] Mitic LL, Anderson JM. Molecular architecture of tight junctions. Annu RevPhysiol,1998,60(10):121–142.。
[7] Furuse M, Sasaki H, Tsukita S. Manner of interaction of heterogeneous CLDNspecies within and between tight junction strands. J Cell Biol,1999,147:891-903.
[8] Liebner S, Fischmann A, Rascher G, et a1. CLDN-1and CLDN-5expressionand tight junction morphology are altered in blood vessels of humanglioblastoma multiforme. Acta Neuropathol(Berl),2000,100:323-331.
[9] Morita K, Sasaki H, Furuse M, Tsukita S. Endothelial CLDN: CLDN-5/TMVCF constitutes tight junction strands in endothelial cells. J Cell Biol.1999Oct4;147(1):185-94.
[10] Liebner S, Kniesel U, Kalbacher H, et al. Correlation of tight junctionmorphology with the expression of tight junction proteins in blood-brainbarrier endothelial cells. Eur J Cell Biol.2000Oct;79(10):707-717.
[11] Turksen K，Troy TC. Barriers built on CLDNs[J].Cell Biol，2004，117：2435-2447.
[12] Anderson J M, CereijidoM. Tight Junctions. Introduction: evolution of ideason the tight junction[M]. Boca Raton: CRC Press,2001:1-18.
[13] Morita K，Furuse M，Fujimoto K，et al. CLDN multigene family encodingfour-transmembrane domain protein components of tight junction strands[J].Cell Biol，1999，2：511-516.
[14] Eveline E. Schneeberger, Robert D. Lynch. The tight junction: amultifunctional complex. Am J Physiol Cell Physiol,2004,286(6):1213-1228.
[15] Miyamoto T., Morita K., Takemoto D., et al. Tight junctions in Schwann cellsof peripheral myelinated axons: a lesson from CLDN19-deficient mice[J].Cell Biol,2005,169:527-538.
[16] Hawkins BT, Davis TP. The blood-brain barrier/neurovascular unit in healthand disease. Pharmacol Rev,2005,57(2):173-185.
[17] Furuse M, Sasaki H, Tsukita S. Manner of interaction of heterogeneous CLDNspecies within and between tight junction strands. J Cell Biol,1999,147(4):891-903.
[18] Mikio F, Hiroyuki S, Shoichiro T. Manner of Interaction of HeterogeneousCLDN Species Within and Between Tight Junction Strands. J Cell Biol,1999,147(4):8912-90.
[19] Furuse M, Furuse K, Sasaki H, et al. Conversion of zonulae occludentes fromtight to leaky strand type by introducing CLDN-2into Madin-Darby caninekidney I cells. J Cell Bio,.2001,153:263–272.
[20] Oscar RC, Christina VI, Christoph R, et al. CLDN extracellular domainsdetermine paracellular charge selectivity and resistance but not tight junctionfibril architecture. Am J Physiol Cell Physiol,2003,284(6):1346-1354.
[21] Van IC, Rahner C, and Anderson JM. Regulated expression of CLDN4decreases paracellular conductance through a selective decrease in sodiumpermeability. J Clin Invest,2001,107:1319–1327.
[22] Colegio OR, Van Itallie CM, McCrea HJ, et al. CLDNs create charge-selective channels in the paracellular pathway between epithelial cells. Am JPhysiol Cell Physiol,2002,283: C142–C147.
[23] Simon D B, Lu Y, Choate K A, et al. Paracellin-1, a renal tight junctionprotein required for paracellular Mg2+resorption. Science,1999,285:103-106.
[24] Weber S, Schneider L, Peters M, et al. Novel paracellin-1mutations in25families with familial hypomagnesaemia with hypercalciuria andnephrocalcinosis. J Am Soc Nephrol,2001,12:1872-1881.
[25] Hirano T, Kobayashi N, Itoh T, et al. Null mutation of PCLN-1/CLDN-16results in bovine chronic interstitial nephritis. Genome Res,2000,10:659-663.
[26] Furuse M, Sasaki H, Tsukita S．Manner of interaction of heterogeneous CLDNspecies within and between tight junction strands．J Cell Biol,1999，147(4)：891-903.
[27] Nitta T., Hata M., Gotoh S., et al. Size-selective loosening of the blood-brainbarrier in CLDN5-deficient mice[J]. Cell Biol,2003,161:653-660.
[28] McLaughlin J., Padfield P.J., Burt J.P.,et al. Ochratoxin A increasespermeability through tight junctions by removal of specific CLDN isoforms[J].Am J Physiol Cell Physiol,2004,287:C1412-1417.
[29] Kyle JH, Rachana A，Patrice JM．The CLDN gene family：expression innormal and neoplastic tissues．BMC Canccr,2006,6:186.
[30] Borlak J,Meier T, Halter R,Spanel R,et al. Epidermal growth factor-inducedhepatocellular carcinoma: gene expression profiles in precursor lesions, earlystage and solitary tumours[J]. Oncogene,2005,24(11):1809-1819.
[31] Al Moustafa AE, Alaoui-Jamali MA, Batist G,et al. Identification of genesassociated with head and neck carcinogenesis by cDNA microarraycomparison between matched primary normal epithelial and squamouscarcinoma cells[J]. Oncogene,2002,21(17):2634-2640.
[32] Chao YC,Pan SH, Yang SC, et al. CLDN-1is a metastasis suppressor andcorrelates with clinical outcome in lung adenocarcinoma[J]. Am J Respir CritCare Med,2009,179(2):123-133.
[33] Paschoud S, Bongiovanni M,Pache JC,et al. CLDN-1and CLDN-5expressionpatterns differentiate lung squamous cell carcinomas from adenocarcinomas[J]. Mod Pathol,2007,20(9):947-954.
[34] Coyne CB, Gambling TM, Boucher RC, et al. Role of CLDN interactions inairway tight junctional permeability[J]. Am J Physiol Lung Cell Mol Physiol,2003,285(5):L1166-1178.
[35] Nunbhakdi-Craig V,Machleidt T, Ogris E, et al. Protein phosphatase2Aassociates with and regulates atypical PKC and the epithelial tight junctioncomplex[J]. Cell Biol,2002,158(5):967-978.
[36] Itoh M, Furuse M, Morita K, et a1. Direct binding of three tightjunction-associated MAGUKs, ZO-l, ZO-2, and ZO-3with the COOH terminiof CLDNs[J]. Cell Bio1,1999, l47:1351-1363.
[37] Ebnet K, Schulz C U, Meyer Zu Brickwedde M K, et a1. Junctional adhesionmolecule interacts with the PDZ domain-containing proteins AF-6and ZO-1.J Biol Chem,2000,275:27979-27988.
[38] Broadwell R D, Charlton H M, Ebert P, et a1. Angiogenesis and theblood-brain barrier in solid and dissociated cell grafts with in the CNS. ProgBrain Res,1990,82:95-101.
[39] Haskins J, Gu L, Wittchen E S, et a1. ZO-3, a novel member of the MAGUKprotein family found at the tight junction, interacts with ZO-1and occludin. JCell Bio1,1998,141:199-208.
[40] Simon D B, Lu Y, Choate K A, et al. Paracellin-1, a renal tight junctionprotein required for paracellular Mg2+resorption. Science,1999,285(5424):103–106.
[41] Konrad M, Schaller A, Seelow D,et al. Mutations in the tight-junction geneCLDN19(CLDN19) are associated with renal magnesium wasting, renalfailure, and severe ocular involvement[J]. Am J Hum Genet,2006,79(5):949–957.
[42] Wolburg H, Wolburg-Buchholz K, Liebner S, et a1. CLDN-l, CLDN-2andCLDN-11are present in tight junction of choroid plexus epithelium of themouse. Neurosci Lett,2001,307:77-80.
[43] Stewart P A, Wiley M J. Developing nervous tissue induces formation ofblood-brain barrier characteristics in invading endothelial cells: a study usingquail-chick transplantation chimeras. Dev Biol,1981,84:183-192.
[44] Janzer R C, Raft M C. Astrocytes induce blood-brain barrier properties inendothelial cells. Nature,1987,325:253-257.
[45] Wilcox E R, Burton Q L, Naz S,et al. Mutations in the gene encoding tightjunction CLDN-14cause autosomal recessive deafness DFNB29[J].Cell,2001,104(1):165–172.
[46] Hadj-Rabia S, Baala L, Vabres P, et al. CLDN-1gene mutations in neonatalsclerosing cholangitis associated with ichthyosis: a tight junction disease[J].Gastroenterology,2004,127(5):1386-1390.
[47] Lu Z, Ding L, Hong H, Hoggard J, Lu Q, Chen YH. CLDN-7inhibits humanlung cancer cell migration and invasion through ERK/MAPK signalingpathway. Exp Cell Res.2011Aug1;317(13):1935-46. doi:10.1016/j.yexcr.2011.05.019.
[48] Moldvay J., Jackel M., Paska C., et al. Distinct CLDN expression profile inhistologic subtypes of lung cancer[J]. Lung Cancer,2007,57:159-167.
[49] Bello I.O., Vilen S.T., Niinimaa A., et al. Expression of CLDNs1,4,5, and7and occludin, and relationship with prognosis in squamous cell carcinoma ofthe tongue[J]. Hum Pathol,2008,39:1212-1220.
[50] Moldvay J., Jackel M., Paska C., et al. Distinct CLDN expression profile inhistologic subtypes of lung cancer[J]. Lung Cancer,2007,57:159-167.
[51] Miyamoto K., Kusumi T., Sato F., et al. Decreased expression of CLDN1iscorrelated with recurrence status in esophageal squamous cell carcinoma[J].Biomed Res,2008,29:71-76.
[52] Takala H., Saarnio J., Wiik H.,et al. CLDNs1,3,4,5and7in esophagealcancer: loss of CLDN3and4expression is associated with metastaticbehavior [J]. APMIS,2007,115:838-847.
[53] Soini Y., Tommola S., Helin H., et al. CLDNs1,3,4and5in gastriccarcinoma, loss of CLDN expression associates with the diffuse subtype[J].Virchows Arch,2006,448:52-58.
[54] Wu Q., Liu Y., Ren Y., Xu X., Yu L., et al. Tight junction protein, CLDN6,downregulates the malignant phenotype of breast carcinoma [J]. Eur J CancerPrev,2010,19(3):186-194.
[55] Osanai M., Murata M., Chiba H., Kojima T., Sawada N. Epigenetic silencingof CLDN6promotes anchorage-independent growth of breast carcinoma cells[J]. Cancer Sci,2007,98(10):1557-1562.
[56] Kominsky SL, Argani P, Korz D et al. Loss of the tight junction proteinCLDN-7correlates with histological grade in both ductal carcinoma in situand invasive ductal carcinoma of the breast. Oncogene.2003,22(13):2021-33.
[57] Park D, Karesen R, Axcrona U et al. Expression pattern of adhesionmolecules (E-cadherin, alpha-, beta-，gamma-catenin and CLDN-7), theirinfluence on survival in primary breast carcinoma, and their correspondingaxillary Lymph node metastasis. APMIS.2007,115(1):52-65.
[58] Sauer T, Pedersen MK, Ebeltoft K et al. Reduced expression of CLDN-7infine needle aspirates from breast carcinomas correlate with grading andmetastatic disease. Cytopathology.2005,16(4):193-198.
[59] TokesAM, KulkaJ, PakuS et al. CLDN-l,-3and-4proteins and mRNAexpression in benign and malignan tbreast lesions: a researeh study. BreastCancerRes.2005,7(2):R296-305.
[60] Soini Y. CLDNs2,3,4, and5inPaget’s disease and breast carcinoma. HumPathol.2004,35(12):1531-6.
[61] Hoevel T, Macek R, Swisshelm K et al. Reexpression of the TJ ProteinCLDN1induces apoptosis in breast tumors Phereoids. Int J Cancer.2004,108(3):374-383.
[62] Rangel LB, Agarwal R, D'Souza T et al. Tight junction proteins CLDN-3andCLDN-4are frequently overexpressed in ovarian cancer but not in ovariancystadenomas. Clin Cancer Res.2003,9(7):2567-2575.
[63] Zhu Y, Brannstrom M, Janson PO et al. Differences in expression patterns ofthe tight junction proteins,CLDN1,3,4and5, in human ovarian surfaceepithelium as compared to epithelia in inclusion cysts and epithelial ovariantumours. Int J Cancer.2006,118(8):1884-1891.
[64] Kreusel KM, Fromm M, Schulzke JD, Hegel U Cl-secretion in epithelialmonolayers of mucus forming human colon cells(HT-29/B6).[J] Am J Physiol,1991，261:C574--C582.
[65] Rahner C, Mitic LL, Anderson JM. Heterogeneity in expression andsubcellular localization of CLDNs2,3,4, and5in the rat liver, pancreas, andgut.[J] Gastroenterology120:411-422.
[66] Amasheh, S; Schmidt,T,Mahn, M, et al. Contribution of CLDN-5to barrierproperties in tight junctions of epithelial cells [J] Cell Tissue Res2005:89-96.
[67] Dhawan P, Singh AB, Deane NG, et al. CLDN-1regulates cellulartransformation and metastatic behavior in colon cancer. J Clin Invest,2005,115(71):1765-1776.
[68] Miwa N, Furuse M, Tsukita S, et al. Involvement of CLDN-1in thebeta-catenin/Tcf signaling parhway and its frequent upregulation in humancolorectal cancers[J]Oncol Res,2001,469-476.
[69] de Oliveira SS, de Oliveira IM, De Souza W, et al. CLDNs upregulation inhuman colorectal cancer[J]. Febs Lett2005,6179-6185.
[70] Kinugasa T, Selective up-regulation of CLDN-l and CLDN-2in colorectalcancer [J].Gastroenterology2008,A446-A447.
[71] Grone J, Weber B, Staub E, et al. Differential expression of genes encodingtight junction proteins in colorectal cancer: frequent dysregulation ofCLDN-1,-8and-12. Int J Colorectal Dis2007,651-659.
[72] Borlak J., Meier T., Halter R., et al. Epidermal growth factor-inducedhepatocellular carcinoma: gene expression profiles in precursor lesions, earlystage and solitary tumours[J]. Oncogene,2005,24:1809-1819.
[73] Christopher D.CLDN-7Immunohistochemistry in Renal Tumors: A CandidateMarker for Chromophobe Renal Cell Carcinoma Identified by GeneExpression Profiling[J].Arch Pathol Lab Med,2007,131:1541-1546.
[74] Yoo Duk Choi, Ki Seung Kim.CLDN-7is Highly Expressed in ChromophobeRenal Cell Carcinoma and Renal Oncocytoma[J]. Korean Med Sci2007,22:305-310.
[75] Lin Li. Expression of CLDN-7in Benign Kidney and Kidney Tumors. Int JClin Exp Pathol,2008,1:57-64.
[76] Moldvay J., Jackel M., Paska C., et al. Distinct CLDN expression profile inhistologic subtypes of lung cancer[J]. Lung Cancer,2007,57:159-167.
[77] Paschoud S., Bongiovanni M., Pache J.C.,et al. CLDN-1and CLDN-5expression patterns differentiate lung squamous cell carcinomas fromadenocarcinomas[J].Mod Pathol,2007,20:947-954.
[78] Schuetz A.N., Rubin B.P., Goldblum J.R.,et al. Intercellular junctions inEwing sarcoma/primitive neuroectodermal tumor: additional evidence ofepithelial differentiation[J]. Mod Pathol,2005,18:1403-1410.
[79] Cohn M.L., Goncharuk V.N., Diwan A.H.,et al. Loss of CLDN1expression intumor-associated vessels correlates with acquisition of metastatic phenotype inmelanocytic neoplasms. J Cutan Pathol,2005,32:533-536.
[80] Soini Y. Expression of CLDNs1,2,3,4,5and7in various types of tumours.[J]Histopathology,2005,46:551-560.
[81] Tokes AM, Kulka J, Paku S et al. CLDN-l,-3and-4proteins and mRNAexpression in benign and malignant breast lesions: a research study[J].BreastCancerRes,2005,7(2):R296-305.
[82] A1Moustafa AE,Alaoui-Jamali MA, Batiste, et al. Identification of genesassociated with head and neck carcinogenesis by cDNAmicroarray comparison betweenmatched primary normal epithelialand squamous carcinoma cells[J].Oncogene,2002,21(17):2634-2640.
[83] Sauer T, Pedersen MK, EbeltoftK, et al. Reduced expression of CLDN-7infine needle aspirates from breast carcinomas correlate with grading andmetastatic disease[J]. Cytopathology,2005,16(4):193-198.
[84] Taddei, A; Giampietro, C; Conti, A, et al. Endothelial adherens junctionscontrol tight junctions by VE-cadherin-mediated upregulation of CLDN-5.[J]Nat Cell Biol2008,923-934.
[85] Michl P,Gress TM.Bacteria and bacterial toxins as therapeutic agents forsolid tumors. Curr Cancer Drug Targets,2004,4:689-702.
[86] Michl P，Buchholz M, Rolke M, et al.CLDN-4:a new target for pancreaticcancer treatment using clostridium perfringens enterotoxin. Gastroenterology，2001，121:678-684.
[87] Hoevel T,Macek R,Swisshelm K, et al induces apoptosis in breast tumorspheroids. Reexpression of the TJ protein CLDN1Int J Cancer2004,108:374-383.
[88] Yamauchi K, Rai T, Kobayashi K,et al. Disease causing mutant WNK4increases paracellular chloride permeability and phosphorylates CLDNs[J].Proc Natl Acad Sci USA,2004,101(13):4690-4694.
[89] Soini Y.CLDNs2,3,4, and5in Paget'sdisease and breastcarcinoma[J]. HumPatho,l2004,35(12):1531-1536.
[90] Rangel LB, Agarwal R, D'Souza T, et al. Tight junction proteins CLDN-3andCLDN-4are frequently overexpressed in ovarian cancer but not in ovariancystadenomas.[J] Clin Cancer Res,2003,9:2567-2575.
[91] Nichols LS, Ashfaq R, Iacobuzio-Donahue CA, et a1. CLDN4proteinexpression in primary andmetastatic pancreatic cancer: sup-port foruse as atherapeutic target[J].Am J Clin Pathol,2004,121(2):226-230.
[92] Tsukahara M, NagaiH, KamiakitoT, et a.1Distinct expression patterns CLDN-1and CLDN-4in intraductal papillary-mucinous tumors of pancreas[J].Pathol Int,2005,55(2):63-69.
[93] Ip, YC; Cheung, ST; Lee, YT, et al. Inhibition of hepatocellular carcinomainvasion by suppression of CLDN-10in HLE cells[J] Molecular CancerTherapeutics2007,6:2858-2867.
[94] IP, YC; Cheung, ST; Lee, YT, et al. CLDN-10enhanced metastatic potentialin hepatocellular carcinoma with MMP activation and modified expressionprofile of other CLDN family members[J] Ejc Supplements2007,5(4):85-86.
[95] Yamauchi K, Rai T, Kobayashi K, et al. Disease-causing mutant WNK4increases paracellular chloride permeability and phosphory-lates CLDNs. ProcNatl Acad Sci USA,2004,101:4690-3694.
[96] Oshima T., Miwa H., Joh T. Aspirin induces gastric epithelial barrierdysfunction by activating P38MAPK via CLDN-7[J]. American Journal ofPhysiology-Cell Physiology,2008,295(3): C800-C806.
[97] Carrozzino F., Pugnale P., Féraille E., Montesano R. Inhibition of basal P38orJNK activity enhances epithelial barrier function through differentialmodulation of CLDN expression [J]. American Journal of Physiology-CellPhysiology,2009,297(3): C775-C787.
[98] Atsumi T., Kato K., Uno K., Iijima K., Koike T., et al. Pathophysiological roleof the activation of P38mitogen-activated protein kinases in poorlydifferentiated gastric cancer [J]. Pathol Int,2007,57(10):635-644.
[99] Oshima T, Miwa H, Joh T, et al. Aspirin induces gastric epithelial barrierdysfunction by activating P38MAPK via CLDN-7[J]. Am J Physiol CellPhysiol,2008,295:800-806.
[100] Zhe Lu, Lei Ding. CLDN-7inhibits human lung cancer cell migration andinvasion through ERK/MAPK signaling pathway[J]. Experimental CellResearch,2011,317:1935-1946.
[101] Chang-Hwan Yoon, Min-Jung Kim, Mei-Hua Yang, et al.CLDN-1Actsthrough c-Abl-Protein Kinase Cδ(PKCδ)Signaling and Has a Causal Role inthe Acquisition of Invasive Capacity in Human Liver Cells. J Biol Chem,2010,285:226-233.
[102] Klover PJ, Muller WJ, Rfeiffer RM, et al. Loss of STAT1from mousemammary epithelium results in an increased Neu-induced tumor burden.Neoplasia,2010,12:899-905.
[103] G R Stark, I M Kerr, et al. How cells respond to interferons[J]. Annu RevBiochem,1998,67:227–264.
[104] G P Dunn, C M Koebel. Interferons,immunity and cancer immunoediting[J].Nat Rev Immunol,2006,6:836–848.
[105] Sultan AS, Xie J, LeBaron, et al. Stat5promotes homotypic adhesion andinhibits invasive characteristics of human breast cancer cells[J]. Oncogene,2005,24:746-760
[106] Chen HH,Chou CY, Chen D, et al. Constitutive STAT5activation correlateswith better survival in cervical cancer patients treated with radiation therapy.Int J Radiat Oncol Biol Phys,2012,82:658-66.
[107] Kataoka TR, Nishizawa Y. Stat4suppresses the proliferation of connectivetissue type mast cells[J]. Lab Invest,2008,88:856-864.
[108] SoiniY.Expression of CLDNs l,2,3,4,5and7in various types of tumours[J]HistoPathology.2005May:46(5):551-560.
[109] Resnick MB，Gavilanez M, Newton E，et al.CLDN expression in gastricadenocarcinomas:a tissue microarray study with prognostic correlation. HumPathol,2005,36:886-892.
[110] Loth C,Szabo E,Holczbauer A,et al.CLDN-4differentiates biliary tractcancers from hepatocellular carcinomas. Mod Pathol,2006,19:460-469.
[111] ResnickMB, GavilanezM, Newton E, et al. CLDN expression in gastricadenocarcinomas: a tissue microarray Hum Patho with prognosticcorrelation[J].2005,36(8):886-892.
[112] Cheung ST,Leung KL，Ip YC,et al.CLDN-10expression level is as-sociated with recurrence of primary hepatocellular carcinoma. Clin CancerRes,2005,11:551-556.
[113] Aldred MA,Huang Y,Liyanarachchi S,et al. Papillary carcinomas showdistinctly different microarray expression and follicular thyroid proliles andcan be distinguished by a minimum of five genes. J Clin Oncol2004,22:3531-3539.
[114] Hoevel T,Macek R, Swisshelm K,et al. Reexpression of the TJ protein CLDN1induces apoptosis in breast tumor spheroids. Int J Cancer.2004Jan20;108(3):374-83.
[115] Offner S,Hekele A,Teichmann U,et al.Epithelial tight junction proteins aspotential antibody targets for pancarcinoma therapy. Cancer ImmunolImmunother2005,54:431-445.
[116] PAEZ JG, JANNE PA, LEE JC, et al. EGFR mutations in lung cancer:correlation with clinical response to gefitinib therapy. Science (New York, NY,2004,304:1497-1500.
[117] CHAN SK, GULLICK WJ, HILL ME. Mutations of the epidermal growthfactor receptor in non-small cell lung cancer-search and destroy[J]. Eur JCancer,2006,42:17-23.
[118] TARON M, ICHINOSE Y, ROSELL R, et al. Activating mutations in thetyrosine kinase domain of the epidermal growth factor receptor are associatedwith improved survival in gefitinib-treated chemorefractory lungadenocarcinomas[J]. Clin Cancer Res,2005,11:5878-5885.
[119] Wu Q, Liu YF, Ren Y, et al. Effects of stable up-regulation of tight junctionprotein CLDN6upon biological phenotypes of breast cancer cell MCF-7.Zhonghua Yi Xue Za Zhi.2010Feb9;90(6):407-12.
[120] Wu Q, Liu Y, Ren Y, et al. Tight junction protein, CLDN6, downregulates themalignant phenotype of breast carcinoma. Eur J Cancer Prev.2010May;19(3):186-94.
[121] Singh A B, Harris R C. Epidermal growth factor receptor activationdifferentially regulates CLDN expression and enhances transepithelialresistance in Madin-Darby canine kidney cells. J Biol Chem,2004,279(5):3543-3552.
[122] Ouyang W, Ma Q, Li J, et al. Cyclin Dl induction tlwough IkappaB kinasebeta/nuclear factor-kappaB pathway is responsible forarsenite-induced increased cell cycle G1-S phase transition in humankeratinocytes. Cancer Res.2005.65(20):9287-9293.
[123] JD Taylor, D Briley, Q Nguyen. Flow cytometric platform for high-throughputsingle nucleotide polymorphism analysis Biotechniques.2001Mar;30(3):661-6,668-9
[124] Sgambato A, Han EK, Zhang YJ, Moon RC, Santella Weinstein IBDeregulated expression of cyclin D1and other cell cycle-related genes incarcinogen-induced rat mammary tumors. Carcinogenesis.1995.16(9):2193-2198.
[125] Lee CC, Yamamoto S, Wanibuchi H, et al. Cyclin D1overexpression in rattwo-stage bladder carcinogenesis and its relationship with oncogenes, tumorsuppressor genes, and cell proliferation. Cancer Res.1997.57(21):4765-4776.
[126] Sanmartin C., Plano D., Sharma A. K., Palop J. A. Selenium compounds,apoptosis and other types of cell death: an overview for cancer therapy. Int JMol Sci,2012,13(8):9649-9672.
[127] Evan G. I., Vousden K. H. Proliferation, cell cycle and apoptosis in cancer.Nature,2001,411(6835):342-348.
[128] Woodhouse E, Chuaqui R, Liotta L. General mechanisms of metastasis.Cancer1997;80:1529-37.
[129] Van Itallie C.M., Anderson J.M.. CLDNs and epithelial paracellulartransport[J]. Annu Rev Physiol,2006,68:403-429.
[130] Furuse M, Sasaki H, Fujimoto K,et.al.A single gene Produet, clandin-1or2,reeonstitutes tight junction strands and recruits occludinin fibroblasts. J CellBiol,1998,143(2):391-401
[131] Furuse M., Sasaki H., Tsukita S. Manner of interaction of heterogeneousCLDN species within and between tight junction strands. J Cell Biol,1999,147:891-903.
[132] McCarthy K.M., Francis S.A., McCormack J.M.,et al. Inducible expression ofCLDN1-myc but not occludin-VSV-G results in aberrant tight junction strandformation in MDCK cells. J Cell Sci,2000,113Pt19:3387-3398.
[133] Van Itallie C., Rahner C., Anderson J.M. Regulated expression of CLDN4decreases paracellular conductance through a selective decrease in sodiumpermeability. J Clin Invest,2001,107:1319-1327.
[134] Wen H., Watry D.D., Marcondes M.C, et al. Selective decrease in paracellularconductance of tight junctions: role of the first extracellular domain ofCLDN5[J]. Mol Cell Biol,2004,24:8408-8417.
[135] Alexandre M.D., Lu Q., Chen Y.H. Overexpression of CLDN7decreases theparacellular Cl-conductance and increases the paracellular Na+conductance inLLC-PK1cells. J Cell Sci,2005,118:2683-2693.
[136] Yu A.S., Enck A.H., Lencer W.I.,et al. CLDN8expression in Madin-Darbycanine kidney cells augments the paracellular barrier to cation permeation. JBiol Chem,2003,278:17350-17359.
[137] Peter Y., Goodenough D. CLDNs [J]. Curr Biol,2004,14(8): R293-294.
[138] Lu Z., Ding L., Hong H., Hoggard J., Lu Q., et al. CLDN-7inhibits humanlung cancer cell migration and invasion through ERK/MAPK signalingpathway [J]. Exp Cell Res,2011,317(13):1935-1946.
[139] Ikari A., Sato T., Watanabe R., Yamazaki Y., Sugatani J. Increase in CLDN-2expression by an EGFR/MEK/ERK/c-Fos pathway in lung adenocarcinomaA549cells [J]. Biochim Biophys Acta,2012,1823(6):1110-1118.
[140] Gong Y., Renigunta V., Himmerkus N., Zhang J., Renigunta A., et al.CLDN-14regulates renal Ca++ transport in response to CaSRsignalling via a novel microRNA pathway [J]. EMBO J.2012Feb28;31(8):1999-2012.
[141] Chiba K., Kondo Y., Yamaguchi K., Miyake H., Fujisawa M. Inhibition ofCLDN-11and Occludin Expression in Rat Sertoli Cells byMono-(2-Ethylhexyl) Phthalate Through p44/42Mitogen-Activated ProteinKinase Pathway. J Androl,2012,33(3):368-374.
[142] Oshima T., Miwa H., Joh T. Aspirin induces gastric epithelial barrierdysfunction by activating P38MAPK via CLDN-7[J]. American Journal ofPhysiology-Cell Physiology,2008,295(3): C800-C806.
[143] Bode JG, Ehlting C, H ussinger D. The macrophage response towards LPS andits control through the P38(MAPK)-STAT3axis. Cell Signal.2012Jun;24(6):1185-94.
[144] Riebe C, Pries R, Schroeder KN, Wollenberg B. Phosphorylation of STAT3inhead and neck cancer requires P38MAPKinase, whereas phosphorylation ofSTAT1occurs via a different signaling pathway. Anticancer Res.2011Nov;31(11):3819-25.
[145] Zhang X, Shan P, Alam J, Fu XY, Lee PJ. Carbon monoxide differentiallymodulates STAT1and STAT3and inhibits apoptosis via a phosphatidylinositol3-kinase/Akt and P38kinase-dependent STAT3pathway duringanoxia-reoxygenation injury. J Biol Chem.2005Mar11;280(10):8714-21.Epub2004Dec7.
[146] BruceE J, PasiA J. Rationale for a dimerization inhibitor, in patients withphase II non-small trial of Pertuzumab, a HER-2cell lung cancer.[J]. ClinCancer Res,2006,12(14Supp1):4436-4440.
[147] Gargova G;Derijard ZB;Wu IH An osmosening signal transduction pathway inmammalian cells. Science.1994Aug5;265(5173):806-8.
[148] Morazzani M;de Carvalbo DD;Kovacic H Monolayer versus aggregate balancein survival process for EGF-induced apoptosis in A431carcinoma cells:Implication of ROS-P38MAPK-integrin alpha2betalpathway.Int-J-Cancer,2004,110(06):788-799.
[149] Uzgare AR, Kaplan PJ, Greenberg NM. Differential expression and/oractivation of P38MAPK, erkl/2, and jnk during the initiation and progressionof prostate cancer[J].Prostate,2003,55(2):128-139.
[150] Kim BJ, Ryu SW, Song B J, et al. JNK-and P38kinase-mediatedphosphorrylation of Bax leads to its activation and mitochond rial translocationand to apoptosis of hum an hepatoma HepG2cells. J Biol Chem,2006,81(30):21256-21265.
[151] Kim MS, Lee EJ, Kim HR. H-Ras-induced cell motility and P38kinase is akey signaling molecule for invasive phenotype in human breast epithelialcells [J]. Cancer Res.2003,63(17):5454-5461.
[152] Bradham C, McClay DR. P38MAPK in development and cancer [J]. CellCycle,2006,5(8):824-828.