微电场对胎盘滋养细胞迁移/侵袭相关信号通路活性的影响

目的：探讨滋养细胞迁移/侵袭相关促分裂原活化蛋白激酶/细胞外信号调节蛋白激酶（MAPK/ERK）,磷酸肌醇-3激酶/丝氨酸苏氨酸蛋白激酶（PI3K/Akt）和蛋白酪氨酸激酶/信号转导和转录活化因子3（JAK/STAT3）信号传导通路在微电场刺激下的活化水平。方法将胎盘滋养细胞用场强为150 mV/mm 的直流微电场加以刺激,通过蛋白质印迹法（Western blot）测定电刺激前和后各时间点滋养细胞 MAPK/ERK、PI3K/Akt 和 JAK/STAT3信号传导通路相关活性分子的活化水平。滋养细胞在加电前分别用 MAPK 抑制剂 PD980599（100μmol/L）和 Akt 抑制剂 LY294002（20μmol/L）处理1 h,在上述相应含抑制剂的培养基中用场强为150 mV/mm 直流微电场刺激5 h,观察抑制剂对微电场刺激细胞行为的影响。结果Western blot 结果显示,滋养细胞在150 mV/mm 微电场作用下,p42/44 MAPK Thr202/Thr204位点、Akt Ser473位点于5 min 开始活化,10-60 min 内逐渐加强,STAT3 S727位点在微电场作用下上述各时间点无明显磷酸化水平改变。相应信号通路抑制剂处理后滋养细胞对微电场刺激的应答反应均受到明显抑制。结论微电场对滋养细胞迁移/侵袭功能的影响与 MAPK/ERK、PI3K/Akt 信号通路的活化有关。

微电场通过非整合素途径活化粘着斑激酶促进滋养细胞迁移/侵袭研究

目的探讨生理性微电场促进体外培养的人胎盘滋养细胞迁移/侵袭功能是否与滋养细胞表面整合素(integrin)表达有关。方法用150 m V/mm的直流微电场刺激滋养细胞,时间分别为5、10和15 h,测定其迁移情况并观察细胞形态变化。Western blot检测刺激前后1 h内粘着斑激酶FAK活化情况和细胞表面integrinα1、integrinα5、integrinαV和integrinα1蛋白表达水平。结果在含有10%胎牛血清的培养基中,150 m V/mm电场刺激下滋养细胞向负极定向迁移,迁移速度和距离较对照组明显增加(P=0.021),胞体拉长,垂直于电场方向排列;胞内FAKTyr397位点于刺激后5、10、30、60 min内迅速活化并逐渐加强(P<0.05);刺激前后滋养细胞表面integrinα1、integrinα5、integrinαV、和integrinα1蛋白表达水平无明显改变(P>0.05)。结论生理性直流微电场可能通过非整合素途径活化FAK从而促进滋养细胞迁移/侵袭功能,但其详细机制仍需进一步研究。

微电场刺激血管内皮细胞培养上清对缺氧培养滋养细胞迁移/侵袭的影响

目的探讨微电场(electric field,EF)刺激血管内皮细胞培养上清对缺氧培养滋养细胞迁移/侵袭能力的影响。方法 200 m V/mm微电场刺激血管内皮细胞(HUVEC)5 h,收集培养上清。Transwell体外侵袭实验和划痕实验观察EF刺激内皮细胞条件培养基对3%O2物理缺氧培养滋养细胞迁移/侵袭功能的影响。采用Western blot和荧光定量RT-PCR检测EF刺激的血管内皮细胞条件培养基与滋养细胞共培养24 h后对缺氧培养滋养细胞MMP-2(Matrix metalloproteinase 2,MMP-2)及MMP-9(Matrix metalloproteinase 9,MMP-9)分子表达的影响。结果 Transwell小室体外侵袭实验和划痕实验结果显示,EF 200m V/mm刺激血管内皮细胞5 h条件培养基(conditioned medium,CM)与滋养细胞共培养,可促进缺氧培养滋养细胞迁移/侵袭功能,其穿膜细胞数目是单纯缺氧的3.62倍(P<0.05),迁移速度是单纯缺氧的1.56倍(P<0.05)。EF刺激的血管内皮细胞条件培养基与滋养细胞共培养24 h,可以促进缺氧培养滋养细胞的MMP-2表达;对MMP-9分子表达的影响不明显。结论EF刺激的血管内皮细胞培养上清能促进缺氧培养滋养细胞的迁移/侵袭能力,可能与内皮细胞作用于滋养细胞、促进MMP-2分子表达增加有关。

Med-19基因对膀胱癌细胞迁移及侵袭能力的影响

目的探讨在体外通过干扰RNA（siRNA）下调Med-19基因的表达对人膀胱癌T24细胞迁移、侵袭能力的影响。方法采用Transwell-移试验、Transwell侵袭实验观察TsiRNA在体外对人膀胱癌T24细胞迁移、侵袭能力的影响，RT—PCR技术检测siRNA处理后Med－19基因与蛋白表达水平的变化。结果siRNAT调Meal－19基因表达可以明显降低T24的迁移、侵袭能力。结论膀胱癌细胞的迁移、侵袭能力与Med－19基因的过表达密切相关，Med－19基因特异性siRNA可以降低T24细胞的迁移、侵袭能力。

间充质干细胞促肿瘤细胞迁移侵袭研究进展

肿瘤转移是大多数癌症患者的重要致死原因,一旦发生转移,肿瘤患者生存率与生存质量明显下降。肿瘤细胞迁移/侵袭能力是转移的关键,其发生机制受到多种因素的影响。大量研究证明,间充质干细胞(mesenchymal stem cells, MSCs)与肿瘤细胞迁移/侵袭之间有着密切关系。本文就MSCs对肿瘤细胞迁移/侵袭能力的影响进行了总结,归纳了MSCs促进肿瘤细胞迁移侵袭的分子机制。有助于加深对癌症转移机制的了解,为进一步研究肿瘤疾病的诊断与治疗提供了科学有效的参考。

Suppression of Human Liver Cancer Cell Migration and Invasion via the GABA_A Receptor

Objective To investigate the roles of the y-aminobutyric acid （GABA） in the metastasis of hepatocellular carcinoma （HCC） and to explore the potential of a novel therapeutic approach for the treatment of HCC. Methods The expression levels of GABA receptor subunit genes in various HCC cell lines and patients＇ tissues were detected by quantitative real-time polymerase chain reaction and Western blot analysis. Transwell cell migration and invasion assays were carried out for functional analysis. The effects of GABA on liver cancer cell cytoskeletal were determined by immunofluorescence staining. And the effects of GABA on HCC metastasis in nude mice were evaluated using an in vivo orthotopic model of liver cancer. Results The mRNA level of GABA receptor subunits varied between the primary hepatocellular carcinoma tissue and the adjacent non-tumor liver tissue. GABA inhibited human liver cancer cell migration and invasion via the ionotropic GABAA receptor as a result of the induction of liver cancer cell cytoskeletal reorganization. Pretreatment with GABA also significantly reduced intrahepatic liver metastasis and primary tumor formation in vivo. Conclusions These findings introduce a potential and novel therapeutic approach for the treatment of cancer patients based on the modulation of the GABAergic system.

Roles of Rap1 signaling in tumor cell migration and invasion

Ras-associated protein-1 （Rapl）, a small GTPase in the Ras-related protein family, is an important regulator of basic cellular functions （e.g., formation and control of cell adhesions and junctions）, cellular migration, and polarization. Through its interaction with other proteins, Rapl plays many roles during cell invasion and metastasis in different cancers. The basic function of Rapl is straightforward; it acts as a switch during cellular signaling transduction and regulated by its binding to either guanosine triphosphate （GTP） or guanosine diphosphate （GDP）. However, its remarkably diverse function is rendered by its interplay with a large number of distinct Rap guanine nucleotide exchange factors and Rap GTPase activating proteins. This review summarizes the mechanisms by which Rap 1 signaling can regulate cell invasion and metastasis, focusing on its roles in integrin and cadherin regulation, Rho GTPase control, and matrix metalloproteinase expression.

Interleukin-8 associates with adhesion,migration,invasion and chemosensitivity of human gastric cancer cells

AIM： To investigate the relationship between Interleu- kin-8 （IL-8） and proliferation, adhesion, migration, inva- sion and chemosensitivity of gastric cancer （GC） cells.

GNAI1 Suppresses Tumor Cell Migration and Invasion and is Post-Transcriptionally Regulated by Mir-320a/c/d in Hepatocellular Carcinoma

Objective To explore the role and regulation of guanine nucleotide-binding protein G（i）, a-1 subunit （GNAI1） in hepatocellular carcinoma （HCC）. Methods Expression of GNAI1 in HCC samples was determined by qRT-PCR and immunohistochemical （IHC） staining. Huh-7 and SNU-387 cells stably expressing GNAI1 were established by the infection of lentivirus transducing unit containing GNAI1. siRNA against GNAI1 was transfected into SMMC-7721 cells to knock down the GNAI1 expression in HCC cells. Mir-320a/c/d mimics were transfected into SMMC-7721 and SK-Hep-1 cells and the expression of GNAll was determined by Western blot. The migration and invasion of Huh-7, SNU-387, SK-Hep-1 and SMMC-7721 cells were investigated by Transwell assays. Results The GNAI1 protein was significantly downregulated in HCC samples without changes in its mRNA levels. GNAI1 could inhibit the migration and invasion of HCC cells in vitro. Further investigations indicated that GNAI1 was a target of miR-320a/c/d in HCC cells. Transwell assays demonstrated that these microRNAs could promote the migratory ability and invasivesess of HCC cells in vitro. Conclusions GNAII is downregulated in HCC and inhibits the migration and invasion of HCC cells. This study is the first to investigate the role of GNAI1 in cancer. Regulation of GNAI1 by miR-320a/c/d indicates new therapeutic avenues for targeting HCC metastasis.

Decreasing Pin1 suppressed the properties of migratory and invasive in colorectal carcinoma SW620 cells

Objective:The aim of our study was to investigate the effect of Pin1 on the expression of MMP-2 and MMP-9 in human colorectal carcinoma SW620 cells. Methods: We constructed a eukaryotic expression vector of RNA interfering (shRNA) for Pin1 gene (pGenesil-1-Pin1), and then observed its expression in SW620 cells by Western blotting. The cells motility were tested by wound healing assay and Boyden chamber assay. The protein levels and activity of MMP-2 and MMP-9 were tested by Western blotting and Gelatin zymography in SW620 cells after transfected with pGenesil-1-PIN1. Results: pGenesil-1-PIN1 was successfully constructed, which was confirmed by sequencing. Silencing the Pin1 by RNAi significantly decreased the cells motility from 96.4±3.9 per field (×10 objective) to 52.7±4.4 per field (P<0.05, Student's t-test) for SW620 cells transfected with pGenesil-1-PIN1 (SW620/p-shRNA) in Boyden chamber assay, and reduced the MMP-2 and MMP-9 expressions and activity in SW620 cells. The protein relative levels of MMP-2 were 0.32±0.04 for SW620/p-shRNA, and 0.76±0.03 for SW620/p-Con; MMP-9 were 0.41±0.09 for SW620/p-shRNA, and 0.94±0.07 for SW620/p-Con (p<0.05). Conclusion: Inhibited Pin1 expression may contribute to the suppression of the invasive and metastatic capacity of colon cancer cells in vitro.

Cutting-Edge FAK-targeting PROTACs:design,synthesis,and biological evaluation

Focal adhesion kinase(FAK)is an intracellular tyrosine kinase that plays a critical role in the occurrence,development,and metastasis of cancer through both its kinase-dependent catalytic functions and kinase-independent scaffolding functions.Current kinase inhibitors target only its catalytic activity,leaving the scaffolding functions unaffected.However,proteolysis targeting chimeras(PROTACs)offers a promising approach by degrading the entire FAK protein,thereby inhibiting both functions simultaneously.In this study,we designed and synthesized novel PROTAC degraders,utilizing a defactinib derivative(compound 12)as the FAK ligand and a lenalidomide analog as the E3 ligase ligand.The structures of these compounds were confirmed through^(1)H NMR,^(13)C NMR,and high-resolution mass spectrometry(HRMS).Among the synthesized compounds,the optimized compound 16b exhibited potent degradation activity against FAK protein in A549 cells,with a DC_(50)of 6.16±1.13 n M,significantly inhibiting the proliferation and colony formation of these cells.Compared to defactinib,16b showed enhanced inhibition of A549 cell migration and invasion.Furthermore,our research demonstrated that the rapid and effective FAK degradation induced by 16b was mediated by a CRBN-dependent proteasome mechanism.

Slit2/Robo1 signaling in glioma migration and invasion

Slit2/Robo1 is a conserved ligand-receptor system,which greatly affects the distribution,migration,axon guidance and branching of neuron cells.Slit2 and its transmembrane receptor Robo1 have different distribution patterns in gliomas.The expression of Slit2 is at very low levels in pilocytic astrocytoma,fibrillary astrocytoma and glioblastoma,while Robo1 is highly expressed in different grades of gliomas at both mRNA and protein levels.Acquisition of insidious invasiveness by malignant glioma cells involves multiple genetic alterations in signaling pathways.Although the specific mechanisms of tumor-suppressive effect of Slit2/Robo1 have not been elucidated,it has been proved that Slit2/Robo1 signaling inhibits glioma cellmigration and invasion by inactivation of Cdc42-GTP.With the research development on the molecular mechanisms of Slit2/Robo1 signaling in glioma invasion and migration,Slit2/Robo1 signaling may become a potential target for glioma prevention and treatment.

组蛋白赖氨酸去甲基化酶4A对结直肠癌进展的影响

目的探讨组蛋白赖氨酸去甲基化酶4A(KDM4A)在结直肠癌发生发展中的作用及可能的机制。方法2019年1月至2019年6月,构建高(KDM4A)、低表达(ShKDM4A#1,ShKDM4A#2)KDM4A的结直肠癌细胞株(购自美国典型菌种保藏中心公司),同时设立对照组(NC,ShNC)。通过四甲基偶氮唑盐微量酶反应比色法(MTT)检测KDM4A对细胞增殖的影响。利用细胞侵袭小室法(Transwell)检测KDM4A对结直肠癌细胞迁移/侵袭能力的影响。通过蛋白质印迹法(Western blot)检测高、低表达KDM4A后细胞周期蛋白D1(Cyclin D1)和基质金属蛋白酶-9(MMP-9)的表达。两组间统计学差异通过双尾t检验进行分析。结果KDM4A高表达组与对照组比较,细胞增殖率明显提高[(50.233±3.430)%和(35.417±4.501)%,t=3.900、2.942,P<0.05]。低表达KDM4A与对照组比较,细胞增殖能力明显受抑制[(58.056±2.750)%和(57.386±8.228)%,t=4.558、4.419,P<0.05]。Transwell实验显示高表达KDM4A后穿孔细胞数[(304.000±62.466)个]比对照组[(64.000±20.833)个]增多(t=5.154,P<0.01);低表达KDM4A后穿孔细胞数[(47.333±13.573)个]较对照组[(93.667±18.874)个]减少(t=2.819,P<0.05)。KDM4A可以正向调节Cyclin D1和MMP-9的蛋白表达。结论KDM4A在结直肠肿瘤发生发展中起到重要作用,其机制可能是通过调节Cyclin D1和MMP-9的表达。

HER-2/EGFR, the major targets for anti-metastasis effect of tetraarsenic oxide on SKBR3 breast cancer cells

Breast cancer is one of the most common female malignant tumors in the world. Although many therapeutic methods for HER-2 positive breast cancer have been developed, the drug resistance and distant metastasis still remain. Tetraarsenic oxide（As_4O_6） has been demonstrated with an anticancer effect on squamous cell carcinoma and cervical cancer. However, there is no report about the relationship between As_4O_6 and HER-2 positive breast cancer. In the present study, we detected the inhibitory efficacy and mechanism of As_4O_6 on the migration and invasion of SKBR3 breast cancer cells using molecular biological methods. The wound-healing assay, matrigel migration assay, transwell invasion assay and cell adhesion assay were used to assess the migration, invasion and adhesion of SKBR3 cells intervened by As_4O_6. Meanwhile, the reverse transcription-PCR and western blotting were performed to investigate the mechanism of As_4O_6 on the migration and invasion of SKBR3 breast cancer cells. The results demonstrated that As_4O_6 could efficiently inhibit the migration and invasion of SKBR3 cells, the HER-2 positive breast cancer cells, and the adhesion of SKBR3 cells was decreased after As_4O_6 treatment. The mechanism revealed that As_4O_6 anticancer efficacy was related to HER-2/EGFR pathways. As_4O_6 exerted its inhibitory effects on migration and invasion in HER-2 positive breast cancer cells by regulating the factors（EGFR, HER-2, Akt, MMP-9） in HER2/ EGFR signaling pathway and other key molecules. In conclusion, the present study indicated that As_4O_6 inhibited the invasion and migration process of HER-2 positive breast cancer SKBR3 cells by negatively regulating the HER-2/EGFR-mediated signaling pathway. These data provided evidence that As_4O_6 might serve as potential anti-metastasis drug for clinical treatment of breast cancer.

Verticillin A inhibits colon cancer cell migration and invasion by targeting c-Met

Verticillin A is a diketopiperazine compound which was previously isolated from Amanita flavorubescens Aik(containing parasitic fungi Hypomyces hyalines(Schw.)Tul.).Here,we initially found,by wound healing assay and Tran swell assay in vitro,that verticilli n A possesses an inhibitory effect agai nst the migrati on and in vasion of the human colon cancer cell.Subsequently,c-mesenchymal,epithelial transition factor(c-Met)was identified as a molecular target of verticillin A by screening key genes related to cell migration.Verticillin A-mediated c-Met suppress!on is at the transcriptio nal level.Further study dem on strated that verticilli n A suppressed c-MET phosphorylation and decreased c-MET protein level.In addition,verticillin A inhibited the phosphorylation of c-MET downstream molecules including rat sarcoma(Ras)-associated factor(Raf),extracellular signal-regulated kinase(ERK),and protein kinase B(AKT).Overexpression of Erk partially reversed the verticillin A-mediated anti-metastasis action in the human colon cancer cell.More importantly,verticillin A also inhibited cancer cell metastasis in vivo.Thus,verticillin A can significantly inhibit the migration and invasion of colon cancer cells by targeting c-Met and inhibiting Ras/Raf/mitogen-activated extracellular signal-regulated kinase(MEK)/ERK signaling pathways.Therefore,we determined that verticillin A is a natural compound that can be further developed as an anti-metastatic drug in human cancers.