Inhibitory Effects of Parthenolide on the Activity of NF-κB in Multiple Myeloma via Targeting TRAF6

This study examined the mechanism of the inhibitory effect of parthenolide(PTL) on the activity of NF-κB in multiple myeloma(MM). Human multiple myeloma cell line RPMI 8226 cells were treated with or without different concentrations of PTL for various time periods, and then MTT assay was used to detect cell proliferation. Cell cycle and apoptosis were flow cytometrically detected. The level of protein ubiquitination was determined by using immunoprecipitation. Western blotting was employed to measure the level of total protein ubiquitination, the expression of IκB-α in cell plasma and the content of p65 in nucleus. The content of p65 in nucleus before and after PTL treatment was also examined with immunofluorescence. Exposure of RPMI 8226 cells to PTL attenuated the level of ubiquitinated Nemo, increased the expression of IκB-α and reduced the level of p65 in nucleus, finally leading to the decrease of the activity of NF-κB. PTL inhibited cell proliferation, induced apoptosis and blocked cell cycle. Furthermore, the levels of ubiquitinated tumor necrosis factor receptor-associated factor 6(TRAF6) and total proteins were decreased after PTL treatment. By using Autodock software package, we predicted that PTL could bind to TRAF6 directly and tightly. Taken together, our findings suggest that PTL inhibits the activation of NF-κB signaling pathway via directly binding with TRAF6, thereby suppressing MM cell proliferation and inducing apoptosis.     

Expression and biological function of N-myc down-regulated gene 1 in human cervical cancer

The expression of N-myc down-regulated gene 1 (NDRG1) has previously been reported to be involved in the proliferation,differentiation,invasion and metastasis of cancer cells,but its role in cervical cancer is still unclear.This study aimed to investigate the expression of NDRG1gene in human cervical cancer and its effect on aggressive tumor behaviors.The NDRG1 expression in cervical tissues and cells was detected by RT-PCR.Specific expression plasmid pEGFP-N1-NDRG1-GFP was used to enhance the expression of NDRG1 in human cervical cancer cell lines.The mRNA and protein level of NDRG1 was assessed by RT-PCR and Western blotting,respectively.Its effects on cell proliferation,migration,invasion,cell cycle and apoptosis were detected by MTT,transwell migration assay and flow cytometry (FCM),respectively.The results showed that the expression of NDRG1 in cervical cancer tissues and cells was significantly lower than in normal cervical tissues (P<0.001).After transfection with pEGFP-N1-NDRG1-GFP,the mRNA and protein expression of NDRG1 was up-regulated in Siha cells,which suppressed cell proliferation (P<0.001),induced cell cycle arrest (P<0.05),reduced invasion and migration of Siha cells (P<0.05),but caused no cell apoptosis.Moreover,vascular endothelial growth factor (VEGF),a tumor-induced angiogenesis factor,was markedly reduced and E-cadherin,a cell adhesion molecule,was increased in the cells transfected with pEGFP-N1-NDRG1-GFP.It was concluded that up-regulated NDRG1 may play a role in the suppression of malignant cell growth,invasion and metastasis of human cervical cancer.     

The role of NF-κB in hepatocellular carcinoma cell

Objective To evaluate the role of nuclear factor-kappaB (NF-κB) and IκBα in hepatocellular cacinoma (HCC) SMMC7721 cells, the consequence of NF-κB inhibition in SMMC7721 cells transfected with mutated IκBα (mIκBα) plasmid and the effect of stable inhibition of NF-κB activity in combination with Doxorubicin.Methods Western blot was used to determine the expression of NF-κB and IκBα in SMMC7721 cells and normal liver cells. Nuclear protein was used to evaluate the binding of the 32P-labeled tandem κB sequence using electrophoretic mobility shift assay and the expression of NF-κB using Western blot between SMMC7721 cells transfected with mIκBα plasmid (SMMC7721-MT) and control cells. Furthermore, cell viability was plotted between SMMC7721-MT and control cells. The binding of κB sequence and cell viability between SMMC7721-MT and control cells at different concentrations of Doxorubicin were also investigated.Results Western blot analysis for nuclear extract showed more P50 (NF-κB1) and P65 (RelA) expression in SMMC7721 cells compared with normal liver cells. The expression of cytosolic IκBα protein in SMMC7721 cells was less than that in normal cells. SMMC7721-MT cells inhibited NF-κB nuclear translocation at 0, 24, 48 and 96 hours. Furthermore, NF-κB cannot be detected in the nuclear protein of SMMC7721-MT cells by Western blot. By calculating cell viability, the proliferation of SMMC7721-MT cells was shown to be suppressed more significantly than that of control cells. NF-κB in untransfected cells was activated by Doxorubicin in a dose-dependent manner, but that in SMMC7721-MT cells was not induced at low concentrations of Doxorubicin. Compared with untransfected cells, the viability of SMMC7721-MT cells was significantly suppressed at the same concentration of Doxorubicin (P&lt;0.01）.Conclusions The present study demonstrates that upregulation of NF-κB and downregulation of inhibitory kappaB (IκBα) in SMMC7721 cells are related with the growth of hepatocellular cacinoma cells. Stable expression of mIκBα in SMMC7721-MT cells can inhibit NF-κB nuclear translocation and suppress cell growth. Furthermore, stable inhibition of NF-κB activity in combination with Doxorubicin can significantly inhibit cell proliferation in SMMC7721-MT cells. Thus, modulation of NF-κB may represent an improvement in the efficacy of HCC therapies and be worthy of further research and investigation.     

The Expression and Activity of MMPs Are Increased in Residual Tumor Tissues after the Termination of Immunotherapy

To investigate the invasive ability of the residual tumor cells after immunotherapy and explore the feasible approach suppressing the invasion, mice were inoculated with B16 cells, and then treated by gene therapy with p4-1BBL/psPD-1 or IFN-γ. The production and activities of MMP-9 and MMP-2 in residual tumor tissues were analyzed with gelatin zymography 1 day and 7 days after the termination of the immunotherapy. The production of MMP-9 and MMP-2 by B16 cells treated with IFN-γ was also analyzed. IFN-γ-treated B16 cells were inoculated to mice via subcutaneous injection. The invasion of tumor to muscular tissue was analyzed. Gene therapy with CH50 was used to suppress the invasive growth of tumor. The results showed that the expression and the activities of MMP-9 and MMP-2 were significantly increased 7 days after the end of immunotherapy. The re- sponse of tumor cells to ECM molecules was intensified after the removal of IFN-γ, resulting in significant increase of both the production and activities of MMP-9 and MMP-2, and the increased invasion of tumor. Gene therapy with CH50 effectively suppressed the invasive growth of tumor. It is concluded that the termination of immunotherapy may result in a higher metastatic potential of residual tumor cells. Suppressing tumor invasion by suitable treatment will improve the efficacy of immunotherapy..     

LPS-induced NF-κB activation requires Ca2+ as a mediator in isolated pancreatic acinar cells of rat

Objective To investigate the effect of Ca2+ on lipopolysaccharide (LPS)-induced NF-κB activation in pancreatic acinar cells and the role of NF-κB in LPS-induced acinar cell injury. Methods Male rat pancreatic acinar cells were isolated by collagenase digestion, then exposed to varying concentrations of LPS (from 1 to 20 mg/L) in the presence or absence of EGTA. At various time points (30 minutes, 1 hour, 2 hours, 4 hours and 10 hours) after treatment with the agents, cell viability was determined by MTT. Nuclear translocation of NF-κB’s subunit p65 was visualized by immunofluorescence staining and nuclei protein was extracted to perform EMSA which was used to assay the activity of NF-κB binding to the DNA sequence containing the recognition site of NF-κB. Results LPS induced cell damage in a time- and concentration-dependent manner while EGTA attenuated LPS-induced cell damage (P&lt;0.05). NF-κB p65 immunofluorescence staining had increased intensity in the cytoplasm and indicated that nuclear translocation occurred within 30 minutes and its zenith was reached at 1 hour after LPS (10 mg/L) treatment. Testing of NF-κB DNA binding activity showed the same alteration phase as p65 immunofluorescence staining. NF-κB activation preceded the pathological alteration of pancreatic acinar cells. The Ca2+ chelator EGTA inhibited LPS-induced NF-κB activation. Conclusions NF-κB activation is an important early event in LPS-induced injury to pancreatic acinar cells. Ca2+ is an important mediator in the process of LPS-induced NF-κB activation.     

Inhibitory Activity of Nuclear Factor-κB Potentiates Cisplatin-induced Apoptosis in A549 Cells

Whether inhibiting the activity of nuclear factor(NF)-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated.The recombinant plasmid pcDNA3.1( )/IκBα expressing IκBα was constructed.The in vitro cultured A549 cells were transfected with pcDNA3.1( )/IκBα alone,or pcDNA3.1( )/IκBα combined with cisplatin.The mito-chondrial membrane potential(?ψm) was determined by rhodamine 123,the activity of caspase-3 was tested by colorimetric assay,and cell apoptosis was detected by flow cytometry with the annexin Ⅴ/propidium iodide assay.The results showed that the activity of NF-κΒ in A549 cells was inhibited by transfecting pcDNA3.1( )/IκΒα.Transfection of pcDNA3.1( )/IκΒα alone did not promote apoptosis.Treatment of cisplatin alone had a little effect on cell apoptosis.Transfection of pcDNA3.1( )/IκΒα combined with cisplatin treatment significantly induced apoptosis of A549 cells.It was concluded that inhibiting the activity of NF-κB potentiated cisplatin-induced apoptosis of A549 cells.     

Inhibitory Activity of Nuclear Factor-κB Potentiates Cisplatin-induced Apoptosis in A549 Cells

Whether inhibiting the activity of nuclear factor (NF)-κB potentiates cisplatin-induced apoptosis in non-small cell lung cell line A549 cells was investigated. The recombinant plasmid pcDNA3.1( )/IκBα expressing IκBα was constructed. The in vitro cultured A549 cells were trans-fected with pcDNA3.1( )/IκBα alone, or pcDNA3.1( )/IκBα combined with cisplatin. The mitochondrial membrane potential (△ψm) was determined by rhodamine 123, the activity of caspase-3 was tested by colorimetric assay, and cell apoptosis was detected by flow cytometry with the annexin V/propidium iodide assay. The results showed that the activity of NF-κB in A549 cells was inhibited by transfecting pcDNA3.1( )/IκBα. Transfection of pcDNA3.1( )/IκBα alone did not promote apoptosis. Treatment of cisplatin alone had a little effect on cell apoptosis. Transfection of pcDNA3.1( )/IκBα combined with cisplatin treatment significantly induced apoptosis of A549 cells. It was concluded that inhibiting the activity of NF-κB potentiated cisplatin-induced apoptosis of A549 cells.     

人涎腺腺样囊性癌细胞系中RUNX3基因5'-CpG岛甲基化及蛋白表达

目的 探讨人涎腺腺样囊性癌细胞株中RUNX3基因启动子5'-CpG岛甲基化及其表达情况.方法 以定量甲基化特异性PCR(qMSP)检测ACC-2、ACC-3和ACC-M细胞在甲基转移酶抑制剂5-氮脱氧胞苷(5-Aza-dc)处理前后RUNX3基因5'-CpG岛甲基化状况,并分别用反转录-聚合酶链反应、免疫荧光化学和Western印迹法分析RUNX3的表达变化.结果 RUNX3在ACC-2和ACC-3细胞中弱表达,在ACC-M中表达缺失.经5-Aza-dc处理后,RUNX3在ACC-2和ACC-3细胞中表达增加,在ACC-M中恢复表达.激光共聚焦发现RUNX3蛋白主要定位在细胞质中,经300 nmol/L5-Aza-dc处理72 h后,在ACC-2和ACC-3细胞核中出现表达,而在ACC-M细胞中仍定位在细胞质中.RUNX3基因启动子区域5'-CpG岛在ACC-2、ACC-M和ACC-3中均为部分甲基化,其甲基化程度分别为50%、75%和33%.5-Aza-dc处理后,RUNX3基因在三株细胞株中均呈现为非甲基化状态.结论 RUNX3甲基化是ACCs中RUNX3表达缺失的主要原因之一,而RUNX3蛋白在ACCs细胞质中的异位表达,也可能与RUNX3蛋白的功能被抑制有关.     

NGF在涎腺腺样囊性癌中的表达及与嗜神经侵袭和疼痛的关系

目的:观察神经生长因子(nerve growth factor,NGF)在涎腺腺样囊性癌(adenoid cystic carcinoma,ACC)的表达情况,探讨NGF与ACC嗜神经侵袭情况及疼痛症状的关系.方法: 以32例ACC标本为研究对象,患者按照有无嗜神经侵袭及疼痛症状进行分组;以5例正常腮腺及3例腺泡细胞癌作为对照,采用免疫组化法对NGF进行检测.结果: NGF在正常腮腺的导管细胞表达为阳性,在腺泡细胞癌为阴性.在ACC中的阳性率为96.9%(31/32),且发现沿神经周围分布的肿瘤细胞染色强度明显高于远离神经者.NGF表达水平的组间对比:存在嗜神经现象组明显高于未见嗜神经现象组(P<0.05),存在疼痛症状组亦显著高于无疼痛症状组(P<0.05).结论: ACC中NGF表达的增高可能是促进其嗜神经侵袭及疼痛症状发生的原因之一.     

沉默WNT4基因对稳定转染PAX2基因大鼠肾小管上皮细胞迁移和侵袭能力的影响

目的：探讨沉默WNT4基因对稳定转染配对盒基因2（PAX2）大鼠肾小管上皮细胞迁移和侵袭能力的影响,阐明PAX2基因是否通过WNT4基因调控细胞生物学活性。方法：选取对数生长期的PAX2基因稳转细胞系,分为稳转组和对照组。稳转组细胞分为稳转对照组（不应用WNTsiRNA沉默）和沉默72 h组（应用WNTsiRNA沉默72h）。Western blotting法检测稳转组和对照组大鼠肾小管上皮细胞中PAX2和WNT4蛋白的相对表达量,Realtime PCR法检测稳转对照组和沉默72 h组大鼠肾小管上皮细胞中WNT4 mRNA相对表达量,细胞划痕实验检测稳转对照组和沉默72 h组大鼠肾小管上皮细胞的细胞迁移率,Transwell实验检测稳转对照组和沉默72 h组大鼠肾小管上皮细胞的穿膜细胞数。结果：Western blotting法检测,稳转组大鼠肾小管上皮细胞中PAX2和WNT4蛋白相对表达量高于对照组（P<0.05）。Real-time PCR法检测,沉默72 h组大鼠肾小管上皮细胞中WNT4 mRNA相对表达量低于稳转对照组（P<0.05）。细胞划痕实验10 h后,沉默72 h组大鼠肾小管上皮细胞的细胞迁移率低于稳转对照组,但组间比较差异无统计学意义（P>0.05）;细胞划痕实验18 h后,沉默72h组大鼠肾小管上皮细胞的细胞迁移率低于稳转对照组（P>0.05）。Transwwell实验,沉默72 h组大鼠肾小管上皮细胞的穿膜细胞数低于稳转对照组（P<0.05）。结论：PAX2基因可能通过WNT4基因调控细胞的生物学活性。     

SIRPα-GFP真核表达载体构建及其在HEK293T细胞中的表达

目的：构建信号调节蛋白α-绿色荧光蛋白（SIRPα-GFP）真核表达载体,转染HEK293T细胞获得稳定表达SIRPα-GFP融合蛋白的细胞系,研究SIRPα-GFP融合蛋白在HEK293T细胞的膜定位。方法：将SIRPα质粒和带有GFP的表达载体分别用限制性内切酶MluⅠ和SgfⅠ进行双酶切,将SIRPα基因克隆到pLenti-GFP载体中,构建pLenti-SIRPα-GFP质粒;将pLenti-SIRPα-GFP质粒转化DH5α大肠杆菌感受态细胞,对重组质粒pLenti-SIRPα-GFP进行DNA测序;采用Lipofectamine 3000转染试剂将pLenti-SIRPα-GFP质粒转染到HEK293T细胞中,通过荧光显微镜观察SIRPα-GFP在稳定转染HEK293T细胞中的表达,采用Western blotting法检测HEK293T细胞中SIRPα-GFP融合蛋白的表达。结果：酶切鉴定和DNA测序,重组质粒pLenti-SIRPα-GFP构建成功。荧光显微镜检测,SIRPα-GFP融合蛋白在HEK293T细胞膜上表达。Western blotting法检测,SIRPα蛋白在pLenti-SIRPα-GFP质粒转染的HEK293T细胞中成功表达。结论：成功构建了pLenti-SIRPα-GFP质粒。通过在HEK293T细胞中转染pLenti-SIRPα-GFP质粒,成功制备了稳定表达SIRPα-GFP的细胞系。SIRPα-GFP蛋白定位于HEK293T细胞的细胞膜上。     

稳定沉默CXCR4表达的肾癌A498细胞株的构建

目的应用RNA干扰技术降低肾癌A498细胞株的CXCR4基因表达水平,并建立稳定转染细胞株。方法针对CXCR4基因设计合成siRNA,转染肾癌A498细胞株,采用RT-PCR法检测CXCR4基因表达变化,根据有效干扰片段结果合成重组shRNA质粒,稳定转染至A498细胞系并进行G418抗性筛选细胞株。采用RT-PCR和Westen印迹法检测CXCR4mRNA及蛋白表达水平,应用流式细胞技术检测CXCR4shRNA诱导A498细胞凋亡的效应,Transwell试验检测细胞侵袭能力的改变。结果将CXCR4重组shRNA质粒成功转染A498细胞后,肾癌细胞内可见绿色荧光,通过G418筛选,获得了CXCR4基因沉默的A498细胞系,RT-PCR及Western印迹检测证实该细胞系的CXCR4水平明显低于对照组的表达水平(P<0.05)。CXCR4shRNA组细胞的早期凋亡率、晚期凋亡率及总凋亡率均显著高于对照组(P<0.05),Tran-swell体外侵袭实验显示CXCR4shRNA能明显抑制A498细胞的体外侵袭力(P<0.05)。结论成功构建稳定沉默CXCR4表达的肾癌A498细胞株,转染后的细胞凋亡率升高,体外侵袭能力降低,为后续研究奠定了基础。     

人体涎腺肿瘤细胞端粒酶活性检测

目的 :探讨端粒酶在涎腺肿瘤发生、发展中的作用 ,为早期诊断和开展涎腺肿瘤的基因治疗奠定实验基础。方法 :用PCR TRAP法 ,聚丙烯酰胺凝胶电泳及银染色法分析PCR产物 ,对人体涎腺腺样囊性癌细胞株ACC M和 2 0例涎腺肿瘤组织的端粒酶活性进行检测 ,并与自身正常涎腺组织相对照。结果 :腺样囊性癌细胞株ACC M的端粒酶活性为阳性 ,相对端粒酶活性为 82 % ,涎腺恶性肿瘤端粒酶活性阳性率最高 (14/ 15 ) ,瘤旁组织次之 (4/ 2 0 ) ,正常对照及良性肿瘤组织最低 (2 / 2 5 )。其相对端粒酶活性 ,涎腺鳞癌端粒酶活性值最高 (87% ) ,涎腺恶性肿瘤明显高于自身正常对照和瘤旁组织以及良性肿瘤 (P <0 .0 1) ,而瘤旁组织也高于相邻的正常组织 (P <0 .0 5 )。结论 :端粒酶可作为涎腺恶性肿瘤诊断的分子指标 ,也可作为阻断恶性转化形成的靶分子     

小干扰RNA沉默组织因子基因对肝癌侵袭转移能力的影响

目的 应用RNA干扰(RNAi)技术抑制人类肝癌细胞内组织因子(TF)基因表达,从而探讨对其侵袭转移能力的影响。方法 本实验以人类肝癌细胞系HepG2细胞株为实验对象,将携带有针对TF小干扰RNA( siRNA)序列的质粒pGPU6/GFP/Neo-TF siRNA,转染HepG2细胞株,以适当浓度的G418筛选获得阳性克隆,通过RT-PCR和蛋白质印迹法比较转染TF siRNA(+)质粒、转染TF siRNA( -)质粒以及未转染任何质粒的HepG2细胞内源性TF在基因及蛋白质表达水平的差异,进而应用Transwell体外侵袭实验检测三者的侵袭转移能力,探讨TF对肿瘤细胞侵袭转移能力的影响。结果 (1) RT-PCR结果 显示,转染TF siRNA(+)质粒的HepG2细胞其内源性TF表达水平低于转染TF siRNA（-）质粒和未转染质粒的表达水平。(2)蛋白质Western印迹法结果 显示,转染TFsiRNA(+)质粒的HepG2细胞其内源性TF蛋白表达水平低于转染TF siRNA( -)质粒和未转染质粒的表达水平。(3) Transwell体外侵袭实验结果 显示,转染TF siRNA(+)质粒的HepG2穿膜细胞数[(14±10)个]少于转染TF siRNA（-）质粒的HepG2穿膜细胞数[(128±18)个],经t检验比较,两组差异有统计学意义（P＜0.05）。这提示应用RNA干扰技术抑制肿瘤细胞TF表达可以使HepG2细胞侵袭转移能力显著下降。结论 TF与肝癌细胞的侵袭转移能力密切相关,应用RNA干扰技术抑制其表达可以明显降低细胞HepG2体外侵袭转移能力。