肿瘤坏死因子相关凋亡诱导配体受体在肾癌中的分布及其意义

目的：研究肿瘤坏死因子相关诱导凋亡配体（TRAIL）受体在肾癌组织中的分布及其意义。方法：采用RT－PCR及NorthernBlot的方法检测TRAIL受体在肾癌组织及正常肾组织,肾癌细胞系GRC－I和正常肾小管细胞系HK－2中的表达。结果：死亡受体DR4、DT4在肾癌组织及正常肾组织、肾癌细胞系GRC－I和正常肾小管细胞系HK－2中强表达;假受体DcR-1在正常肾组织和正常肾小管细胞系HK－2中强表达;假受体DcR-2未见表达。结论TRAIL基因在肾癌肿瘤细胞的凋亡机制中可能发挥重要的作用。     

肿瘤坏死因子相关凋亡诱导配体联合化疗药物杀伤胰腺癌细胞的研究

目的研究肿瘤坏死因子相关凋亡诱导配体以及联合应用亚毒性剂量的化疗药物对胰腺癌的治疗作用。方法半定量 RT-PCR 检测 TRAILR mRNA 在胰腺癌细胞株 Canpan-2中的表达。DNA 琼脂糖凝胶电泳检测细胞凋亡情况。应用不同浓度的 TRAIL 及联合亚毒性剂量的氟尿嘧啶(5-fluorouracil)、吉西他滨(gemcitabin)处理胰腺癌细胞,通过 MTT 法检测细胞毒性作用,流式细胞仪分析细胞凋亡率。结果死亡受体 DR4、DR5及诱骗受体 DcR1、DcR2在胰腺癌细胞株 Canpan-2中均有表达。DNA 琼脂糖凝胶电泳可见到典型的凋亡梯形带。TRAIL100 ng/ml 作用胰腺癌细胞24h 后,细胞杀伤率为(29.5±1.2)%,且其作用存在浓度依赖性;联合应用亚毒性剂量的氟尿嘧啶、吉西化滨能够大大提高TRAIL 的细胞毒活性,杀伤率分别为 TRAIL+氟尿嘧啶(43.7±1.4)%,TRAIL+吉西化滨(49.8±1.2)%,联合用药前后差异有显著性(P<0.01)。结论 TRAIL 受体在胰腺癌细胞普遍表达,并存在受体类型的表达差异;TRAIL 与化疗药物氟尿嘧啶、吉西他滨有协同杀伤胰腺癌细胞的作用。     

胡黄连苷Ⅱ通过线粒体途径诱导肾癌细胞凋亡的实验研究

目的研究胡黄连苷Ⅱ(picrosideⅡ)对人肾癌ACHN细胞凋亡的影响及作用机制。方法将体外培养的肾癌ACHN细胞分为对照组、不同浓度胡黄连苷Ⅱ(1μg/mL、10μg/mL及100μg/mL)组,采用CCK-8法测定细胞增殖率,采用Hoechst 33258核染色法和Annexin V/PI检测细胞凋亡,采用Western blot法检测肾癌ACHN细胞内Bax及Bcl-2蛋白表达。结果不同浓度的胡黄连苷Ⅱ能抑制肾癌ACHN细胞生长并诱导细胞凋亡,随着胡黄连苷Ⅱ作用浓度的递增,肾癌ACHN细胞凋亡率呈剂量依赖性递增;胡黄连苷Ⅱ作用后,Bax蛋白表达增加,Bcl-2蛋白表达下降。结论胡黄连苷Ⅱ通过改变线粒体凋亡途径中凋亡相关信号分子Bax及Bcl-2的表达,抑制肾癌ACHN细胞增殖并促进细胞凋亡。     

姜黄素抑制肾癌ACHN细胞增殖及促凋亡的实验研究

目的观察姜黄素对人肾癌ACHN细胞株增殖及细胞凋亡的影响,探讨姜黄素诱导ACHN细胞株凋亡的作用机制。方法不同浓度姜黄素作用人肾癌ACHN细胞24 h后,应用MTT比色法检测姜黄素对人肾癌ACHN细胞的增殖抑制率;流式细胞术检测姜黄素诱导细胞的凋亡率;RT-PCR检测姜黄素对ACHN细胞Bcl-2、Bax、NF-κBP65 mRNA表达的影响;Western blot方法检测其对细胞Bcl-2、Bax、NF-κBP65I、κB蛋白表达的影响。结果姜黄素对人肾癌ACHN细胞有明显的抑制作用,可引起细胞凋亡,并且存在剂量和时间依赖;不同浓度姜黄素作用细胞24 h后,Bcl-2、NF-κBP65 mRNA水平下降,Bax mRNA水平升高(P0.05),Bcl-2、NF-κBP65蛋白表达量下降,BaxI、κB蛋白表达量升高(P0.05)。结论姜黄素通过上调IκB,下调NF-κB活性,调控凋亡基因Bcl-2/Bax的表达,抑制人肾癌ACHN细胞的增殖,诱导人肾癌ACHN细胞的凋亡。     

线粒体途径在肿瘤坏死因子凋亡诱导配体诱导结肠癌细胞凋亡中的调节作用

目的探讨线粒体途径在肿瘤坏死因子凋亡诱导配体(TRAIL)诱导结肠癌细胞凋亡过程中的调节作用,为临床合理用药提供理论指导。方法采用流式细胞仪技术、荧光显色技术和Western印迹技术检测TRAIL处理结肠癌细胞SW1116后,在不同时间细胞凋亡情况、线粒体完整性改变(ΔΨm、cardiolipin情况)以及线粒体下游通路细胞色素C和Caspase-9的表达情况。结果TRAIL诱发结肠癌细胞凋亡,于4 h达凋亡高峰,凋亡指数为32．98%；在4 h出现线粒体ΔΨm下降和cardiolipin丢失增加,造成其内膜损伤；细胞色素C表达及Caspase-9酶活性随时间的延长而增加,24 h酶活性达到最大峰值为(48．12±2．21)μmol·L~(-1)·h~(-1)·mg~(-1)蛋白。TRAIL诱导的线粒体损伤可被Caspase抑制剂Z-VAD．fmk所抑制。结论线粒体途径参与TRAIL诱导结肠癌细胞的凋亡过程,以Caspase依赖方式引发线粒体ΔΨm和cardiolipin丢失,造成内膜损伤,导致细胞色素C释放和Caspase-9激活,诱发凋亡。     

肿瘤坏死因子凋亡诱导配体诱导结肠癌细胞凋亡的机制

目的探讨肿瘤坏死因子凋亡诱导配体(TRAIL)诱导结肠癌细胞发生凋亡的机制。方法流式细胞仪技术、酶联免疫吸附技术、荧光显色技术检测500μg／L TRAIL和／或Caspase抑制剂Z-VAD．fmk处理后,结肠癌细胞SW1116在不同时点(0、2、4、6、24 h)凋亡情况、Caspase-3酶活性及4 h线粒体△ψm、cardiolipin、细胞ROS变化。结果 TRAIL可引起结肠癌细胞凋亡,并于 4 h达凋亡高峰,凋亡指数为32．98％;并出现线粒体△ψm下降、cardiolipin丢失增加及Caspase-3酶活性增加,4 h达到最大峰值为(37．56±2．572)μmol／L·hr-1·mg-1蛋白。但TRAIL所诱导的细胞凋亡作用可被Caspase抑制剂Z-VAD．fmk所抑制。同时证实TRAIL所诱导的结肠癌细胞凋亡与细胞氧自由ROS的生成无关。结论 TRAIL通过Caspase依赖方式引起线粒体△ψm下降、cardi- olipin丢失增加导致线粒体内膜损伤,从而诱导细胞发生凋亡。     

TRALL联合IFN-γ诱导骨肉瘤细胞凋亡实验研究

目的观察干扰素(IFN)-γ协同肿瘤坏死因子相关凋亡诱导配体(TRAIL)诱导骨肉瘤细胞(MG-63)凋亡的作用,初步探讨协同作用机制,并观察联合用药时对成纤维细胞的毒性作用。方法应用相差显微镜和电镜观察MG-63细胞经TRAIL、IFN-γ及TRAIL联合IFN-γ作用后细胞形态学变化;进行四唑盐比色试验观察不同实验组MG-63细胞和成纤维细胞抑制率;流式细胞仪观察细胞凋亡情况;逆转录-聚合酶链反应法观察IFN-γ作用后TRAIL膜受体表达情况。结果TRAIL联合IFN-γ应用时,随着IFN-γ作用时间的延长,细胞悬浮、空泡化等现象逐渐明显,且在作用24小时最为显著,电镜显示细胞凋亡的特征性变化;联合用药组细胞抑制率较单独TRAIL组增加,两者有显著性差异(P<0.01);联合用药组细胞凋亡率显著增加(P<0.01);应用IFN-γ前后,TRAIL死亡受体(DR)4的相对表达量增加,IFN-γ作用24小时表达量最大,两者有显著性差异(P<0.01);联合用药组对成纤维细胞的毒性,与单独TRAIL组相比没有显著增加(P>0.05)。结论IFN-γ可协同TRAIL诱导MG-63细胞凋亡,DR4表达量增加可能是协同作用的缘故。联合用药不会增加对成纤维细胞的毒副反应。     

血管新生抑制剂TNP-470对鼠ACHN肾细胞癌增殖与凋亡的影响

目的 :探讨血管新生抑制剂TNP 4 70对ACHN肾细胞癌增殖与凋亡状态的影响。方法 :采用TNP 4 70 (4 0mg·kg-1·0 .2ml-1)皮下注射治疗ACHN肾细胞癌荷瘤裸鼠 ,观察肿瘤生长情况 ,进行细胞增殖与细胞凋亡的研究。结果 :TNP 4 70治疗组与对照组相比 ,ACHN肾癌生长明显减慢 ,肿瘤细胞凋亡指数明显增加 (P <0 .0 1) ,增殖指数无显著变化 (P >0 .0 5 )。肿瘤体积与凋亡指数间存在负相关 (r =- 0 .85 4 0 ,P <0 .0 1)。结论 :TNP 4 70对ACHN肾癌有显著的抑制肿瘤生长作用 ,可能与肾癌细胞凋亡增加有关     

肿瘤相关凋亡诱导配体、Survivin、Caspase-3和NF-κB在大肠癌中的表达

肿瘤相关凋亡诱导配体（TRAIL）与FasL有较高的同源性，其选择性地诱导肿瘤细胞发生凋亡，从而使正常细胞逃逸其杀伤作用。有研究结果表明，许多肿瘤对TRAIL并不敏感．Sunivin、NF-κB和Caspase．3是TRAIL诱导细胞凋亡通路中三个关键性的影响因子。我们通过其在大肠癌中的表达的研究以期阐明TRAIL的效能与Survivin、Caspase．3和NF-κB表达的关系。     

Caspase-8和Fas抗原调控化疗诱导的人肝癌细胞凋亡

目的　探讨Fas抗原和Caspase 8在化疗诱导人肝癌细胞凋亡中的调控作用。方法　用 1× 10 -2 mol/L浓度的氟尿嘧啶处理HepG2细胞 ,分别作用 4、8、16、2 4h。免疫细胞化学法检测Fas抗原的表达。用荧光试剂盒检测Caspase 8的活性。流式细胞仪检测氟尿嘧啶或抗 Fas 抗体诱导的肝癌细胞凋亡百分率 ,以及加入Caspase 8活性抑制剂IETD FMK后凋亡百分率的变化。 结果　氟尿嘧啶诱导HepG2细胞凋亡后 ,与对照组比较 ,Fas抗原表达强度增加 (P <0 0 1) ,Caspase 8活性升高 (P <0 0 1)。Fas抗原表达和Caspase 8活性随着氟尿嘧啶作用时间的延长而逐步升高 ,至 16h后达到高峰 ,然后下降 ,但仍显著高于对照组 (P <0 0 1)。Fas抗原的表达与Caspase 8活性变化呈显著正相关 (r =0 96 9,P <0 0 1)。表达增强的Fas抗原具有转导凋亡信号的功能 ,借此抗 Fas 抗体增强了氟尿嘧啶诱导的HepG2细胞凋亡。Caspase 8活性抑制剂IETD FMK能阻断Caspase 8活化而抑制氟尿嘧啶或抗 Fas 抗体诱导的HepG2细胞凋亡 ,实验组和抑制剂组比较 ,细胞凋亡百分率有显著性差异 (P <0 0 1)。结论　氟尿嘧啶诱导HepG2细胞经Fas依赖途径凋亡。Caspase 8活性上调在该凋亡过程中发挥重要作用。     

Sensitization of human renal cell carcinoma cell lines to TRAIL-induced apoptosis by anthracyclines

BACKGROUND: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new member of the tumor necrosis factor family. The present study investigated whether anthracyclines enhance TRAIL-induced apoptosis and cytotoxicity in renal cell carcinoma (RCC) cells. METHODS: Cytotoxicity was measured using the microtiter assay. Apoptosis was monitored using DNA ladder analysis. Caspase activity was determined using a quantitative colorimetric assay. RESULTS: Treatment of ACHN and Caki-1 human RCC lines with TRAIL, in combination with subtoxic concentrations of epirubicin (EPI) or pirarubicin (THP), enhanced induction of apoptosis and cytotoxicity. Sequential treatment with EPI followed by TRAIL induced significantly more cytotoxicity than the inverse treatment. The combined cytotoxicity of TRAIL and EPI was significantly inhibited by the TRAIL-neutralizing fusion protein DR5:Fc, although EPI did not affect the mRNA expression of DR4, DR5, DcR1 or DcR2. The combination treatment with TRAIL and EPI activated caspase-6 and -3, which were downstream molecules of the death receptor. Furthermore, the combined cytotoxicity of TRAIL and EPI was almost completely inhibited by Z-VAD-FMK, and partly inhibited by Ac-DMQD-CHO. CONCLUSION: These findings indicate that anthracyclines sensitize RCC cells to TRAIL-induced apoptosis and cytotoxicity through activation of caspases, suggesting that TRAIL, in combination with anthracyclines, has a therapeutic potential in the treatment of RCC.     

Enhancement of death receptor 4 mediated apoptosis and cytotoxicity in renal cell carcinoma cells by subtoxic concentrations of doxorubicin

PURPOSE: TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) triggers apoptosis in various tumor cells by engaging death receptors 4 and 5. We investigated the effect of chemotherapeutic agents on death receptor 4 mediated apoptosis in human renal cell carcinoma cells using HGS-ETR1, which is a human monoclonal agonistic antibody specific for death receptor 4. MATERIALS AND METHODS: Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Synergy was assessed by isobolographic analysis. RESULTS: Treatment of the ACHN human renal cell carcinoma cell line with HGS-ETR1 combined with 5-fluorouracil, vinblastine or gemcitabine did not overcome resistance to these agents. However, treatment with HGS-ETR1 combined with doxorubicin had a synergistic cytotoxic effect. Synergy was also achieved in another human renal cell carcinoma cell line, Caki-1, and in 5 freshly derived renal cell carcinoma cell cultures. A synergistic effect was also observed with HGS-ETR1 combined with the doxorubicin derivatives epirubicin, pirarubicin or amrubicin. The synergy achieved in cytotoxicity with HGS-ETR1 and doxorubicin was also achieved in apoptosis. Sequential treatment with doxorubicin followed by HGS-ETR1 induced significantly more cytotoxicity than reverse treatment or simultaneous treatment (p<0.05). Doxorubicin remarkably increased the cell surface expression of death receptor 4 in renal cell carcinoma cells. The combination of doxorubicin and HGS-ETR1 significantly activated the caspase cascade, including caspase-8, 9, 6 and 3, which are the downstream molecules of death receptors. CONCLUSIONS: These findings indicate that doxorubicin sensitizes renal cell carcinoma cells to death receptor 4 mediated apoptosis through the induction of death receptor 4 and the activation of caspases, suggesting that combination therapy of doxorubicin and HGS-ETR1 might be effective as renal cell carcinoma therapy.     

Verotoxin induces rapid elimination of human renal tumor xenografts in SCID mice

PURPOSE: Verotoxins (VTs) are subunit toxins produced by enteropathogenic Escherichia coli. The VT receptor glycolipid Gb3, which mediates the cytotoxicity of VTs, has been reported to be elevated on the surface of several tumor cell lines. In this study the effect of VT1 as an antineoplastic agent was assessed using various human urological cancer cell lines. MATERIALS AND METHODS: The expression of Gb3 on human cancer cell lines originating from renal cell carcinoma (ACHN, A-704, CAKI-1 and CAKI- 2), prostate cancer (LNCaP and PC3) and testicular tumor (2102Ep) were examined by FACScan (Becton Dickinson, Sunnyvale, California). These cell lines were cultured with various concentrations of VT1 and subjected to microculture tetrazolium dye assay for determination of cell viability. Furthermore, ACHN cells were inoculated into the backs of SCID mice and intratumor injection of VT1 was performed. Pathological samples were examined by hematoxylin and eosin staining as well as by TUNEL assay. RESULTS: The growth of ACHN, CAKI-1, A-704, 2102Ep and LNCaP but not CAKI-2 and PC3 was significantly inhibited by co-incubation with VT1, as determined by microculture tetrazolium dye assays, consistent with FACScan results for Gb3 expression. When mice bearing ACHN tumors were injected with VT1, rapid reduction in the size of subcutaneous tumors was observed with complete regression within 5 to 7 days. Pathological examination by the TUNEL method indicated that the cytotoxicity of VT1 was mediated by apoptosis. CONCLUSIONS: These results suggest that VTs could be candidates for antineoplastic agents against Gb3 expressing tumors for clinical use.     

Lysis of autologous tumor cells by peripheral blood lymphocytes treated with IFN-alpha in patients with renal cell carcinoma]

Immunotherapy, consisting mainly of interferon (IFN) therapy, has been used to treat renal cell carcinoma refractory to radiotherapy and chemotherapy ever since IFN was reported to be effective against renal cell carcinoma in 1982. The efficacy of IFN is low, with the response rate being only about 20% and the method and duration of its administration have yet to be established. Using viable tumor cells obtained at nephrectomy, we discovered that IFN-alpha induced killer cells have a cytocidal effect on autologous renal cell carcinoma cells and that assaying the cellular immunity of the peripheral blood lymphocytes of 25 patients with renal cell carcinoma, using ACHN derived from human renal cell carcinoma as the target cells, is useful as a monitoring method during IFN-alpha therapy. Augmentation of the cytotoxicity of peripheral blood lymphocytes in response to administration of IFN-alpha was observed in all 25 cases. Cytotoxicity was activated to 5-426 LU compared with the control value of 1 LU or less before IFN therapy, when autologous renal cell carcinoma cells were used as the target cells. Cytotoxicity for ACHN cells in the control value before IFN-alpha therapy and 4 weeks after the institution of IFN-alpha administration and autologous tumor lysis in the induction assay were more strongly correlated than in the case of K562 cells. A strong correlation was also found between cytotoxicity for ACHN cells in the induction assay and 4 weeks after the institution of IFN-alpha administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in cell kinetic changes among renal cancer cell lines treated with interferon-α

BACKGROUND: Interferon (IFN)-alpha shows certain clinical effects on the treatment of renal cell carcinoma. The purpose of the present study was to investigate its direct effects and to compare the responses among different human renal cancer cell lines. METHODS: Three cell lines, ACHN, RCC10RGB and OS-RC-2, were incubated with IFN-alpha and evaluated using MTT assay for cell proliferation and two-color flow cytometry for cell-cycle-specific cyclin expressions coupled with DNA ploidy analysis. RESULTS: Interferon-alpha inhibited cell proliferation and caused cell accumulation at S and G2/M phases. However, IFN-alpha induced no significant change in cyclins D1, E, A or B1 expression. Interestingly, cell kinetic changes caused by IFN-alpha were different among cell lines. Cell proliferation was suppressed most in ACHN, then RCC10RGB and least in OS-RC-2. Comparing DNA histograms, ACHN showed distinct increase of G2/M cells associated with elevation of late S cells. RCC10RGB showed a predominant increase of whole S cells accompanied with a slight increase of G2/M. OS-RC-2 showed a modest increase of S cells with a little change of G2/M cells. Chronological observation revealed that S-phase increase and proliferative inhibition appeared on day 1 and day 3, respectively, in ACHN and RCC10RGB, and on day 5 in OS-RC-2. CONCLUSIONS: Interferon-alpha induced substantial cell kinetic interference directly in the tested human renal carcinoma cell lines. The degree of change was different according to the nature of the cell line. It may partly indicate the variety of the efficacy of IFN-alpha treatment against renal cancers.     

Prominent induction of cyclin B1 in G2/M renal cancer cells with butyrolactone 1

BACKGROUND: Butyrolactone 1 (BL) is a cyclin dependent kinase (CDK) inhibitor derived from Aspergillus terreus. None of the present drugs are effective for the treatment of renal cell carcinoma. The use of BL is expected to promote a new type therapy of renal cancer. METHODS: We investigated three human renal cancer cell lines: ACHN, OS-RC-2 and RCC10RGB, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and two-color flow cytometry. Simultaneous measurements of DNA content and cyclin expression allowed us to perform cell cycle specific analysis. Western blot analysis was performed using ACHN to represent cell lines. RESULTS: BL inhibited cell proliferation and caused cell accumulation at G2/M phase associated with the emergence of the third peak. Moreover, BL induced cyclin B1 over-expression in G2/M cells. These changes were quite definite, whereas cyclins D1, E and A showed no changes at all. Cyclin B1 accumulation was confirmed by western blot analysis. The chronological observation revealed that the emergence of the third peak preceded the regression of the increased cyclin B1 positive G2/M cells. These results suggested that BL accelerated cyclin B1 accumulation in G2/M cells, which then shifted to G1 phase without cell division. New G1 cells started DNA synthesis most likely as endoreduplication to form the third peak and the mechanism of cyclin B1 accumulation converted into down-regulation. CONCLUSION: BL induced significant cell kinetic interference in the tested human renal carcinoma cell lines. This might indicate the possibility of a new medical treatment modality for renal cancer.     

siRNA沉默VEGF基因对肾癌细胞增殖与凋亡的影响

目的：探讨siRNA干扰血管内皮生长因子（VEGF）基因对人肾细胞癌细胞株（ACHN）细胞增殖与凋亡的影响.方法：化学合成针对VEGF的小干扰RNA,通过脂质体转染至ACHN中,利用Western印迹法检测细胞内VEGF的表达,采用台盼蓝拒染法测定细胞生长曲线,用MTT比色分析法检测细胞增殖抑制率（IR）,用TUNEL方法检测细胞凋亡率（AR）.结果：生长曲线提示,与空白对照组及阴性对照组相比,siRNA1组、siRNA2组ACHN细胞的生长明显减慢 在24 h、48 h、72 h,siRNA1的增殖抑制率为10.6%、18.0%、27.1%,siRNA2增殖抑制率为18.9%、32.7%、40.3%,均高于空白对照组及阴性对照组（P〈0.05） siRNA1组的细胞凋亡率为10.7%、15.2%、20.3%,siRNA2组的细胞凋亡率为17.3%、26.2%、37.4%,均高于空白对照组及阴性对照组（P〈0.05） siRNA1组、siRNA2组VEGF蛋白表达水平明显低于空白对照组及阴性对照组,其中siRNA2对ACHN细胞的IR、AR和VEGF蛋白表达的抑制作用均显著高于siRNA1组（P〈0.05）.结论：VEGF在肾癌的发生发展中起着重要作用,化学合成的VEGF-siRNA能特异性抑制肾细胞癌ACHN细胞株中VEGF的表达,抑制细胞生长增殖,促进细胞凋亡.对于VEGF基因高表达的肾细胞癌患者,针对VEGF的RNAi技术有望成为肾细胞癌新的基因治疗手段.