细胞表面热休克蛋白90在人前列腺癌细胞中的表达及其对侵袭能力的影响

目的 观察细胞表面热休克蛋白90(HsPgo)在高侵袭性人前列腺癌细胞PC3中的表达,及其对细胞侵袭能力的影响,探讨其可能的作用机制.方法 采用免疫荧光染色和膜蛋白生物素标记法,检测HSPgO在PC3细胞表面的表达;采用Boyden小室侵袭实验观察细胞侵袭能力的改变;采用Western blot和免疫共沉淀法,检测局部黏着斑激酶(FAK)、c-Src蛋白磷酸化水平及其两者相互作用的变化;采用RNA干扰技术下调FAK蛋白表达,应用抑制剂PP2抑制Src激酶活性,观察细胞FAK/Src信号通路及其侵袭能力的改变.结果 PC3细胞表面表达HSP00.HSPg0特异性抗体可显著降低PC3细胞体外侵袭能力,且伴有FAK tyr 397、576、577、925及c-Src tyr 416磷酸化水平的显著下降,以及FAK与c-Src蛋白相互结合的明显减弱.经小分子干扰RNA(siRNA)有效干扰FAK表达,或采用PP2抑制Src激酶活性,均可显著抑制PC3细胞的侵袭性,但同时联合应用抗HSPg0抗体,未有协同作用.小室侵袭实验结果显示,PC3细胞经抗HSPg0抗体作用后,穿膜细胞数为(39.57 4±7.54)个,而对照组和lgG对照组分别为(105.70±12.16)个和(110.60 4±11.61)个,差异均有统计学意义(P<0.001).结论 细胞表面HSP90可经FAK/c-Src信号通路促进体外培养前列腺癌细胞的侵袭,提示其可能是潜在的对肿瘤转移实施防治的一个靶点.

Abstract：

Objective To investigate the expression of heat shock protein 90 (HSP90) on the cell surface of highly invasive human prostate cancer cells PC3 and its possible molecular mechanisms of its effect on cell invasion through analyzing FAK/Src signaling pathway. Methods The expression of cell surface HSP90 on PC3 cells was studied by immunofluorescence staining and surface biotinylation assay respectively. A specific HSP90 antibody was used to inhibit the cell surface HSP90. In vitro cell invasion was assessed by modified Boyden chambers. Phosphorylated FAK on tyr 397, 576, 577 and 925, and phosphorylated c-Src on tyr 416 were examined by Western blot assay. The association between FAK and c-Src was analyzed by immunoprecipitation. The effects of FAK knockdown by siRNA or Src kinases inhibitor PP2, with or without anti-HSP90 antibody, on PC3 cell invasion were also evaluated. Results A pool of HSP90 was detected on the cell surface of PC3 cells. A specific HSP90 antibody significantly retarded tumor cell invasion. Concomitant with this finding, targeting cell surface HSP90 significantly inhibited the phosphorylations of FAK and c-Src, and also the interactions between FAK and c-Src. FAK knockdown or PP2 dramatically suppressed cell invasion, however, anti-HSP90 antibody didn't further inhibit cell invasion. Conclusions Cell surface HSP90 promotes human prostate cancer cell invasion through a FAK/c-Src signaling, with may be a novel therapeutic target against metastatic tumors.     

细胞表面热休克蛋白90在人前列腺癌细胞中的表达及其对侵袭能力的影响

目的 观察细胞表面热休克蛋白90(HsPgo)在高侵袭性人前列腺癌细胞PC3中的表达,及其对细胞侵袭能力的影响,探讨其可能的作用机制.方法 采用免疫荧光染色和膜蛋白生物素标记法,检测HSPgO在PC3细胞表面的表达;采用Boyden小室侵袭实验观察细胞侵袭能力的改变;采用Western blot和免疫共沉淀法,检测局部黏着斑激酶(FAK)、c-Src蛋白磷酸化水平及其两者相互作用的变化;采用RNA干扰技术下调FAK蛋白表达,应用抑制剂PP2抑制Src激酶活性,观察细胞FAK/Src信号通路及其侵袭能力的改变.结果 PC3细胞表面表达HSP00.HSPg0特异性抗体可显著降低PC3细胞体外侵袭能力,且伴有FAK tyr 397、576、577、925及c-Src tyr 416磷酸化水平的显著下降,以及FAK与c-Src蛋白相互结合的明显减弱.经小分子干扰RNA(siRNA)有效干扰FAK表达,或采用PP2抑制Src激酶活性,均可显著抑制PC3细胞的侵袭性,但同时联合应用抗HSPg0抗体,未有协同作用.小室侵袭实验结果显示,PC3细胞经抗HSPg0抗体作用后,穿膜细胞数为(39.57 4±7.54)个,而对照组和lgG对照组分别为(105.70±12.16)个和(110.60 4±11.61)个,差异均有统计学意义(P<0.001).结论 细胞表面HSP90可经FAK/c-Src信号通路促进体外培养前列腺癌细胞的侵袭,提示其可能是潜在的对肿瘤转移实施防治的一个靶点.     

热休克蛋白90在食管鳞癌中的表达及临床意义

食管鳞状细胞癌(Esophagus squamous cell carcinoma,ESCC)是我国食管癌的主要病理分型,它起病隐匿、转移速度快,且给予治疗时产生耐药的速度快,导致预后不理想。热休克蛋白90(Heat shock protein 90,HSP90)调控的众多下游蛋白参与细胞的增殖等过程,所以在恶性肿瘤的发生发展中常扮演重要角色。越来越多的研究证实HSP90在多种恶性肿瘤中高表达并可影响肿瘤耐药性。近年来,对HSP90在ESCC中相关作用的探索也收获了许多值得关注的成果,本文将对HSP90在ESCC中的表达及临床意义做一综述。     

HSP90基因在前列腺癌细胞凋亡中的相关研究

目的：克隆全反式维甲酸诱导前列腺癌细胞系DU-145细胞产生凋亡的相关基因。探讨前列腺癌细胞产生凋亡的分子机制。方法：应用全反式维甲酸诱导前列腺癌DU-145细胞凋亡，建立细胞凋亡模型，采用基于PCR的改良消减杂交技术克隆与凋亡相关的基因。结果：在前列腺癌DU-145细胞凋亡过程中，克隆出包括HSP90基因在内的多个基因序列。结论：全反式维甲酸诱导的前列腺癌细胞DU-145凋亡有多基因参与其中．其中HSP90基因可能与前列腺痛细胞凋亡关系密切。     

细胞表面热休克蛋白90与肿瘤转移

热休克蛋白90(Hsp90)调控其多种底物蛋白的稳定及功能,是抗肿瘤治疗的独特靶蛋白.Hsp90不仅位于细胞表面,甚至分泌至细胞外发挥一定功能,且与肿瘤高侵袭性及转移密切相关.     

热休克蛋白90α的表达及其对体内肿瘤生长的影响

目的 探讨ｈｓｐ９０α对体内肿瘤生长的影响及机理。方法 利用克隆技术构建了ｈｓｐ９０α融合表达质粒ｐＭａｌ－ｈｓｐ９０α。ＤＢＡ／２小鼠接种肿瘤细胞前,皮下注射融合蛋白,每隔１天测肿瘤大小,２５天后,＾５１Ｃｒ释放法测ＮＫ细胞活性。结果 注射热休克帽白组小鼠抗ｈｓｐ９０ａ抗体升高,肿瘤生长快,ＮＫ细胞活性低,结论 ｈｓｐ９０α对肿瘤生长的影响与不鼠体内的ＮＫ细胞活性降低有关。     

细胞表面热休克蛋白90与肿瘤转移

热休克蛋白90(Hsp90)调控其多种底物蛋白的稳定及功能,是抗肿瘤治疗的独特靶蛋白.Hsp90不仅位于细胞表面,甚至分泌至细胞外发挥一定功能,且与肿瘤高侵袭性及转移密切相关.     

细胞表面热休克蛋白90与肿瘤转移

热休克蛋白90(Hsp90)调控其多种底物蛋白的稳定及功能,是抗肿瘤治疗的独特靶蛋白.Hsp90不仅位于细胞表面,甚至分泌至细胞外发挥一定功能,且与肿瘤高侵袭性及转移密切相关.     

热休克蛋白90与肿瘤

热休克蛋白９０（ＨＳＰ９０）是一种非常重要的应激蛋白，作为分子伴侣参与蛋白质的折叠，运输等过程。在肿瘤细胞中ＨＳＰ９０表达异常，ＨＳＰ９０能与许多癌基因，抑癌基因产物结合，产与肿瘤细胞的细胞周期调控，肿瘤免疫以及肿瘤细胞的发生发展有着密切关系，本文仅就ＨＳＰ９０与肿瘤的关系作一综述。     

热休克蛋白90α在人类肝癌细胞外的表达及其对侵袭、迁移能力的影响

目的探讨细胞外热休克蛋白90α（HSP90α参与肝癌细胞转移的可能分子机制。方法采用免疫印迹法检测HSP90α在低、高转移性肝癌MHCC97-L和MHCC97-H细胞外的表达。选取高转移潜能MHCC97-H细胞,应用不穿透细胞膜的小分子抑制剂抑制细胞外HSP90α表达,体外侵袭实验观察细胞侵袭、迁移能力的变化,明胶酶谱法分析基质金属蛋白酶2（MMP-2）活性的改变。免疫印迹和免疫共沉淀法检测细胞外辅助分子伴侣HSP70和MMP-2的表达及其与HSP90d的相互作用。利用RNA干扰技术下调细胞HSP70表达,观察细胞外HSP90仅与MMP-2相互作用及其细胞转移潜能的变化。结果HSP90d在不同转移潜能肝癌细胞内外均表达,且与肝癌细胞转移潜能呈正相关。MHCC97-H细胞经特异性HSP90抑制剂二甲氨基乙氨基-17-去甲氧基格尔德霉素-N-氧化物（DMAG—N—oxide）作用24h后,穿过上室基底膜的细胞数较未处理组和空白对照组明显下降（P〈0．01）,分别为（28．11±3．56）、（80．12±4．16）、（82．24±4．12）个。体外侵袭实验显示,穿过Matrigel人工基底膜的细胞数低于未处理组和空白对照组,分别为（36．54±4．12）、（95．12±3．48）、（101．11±3．36）个,差异有统计学意义（P=0．017）,并伴有细胞外MMP-2活性减低。HSP70和MMP-2均可在MHCC97-H细胞外表达,且能与HSP90α相互结合。小分子干扰RNA（siRNA）能有效抑制细胞HSP70表达,MHCC97-H细胞外HSP90d和MMP-2相互作用减弱,MMP-2活性下降,细胞侵袭、迁移能力受抑制。同时联合应用MMP-2抑制剂,具有协同抑制效应。结论细胞外HSP90饯可能通过与HSP70形成伴侣复合体介导MMP-2成熟活化,增强肝癌细胞侵袭、迁移运动,提示其可能是潜在的对肝癌转移实施防治的靶点。     

HSP70单抗联合反义寡核苷酸诱导前列腺癌细胞凋亡的研究

目的：探讨ＨＳＰ７０单抗及反义寡核苷酸对前列腺癌细胞株ＬＮＣａＰ和ＰＣ－３ｍ调亡在诱导作用及差异。方法：用流式细胞术分析了ＨＳＰ７０单抗（２μｌ）、反义寡核苷酸（１０μｍｏｌ／Ｌ）及单抗联合反义寡核苷酸分别处理ＬＮＣａＰ和ＰＣ－３ｍ２４、４８、７２、９６ｈ后,细胞凋亡的发生率。结果：在４８、７２、９６ｈ时单抗联合反义寡核苷酸诱导的凋亡发生率高于单抗或反义寡核苷酸单独诱导时的调亡率,差异显著（分别为     

c-Src antisense complexed with PAMAM denderimes decreases of c-Src expression and EGFR-dependent downstream genes in the human HT-29 colon cancer cell line

c-Src is one member of non-receptor tyrosine kinase protein family that has over expression and activation in many human cancer cells. It has been shown that c-Src is implicated in various downstream signaling pathways associated with EGFR-dependent signaling such as MAPK and STAT5 pathways. Transactivation of EGFR by c-Src is more effective than EGFR ligands. To inhibit the c-Src expression, we used c-Src antisense oligonucleotide complexed with PAMAM Denderimes. The effect of c-Src antisense oligonucleotide on HT29 cell proliferation was determined by MTT assay. Then, the expression of c-Src, EGFR and the genes related to EGFR-depended signaling with P53 was applied by real time PCR. We used western blot analysis to elucidate the effect of antisense on the level of c-Src protein expression. The results showed, c-Src antisense complexed with PAMAM denderimers has an effective role in decrease of c-Src expression and EGFR-dependent downstream genes.     

Combined inhibition of heat shock proteins 90 and 70 leads to simultaneous degradation of the oncogenic signaling proteins involved in muscle invasive bladder cancer

Heat shock protein 90 (HSP90) plays a critical role in the survival of cancer cells including muscle invasive bladder cancer (MIBC). The addiction of tumor cells to HSP90 has promoted the development of numerous HSP90 inhibitors and their use in clinical trials. This study evaluated the role of inhibiting HSP90 using STA9090 (STA) alone or in combination with the HSP70 inhibitor VER155008 (VER) in several human MIBC cell lines. While both STA and VER inhibited MIBC cell growth and migration and promoted apoptosis, combination therapy was more effective. Therefore, the signaling pathways involved in MIBC were systematically interrogated following STA and/or VER treatments. STA and not VER reduced the expression of proteins in the p53/Rb, PI3K and SWI/SWF pathways. Interestingly, STA was not as effective as VER or combination therapy in degrading proteins involved in the histone modification pathway such as KDM6A (demethylase) and EP300 (acetyltransferase) as predicted by The Cancer Genome Atlas (TCGA) data. This data suggests that dual HSP90 and HSP70 inhibition can simultaneously disrupt the key signaling pathways in MIBC.     

Slit/Robo信号通路在胰腺癌细胞中的表达及其对胰腺癌细胞生长的影响

目的 探讨Slit/Robo信号通路在胰腺癌细胞中的表达情况,及其对胰腺癌细胞增殖的影响.方法 分别采用RT-PCR、免疫细胞化学、免疫荧光方法 检测胰腺癌细胞株BxPC-3、Panc-1中Slit和Robo的表达;通过Slit/Robo信号通路阻断剂RoboN阻断该通路,采用MTT法观察其对细胞生长的影响.结果 胰腺癌细胞株Bx-PC-3、Panc-1中存在着Slit2、Robo1基因和蛋白表达;在RoboN的作用下,胰腺癌细胞体外生长受到抑制,增殖能力下降.结论 胰腺癌细胞中可能存在着Slit2/Robo1信号通路,它能够负性调节胰腺癌细胞的生长,提示Slit/Robo信号通路可能参与了胰腺癌的发生发展的过程.     

硼替佐米体外对前列腺癌细胞迁移和侵袭的影响及其机制

目的 探讨不同剂量的硼替佐米对前列腺癌DU145细胞迁移和侵袭的影响及其可能机制。方法 0、10、20 nmol／L硼替佐米处理DU145细胞24 h后,采用Transwell小室检测DU145细胞迁移,Matrigel侵袭实验检测细胞侵袭能力。采用Western blot技术检测黏附相关蛋白黏着斑激酶（FAK）及其自主磷酸化位点Tyr397的表达水平。结果 10、20 nmol／L硼替佐米作用24 h后,DU145细胞的迁移指数为（69.05±10.56）、（52.55±6.98）个,明显低于未经硼替佐米处理组的（81.55±10.56）个（均P＜0.05）;10、20 nmol／L硼替佐米处理组细胞侵袭指数分别为（39.35±6.45）、（32.05±4.22）个,明显低于未经硼替佐米处理组的（58.75±5.41）个（均P＜0.05）;硼替佐米处理组同时伴有Try397表达水平的下调,但FAK总蛋白的表达不受影响。结论 蛋白酶体通路抑制剂硼替佐米可能通过下调FAK Tyr397的表达,抑制前列腺癌DU145细胞迁移和侵袭能力。     

HSP90 inhibitors in the context of heat shock and the unfolded protein response: effects on a primary canine pulmonary adenocarcinoma cell line*

Background: Agents targeting HSP90 and GRP94 are seldom tested in stressed contexts such as heat shock (HS) or the unfolded protein response (UPR). Tumor stress often activates HSPs and the UPR as pro-survival mechanisms. This begs the question of stress effects on chemotherapeutic efficacy, particularly with drugs targeting chaperones such as HSP90 or GRP94. We tested the utility of several HSP90 inhibitors, including PU-H71 (targeting GRP94), on a primary canine lung cancer line under HS/UPR stress compared to control conditions.

Methods: We cultured canine bronchoalveolar adenocarcinoma cells that showed high endogenous HSP90 and GRP94 expression; these levels substantially increased upon HS or UPR induction. We treated cells with HSP90 inhibitors 17-DMAG, 17-AAG or PU-H71 under standard conditions, HS or UPR. Cell viability/survival was assayed. Antibody arrays measured intracellular signalling and apoptosis profiles.

Results: HS and UPR had varying effects on cells treated with different HSP90 inhibitors; in particular, HS and UPR promoted resistance to inhibitors in short-term assays, but combinations of UPR stress and PU-H571 showed potent cytotoxic activity in longer-term assays. Array data indicated altered signalling pathways, with apoptotic and pro-survival implications. UPR induction?+?dual targeting of HSP90 and GRP94 swayed the balance toward apoptosis.

Conclusion: Cellular stresses, endemic to tumors, or interventionally inducible, can deflect or enhance chemo-efficacy, particularly with chaperone-targeting drugs. Stress is likely not held accountable when testing new pharmacologics or assessing currently-used drugs. A better understanding of stress impacts on drug activities should be critical in improving therapeutic targeting and in discerning mechanisms of drug resistance.