靶向VEGF发夹状RNA对宫颈癌HeLa细胞抑制作用的研究

目的:运用RNAi技术下调血管内皮生长因子(VEGF)在HeLa细胞中的表达,观察其对肿瘤细胞凋亡的影响,为人宫颈癌治疗提供理论依据。方法:设计并构建针对VEGF的携带绿色荧光蛋白(GFP)发夹状RNA(shRNA)质粒表达载体(PGPU6/GFP/Neo-shRNA),脂质体法转染HeLa细胞;荧光显微镜观察GFP的表达,并计算转染效率;RT-PCR检测HeLa细胞VEGF的表达,筛选出靶序列;再用流式细胞仪法检测细胞凋亡。结果:构建的PGPU6/GFP/Neo载体成功转入HeLa细胞;转染48h后,HeLa-shVEGF1组HeLa细胞VEGFmRNA表达的抑制率为75.0%;与HeLa组和HeLa-shNC组相比,HeLa-shVEGF1组HeLa细胞凋亡率明显增加,P<0.01。结论:本研究构建的PGPU6-shRNA表达载体携带GFP便于观察细胞的转染情况,且不影响U6启动子的转录,同时有效沉默了VEGF基因,明显增加HeLa细胞的凋亡,为未来肿瘤的治疗提供新途径。     

Trace element concentrations in renal cell carcinoma.

Cadmium, zinc, copper levels and zinc-copper, zinc-bromine, iorn-zinc, iron-copper and iron-bromine ratios are measured in neoplastic and normal kidney samples from humans by the particle induced X-ray emission analysis (PIXE) technique. It is found that cadmium which is normally present in the tubular cells of kidney is not detectable in tumor samples. It is also observed that the zinc-copper ratios in all neoplastic kidney tissues are decreased, but this observation cannot be extended to other element ratios.     

Flow cytometric DNA analysis of interleukin-2 responsive renal cell carcinoma

Adoptive immunotherapy using interleukin-2 (IL-2) based therapy can result in marked tumor regression in some patients with metastatic renal cell carcinoma. DNA flow cytometry has not been previously studied as a predictor of outcome of this therapy. Archival paraffin embedded tumors were studied in 23 IL-2 treated patients with metastatic renal cell carcinoma. Eleven patients were complete responders (CR) and 12 were nonresponders (NR). In the CR group, 4/11 (40%) were diploid and 7/11 (60%) were aneuploid. In the NR group, 9/12 (75%) were diploid and 3/12 (25%) were aneuploid. Although there was a trend that patients with an aneuploid DNA pattern were more likely to undergo a complete response, ploidy pattern alone was not significantly predictive of response (p² = 0.10, Fischer's exact test). When combining ploidy pattern with other variables that were predictive for complete response, such as good performance status and a higher pretreatment weight, prediction of complete response was not improved by including ploidy. This preliminary report suggests that DNA ploidy does not appear to provide any additional information concerning responsiveness to IL-2 based immunotherapy beyond that obtained by performance status and pretreatment weight in this patient population. © 1993 Wiley-Liss, Inc.     

The place of VEGF inhibition in the current management of renal cell carcinoma

Vascular endothelial growth factor (VEGF) is overexpressed in around 80% of patients with clear cell carcinoma of the kidney owing to the inactivation of von Hippel Lindau gene activity. VEGF stimulates angiogenesis and acts as an autocrine growth factor. A number of different agents are now available which target VEGF and its signalling pathways. A significant body of evidence has accumulated demonstrating that antagonism of VEGF and its downstream pathways is clinically useful in a significant proportion of patients with metastatic clear cell carcinoma of the kidney. Enough data is now available to recommend that patients with metastatic clear cell carcinoma of the kidney should at some point during the course of their disease be offered entry into a clinical trial enabling exposure to a targeted inhibitor of VEGF or its signalling pathways. Assuming early clinical trial data is substantiated by ongoing registration studies, efforts should be made to minimise the time taken between licensing and general availability of these active agents.     

Growth inhibition and enhanced chemosensitivity induced by down-regulation of Aurora-A in human renal cell carcinoma Caki-2 cells using short hairpin RNA

The objective of this study was to investigate the inhibitory effects of Aurora-A expression on the growth and chemosensitivity of Caki-2 cells in human renal cell carcinoma (RCC). Caki-2 cells were established, in which an expression vector containing short hairpin RNA (shRNA) targeting Aurora-A was introduced (Caki-2/sh-A). The growth and sensitivity of chemotherapeutic agents in Caki-2/sh-A cells were compared to those in Caki-2 cells transfected with control vector alone (Caki-2/C). The expression levels of both Aurora-A mRNA and protein in Caki-2/sh-A cells were less than 10% of those in Caki-2/C cells. The in vitro growth of Caki-2/sh-A cells was significantly inferior to that of Caki-2/C cells, and the proportion of Caki-2/sh-A cells in the G2-M phase was significantly greater compared to that of Caki-2/C cells. In addition, the expression level of Bax in Caki-2/sh-A cells was significantly higher as compared to that in Caki-2/C cells, while phosphorylated Akt in Caki-2/sh-A cells was markedly down-regulated compared to that in Caki-2/C cells. Among several chemotherapeutic agents examined, the most significant difference between Caki-2/sh-A and Caki-2/C cells was observed in the sensitivity to docetaxel. Thus, the IC(50) value of docetaxel in Caki-2/sh-A cells was decreased by approximately 90% compared to that in Caki-2/C cells. Treatment of Caki-2/sh-A cells, but not Caki-2/C ones, with 5 nM docetaxel resulted in the induction of apoptotic cell death accompanying the induction of p53. The findings suggest that the suppression of Aurora-A expression using shRNA is a useful therapeutic strategy against RCC through growth inhibition as well as enhanced chemosensitivity.     

VHL-mediated hypoxia regulation of cyclin D1 in renal carcinoma cells

Renal cell carcinoma is associated with mutation of the von Hippel-Lindau (VHL) tumor suppressor gene. Cell lines derived from these tumors cannot exit the cell cycle when deprived of growth factors, and the ability to exit the cell cycle can be restored by the reintroduction of wild-type protein VHL (pVHL). Here, we report that cyclin D1 is overexpressed and remains inappropriately high in during contact inhibition in pVHL-deficient cell lines. In addition, hypoxia increased the expression of cyclin D1 specifically in pVHL-negative cell lines into which pVHL expression was restored. Hypoxic-induction of cyclin D1 was not observed in other pVHL-positive cell lines. This suggests a model whereby in some kidney cell types, pVHL may regulate a proliferative response to hypoxia, whereas the loss of pVHL leads to constitutively elevated cyclin D1 and abnormal proliferation under normal growth conditions.     

Suppression of MMP-9 activity by NDRG2 expression inhibits clear cell renal cell carcinoma invasion

Members of the NDRG (N-Myc downstream-regulated) gene family have been shown to play a variety of roles in human malignancies. Recently, it was shown decreased expression in clear cell renal cell carcinoma (CCRCC) and inhibited cell proliferation, but the role of the NDRG2 in CCRCC invasion has not been described. We examined the expression of NDRG2 protein in CCRCC samples and the association between NDRG2 expression and CCRCC patients survival. Real-time RT-PCR and immunohistochemical analysis were used to measure NDRG2 expression in 60 paired CCRCC and adjacent normal tissues. Changes in cell invasion were detected by up- or down-regulating NDRG2 by adenovirus or siRNA. We found that NDRG2 expression is significantly down-regulated in CCRCC at mRNA and protein levels in a manner negatively associated with aggressive tumor behaviors, such as TNM stage (P?=?0.003), Fuhrman??s grade (P?=?0.024), tumor invasion (P?=?0.001) and tumor recurrence (P?=?0.004), as well as shorter patient survival rates (P?=?0.0041). Furthermore, NDRG2 could suppress CCRCC cell invasion through regulating MMP-9 expression and activity. So, these results suggest that NDRG2 can inhibit extracellular matrix-based tumor cell invasion and thereby play important roles in suppressing tumor metastasis in CCRCC. NDRG2 expression may also be a significant prognostic indicator for CCRCC.     

Enhancement of Fas-mediated apoptosis in renal cell carcinoma cells by adriamycin

Anti-Fas monoclonal antibody (mAb) kills Fas-expressing cells by apoptosis. Several anticancer agents also mediate apoptosis and may share common intracellular pathways leading to apoptosis with Fas. Thus, we reasoned that combination treatment of drug-resistant cells with anti-Fas mAb and drugs might overcome their resistance. We investigated whether anticancer agents enhance Fas-mediated apoptosis and cytotoxicity against renal cell carcinoma (RCC) cells. Treatment of ACHN RCC cells with anti-Fas mAb in combination with 5-fluorouracil, vinblastine, IFN-alpha, or IFN-gamma did not overcome resistance to these agents. However, combination treatment with anti-Fas mAb and Adriamycin (ADR) resulted in a synergistic cytotoxic effect. Furthermore, synergy was also obtained even when the exposure time was shortened from 24 h to 8 or 2 h. Synergy was also achieved in four other RCC cell lines and five freshly derived human RCC cells. Treatment with anti-Fas mAb in combination with epirubicin or pirarubicin also resulted in a synergistic cytotoxic effect on ACHN cells. Similar results were achieved with a combination of humanized anti-Fas mAb and ADR. Incubation of ACHN cells with ADR augmented the expression of Fas and p53, but not Bcl-2, Bax, or caspase-3. However, the activity of caspase-3 itself was apparently enhanced after treatment with ADR alone or combined treatment with anti-Fas mAb. The synergy obtained in cytotoxicity with anti-Fas mAb and ADR was also achieved in apoptosis. Exposure of ACHN cells and freshly derived RCC cells to ADR enhanced their susceptibility to lysis by peripheral blood lymphocytes and tumor-infiltrating lymphocytes. This study demonstrates that combination treatment of RCC cells with anti-Fas mAb and ADR might overcome their resistance. The sensitization required a low concentration of ADR and a short exposure time, thus supporting the potential in vivo application of a combination of ADR and anti-Fas mAb or immunotherapy in the treatment of ADR- and/or immunotherapy-resistant RCC.     

肾癌组织MMP-10和VEGF表达及其临床意义的研究

目的:研究MMP-10和VEGF 在肾癌组织中的表达及其临床意义.方法:采用免疫组织化学技术SP法检测56例肾细胞癌(其中20例伴发淋巴结转移)及10例距癌旁＞2 cm正常肾组织的MMP-10和VEGF表达,并分析其与MVD等临床病理参数的关系.结果:56例肾细胞癌组织中MMP-10和VEGF的阳性表达率分别为66.1...     

肾细胞癌干细胞标志物研究进展

肾细胞癌干细胞决定了肾细胞癌的生长和增殖,目前已发现的肾细胞癌潜在标志物包括八聚体序列结合因子4（Oct4）、CD133、CD105、ATP结合盒B亚家族成员1转运蛋白基因（ABCB1）、趋化因子受体4（CXCR4）,但这些分子标志物仍然存在争议。因此,迫切需要研究证实普遍适用的肾细胞癌标志物,以针对其更加有效地治疗肾细胞癌。     

Phenotypic switching of VEGF and collagen XVIII during hypoxia in head and neck squamous carcinoma cells

The present study sought to determine the potential role of stress activated MAPK and phosphatidylinositol 3-kinase (PI3K) signaling pathways in mediating phenotypic switching between angiogenic and angiostatic elements among squamous cell carcinoma (SCC) cell lines. In particular, we investigated the effects of hypoxia and those of cobalt chloride (CoCl₂), which mimics the hypoxic response including the production of reactive oxygen species, on such phenotypic shifts. The expression and production of collagen XVIII, and CBP2/Hsp47 provided a measure of an angiostatic phenotype, while vascular endothelial growth factor (VEGF) expression was used to assess potential angiogenic states. These studies revealed that hypoxia produced a slight up-regulation of collagen XVIII and CBP2/Hsp47 that was inhibited by the stress kinase inhibitor SB203580 but was unaffected by N-acetylcysteine (NAC). In addition, VEGF expression was increased following hypoxia and this effect was reversed with inhibition of by SB203580. Conversely, CoCl₂ significantly diminished the expression of both collagen XVIII and CBP2/Hsp47 and enhanced VEGF expression. These changes were reversed by the PI3K inhibitor wortmannin and by treating cells with NAC. These studies show that phenotypic switching between collagen XVIII and VEGF is controlled by stress activated kinases under hypoxia, and PI3K signaling pathways as well as reactive oxygen species (ROS) following CoCl₂ treatment. Furthermore, modulation of the angiogenic switch is most profound during Akt activation than during activation of stress activated kinases.     

MYC pathway is activated in clear cell renal cell carcinoma and essential for proliferation of clear cell renal cell carcinoma cells

Bone morphogenetic protein 7 (BMP7) is a signaling molecule originally identified based on its ability to form bone. It is essential during development, and more recently has also been implicated in cancer pathogenesis. We have recently shown that BMP7 is overexpressed in breast cancer, and that this increased expression is associated with early bone metastasis formation. In the present study, we explored the possible contribution of BMP7 function to the breast cancer cell phenotype. A two-way approach was applied in which BMP7 was silenced using RNA interference in three cell lines with high endogenous expression or, conversely, exogenous BMP7 was added to the growth medium of five cell lines with low or no BMP7 expression. These manipulations led to diverse cell line-specific phenotypic responses. BMP7 manipulation increased cell growth in two cell lines (BT-474, MDA-MB-231), and BMP7 treatment led to reduced growth in four cell lines (HCC1954, MDA-MB-361, T-47D, and ZR-75-30). Growth changes were due to distinct mechanisms since BMP7 silencing led to growth inhibition via G1 arrest in BT-474 cells, whereas BMP7 treatment protected MDA-MB-231 cells from apoptosis. Furthermore, BMP7 stimulation altered the MDA-MB-231 phenotype by inducing a distinct 2.3-fold increase in cell migration and an even more dramatic 3.9-fold increase in cell invasion. In conclusion, BMP7 can promote and inhibit cell growth in breast cancer cell lines and, in a suitable environment, can also considerably induce breast cancer cell migration and invasion.     

联合靶向mTOR和Gli1/2信号通路的药物对肾癌细胞生长的抑制效应

目的:探讨联合靶向mTOR和Gli1/2信号通路的药物对肾癌细胞生长的抑制效应。方法:采用Real time-PCR的方法检测SHH/Gli信号通路的核心效应成分Gli1和Gli2在20对正常肾脏和肾癌组织中的表达情况;采用Western blot技术验证4对正常肾脏和配对的肿瘤中Gli1和Gli2蛋白的表达水平。单独或联合使用不同浓度的mTOR抑制剂与Gli1/2抑制剂于肾癌细胞后,采用MTT的方法检测细胞的体外增殖能力,然后采用Western blot技术检测联合应用低剂量mTOR抑制剂与Gli1/2抑制剂的肾癌细胞内细胞周期、凋亡相关蛋白的变化情况。结果:Gli1与Gli2在肾癌组织中的表达高于对照的正常肾脏组织。低剂量（5 μmol/L）Gli1/2抑制剂Gant61并不能显著抑制肾癌细胞的生长,高剂量（10 μmol/L）的Gant61能达到有效的抑制效应;但5 μmol/L Gant61与低剂量mTOR抑制剂Rapamycin联用时,肾癌细胞的生长显著被抑制,且诱导细胞凋亡,阻滞细胞周期。结论:联合靶向mTOR和Gli1/2信号通路显著抑制肾癌细胞生长,为潜在的治疗策略。     

Enhancement of arsenic trioxide-induced apoptosis in renal cell carcinoma cells by L-buthionine sulfoximine

Arsenic trioxide (As2O3) induces clinical remission in acute promyelocytic leukemic patients and apoptosis in various tumor cells in vitro. To develop As2O3-based combination chemotherapy for renal cell carcinoma (RCC), we investigated the cytotoxic effects of As2O3 in combination with chemotherapeutic agents or L-buthionine sulfoximine (BSO), a glutathione (GSH) synthesis inhibitor. Cytotoxicity and synergy were assessed by the MTT assay and isobolographic analysis, respectively. Apoptosis was monitored by Hoechst 33342 staining, flow cytometrical analysis, and DNA fragmentation assay. Treatment of ACHN cells with As2O3 in combination with adriamycin, vinblastine, or 5-fluorouracil induced an antagonistic effect. However, combination treatment with As2O3 and BSO resulted in a synergistic cytotoxic effect. Synergy was also obtained in Caki-1, Caki-2, NC65 cells and freshly derived RCC cells from 6 patients. Simultaneous treatment of ACHN cells with As2O3 and BSO caused significantly more cytotoxicity than the As2O3 first BSO second or the reverse treatment. We further explored the mechanisms underlying this synergistic effect and found that the synergistic cytotoxicity of As2O3 and BSO was realized by inducing apoptosis. This combination markedly decreased intracellular GSH content and GSH-S-transferase (GST) activity. However, neither the intracellular GSH nor GST was decreased by As2O3 with adriamycin, vinblastine, or 5-fluorouracil. Furthermore, the GSH-increasing agents N-acetylcysteine and lipoic acid significantly inhibited the combined cytotoxicity of As2O3 and BSO. These findings indicate that BSO sensitizes RCC cells to As2O3-induced apoptosis through the down-regulation of the intracellular GSH redox system, suggesting the potential application of a combination of As2O3 and BSO for the treatment of RCC.     

Inhibition of renal cell carcinoma angiogenesis and growth by antisense oligonucleotides targeting vascular endothelial growth factor

Angiogenesis is critical for growth and metastatic spread of solid tumours. It is tightly controlled by specific regulatory factors. Vascular endothelial growth factor has been implicated as the key factor in tumour angiogenesis. In the present studies we evaluated the effects of blocking vascular endothelial growth factor production by antisense phosphorothioate oligodeoxynucleotides on the growth and angiogenic activity of a pre-clinical model of renal cell carcinoma (Caki-1). In vitro studies showed that treating Caki-1 cells with antisense phosphorothioate oligodeoxynucleotides directed against vascular endothelial growth factor mRNA led to a reduction in expressed vascular endothelial growth factor levels sufficient to impair the proliferation and migration of co-cultured endothelial cells. The observed effects were antisense sequence specific, dose dependent, and could be achieved at a low, non-toxic concentration of phosphorothioate oligodeoxynucleotides. When vascular endothelial growth factor antisense treated Caki-1 cells were injected into nude mice and evaluated for their angiogenic potential, the number of vessels initiated were approximately half that induced by untreated Caki-1 cells. To test the anti-tumour efficacy of vascular endothelial growth factor antisense, phosphorothioate oligodeoxynucleotides were administrated to nude mice bearing macroscopic Caki-1 xenografts. The results showed that the systemic administration of two doses of vascular endothelial growth factor antisense phosphorothioate oligodeoxynucleotides given 1 and 4 days after the tumours reached a size of approximately 200 mm(3) significantly increased the time for tumours to grow to 1000 mm(3).     

siRNA抑制VEGF基因表达对鼻咽癌细胞生物学行为的影响

目的:应用RNA干扰(RNAi)技术抑制人鼻咽低分化鳞状上皮细胞癌细胞株CNE-2中血管内皮生长因子(VEGF)的表达,研究VEGF对鼻咽癌细胞生物学行为的影响.方法:构建针对VEGF的siRNA真核表达载体pU-VEGF-siRNA,将其表达载体经脂质体LipofectamineTM2000转染至CNE-2细胞,荧光显微镜下观察转染效率,采用RT-PCR、Western blot方法检测转染后CNE-2细胞中VEGF mRNA和蛋白的表达.通过流式细胞仪分析细胞周期的分布,并应用平板克隆形成实验、Transwell侵袭小室模型观察转染后CNE-2细胞恶性生物学行为的变化.结果:pU-VEGF-siRNA转染后CNE-2细胞株中VEGF mRNA和蛋白表达较pU-siCONT组、空白对照组显著下降(P＜0.05),细胞周期被阻滞在G1期,细胞生长缓慢,体外侵袭能力下降.结论:通过RNAi技术阻断VEGF的表达,可抑制CNE-2细胞的生长、增殖、迁徙,提示VEGF在鼻咽癌的发生、发展过程中起重要作用.     

RNAi抑制肾癌细胞MDR1基因表达及对顺铂化疗耐药的逆转

目的:研究RNA干扰(RNA interference,RNAi)对肾癌细胞多药耐药基因1(multidrug resistance,MDR1)表达的抑制作用,并分析干扰前后肾癌细胞对顺铂(cisplatin)敏感度的变化.方法:根据MDR1基因序列设计3条小干扰RNA(small interfering RNA,siRNA),转染肾癌A498细胞;RT-PCR法检测转染前后MDR1 mRNA表达水平的变化,筛选出干扰效率最高的siRNA序列;进而利用慢病毒包装siRNA重组质粒,感染A498细胞,RT-PCR法筛选沉默效果最好的细胞株进行克隆;Western印迹法检测MDR1蛋白表达水平;MTT法检测干扰前后顺铂对半数细胞抑制浓度(half inhibitory concentration,IC_(50))的变化.结果:3条siRNA序列均能不同程度地抑制细胞MDR1基因的表达,其中siRNA-1序列能更有效地封闭MDR1基因,使MDR1 mRNA表达水平下降67%;筛选得到稳定转染的细胞株与未干扰细胞株相比,MDR1蛋白表达量明显下降(P<0.01),并使顺铂对细胞的IC_(50)值降低约83.37%(P<0.01).结论:RNAi能有效抑制肾癌A498细胞MDR1基因的表达,并可显著增加其对顺铂的敏感度,从而使肾癌细胞化疗耐药逆转成为可能.