REGγ基因下调对HaCat细胞的生物学影响

为了研究蛋白酶体激活因子REGγ对HaCat细胞增殖、凋亡、周期的影响,该研究通过瞬时转染建立了siN、siR细胞系,并利用流式细胞术、MTT、Western blot等实验技术检测细胞的处理效果。研究结果显示,通过小干扰RNA使REGγ低表达后,HaCat细胞内抑癌基因蛋白p53、细胞周期依赖性激酶抑制因子p21表达量增加,进而抑制了细胞周期和增殖、促进了细胞凋亡。     

p53在蓝莓花青素诱导Lovo细胞凋亡中的作用

[目的]研究蓝莓花青素(BA)对人结肠癌Lovo细胞增殖及凋亡的影响,分析凋亡相关基因—p53基因mRNA和蛋白表达的变化。[方法]分别用浓度为50μg/ml、100μg/ml、200μg/ml的BA处理结肠癌Lovo细胞24h,采用MTT法、免疫荧光法和Real-time PCR法和免疫细胞组织化学分析BA对Lovo细胞增殖、细胞凋亡、p53 mRNA和蛋白表达的影响。[结果]BA呈剂量依赖的方式抑制Lovo细胞增殖;能诱导细胞发生晚期凋亡;与对照组相比,不同浓度BA处理后p53基因mRNA表达水平分别上调0.602、0.697、0.541倍(P0.05),但BA不同剂量组间并无明显的差异(P0.05);免疫细胞组化结果表明随着BA浓度的增加,p53表达增强。[结论]BA可以诱导结肠癌Lovo细胞发生晚期凋亡,p53基因mRNA表达上调,但与BA的浓度无相关性。     

ASPP2增强结肠癌HCT116细胞对奥沙利铂的化疗敏感性

为研究ASPP2对奥沙利铂诱导的结肠癌细胞系HCT116 p53+/+(野生型)凋亡及周期的影响.利用ASPP2（rAd-ASPP2）及p53腺病毒（rAd-p53）感染HCT116 p53+/+细胞,经奥沙利铂50 μmol/L诱导细胞凋亡及周期改变.Western印迹检测ASPP2及p53的表达水平;MTT法检测ASPP2腺病毒对奥沙利铂诱导的HCT116细胞活性的影响;Calcein/PI吸收试验检测细胞凋亡情况;流式细胞术分析细胞周期分布. 结果显示,ASPP2、p53共同过表达,或者ASPP2单独过表达均能增强奥沙利铂诱导的HCT116 p53+/+细胞增殖抑制,以及S期抑制并伴有细胞凋亡水平的升高;而无奥沙利铂诱导时,ASPP2对HCT116 p53+/+细胞的活性、细胞周期及细胞凋亡水平的影响无统计学意义. 上述结果表明,ASPP2能够增强奥沙利铂诱导HCT116 p53+/+细胞的增殖抑制、细胞周期抑制和细胞凋亡.     

苦参碱对鼻咽癌细胞凋亡及凋亡相关基因p53 的表达研究

目的：探讨苦参碱对体外培养的人鼻咽癌细胞增殖、凋亡及凋亡相关基因p53 mRNA 和蛋白表达的影响,初步探讨苦参碱 诱导人鼻咽癌细胞凋亡的可能机制。方法：采用MTT 法检测不同浓度苦参碱（0、0.25、0.5、1、1.5、2 mg/ml）对CNE1、CNE2 细胞增 殖的影响;采用荧光定量PCR 法检测这些浓度的苦参碱处理48 h后CNE2 细胞p53 mRNA的变化; Western Blot 检测其蛋白的 变化情况。结果：MTT结果显示苦参碱具有抑制CNE1、CNE2 细胞体外增殖作用,其抑制率存在浓度、时间依赖性。荧光定量 PCR及Western Blot 检测结果显示,苦参碱抑制CNE2细胞p53 mRNA 和蛋白的表达,且亦呈浓度依赖性。结论：苦参碱抑制 CNE2 细胞的增殖,诱导细胞凋亡,呈现浓度、时间依赖性,其作用与抑制CNE2 细胞中p53 基因和蛋白的表达密切相关。     

苦参碱对鼻咽癌细胞凋亡及凋亡相关基因p53的表达研究

目的：探讨苦参碱对体外培养的人鼻咽癌细胞增殖、凋亡及凋亡相关基因p53 mRNA和蛋白表达的影响,初步探讨苦参碱诱导人鼻咽癌细胞凋亡的可能机制。方法：采用MTT法检测不同浓度苦参碱（0、0.25、0.5、1、1.5、2 mg/ml）对CNE1、CNE2细胞增殖的影响;采用荧光定量PCR法检测这些浓度的苦参碱处理48 h后CNE2细胞p53 mRNA的变化;Western Blot检测其蛋白的变化情况。结果：MTT结果显示苦参碱具有抑制CNE1、CNE2细胞体外增殖作用,其抑制率存在浓度、时间依赖性。荧光定量PCR及Western Blot检测结果显示,苦参碱抑制CNE2细胞p53 mRNA和蛋白的表达,且亦呈浓度依赖性。结论：苦参碱抑制CNE2细胞的增殖,诱导细胞凋亡,呈现浓度、时间依赖性,其作用与抑制CNE2细胞中p53基因和蛋白的表达密切相关。     

TGF-β信号通路抑制剂LY364947对急性髓系白血病细胞增殖、凋亡和侵袭的影响

该文旨在探讨抑制TGF-β信号通路对人急性髓系白血病(acute myeloid leukemia,AML)细胞体外增殖、凋亡和侵袭能力的影响。使用不同浓度TGF-β信号通路抑制剂LY364947处理AML细胞系(KG1a、OCI-AML3)后,采用CCK-8实验检测细胞体外增殖能力;流式细胞术检测细胞周期分布及凋亡情况; Western blot检测细胞周期调控因子Cyclin D1/p21、凋亡相关蛋白Bcl-2/Bax以及上皮细胞间质转化相关蛋白E-cadherin、N-cadherin和vimentin的表达; Transwell实验测定AML细胞迁移及侵袭能力的变化。结果显示:LY364947作用后,白血病细胞生长明显受抑制;细胞周期阻滞在G1期,伴有Cyclin D1表达下调和p21表达上调;细胞凋亡率增加,同时细胞抗凋亡蛋白Bcl-2的表达水平下降,促凋亡蛋白Bax表达增高;细胞体外迁移和侵袭能力减弱。此外, E-cadherin表达增高, N-cadherin和vimentin表达下降。该研究结果提示,抑制TGF-β信号通路能够抑制白血病细胞的体外增殖,诱导细胞凋亡,降低细胞迁移及侵袭能力。     

hDaxx与细胞肿瘤抑制子p53在体内外的相互作用

与急性早幼粒细胞性白血病蛋白(promyelocytic leukemia protein,PML)在体内相互作用共定位于细胞核PML致癌结构城(PML oncogenic domains,PODs)的人Daxx(human Daxx,hDaxx),能结合Fas死亡结构域诱导细胞凋亡.细胞肿瘤抑制子p53抑制细胞及病毒转录,提高细胞内Fas的表达并调节细胞凋亡.为了探索hDaxx与p53在诱导细胞凋亡中有无相互作用及其作用效果,利用酵母双杂交体系测定发现p53通过C端与hDaxx结合,共免疫沉淀反应及Western blot结果显示hDaxx与p53能在体内外直接结合.hDaxx与p53结合并发生相互作用可能对细胞周期有一定的调节作用.     

雷氏大疣蛛毒液对人肝癌HepG2细胞p21基因表达的影响

本文研究了雷氏大疣蛛毒液对人肝癌细胞株HepG2增殖抑制作用及其分子机制。采用XTT法观察到雷氏大疣蛛毒液剂量依赖抑制HepG2细胞增殖;流式细胞仪检测发现,经过雷氏大疣蛛毒液作用的HepG2细胞周期发生明显的选择性改变;RT-PCR方法检测到p21基因表达增强;Western blot检测发现,p21蛋白表达增加。结果提示,雷氏大疣蛛毒液抑制人肝癌细胞HepG2增殖的可能机制之一是使p21基因和蛋白表达增加,G2IM细胞周期被阻滞,从而诱导细胞凋亡。     

毛蕊异黄酮抑制SIRT1促乳腺癌细胞MCF-7凋亡

目的:探讨毛蕊异黄酮促乳腺癌细胞MCF-7凋亡的机制。方法:MTT检测低、中、高(10μM,50μM,100μM)剂量的毛蕊异黄酮对细胞活力的影响;Tunel检测毛蕊异黄酮对细胞凋亡的影响;Western blot检测SIRT1,p53和cleaved caspase-3的蛋白表达;Real-time PCR检测caspase-3 mRNA的表达。结果:毛蕊异黄酮能够剂量依赖性地降低细胞活力,100μM剂量组的毛蕊异黄酮显著地促进肿瘤细胞凋亡并降低SIRT1,增加p53和cleaved caspase-3的蛋白表达。SIRT1抑制剂烟酰胺(Nicotinamide,NAM,300μM)组与毛蕊异黄酮处理组相比显著地抑制SIRT1的蛋白表达,p53和cleaved caspase-3蛋白表达水平进一步增加;SRT1720(SIRT1特异性激动剂)与毛蕊异黄酮共孵育组逆转SIRT1蛋白表达,降低p53和cleaved caspase-3的蛋白水平。结论:毛蕊异黄酮促进肿瘤细胞MCF-7的凋亡,部分可能是通过降低SIRT1的表达水平,从而增加p53和cleaved caspase-3的蛋白表达促进细胞凋亡。     

中国野生蓝莓总花青素对A549细胞活力和周期改变的影响

为了进一步探究中国野生蓝莓的功能特性,本研究从中国野生蓝莓中提取总花青素,并使用XAD-7大孔树脂进行纯化,考察纯化后总花青素提取物对A549细胞平板克隆、增殖、细胞周期、早期凋亡和ROS释放的影响,并初步探讨其作用机理。研究结果表明,中国野生蓝莓总花青素对A549细胞活力有明显的抑制作用,对其增殖抑制的IC50值为176.652μg/m L,对细胞周期也有明显影响并能引起早期凋亡现象以及ROS释放。     

MicroRNA-205 suppresses the oral carcinoma oncogenic activity via down-regulation of Axin-2 in KB human oral cancer cell

MicroRNA (miRNA) is a small noncoding RNA molecule, 19–25 nucleotides in length, which regulates several pathways including cell development, cell proliferation, carcinogenesis, apoptosis, etc. In this study, the over-expression of microRNA-205 (miR-205) increased the number of apoptotic cells by at least 4 times compared to the control. In addition, over-expressed miRNA in KB oral cancer cells triggered apoptosis via the caspase cascade, including the cleavage of caspase-9, caspase-7, caspase-3, and PARP. Flow cytometry showed that apoptotic cell death was increased significantly by 35.33 % in KB oral cancer cells with over-expressed miR-205 compared to the control. The microarray data showed that axis inhibitor protein 2 (Axin2) was down-regulated in KB oral cancer cells transfected with miR-205. In addition, Axin2 was down-regulated by approximately 50 % by over-expressed miR-205 at both the mRNA and protein levels. Interestingly, Axin2 was up-regulated in KB oral cancer compared to human normal oral keratinocytes. Furthermore, the cell cytotoxicity and apoptotic population of KB oral cancer cells were increased significantly after Axin2 siRNA transfection. These results suggest that Axin2 is might be as potential oncogene in KB oral cancer cells. The luciferase assay showed that over-expressed miR-205 in KB oral cancer cells suppressed AXIN2 expression through an interaction with its own binding site at AXIN2 3′UTR (64–92). These results suggest that miR-205 is a novel anti-oncogenic miRNA in KB oral cancer cells, and may have potential applications in oral cancer therapy.     

Transplantation of bone marrow stromal cells enhances nerve regeneration of the corticospinal tract and improves recovery of neurological functions in a collagenase-induced rat model of intracerebral hemorrhage

MicroRNA (miRNA) is a form of small noncoding RNA that regulates the expression of genes either by inhibiting mRNA translation or by inducing its degradation. Small microRNA play important roles in regulating a large number of cellular processes, including development, proliferation and apoptosis. This study examined the biological functions of miR-205 as a tumor suppressor in KB oral cancer cells. The results showed that miR-205 expression was significantly lower in KB oral cancer cells than in human normal oral keratinocytes. Furthermore, the miR-205 over-expressed in KB oral cancer cells increased the cell cytotoxicity and induced apoptosis through the activation of caspase-3/-7. The transfection of miR-205 into KB oral cancer cells strongly induced IL-24, a well known cytokine that acts as a tumor suppressor in a range of tumor tissues. In addition, miR-205 targeted the IL-24 promoter directly to induce gene expression. Overall, miR-205 has significant therapeutic potential to turn on silenced tumor suppressor genes by targeting them with miRNA.     

Cytosolic protection against ultraviolet induced DNA damage by blueberry anthocyanins and anthocyanidins in hepatocarcinoma HepG2 cells

UV-induced DNA damage plays a key role in the etiology of certain diseases. The ability of blueberry anthocyanins and anthocyanidins (BA) to protect cellular DNA from UV-induced damage was investigated. BA were extracted by water (BAW), ethanol (BAE) or methanol (BAM). These extracts partially restored proliferation of UV-irradiated HepG2 cells as shown by MTT assay. Treatment with BA extracts at 75 μg/ml decreased reactive oxygen species and decreased DNA damage by tail moment of comet assay and expression of γH2AX in situ. BAM significantly decreased gene and protein expression of p53, phospho-p53 (Ser15), and p21 in UV-irradiated HepG2 cells. BA thus efficiently protects cells from DNA damage in vitro. Blueberry may potentially be used as a good source of naturally radioprotective agents.     

Quantitative phosphoproteomic analysis reveals γ‐bisabolene inducing p53‐mediated apoptosis of human oral squamous cell carcinoma via HDAC2 inhibition and ERK1/2 activation

γ‐Bisabolene, one of main components in cardamom, showed potent in vitro and in vivo anti‐proliferative activities against human oral squamous cell carcinoma (OSCC). γ‐Bisabolene activated caspases‐3/9 and decreased mitochondrial memebrane potential, leading to apoptosis of OSCC cell lines (Ca9‐22 and SAS), but not normal oral fibroblast cells. Phosphoproteome profiling of OSCC cells treated with γ‐bisabolene was identified using TiO2‐PDMS plate and LC‐MS/MS, then confirmed using Western blotting and real‐time RT‐PCR assays. Phosphoproteome profiling revealed that γ‐bisabolene increased the phosphorylation of ERK1/2, protein phosphatases 1 (PP1), and p53, as well as decreased the phosphorylation of histone deacetylase 2 (HDAC2) in the process of apoptosis induction. Protein–protein interaction network analysis proposed the involvement of PP1‐HDAC2‐p53 and ERK1/2‐p53 pathways in γ‐bisabolene‐induced apoptosis. Subsequent assays indicated γ‐bisabolene eliciting p53 acetylation that enhanced the expression of p53‐regulated apoptotic genes. PP1 inhibitor‐2 restored the status of HDAC2 phosphorylation, reducing p53 acetylation and PUMA mRNA expression in γ‐bisabolene‐treated Ca9‐22 and SAS cells. Meanwhile, MEK and ERK inhibitors significantly decreased γ‐bisabolene‐induced PUMA expression in both cancer cell lines. Notably, the results ascertained the involvement of PP1‐HDAC2‐p53 and ERK1/2‐p53 pathways in mitochondria‐mediated apoptosis of γ‐bisabolene‐treated cells. This study demonstrated γ‐bisabolene displaying potent anti‐proliferative and apoptosis‐inducing activities against OSCC in vitro and in vivo, elucidating molecular mechanisms of γ‐bisabolene‐induced apoptosis. The novel insight could be useful for developing anti‐cancer drugs.     

Computer-assisted analysis of p53 and PCNA expression in oral lesions infected with human papillomavirus

OBJECTIVE: To carry out a retrospective study to determine whether human papillomavirus (HPV) infection and immunohistochemical expression of p53 and proliferating cell nuclear antigen (PCNA) are related to the risk of oral cancer. STUDY DESIGN: Fifty-seven oral biopsies, consisting of 30 oral squamous papillomas (OSPs) and 27 oral squamous cell carcinomas (OSCCs) were tested for the presence of HPV 6/11 and 16/18 by in situ hybridization using catalyzed signal amplification and in situ hybridization. p53 And PCNA expression was analyzed by immunohistochemistry and evaluated quantitatively by image analysis. RESULTS: Nineteen of the 57 oral lesions (33.3%) were positive for HPV. HPV 6/11 was found in 6 of 30 (20%) OSPs and 1 of 27 (3.7%) OSCCs. HPV 16/18 was found in 10 of 27 (37%) OSCCs and 2 of 30 (6.7%) OSPs. Sixteen of the 19 HPV-positive cases (84.2%) were p53 negative; 5 (9%) were HPV 6/11 and 11 (19%) HPV 16/18, with an inverse correlation between the presence of HPV DNA and p53 expression (P = .017, P < .05). PCNA expression appeared in 18 (94.7%) of HPV positive cases, showing that HPV 16/18 was associated with intensity of PCNA expression and with OSCCs (P = .037, P < .05). CONCLUSION: Quantitative evaluation of p53 by image analysis showed an inverse correlation between p53 expression and HPV presence, suggesting protein degradation. Image analysis also demonstrated that PCNA expression was more intense in HPV DNA 16/18 OSCCs. These findings suggest involvement of high-risk HPV types in oral carcinogenesis.     

Radiation response and cell cycle regulation of p53 rescued malignant keratinocytes

Mutations in the tumor suppressor gene p53 were found in more than 90% of all human squamous cell carcinomas (SCC). To study the function of p53 in a keratinocyte background, a tetracycline-controlled p53 transgene was introduced into a human SCC cell line (SCC15), lacking endogenous p53. Conditional expression of wild-type p53 protein upon withdrawal of tetracycline was accompanied with increased expression of p21(WAF1/Cip1) resulting in reduced cell proliferation. Flow-cytometric analysis revealed that these cells were transiently arrested in the G1/S phase of the cell cycle. However, when SCC15 cells expressing p53 were exposed to ionizing radiation (IR), a clear shift from a G1/S to a G2/M cell cycle arrest was observed. This effect was greatly depending on the presence of wild-type p53, as it was not observed to the same extent in SCC15 cells lacking p53. Unexpectedly, the p53- and IR-dependent G2/M cell cycle arrest in the keratinocyte background was not depending on increased expression or stabilization of 14-3-3sigma, a p53-regulated effector of G2/M progression in colorectal cancer cells. In keratinocytes, 14-3-3sigma (stratifin) is involved in terminal differentiation and its cell cycle function in this cell type might diverge from the one it fulfills in other cellular backgrounds.     

Reduction of Orc6 Expression Sensitizes Human Colon Cancer Cells to 5-Fluorouracil and Cisplatin

Previous studies from our group have shown that the expression levels of Orc6 were highly elevated in colorectal cancer patient specimens and the induction of Orc6 was associated with 5-fluorouracil (5-FU) treatment. The goal of this study was to investigate the molecular and cellular impact of Orc6 in colon cancer. In this study, we use HCT116 (wt-p53) and HCT116 (null-p53) colon cancer cell lines as a model system to investigate the impact of Orc6 on cell proliferation, chemosensitivity and pathways involved with Orc6. We demonstrated that the down regulation of Orc6 sensitizes colon cancer cells to both 5-FU and cisplatin (cis-pt) treatment. Decreased Orc6 expression in HCT-116 (wt-p53) cells by RNA interference triggered cell cycle arrest at G1 phase. Prolonged inhibition of Orc6 expression resulted in multinucleated cells in HCT-116 (wt-p53) cell line. Western immunoblot analysis showed that down regulation of Orc6 induced p21 expression in HCT-116 (wt-p53) cells. The induction of p21 was mediated by increased level of phosphorylated p53 at ser-15. By contrast, there is no elevated expression of p21 in HCT-116 (null-p53) cells. Orc6 down regulation also increased the expression of DNA damaging repair protein GADD45β and reduced the expression level of JNK1. Orc6 may be a potential novel target for future anti cancer therapeutic development in colon cancer.