胃癌组织中p21、p27、p53和Rb的表达及临床意义

目的探讨p21、p27、p53和Rb蛋白表达在胃癌发生、发展中的作用。方法应用免疫组化EnVision法检测111例胃癌,38例不典型增生4、7例肠上皮化生2、5例慢性萎缩性胃炎和53例正常胃黏膜组织中p21、p27、p53和Rb蛋白的表达。结果 p21和p27蛋白表达水平在不典型增生组织最高,其次是胃癌组织,与正常胃黏膜组织相比差异均有显著性(P<0.001)。在胃癌组织中p21、p27、p53和Rb蛋白表达均高于正常胃黏膜组织(P均<0.01)。p21、p27蛋白表达与胃癌的类型和分化相关(P均<0.01),p53表达水平与患者的年龄、性别、胃癌的类型和患者的生存率相关。女性患者Rb蛋白表达明显高于男性,差异有显著性(P<0.05)。p21、p27、p53和Rb蛋白表达水平之间均呈正相关。结论 p21、p27、p53和Rb蛋白表达可以作为胃癌的辅助诊断指标,四种蛋白在判断胃癌的生物学行为上具有协同作用。

细胞衰老的重要通路:p16^(INK4a)/Rb和p19^(ARF)/p53/p21^(Cip1)信号途径

细胞周期蛋白依赖激酶(cyclin-dependent kinase,CDK)抑制因子p16^(INK4a)、p21^(Cipl)等是细胞衰老的关键效应物。本文对涉及这些抑制物的两条衰老诱导途径作一综述,它们是pl6^(INK4a)/Rb和p19^(ARF)/p53/p21^(Cipl)信号途径。其中,几个抑癌基因的产物Rb、p16^(INK4)a、p53及p19^(ARF)处于两条途径的核心位置。

卵巢癌和乳腺癌P53、P21、P16和Rb基因表达水平的研究

目的 :研究肿瘤组织癌相关基因P53、Rb、P16、和P21表达水平的临床意义。 方法:用流式细胞仪(FCM)检测30例卵巢癌和乳腺癌瘤体中心灶P53、Rb、P16、和P21异常表达的阳性细胞百分率。 结果: 卵巢癌和乳腺癌的P53、Rb、P16、和P21基因蛋白表达水平及异常表达率均无明显差异(P>0.05)。30例肿瘤的4种基因异常表达率分别为 :P5340 % ;Rb53.3 % ;P2166.7 %和P1653.3 %。96.7 %(29/30)的肿瘤存在至少一种以上癌相关基因的异常表达。异常表达两个以上癌相关基因的患者较易出现临床转移。 结论:联合检测实体肿瘤P53、Rb、P16、和P21基因蛋白表达水平并综合分析这些指标 。

p185、ras p21、p53、p21、Rb蛋白在子宫内膜癌组织中的表达及其意义

目的 研究与细胞周期G1→S调控点相关的p185、rasp2 1、p5 3、p2 1、Rb蛋白在子宫内膜癌组织中的表达及相互间的关系 ,并探讨其临床应用价值。方法 应用LSAB免疫组化法 ,检测 2 1例正常子宫内膜、19例子宫内膜上皮内瘤样病变 (EIN )及45例子宫内膜癌组织中 p185、rasp2 1、p5 3、p2 1、Rb蛋白的表达。 结果 p185、rasp2 1、p5 3蛋白在正常子宫内膜、EIN至内膜癌组织中的表达率依次增高 ,p2 1、Rb蛋白表达率则依次降低 ;在子宫内膜癌组织中 ,p185蛋白表达与rasp2 1蛋白表达呈正相关 (γ =0 .36 3) ,与 p5 3、p2 1表达呈负相关 (γ =-0 .448;γ =-0 .30 3) ,rasp2 1,p5 3蛋白表达与p2 1蛋白表达均呈负相关 (γ =-0 .36 5 ;γ= -0 .32 2 ) ,p2 1蛋白与Rb蛋白表达呈正相关 (γ =0 .441)。p185蛋白的表达与子宫内膜癌病理分级有关 ,单因素分析其为 1个预后保护性因素 (P <0 .0 5 ) ;p5 3蛋白的表达与各临床病理因素均有关 ,其单因素及多因素分析均提示预后差 (P <0 .0 5 )。 结论 与细胞周期G1→S调控相关的 p185、rasp2 1、p5 3、p2 1、Rb蛋白均参与子宫内膜癌的发生、发展过程 ,且部分基因相互关联 ;p5 3可作为其独立的预后因素。

Pien Tze Huang Inhibits Proliferation of Colorectal Cancer Cells through Suppressing PNO1 Expression and Activating p53/p21 Signaling Pathway

ObjectiveTo explore the regulatory effect of Pien Tze Huang (PZH) on targeting partner of NOB1 (PNO1) and it’s down-stream mediators in colorectal cancer (CRC) cells.MethodsQuantitative polymerase chain reaction was performed to determine mRNA levels of PNO1, TP53, and CDKN1A. Western blotting was performed to determine protein levels of PNO1, p53, and p21. HCT-8 cells were transduced with a lentivirus over-expressing PNO1. Colony formation assay was used to detect cell survival in PNO1 overexpression of HCT-8 cells after PZH treatment. Cell-cycle distribution, cell viability and cell apoptosis were performed to identify the effect of PNO1 overexpression on cell proliferation and apoptosis of HCT-8 cells after PZH treatment. Xenograft BALB/c nude mice bearing HCT116 cells transduced with sh-PNO1 or sh-Ctrl lentivirus were evaluated. Western blot assay was performed to detect PNO1, p53, p21 and PCNA expression in tumor sections. Terminal deoxynucleotidyl transferase dUTP nick end labling (TUNEL) assay was used to determine the apoptotic cells in tissues.ResultsPZH treatment decreased cell viability, down-regulated PNO1 expression, and up-regulated p53 and p21 expressions in HCT-8 cells (P<0.05). PNO1 overexpression attenuated the effects of PZH treatment, including the expression of p53 and p21, cell growth, cell viability, cell cycle arrest and cell apoptosis in vitro (P<0.05). PNO1 knockdown eliminated the effects of PZH treatment on tumor growth, inhibiting cell proliferation inhibition and apoptosis induction in vivo (P<0.05). Similarly, PNO1 knockdown attenuated the effects of PZH treatment on the down-regulation of PNO1 and up-regulation of p53 and p21 in vivo (P<0.05).ConclusionThe mechanism by which PZH induces its CRC anti-proliferative effect is at least in part by regulating the expression of PNO1 and its downstream targets p53 and p21.

p53/Rb细胞转导通路相关基因和蛋白在衰老大鼠睾丸组织的表达

目的通过检测p53、Rb、p16、p19、p21在衰老大鼠睾丸组织的表达,探讨p53/Rb细胞转导通路相关基因和蛋白在睾丸组织衰老中的意义。方法选用8周龄雄性SD大鼠30只,体重180～220g,随机分为正常对照组和模型组,每组15只。模型组大鼠采用D-半乳糖连续腹腔注射建立亚急性衰老大鼠模型。采用RT-PCR方法检测p53、Rb基因在大鼠睾丸组织的表达;Western blotting法检测大鼠睾丸磷酸化Rb、p16、p19、p21蛋白的表达。结果模型组睾丸组织p53、Rb基因表达明显高于正常对照组(P<0.01);模型组睾丸组织与正常组比较:p16、p19、p21蛋白的表达明显升高,而磷酸Rb蛋白表达显著下降(P<0.01)。结论Rb/p53细胞转导通路相关基因及蛋白在衰老大鼠睾丸组织发生明显改变,Rb/p53细胞转导通路阻滞可能在睾丸组织的衰老过程中起着一定作用。

Rb2/p130基因编码蛋白在非小细胞肺癌中的表达及临床意义

目的:联合检测Rb2/p130、p53和p21基因编码蛋白在非小细胞肺癌中的表达,分析其与预后的关系。方法:采用免疫组织化学方法,检测87例非小细胞肺癌病人的手术标本Rb2/p130、p53和p21基因编码蛋白的表达,并与病理组织学、临床分期、生存时间进行统计学分析。结果:Rb2/p130和p21蛋白表达阴性者分别为34例(39.1%)和42例(48.3%)。p53蛋白阳性表达者为45例(51.7%)。Rb2/p130表达缺失与肿瘤分化程度、临床分期均呈负相关(P<0.05)。p53表达阳性率与肿瘤分化程度相关(P<0.05),与临床分期无关。p53表达和Rb2/p130表达负相关,同时p53表达阳性和Rb2/p130表达缺失者预后差,术后生存期短(P<0.05)。结论:在非小细胞肺癌中,Rb2/p130表达缺失和p53表达阳性提示恶性程度高,同时检测p53和Rb2/p130的表达对判断非小细胞肺癌的预后可能具有一定的临床意义。

Growth arrest-specific gene 2 suppresses hepatocarcinogenesis by intervention of cell cycle and p53-dependent apoptosis

BACKGROUND Growth arrest-specific gene 2(GAS2)plays a role in modulating in reversible growth arrest cell cycle,apoptosis,and cell survival.GAS2 protein is universally expressed in most normal tissues,particularly in the liver,but is depleted in some tumor tissues.However,the functional mechanisms of GAS2 in hepatocellular carcinoma(HCC)are not fully defined.AIM To investigate the function and mechanism of GAS2 in HCC.METHODS GAS2 expression in clinic liver and HCC specimens was analyzed by real-time PCR and western blotting.Cell proliferation was analyzed by counting,MTS,and colony formation assays.Cell cycle analysis was performed by flow cytometry.Cell apoptosis was investigated by Annexin V apoptosis assay and western blotting.RESULTS GAS2 protein expression was lower in HCC than in normal tissues.Overexpression of GAS2 inhibited the proliferation of HCC cells with wide-type p53,while knockdown of GAS2 promoted the proliferation of hepatocytes(P<0.05).Furthermore,GAS2 overexpression impeded the G1-to-S cell cycle transition and arrested more G1 cells,particularly the elevation of sub G1(P<0.01).Apoptosis induced by GAS2 was dependent on p53,which was increased by etoposide addition.The expression of p53 and apoptosis markers was further enhanced when GAS2 was upregulated,but became diminished upon downregulation of GAS2.In the clinic specimen,GAS2 was downregulated in more than 60%of HCCs.The average fold changes of GAS2 expression in tumor tissues were significantly lower than those in paired non-tumor tissues(P<0.05).CONCLUSION GAS2 plays a vital role in HCC cell proliferation and apoptosis,possibly by regulating the cell cycle and p53-dependent apoptosis pathway.

Network pharmacology analysis combined with experimental verification of the molecular mechanism of Xihuang pill in treating liver cancer

Background:Xihuang pill is a kind of traditional Chinese medicine,which has been widely used in the treatment of kinds of cancer.However,there is still a lack of systematic understanding of the molecular mechanism of Xihuang pill in the treatment of liver cancer.In this work,we aim to explore the molecular mechanism of Xihuang pill in treating liver cancer.Methods:The functional components in Xihuang pill were collected from Traditional Chinese Medicine Database and Analysis Platform.The target genes of these components were also collected using Traditional Chinese Medicine Database and Analysis Platform.The target genes of liver cancer were predicted using GeneCards database.The intersecting genes were then analyzed with Venn diagrams.Kyoto Encyclopedia of Genes and Genomes and Database for Annotation,Visualization,and Integrated Discovery were used to analyze the pathway.Then,cell counting kit-8 was used to measure the half-maximal inhibitory concentration of Xihuang pills.The living dead cell staining method was used to observe the survival of cells.HepG2 cell apoptosis was tested by flow cytometry with fluorescein isothiocyanate/propidium iodide double staining method,and then the mitochondrial damage was also detected by flow cytometry.The expression of target genes was detected by quantitative real-time polymerase chain reaction.Results:A total of 130 compounds and 198 genes were identified as potential active ingredients and putative liver cancer‑related targets.We obtained 1,899 disease targets and 297 transcriptome targets from the database.Six drug-disease intersecting genes,CCNB1,BIRC5,TOP2A,ESR1,IGF2 and IGFBP3 were obtained.They are enrichment in apoptosis,PI3K-AKT signaling pathway,MAPK signaling pathway,pathways in cancer and p53 signaling pathway.Besides,it was found that the apoptosis rate of the HepG2 cells in Xihuang pill treated group was significantly higher than that of the control group.And the apoptosis rate gradually increased in a dose dependent manner of Xihuang pill treatment.Xihuang pill also induced the mitochondrial membrane potential damage.Compared with the control group,the expression level of CCNB1 and BIRC5 was induced,while the expression level of IGF2 was reduced after Xihuang pill treatment.Conclusion:Xihuang pill may act on six proteins(CCNB1,BIRC5,TOP2A,ESR1,IGF2 and IGFBP3)and cover multiple pathways to form a therapeutic network to treat liver cancer.

MAPK/ERK regulation of P53 in human epidermoid carcinoma cell line A431

Objective:To observe the impact of activation and inhibition of mitogen activated protein kinases(MAPK)/extracellular signalregulated protein kinase(ERK)signaling pathway on the proliferation and apoptosis of cutaneous squamous cell carcinoma(SCC).cells and investigate the interaction mechanism between MAPK/ERK signaling pathway and tumor suppressor gene P53 in SCC.Methods:Human A431 cells were cultured and divided into MAPK/ERK inhibition groups with low-,medium-and highconcentration of inhibitors(PD98059+DMSO),MAPK/ERK activation groups with low-,medium-and high-concentration of stimuli(IGF+PBS)and blank control group(DMSO).The cell proliferation in vitro was detected by MTT assay,with the cell apoptosis detected by flow cytometry(FCM)and the protein expression of P-ERK and P53 detected by western blot in each group.Results:The A431 cell proliferation was inhibited by different concentrations of PD98059 with a clear concentration-effect and time-effect relationship(p<.05);and the cell proliferation was promoted by the different concentrations of IGF with a clear concentration-effect and time-effect relationship(p<.05).The FCM results showed a significant increase in the apoptosis rate of A431 cells which were treated with PD98059,with a clear concentration-effect relationship(p<.05);while the apoptosis rate was decreased significantly after A431 cells were treated with IGF,also with a concentration-effect relationship(p<.05).The western blot results showed that the expression of P-ERK protein was decreased but the expression of P53 was increased after A431 cells were treated with PD98059.With the concentration of PD98059 going up,the decrease in P-ERK and the increase in P53 were more significant(p<.05);while the expression of P-ERK protein was increased but the expression of P53 was decreased after A431 cells were treated with IGF.With the concentration of IGF going up,the increase in P-ERK and the decrease in P53 were more significant(p<.05).According to Pearson correlation analysis,the expression of P53 was negatively correlated to that of P-ERK(p<.05).Conclusions:After MAPK/ERK signaling pathway was activated by IGF in A431 cells,the expression of pro-apoptotic factor P53 was decreased with the ability of cell proliferation enhanced and the ability of apoptosis reduced.However,after the inhibition of MAPK/ERK signaling pathway,the expression of pro-apoptotic factor P53 was increased with the ability of cell proliferation reduced and the ability of apoptosis increased.

Extract from Zanthoxylum piperitum Induces Apoptosis of AGS Gastric Cancer Cells through Akt/MDM2/p53 Signaling Pathway

Objective:To determine the effect of Zanthoxylum piperitum extracet(ZPE)on apoptosis and analyze anticancer substances in ZPE,changes in proteins related to apoptosis,and pathological changes in tumors in mouse.Methods:Fifteen 4-week-old female BALB/c nu/nu mice were divided into 3 groups depending on ZPE dose,with 5 in each group.AGS gastric carcinoma cells(1 x 10^(6) cells/200 jxL)were subcutaneously injected into the flank of each mouse.One week after the injection of AGS cells,ZPE was administered to the skin tissue[10 or 50 mg/(kg-d)]in the low-and high-dose groups,respectively for 20 days.Control animals were injected with vehicle only.After 3 weeks,the tumor was extracted and carried out for immunohistochemistry,the tendency of apoptosis and p53 in the body was checked using TdT-mediated dUTP nick-end labeling(TUNEL)assay.For 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,annexin V dead cell staining,cell cycle arrest and Western blotting,AGS gastric carcinoma cells were incubated with various concentrations of ZPE for 24 h.Cell survival rates were analyzed by MTT assays.Apoptosis was analyzed using annexin V dead cell staining and cell cycle arrest and measured using Muse cell analyzer.Results:High performance liquid chromatography(HPLC)analysis showed that ZPE contained organic sulfur compounds such as alliin and S-allylcysteine.MTT assay results revealed that ZPE(10-85»xg/mL)could effectively inhibit the growth of AGS gastric cancer cells at higher concentrations(P<0.05,P<0.01).The annexin V&dead cell staining assay and cell cycle arrest assay confirmed a dose-dependent increase in the apoptosis rate and G!phase in ZPE(10-70 jig/mL)groups.ZPE decreased the expression of anti-apoptotic proteins(p-Akt,p-MDM2,Bcl-2),while increased pro-apoptotic proteins(cleaved PARP,p53,pro-Caspase 3,Bax).TUNEL assays revealed an increase in cell apoptosis.Immunohistochemistry staining confirmed the involvement of p53.Conclusion:ZPE decreases AGS cell proliferation and induces apoptosis by inhibiting Akt and MDM2 expression.

灵芝miRNA对人皮肤成纤维细胞衰老的作用研究

为探究灵芝中miRNA对细胞衰老的干预作用及影响,利用过氧化氢(H_(2)O_(2))诱导人皮肤成纤维细胞(Human skin fibroblasts,HSF)构建了细胞衰老模型,将灵芝特有的miRNA Glu-miR-01和Glu-miR-03转染HSF衰老细胞,分析细胞衰老相关氧化应激指标,并通过Western blotting检测衰老相关蛋白表达水平的变化。结果显示:构建HSF细胞衰老模型的最适条件为H_(2)O_(2)浓度0.8mmol/L,诱导时间3h;Glu-miR-01和Glu-miR-03均能促使衰老细胞中超氧化物歧化酶(Superoxide dismutase,SOD)和总抗氧化能力(Total antioxidant capacity,T-AOC)水平上升,丙二醛(Malondialdehyde,MDA)含量下降,细胞衰老相关β-半乳糖苷酶(Senescence associated-β-Galactosidase,SA-β-gal)染色阳性率降低,线粒体膜电位升高;衰老信号通路中P53、P21和P16蛋白的表达显著降低,Rb蛋白表达显著升高。该结果表明灵芝中Glu-miR-01和Glu-miR-03对HSF衰老细胞具有保护作用,并对衰老相关的p53/p21/Rb信号通路具有调控作用,为阐明灵芝miRNA对细胞衰老的分子作用机制提供了理论依据。