Apoptosis,proliferation and p53 gene expression of H.pylori associated gastric epithelial lesions

AIM: To study the relationship between Helicobacter pylori (H. pylori) and gastric carcinoma and its possible pathogenesis by H. pylori. METHODS: DNEL technique and immunohistochemical technique were used to study the state of apoptosis, proliferation and p53 gene expression. A total of 100 gastric mucosal biopsy specimens, including 20 normal mucosa, 30 H. pylori-negative and 30 H. pylori-positive gastric precancerous lesions along with 20 gastric carcinomas were studied. RESULTS: There were several apoptotic cells in the superficial epithelium and a few proliferative cells within the neck of gastric glands, and no p53 protein expression in normal mucosa. In gastric carcinoma, there were few apoptotic cells, while there were a large number of proliferative cells, and expression of p53 protein significantly was increased. In the phase of metaplasia, the apoptotic index (AI, 4.36%+/-1.95%), proliferative index (PI, 19.11%+/-6.79%) and positivity of p53 expression (46.7%) in H. pylori-positive group were higher than those in normal mucosa (P【0.01). AI in H. pylori-positive group was higher than that in H. pylori-negative group (3.81%+/-1.76%), PI in H. pylori-positive group was higher than that in H. pylori-negative group (12.25%+/-5.63%, P【0.01). In the phase of dysplasia, AI (2.31%+/-1.10%) in H. pylori-positive group was lower (3.05%+/-1.29%) than that in H. pylori-negative group, but PI (33.89%+/-11.65%) was significantly higher (22.09+/-8018%, P【0.01). In phases of metaplasia, dysplasia and gastric cancer in the H. pylori-positive group, AIs had an evidently graduall decreasing trend (P【0.01), while PIs had an evidently gradual increasing trend (P【0.05 or P【0.01), and there was also a trend of gradual increase in the expression of p53 gene. CONCLUSION: In the course of the formation of gastric carcinoma, proliferation of gastric mucosa can be greatly increased by H. pylori, and H. pylori can induce apoptosis in the phase of metaplasia, but in the phase of dysplasia H. pylori can inhibit cellular apoptosis. And H. pylori infection can strengthen the expression of mutated p53 gene.

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.

Down Regulation of Survivin Gene and Up Regulation of p53 Gene expression by siRNA Induces Apoptosis in human Hepatocellular Carcinoma cell Line HepG2

Survivin gene may be a good target for cancer gene therapy because it is over expressed in a variety of human tumors including human hepatocellular carcinoma but not in differen- tiated adult tissues. To explore the effects of the siRNA of survivin gene inducing apoptosis in human hepatocellular cancer cells, three siRNAs cpusiRNA1, cpusiRNA2 and cpusiRNA3 were designed and transferred into human hepatocellular carcinoma cell line HepG2 (HepG2) by lipofection. MTT test showed that the growth of HepG2 decreased when it was transfected with 25nM, 50nM, 100nM, 150nM, 200nM, 400nM siRNA respectively after 48 hours. And the change of mRNA and protein of survivin gene and p53 gene had been detected by RT-PCR and Western blot. Cells presented an increase in apoptosis index was assayed by flow cytometry. Small interfering RNA can exert a knockdown of survivin gene expression and up regulation of p53 gene to induce apoptosis and to inhibit the growth of HepG2.

SUMO-1 Enhancing the p53-induced HepG2 Cell Apoptosis

In order to investigate the effect of small ubiquitin-like modifier-1 (SUMO-1) on the p53-induced HepG2 cell apoptosis, HepG2 cells were transfected by recombinant plasmids as pwtp53, pMDM2 and pSUMO-1 respectively. Western blot was employed to detect the protein expression of the transfected recombinant plasmids and the rate of apoptosis was measured by flow cytometry. The results showed that in cells transfected with pwtp53 and pwtp53+pSUMO-1, the apoptosis rate was (16.79±1.62) % and (18.15±1.36) % respectively, while transfected with pwtp53+pMDM2, the rate was decreased to (5.17±1.23) %. The apoptosis rate was (14.06±1.84) % in the cells transfected with pwtp53+pMDM2+pSUMO-1, significantly higher than that in the cells Transfected with pwtp53+pMDM2 (P<0.01). The apoptosis rates in the cells were all less than 2 % and had no significant difference among the groups. It was suggested that in the HepG2 cells, SUMO-1 can increase the apoptosis induced by wild-type p53 through binding to p53 protein, post-translational modification and inhibiting the p53 degradation by MDM2.

Inhibitory effect of tumor suppressor p33^(ING1b) and its synergy with p53 gene in hepatocellular carcinoma

AIM: To investigate the inhibitory effect of tumor suppressor p33ING1b and its synergy with p53 gene in hepatocellular carcinoma (HCC).METHODS: Recombinant sense and antisense p33ING1b plasmids were transfected into hepatoma cell line HepG2 with lipofectamine. Apoptosis, G0/G1 arrest, cell growth rate and cloning efficiency in soft agar of HepG2 were analyzed after transfection. In three hepatoma cell lineswith different endogenous p53 gene expressions, the synergistic effect of p33ING1b with p53 was analyzed by flow cytometry and luciferase assay was performed to detect the activation of p53 downstream gene p21WAF1/CIP1. In addition, the expression and mutation rates of p33ING1b in HCC tissues were measured by immunohistochemistry and polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP).RESULTS: Overexpression of p33ING1b inhibited cell growth of HepG2, induced more apoptosis and protected cells from growth in soft agar. Combined transfer of p33ING1b and p53 gene promoted hepatoma cell apoptosis, G0/G1 arrest and elevated expression of p21WAF1/CIP1. Immunostaining results showed co-localized P33ING1b with P53 protein in HCC tissues and there was a significant relation between protein expression rates of these two genes (P＜0.01).Among 28 HCC samples, p33ING1b presented a low gene mutation rate (7.1%).CONCLUSION: p33ING1b collaborates with p53 in cell growth inhibition, cell cycle arrest and apoptosis in HCC. Loss or inactivation of p33ING1b normal function may be an important mechanism for the development of HCC retaining wildtype p53.

ADENOVIRUS-MEDIATED P53 GENE TRANSFER INCREASES THE THERMOSENSITIVITY OF HUMAN GASTRIC CARCINOMA CELL LINES(IN VITRO AND IN VIVO)

Objective: To evaluate the effect of adenovirus-mediated p53 gene (Adp53) on apoptosis andradiosensitivity of human gastric carcinoma cell lines.Methods: Recombinant adenovirus expressing wild-type p53 gene was transferred into four human gastric carcinoma cell lines with different p53 genetic status. p53 proteinexpression was detected by immunohistochemistry assayand western blot assay. Cell survival was assessed using a clonogenic assay. TUNEL assay was used in determination of apoptosis. Four human gastric carcinoma cells infectedwith Adp53 were irradiated with 4Gy and cell cycle distribution and Sub-G1 peak were assayed by flowcytometry. Results: G2/M arrest, apoptosis and inhibition of tumor cell proliferation were induced by infection atAdp53 at 100 MOI which caused high transfer rate ofwild-type p53 and strong expression of p53 protein in four human gastric carcinoma cells. The radio-enhancement ratio of Adp53 at 4Gy were 3.0 for W cell, 3.6 for M cell, 2.2 for neo cell and 2.5 for 823 cell in vitro. Conclusion: Thisstudy demonstrated that Adp53 transfer increased cellularapoptosis and radiosensitivity of human gastric carcinoma cell lines in vitro independently on cellular intrinsic p53status thus supporting the combination of p53 gene therapy with radiotherapy in clinical trials.

RADIATION-INDUCED APOPTOSIS OF TWO NASOPHARANGEALCARCINOMA CELL LINES

Objective: To study apoptosis induced by radiation in two nasopharyngeal carcinoma (NPC) cell lines, CNE and CNE-2. Methods: Hoechst 33342 staining, immuno-histochemical staining, RT-PCR, DNA dot blotting and Southern blotting were used to identify apoptosis. Results: A single dose of X-irradiation resulted in apoptosis, the apoptotic index (AI) was time- and dose-dependent. Different apoptotic responses existed in the two cell lines. Immunohistochemical staining showed that bcl-2 protein was strongly positive in CNE but negative in CNE-2. However, RT-PCR revealed p53 mRNA in CNE-2 but not in CNE. P53 and bcl-2 genes were both present in the two cell lines as shown by DNA blotting, but the 2.8 kb fragment of the p53 gene was much lower than the 5.6 kb fragment on CNE which was clearly shown in Southern hybridization, suggestive of partial deletion of p53 gene in CNE. Conclusion: Apoptotic response to radiation is different in two NPC cell lines. CNE is more radioresistant than CNE-2. Overexpression of bcl-2 protein and partial deletion of p53 gene may explain their difference in radiosensitivity.

Detection of apoptotic cells and immunohistochemical study of bcl-2 and p53 gene protein in primary gastric mucosa-associated lymphoid tissue (MALT) lymphoma

To identify the apoptotic cells in gastric MALT lymphoma and its relationship between bcl-2 and p53 gene expression. Methods: TdT-mediated dUTP biotin Nick End labeling (TUNEL) and immuno-histochemistry ABC method were used to display apoptotic cells and the gene protein expression of bcl-2 and p53 independently. Results: Apoptotic indices (AI) in high-grade MALT lymphomas were significantly higher than in mixed-grade group and low-grade group (P<0.05). Bcl-2 was expressed in 83% of low-grade tumors, 61.6% of the median-grade tumors and 43.7% of high-grade tumors. An inverse correlation was observed between the expression of bcl-2 and apoptotic indices. Only 27 cases were p53 positive. The frequency of p53 positivity was significantly increased as the histologic grade advanced (P<0.05). There was also an inverse correlation between the expression of bcl-2 and p53. Conclusion: Apoptosis may be important in tumors development and transmission. p53 and bcl-2 were important regulatory genes of apoptosis and may be associated with transformation from low- grade to high-grade lymphomas.

康尔爱片诱导人胃腺癌SGC-7901细胞凋亡及对P^(53)基因表达影响的研究

目的 :观察康尔爱片诱导人胃腺癌SGC - 90 1细胞凋亡及对P5 3基因表达的影响。方法 :应用流式细胞仪检测康尔爱片体外给药后人胃腺癌SGC - 790 1细胞DNA含量变化和P5 3基因表达情况。结果 :康尔爱片中、大剂量组细胞凋亡百分率分别为 9.0 6 %、96 .32 % ,与对照组相比有明显差异 (P <0 .0 1) ;小、中、大 3个剂量组P5 3基因表达率分别为 95 .0 9%、89.47%、5 2 .0 4% ,与对照组相比有明显差异 (P <0 .0 1)。结论 :康尔爱片中、大剂量组可以明显诱导人胃腺癌SGC - 790 1细胞凋亡 ,且小、中、大 3个剂量组均可降低突变型P5 3基因表达率。

胰腺癌Bcl-2,P53蛋白表达和细胞凋亡

目的 探讨ｂｃｌ２ ，ｐ５３ 基因和细胞凋亡在胰腺癌发病机制中的作用以及它们之间相互关系．方法 应用ＡＢＣ 免疫组化技术检测５０ 例胰腺癌中Ｂｃｌ２ 和Ｐ５３ 蛋白表达，运用原位末端标记法观察肿瘤中细胞凋亡数量．结果 Ｐ５３ 蛋白表达阳性率为５４ ％ ，临床Ⅰ期阳性率（２６－７ ％ ）却显著低于Ⅱ期（６１－１ ％ ） 和Ⅲ＋ Ⅳ期（７０－６ ％ ，Ｐ＜ ０－０５） ；Ｂｃｌ２蛋白表达阳性率为６４ ％ ，临床Ⅰ期阳性率（９３－３ ％ ） ，显著高于Ⅱ期（５５－６ ％ ） 和Ⅲ＋ Ⅳ期（４７－１ ％ ，Ｐ＜ ０－０５） ；组织学Ⅲ级癌细胞中凋亡指数明显高于Ⅰ，Ⅱ级（ Ｐ＜ ０－０５） ，Ｂｃｌ２ 蛋白阴性病例中凋亡指数明显高于Ｂｃｌ２ 阳性者（ Ｐ＜ ０－０１） ．结论 Ｂｃｌ２ 是通过抑制细胞凋亡参与肿瘤的生长过程，Ｂｃｌ２和Ｐ５３ 蛋白表达之间 存在密切负相关（τ＝ － ０－１７４７ ，Ｐ＜ ０－０５） ．

腺病毒介导的p53和顺铂的联合应用对胆管癌细胞系QBC939的生长抑制作用

目的 :研究 p5 3基因和顺铂联合应用对胆管癌细胞的作用。方法 :将重组体腺病毒p5 3和顺铂联合作用于人胆管癌细胞QBC939,对 p5 3基因的表达、细胞的生长抑制及机制进行分析。结果 :用Ad -LacZ进行重组腺病毒转导效率的检测 ,发现当MOI为 10 0以上时 ,重组腺病毒可使 90 %以上的培养的人胆管癌QBC939细胞被传导。用RT -PCR方法检测 ,在胆管癌QBC939细胞系中 p5 3无表达。重组体腺病毒能介导外源基因 p5 3在胆管癌QBC939细胞系中高效表达。重组体腺病毒介导的 p5 3在QBC939细胞中表达 ,能抑制QBC939细胞的生长和集落形成。其与顺铂联合应用对QBC939细胞的生长抑制具有明显作用。流式细胞计数证实 p5 3能诱导QBC939细胞发生凋亡并导致其发生G1期阻滞 ,顺铂能诱导QBC939细胞发生凋亡并导致细胞发生明显的G2期阻滞。结论 :p5

胰腺癌P^(53)与细胞凋亡相关性研究

目的研究胰腺癌P53 基因表达与细胞凋亡的关系及其临床意义。方法采用原位DNA末端标记技术和SP免疫组织化学方法分别对 15 0例异常和正常胰腺组织石蜡包埋标本 (其中包括 97例胰腺癌、32例胰腺炎和 2 1例正常胰腺组织 )进行P53 基因表达和细胞凋亡检测。结果 (1)P53 基因阳性表达率在胰腺癌、胰腺炎和正常胰腺组织中分别为 5 9 8%、3 1%和 0 % ,P53 基因表达在胰腺癌组织中明显高于胰腺炎和正常胰腺组织 (P <0 0 5 ) ,细胞凋亡平均指数分别为 13 15± 8 3、14 2 1± 9 0和 11 6 7± 7 9,三种组织内细胞凋亡指数无显著性差异 (P >0 0 5 )。 (2 )P53 基因表达与肿瘤分化程度、转移和TNM分期相关 ,而与病人年龄、性别、肿瘤部位和大小无关。低分化、转移和TNMⅢ与Ⅳ期肿瘤细胞的P53 基因阳性表达率显著高于高分化、未转移和TNMⅠ与Ⅱ期肿瘤病人的胰腺癌细胞P53 基因阳性率 (P <0 0 5 ) ,P53 基因阳性表达胰腺癌患者术后平均生存期为 2 8 7± 6 9个月 ,明显低于P53 基因阴性表达患者 (39 5± 3 4个月 ) ,P <0 0 5。高分化、TNMⅠ与Ⅱ期肿瘤细胞凋亡指数明显高于低分化和TNMⅢ期与Ⅳ期病人细胞凋亡指数 (P <0 0 5 )。 (3)胰腺癌P53 基因表达与细胞凋亡指数呈负相关关系 (r = 0 5 0 16 ,P <0 0 5 )。

逆转录病毒介导野生型P^53与反义mdm2融合基因对Mc3细胞凋亡的影响

目的探讨P^53与反义mdm2 cDNA序列真核表达载体诱导人黏液表皮样癌高转移细胞株Mc3细胞凋亡的影响。方法将P^53与反义mdm2 cDNA序列真核表达载体通过脂质体转染Mc3细胞，采用形态学观察凋亡细胞；琼脂糖凝胶电泳检测“梯状条带”；末端转移酶标记法（TUNEL）检测凋亡指数；透射电镜检测凋亡小体。结果转染48h后形态学观察到细胞体积缩小；核浓染碎裂成大小不等的碎片；核染色质成新月形，琼脂糖凝胶电泳可见180～200bp典型的凋亡带，TUNEL检测凋亡指数为25．22±1．01（转染后），与对照组2．01±1．10（转染前）比较差异有统计学意义（P〈0．05），转染空载体（2．02±1．11）与对照组比较差异无统计学意义（P〉0．05），透射电镜可见染色质浓集分布不均，形成由核膜包裹断裂的核碎片的凋亡小体。结论P^53与反义mdm2基因融合体真核表达载体能诱导和促使体外培养的人黏液表皮样癌高转移细胞株Mc3细胞发生凋亡。

人骨肉瘤细胞凋亡及P^(53)PCNA的表达研究

目的：研究人骨肉瘤细胞凋亡及P53、PCNA的表达情况，并初步探讨其临床及生物学意义。方法：采用原位DNA末端标记法及免疫组化方法检测了39例骨肉瘤中细胞凋亡及P53、PCNA的表达情况，并统计其阳性反应率。结果：本组病例中细胞凋亡阳性率61．5％，P53呈阳性反应29例，阳性率74．3％，PCNA呈阳性反应34例，阳性率87．2％。结论：骨肉瘤组织中有P53基因和PCNA的高表达，与肿瘤的预后相关。在骨肉瘤中细胞凋亡是一种自然现象，其发生情况各不相同，研究初步提示了其与预后相关，并应与P53蛋白和PCNA的表达相结合而综合判断。

The effect of adenovirus expressing wild-type p53 on 5-fluorouracil chemosensitivity is related to p53 status in pancreatic cancer cell lines

AIM:There are conflicting data about p53 function on cellular sensitivity to the cytotoxic action of 5-fluorouracil (5-FU). Therefore the objective of this study was to determine the combined effects of adenovirus-mediated wild-type (wt) p53 gene transfer and 5-FU chemotherapy on pancreatic cancer cells with different p53 gene status. METHODS:Human pancreatic cancer cell lines Capan-1^(p53mut), Capan-2^(p53wt),FAMPAC^(p53mut),PANC1^(p53mut),and rat pancreatic cancer cell lines AS^(p53wt) and DSL6A^(p53null) were used for in vitro studies.Following infection with different ratios of Ad- p53-particles (MOI) in combination with 5-FU,proliferation of tumor cells and apoptosis were quantified by cell proliferation assay (WST-1) and FACS (PI-staining).In addition,DSL6A syngeneic pancreatic tumor cells were inoculated subcutaneously in to Lewis rats for in vivo studies. Tumor size,apoptosis (TUNEL) and survival were determined. RESULTS:Ad-p53 gene transfer combined with 5-FU significantly inhibited tumor cell proliferation and substantially enhanced apoptosis in all four cell lines with an alteration in the p53 gene compared to those two cell lines containing wt-p53.In vivo experiments showed the most effective tumor regression in animals treated with Ad-p53 plus 5-FU.Both in vitro and in vivo analyses revealed that a sublethal dose of Ad-p53 augmented the apoptotic response induced by 5-FU. CONCLUSION:Our results suggest that Ad-p53 may synergistically enhance 5-FU-chemosensitivity most strikingly in pancreatic cancer cells lacking p53 function.These findings illustrate that the anticancer efficacy of this combination treatment is dependent on the p53 gene status of the target tumor cells.

Effect of basic fibroblast growth factor and danshen on bcl-2 and p53 mRNA expression in the brain of rats exposed to repeated, high, positive acceleration (+Gz)

BACKGROUND： Both animal experiments and clinical studies have shown that basic fibroblast growth factor （bFGF） and danshen （Salvia miltiorrhiza） can exhibit protective effects on ischemia-reperfusion cerebral injury. OBJECTIVE： To test whether bFGF and danshen can protect cerebral injury induced by exposure to repeated, high, positive acceleration （＋Gz） in an animal model and to analyze the possible mechanisms. DESIGN, TIME AND SETTING： Randomized controlled animal study. The experiment was performed at the Research Center for Molecular Biology, Air-force General Hospital of Chinese PLA from April to August 2000. MATERIALS： A total of 20 clean grade, healthy, Sprague Dawley rats of both genders, weighing （200 ± 15） g, were provided by our experimental animal center. Rats were randomly divided into 5 groups： the control group, ＋Gz exposure group, bFGF group, danshen group, and saline group, with 4 animals per group. bFGF （Beijing Bailuyuan Biotechnology Co. Ltd.） and danshen solution （Shanghai Zhongxi Pharmaceutical Co. Ltd.） were used. METHODS： All rats were fixed on a rotary arm of a centrifugal apparatus （2 m in radius） with their heads oriented towards the center of the apparatus. Except for rats in the control group, the value of ＋Gz exposure was ＋14 Gz with an acceleration rate of 1.5 G/s. The peak force lasted for 45 seconds. ＋Gz exposure was performed three times with intervals of 30 minutes. Rats in the control group received the same ＋Gz procedure, but the G value was ＋1 Gz. Rats in bFGF group and danshen group were intraperitoneally injected with 100 μg/kg bFGF or 15 g/kg danshen solution, respectively, at 30 minutes prior to centrifugation and immediately after centrifugation. Rats in saline group were injected with the same volume of saline. Six hours after exposure, rats were decapitated. One hemisphere was preserved in liquid nitrogen for RNA extraction and the other was processed for apoptosis detection. MAIN OUTCOME MEASURES： mRNA levels of bcl-2 and p53 were measured by semi-quantitative reverse-transcription polymerase chain reaction. Apoptotic cell death was detected by terminal deoxynuleotidyl transferase-mediated dUTP nick end labeling. RESULTS： Changes in mRNA expression of bcl-2 and p53 and apoptotic cells were observed in rat brain six hours after repeated ＋Gz exposures, bFGF and danshen were able block the changes of bcl-2 and p53 expression and inhibit apoptotic cell death. CONCLUSION： The data suggest that apoptosis and changes in bcl-2 and p53 expression in the rat brain can be induced by repeated ＋Gz exposures. Apoptosis is, therefore, one of the molecular mechanisms of brain damage induced by repeated ＋Gz exposures, bFGF and danshen were of the equal potency in preventing brain injury induced by repeated ＋Gz exposures.

2-hydroxy-3-methyl anthraquinone promotes apoptosis and inhibits invasion of human hepatocellular carcinoma cells by targeting nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-1/cellular tumor antigen p53 signaling pathway

OBJECTIVE: To investigate the anti-liver cancer effect of 2-hydroxy-3-methyl anthraquinone(HMA) and the specific mechanism based on nicotinamide adenine dinucleotidedependent protein deacetylase sirtuin-1(SIRT1)/cellular tumor antigen p53(p53) pathway. METHODS: Cell counting kit-8 method was used to observe the effect of HMA on the activity of human hepatocellular carcinoma cells(Hep G2) cells. At 72 h and 80 μL HMA, the apoptosis rate of Hep G2 cells in each group was measured by flow cytometry. Transwell was used to assay for cell invasion. The protein expression levels of SIRT1, p53, B-cell lymphoma-2(Bcl-2), Bcl-2 associated X protein(Bax), caspase-9(CASP9) and caspase-3(CASP3) were detected by Western Blot. RESULTS: HMA significantly inhibited the proliferation of Hep G2 cells, The half inhibiting concentration(IC50) of the HMA at 24, 48 and 72 h were examined and it were 126.3, 98.6, and 80.55 μM, respectively. Compared with the control group, the apoptosis rate of HMA, Selisistat(EX527), and HMA+ EX527 groups enhanced, while the apoptosis rate of SRT1720 diminished, demonstrating that inhibition of SIRT1 can lead to apoptosis of Hep G2 cells. HMA+ EX527 group had the highest apoptosis rate, the lowest expression of SIRT1 and Bcl-2, and the highest expression of p53, Bax, CASP9 and CASP3. The number of invasions of Hep G2 was significantly reduced after HMA and EX527 intervened. Western blot shows HMA could inhibit SIRT1, promote the expression of p53, and decrease the ratio of Bcl-2/Bax. CONCLUSIONS: HMA induced apoptosis in Hep G2 cells, while inhibiting proliferation and invasion. The mechanism of HMA against HCC may be related to the SIRT1/p53 pathway.

The prognostic molecular markers in hepatocellular carcinoma

The prognosis of hepatocellular carcinoma (HCC) still remains dismal, although many advances in its clinical study have been made. It is important for tumor control to identify the factors that predispose patients to death. With new discoveries in cancer biology, the pathological and biological prognostic factors of HCC have been studied quite extensively. Analyzing molecular markers (biomarkers) with prognostic significance is a complementary method. A large number of molecular factors have been shown to associate with the invasiveness of HCC, and have potential prognostic significance. One important aspect is the analysis of molecular markers for the cellular malignancy phenotype. These include alterations in DNA ploidy, cellular proliferation markers (PCNA, Ki-67, Mcm2, MIB1, MIA, and CSE1L/CAS protein), nuclear morphology, the p53 gene and its related molecule MD M2, other cell cycle regulators (cyclin A, cyclin D, cyclin E, cdc2, p27, p73), oncogenes and their receptors (such as ras, c-myc, c-fms, HGF, c-met, and erb-B receptor family members), apoptosis related factors (Fas and FasL), as well as telomerase activity. Another important aspect is the analysis of molecular markers involved in the process of cancer invasion and metastasis. Adhesion molecules (E-cadherin, catenins, serum intercellular adhesion molecule-1, CD44 variants), proteinases involved in the degradation of extracellular matrix (MMP-2, MMP-9, uPA, uPAR, PAI), as well as other molecules have been regarded as biomarkers for the malignant phenotype of HCC, and are related to prognosis and therapeutic outcomes. Tumor angiogenesis is critical to both the growth and metastasis of cancers including HCC, and has drawn much attention in recent years. Many angiogenesis-related markers, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived endothelial cell growth factor (PD-ECGF), thrombospondin (TSP), angiogenin, pleiotrophin, and endostatin (ES) levels, as well as intratumor microvessel density (MVD) have been evaluated and found to be of prognostic significance. Body fluid (particularly blood and urinary) testing for biomarkers is easily accessible and useful in clinical patients. The prognostic significance of circulating DNA in plasma or serum, and its genetic alterations in HCC are other important trends. More attention should be paid to these two areas in future. As the progress of the human genome project advances, so does a clearer understanding of tumor biology, and more and more new prognostic markers with high sensitivity and specificity will be found and used in clinical assays. However, the combination of some items, i.e., the pathological features and some biomarkers mentioned above, seems to be more practical for now.

Huanglian decoction suppresses the growth of hepatocellular carcinoma cells by reducing CCNB1 expression

BACKGROUND Hepatocellular carcinoma(HCC)is one of the most prevalent cancers in human populations worldwide.Huanglian decoction is one of the most important Chinese medicine formulas,with the potential to treat cancer.AIM To investigate the role and mechanism of Huanglian decoction on HCC cells.METHODS To identify differentially expressed genes(DEGs),we downloaded gene expression profile data from The Cancer Genome Atlas Liver Hepatocellular Carcinoma and Gene Expression Omnibus(GSE45436)databases.We obtained phytochemicals of the four herbs of Huanglian decoction from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform.We also established a regulatory network of DEGs and drug target genes and subsequently analyzed key genes using bioinformatics approaches.Furthermore,we conducted in vitro experiments to explore the effect of Huanglian decoction and to verify the predictions.In particular,the CCNB1 gene was knocked down to verify the primary target of this decoction.Through the identification of the expression levels of key proteins,we determined the primary mechanism of Huanglian decoction in HCC.RESULTS Based on the results of the network pharmacological analysis,we revealed 5 bioactive compounds in Huanglian decoction that act on HCC.In addition,a protein-protein interaction network analysis of the target genes of these five compounds as well as expression and prognosis analyses were performed in tumors.CCNB1 was confirmed to be the primary gene that may be highly expressed in tumors and was significantly associated with a worse prognosis.We also noted that CCNB1 may serve as an independent prognostic indicator in HCC.Moreover,in vitro experiments demonstrated that Huanglian decoction significantly inhibited the growth,migration,and invasiveness of HCC cells and induced cell apoptosis and G2/M phase arrest.Further analysis showed that the decoction may inhibit the growth of HCC cells by downregulating the CCNB1 expression level.After Huanglian decoction treatment,the expression levels of Bax,caspase 3,caspase 9,p21 and p53 in HCC cells were increased,while the expression of CDK1 and CCNB1 was significantly decreased.The p53 signaling pathway was also found to play an important role in this process.CONCLUSION Huanglian decoction has a significant inhibitory effect on HCC cells.CCNB1 is a potential therapeutic target in HCC.Further analysis showed that Huanglian decoction can inhibit HCC cell growth by downregulating the expression of CCNB1 to activate the p53 signaling pathway.

老年性白内障晶状体上皮细胞凋亡及相关基因蛋白的表达

目的 探讨老年性白内障与晶状体上皮细胞凋亡的关系。方法 透射电镜下观察老年性白内障晶状体上皮细胞的超微结构 ;Tunel法检测凋亡细胞百分率 ;并对其晶状体上皮细胞DNA进行琼脂糖凝胶电泳 ;免疫组化法检测P5 3 、bcl 2在老年性白内障晶状体上皮细胞中的蛋白表达。结果 透射电镜下发现老年性白内障晶状体上皮细胞中有凋亡细胞 ;凋亡细胞百分率为 8 4%～ 3 7 8% ,琼脂糖凝胶电泳出现梯状条带 ;P5 3 在老年白内障晶状体上皮细胞中蛋白表达率为 16 9%～ 19 1% ,bcl 2无蛋白表达。