Antitumor activity of 2'-deoxy-2'-methylidenecytidine, a new 2'-deoxycytidine derivative

The antitumor activity of 2'-deoxy-2'-methylidenecytidine (DMDC), an inhibitor of DNA synthesis, was examined and compared with that of 1-beta-D-arabinofuranosylcytosine (ara-C) against various murine tumors and human tumor xenografts. Against P388 murine leukemia, repeated treatments of DMDC were more effective than its single administration. Interestingly, DMDC was effective against colon 26 murine carcinoma, M5076 murine reticulum cell sarcoma, LX-1 human lung cancer xenograft, and SK-Mel-28 human melanoma xenograft, which are less sensitive or refractory to ara-C, while DMDC was not more potent against murine leukemias P388 and L1210 than ara-C. The in vitro cytotoxic effects of DMDC and ara-C against L1210 leukemia cells were prevented dose dependently by deoxycytidine, suggesting that DMDC, like ara-C, may require phosphorylation by deoxycytidine kinase for antitumor activity. DMDC was effective against human and murine experimental tumor models, especially nonleukemic tumors refractory to ara-C, suggesting that DMDC will be a promising agent for the treatment of cancer.     

转染和干扰Runx2基因对K7M2细胞的影响

 目的探讨干扰和转染Runx2基因后对K7M2骨肉瘤细胞体外生物学活性的影响。方法分别将阴性对照质粒、携带Runx2和siRNA Runx2的质粒转染到K7M2细胞中。应用Western blot检测转染和干扰Runx2基因对其蛋白表达的影响,并检测细胞体外的增殖能力(MTT )、侵袭性、迁移性和黏附性的变化,并应用流式细胞仪（FCM）检测相应细胞的凋亡率和细胞周期分布的变化。结果与空白对照组比,转染Runx2和siRunx2组的K7M2细胞中Runx2蛋白表达分别明显增强和减弱,而阴性对照质粒组没有明显改变。转染siRunx2组的细胞,MTT结果显示增殖能力明显下降,迁移性、侵袭性和增殖指数（PI）也明显下降,与空白对照组比差异有统计学意义（P<0.05）,黏附性和凋亡率却没有明显变化。转染Runx2组K7M2的细胞增殖能力、迁移性、侵袭性和PI明显增高,与空白对照组比差异有统计学意义（P<0.05）,黏附性和凋亡率没有明显变化。阴性对照组和空白对照组比上述指标没有明显改变。结论Runx2可以促进K7M2细胞增殖,增强细胞的侵袭、迁移能力,其机制可能是和改变细胞周期的分布,使细胞更快的进入G₂/M期有关。     

Identification of CYP2C9*2 Allele in HepG2 Cell Line

Background

Hepatoma is caused by many factors including alcohol, chemicals, viral infection, and chronic inflammation. Cytochrome P450 polymorphism plays an important role in its pathogenesis. CYP2D6, CYP2E1, and CYP1A1 have been identified to be related with hepatic carcinogenesis and tumor size and stage. However, no studies have been performed on CYP2C9, a major CYP in the liver and hepatoma.

Aim of the study

To identify if there is polymorphism of CYP2C9 in a HepG2 cell line.

Methods

A pair of primers was used to clone CYP2C9 exon 3 region and subsequently sequenced. The sequence was compared to normal CYP2C9 for identification of any mutation.

Results

A point mutation was identified. It was located in the amino acid number 144 of CYP2C9 protein with the change of normal amino acid arginine into cysteine, which is the same as identified in poor metabolism patients as homozygous CYP2C9*2.

Conclusions

There is a mutation (CYP2C9*2/ CYP2C9*2) in a HepG2 cell line. Thus, polymorphism of CYP2C9 may also be involved in the carcinogenesis of hepatoma as CYPs2D6 and 2E1.     

Cyclooxygenase-2 (COX-2)-independent anticarcinogenic effects of selective COX-2 inhibitors

Nonsteroidal antiinflammatory drugs (NSAIDs) appear to reduce the risk of developing cancer. One mechanism through which NSAIDs act to reduce carcinogenesis is to inhibit the activity of cyclooxygenase-2 (COX-2), an enzyme that is overexpressed in various cancer tissues. Overexpression of COX-2 increases cell proliferation and inhibits apoptosis. However, selective COX-2 inhibitors can also act through COX-independent mechanisms. In this review, we describe the COX-2-independent molecular targets of these COX-2 inhibitors and discuss how these targets may be involved in the anticarcinogenic activities of these selective COX-2 inhibitors. We also compare the concentrations of these inhibitors used in in vitro and in vivo experiments and discuss the implications of the in vitro studies for clinical management of cancer with these drugs.     

Evaluation of the antitumor activity of gemcitabine (2',2'-difluoro-2'-deoxycytidine)

A new pyrimidine antimetabolite, 2',2'-difluorodeoxycytidine, Gemcitabine (LY188011, dFdCyd) has been synthesized and evaluated in experimental tumor models. dFdCyd is a very potent and specific deoxycytidine analogue. The concentration required for 50% inhibition of growth is 1 ng/ml in the CCRF-CEM human leukemia cell culture assay. Concurrent addition of deoxycytidine to the cell culture system provides about a 1000-fold decrease in biological activity. The inhibition of growth of human leukemia cells in culture led to the in vivo evaluation of this compound as a potential oncolytic agent. Maximal activity in vivo was seen with dFdCyd when administered on an every third day schedule. 1-beta-D-Arabinofuranosylcytosine, administered on a daily for 10-day schedule, was directly compared to dFdCyd in this evaluation. dFdCyd demonstrated good to excellent antitumor activity in eight of the eight murine tumor models evaluated. 1-beta-D-Arabinofuranosylcytosine was substantially less active or had no activity in these same tumor models. This in vivo activity against murine solid tumors supports the conclusion that dFdCyd is an excellent candidate for clinical trials in the treatment of cancer.     

Cell cycle-dependent phosphorylation of Disabled-2 by cdc2

Disabled-2 (Dab2; also known as p96 and DOC-2) is a signal transduction protein that has been implicated in the control of cell growth. Dab2 is known to be a phosphoprotein, but little is known about the kinases that phosphorylate Dab2. We have found that Dab2 phosphorylation is markedly increased during the mitosis phase of the cell cycle. This phosphorylation is blocked by roscovitine, a selective inhibitor of cyclin-dependent kinases. Dab2 robustly coimmunoprecipitates from cells with the cyclin-dependent kinase cdc2, and purified cdc2 can phosphorylate purified Dab2 fusion proteins in vitro on multiple sites. Cellular phosphorylation of Dab2 by cdc2 promotes the association of Dab2 with Pin1, a peptidylprolyl isomerase that regulates the rate of Dab2 dephosphorylation. These findings reveal that Dab2 is differentially phosphorylated during the cell cycle by cdc2 and provide a potential feedback mechanism by which Dab2 inhibition of cell growth and proliferation may be regulated.     

Polymorphisms in prostaglandin synthase 2/cyclooxygenase 2 (PTGS2/COX2) and risk of colorectal cancer

Inflammation plays a key role in the development of colorectal cancers. We have investigated the relationship between PTGS2 (COX2) polymorphisms and colorectal cancer risk in a hospital based case-control study. We recruited 292 patients with colorectal cancer and 274 controls from new patients admitted to Bellvitge Hospital, Barcelona, Spain, from 1996 to 1998. Subjects responded to a questionnaire on risk factors. Genotypes of the eight more frequent polymorphisms of PTGS2 were determined. Two polymorphisms are located in the promoter sequence, one in the untranslated region of exon 1, one in exon 3, one in intron 5, two in the untranslated region of exon 10, and one downstream of the last polyadenylation (poly-A) signal. Associations were analysed with logistic regression models assuming a dominant effect for rare variants to increase statistical power. An association was detected between colorectal cancer and a polymorphism in the untranslated region of exon 10 of PTGS2, with an odds ratio (OR) of 2.49, 95% confidence interval (CI) of 1.17-5.32, P=0.01. A nearby polymorphism downstream of the last poly-A signal also showed a nonsignificant increase in risk (OR 2.17, 95% CI 0.99-4.78, P=0.05). Analysis of haplotypes confirmed that individuals with these variants were at increased risk of colorectal cancer (OR compared to the most frequent haplotype: 2.17, 95% CI 0.97-4.84, P=0.06) Interactions between PTGS2 genotype and use of nonsteroidal anti-inflammatory drugs and risk of colorectal cancer were also explored.     

MMP-2、TIMP-2在口腔鳞状细胞癌中表达的临床病理研究

[目的]研究口腔鳞状细胞癌（OSCC）组织中基质金属蛋白酶-2（MMP-2），基质金属蛋白酶组织抑制因子-2（TIMP-2）的表达。[方法]采用免疫组化SP法检测60例OSCC、35例非典型增生、20例止常口腔黏膜中MMP-2、TIMP-2的表达情况。[结果]OSCC中MMP-2、TIMP-2的表达均高于非典划增生组（P〈0．05），在非典型增生组中表达高于正常口腔黏膜组（R＜0．05）。MMP-2的表达与肿瘤细胞分化程度、淋巴结转移密切相关（P〈0．05），与年龄、肿瘤大小、TNM分期无关（P〉0．05）。TIMP-2与肿瘤的淋巴结转移密切相关（P〈0．05），与年龄、肿瘤大小、肿瘤细胞分化程度、TNM分期无关（P〉0．05）。淋巴结转移组中MMP-2／TIMP-2比值显著高于无淋巴结转移组（P〈0．05）。[结论]MMP-2、TIMP-2是OSCC转移的重要生物学标志，MMP-／TIMP-2比值对评估淋巴结转移的潜能有重要意义。     

Interference of E2-2-mediated effect in endothelial cells by FAM96B through its limited expression of E2-2

The basic helix–loop–helix protein E2-2 is known to play a role in quiescence of endothelial cells (ECs). However, it is unclear how the activity of E2-2 is controlled in the cells. In this study, we identified FAM96B as an interaction partner of E2-2. FAM96B interfered with E2-2-mediated effects on luciferase reporter activities. Furthermore, the suppression of vascular endothelial growth factor receptor 2 promoter activity by E2-2 was rescued by the expression of FAM96B in a dose-dependent manner. Interestingly, FAM96B decreased the expression of ectopic and endogenous E2-2 proteins. Mutational analysis revealed that the middle region of FAM96B is required for the limited expression of E2-2 protein. When FAM96B was expressed in ECs, the EC migration, proliferation, and tube formation were potentiated. Taken together, these findings suggest that FAM96B acts as a regulator of E2-2 through the control of its protein expression.     

The role of cell cycle progression in radiosensitization by 2',2'-difluoro-2'-deoxycytidine

Gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdCyd) has been shown to be a potent radiosensitizer in tumor cells both in vitro and in vivo. We evaluated the ability of dFdCyd to enhance the radiosensitivity of two human glioblastoma cell lines. The results demonstrated that U251 cells were more sensitive to the cytotoxicity of dFdCyd, and that dFdCyd was able to radiosensitize these cells. In contrast, D54 cells were more resistant to the cytotoxic effect of dFdCyd, and no radiosensitization occurred at any concentration of dFdCyd tested. Because radiosensitization by dFdCyd has been correlated with its ability to deplete dATP pools through inhibition of ribonucleotide reductase by dFdCyd diphosphate, we evaluated the metabolism of dFdCyd in both cell lines. At equitoxic concentrations of dFdCyd, both cell lines accumulated similar levels of the cytotoxic metabolite, dFdCyd triphosphate, as well as similar levels of dFdCyd monophosphate in DNA. In U251 cells, radiosensitizing concentrations of dFdCyd (10 or 25 nM; IC10 or IC50) depleted dATP by approximately 80% within 4 h. In contrast, 80 nM (IC50) was unable to deplete dATP by >30% within 4 h in D54 cells. Higher concentrations of dFdCyd or hydroxyurea, an inhibitor of ribonucleotide reductase that depleted dATP >90%, also did not produce radiosensitization in D54 cells. D54 cells were not resistant to radiosensitization because bromodeoxyuridine was able to induce radiosensitization. Because D54 cells express wild-type p53, whereas U251 cells express a mutant p53, the effect of dFdCyd and ionizing radiation on cell cycle progression was evaluated. Radiation alone produced a G1 block in D54 cells and a transient G2-M block in U251 cells. After a 24 h incubation with dFdCyd alone or in combination with ionizing radiation, U251 cells readily accumulated in S-phase, which remained elevated for at least 72 h, consistent with previous results in other mutant p53 cell lines. In addition, radiation enhanced the ability of dFdCyd to induce S-phase-specific cell death in U251 cells. In contrast, D54 cells showed a G1 block after dFdCyd and radiation exposure, with fewer cells in S-phase for at least 48 h after drug washout/irradiation. Furthermore, treatment with dFdCyd and/or radiation did not increase the amount of S-phase-specific cell death in D54 cells compared with control cells. These results suggest that the G1 block in D54 cells resulting from wild-type p53 induction prevented radiosensitization by dFdCyd.     

Expression of Bcl-2 and c-ErbB-2 in colorectal neoplasia

Several studies have been demonstrated the value of c-ErbB-2 and Bcl-2 in predicting the biological behaviour of tumors. The aim of this study was to investigate Bcl-2 and c-ErbB-2 expression in colorectal carcinomas and the correlation between their presence and other clinicopathologic parameters. Eighty-six colorectal carcinomas and 17 adenomas were stained with Bcl-2 and c-ErbB-2 immunohistochemically. Staining patterns were assessed semi-quantitatively and correlated with tumor size, Duke s classification, tumor differentiation, mucinous characteristic and anatomic locations. We detected Bcl-2 expression in 10 of 17 adenomas (58.8 %) and 31 of 86 carcinomas (36.04 %). Positive staining in normal mucosa was observed only in the compartment of cryptic cells. However neither the difference in the rates of Bcl-2 positivity in adenoma and carcinoma groups, nor the correlation with other mentioned clinicopathological parameters, were found statistically significant. Bcl-2 expression was found to be significantly high in mucinous carcinomas. Expression of c-ErbB-2 was observed in 12 of 86 (13.95 %) carcinomas. It was not detected in adenomas and normal mucosa. Although the incidence of c-ErbB-2 in nonmucinous carcinoma was higher than that of mucinous carcinoma, this was not significant. In addition we were unable to show any significant relation between c-ErbB-2 expression and other clinicopathologic features. Our result suggest that c-ErbB-2 protein expression in colorectal carcinomas, is not very frequent event. There is no correlation between c-ErbB-2 expression and malignant potential of colorectal carcinomas. Higher expressions of Bcl-2 in adenomas than carcinomas suggest us a possible role of Bcl-2 in early carcinogenesis of colon. However since we were unable to find any significant correlation between Bcl-2 expression and other parameters the impact of this gene on biological behavior is still unclear for us.     

致癌基因CUG2的研究进展

CUG2蛋白,又称着丝粒蛋白W,是一种近期发现的核蛋白,研究发现CUG2为细胞分裂、细胞凋亡等过程所必需,在许多癌组织中都存在高表达,如结肠直肠癌及胃癌,被预测是一种致癌基因。它位于核仁上或者着丝粒上,能和着丝粒蛋白T相互结合,共同参与着丝粒染色质结构的形成。现概要介绍CUG2基因的发现、结构特点、生物学功能及其与肿瘤之间关系的最新进展。     

过氧化氢对不同时相HepG2细胞氧化作用的研究

背景与目的:研究过氧化氢(H2O2)对不同时相HepG2细胞作用的选择性,以进一步探讨H2O2:诱导肿瘤细胞凋亡的机制.材料与方法:以胸腺嘧啶核苷(TdR)阻断法获得不同细胞周期时相的同步化细胞,分别加入800 μmol/L的H2O2作用3 h,并检测各组细胞的丙二醛(MDA)含量、黄嘌呤氧化酶(XOD)和5'核苷酸酶(5'NT)的活性及抗羟自由基(抗·OH)和抗超氧阴离子自由基(抗O2)的活性.结果:经H2O2处理的各组MDA含量和XOD、抗·OH、抗O2活性显著高于对照组,其差异均具有统计学意义(P均＜0.01),5'NT活性低于对照组(P＜0.01).与未同步化组比较,同步化于S期和G2/M期的细胞MDA含量和XOD、5'NT、抗·OH和抗O2活性显著升高,其差异均具有统计学意义(P均＜0.01),而同步化于G1期的细胞MDA含量、XOD、5'NT和抗O2-活性显著降低,差异均具有统计学意义(P均＜0.01).结论:H2O2作用同步化HepG2细胞后,代谢产生自由基的酶活性,脂质过氧化产物含量以及抗氧化水平均表现出细胞周期特异性,这可能是过氧化氢对不同时相HepG2细胞损伤作用不同的原因.     

己酮可可碱增强（E）—（2‘）—脱氧—氟亚甲基胞苷的细胞毒性和对宫颈癌细胞的放射增敏作用

目的观察己酮可可碱(pentoxifylline,PTX)对(E)－(2')－脱氧－氟亚甲基胞苷[(E)－2－deoxy-2-(fluoromethylene)cytidine,FMdC]的细胞毒性和放射增敏作用的影响。方法在人类宫颈癌细胞系C33-A和C4-I,FMdC和PTX的细胞毒性和放射增敏作用应用MTT和克隆形成分析。两种药物的细胞毒性相互作用应用Isoborogram分析。常规照射剂量2Gy时的放射增敏比（sensitive enhancement ratio,SER）定义为2Gy时对照组存活分数（survival fraction,SF）和药物处理组SF之比(SER2Gy)。结果PTX增加FMdC对C33-A和C4-I细胞的细胞毒性,并呈剂量依赖关系。单药FMdC对C4-I和C33-A的50％抑制浓度（50％inhibition concentration,IC50）分别为139.5nmol/L和46.7nmol/L。在C4-I细胞,0.25mmol/L、0.5mmol/L和1.0mmol/LPTX降低FMdC的IC50至58.0nmol/L、40.6nmol/L和20.9nmol/L。在C33-A细胞,0.25mmol/L、0.5mmol/L和1.0mmol/LPTX降低FMdC的IC50至32.1nmol/L、23.3nmol/L和9.1nmol/L。Isoborogram分析表明,FMdC和PTX的细胞毒性效应为相乘作用。应用克隆形成分析,照射前用30nmol/LFMdC处理指数生长期C33-A和C4-I细胞48h或照射后立即单用0.25～1.0mmol/LPTX,均能观察到各自的放射增敏作用;如果两药联合应用,细胞放射敏感性明显增加。在C4-I细胞系,单药30nmol/LFMdC和0.5mmol/LPTX的SER2Gy分别为2.08±0.66和1.76±0.30,而两药联合时的SER2Gy为3.18±1.32。在C33-     

MMP-2与TIMP-2在大肠癌中的表达

目的探讨大肠癌组织中基质金属蛋白酶(Matrix Meralloproteinases,MMPS)和金属蛋白酶组织抑制物(Tissue Inhibitors of Meralloproteinases,TIMPS)基因表达与肿瘤发生,浸润深度,转移及预后之间的关系.方法采用免疫组化S-P法对98例大肠癌组织及40例大肠良性病变组织进行MMP-2和TIMP-2的检测.结果98例大肠癌组织中MMP-2、TIMP-2的表达分别为75.51%(74/98)、57.14%(56/98),而在癌旁组织及良性病变中大部分不着色,少部分有微弱着色,二者的表达与肿瘤的发生明显相关;MMP-2的高表达与肿瘤的浸润深度、淋巴结转移及临床预后均呈明显的正相关(P＜0.05),TIMP-2的高表达与淋巴结转移及预后呈明显的负相关(P＜0.05).结论大肠癌组织中MMP-2的高表达预示着病人预后更差,评价MMP-2和TIMP-2之间的平衡比单独采用MMP-2指标更有利于了解肿瘤的进展及预后.