hMAM mRNA和CEA mRNA RT-PCR检测乳腺癌外周血微转移

目的:应用RT-PCR法检测乳腺癌患者外周血中的hMAM mRNA和CEA mRNA表达情况.方法:用RT-PCR法,检测52例乳腺癌患者外周血中hMAM mRNA和CEA mRNA的表达情况,另抽取28例乳腺良性疾病患者和10例健康志愿者外周血标本作为对照.结果:52例乳腺癌患者外周血中hMAM mRNA和CEA mRNA的阳性率分别为30.8%、34.6%.28倒乳腺良性疾病患者和10例健康志愿者外周血中hMAM mRNA、CEA mRNA表达均为阴性.二者的阳性表达与患者的腋淋巴结状况、TNM分期差异均有显著性意义(P＜0.05),而与患者的年龄、原发肿瘤大小、病理类型以及激素受体状况等差异均无显著性(P＞0.05).结论:hMAM mRNA、CEA mRNA均可作为标志物来检测乳腺癌患者外周血中微转移,联合检测可增加外周血中循环肿瘤细胞的检出率.     

应用RT-PCR检测乳腺癌患者外周血CEA mRNA和CK-19 mRNA的研究及意义

目的：检测乳腺癌患者外周血中CEA mRNA和CK-19 mRNA的表达，建立取材于外周血早期诊断乳腺癌微转移的方法。方法：65例乳腺癌和37例乳腺良性疾病患者的外周血，分离其有核细胞后进行细胞总RNA的抽提，运用果式RT-PCR技术进行CEA mRNA和CK-19 mRNA的检测。结果：以     

RT-PCR法检测非小细胞肺癌患者外周血CK19 mRNA的临床意义

目的:检测非小细胞肺癌患者外周血CK19mRNA的表达情况,探讨其表达的临床意义。方法:采用逆转录PCR(RT-PCR)方法,分别检测非小细胞肺癌患者和对照组患者外周血中CK19mRNA的表达。结果:43例非小细胞肺癌患者外周血中CK19mRNA表达阳性率为56%,对照组阳性率为5%,两组外周血CK19mRNA阳性率比较有显著性差异(P<0.01);外周血CK19mRNA阳性率与病理类型无相关性;淋巴结转移阳性和阴性患者的外周血中CK19mRNA阳性率分别为66%(21/32)和18%(2/11),二者差异显著(χ²=6.65,P<0.05)。随TNM分期上升,CK19mRNA阳性率增加(χ²=18.03,P<0.01)。结论:CK19mRNA可作为RT-PCR法检测非小细胞肺癌患者外周血微转移的分子标志物,该方法敏感性高,有助于早期诊断肺癌血行转移,指导临床分期和治疗。     

非小细胞肺癌术中外周血CEA mRNA的检测及意义

目的: 研究手术对非小细胞肺癌(NSCLC)外周血微转移的影响。 方法: 对70例NSCLC患者、18例肺部良性疾病患者于手术开始时、结扎肺静脉时和结扎肺静脉后1小时取外周静脉血,采用逆转录巢式聚合酶链反应(nested-RT-PCR)和微流控芯片(micro-fluid-chip)方法对外周血CEA mRNA进行检测。 结果: CEA mRNA检测阳性率在手术开始时、结扎肺静脉时和结扎肺静脉后1小时的CEA mRNA检测阳性率分别为50.0%、62.8%和57.1%;经χ²检验,手术开始时和结扎肺静脉时阳性率比较,统计学有显著性差异(χ²=7.114,Plt;0.05);结扎肺静脉后1小时阳性率高于手术开始时,结扎肺静脉时阳性率高于结扎肺静脉后1小时,但统计学无显著性差异(P>0.05)。 结论: 手术操作会促进肿瘤细胞进入血液循环,先结扎并尽早结扎肺静脉可能会减少术中进入血液循环的肿瘤细胞数。     

CPT-11 May Provide Therapeutic Efficacy for Esophageal Squamous Cell Cancer and the Effects Correlate with the Level of DNA Topoisomerase I Protein

CPT-11 is a potent anti-cancer drug and a specific inhibitor of DNA topoisomerase I (Topo I). In this study, we aim to evaluate the effects of CPT-11 on esophageal squamous cell cancers (ESCC) and to determine the correlation between the effects and the levels of Topo I expression. We examined the growth-inhibitory effect caused by SN-38, an active metabolite of CPT-11, in 14 human ESCC cell lines established from 10 primary and 4 metastatic lesions. CPT-11 was considered effective against 5 cell lines from primary lesions and one from metastatic lesions, and thus may show therapeutic efficacy against both primary and metastatic ESCC tumors. Although Topo I mRNA levels in these 14 ESCC cell lines, as quantitated by northern blot analysis, showed no correlation with the IC₅₀ values, Topo I protein levels, as quantitated by western blot analysis, showed an inverse correlation with the IC₅₀ values. Topo I protein levels could be an indicator of sensitivity to CPT-11. We also determined Topo I protein levels in 40 ESCC tumors and matched normal mucosae. Thirty-four tumors showed 1.2-22.3-fold increases in Topo I levels. Two patients receiving pre-operative chemotherapy and one receiving radiotherapy exhibited increased Topo I protein levels in their tumor lesions. It appeared that CPT-11 could provide selective therapeutic efficacy against ESCC tumors. CPT-11 may be effective for the treatment of metastatic ESCC tumors and as a second-line anti-cancer drug for ESCC.     

p16INK4 Expression Is Associated with the Increased Sensitivity of Human Non-small Cell Lung Cancer Cells to DNA Topoisomerase I Inhibitors

Inactivation of p16^INK4, an inhibitor of cyclin-dependent kinases 4 (CDK4) and 6 (CDK6), may be essential for ontogenesis in non-small cell lung cancer (NSCLC). We examined the sensitivity of two clones of P16^INK4 -transfected NSCLC cell line with homozygous deletion of p16^INK4, A549/pl6-l and 2, to DNA topoisomerase I (topo I) inhibitors. A549/pl6-l and -2 showed 7.7- and 9.1-fold increases in sensitivity to CPT-11 (11,7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyloxycamptothecin), respectively, compared with A549 cells. Ectopic p16^INK4-expressing cells also showed ∼4.0-fold increase in sensitivity to SN-38 (7-ethyl-10-hydroxycamptothecin), the active metabolite of CPT-11, compared to the parent cells. The topo I-mediated DNA relaxation activities of ectopic p16^INK4-expressing cells were approximately 5 times higher than those of the parent cells. Northern and western blot analyses indicate that these increased topo I activities of ectopic p16^INK4-expressing cells were due to an elevated topo I mRNA level and an increase in topo I protein. The chemosensitivity to topo I inhibitors, topo I mRNA level, protein content and activity of a pl6^INK4 revertant, lacking functional p16^INK4, tended to be restored toward those of the parental phenotype to some extent. These results suggest that p16^1NK4 expression is closely associated with the increased sensitivity of ectopic pl6^INK4-expressing NSCLC cells to topo I inhibitors. The up-regulation of topo I mRNA level, protein content and activity may he responsible for this hypersensitivity.     

Increase in the Level of P-Glycoprotein mRNA Expression in Multidrug-resistant K562 Cell Lines Treated with Sodium Butyrate Is Not Accompanied with Erythroid Differentiation

We examined the effects of hemin, sodium butyrate and mitomycin C on levels of P-glycoprotein mRNA in human myelogenous K562 cells by northern blot analysis. After treatment with sodium butyrate a dose-dependent increase of P-glycoprotein mRNA expression was observed in the adriamycin-resistant K562 and vincristine-resistant K562 lines. With 10 m M sodium butyrate, the level of P-glycoprotein mRNA reached 20 times that of control adriamycin-resistant K562 and with 30 m M sodium butyrate, it exceeded 5 times that of control vincristine-resistant K562. In contrast, hemin and mitomycin C had almost no effect on P-glycoprotein mRNA. In this experiment, since expression of P-glycoprotein mRNA was not necessarily accompanied with induction of erythroid differentiation, the increased amount of P-glycoprotein mRNA is unlikely to be a result of differentiation.     

Cytotoxicity of Cratoxylum Formosum Subsp. Pruniflorum Gogel Extracts in Oral Cancer Cell Lines

Background: Oral cancer is a health problem in Thailand. Cratoxylum formosum subsp. pruniflorumGogel (Teawdang), normally consumed in northeast Thailand, has proven cytotoxic to cervical cancer celllines including HeLa, SiHa and C-33A. Recently, Asian oral cancer cell lines, ORL-48 and ORL-136, wereestablished. Therefore, we aimed to study cytotoxicity of Teawdang in these. Total phenolic (TPC) and flavonoidcontent (TFC), and antioxidant activity of Teawdang were also determined. Materials and Methods: Teawdangwas purchased from Khon Kaen market during June-October 2013. Hexane (CHE), ethyl acetate (CEE) andmethanol (CME) extracts of its edible part were analyzed for TPC by the folin-ciocalteau method and for TFCby an aluminium colorimetric method. Antioxidant activity and cytotoxicity in normal Vero cells and oral cancercells were investigated. Cell viability was assessed using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazoliumbromide (MTT) assays. Results: CME and CEE had higher TPC and TFC and antioxidant activity than CHE.Both CME and CEE, at 200 μg dry wt/mL, were cytotoxic to the studied oral cancer cell lines. However, CMEwas cytotoxic to Vero cells whereas CEE was not. Compared to Vero cells, CEE significantly inhibited ORL-48and ORL-136 growth (p=0.03 and p=0.02, respectively). Conclusions: CEE exhibited cytotoxic effects on thestudied oral cancer cell lines but not normal Vero cells. The bioactive compounds in CEE should be furtherpurified and elucidated for their mechanisms of action for development as anticancer agents.     

Expression of Fas Antigen and Its Mediation of Apoptosis in Human Gastric Cancer Cell Lines

Fas, a member of the tumor necrosis factor receptor/nerve growth factor receptor family, induces apoptosis by crosslinking with Fas ligand or anti-Fas antibody in a variety of cultured cells. We examined the expression of Fas antigen and its mediation of apoptosis in six human gastric carcinoma cell lines. Flow cytometric analysis and western blotting revealed relatively high expression of Fas antigen in MKN-74 (wild-type p53 gene) and MKN-45 (wild-type), followed by MKN-1 (mutated), MKN-7 (mutated) and KATO-III (deleted). MKN-28 (mutated) showed minimal expression of the antigen. The expression was apparently enhanced by interferon-γ, except for MKN-1 and MKN-28. Anti-Fas antibody (100 ng/ml) induced nuclear fragmentation characteristic of apoptosis. Apoptosis occurred in a delayed fashion and the apoptotic index at 72 h was approximately 60% in MKN-74, 35% in MKN-45, and 20% in MKN-1 and KATO-III. A DNA ladder was noted in MKN-74 at 72 h. Expression levels of P53 and P21^Wafl did not change for up to 48 h in MKN-74. The biological effects did not correlate with endogenous Bcl-2 expression. These results indicated that a) Fas antigen is variably expressed in human cultured gastric carcinoma cells, b) the protein transduces an apoptotic signal which leads to delayed cell death, and c) susceptibility to the antibody correlates well with the expression level of Fas antigen.     

Apoptosis Induction,Cell Cycle Arrest and in Vitro Anticancer Activity of Gonothalamin in a Cancer Cell Lines

Cancer is one of the major health problems worldwide and its current treatments have a number of undesiredadverse side effects. Natural compounds may reduce these. Currently, a few plant products are being used totreat cancer. In this study, goniothalamin, a natural occurring styryl-lactone extracted from Goniothalamusmacrophyllus, was investigated for cytotoxic properties against cervical cancer (HeLa), breast carcinoma(MCF-7) and colon cancer (HT29) cells as well as normal mouse fibroblast (3T3) using MTT assay. Fluorescencemicroscopy showed that GTN is able to induce apoptosis in HeLa cells in a time dependent manner. Flow cytometryfurther revealed HeLa cells treated with GTN to be arrested in the S phase. Phosphatidyl serine propertiespresent during apoptosis enable early detection of the apoptosis in the cells. Using annexin V/PI double stainingit could be shown that GTN induces early apoptosis on HeLa cells after 24, 48 and 72 h. It could be concludedthat goniothalamin showing a promising cytotoxicity effect against several cancer cell lines including cervicalcancer cells (HeLa) with apoptosis as the mode of cell death induced on HeLa cells by Goniothalamin was.     

RRM1 mRNA 表达水平与晚期 NSCLC 吉西他滨维持治疗疗效的相关性分析

目的：探讨晚期NSCLC组织中RRM1 mRNA表达水平及其与吉西他滨维持治疗疗效的相关性。方法通过液相芯片法检测40例晚期NSCLC患者肿瘤组织中RRM1 mRNA表达水平,分析RRM1 mRNA表达水平与患者临床特征、吉西他滨维持治疗有效率、无进展生存及总生存时间的相关性。结果 RRM1 mRNA低表达与高表达患者在各临床特征方面无显著差别,两组总生存时间比较无统计学差异（15．6个月 vs 12．1个月,P＝0．582）,但RRM1 mRNA低表达患者具有更高的疾病控制率（85．7％ vs 52．6％,P＝0．023）和更长的无进展生存时间（7．0个月 vs 4．5个月,P＝0．025）。结论晚期NSCLC 肿瘤组织中RRM1 mRNA低表达患者吉西他滨维持治疗疾病控制率更高;无进展生存时间更长,RRM1 mRNA是独立的预后因素,适合作为筛选吉西他滨维持治疗获益人群的指标。     

三氯乙烯对B6C3F1雄性小鼠肝脏和肾脏中细胞增殖相关基因表达和DNA甲基化的影响

目的:三氯乙烯(TCE)是环境中广泛存在的工业污染物,可引起小鼠肝癌,但对肾癌发生未见显著影响。本研究通过检测TCE对小鼠肝脏和肾脏细胞增殖和DNA甲基化调控相关基因表达以及对DNA甲基化的影响,探讨TCE引起小鼠肝癌的分子机制。方法:将6周龄B6C3F1雄性小鼠随机分成3组,每组4只,分别以0、500和1 000 mg/kg剂量的TCE连续灌胃5 d。以荧光定量PCR方法检测TCE染毒小鼠肝脏、肾脏中与细胞增殖以及DNA甲基化调控相关基因的mRNA表达水平,以结合重亚硫酸盐的限制性内切酶方法检测Cdkn1a启动子区和重复序列的DNA甲基化水平。结果:与对照组相比,TCE可引起小鼠肝脏中细胞增殖相关基因Cdkn1a、Jun和Mki67的mRNA水平显著升高(P均<0.05),且呈剂量反应关系。同时1 000 mg/kg TCE染毒小鼠肝脏中主要DNA甲基化调控基因Dnmt3a、Dnmt3b和Tet2的mRNA水平降低(P均<0.05),Uhrf1 mRNA的表达升高(P均<0.05)。TCE染毒还导致肝脏内Cdkn1a启动子区的DNA甲基化水平降低,但对肾脏中相关基因及DNA甲基化水平无显著影响。结论:TCE引起的细胞增殖相关基因表达升高及DNA甲基化异常可能在其促进小鼠肝癌发生中起重要作用。     

Features of DNA Oligonucleosomal Fragmentation in Human Tumor Cell Lines and Its Detection by Flow Cytometry: Utility and Limitations

Cultured HL-60, HeLa S3 and WiDr cells were treated with various doses of ethanol, then subjected to flow cytometry and gel electrophoresis of cellular DNA. On electrophoresis of DNA from HL-60 cells treated with 0.5 or 1.0 mM ethanol, a ladder pattern was recognized after 3 h. At higher doses of ethanol (2.0 and 5.0 mM), a smear pattern resulted. On flow cytometry, however, A₀ cells (lower fluorescence level than G₀+ G₁ cells) were noted from 0.5 to 5,0 m M ethanol. The observation of A₀ cells at higher doses indicated loss of DNA after random DNA degradation. HeLa S3 and WiDr cells were partially detached from flasks after administration of ethanol and separated into adherent and non-adherent categories. In DNA from non-adherent HeLa S3 cells treated with 0.5 m M ethanol, a ladder pattern was observed after 24 h. On flow cytometry, prior to the appearance of A₀ cells, an accumulation in the G₂+M-phase became obvious after 3 h. Increased mitotic indices indicated that this phenomenon was due to M-phase arrest. Adherent HeLa S3 cells showed no DNA Oligonucleosomal fragmentation or A₀ cells. These findings indicate that detection of A₀ cells by flow cytometry is not proof of cell death by DNA Oligonucleosomal fragmentation.     

Revisiting Clinical Trials Using EGFR Inhibitor-Based Regimens in Patients with Advanced Non-Small Cell Lung Cancer: A Retrospective Analysis of an MD Anderson Cancer Center Phase I Population

Purpose

Single-agent EGFR inhibitor therapy is effective mainly in patients with lung cancer and EGFR mutations. Treating patients who develop resistance, or who are insensitive from the outset, often because of resistant mutations, other aberrations or the lack of an EGFR mutation, probably requires rational combinations. We therefore investigated the outcome of EGFR inhibitor-based combination regimens in patients with heavily-pretreated non-small cell lung cancer (NSCLC) referred to a Phase I Clinic.

Methods

We reviewed the electronic records of patients with NSCLC treated with an EGFR inhibitor-based combination regimen: erlotinib and cetuximab; erlotinib, cetuximab and bevacizumab; erlotinib and dasatinib; erlotinib and bortezomib; or cetuximab and sirolimus.

Results

EGFR mutations were detected in 16% of patients (21/131). EGFR inhibitor-based combination regimens were administered to 15 patients with EGFR-mutant NSCLC and 24 with EGFR wild-type disease. Stable disease (SD) ≥6 months/partial remission (PR) was attained in 20% of EGFR-mutant patients (3/15; two with sensitive mutations and secondary resistance to prior erlotinib, and one with a resistant mutation), as well as 26% of evaluable patients (5/19) with wild-type disease. One of three evaluable patients with squamous cell histology achieved SD for 26.5 months (EGFR wild-type, TP53-mutant, regimen=erlotinib, cetuximab and bevacizumab).

Conclusions

Eight of 34 evaluable patients (24%) with advanced, refractory NSCLC evaluable for response achieved SD ≥6 months/PR (PR=3; SD ≥6 months=5) on EGFR inhibitor-based combination regimens (erlotinib, cetuximab; erlotinib, cetuximab and bevacizumab; and, erlotinib, bortezomib), including patients with secondary resistance to single-agent EGFR inhibitors, resistant mutations, wild-type disease, and, squamous histology.     

Molecular Cytogenetic Analysis of 17 Renal Cancer Cell Lines: Increased Copy Number at 5q31-33 in Cell Lines from Nonpapillary Carcinomas

Comparative genomic hybridization (CGH) was used to screen for genomic imbalances in cell lines derived from 13 nonpapillary renal-cell carcinomas (RCCs), two papillary RCCs, one renal squamous-cell carcinoma, and one transitional-cell carcinoma of the renal pelvis. Aberrations were found in all 17 lines. The most frequent changes in nonpapillary RCC cell lines were gains of 5q (85%), 7q (69%), 8q (69%) and 1q (54%) and losses of 3p (92%), 8p (77%), 4q (62%) and 14q (54%). High-level gains (HLGs) were detected at 4q12, 5p, 5q23-33, 7q22-qter, 8q23-24, 10q21-qter, 12p and 12q13-22. By means of fluorescence in situ hybridization (FISH) we narrowed the smallest common region involving 5q gains to the genomic segment between D5S642 and D5S673, and found that the HLG at 4q12 possibly involved amplifications of c-kit and PDGFRA . Two papillary RCC cell lines showed gains of entire chromosomes 7, 12 and 17. The CGH data reported here should help to facilitate the choice of individual renal-tumor cell lines for exploring target genes in regions of interest.