益气化瘀解毒方对MRP、GST-π和Topo Ⅱ基因在Sorafenib获得性耐药人肝癌QGY7702细胞表达的干预研究

目的探讨益气化瘀解毒方干预后对Sorafenib获得性耐药人肝癌QGY7702细胞(QGY7702/Sora)增殖及MRP、GST-π和Topo Ⅱ基因表达的影响。方法培养QGY7702/Sora细胞和QGY7702细胞,利用Cell Counting Kit-8(CCK-8)法检测Sorafenib对细胞的半数抑制率浓度(IC50值),计算耐药指数RI;观察益气化瘀解毒方对耐药细胞的增殖影响;采用荧光定量PCR检测药物干预前后2种细胞中MRP、GST-π和Topo Ⅱ基因表达水平。结果亲本细胞和耐药细胞Sorafenib的IC50值分别为(7.993±0.522)μmol/L和(19.651±1.216)μmol/L,RI约为2.5。益气化瘀解毒方可抑制耐药细胞的增殖活性。2种细胞的MRP、GST-π、Topo Ⅱ表达量无明显差异(P>0.05)。Sorafenib组可促进耐药细胞MRP 、GST-π基因的过表达(P<0.05),益气化瘀解毒方组可抑制GST-π基因的过表达(P<0.01),且联合Sorafenib可显著提高Topo Ⅱ基因的表达量(P<0.01)。结论 QGY7702/Sora细胞MRP、GST-π和Topo Ⅱ的表达水平与亲本细胞无显著差异。耐药细胞对Sorafenib敏感性降低与MRP、GST-π过表达相关,而益气化瘀解毒方拮抗Sorafenib耐药与抑制GST-π过表达相关。     

喜树中两个DNA拓扑异构酶Ⅰ抑制剂

目的：以生物活性为向导在喜树中寻找天然的DNA拓扑异构酶Ⅰ抑制剂。方法：用各种层析方法和波谱方法从喜树中分离并鉴定了14个化合物。结果：其结构分别鉴定为喜树碱(1)，肌醇(2)，喜果苷(3)，3’甲基3，4O，O-亚甲基鞣花酸-4’-D-β-D-吡喃葡萄糖苷(4)，2-氧-1，2-二氢．喹啉4酸(5)，马钱子酸(6)，4-甲基-1，2-环己烷二甲醇(7)，strictosamide(8)，獐芽菜苷(9)，乙酰胺(10)，氯原酸(11)，strictosidinic acid(12)，1-咖啡酰基奎宁酸(13)和10-羟基喜树碱(14)。结论：化合物4-13为首次从该植物中分得，化合物4的核磁数据在文献中有误，本文对其进行了重新归属。经体外活性测试，化合物1对DNA拓扑异构酶Ⅰ有中等程度的抑制作用，化合物4，14对DNA拓扑异构酶Ⅰ有强的抑制作用。     

Therapeutic strategies in male breast cancer: Clinical implications of chromosome 17 gene alterations and molecular subtypes

Male breast cancer (MBC) is a rare disease. To date, therapy is mainly based on studies and clinical experiences with breast cancer in women. Only little is known about molecular typing of MBC, particularly with regard to potential biological predictors for adjuvant therapy. In female breast cancer tumors with chromosome 17 centromere (CEP17) duplication, HER2 and/or Topoisomerase II alpha (Topo II-α) gene alterations have been suggested to be associated with poor prognosis and increased sensitivity to anthracycline-containing regimens.In a well characterized cohort of 96 primary invasive MBC, we studied CEP17, HER2 and Topo II-α alterations by fluorescence in-situ hybridization (FISH), and expression of hormone receptors (HR), HER2 and Ki67 by immunohistochemistry to define molecular subtypes. Tumor characteristics and follow-up data were available and correlated with molecular findings.HER2 amplification and Topo II-α amplification/deletion were exceptionally rare in MBC (6.3% and 3.1%, respectively). CEP17 polysomy were found in 9.4% of tumors. HER2, Topo II-α and CEP17 gene alterations were not correlated to patients outcome. 96.9% of our cases were HR positive. Triple negative tumors were found in only 3.1% of the cases. In nodal negative tumors luminal A subtypes were significantly associated with better overall survival.Our results provide evidence for a predominant male breast cancer phenotype, characterized by HR expression and a lack of HER2/Topo II-α alterations and CEP17 duplicates. Therefore, the impact of anthracycline sensitivity linked to HER2/Topo II-α alterations as found in female breast cancer has low clinical significance for this specific male breast cancer phenotype.     

G226, a new epipolythiodioxopiperazine derivative,triggers DNA damage and apoptosis in human cancer cells in vitro via ROS generation

Aim:

G226 is a novel derivative of epipolythiodioxopiperazines with potent inhibitory activity against cancer cells. Here, we sought to identify potential targets involved in the anti-cancer activity of G226.

Methods:

Cell proliferation assay was conducted in a panel of 12 human cancer cell lines. The activities of topoisomerase I (Topo I) and Topo II were studied using supercoiled pBR322 DNA relaxation and kDNA decatenation assays. ROS production was assessed with probes DCFH-DA and H&E. Western blot analysis and flow cytometry were used to examine DNA damage, apoptosis and cell cycle changes.

Results:

G226 displayed potent cytotoxicity in the 12 human cancer cell lines with a mean IC₅₀ value of 92.7 nmol/L. This compound (1–100 μmol/L) selectively inhibited the activity of Topo II, and elevated the expression of phosphorylated-H2AX in a dose-dependent manner. In Topo II-deficient HL60/MX2 cells, however, G226-induced DNA damage, apoptosis and cytotoxicity were only partially reduced, suggesting that Topo II was not essential for the anti-tumor effects of G226. Furthermore, G226 (0.125–2 μmol/L) dose-dependently elevated the intracellular levels of H₂O₂ and in the cancer cells, and pretreatment with GSH, NAC or DTT not only blocked G226-induced intracellular accumulation of ROS, but also abrogated G226-mediated phosphorylation of H2AX, apoptosis and cytotoxicity.

Conclusion:

G226-mediated ROS production contributes to the anti-cancer activity of this compound.     

A study of proliferative markers and tumor suppressor gene proteins in different grades of ependymomas

Ependymomas are CNS tumors that originate from the spinal canal and walls of the ventricular system. Considerable controversy continues to exist with regard to their prognostic factors; age and extent of resection are the only statistically significant prognostic factors yet identified. The authors report a retrospective study of a homogenous population of 119 patients harbouring ependymomas between 1991 and 2002. All clinico‐radiological and follow‐up data were analyzed and a pathologic review was performed by two pathologists. Immunohistochemical staining for MIB‐1, Topo IIα, p53 and MDM2 was performed. Histopathologic grades show relationship with MIB1 and Topo IIα labelling indices and cut‐off values of 5% can differentiate between anaplastic and lower grades. p53 and MDM2 proteins expression are not common in ependymomas; however, they are seen in higher grades only and may be involved in the tumor progression.     

P-gp、TopoⅡ、GSTπ在胃癌中的表达及临床意义

目的 探讨胃癌组织中耐药基因P-糖蛋白(P-gP)、DNA拓扑异构酶(Topo)Ⅱ和谷胱甘肽转移酶(GST)π的表达以及与胃癌组织学类型、临床分期、淋巴结转移的关系及临床意义.方法 用S-P免疫组化二步法对216例胃癌手术标本进行检测.结果 胃癌组织中P-gp、Topo Ⅱ、GSTπ阳性表达率分别为56.90%、56.90%、60.20%,P-gp表达与胃癌组织学类型和临床分期有关(均P<0.05);Topo Ⅱ表达与胃癌组织学类型、临床分期、淋巴结转移有关(均P<0.05);GSTπ表达与临床分期有关(P<0.05).结论 P-gP、Topo Ⅱ、GST π在胃癌原发性多药耐药中起重要作用.联合检测P-gP、Topo Ⅱ、GSTπ对于胃癌化疗方案选择具有一定的指导意义.     

Metastatic Breast Cancer Survival according to HER2 and Topo2a Gene Status

The aim of this study was to determine the relationship between amplification of HER2 (Human epidermal growth factor receptor 2) and Topo2a (topoisomerase 2a) and their influence on prognosis in metastatic breast cancer (MBC) patients. Amplification of both HER2 and Topo2a genes was determined by chromogenic in situ hybridization (CISH) in primary tumor tissue of 71 MBC patients. Starting point for follow-up was the time of diagnosis of metastatic disease. Although there was significant correlation between HER2 amplification and Topo2a alterations, Topo2a amplification was not strictly related to HER2 amplification. Follow-up of patients showed that there was no difference in MBC survival between HER2-nonamplified and HER2-amplified patients for subgroup as whole, but there was significant difference in MBC survival between patients with and without Topo2a amplification. HER2 amplification showed prognostic value in subgroups of patients, as well as Topo2a. Combination of these two genes with different status (nonamplified, amplified, coamplified) indicated that they might have additive effect. Also, it has been shown that Topo2a-amplified cases have poorer survival than Topo2a-nonamplified, when treated with CMF therapy.Topo2a amplification seems to be more promising biomarker of MBC survival, than HER2, and potential marker of resistance to CMF therapy.     

Involvement of CPP32/Yama-like protease in CPT-11-induced death signal transduction pathway

CPT-11, a derivative of camptothecin (CPT) that interacts with type-I DNA topoisomerase, induced apoptosis in HL60 and Daudi cells in vitro. This cytotoxic activity was time and dose dependent, and was prevented by cycloheximide (CHX), a protein synthesis inhibitor, indicating the requirement of new protein synthesis for CPT-11-induced apoptotic cell death. Ac-Tyr-Val-Ala-Asp-aldehyde (YVAD) and Ac-Asp-Glu-Val-Asp-aldehyde (DEVD), synthesized tetrapeptide inhibitors of interleukin_1β-converting enzyme (ICE)- and CPP32/Yama-like proteases, were used to examine the CPT-11-induced death signal transduction. These inhibitors blocked CPT-11-induced cytotoxicity in a time- and dose-dependent manner. Cytotoxic activity of SN-38, an active metabolite of CPT-11, was about 1000-fold that of CPT-11 and was also prevented by CHX, YVAD and DEVD. The doses of YVAD, however, were a little too high; the prevention by YVAD is then thought to be non-specific. In addition, lymphocytes obtained from normal and lpr^cg mutant mice showed similar susceptibility to CPT-11 cytotoxicity. These results indicate the direct involvement of CPP32/Yama-like protease in the CPT-11-induced death signal transduction pathway, and no involvement of Fas antigen in the pathway.     

TopoⅡ及GST-π在胃癌和结直肠癌中的表达及其临床意义

目的：探讨拓扑异构酶（ TopoⅡ）和谷胱甘肽转移酶π（ GST-π）在胃癌和结直肠癌组织中表达的临床意义。方法：采用免疫组化二步法检测TopoⅡ和GST-π在40例胃癌组织和60例结直肠癌组织中的表达情况,探讨其与临床病理参数间的关系。结果：在40例胃癌组织中TopoⅡ和GST-π阳性率分别为42.5％和82.5％,TopoⅡ在高中分化腺癌中的阳性率低于低分化腺癌（ P＜0.01）,而GST-π在高分化腺癌中阳性率高于低分化癌（ P＜0.05）,但与浸润深度及淋巴结转移无关。TopoⅡ和GST-π在结直癌中阳性率分别为43.3％和93.3％,与肿瘤的分化程度、浸润深度以及有无淋巴结转移均无关。结论：中晚期胃癌和结直肠癌广泛存在耐药性,TopoⅡ和GST-π的表达水平可作为判断胃癌恶性程度的指标,还可以指导临床对胃癌和结直肠癌化疗药物的选择。     

GRP78和TopoⅡ在胃腺癌组织中的表达及意义

目的：研究GRP78和TopoⅡ蛋白在胃腺癌组织中的表达及两者的相关性,探讨两者在胃腺癌耐药性中的作用。方法：运用免疫组化法检测60例胃腺癌和20例癌旁组织中GRP78和TopoⅡ的表达。结果：GRP78在癌旁组织呈弱阳性表达,在胃腺癌组织中呈中、强阳性表达,且在腺癌组织中的血管内皮细胞也有表达。GRP78在腺癌中的表达与性别、年龄等无关,而与肿瘤大小、分化程度、浸润深度、有无淋巴结转移密切相关（ P＜0.01）。TopoⅡ在高中分化腺癌中表达高于低分化腺癌（ P＜0.05）,而与性别、年龄、肿瘤大小、浸润深度、有无淋巴结转移均无关（P ＞0.05）。GRP78与 TopoⅡ的表达无明显相关性（P ＞0.05）。结论：GRP78在胃腺癌中高表达,对胃癌耐药性有一定影响,GRP78在胃腺癌中是否降低TopoⅡ的表达有待进一步研究。