Combinatorial treatment of ovarian cancer cells with harringtonine and cisplatin results in increased cisplatin-DNA adducts

The current studies represent the first step in assessing the utility of harringtonine in combination with cisplatin as an improved approach for treating ovarian cancer. Three ovarian cancer cell lines, platinum-sensitive A2780, and platinum-resistant A2780/CP70 and OvCar-3, were exposed to their respective IC(50) dose of cisplatin for 1 h with or without a 24-h pretreatment with harringtonine. The level of platinum-DNA adducts was determined by atomic absorption spectrometry (AAS). These studies show for the first time that harringtonine pretreatment significantly increased the amount of platinum-DNA adducts in all ovarian cancer cell lines by 2-4 fold, immediately following 1-h exposure to cisplatin. Moreover, the level of cisplatin-DNA adducts in harringtonine-pretreated cells remained elevated by 3-4.7-fold for at least 6 h after cisplatin was removed, relative to cells only exposed to cisplatin. In all three cell lines the removal (repair) of platinum-DNA adducts was not significantly altered by harringtonine. In addition, the extent to which harringtonine altered the expression of select DNA damage response genes (p53, P16, ERCC1 and XPB) was determined using RT-PCR and Southern hybridization in A2780 and A2780/CP70 cells. The expression of ERCC1 and XPB RNAs were only modestly altered by harringtonine, consistent with a lack of effect of harringtonine on repair of cisplatin-DNA damage. However, harringtonine altered expression of p53 and P16 RNAs in both cell lines, although the down-regulation of p53 and P16 RNAs by harringtonine were more pronounced in A2780 cells. The novel observation that harringtonine augments platinum-DNA adducts in both platinum-sensitive and -resistant ovarian cancer cells indicates this combination of drugs may have utility in treating ovarian cancer and may be especially useful in managing platinum-resistant cancers. Additional studies are required to determine which sequence of these drugs is most beneficial, as well as the mechanism by which harringtonine increases cisplatin-DNA damage in ovarian cancer cells.     

Induction and removal of cisplatin-DNA adducts in human cells in vivo and in vitro as measured by immunochemical techniques

The same spectrum of cisplatin adducts was detected in DNA isolated from white blood cells of a cisplatin-treated cancer patient as had been found in cisplatin-treated DNA in vitro. The adducts were quantified in femtomole amounts by competitive enzyme-linked immunosorbent assay (ELISA) with three antisera raised against synthetic cisplatin-containing (oligo)nucleotides. For this assay, DNA samples digested with nucleases were fractionated by ion-exchange chromatography; the fractions were used as inhibitors of antibody binding. Determinations of the main adduct formed, cis-Pt(NH3)2d(pGpG), in patients immediately after a first treatment with equal doses of cisplatin showed interindividual differences in the platination levels of the white blood cells. These differences were found to correlate with those found after in-vitro exposure to cisplatin of blood samples taken from patients before treatment. In vivo, about 75% of the adducts formed after the first treatment were removed within 24 h. During a five-day course, the amounts of the main adduct increased after the first three administrations; no increase was seen on day 4 or 5. By day 6, considerable removal of adducts had occurred. Analysis of the formation and repair of the cis-Pt(NH3)2d(pGpG) adducts in cultured cells, i.e., human fibroblasts with different DNA repair capacities and one bladder and two testicular human cancer cell lines, indicated that both the amounts of adducts formed and the ability of the cells to repair the adducts can differ. These differences appear to determine the susceptibility of the cells for the cytotoxic action of cisplatin.     

Repair of etheno DNA adducts by N-glycosylases

After incubation of chloroacetaldehyde-treated DNA with cell-free homogenates, the excision of N2,3-ethenoguanine and 1,N6-ethenoadenine was observed with a rat brain tumour cell line. The repair mechanism was that of an N-glycosylase. The high specificity of all known DNA N-glycosylases and some unique properties of the enzymatic reaction indicate the existence of N-glycosylases specific for the repair of etheno, or similar, adducts.     

Autotaxin在人卵巢癌基因及蛋白水平表达的研究及意义

目的:探讨自分泌运动因子(Autotaxin,ATX)mRNA在人卵巢癌中的表达及其与卵巢癌临床病理特征之间的关系。方法:应用半定量逆转录聚合酶链反应(RT-PCR)、Western blot研究8例正常卵巢组织、18例良性卵巢上皮性肿瘤(以下简称良性卵巢肿瘤)、50例卵巢上皮性癌(以下简称卵巢癌)组织、23例大网膜转移灶中ATX mRNA及蛋白的表达。结果:8例正常卵巢组织、18例良性卵巢肿瘤、50例卵巢癌组织、23例大网膜转移灶中均有ATX mRNA表达;但ATX基因表达值在癌组织和大网膜转移灶中显著高于良性卵巢肿瘤和正常卵巢组织(P<0.001),分别为(89.31±10.15)%、(88.91±11.05)%、(40.18±16.71)%、(35.29±13.82)%。Western blot显示ATX蛋白在卵巢癌细胞中表达,在相对分子质量55×103、60×103大小的位置可见清楚的显色条带。ATX mRNA在卵巢癌细胞亦可见明显扩增条带。高表达ATX基因与卵巢癌手术分期和病理分化程度有关,而与年龄、病理类型等无关。结论:卵巢癌细胞中ATX分子在核酸与蛋白水平均有表达,两者结果一致;ATX基因过表达可能与卵巢癌的进展、转移有关。     

IL-1 and IL-6 release by tumor-associated macrophages from human ovarian carcinoma

Our study was designed to investigate the production of interleukin-1 (IL-1) and IL-6 in tumor-associated macrophages (TAM) isolated from ascites (18 cases) or solid (7 cases) human ovarian carcinoma. These are pleiotropic monokines which, in addition to affecting proliferation and differentiation of lymphocytes, act on various targets, including vascular cells and liver, and may therefore be involved in the pathogenesis of certain manifestations of malignancy. IL-1 was measured by the thymocyte co-stimulator assay, under conditions in which IL-6 was inactive, and, in 8 cases, by radioimmunoassay (RIA). IL-6 was measured as hybridoma growth factor (HGF) on the 7TD1 cell line. TAM did not release appreciable levels of IL-1 spontaneously and, upon LPS stimulation, were poor producers of this monokine compared to blood monocytes. In contrast, TAM supernatants contained a high level of HGF in the absence of deliberate stimulation, and exposure to LPS either did not affect or further augmented production of this monokine. HGF activity of TAM supernatants was completely blocked by anti-IL-6 antibodies. Ascites fluid from 8 ovarian-carcinoma patients contained high levels of HGF activity, blocked by anti-IL-6 antibodies. Thus, TAM exhibit a dissociation in their capacity to release the functionally related monokines IL-1 and IL-6. IL-6 produced by TAM may account for the elevation of liver-derived acute-phase proteins associated with malignancy.     

肺组织粗提物影响人原发性肝癌细胞侵袭转移机制的初步探讨

背景与目的：转移复发是原发性肝癌治疗失败的主要原因,而肺组织是原发性肝癌远处转移的好发部位。在小鼠不同组织粗提物诱导高转移潜能的人原发性肝癌细胞的体外趋化侵袭实验中,肺组织提取物具有最强的诱导能力。本研究旨在探讨小鼠肺组织粗提物促进人肝癌高转移细胞株移动侵袭的机制。方法：采用F型肌动蛋白聚合实验和流式细胞仪,分析C57BL／6小鼠肺组织粗提物诱导高转移潜能人肝癌细胞株MHCC97-H细胞骨架的变化。肺组织粗提物与MHCC97-H细胞孵育后,利用双重荧光染色分析F型肌动蛋白和基质金属蛋白酶-9(matrix metalloproteinase-9,MMP-9)表达的关系。结果：经无血清培养液或脾组织粗提物孵育后,MHCC97-H细胞呈梭型或多边形。与肺组织粗提物孵育后,随时间的增加,MHCC97-H细胞片层样或丝状伪足明显增多,流式细胞仪分析显示F型肌动蛋白在30s内增加1．9倍,激光扫描共聚焦显微镜观察MHCC97-H细胞F型肌动蛋白从细胞外周浓染到重新分布于细胞的导引侧。无血清培养基孵育MHCC97-H细胞后,MMP-9和F型肌动蛋白主要位于细胞的核周池;经肺组织粗提物孵育后,MHCC97-H细胞MMP-9和F型肌动蛋白则定位于细胞伪足的前端。结论：肺组织粗提物可能通过诱导MHCC97-H细胞形成伪足和改变MMP-9运送方式,从而促进MHCC97-H细胞移动侵袭。肺组织粗提物可能与人肝癌细胞器官特异性转移有关。     

Smoking-related DNA and protein adducts in human tissues

Tobacco smoking causes not only lung cancer but also cancer of the oral and nasal cavities, oesophagus, larynx, pharynx, pancreas, liver, kidney, stomach, urinary tract and cervix. Tobacco smoke contains many carcinogens that exert their biological effects through interaction of reactive intermediates with DNA to form DNA adducts. The same electrophilic species also react with cellular proteins. The effects of smoking are evident by the detection of elevated levels of carcinogen-DNA adducts in many human tissues and of carcinogen-protein adducts in blood. Components of tobacco smoke also induce oxidative DNA damage. Systemic exposure to tobacco-derived carcinogens is demonstrated by the observation of elevated levels of DNA adducts in tissues not directly exposed to tobacco smoke. For many of these tissues there is epidemiological evidence, varying from comprehensive to preliminary, that smoking is a causative factor in cancer of that site. The effects of passive smoking, which also causes lung cancer in non-smokers, is also evident in elevated levels of protein adducts in exposed non-smokers so exposed, relative to non-exposed non-smokers. This paper reviews the literature on smoking-related DNA and protein adducts in human tissues and shows how such studies have provided mechanistic insight into the epidemiological associations between smoking and cancer.     

Pharmacokinetics and tissue distribution of cisplatin in nude mice: platinum levels and cisplatin-DNA adducts

The pharmacokinetics of platinum (Pt) and cisplatin (CDDP)-DNA adducts were studied in nude mice after single-dose CDDP treatments. Whole blood, serum, kidney, lever, testis, brain, and tumor were collected at different intervals after injection of CDP at different dose levels. Pt was measured with flameless atomic absorption spectrometry (FAAS) or adsorptive voltammetry (AdV) and CDDP-DNA adducts with quantitative immunohistochemistry. The drug was immediately absorbed into the blood circulation (peak serum Pt levels were reached within 5 min) after i.p. CDDP administration, and distribution into most tissues also occurred rapidly (tissue Pt levels peaked at 15 min). With a sampling period of 7 days there was a biphasic elimination of Pt from blood, serum, and tissues. In the brain the pharmacokinetics differed with a gradual accumulation of Pt occurring during the 1st week. Formation of CDDP-DNA adducts in tissues was a slower process, with maximal levels being achieved at between 30 min and 4 h after drug administration, followed by a steady state lasting for at least 24 h. Each tissue type had its specific immunohistochemical staining pattern of adducts. With escalating CDDP doses there was a linear, or almost linear, increase in Pt concentrations and CDDP-DNA adduct levels in all sample types examined. These results suggest that a fair estimation of the amount of drug in tumor and normal tissues can be made from analysis of serum Pt at a fixed time point after a single dose of CDDP.     

Reduced expression of death-associated protein kinase in human uterine and ovarian carcinoma cells

The expression of the death-associated protein kinase (DAPK) protein and promoter methylation in 11 human uterine and ovarian carcinoma cell lines originally established from histopathologically-different carcinoma tissues were examined to investigate the relationship between DAPK and carcinogenesis in female reproductive tissues. The 11 cell lines included three cervical carcinomas, three endometrial carcinomas and five ovarian carcinomas. Western blot analysis showed no detectable expression of DAPK protein in 4 cell lines (ME180, HOKUG, MCAS and OVK-18) while moderate levels of DAPK protein were readily detected in normal human endometrium, and normal murine uterus and ovary. Methylation-specific PCR of the 11 cell lines revealed that 5 carcinoma cell lines (ME180, HOKUG, MCAS, OVK-18 and HEC-1) expressed hypermethylated promoters in the DAPK genes, while DAPK promoters in the other 6 carcinoma cell lines remained unmethylated. These results indicate that DAPK protein expression is reduced or silenced in some human uterine and ovarian carcinoma cells by methylation of the DAPK gene promoter region. Therefore, reduced DAPK expression and methylation of the DAPK promoter may be involved in carcinogenesis of human uterine and ovarian tissues.     

DNA and protein adducts in human tissues resulting from exposure to tobacco smoke

Tobacco smoke contains a variety of genotoxic carcinogens that form adducts with DNA and protein in the tissues of smokers. Not only are these biochemical events relevant to the carcinogenic process, but the detection of adducts provides a means of monitoring exposure to tobacco smoke. Characterization of smoking-related adducts has shed light on the mechanisms of smoking-related diseases and many different types of smoking-derived DNA and protein adducts have been identified. Such approaches also reveal the potential harm of environmental tobacco smoke (ETS) to nonsmokers, infants and children. Because the majority of tobacco-smoke carcinogens are not exclusive to this source of exposure, studies comparing smokers and nonsmokers may be confounded by other environmental sources. Nevertheless, certain DNA and protein adducts have been validated as biomarkers of exposure to tobacco smoke, with continuing applications in the study of ETS exposures, cancer prevention and tobacco product legislation. Our article is a review of the literature on smoking-related adducts in human tissues published since 2002.     

Tumor necrosis factor expression by human ovarian carcinoma in vivo

Tumor necrosis factor (TNF) is a cytokine produced by monocytes and other cells with selective cytolytic activity against some but not all tumor cells. Cellular resistance to the cytolytic effects of TNF has been reported to be associated with autocrine production of TNF by the target cells. The purpose of this study was to determine whether or not human tumors produce tumor necrosis factor in vivo. Ovarian carcinoma tissue from 25 patients with ovarian carcinoma was examined for the presence of TNF. Four of 5 ascites fluid specimens and tissue sections of 16 of 20 patients were positive for TNF by immunoperoxidase staining. The source of the immunoreactive protein was further examined by in situ hybridization studies. TNF mRNA was detectable in each of the ascites specimens and 7 of 16 tissue sections that were positive by immunoperoxidase staining. These findings suggest that TNF is produced by some human tumors in vivo and that the association between TNF production and resistance to TNF antitumor action may be clinically relevant.     

Cerebral metastases from ovarian carcinoma

Apart from choriocarcinoma, involvement of the central nervous system (CNS) by gynecologic malignancy is rare. A 10-year retrospective review at the University of Washington Medical Center (Seattle, WA) and Swedish Hospital and Medical Center Tumor Registry (Seattle, WA) identified 14 patients with cerebral metastases from ovarian carcinoma. Median age at diagnosis of cerebral metastases was 52.5 years. Median interval from the diagnosis of ovarian carcinoma to the diagnosis of CNS metastases was 14.5 months. Seven patients had received cisplatin therapy before CNS relapse. Seven patients underwent second-look procedures before developing CNS metastases; in three, results were negative. Eight patients had evidence of extraperitoneal spread to other sites at the time of CNS relapse. Clinical manifestations included motor weakness, seizures, headache, confusion, and speech disturbance. All lesions were contrast enhancing on computed tomography (CT) scans and were located in the cerebral hemispheres. Nine patients had single lesions, five of whom underwent surgical resection of the lesion with histologic confirmation of metastases from the primary site. Median survival was 2 months in patients receiving radiation therapy alone and 17 months in patients who received surgery and radiation. Median survival of the entire series was 3 months. The presence of multiple cerebral metastases or evidence of extraperitoneal spread elsewhere in the body was adversely associated with survival. The prognosis of patients with cerebral metastases from ovarian carcinoma appears poor. However, early diagnosis by routine CT scanning followed by surgical resection and radiation may improve overall survival in a select group of patients.     

Deficient nucleotide excision repair activity in protein extracts from normal human lymphocytes

DNA repair activity in human peripheral blood lymphocytes (PBL)has been investigated by various techniques. Here, we reportthe use of an in vitro assay in order to assess nucleotide excisionrepair activity (NER). The mechanism of this major repair processrelies on two broad steps: first, recognition, incision andexcision of the damaged DNA; second, repair synthesis on thegapped DNA. Briefly, damaged plasmids were incubated with wholecell extracts which allows one to quantify DNA repair synthesis.When NER was determined on plasmid DNA damaged with UV-lightor cisplatin, PBL extracts showed no repair synthesis for unstimulatedlymphocytes. Using a new in vitro assay measuring only the damage-specificDNA incision activity in cell extracts, we found that the incisionstep in the repair reaction was blocked in unstimulated PBL.By mixing PBL with XP (group A, B, C, D) extracts, no restorationof NER activity was observed. In addition, these lymphocytesalso lacked DNA replication activity as determined with pre-incisedplasmid substrate. However, a phytohemagglutinin treatment ofPBL led to an extent of repair synthesis similar to that observedwith extracts from lymphoblastoid cells. When lymphocytes wereincubated in 20% serum medium with and without phytohemagglutinin,the repair activity increased dramatically after 24 h. Duringthe activation of lymphocytes, the extent of repair synthesiswas proportional to the percentage of cells in S phase of thecell cycle. Our results suggest that the blockage of the cellcycle in G0/G1 in PBL may be responsible for their lack of NERactivity.     

Epidermal growth factor reduces HER-2 protein level in human ovarian carcinoma cells.

Over-expression of the proto-oncogene HER-2 (c-erbB-2/neu) in ovarian, endometrial, and mammary carcinoma is an indicator of poor prognosis. Interactions between the epidermal growth factor (EGF) receptor and the HER-2 protein have been described. The aim of this study was to elucidate the effects of EGF on HER-2 expression. In the human ovarian carcinoma cell lines HTB-77, OVCAR-3, 2780, SKOV-6, SKOV-8 and 2774, and the human mammary tumor cell line SKBR-3, total cellular p185HER-2 was determined by an ELISA, whereas the surface p185HER-2 was measured with a living-cell RIA. Stimulation of these cell lines with either EGF (0.1-30 nM) or TGF-alpha (0.1-30 nM) led to a significant reduction in p185HER-2 expression. The effect was more pronounced in cells with normal HER-2 expression. A reduction of mRNA levels for p185HER-2 by EGF was observed in OVCAR-3 cells but not in the over-expressing lines HTB-77 and SKBR-3. Interestingly, the EGF-induced effect was not always associated with growth stimulation and was not correlated with the number of EGF binding sites detected by a radioligand assay. Our data indicate that EGF treatment results in a down-regulation of p185HER-2.     

ras oncogene activation in human ovarian carcinoma

Samples of 37 fresh human ovarian tumor biopsies were screened to detect proto-oncogene amplification and ras mutations. Three samples showed c-K-ras2 amplification; none of the other oncogenes tested revealed any gene amplification. 5-, 25-, and 120-fold amplifications were assessed by dilution experiments and soft laser densitometry. Corresponding elevated levels of c-K-ras2 mRNA and p21 ras protein were found in the three tumors. Analysis by the polymerase chain reaction method to detect point mutations of codon 12 or codon 61 of Harvey-, Kirsten-, or N-ras showed only the wildtype sequence in all specimens. No correlation was found between ras activation and degree of tumor progression or histological subtype. DNA from one of the tumors with c-K-ras2 amplification proved to have high transforming activity in the NIH 3T3 tumorigenicity assay, but the transforming gene was not c-K-ras2.     

Measurement of self-renewal in culture of clonogenic cells from human ovarian carcinoma.

To test the identity of human tumour clonogenic cells and stem cells, a procedure was developed to allow quantitation of self-renewal capacity of human ovarian carcinoma clonogenic cells. Primary colonies grown from malignant effusions of 10 patients were disaggregated and replated; secondary colonies were observed to be similar to primary colonies in size, morphology and culture requirements. Density-gradient separation of tumour-cell populations demonstrated that not all primary clonogenic cells are capable of self-renewal during clonal expansion. Patient-to-patient variation in self-renewal capacity was shown to be significantly correlated with the concentration of the tumour-cell population in the effusion fluid, and preliminary evidence of a progressive increase in self-renewal was found in one patient. It was concluded that some, but not all, ovarian-tumour clonogenic cells have the stem-cell property of self-renewal, and that quantitation of such a property may identify an important prognostic variable.     

Maintenance on extracellular matrix and expression of heparanase activity by human ovarian carcinoma cells from biopsy specimens

A routine procedure has been developed for the isolation and maintenance in culture of human ovarian carcinoma cells derived from biopsy specimens. Cell attachment, plating efficiency and initial outgrowth were greatly improved by seeding the cells on a basement-membrane-like extracellular matrix (ECM) deposited by cultured corneal endothelial cells. These effects were most significant in serum-free conditions which markedly reduced the rate of cell attachment and growth on regular tissue culture plastic. In 60-80% of the cases and regardless of the patient's age, cells cultured on ECM in the absence of serum divided actively and formed a tightly packed epithelial cell monolayer. Fibroblast overgrowth and cell detachment often occurred on ECM in the presence of serum. Incubation of the human ovarian carcinoma cells with sulfate-labelled ECM, resulted in the release of heparan sulfate degradation fragments, 4- to 7-fold smaller than intact heparan sulfate side chains. This degradation was brought about by endoglycosidase (heparanase) activity expressed to a higher extent by cells that were first maintained in primary cultures as compared with cell aggregates taken directly from the biopsy specimen. In most cases, cells derived from metastatic tumors expressed a higher heparanase activity than cells from the primary ovarian tumor. This result corroborates previous studies, performed with cell lines, on the possible involvement of heparanase in tumor cell invasion and metastasis.     

Inhibition of cell motility by troglitazone in human ovarian carcinoma cell line

Background  

Troglitazone (TGZ) is a potential anticancer agent. Little is known about the effect of this agent on cancer cell migration.