Extracellular lipid-mediated signaling in tumor-cell activation and pseudopod protrusion

We have pioneered an in vitro pseudopod-generation model wherein suspended tumor cells are stimulated to form pseudopods into glass micropipettes in response to soluble collagen type IV (CIV). Pertussis toxin and removing intracellular calcium were found previously to be inhibitory to that process. We now extend those observations to dissect the roles of transmembrane calcium influx and circulating fatty acids on pseudopod extension. Removal of fatty acids from BSA in basal media resulted in abrogation of pseudopod formation, while reconstitution of free fatty acids restored cell pseudopod protrusion. We thus hypothesized that fatty acids may provide necessary pseudopod stimulatory signals. Addition of lysophosphatidic acid (LPA) to the fatty acid-free CIV solution or in an opposite pipette without CIV permitted approximately 50% pseudopod recovery in all pipette directions in a dose-dependent fashion. Thapsigargin (TG), an agent that releases internal calcium stores and causes opening of store-operated calcium channels, restored pseudopod protrusion up to 80% in CIV with fatty acid-free albumin. [Ca(2+)](i) release was non-additive when cells were stimulated by TG and LPA, suggesting overlapping [Ca(2+)](i) stores. The combination of TG and LPA in fatty acid-free albumin fully restored the pseudopod response to CIV. Addition of EGTA to chelate stimulatory media calcium blocked the pseudopod response to CIV in the presence of fatty acids. This indicates that pseudopod protrusion requires transmembrane calcium entry. Thus, extracellular lipids and calcium mobilization are required to complement CIV in pseudopod protrusion from suspended cells.     

Rabbit blastocoelic fluid regulation of tumor-cell proliferation in vitro

To determine whether rabbit blastocoelic fluid could inhibit tumor-cell proliferation, day-9 and day-12 embryonic fluids, together with autologous and homologous sera, were collected from pregnant or pseudopregnant rabbits and tested against 13 different cell lines and on human carcinoma cells in primary culture. An inhibitory effect on cell proliferation was observed in the presence of blastocoelic fluids, but not with homologous or heterologous sera. This suppression was higher with samples collected at day 12 than at day 9 of pregnancy. No such inhibition could be detected on one-cell rabbit embryos or on freshly prepared uterine stromal or myometrial cells. In addition, the inhibitory activity on tumor cells was completely reversible upon removal of the fluids. Incorporation of 3H-thymidine, 3H-uridine and 35S-methionine revealed that, in the presence of blastocoelic fluids, both DNA and RNA syntheses were rapidly inhibited. Inhibition of protein synthesis did not occur before 24 hr of treatment. We conclude that rabbit blastocoelic fluid suppresses the proliferation of tumor cells via inhibition of RNA and DNA synthesis by a process which may involve the expression of growth-suppression gene(s).     

Effect of autologous mammary tumour extracts on human leukocyte migration in vitro

In eight out of 22 patients with mammary carcinoma, extract of tumour tissue induced inhibition of autologous leukocyte migration in vitro, demonstrating a state of cellular (delayed type) hypersensitivity. Non-tumorous tissue did not inhibit migration. The effect of the carcinoma extracts was not due to non-specific toxicity, since the migration of leukocytes from matched controls was not affected by the extracts. In nine cases of benign mammary lesions, extracts of autologous mammary tissue did not induce inhibition of migration. Preliminary results of follow-up studies in some of the carcinoma patients are reported.     

Effect of cimetidine on human leukocyte function

In vitro studies of the effects of cimetidine on neutrophil and monocyte chemotaxis revealed no differences between the treated cells and control cells. In vitro studies were performed on neutrophils and monocytes from 20 patients who were treated with oral cimetidine for duodenal ulcer disease. Neutrophil studies from these patients revealed no effect by cimetidine. A significant augmentation of chemotaxis was found in monocytes from patients receiving the drug. Studies of neutrophil phagocytosis from cimetidine-treated patients were exactly identical to control values. These studies indicate cimetidine to have a stimulatory effect on monocyte chemotaxis and no effect on neutrophil function.     

DNA binding and adduct formation of 7,12-dimethylbenz[a]-anthracene by rat mammary epithelial cell aggregates in vitro

Freshly isolated mammary epithelial cell aggregates from femaleSprague-Dawley rats metabolized 7,12-dimethylbenz-[a]anthracene(DMBA) to bay-region anti- and syn-dihydro-diolepoxides thatbound to deoxyguanosine and deoxyadenosine residues in cellularDNA. After 24 h of incubation 68% of the DMBA (0.4 µg/ml)was metabolized and 58% of the extracellular metabolites werewater-soluble. DMBA - DNA binding increased rapidly during theinitial 24 h of incubation. Formation of the bay-region syn-dihydrodiolepoxide:deoxyadenosineadduct increased linearly throughout the 24 h, whereas formationof deoxyadenosine and deoxyguanosine adducts with the bay-regionanti-dihydrodiolepoxide increased rapidly following a delayof 12 h.     

Effect of lime composition on the formation of reactive oxygen species from areca nut extract in vitro

Lime, representative of that used by betel quid chewers, was collected in a region of Papua New Guinea where the incidence of oral cancer is high. The free calcium hydroxide content and pH of 25 lime samples were highly correlated with the generation of reactive oxygen species from areca nut extract in vitro, and DNA damage in vitro, measured as 8-hydroxy-2'-deoxyguanosine. Fe2+ and Mg2+ levels in the lime samples were too low to modify formation of reactive oxygen species, but hydrogen peroxide formation was almost entirely inhibited by addition of Mg2+ to the reaction mixture. These results suggest that the calcium hydroxide content of lime in the presence of areca nut is primarily responsible for the formation of reactive oxygen species which might cause oxidative damage in the DNA of buccal mucosa cells of betel quid chewers.     

天然花色素类诱导肿瘤细胞凋亡机制研究进展

天然花色素类可以诱导多种肿瘤细胞凋亡。其作用机制是通过JNK信号传导通路、活化CASPases、线粒体途径、产生活性氧及阻止细胞增殖周期等多种途径实现的。现将天然花色素类诱导肿瘤细胞凋亡的主要作用机制作一综述。     

天然花色素类诱导肿瘤细胞凋亡机制研究进展

天然花色素类可以诱导多种肿瘤细胞凋亡。其作用机制是通过JNK信号传导通路、活化CASPases、线粒体途径、产生活性氧及阻止细胞增殖周期等多种途径实现的。现将天然花色素类诱导肿瘤细胞凋亡的主要作用机制作一综述。     

多糖诱导肿瘤细胞凋亡机制的研究进展

多糖是普遍存在于动物、植物和微生物中的大分子物质,在细胞识别、信号传导、调节肌体免疫及细胞的迁移、增殖、分化和代谢等方面均有十分重要的作用。近年来众多研究结果表明,多糖可抗肿瘤活性,抑制多种肿瘤细胞生长。究其机制,多糖除可诱导免疫因子生成,增强机体免疫功能外,还能直接杀死肿瘤细胞。通过阐述动物、植物和微生物多糖可以诱导多种肿瘤细胞凋亡,说明多糖具有广谱抑制肿瘤细胞生长的作用;初步探讨了多糖诱导肿瘤细胞凋亡的机制,例如激活死亡受体途径和线粒体途径,调控凋亡基因,调节端粒酶和拓扑酶等。     

Effect of Fluosol-DA/O2 on tumor-cell and bone-marrow cytotoxicity of nitrosoureas in mice bearing FSA-II fibrosarcoma

The perfluorochemical emulsion, Fluosol-DA, combined with carbogen breathing, potentiates the effects of radiation and a number of chemotherapeutic agents in several rodent tumors. The interaction of Fluosol-DA with drugs may be quite complex. In addition to increasing the oxygen supply in the tumor, Fluosol-DA may alter the pharmacokinetics of the drug and function as a drug delivery system. A series of 4 nitrosoureas of varying lipophilicity were administered as single doses intravenously (i.v.) to C3H/Be/FeJ mice bearing subcutaneous FSa-IIC fibrosarcomas. Doses of 40 mg/kg of CCNU, 15 mg/kg of BCNU, 20 mg/kg of MeCCNU and 15 mg/kg of chlorozoticin followed by 2 hr of breathing 95% oxygen produced tumor growth delays of 7.5, 4.0, 3.8 and 2.7 days, respectively. When the drug injection was followed immediately by 0.3 ml of Fluosol-DA and 2 hr of breathing 95% oxygen, the tumor growth delay produced by CCNU, BCNU, MeCCNU and chlorozoticin increased 2-fold, 10-fold, 4.5-fold and 3.5-fold, respectively. Administration of the drugs in Fluosol-DA followed by 2 hr of 95% oxygen breathing resulted in a 3.5-fold increase in tumor growth delay with CCNU, a 17-fold increase with BCNU, a 12.5-fold increase with MeCCNU and a 6-fold increase with chlorozoticin compared to drug and 95% oxygen breathing. These results are quantified in terms of cell survival by the tumor excision assay. Effects on the bone marrow from each treatment were measured using the granulocyte-monocyte colony-forming units (CFU-GM) assay. There was no correlation between the lipophilicity of the nitrosoureas tested and the tumor growth delay produced by each treatment.     

The role of tumor-cell surface carbohydrate in experimental metastasis

Quantitative studies on the binding of concanavalin A (Con A) and wheat-germ agglutinin (WGA) to a series of rat hepatocarcinoma metastatic variants revealed a positive correlation between the amount of cell-surface-bound lectin and lung colonization potential. Scatchard analysis of Con A and WGA binding to 10 individual clones isolated from a subcutaneous (s.c.) tumor transplant and to tumor-cell isolates from 10 individual spontaneous lung metastases from the same animal showed diverse binding characteristics for these cell populations. Nevertheless, the expression of Con A receptor sites accurately predicted the lung colonization potential of 3 isolates from the lung metastases. Higher lectin binding curves were observed for the clones from the subcutaneous tumor than for the isolates from lung metastases. These data suggest that a high Con-A binding potential is indicative of a high lung colonization potential for these hepatocarcinoma cells, but that this phenotype may be rapidly lost during tumor outgrowth in the lungs. The binding of tumor cells to vascular endothelial cell monolayers was inhibited in the presence of Con A; however, no inhibition was observed with 2 other lectins. Attachment of tumor cells to endothelial cell monolayers was also inhibited by the monosaccharides methyl alpha-D-mannopyranoside and N-acetyl-D-galactosamine. Other monosaccharides tested did not alter the attachment of tumor cells to endothelial cell monolayers.     

Comparison of DNA adduct formation by aristolochic acids in various in vitro activation systems by 32P-post-labelling: evidence for reductive activation by peroxidases

Aristolochic acid I (AAI) and aristolochic acid II (AAII), the two major components of the carcinogenic plant extract aristolochic acid (AA), are known to be mutagenic and to form DNA adducts in vivo. According to the structures of the major DNA adducts identified in animals and humans, nitroreduction is the crucial pathway in the metabolic activation of these naturally occurring nitroarenes to their ultimate carcinogenic species. Using the nuclease P1-enhanced version of the 32P-post-labelling assay we investigated the formation of DNA adducts by AAI and AAII in different in vitro activation systems in order to determine the most suitable in vitro system mimicking target tissue activation. Although DNA adducts resulting from oxidative activation of AAs have not yet been identified both reductive and oxidative in vitro systems were employed. In vitro incubations were conducted under standardized conditions (0.3 mM AAs; 4 mM dNp as calf thymus DNA) using rat liver microsomes, xanthine oxidase (a mammalian nitroreductase), horseradish peroxidase, lactoperoxidase and chemical reduction by zinc. Enzymatic incubations were performed under aerobic and anaerobic conditions. A combination of two independent chromatographic systems (ion-exchange chromatography and reversed-phase HPLC) with reference compounds was used for the identification of DNA adducts detected by the 32P-post-labelling assay. The two known major adducts of AAI or AAII found in vivo were generated by all in vitro systems except for incubations with AAII and horseradish peroxidase where two unknown adducts predominated. Irrespective of the in vitro activation system used, the majority of adduct spots obtained were identified as the previously characterized four AA-DNA adducts: dA-AAI, dA-AAII, dG-AAI and dG-AAII. This indicates that both reductive and peroxidative activation of AAI or AAII resulted in chromatographically indistinguishable DNA adducts. Thus, peroxidase mediated activation of AAs led to the formation of the same adducts that had been observed in vivo and upon reductive activation in several in vitro systems. Quantitative analyses of individual adducts formed in the various in vitro systems revealed relative adduct labelling (RAL) values over a 100,000-fold range from 4 in 10(3) for activation of AAII to deoxyadenosine adducts by zinc to only 3 in 10(8) for activation of AAII by lactoperoxidase. The extent of DNA modification by AAI was higher than by AAII in all enzymatic in vitro systems. Only activation by zinc resulted in higher total binding to exogenous DNA by AAII than by AAI. Aerobic incubations with rat liver microsomes generated AAI- and AAII-DNA adduct profiles reproducing profiles in target tissue (forestomach) of rats, thus providing the most appropriate activation among the in vitro systems tested.      

Effect of human leukocyte interferon on malignant brain tumors

The antitumor effect of human leukocyte interferon was investigated on ten patients with malignant brain tumor. In eight cases of primary tumor, IFN alone was administered when their recurrent sign was evident. A dose of 3 X 10(6) IU or 1 X 10(6) IU of IFN was injected intramuscularly two or three times a week in high-dose group, while a dose of 5 X 10(4) IU once a week in low-dose group. No remarkable side effects including bone marrow depression were noted. Natural killer activity was enhanced and immunologic skin reaction manifested. Partial remission of more than 50% decrease of tumor volume calculated on CT scan was seen in two cases in the low-dose group for about 3-6 months. Complete remission could not be obtained by IFN alone. Our pilot study has shown that IFN alone will not be effective against progressive malignant brain tumors by general administration. Further investigation should be carried out to improve the use of IFN therapy in malignant brain tumor.     

Involvement of disulfide bond formation in the activation of heparanase

Heparanase is overexpressed in many solid tumor cells and is capable of specifically cleaving heparan sulfate, and this activity is associated with the metastatic potential of tumor cells; however, the activation mechanism of heparanase has remained unknown. In this study, we investigated the link between disulfide bond formation and the activation of heparanase in human tumor cells. Mass spectrometry analysis of heparanase purified from a conditioned medium of human fibrosarcoma cells revealed two disulfide bonds, Cys127-Cys179 and Cys437-Cys542, and one S-cysteinylation at the Cys211 residue. It was shown that, although the formation of the Cys127-Cys179 bond and S-cysteinylation at Cys211 have little effect on heparanase function, the disulfide bond between Cys437 and Cys542 is necessary for the secretion and activation of heparanase. Thus, the present findings will provide a basis for the further refinement of heparanase structural studies and for the development of novel heparanase inhibitors.     

Initial formation of IGROV1 ovarian cancer multicellular aggregates involves vitronectin

Ovarian cancer progression is frequently associated with the development of malignant ascites. Multicellular aggregates of carcinoma cells (spheroids) found within ascites are thought to be able to promote peritoneal carcinomatosis. We have previously demonstrated the involvement of the vitronectin/αv integrin adhesive system in the dissemination of ovarian cancer cells and continue to investigate the influence of these molecules by studying their role(s) in spheroid behavior. The aim of this study was to generate ovarian cancer multicellular aggregates and to focus on the role of vitronectin and αv integrins in their initiation. IGROV1 cancer cells cultured in the absence of adhesive substratum formed multicellular aggregates comparable to spheroids. After 21 days, a fraction of the cells within clusters remained viable and proliferated recurrently. Within the multicellular aggregates, vitronectin and αv integrins were co-localized at intercellular sites, suggesting their involvement in cell-cell interactions. Initial formation of IGROV1 aggregates was inhibited using anti-vitronectin and anti-αv integrin blocking antibodies or the cyclic peptide cRGDfV. Vitronectin expression persisted during cluster disaggregation on fibronectin. These results demonstrate the ability of IGROV1 cells to generate multicellular aggregates and point to a contributory role for the vitronectin/αv integrin system in the initial step of this process. These events could represent a prerequisite for further dissemination.