Effects of alkylating antineoplastics alone or in combination with 3-aminobenzamide on genotoxicity, antitumor activity, and NAD levels in human lymphocytes in vitro and on Ehrlich ascites tumor cells in vivo

Enhanced cytogenetic damage by the homo-aza-steroidal ester of p-bis(2-chloroethyl)-aminophenylacetic acid (ASE) was observed when human lymphocytes in vitro or Ehrlich ascites tumor (EAT) cells in vivo were exposed to nontoxic concentrations of 3-amino-benzamide (3-AB). 3-AB at these concentrations was found to enhance synergistically the cytogenetic damage induced in vivo by cyclophosphamide (CP), a metabolically activated chemotherapeutic, or chlorambucil (CBC) in EAT cells. One hour before i.p. injection of 5-bromodeoxyuridine (BrdUrd) adsorbed to activated charcoal, EAT-bearing mice treated i.p. with ASE or CP showed a dose-dependent increase in sister chromatid exchange (SCE) rates and cell division delays. The treatment of human lymphocytes in vitro with ASE led to the depletion of cellular NAD, and addition of 3-AB, a potent inhibitor of poly(ADP-ribose)polymerase [P(ADPR)polymerase], to ASE-treated human lymphocytes prevented the drop of NAD, which remained at approximately control levels. Also, the in vivo treatment of EAT cells with CBC, ASE, or CP led to the depletion of NAD, whereas addition of 3-AB to CBC-, ASE- or CP-treated cells prevented the drop of NAD, which remained at nearly control levels. 3-AB in conjunction with CBC, ASE, or CP increased the survival time of the EAT-bearing mice and markedly reduced the ascitic volume. Thus cytogenetic damage induced by ASE plus 3-AB in vitro and by CBC, ASE, or CP plus 3-AB in vivo correlates well with 1) the prevention of NAD depletion in the presence of 3-AB in cells treated with the same alkylating agents in vitro or in vivo and 2) the in vivo antitumor effect by ASE, CBC, or CP in combination with 3-AB.     

含人Fc重组凋亡融合蛋白体内外抗肿瘤活性初步研究

目的初步研究含人抗体Fc片段的肿瘤坏死因子相关凋亡诱导配体（TRAIL-Fc）重组融合蛋白的体内外抗肿瘤活性。方法①体外实验：将人急性T淋巴细胞白血病Jurkat细胞、人结肠癌LOVO细胞、人胃癌MKN45细胞、人表皮癌A431细胞、人肝癌HEPG2细胞、人乳腺癌MCF-7细胞和小鼠肝癌H22细胞均随机分为TRAIL-Fc重组融合蛋白组、对照组和空白对照组,分别加入浓度为1000、250、62.5、15.6、3.9、0.97ng/ml的TRAIL-Fc重组融合蛋白和TRAIL对照品以及完全培养液,采用MTT法检测TRAIL-Fc重组融合蛋白对7种肿瘤细胞株的体外生长抑制率。②体内实验：腹腔和腋下接种H22细胞建立小鼠肝癌模型,据治疗药物的不同将小鼠随机分为空白对照组、5-Fu组和TRAIL-Fc重组融合蛋白组。测量记录腹腔接种小鼠的腹围和体重以及腋下接种小鼠的肿瘤体积,15d后取外周血处死各组小鼠,计算抑瘤率并检测血清中AST、ALT含量。结果体外实验结果显示,TRAIL-Fc重组融合蛋白对7种肿瘤细胞株的生长均有不同程度的抑制作用,且抑制作用呈剂量依赖效应;其中对MCF-7细胞的抑制作用最强,其最大生长抑制率为82.5%,其次依次为LOVO（81.9%）、MKN45（52.3%）、H22（51.2%）、Jurkat（50.9%）、HEPG2（35.4%）和A431细胞（20.3%）。与对照品相比,TRAIL-Fc重组融合蛋白对LOVO（IC50为83.5）、MKN45（IC50为243.2）、MCF-7（IC50为84.6）细胞更敏感。体内实验结果显示,空白对照组小鼠腹部明显膨出,后期体重略有下降,肿瘤体积持续显著增加;TRAIL-Fc重组融合蛋白组小鼠腹部轻微膨胀,体重维持较好,肿瘤体积后期略有增加;5-Fu组小鼠腹部无膨胀,体重急剧下降,肿瘤体积明显缩小。TRAIL-Fc重组融合蛋白的抑瘤率为45.79%,AST和ALT检测结果显示其对小鼠肝功能无明显影响。结论 TRAIL-Fc重组融合蛋白具有较强的抑制肿瘤细胞生长的能力,且在体内半衰期明显延长。     

An in vitro model of pancreatic carcinoma. Morphology and in vivo growth.

Pancreas rudiments from 13-day rat embryos were cultured in the presence of dimethylnitrosamine (DMN) for up to 10 weeks. Pancreas morphogenesis and differentiation occurred during the first week of culture. Acinar cell degeneration and necrosis began on the fifth day of culture and resulted in almost complete loss of acinar cells, islet cells, and fibroblasts by the end of the third week. This was associated with proliferation of cells without zymogen granules (centroacinar, ductal, or undifferentiated?). Theses cells formed glandular structures which extended to the surface of the explant. By the end of the fourth week, explants resembled ductal hyperplasia with foci of carcinoma in situ. The distribution pattern of neoplasia in 343 explants examined after 10 weeks of DMN treatment was as follows: 79% resembled ductal cell carcinoma; 9%, ductal hyperplasia; and 3%, acinar cell carcinoma. Nude mice injected with cell suspensions prepared from 10-week-old culture developed subcutaneous nodules. These nodules resembled duct cell carcinoma with desmoplastic reaction.     

Comparative study of in vitro and in vivo drug effects on cell-mediated cytotoxicity.

Cell-mediated cytolysis (CMC) was assayed in a system using spleen cells from mice (C57BL/6) sensitized with allogeneic tumour cells (DBA/2 mastocytoma P-815). Anti-inflammatory drugs, immunosuppressives, inhibitors of cell division and other agents were investigated for their capacity to inhibit CMC in three different ways. First, inhibition of CMC after in vitro addition of drug was observed with corticosteroids, some immunosuppressives and inhibitors of cell division. Secondly, suppression of CMC after a single drug administration to sensitized mice shortly before being killed was found with corticosteroids, several immunosuppressives and irradiation. Thirdly, prevention of development of CMC by repeated drug treatment (immunosuppressive schedule) was achieved with most immunosuppressives and cytostatic drugs. Non-steroidal anti-inflammatory drugs were inactive in these tests. Correlation of effects between the three procedures was very poor and it is suggested that various mechanisms may be involved in the different assays.     

Abortive myxovirus infection of Ehrlich ascites carcinoma cells. Analysis of virus-specific structures in the ascitic fluid]

In the course of classical fowl plague virus reproduction in Ehrlich ascites carcinoma cells both hemagglutinins and S-antigen accumulate and titers of the infectious activity increase. However virus reproduction does not terminate in formation of virus, and subviral structures are found in the liquid fraction of the infected cells. Analysis of these structures has shown them to have a sedimentation coefficient of 350-370S and buovant density 1.29 g/ml. The rapidly sedimenting structure has complement-fixing hemagglutinating activity but bow infectivity.     

Inhibition of T cell activity in vivo: a test model for quantitative evaluation.

A test model is presented which, in comparison with the conventional models of skin transplantation or graft-versus-host (GvH) reaction in mice, permits a more sensitive quantitative evaluation of T cell inhibition in vivo. Prospective donors (type AA) are immunized with prospective recipient material (type AB); the resulting T cell reaction of A versus B is inhibited by consecutive treatment. Extent of inhibition can be evaluated after transfer of the pretreated AA material onto AB recipients by calculation of remaining GvH reactivity, if compared to adequate control tranfers. In this model the target animal for T cell reactivity (the AB recipient) remains untouched from immunosuppressive regimen.     

Biological time and the effects of hydroxyurea on DNA synthetic activity of bone marrow and tumor cells in mice bearing the Ehrlich ascites carcinoma

The objective of this experiment was to attempt to induce, with hydroxyurea (HU), significant quantitative differences in the level of DNA-synthetic activity (DNA-SA) between a neoplastic cell population (the Ehrlich ascites carcinoma or EAC) and bone marrow in the same animal. Mice bearing a 5-day-old EAC were standardized to and kept on an LD 12:12 cycle (light 0600–1800 hr). They were treated with 500 mg/kg HU at 0500 hr (23 hr after lights on, or HALO) or at 1700 hr (11 HALO). DNA-SA was determined by liquid scintillation counting of ³H-thymidine incorporation into chemically isolated DNA. DNA-SA in bone marrow and EAC cells was monitored over the next 60 hr with subgroups of ten mice each killed every 3 hr beginning 3 hr after treatment with HU. The circadian system of the host influenced the response of the bone marrow to HU; i.e., the response to HU administered at 0500 hr was different both qualitatively and quantitatively from that for HU given at 1700 hr. Comparisons of DNA-SA in bone marrow and EAC from the same animal revealed time points after treatment with HU when DNA-SA in the EAC was high, but DNA-SA in bone marrow was low. These differences in the level of DNA-SA between a tumor cell population and bone marrow should be of therapeutic value; i.e., executor doses of anti-DNA-SA drugs such as cytosine arabinoside could be given at that point in time after treatment with HU when DNA-SA in the tumor was high, but DNA-SA in the bone marrow was low.     

Gimatecan exerts potent antitumor activity against gastric cancer in vitro and in vivo via AKT and MAPK signaling pathways

Background

We investigated antitumor activity and underlying mechanisms of DNA topoisomerase I (TopI) inhibitor gimatecan and irinotecan in gastric cancer (GC) in vitro cell lines and in vivo patient-derived xenograft (PDX) models.

Methods

GC cell lines SNU-1, HGC27, MGC803 and NCI-N87 were used to evaluate cell viability and apoptosis after gimatecan or irinotecan treatment, using a cell proliferation assay and flow cytometry, respectively. DNA TopI expression and critical molecules of PI3K/AKT, MAPK and apoptosis signaling pathways were analyzed with western blot. For in vivo studies, five PDXs models were treated with gimatecan or irinotecan to assess its antitumor activity. Immunohistochemistry staining of Ki-67 was performed after mice were sacrificed.

Results

Gimatecan inhibited the proliferation of GC cells in vitro in a dose- and time-dependent manner by inducing apoptosis, and gimatecan had greater inhibitory effects than irinotecan. In addition, both gimatecan and irinotecan demonstrated significant tumor growth inhibition in in vivo PDX models. Gimatecan treatment significantly inhibited the expression of DNA TopI, phosphorylated AKT (pAKT), phosphorylated MEK (pMEK) and phosphorylated ERK (pERK). Meanwhile, gimatecan could also activate the JNK2 and p38 MAPK pathway as indicated by upregulation of phosphorylated p38 MAPK (p-p38) and phosphorylated JNK2 (pJNK2).

Conclusions

For the first time, we have shown that the antitumor activity of gimatecan in GC via suppressing AKT and ERK pathway and activating JNK2 and p38 MAPK pathway, which indicated that gimatecan might be an alternative to irinotecan in the treatment of GC.

     

Human hepatocellular carcinoma expresses specific PCNA isoforms: an in vivo and in vitro evaluation

Proliferating cell nuclear antigen (PCNA) is a 36 kDa protein involved in several cellular mechanisms, including DNA synthesis and repair, cell cycle regulation and apoptosis. An alteration in PCNA structure might contribute to DNA-damage accumulation in cancer cells. This study was aimed to evaluate the PCNA pattern of expression, in terms of aggregation status, isoforms and post-translational modifications, in human hepatocellular carcinoma (HCC) and cirrhosis as well as in HCC cell lines. Twelve HCCs and surrounding cirrhotic tissues were analysed, along with HepG2, Hep3B and SNU-398 cell lines. Normal liver specimens and cirrhosis without HCC were included as controls. Both DNA-bound and DNA-unbound PCNA fractions were analysed, and PCNA pattern of expression was displayed on two-dimensional gel electrophoresis followed by western blot. Results were confirmed by mass spectrometry. To compare HCCs vs surrounding tissues, immunolabelling and immunostaining were performed. In 6 of 12 HCCs and in cell lines, we found three major PCNA acidic forms, corresponding to monomers, probably dimers and trimers, and a basic isoform. In the six remaining HCCs, only a PCNA acidic form associated with multiple basic isoforms was detected. Importantly, the PCNA basic form was not found in cirrhotic tissues. To clarify the nature of the detected PCNA isoforms, ubiquitin-specific immunoblotting as well as phosphatase treatment were employed. A PCNA-ubiquitylated form in cell lines and PCNA-phosphorylated isoforms in 6 of 12 HCCs were detected. Finally, in the DNA-bound fraction we detected only an acidic PCNA monomeric form. We conclude that human hepatocellular carcinoma expresses specific PCNA isoforms compared to those found in cirrhosis, implicating a role for PCNA functional alterations in hepatocarcinogenesis.     

Comparison of IgE and IgG antibody-dependent cytotoxicity in vitro and in a SCID mouse xenograft model of ovarian carcinoma

Allergic reactions are mediated by IgE antibodies bound to high-affinity receptors on mast cells in peripheral tissues and are characterized by their immediacy and hypersensitivity. These properties could also be advantageous in immunotherapy against cancer growth in peripheral tissues. We have constructed chimeric IgE and IgG1 antibodies with murine V regions and human C regions corresponding to the MOv18 monoclonal antibody against the human ovarian tumor-associated antigen, folate binding protein. The antibodies exhibited the expected binding affinities for antigen and Fc receptors, and effector activities with human basophils and platelets in vitro. The protective activities of MOv18-IgE and MOv18-IgG1 were compared in a SCID mouse xenograft model of ovarian carcinoma. The beneficial effects of MOv18-IgE were greater and of longer duration than those of MOv18-IgG1. Our results suggest that the allergic reaction could be harnessed for the suppression of ovarian tumors.     

In vitro cytotoxicity and in vivo biocompatibility of poly(propylene fumarate-co-ethylene glycol) hydrogels.

The in vitro cytotoxicity and in vivo biocompatibility of poly(propylene fumarate-co-ethylene glycol) [P(PF-co-EG)] hydrogels were assessed in order to investigate the influence of poly(ethylene glycol) molecular weight and copolymer composition. These materials have application as injectable cardiovascular implants; cytotoxicity due to leachable products, as well as inflammation caused by the biomaterial itself, may ultimately affect the biocompatibility of the implant. We utilized a 7-day in vitro cytotoxicity assay to quantify cell density and cellular proliferation in the presence of copolymer films. The copolymer films exhibited slight to moderate cytotoxicity toward cultured endothelial cells, showing 20-86% viability relative to controls. Cell viability increased with an increasing weight percent of PEG or, to a lesser extent, the molecular weight of PEG. In vivo biocompatibility was assessed using a cage implantation model over a 21-day time period. This system was used to characterize the local cellular and humoral inflammatory response in the surrounding exudate, as well as the size and density of macrophages adherent to the material itself. All copolymer formulations exhibited excellent biocompatibility relative to controls with no significant differences in total leukocyte count among the different formulations. The in vivo inflammatory reaction displayed normal wound healing over 21 days as shown by a progressive decrease in both leukocyte concentration and enzymatic activity. The surface coverage of the copolymer films remained relatively constant from 7 to 21 days. There were no cells larger than 0.003 mm2, which was previously shown to be the threshold value for foreign-body giant cells. These data suggest that P(PF-co-EG) hydrogels have potential for use as injectable biomaterials.     

In vitro and in vivo antitumor activity of crude extracts obtained from Brazilian Chromobacterium sp isolates

Natural products produced by microorganisms have been an important source of new substances and lead compounds for the pharmaceutical industry. Chromobacterium violaceum is a Gram-negative β-proteobacterium, abundant in water and soil in tropical and subtropical regions and it produces violacein, a pigment that has shown great pharmaceutical potential. Crude extracts of five Brazilian isolates of Chromobacterium sp (0.25, 2.5, 25, and 250 µg/mL) were evaluated in an in vitro antitumor activity assay with nine human tumor cells. Secondary metabolic profiles were analyzed by liquid chromatography and electrospray ionization mass spectrometry resulting in the identification of violacein in all extracts, whereas FK228 was detected only in EtCE 308 and EtCE 592 extracts. AcCE and EtCE 310 extracts showed selectivity for NCI/ADR-RES cells in the in vitro assay and were evaluated in vivo in the solid Ehrlich tumor model, resulting in 50.3 and 54.6% growth inhibition, respectively. The crude extracts of Chromobacterium sp isolates showed potential and selective antitumor activities for certain human tumor cells, making them a potential source of lead compounds. Furthermore, the results suggest that other compounds, in addition to violacein, deoxyviolacein and FK228, may be involved in the antitumor effect observed.     

Anti-neoplastic activities of sepia officinalis ink and coelatura aegyptiaca extracts against Ehrlich ascites carcinoma in Swiss albino mice

Objectives: With the development of sophisticated instruments for the isolation and elucidation of natural products structures from marine and freshwater organisms, major advances have been made in the discovery of aquatic derived therapeutics. Present investigations were carried out to evaluate cuttlefish (Sepia officinalis) ink extract (IE) and freshwater clam (Coelatura aegyptiaca) extract (CE) for their anticancer and antioxidant activities as compared to 5-flurouracil (5-Fu), in Ehrlich ascites carcinoma (EAC). Methods: Sixty female Swiss albino mice were divided into five groups (n = 12). All groups except group I received EAC cells (5 × 10⁶ cells/mouse i.p.) and this was taken as the 0th day. Group I served as saline control (5 ml/kg 0.9% NaCl w/v p.o). Group II served as EAC control. Rats of groups III, IV and V received IE, CE (200 mg/kg body weight i.p.), and reference drug (5-Fu, 20 mg/kg body weight i.p.), respectively. Results: The reduction in tumor volume, packed cell volume, tumor cell counts and increase in median survival time and percentage increase in life span in treated animals were observed. There was a significant increase in RBC count; Hb content in treated animals and reduction in total WBC count. There was a significant decrease in AST, ALT, ALP and liver MDA levels and increase in GSH, SOD and NO levels were observed in all treated animals. Conclusion: Both IE and CE were effective in inhibiting the tumor growth in ascitic tumor models. The biochemical, antioxidants and histopathological studies were also supported their antitumor properties.     

人肺癌浸润淋巴细胞抗肿瘤活性的诱导及其实验性扩增

人们早就发现,肿瘤组织并非仅由肿瘤细胞所组成。除支持肿瘤生长的血管和间质细胞外,还有相当数量的浸润淋巴细胞(TIL)。近2、3年,人们对TIL有了不少新的认识,但TIL的确切功能、活性调节、增殖特征等都还未完全明了,有关人肺癌TIL的研究更是少见。本工作的目的在于研究人肺癌TIL抗肿瘤活性的诱导、体外抗肿瘤的特性、体外实验性扩增及抗肿瘤活性的变动情况,探讨利用TIL进行临床抗肿瘤抗转移免疫过继疗法的可行性。     

In vitro and in vivo evaluation of ribavirin and pleconaril antiviral activity against enterovirus 71 infection

Enterovirus 71(EV71) causes recurring outbreaks of hand, foot and mouth disease and encephalitis leading to complications or death in young children. More effective antiviral drugs are needed to prevent or reduce EV71-related disease and complications. However, there are no standard models currently in use to evaluate activity against EV71 infection both in vitro and in vivo. In this study, the activity of ribavirin and pleconaril against EV71 infection was evaluated in two models. An in vitro EV71 infection model was developed in RD cells, and an in vivo EV71 infection model was applied. Ribavirin and pleconaril effectively increased the viability of infected cells. Pleconaril reduced the morbidity and mortality of one-day-old infected mice, but ribavirin did not protect the infected mice. In all, the results demonstrated that infected cells and infected mice can be used to evaluate antiviral activity of ribavirin and pleconaril against EV71 infection in vitro and in vivo.