Decrease in tumorigenic activity of murine hepatoma cells after treatment with antioxidants and melatonin

We studied the effect of antioxidants such as N-acetylcysteine (NAC, 10 mM) and alpha-lipoic acid (ALA, 1.25 mM) and of the hormone melatonin (1 microM) on the ability of murine hepatoma cells MH22a to develop tumors in syngenic mice (C3HA) after subsutaneous injection. Tumor formation and development slowed down and mouse mortality decreased when the injected cells were pretreated by NAC, ALA or melatonin during 20 h. Melatonin had the most marked effect. Tumors appeared in 100 % cases after 10 days in control mice when untreated cells had been injected; injection of cells pretreated by NAC or ALA resulted in tumor formation only in 40 and 53 % of mice, respectively. When cells were pretreated with melatonin the tumors appeared only in 18-20 days after injection. Until the end of the observation (36 days) 67 % of control mice died, but when the cells were pretreated by NAC or ALA mouse death-rate was 20 and 53 %, respectively. In the case of melatonin we did not observed any dead mice at all. We showed that treatment by antioxidants delayed (NAC) or completely inhibited (ALA) cell cycle of hepatoma cells. Cell cycle was restored after removal of the antioxidants. Melatonin did not change cell cycle phase distribution. We conclude that there is no direct correlation between loss of tumorigenic properties and changing of proliferative activity of hepatoma cells. Different mechanisms of antioxidants and melatonin action resulting in transient tumor phenotype normalization are discussed.     

The effect of radiation with polychromatic visible and infrared light on the tumorigenicity of murine hepatoma 22A cells and their sensitivity to lysis by natural killers

The tumorigenicity of murine hepatoma cells (MH-22a) and their sensitivity to lysis by natural killers (NKs) have been studied after exposure to polychromatic visible and infrared light (VIS-IR, 480–3400 nm, 40 mW/cm²), similar to the terrestrial solar spectrum without its minor UV component, with the aim of clarifying the participation of this important environmental and physiotherapeutic factor in regulation of antitumor protective system. MH-22 cells were exposed in vitro to VIS-IR light and their sensitivity to lytic activity of NKs was evaluated. It was found that, after exposure to VIS-IR light at a dose of 4.8 J/cm², the sensitivity of MH-22a cells to lysis by NKs increased by 1.5–2 times, while after exposure at a dose of 9.6 J/cm² it did not change at all the ratios of the NKs-number (effectors) to that of hepatoma cells — targets (1 : 5–1 : 50). An increase of the hepatoma cell sensitivity to NKs was accompanied by structural changes of cell surface: the capability of supramembranous glycoproteins (glycocalyx) to sorb the vital dye alcian blue (AB) was significantly lower than in the case of unexposed cells of the control group. However, no changes in AB sorption was revealed in hepatoma cells exposed to light at a dose of 9.6 J/cm². The tumorigenicity of photoirradiated MH-22a cells has been studied in the experiments in vitro. For 25 days after transplantation of light-exposed hepatoma cells to C3HA syngene mice, the tumor volume proved to be smaller after exposure to light at both doses of 4.8 and 9.6 J/cm² than in the control group (by 4–4.5 times and 2.5–4 times, respectively), which correlated with an increase of sensitivity to lysis by NKs and with a decrease of AB sorption after light exposure only at a dose of 4.8 J/cm². Using the flow-cytometry method, we could show that VIS-IR light at the doses used did not interfere with the distribution of hepatoma cells over the cell-cycle phases and, thus, deceleration of the tumor growth was not associated with a cytostatic effect of the VIS-IR light. To evaluate the effect of polychromatic light on growth of the preformed tumors, a 5-day course of daily light exposure of C3HA tumor-bearing mice was performed on the 10th day after subcutaneous transplantation of 2 × 10⁵ cells of syngene hepatoma, when tumors developed in all (100%) animals. As in the case of transplantation of light-exposed cells, irradiation of tumor-bearing mice at doses 4.8–9.6 J/cm² resulted in a deceleration of tumor growth (by 2.1–2.9 and 2,2 times, respectively) for 4 weeks compared with unirradiated mice.     

Changes in the lectin binding capacities of hepatoma cells after treatment with chondroitinase.

Binding capacities of cells of ascites hepatoma, AH 130 FN, towards lectins were examined before and after treatment with chondroitinase AC. Chondroitin sulfate A was removed from the cells by the enzyme treatment, and the binding capacity of the cells towards $\text{Ricinus communis}$ agglutinin (RCA) increased remarkably while the binding constant was unchanged, whereas that towards Concanavalin A (ConA) remained practically unchanged.     

H-2Kb antigen expression has no effect on natural killer susceptibility and tumorigenicity of a murine hepatoma

Recent reports suggested a correlation between decreased expression of tumor cell MHC class I Ag and increased susceptibility to NK cells. These studies led to the hypothesis that tumor cells displaying reduced levels of MHC class I Ag have reduced tumorigenicity in vivo because they are eliminated from the host by endogenous NK cells. The present studies use the murine hepatoma BW7756 and a spontaneous H-2Kb loss variant, Hepa-1, to test this hypothesis. The parental BW7756 tumor is highly malignant in syngeneic C57L/J hosts while Hepa-1 cells do not give rise to tumors, suggesting that the loss of H-2Kb Ag expression correlates with decreased tumorigenicity and NK susceptibility. Hepa-1 cells were therefore transfected with an H-2Kb gene to generate H-2Kb Ag expressing clones. The resulting clones were tested for tumorigenicity. Syngeneic or NK-deficient C57BL/6-beige/beige mice challenged with Hepa-1 or the H-2Kb transfectants rejected the cells, suggesting that reexpression of H-2Kb Ag does not restore tumorigenicity and that NK cells are not involved in Hepa-1 rejection. In vitro H-2Kb Ag-negative and -positive Hepa-1 cells are equally susceptible to tilorone-boosted NK cells, indicating that MHC class I Ag expression also does not affect in vitro NK susceptibility. Tumor challenged athymic nude and sublethally irradiated syngeneic mice develop tumors demonstrating that T cells are probably responsible for rejection of the Hepa-1 tumor, and that H-2Kb Ag expression has no effect on rejection. Inasmuch as the expression of H-2Kb Ag on Hepa-1 cells does not effect tumorigenicity or in vitro NK susceptibility, the previously reported association between reduced MHC class I Ag levels and increased NK susceptibility is not universally applicable.     

Suppression of tumorigenicity in transformed cells after transfection with vinculin cDNA

Transfection of chicken vinculin cDNA into two tumor cell lines expressing diminished levels of the endogenous protein, brought about a drastic suppression of their tumorigenic ability. The SV-40-transformed Balb/c 3T3 line (SVT2) contains four times less vinculin than the parental 3T3 cells, and the rat adenocarcinoma BSp73ASML has no detectable vinculin. Restoration of vinculin in these cells, up to the levels found in 3T3 cells, resulted in an apparent increase in substrate adhesiveness, a decrease in the ability to grow in soft agar, and suppression of their capacity to develop tumors after injection into syngeneic hosts or nude mice. These results suggest that vinculin, a cytoplasmic component of cell-matrix and cell-cell adhesions, may have a major suppressive effect on the transformed phenotype.     

Protective effects of antioxidants on deoxynivalenol-induced damage in murine lymphoma cells

As contradictory results have been reported on the immunotoxic properties of deoxynivalenol (DON) in animal studies, we introduced a lymphoblast cell culture model in order to examine the effects of DON on lymphoblastic cell growth and metabolism as well as the preventive properties of free radical scavenger molecules against the DON-induced cell damage. Murine YAC-1 lymphoma cells were used because lymphoblasts have been shown to be sensitive to DON-induced immunotoxicity. Cells were quantified and their proliferative activity was measured by a proliferation test. Lipid peroxidation and protein oxidation were determined using assays quantifying thiobarbituric acid reactive substances (TBARS) and carbonylated proteins. Severely reduced cell counts were detected in DON-treated samples, confirmed by a 5–10 times lower proliferative activity. Significant increases in lipid peroxidation and protein oxidation were found in parallel incubated samples. The pre-incubation with free radical scavengers significantly reduced DON-induced changes to proteins and lipids as well as the tarnished cell viability and cell proliferation. These results suggest that YAC-1 lymphoma cells are a suitable model to investigate and elucidate the basic molecular and cellular mechanisms for possible immunotoxic effects of DON. With regard to the impact of free radical scavengers, the applied in-vitro model might enable the investigation of potential prophylactic and therapeutic effects before or even without harmful animal experiments and cost- and time-intensive expenses.     

Reduced tumorigenicity of A-10 adenocarcinoma in the presence of immunogenic A-10 tumor cells

Summary The highly tumorigenic A-10 adenocarcinoma caused progressive tumor growth and death of syngenic A/J mice, whereas cultured A-10 cells did not cause tumors in all mice. The mixture of in vivo-grown and cultured A-10 tumor cells resulted in either progressive tumor growth or tumor rejection, depending upon the relative proportion of the two cell types. Expression of cell-surface carbohydrates was quantitated using cell-microbead, lectin-induced agglutination. The immunogenic, cultured A-10 tumor possessed a marked increase in agglutinability, compared to the in vivo-grown tumor. The results are consistent with the concept that the immunogenic properties of murine adenocarcinoma are closely associated with properties of the cell-surface.Supported, in part, by a research contract from the National Cancer Institute, USA     

Cholera toxin treatment stimulates tumorigenicity of Rous sarcoma virus-transformed cells.

Chinese hamster ovary cells transformed by Rous sarcoma virus form tumors poorly in nude mice. Tumorigenicity was markedly stimulated by pretreatment of the cells with cholera toxin, which raises cyclic AMP levels and activates cyclic AMP-dependent protein kinase. Increased tumorigenicity was manifested by a severalfold increase in the rate of tumor formation, as well as earlier appearance and more rapid growth of tumors. In contrast, spontaneously transformed Chinese hamster ovary cells showed decreased tumorigenicity after cholera toxin treatment. The activation of tumorigenic potential in Rous sarcoma virus-transformed Chinese hamster ovary cells by cholera toxin correlated with increased phosphorylation of the viral oncogene product pp60src and stimulation of its tyrosine kinase activity.     

褪黑素对小鼠MFC前胃癌细胞的survivin、caspase-3表达的影响

目的探讨褪黑素（Melatonin,MLT）对小鼠前胃癌细胞（murine foregastric carcinoma,MFC）的生存素survivin和半胱天冬酶-3（cysteinyl aspartate-specific protease-3,caspase-3）表达的影响。方法使用不同浓度褪黑素处理培养的胃癌细胞,运用细胞免疫组化、实时荧光定量PCR、免疫印迹法、分光光度法等方法检测褪黑素在抑制小鼠MFC前胃癌细胞增殖过程中对细胞survivin、caspase-3的表达影响。结果与空白对照组相比,6mmol/L浓度褪黑素干预组的胃癌细胞survivin表达下调,caspase-3蛋白表达上调,并呈剂量依赖性。结论褪黑素能够降低胃癌细胞survivin的表达,进而激活caspase-3的活性,是褪黑素体外抑制肿瘤细胞增殖的作用机制之一。     

Changed murine lymphocyte trapping after treatment with Propionibacterium granulosum

Summary The trapping of lymphocytes occurring at different times after a single injection of Propionibacterium granulosum was studied. Labeled syngeneic lymphocytes injected into Propionibacterium-treated recipients showed a different pattern of localization from that observed in untreated animals. A pronounced decrease in homing to the lymph nodes and spleen and an increase in localization in the liver were found. The extent of trapping in the liver corresponded to the increase in weight of this organ. Whole-body irradiation with 400 R, used to achieve autologic lymphocyte depletion, did not change the localization of labeled cells. However, macrophage damage caused by silica resulted in diminished trapping in the liver in Propionibacterium-treated animals and increased accumulation of labeled lymphocytes in the spleen and lymph nodes.     

Morphometrical studies of reproductive system of birds after treatment with dopamine receptor blockers and melatonin

Male Japanese quails were treated with melatonin alone or melatonin combined with D1 and D2 dopamine receptor blockers. Following the treatment, hypothalamus, pituitary glands and testes were analyzed morphometrically. The results suggest the existence of an interaction between melatonin and dopaminergic system in the brain in the regulation of reproductive processes in immature birds. The character of this interaction alters according to the time of the treatment (morning, afternoon, evening, night).     

Suppression of tumorigenicity and metastasis in murine UV-2237 fibrosarcoma cells by infection with a retroviral vector harboring the interferon-beta gene

 In this study, we endeavored to determine the effectiveness of interferon β (IFNβ) gene therapy against highly metastatic murine UV-2237m fibrosarcoma cells. UV-2237m cells were engineered to produce murine IFNβ constitutively following infection by a retroviral vector harboring the murine IFNβ gene. Parental (UV-2237m-P), control-vector-transduced (UV-2237m-Neo), and IFNβ-transduced (UV-2237m-IFNβ) cells were injected subcutaneously (s.c.) or intravenously (i.v.) into syngeneic mice. Parental and control-transduced cells produced rapidly growing tumors, whereas IFNβ-transduced cells did not. The tumorigenicity of IFNβ-sensitive or -resistant parental cells was significantly suppressed when they were injected s.c. together with IFNβ-transduced cells. The IFNβ-transduced cells did not inhibit growth of parental cells injected s.c. at a distant site. UV-2237m-IFNβ cells produced s.c. tumors in nude, SCID/Beige, and natural killer(NK)-cell-compromised syngeneic mice. The IFNβ-transduced cells were more sensitive to in vitro splenic cell-mediated lysis than were the parental or control-transduced cells. Pretreatment of C3H/HeN mice with the NK-cell-selective antiserum (anti-asialoGM1) partially abrogated the cytotoxic activity of the cells. Cytotoxic activity was not observed in mixed culture of UV-2237m-IFNβ cells and splenic cells from SCID/Beige mice. Significant cytotoxicity against UV-2237m-IFNβ cells was mediated by macrophages activated by either IFNγ, lipopolysaccharide, or a combination of both. Our data led us to conclude that the constitutive expression of IFNβ can suppress tumorigenicity and metastasis of UV-2237m cells, which is due, in part, to activation of host effector cells. Received: 12 November 1997 / Accepted: 30 January 1998     

Synthesis of alpha-fetoprotein, albumin and transferrin by long-term cultured cells of murine hepatoma]

The capacity of continuous cell of XXIIa mouse hepatoma (strain MHXXIIa) to synthesize alpha-fetoprotein, albumin and transferrin was studied by immunoautoradiography. Albumin and transferrin were detected in the polyethylene glycol concentrated growth medium of hepatoma cells on the 5th year (the 55th month) of their cultivation. alpha-fetoprotein was not found. Only transferrin was revealed in the growth medium of hepa toma cells of the 8th year (the 92d month) of cultivation. Two clonal cultures obtained on the 8th year of hepatoma cell cultivation were also characterized by the ability to synthesize transferrin. The continuous mouse hepatoma cells retained their malignancy. The agar micro-precipitation reaction showed the presence of alpha-fetofetoprotein in lyfogel concentrated serum of mice with tumors formed after inoculation of the hepatoma cells of the 5th year of cultivation. However, alpha-fetoprotein was not detected in the serum of mice with tumors induced by inoculation of the hepatoma cells of the 8th year of cultivation.     

Suppression of in vivo tumorigenicity of rat hepatoma cell line KDH-8 cells by soluble TGF-beta receptor type II

We have previously reported that transforming growth factor beta (TGF-beta) produced by rat hepatoma cell line KDH-8 cells suppressed the interleukin-2 (IL-2) production of T cells and the tumoricidal activity of macrophages in KDH-8 tumor-bearing rats and that the inhibition of TGF-beta production by low-dose bleomycin restored these activities significantly. In this study, we established three transfectant clones with stable expression of soluble TGF-beta receptor type II (sTRII), namely KT1, KT2 and KT3, and one with an empty vector used as control vector (KV), and then investigated the effects of sTRII on the tumorigenicity of KDH-8 cells and immune responses in syngeneic Wistar King Aptekman/Hok (WKAH) rats. We found that sTRII expressed in sTRII transfectants could abolish growth inhibition of Mv1Lu cells by TGF-beta1 produced by the cells themselves, and that tumor growth of KT2 and KT3 clones in vivo was suppressed significantly compared with that of parent, KV and KT1 clones. Furthermore, we demonstrated that IL-2 production of splenocytes and IL12p40 mRNA expression in tumor tissues were restored in rats inoculated with KT2 and KT3 clones, whereas such restoration was not observed in rats inoculated with parent, KV and KT1 clones. Combined with a low expression of sTRII in KT1 tumor tissues, these results suggest that sTRII may to some extent be able to abolish the tumor-promoting activity of TGF-beta, and imply that sTRII might have a therapeutic effect on TGF-beta-producing tumors.     

Potential anticarcinogenic action of melatonin and other antioxidants mediated by antioxidative mechanisms

The complex process of carcinogenesis is, to a large extent, due to oxidative stress. Numerous indicators of oxidative damage are enhanced in the result of the action of carcinogens. Several antioxidants protect, with different efficacy, against oxidative abuse, exerted by carcinogens. Recently, melatonin (N-acetyl-5-methoxytryptamine) and some other indoleamines have gained particular meaning in the defense against oxidative stress and, consequently, carcinogenesis. Some antioxidants, like ascorbic acid, play a bivalent role in the antioxidative defense, revealing, under specific conditions, prooxidative effects. Among known antioxidants, melatonin is particularly frequently applied in experimental models of anticarcinogenic action. In the numerous studies, examining several parameters of oxidative damage and using several in vitro and in vivo models, this indoleamine has been shown to protect DNA and cellular membranes from the oxidative abuse caused by carcinogens. When either preventing or decreasing the oxidative damage to macromolecules, melatonin also protects against the initiation of cancer. The protection provided by melatonin and some other antioxidants against cellular damage, due to carcinogens, make them potential therapeutic supplements in the conditions of increased cancer risk.     

Biochemical changes in cultured murine fibroblasts after treatment with hydrazinophthalazines

Fibroblast cultures exposed to the drugs inducing a collagen-like syndrome (hydralazine and binazine) displayed growth inhibition and decrease in cellular protein content in a dose-dependent manner compared with control cultures. This was accompanied by the inhibitory effect of the drugs on DNA synthesis. The changes in the basic biochemical parameters of fibroblasts testify to the toxicity of hydrazinophthalazines in the connective tissue.     

Immunological and parasitological parameters after treatment with dexamethasone in murine Schistosoma mansoni

This work aimed to evaluate the effect of diphenyl dimethyl bicarboxylate (DDB) and dexamethasone alone and in combination with praziquantel on various parasitological, immunological and pathological parameters reflecting disease severity and morbidity in murine schistosomiasis. DDB and dexamethasone had no effect on worm burden but altered tissue egg distribution. This indicates that, under the schedule used, neither drug interfered with the development of adult worms or oviposition, but both can modulate liver pathology. Dexamethasone resulted in a greater reduction in granuloma size than did DDB. Dexamethasone-treated mice also showed lower levels of serum gamma interferon (IFN-γ), interleukin-12 (IL-12) and IL-4, together with higher IL-10 levels, than infected untreated control animals. These data suggest that dexamethasone is a convenient and promising coadjuvant agent that results in decreased morbidity in murine schistosomiasis.     

Synergistic induction of apoptosis in murine hepatoma Hepa1-6 cells by IFN-gamma and TNF-alpha

In this study, we examined the susceptibility of murine hepatoma Hepa1-6 cells to undergo IFN-gamma- and/or TNF-alpha-induced apoptosis. IFN-gamma or TNF-alpha alone had no demonstrable cytotoxic effects, whereas IFN-gamma and TNF-alpha in combination induced apoptosis drastically in Hepa1-6 cells. During this apoptosis, an increase in caspase-3- and -8-like protease activities and activation of caspase-3, identified by the appearance of its p17 fragment, were observed. Moreover, the cytotoxic induction and caspase-3 activation were effectively inhibited by Z-Asp-CH(2)-DCB (Z-Asp), a caspase inhibitor. Further, an elevation of cytochrome c in the cytosol, in a parallel to activation of caspase-3, was observed in a time-dependent manner. Concurrently, up-regulation of caspase-11 gene expression and processing of procaspase-11 were detected during this apoptosis. These results suggest that the caspase-3 activation, the release of cytochrome c from mitochondria, and increased caspase-11 gene expression involve in synergistic induction of apoptosis in Hepa1-6 cells by IFN-gamma and TNF-alpha.