Mark Micallef

In vivo antitumor effects of murine interferon- γ -inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites

Abstract Interferon-γ-inducing factor/interleukin-18 is a novel cytokine that reportedly augments natural killer (NK) activity in human and mouse peripheral blood mononuclear cell cultures in vitro and has recently been designated IL-18. In this study, IL-18 exhibited significant antitumor effects in BALB/c mice challenged intraperitoneally (i.p.) with syngeneic Meth A sarcoma when administered i.p. on days 1, 2 and 3 after challenge. Intravenous (i.v.) administration also induced antitumor effects in the tumor-bearing mice; however, subcutaneous (s.c.) administration did not. When mice were twice pretreated with 1 μg IL-18 3 days and 6 h before tumor challenge, all mice survived whereas control mice died within 3 weeks of challenge. Inhibitory effects on Meth A cell growth in vitro were not observed with either IL-18 or interferon γ. The effects of IL-18 pretreatment were abrogated by abolition of NK activity after mice had been injected with anti-asialo GM1 antibody 48 h before and, 24 h and 72 h after tumor challenge. Mice pretreated with IL-18 and surviving tumor challenge resisted rechallenge with Meth A cells but could not reject Ehrlich ascites carcinoma, and spleen cells from the resistant mice, but not control mice, exhibited cytotoxic activity against Meth A cells in vitro after restimulation with mitomycin C-treated Meth A cells for 5 days. The effector cells in the spleen cell preparations from resistant mice appear to be CD4+ cells because cytolytic activity was significantly inhibited after depletion of this subset by monoclonal antibodies and complement. In conclusion, IL-18 exhibits in vivo immunologically (primarily NK) mediated antitumor effects in mice challenged with syngeneic Meth A sarcoma and induces immunological memory and the generation of cytotoxic CD4+ cells.

Synergistic antitumor effects of interleukin-12 gene transfer and systemic administration of interleukin-18 in a mouse bladder cancer model

Abstract We introduced the interleukin-12 (IL-12) gene into the mouse bladder cancer cell line (MBT2) to establish sublines that secrete bioactive IL-12. IL-12-secreting MBT2 (MBT2/IL-12) sublines were completely rejected when subcutaneously implanted into immunocompetent syngeneic C3H mice. Although this antitumor effect did not change when IL-12-secreting cells were injected into immunodeficient mice whose CD8+ T or CD4+ T cells had been depleted by the corresponding antibody, it was abrogated when natural killer cells were depleted by anti-asialoGM1 antibody. In addition, when parental MBT2 cells mixed with MBT2/IL-12 cells were subcutaneously injected into mice, admixed MBT2/IL-12 inhibited the growth of the parental tumor. Furthermore, this antitumor effect was enhanced by systemic IL-18 administration. This synergism was abrogated when the mice were treated with interferon-γ-neutralizing antibody in vivo. In conclusion, local secretion of IL-12 led to effective antitumor activity that was enhanced by systemic administration of IL-18. Interferon-γ plays an important role in the synergism of IL-12 gene transduction and systemic administration of IL-18.

Effectiveness of cancer vaccine therapy using cells transduced with the interleukin-12 gene combined with systemic interleukin-18 administration

We introduced the interleukin-12 (IL-12) gene into mouse renal cell carcinoma (RenCa) cells to develop a tumor vaccine and to examine mechanisms of tumor rejection. IL-12-secreting RenCa (RenCa/IL-12) cells were completely rejected when implanted into syngeneic BALB/c but not athymic nude mice, suggesting that T cells were involved in this antitumor effect. Depletion of natural killer (NK) cells in nude mice did not affect the tumor growth of RenCa/IL-12. The simultaneous injection of mitomycin C-treated RenCa/IL-12 inhibited the tumor growth of parental RenCa injected at a distant site, whereas injection of mitomycin C-treated parental RenCa did not. The antitumor effect of RenCa/IL-12 as a cancer vaccine was induced by CD8+ T cells and NK cells and was inhibited by CD4+ T cells. Although the systemic administration of recombinant IL-18 (rIL-18) alone did not inhibit the tumor growth, it did enhance the cancer vaccine effect of RenCa/IL-12. The combination therapy of RenCa/IL-12 and the systemic administration of rIL-18 retarded even the growth of established tumors. The effector cells of this combination therapy consist not only of CD8+ T cells and NK cells but also of CD4+ T cells. This synergistic cancer vaccine effect of in situ secretion of IL-12 and the systemic administration of rIL-18 may be attributed to a functional change of CD4+ T cells.

Cell differentiation and apoptosis of monocytic and promyelocytic leukemia cells (U-937 and HL-60) by tryptanthrin, an active ingredient of Polygonum tinctorium Lour.

Tryptanthrin, a bioactive ingredient of Polygonum tinctorium Lour., is a member of the Indigo plant family and has potent cytocidal effects on various human leukemia cells in vitro. At low concentrations, tryptanthrin enhanced the expression of cell differentiation (CD) markers in human monocytic (U‐937) and promyelocytic (HL‐60) leukemia cells indicative of differentiation to monocytes/macrophages. Furthermore, nitroblue tetrazolium (NBT) reductive and α‐naphthyl butyrate esterase (NBE) activities were markedly increased after treatment. Tryptanthrin was more potent than dimethyl sulfoxide (DMSO) at inducing U‐937 cell differentiation into monocytes/macrophages. After treatment with higher concentrations of tryptanthrin for 24 h, cytoplasmic vacuolation and destruction of mitochondria were observed. The leukemia cells died via apoptosis 48 h after treatment. Cytoplasmic vacuolation and apoptotic changes correlated with the dysfunction of mitochondria. Electron microscopic observations revealed marked swelling and destruction of mitochondria after exposure of the leukemia cells to tryptanthrin. Exposure to tryptanthrin enhanced Fas‐induced apoptosis and increased caspase‐3 activity before induction of apoptosis. These results show that low concentrations of tryptanthrin can induce differentiation of leukemia cells but higher concentrations will kill leukemia cells through apoptosis, possibly through a caspase‐3/Fas antigen pathway.

Renal carcinogenesis induced by ferric nitrilotriacetate in mice, and protection from it by Brazilian propolis and Artepillin C

The protective effect of Brazilian propolis and its extract Artepillin C against ferric nitrilotriacetate (Fe‐NTA)‐induced renal lipid peroxidation and carcinogenesis was studied in male ddY mice. Fe‐NTA‐induced renal lipid peroxidation leads to a high incidence of renal cell carcinoma (RCC) in mice. Administration of propolis by gastric intubation 2 h before or Artepillin C at either the same time, 2 h, or 5 h before the intraperitoneal injection of Fe‐NTA (7 mg Fe/kg) effectively inhibited renal lipid peroxidation. This was evaluated from the measurement of renal thiobarbituric acid‐reactive substances (TBARS) or histochemical findings of 4‐hydroxy‐2‐nonenal (4‐HNE)‐modified proteins and 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG). Repeated injection of Fe‐NTA (10 mg Fe/kg per day, twice a week for a total of 16 times in 8 weeks) caused subacute nephrotoxicity as revealed by necrosis and pleomorphic large nuclear cells in the renal proximal tubules, and gave rise to RCC 12 months later. A protective effect from carcinogenicity was observed in mice given propolis or Artepillin C. Furthermore, the mice given Fe‐NTA only developed multiple cysts composed of precancerous lesions with multilayered and proliferating large atypical cells. Mice treated with propolis and Artepillin C also had cysts, but these were dilated and composed of flat cells. These results suggest that propolis and Artepillin C prevent oxidative renal damage and the carcinogenesis induced by Fe‐NTA in mice.

Differential requirements for JAK2 and TYK2 in T cell proliferation and IFN‐γ production induced by IL‐12 alone or together with IL‐18

IL‐12 activates TYK2 and Janus kinase (JAK)‐2 to induce the phosphorylation of various signal transducers and activators of transcription (STAT) proteins. However, little is known regarding how these JAK exhibit the distinct biological effects of IL‐12. Using two JAK inhibitors, tyrphostin A1 (A1) for TYK2 and tyrphostin B42 (B42) for JAK2, we investigated the involvement of JAK2 and TYK2 in IL‐12‐induced T cell proliferation and IFN‐γ production. B42, but not A1, inhibited T cell proliferation along with down‐regulation of IL‐12‐induced c‐Myc expression and STAT5 phosphorylation. In contrast, A1 but not B42 inhibited STAT4/STAT3 phosphorylation and IFN‐γ production. IL‐18, but not IL‐12, induced activator protein‐1 (AP‐1) responsible for high levels of IFN‐γ promoter activation. However, this IL‐18 effect depended on the interaction of AP‐1 with STAT4. A1 prevented AP‐1 binding by inhibiting STAT4 involvement and down‐regulated synergistic IFN‐γ promoter activation. These results indicate that JAK2 activation is required for IL‐12‐mediated T cell growth, whereas the TYK2‐STAT4 signaling pathway is critical for IFN‐γ expression that is mediated by IL‐12 alone and enhanced synergistically by combination with IL‐18.

The natural plant product tryptanthrin ameliorates dextran sodium sulfate-induced colitis in mice.

The therapeutic effects of tryptanthrin (TRYP), a natural product from the medicinal plant Polygonum tinctorium, were examined in a murine model of inflammatory bowel disease (IBD). Colitis was induced by 5% dextran sodium sulfate (DSS) in drinking water for 7 days from day 0. TRYP (100 mg/kg) was administered orally suspended in 5% arabia gum everyday from day 3 for 5 days. Histopathological analysis showed reduced colon damage in TRYP-treated mice on day 6; however, colon injury resumed after treatment was stopped. The production of prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) by untreated and treated mouse colon tissues cultured in vitro were mostly unchanged by TRYP treatment. However, mitogen-stimulated spleen cells from TRYP-treated colitic mice produced less interleukin 2 (IL-2) and less interferon-gamma (IFN-gamma) than untreated colitic mouse spleen cells, early after induction of colitis. When colitis was induced with 5% DSS for 7 days and TRYP was given to the mice for 8 days from day 3, TRYP enhanced the survival of the mice but results were not significant. A significant reduction of weight loss was observed in TRYP-treated mice with colitis induced by 5% DSS for 4 days as compared to control mice. Remarkably, whereas 90% of the vehicle-treated mice died from wasting disease, all the TRYP-treated mice survived, suggesting that TRYP may have a therapeutic effect on colitis.

Simultaneous exposure to interleukin-18 and interleukin-10 in vitro synergistically augments murine spleen natural killer cell activity.

Abstract Interleukin-18 (IL-18) enhances interferon γ (IFNγ) production and natural killer (NK) cell activity, and elicits protective antitumor effects in vivo. IL-18 and IL-12 synergistically augment IFNγ production reportedly because IL-12 enhances IL-18 receptor (IL-18R) expression. We now show that IL-18 also synergizes with IL-10 to augment murine splenic NK activity against Yac-1 cells in a standard 4-h chromium-release assay, but IFNγ production is only slightly enhanced. This pattern of NK activity was also observed with severe combined immunodeficient (SCID) mouse spleen cells indicating that the cytokines were not acting on T or B cells. The cytokines had no priming activity on the spleen cells and, when cells were left unstimulated for 24 h in culture, little NK activity was induced when IL-18 was added for the next 24 h. The reverse transcriptase/polymerase chain reaction revealed that IL-18 receptor (IL-18R) mRNA was expressed early during in vitro spleen cell culture but none was expressed after culture for 24 h regardless of the stimulus. Binding of 125I-labeled IL-18 revealed that exposure to IL-10 only slightly increased IL-18R expression. Expression of perforin mRNA was constitutive and was unaffected by the cytokines; however, Fas ligand (FasL) mRNA expression was strong in cultures with IL-18 alone or combined with IL-10. When Fas-expressing cells and their parental cells were used as targets, weak Fas-mediated cytolytic activity was observed after exposure to IL-18, and this was further enhanced by combination with IL-10. Finally, the augmentation of NK activity was abrogated by the inhibitor concanamycin A, indicating that the enhanced NK activity is perforin-dependent.

An extract from Ricinus communis L. leaves possesses cytotoxic properties and induces apoptosis in SK-MEL-28 human melanoma cells

Plants are an important source of several clinically useful anti-cancer agents. A volatile extract was obtained from Ricinus communis L. (Euphorbiaceae) leaves by standard hydrodistillation and subsequent extraction of the cohobated water in chloroform. GC-MS identified three monoterpenoids: 1,8-cineole, camphor and α-pinene, and a sesquiterpenoid: β-caryophyllene, as the main constituents. The leaf extract is cytotoxic to several human tumour cell lines in a dose-dependent fashion, with IC50 values ranging between 10–40 µg mL−1. Apoptosis was shown to be induced in SK-MEL-28 human melanoma cells at a concentration of 20 µg mL−1, as identified by means of morphological examination, nuclear staining and flow cytometric analysis of DNA content. Translocation of phosphatidyl serine to the cell membranes external surface and loss of mitochondrial membrane potential were also detected. This study provides further insight into the potential use of mixtures of terpenoids as they occur in nature, as inducers of apoptosis in cancer cells.

Antitumor activity of interleukin-18 against the murine T-cell leukemia/lymphoma EL-4 in syngeneic mice.

Interleukin (IL)-18 induces interferon (IFN)-&ggr; production by T cells and natural killer (NK) cells, and augments NK cell activity in mouse spleen cell cultures. It has recently been demonstrated that in vivo administration of IL-18 to mice results in considerable antitumor effects against syngeneic Meth A sarcoma. In this study, the antitumor effects of IL-18 against murine T-cell leukemia (EL-4) were evaluated. EL-4 proliferation was resistant in vitro to IL-18 and IFN-&ggr;. When 4 × 106 EL-4 cells were transplanted intravenously, the antitumor effects of IL-18 were not pronounced, and only a slight prolongation of the mean survival times was observed. The antitumor effects of IFN-&ggr; were even less apparent than those of IL-18. However, when mice were transplanted intravenously with 5 × 105 EL-4 cells, the extent of experimental visceral dissemination of EL-4 was markedly reduced in mice treated subcutaneously with IL-18, resulting in an increase in survival time with some mice even cured. Although IL-18 was highly effective at inhibiting the development of EL-4 lymphoma dissemination in C57BL/6 mice, it could not inhibit the development of dissemination in mutant C57BL/6 beige (bg/bg) mice lacking NK cell activity. The efficacy of IL-18 was also significantly reduced in nude mice lacking T cells. These results suggest that antitumor efficacy of IL-18 is mediated primarily by NK cells, but that T cells are also required for the complete antitumor efficacy of IL-18.