Adam Giermasz

Lovastatin and tumor necrosis factor‐α exhibit potentiated antitumor effects against Ha‐ras‐transformed murine tumor Via inhibition of tumor‐induced angiogenesis

Lovastatin, a drug commonly used in the treatment of hypercholesterolemia, has previously been reported to exert potentiated antitumor activity when combined with either tumor necrosis factor‐α (TNF‐α), cisplatin or doxorubicin in a melanoma model in mice. Since lovastatin interferes with the function of ras oncogene‐encoded (Ras) proteins, we have investigated the antitumor activity of lovastatin and TNF‐α using a Ha‐ras‐transformed murine tumor model. In in vitro studies, lovastatin inhibited the growth of cells transformed with Ha‐ras oncogene (Ras‐3T3 and HBL100‐ras cells) more effectively than control NIH‐3T3 and HBL100‐neo cells. In in vivo experiments, the Ras‐3T3 tumor demonstrated significantly increased sensitivity to combined treatment with both lovastatin (50 mg/kg) and TNF‐α (1 μg/day) compared with either agent alone. Combined treatment with both agents also resulted in greater inhibition of blood‐vessel formation. Ras‐3T3 tumor cells produced increased amounts of vascular endothelial growth factor (VEGF) and lovastatin effectively suppressed VEGF production by these cells. Our results suggest that lovastatin increases antitumor activity of TNF‐α against tumor cells transformed with v‐Ha‐ras oncogene via inhibition of tumor‐induced blood‐vessel formation. Int. J. Cancer 81:560–567, 1999.

Effective chemo-immunotherapy of L1210 leukemia in vivo using interleukin-12 combined with doxorubicin but not with cyclophosphamide, paclitaxel or cisplatin

It has been well established that chemo‐immunotherapy using cytotoxic drugs and appropriate cytokines offers a new approach to increasing the therapeutic index in the treatment of neoplastic diseases. This study investigates the efficacy of combinations of interleukin‐12 with cyclophosphamide, paclitaxel, cisplatin or doxorubicin in the murine L1210 leukemia model. Mice inoculated i.p. with 1 × 103 or 1 × 105 leukemia cells were treated with interleukin‐12 and/or chemotherapeutics, and were observed daily for survival. Immunosuppression with X‐irradiation or macrophage depletion with injections of silica were used to examine the dependence of the therapeutic effects on the efficiency of the immune system. Treatment with interleukin‐12 or one of the studied chemotherapeutics given alone resulted in moderate anti‐leukemic effects. Combination of interleukin‐12 with cyclophosphamide or paclitaxel produced no augmentation of anti‐leukemic effects in comparison with these agents given alone. Combination of interleukin‐12 with cisplatin resulted in prolongation of the survival time; however, in the experiment with mice inoculated with 1 × 105 leukemia cells, no long‐term survivors (>60 days) were observed; on the contrary, combination of interleukin‐12 with doxorubicin resulted in 100% long‐term survivors. This effect was completely abrogated either by X‐irradiation of mice or by macrophage depletion. We also found that doxorubicin augments IL‐12‐stimulated production of interferon‐γ in vivo. Our observations demonstrating potentiation of the anti‐leukemic effects of the IL‐12 and doxorubicin combination suggest that the combined use of these 2 agents could be beneficial in leukemia therapy. Int. J. Cancer 77:720–727, 1998.

Potentiation of the anti-tumour effects of Photofrin®-based photodynamic therapy by localized treatment with G-CSF

Photofrin®-based photodynamic therapy (PDT) has recently been approved for palliative and curative purposes in cancer patients. It has been demonstrated that neutrophils are indispensable for its anti-tumour effectiveness. We decided to evaluate the extent of the anti-tumour effectiveness of PDT combined with administration of granulocyte colony-stimulating factor (G-CSF) as well as the influence of Photofrin®and G-CSF on the myelopoiesis and functional activity of neutrophils in mice. An intensive treatment with G-CSF significantly potentiated anti-tumour effectiveness of Photofrin®-based PDT resulting in a reduction of tumour growth and prolongation of the survival time of mice bearing two different tumours: colon-26 and Lewis lung carcinoma. Moreover, 33% of C-26-bearing mice were completely cured of their tumours after combined therapy and developed a specific and long-lasting immunity. The tumours treated with both agents contained more infiltrating neutrophils and apoptotic cells then tumours treated with either G-CSF or PDT only. Importantly, simultaneous administration of Photofrin®and G-CSF stimulated bone marrow and spleen myelopoiesis that resulted in an increased number of neutrophils demonstrating functional characteristics of activation. Potentiated anti-tumour effects of Photofrin®-based PDT combined with G-CSF observed in two murine tumour models suggest that clinical trials using this tumour therapy protocol would be worth pursuing.

Potentiation of antitumor effects of tumor necrosis factor α and interferon γ by macrophage-colony-stimulating factor in a MmB16 melanoma model in mice

The efficacy of systemic infusion of recombinant human macrophage-colony-stimulating factor (M-CSF) in combination with local treatment with human recombinant tumor necrosis factor (TNF) α and mouse recombinant interferon (IFN) γ was studied in vivo on a subclone of B16 melanoma (MmB16) in mice. Short-term intravenous administration of M-CSF at a dose of 106 units daily had no antitumor effect in vivo. Similarly, local treatment of tumor with TNFα (5 μg daily) did not produce any therapeutic effect. However, simultaneous administration of the same dose of TNFα with IFNγ (1000 units daily) resulted in a synergistic effects manifested by the retardation of tumor growth. Addition of systemic infusion of M-CSF to the local therapy with TNFα and IFNγ induced further augmentation of antitumor efficacy and delayed progression of MmB16 melanoma. The strengthened antitumor effect of combination therapy including M-CSF, TNFα and IFNγ was most probably due to the increased release of monocytes from the bone marrow, their recruitment into the site of tumor growth and subsequent local stimulation of their antitumor activity.

Lovastatin and simvastatin are modulators of the proteasome

Lovastatin and simvastatin are HMG-CoA reductase inhibitors widely used as antihyperlipidemic drugs, which also display antiproliferative properties. In the present paper, we provide evidence that both lovastatin and simvastatin are modulators of the purified bovine pituitary 20 S proteasome, since they mildly stimulate the chymotrypsin-like activity and inhibit the peptidylglutamylpeptide hydrolyzing activity without interfering with the trypsin-like activity. However, those effects are only observed when the closed ring forms of the drugs are used, while the opened ring form of lovastatin acts as a mild inhibitor of the chymotrypsin like activity. The closed ring form of lovastatin is much more potent as a cytotoxic agent on the Colon-26 (C-26) colon carcinoma cell line than the opened ring form, which is only mildly cytostatic. Moreover, neither the cytotoxic effects nor the effects on 20 S proteasome activities are prevented by mevalonate, which by itself inhibits the trypsin-like activity of the proteasome. Neither the opened ring nor the closed ring form of lovastatin induces an accumulation of ubiquitin-protein conjugates, which is observed after treatment with lactacystin, a selective proteasome inhibitor. In contrast with the opened ring form of lovastatin, the closed ring form induces the disappearance of detectable p27(kip1) from C-26 cells. Altogether, our results indicate that the closed ring form of lovastatin induces cytotoxic effects independent of its HMG-CoA inhibiting activity, however, those effects are mediated by a complex modulation of proteasome activity rather than by inhibition of the 20 S proteasome.

Potentiation of the anti-tumor effect of actinomycin D by tumor necrosis factor α in mice : Correlation between in vitro and in vivo results

The anti‐tumor effects of actinomycin D (Act D) and recombinant human tumor necrosis factor (TNF)‐α have been studied on 4 established murine tumor cell lines: MmB16 melanoma, Lewis lung (LL/2) carcinoma, L1 sarcoma and L1210 leukemia. During short‐term incubation (24 hr) Act D produced dose‐dependent cytostatic/cytotoxic effects against MmB16, LL/2 and L1 tumor cells but did not reduce the viability of these cells even at high concentration (10 μg/ml), below a threshold of 30–60%. However, L1210 leukemic cells were highly susceptible to Act D, and no viable cells were detected in cultures incubated with 1 μg/ml of Act D. TNF‐α alone, when used under the same culture conditions, had only a negligible effect on all cell lines tested. However, the combination of this cytokine with Act D produced synergistic cytotoxic effects against MmB16, LL/2 and L1 cells but not against L1210 leukemia cells. In an in vivo model of regional therapy in which tumor‐bearing mice were treated with Act D and TNF‐α, a correlation with in vitro results was observed. In mice bearing MmB16 melanoma, LL/2 carcinoma and L1 sarcoma, the most potent anti‐tumor effects were observed in mice treated with Act D and TNF‐α together. This treatment led to a delay of tumor growth and induced complete tumor regression in some cases. On the contrary, TNF‐α did not enhance the effect of Act D in mice injected with L1210 leukemia cells. Our results show that TNF‐α can potentiate the anti‐tumor effects of Act D against tumors weakly susceptible to Act D and may be a useful adjuvant to chemotherapy in the local treatment of neoplasia.

Apoptosis induced in L1210 leukaemia cells by an inhibitor of the chymotrypsin-like activity of the proteasome.

Of a number of factors involved in apoptosis, protease activity may play a crucial role. We show that N-benzyloxycarbonyl-Ile-Glu(O-t-butyl)-Ala-leucinal (PSI), a selective inhibitor of the chymotrypsin-like activity of the proteasome, induces massive apoptosis in murine leukaemia L1210 cells. At 50 nM concentration, PSI induces a block of cytokinesis, while higher concentrations (500 nM) cause S phase block and massive apoptosis. Z-Leu-leucinal, a specific calpain inhibitor, did not induce apoptosis. In contrast to previous reports, TNF-α did not enhance apoptosis when combined with PSI. Our results suggest that proteasome inhibitors may be considered as potential anti-neoplastic agents.

Potentiating antitumor effects of a combination therapy with lovastatin and butyrate in the Lewis lung carcinoma model in mice

Lovastatin, the drug used for the treatment of hypercholesterolemia, has previously been reported to exert antitumor activity in experimental murine models. Butyrate and butyric acid derivatives are well known to induce differentiation and apoptosis of tumour cells and also have recently gained acceptance as potential anticancer agents. In this study, we examined the antitumor effects of the combination of lovastatin and butyrate or its prodrug tributyrin in vitro and in vivo against a murine Lewis lung carcinoma (3LL). This combination therapy showed synergistic antitumor activity against 3LL cells in vitro. These effects were at least in part due to apoptosis induction that occurred after 12 hr of incubation with lovastatin and butyrate and was preceded by changes in cell cycle distribution of treated cells and expression of p21, p53 and cyclin D1. Remarkably, a systemic treatment of syngeneic mice inoculated with 3LL cells with both drugs resulted in significant tumour growth retardation.

Interleukin 12 and indomethacin exert a synergistic, angiogenesis-dependent antitumor activity in mice

Nonsteroidal anti-inflammatory drugs have been shown to reduce the incidence and mortality from colorectal cancer. It has recently been demonstrated that these drugs are capable of suppressing the production of pro-angiogenic factors from tumor cells. The mechanisms of antitumor action of interleukin 12 include the enforced secretion of anti-angiogenic factors and stimulation of antitumor immunity. Therefore, we hypothesized that the combination of a model nonsteroidal anti-inflammatory drug--indomethacin and interleukin 12--would result in enhanced angiogenesis-dependent antitumor effects against a colon-26 carcinoma cells transplanted into syngeneic mice. As expected the combined administration of both agents simultaneously resulted in a strengthened antitumor activity that was manifested as a retardation of tumor growth and prolongation of mouse survival. Importantly some mice were completely cured after the combined treatment. As administration of interleukin 12 and indomethacin resulted in enhanced inhibition of angiogenesis it seems possible that prevention of new blood vessel formation is one of the mechanisms responsible for the observed antitumor effects.

Potentiatied antitumor effectiveness of combined chemo-immunotherapy with interleukin-12 and 5-fluorouracil of L1210 leukemia in vivo

In this study we investigated the efficacy of a combination of IL-12 and 5-FU, a chemotherapeutic exerting several immunomodulatory effects, in murine L1210 leukemia. Mice inoculated with 1 × 105 leukemia cells were treated with a single dose of 5-FU (50 mg/kg) and seven daily doses of IL-12 (100 ng/dose), and were observed for survival. Treatment with IL-12 or 5-FU given alone produced moderate anti-leukemic effects. However, combination of both drugs resulted in a significant prolongation of mouse survival time. Importantly, there were 70% of long-term (>60 days) survivors among mice treated with both agents simultaneously. Moreover, we observed 100% of long-term survivors when mice were treated with a minimally increased dose of IL-12 (170 ng) in combination with 5-FU (50 mg/kg). The antileukemic effects were completely abrogated in scid/scid mice and in mice depleted of peritoneal macrophages and significantly decreased after administration of anti-CD3+, anti-CD4+ or anti-CD8+ monoclonal antibodies. Administration of anti-NK1.1 antibodies did not decrease the antileukemic effects indicating that NK cells are not important effectors of this treatment regimen. Collectively, these results indicate that the combination of IL-12 and 5-FU is inducing strong antileukemic responses that are dependent on the presence and activity of macrophages and T lymphocytes and warrant further studies of combined chemo-immunotherapy with IL-12.