rIFN-γ-activated rat neutrophils induce tumor cell apoptosis by nitric oxide

We have previously shown that 1) neutrophils activated with various cytokines, including rat recombinant interferon γ (rIFN-γ), inhibit tumor cell growth and that 2) nitric oxide (NO) is the effector molecule in tumor inhibition by rIFN-γ-stimulated rat peritoneal exudate neutrophils. In this study, we examined the nature of tumor cell death induced by rat peritoneal neutrophils activated by rIFN-γ in order to clarify the mechanism of apoptosis in neoplastic tumor cell death. DNA of 3 syngeneic rat tumor cell lines was significantly fragmented within 3 hr of incubation in the presence of rIFN-γ-activated neutrophils, and this effect was dependent on both the concentration of rIFN-γ and the number of neutrophils. This DNA fragmentation was inhibited by L-N-(I-iminoethyl)-ornithine (L-NIO), a NO synthase inhibitor, but not by superoxide dismutase (SOD). Tumor cells treated with the activated neutrophils were shown by electron microscopy to be apoptotic, exhibiting necrotic features with a longer incubation. On the other hand, cytolysis of tumor cells, as determined by a [³H]-uridine release assay, was first observed only at 24 hr of incubation with the rIFN-γ-activated neutrophils. Taken together, our results suggest that tumor cell apoptosis by activated neutrophils is NO-dependent and that apoptotic tumor cells undergo necrosis as a secondary process. We suggest that tumor cell apoptosis induced by activated neutrophils plays an important role in regulation of neoplastic tumor cell growth and death in vivo. Int. J. Cancer 71:231–236, 1997. © 1997 Wiley-Liss, Inc.     

Nitric oxide is an effector molecule in inhibition of tumor cell growth by rIFN-γ-activated rat neutrophils

This study was designed to determine the effector molecule responsible for the tumor-inhibitory activity of rat neutrophils treated with rat recombinant interferon gamma (rIFN-γ) in vitro. The results show that nitric oxide (NO) production by neutrophils is dependent on rIFN-γ concentration, and that neutrophil-mediated tumor cytostasis is in turn dependent on the amount of NO. NO production and tumor cytostatis by rIFN-γ-activated neutrophils were inhibited completely by N^G monomethyl-L-arginine (N^GMMA), a specific competitive NO production inhibitor. Tumor cytostasis was also inhibited by oxyhemoglobin (HbO₂), an NO scavenger. An extracellular oxygen radical scavenger, superoxide dismutase (SOD), was found to increase tumor cell inhibition by rIFN-γ-activated neutrophils by a factor of 4. This SOD-enhanced cytostasis was not even inhibited by catalase. Tumor cytostasis was slightly increased by a hydroxyl radical-(-OH) scavanger, dimethylthiourea (DMTU), which did not affect NO production by rIFN-γ-activated neutrophils. Our findings suggest that tumor cytostasis of neutrophils activated by rIFN-γ is mediated by L-arginine-derived nitrogen oxidation products, and that O₂⁻ produced by these neutrophils reduces NO-mediated tumor cytostasis at low NO concentrations. Int. J. Cancer 71:223-230, 1997. © 1997 Wiley-Liss, Inc.     

Pulse treatment of human vascular endothelial cells with high doses of tumor necrosis factor and interferon-gamma results in simultaneous synergistic and reversible effects on proliferation and morphology

Regional administration of high doses of tumor necrosis factor (TNF), interferon gamma (IFNγ) and melphalan to patients with advanced cancers of the limbs, results in rapid and specific tumor necrosis, while the normal adjacent tissues remain unaffected. The tumor vasculature is selectively destroyed by this treatment, and neovascular endothelial cells appear to be an early and specific target of TNF and IFNγ. To further understand some of the cellular events underlying these in vivo effects, we have investigated the response of human macro- and microvascular endothelial cells in vitro, after exposure to high doses of TNF and IFNγ (up to 40 × 10³ U/ml each). TNF and IFNγ synergistically inhibited endothelial-cell proliferation by up to 80% after 72 hr of treatment. Achievement of synergy required the simultaneous presence of both cytokines. A cytokine pulse as short as 30 min was sufficient to induce maximal growth inhibition measured after 48 hr. Both cytokines also induced progressive and dose-dependent elongation of the endothelial-cell morphology. The effects on endothelial-cell proliferation and morphology were reversible upon removal of the cytokines. Moreover, replating of treated cells onto a fresh substrate immediately resulted in re-acquisition of their normal shape. In contrast to the effect on cell proliferation, there was little or no effect on the rate of endothelial-cell apoptosis. The presented data extend reports on the effects of TNF and IFNγ on human endothelial cells in vitro, and suggest that the in vivo disruption of the tumor vasculature caused by high doses of TNF and IFNγ is not due to a direct cytotoxic effect on endothelial cells but occurs through an indirect mechanism. Int. J. Cancer 77:592–599, 1998. © 1998 Wiley-Liss, Inc.     

in vitro effects of recombinant human interferon gamma on human mesothelioma cell lines

Malignant mesothelioma is a tumor arising from serous surfaces and often related to asbestos exposure. Malignant mesothelioma is resistant to various forms of therapy. Radiotherapy, surgery or chemotherapy only slightly improve prognosis. IFN-γ produces complete or partial responses in stage-l patients with malignant mesothelioma. The in vitro biological effect of IFN-γ on malignant mesothelioma cells remains poorly elucidated. In the present study, 32 well-characterized human mesothelioma cell lines (HMCL) were treated with r-hu IFN--γ at 4 doses and cell growth was determined by a colorimetric method (MTT assay). Among the 32 HMCLs tested, 11 exhibited significant cell-growth inhibition; 16 were insensitive to r-hu IFN-γ, and 5 were slightly inhibited. The sensitive cell lines were strongly inhibited by r-hu IFN-γ. Our results show that HMCL exhibit a large range of responses to r-hu IFN-γ, some of which can be compared with those obtained in vivo in humans.     

Interferons alpha and gamma differ in their ability to cause tumour stasis and regression in vivo

We have studied the antitumour activity of human lymphoblastoid interferon-α [HuIFN-α(N)] and human recombinant interferon-γ (rHuIFN-γ) on 12 early passage (passages 2–7) human tumour xenografts derived from primary malignancies. Systemic daily therapy of established (approx. 0.5 cm diameter) subcutaneous xenografts with HuIFN-α(N) resulted in significant tumour stasis, and occasionally regression, in nine of 12 breast, bowel, and ovarian cancers studied. A significant decrease in tumour mitotic index was seen in three HuIFN-α(N) sensitive breast tumours. In contrast, none of nine of the same tumours responded significantly to rHuIFN-γ therapy. Direct administration of rHuIFN-γ into the tumour did not improve its therapeutic efficacy. However, when tumour cells from xenografts were dissociated and grown as colonies in soft agar, both IFNs, used at doses that are found circulating in vivo after therapy, inhibited colony development in three of three lines tested. Combinations of rHuIFN-γ and HuIFN-α(N) had no synergistic benefit in four of four xenografts studied.     

ENHANCED METASTASIS OF AMOUSE MAMMARY ADENOCARCINOMA AFTER INVITRO TREATMENT WITHγ－INTERFERON

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Tumor markers of bladder cancer: the schistosomal bladder tumors versus non-schistosomal bladder tumors

Recent data have redefined the concept of inflammation as a critical component of tumor progression. However, there has been little development on cases where inflammation on or near a wound and a tumor exist simultaneously. Therefore, this pilot study aims to observe the impact of a wound on a tumor, to build a new mouse tumor model with a manufactured surgical wound representing acute inflammation, and to evaluate the relationship between acute inflammation or wound healing and the process of tumor growth. We focus on the two phases that are present when acute inflammation influences tumor. In the early phase, inhibitory effects are present. The process that produces these effects is the functional reaction of IFN-γ secretions from a wound inflammation. In the latter phase, the inhibited tumor is made resistant to IFN-γ through the release of TGF-β to balance the inflammatory factor effect on the tumor cells. A pair of cytokines IFN-γ/TGF-β established a new balance to protect the tumor from the interference effect of the inflammation. The tumor was made resistant to IFN-γ through the release of TGF-β to balance the inflammatory effect on the tumor cells. This balance mechanism that occurred in the tumor cells increased proliferation and invasion. In vitro and in vivo experiments have confirmed a new view of clinical surgery that will provide more detailed information on the evaluation of tumors after surgery. This study also provides a better understanding of the relationship between tumor and inflammation, as well as tumor cell attacks on inflammatory factors.     

Anti-tumor activity of cytokines against opportunistic vascular tumors in mice

Polyoma middle T (PmT)-transformed endothelial cells may represent a unique murine model for human opportunistic vascular tumors. The present study was designed to evaluate the anti-tumor potential of a panel of 13 cytokines against murine PmT-transformed endothelial cells. Interferon gamma (IFNγ) and transforming growth factor beta 1 (TGFβ1) substantially decreased in a dose-dependent manner the proliferation of a panel of 6 PmT-transformed cell lines. IFNα and tumor necrosis factor α (TGFα) had marginal anti-proliferative activity, whereas other molecules (interleukins-1, -2, -4, -6 and -13, IFNβ, leukemia inhibitory factor, oncostatin M, granulocyte-macrophage colony-stimulating factor) caused no growth inhibition. IFNγ and TGFβ1 were therefore selected for further analysis of their mechanism of action and in vivo relevance. IFNγ and TGFβ1 reduced the activity of phosphatidylinositol-3-kinase and the production of phosphatidylinositol 3,4-biphosphate, without modifying the tyrosine kinase(s) activity associated with PmT. IFNγ and TGFβ1 were also tested for their ability to modify the in vivo growth of the PmT-transformed endothelial cells H5V in syngeneic C57B1/6 mice. Treatment with IFNγ and TGFβ1 significantly delayed tumor growth and increased survival time. In contrast, treatment with IFNα and TNFα failed to prolong survival. In nude mice, IFNγ and TGFβ1 had a transient effect on tumor growth but no effect on survival, suggesting a contribution of T cells to the in vivo anti-tumor activity of these cytokines. © 1996 Wiley-Liss, Inc.     

Prostaglandin d synthase is a potential novel therapeutic agent for the treatment of gastric carcinomas expressing PPARγ

The antitumor activity of prostaglandin (PG) D₂ has been demonstrated against some types of cancer, including gastric cancer. However, exogenous PGD₂ is not useful from a clinical point of view because it is rapidly metabolized in vivo. The aim of this study was to clarify the antitumor efficacy of an alternative, PGD synthase (PGDS), on gastric cancer cells. The effects of PGD₂ and PGDS on the proliferation of gastric cancer cells were examined in vivo and in vitro. The expression levels of PGD₂ receptors and peroxisome proliferator‐activated receptor γ (PPARγ) were evaluated by RT‐PCR. The effects of a PPARγ antagonist or siPPARγ on the proliferation of cancer cells and the c‐myc and cyclin D1 expression were examined in the presence or absence of PGD₂ or PGDS. PPARγ was expressed in gastric cancer cell lines, but PGD₂ receptors were not. PGD₂ and PGDS significantly decreased the proliferation of gastric cancer cells that highly expressed PPARγ. PGDS increased the PGD₂ production of gastric cancer cells. A PPARγ antagonist and siPPARγ transfection significantly suppressed the growth‐inhibitory effects of PGD₂ and PGDS. Expression of c‐myc and cyclin D1 was significantly decreased by PGD₂; this inhibitory effect was suppressed by PPARγ antagonist. Both PGD₂ and PGDS significantly decreased subcutaneous tumor growth in vivo. Tumor volume after PGDS treatment was significantly less than PGD₂ treatment. These findings suggest that PGDS and PGD₂ decrease the proliferation of gastric cancer cells through PPARγ signaling. PGDS is a potentially promising therapeutic agent for gastric cancers that express PPARγ.     

Recombinant tumor necrosis factor: its effect and its synergism with interferon-γ on a variety of normal and transformed human cell lines

Tumor Necrosis Factor (TNF), released by induced macrophages, causes tumor necrosis in animals, and preferentially kills transformed cells in vitro. Using pure, recombinant human TNF, we report here its cytotoxic action on several human transformed and non-transformed cell lines. Furthermore, remarkable synergism between TNF and interferon-γ (IFN-γ) was observed in a large number of human cell lines, especially breast, cervix and colon carcinomas. Some other human cell lines, not sensitive to TNF alone, became highly sensitive when IFN-γ was present as well. We could not demonstrate a synergism between TNF and IFN-γ on any of the lymphoma/leukemia cell lines tested. All normal human, non-transformed diploid cell lines were insensitive to TNF even in the presence of IFN-γ. This study also confirms the observation that inhibition of protein synthesis by metabolic drugs (e.g. actinomycin D) remarkably enhances the sensitivity of several target cells lines to cytolysis by TNF.     

Effect of in vivo administration of interferon gamma on expression of MHC products and tumour associated antigens in patients with metastatic melanoma

IFN-γ is an effective inducer of MHC class II antigen expression in cell lines of malignant melanoma. To investigate the possibility that IFN-γ may increase MHC class I and II and melanoma tumour associated antigens in vivo, immunohistochemical analyses of biopsies from six patients with metastatic disease undergoing IFN-γ treatment were performed. Before IFN-γ treatment, the melanomas were class I positive and class II negative. After treatment, class I expression was neither enhanced nor class II expression induced in any tissue sample regardless of biopsy time or dose of IFN-γ. TAA was similarly unchanged. However, in one of the six patients a primary cell line was established and IFN-γ induced expression of MHC products. The possible reasons for lack of MHC induction are discussed, although qualitative changes in antigen expression cannot be excluded on the basis of qualitative immunocytochemical techniques alone.     

Peroxisome proliferator-activated receptor γ-dependent activity of indole ring-substituted 1,1-bis(3′-indolyl)-1-(p-biphenyl)methanes in cancer cells

Purpose

1,1-Bis(3-indolyl)-1-(p-substituted phenyl)methanes (C-DIMs) substituted in the phenyl ring with a para-, t-butyl, trifluoromethyl (DIM-C-pPhCF₃) or phenyl (DIM-C-pPhC₆H₅) group activate peroxisome proliferator-activated receptor γ (PPARγ) in several cancer cell lines, and DIM-C-pPhCF₃ also activates the orphan receptor Nur77. In this study, we have examined the effects of 5,5′-dihydroxy, 5,5′-dimethyl, 5,5′-dibromo, 5,5′-dinitro and 5,5′-dimethoxyindole ring-substituted analogs of DIM-C-pPhC₆H₅ on their activity as PPARγ agonists.

Methods

Various substituted C-DIM analogs were used to investigate their growth-inhibitory activities and activation of PPARγ-mediated transactivation in colon and pancreatic cancer cells. Their structure-dependent induction of putative PPARγ-responsive genes/proteins including p21, KLF-4 and caveolin1 were also determined by Western and Northern blot analysis.

Results

Introduction of the 5,5′-dihydroxy and 5,5′-dimethyl substituents enhanced activation of PPARγ in colon and pancreatic cancer cells. However, activation of p21 in Panc28 pancreatic cancer cells and induction of caveolin-1 and KLF4 in colon cancer cells by the C-DIM compounds were structure- and cell context-dependent.

Conclusions

The results demonstrate that DIM-C-pPhC₆H₅ and indole ring-substituted analogs are selective PPARγ modulators.     

Peroxisome proliferator-activated receptor-γ and growth inhibition by its ligands in prostate cancer

Background: Peroxisome proliferator-activated receptor-γ (PPAR-γ) is expressed in certain human cancers. Ligand-induced PPAR-γ activation can result in growth inhibition and differentiation in these cancer cells; however, the precise mechanism for the anti-proliferative effect of PPAR-γ ligands is not clear. Methods: In this study, we examined the expression of PPAR-γ in human prostate cancer and the effect of two PPAR-γ ligands, 15 deoxy-Δ^12,14-prostaglandin J2 (15d-PGJ2) and troglitazone, on prostate cancer cell growth. Results: PPAR-γ is frequently over-expressed in androgen independent prostate cancer cell lines and human prostate cancer tissues (22 of 47; 47%). Both 15d-PGJ2 and troglitazone inhibited proliferation and DNA synthesis of prostate cancer cell lines in a dose-dependent manner, and slightly increased the proportion of cells with S-phase DNA content. Prostate specific antigen (PSA) promoter reporter assays showed that troglitazone and 15d-PGJ2 down-regulated androgen stimulated reporter gene activity in prostate cancer cell lines LNCaP. Interestingly, LNCaP with troglitazone dramatically suppressed PSA protein expression without suppressing AR expression. Conclusions: Taken together, these results suggest that PPAR-γ ligands may be a useful therapeutic agent for the treatment of prostate cancer.     

Peroxisome proliferator-activated receptor-gamma and growth inhibition by its ligands in prostate cancer

Background: Peroxisome proliferator-activated receptor-γ (PPAR-γ) is expressed in certain human cancers. Ligand-induced PPAR-γ activation can result in growth inhibition and differentiation in these cancer cells; however, the precise mechanism for the anti-proliferative effect of PPAR-γ ligands is not clear. Methods: In this study, we examined the expression of PPAR-γ in human prostate cancer and the effect of two PPAR-γ ligands, 15 deoxy-Δ^12,14-prostaglandin J2 (15d-PGJ2) and troglitazone, on prostate cancer cell growth. Results: PPAR-γ is frequently over-expressed in androgen independent prostate cancer cell lines and human prostate cancer tissues (22 of 47; 47%). Both 15d-PGJ2 and troglitazone inhibited proliferation and DNA synthesis of prostate cancer cell lines in a dose-dependent manner, and slightly increased the proportion of cells with S-phase DNA content. Prostate specific antigen (PSA) promoter reporter assays showed that troglitazone and 15d-PGJ2 down-regulated androgen stimulated reporter gene activity in prostate cancer cell lines LNCaP. Interestingly, LNCaP with troglitazone dramatically suppressed PSA protein expression without suppressing AR expression. Conclusions: Taken together, these results suggest that PPAR-γ ligands may be a useful therapeutic agent for the treatment of prostate cancer.     

IFN-γ selectively exerts pro-apoptotic effects on tumor-initiating label-retaining colon cancer cells

Label-retaining cancer cells (LRCCs) represent a novel population of stem-like cancer cells exhibiting slow cycling, chemoresistance and tumor-initiating capacities; however, their properties remain unclear, and approaches to eradicate LRCCs remain elusive. Here, we report that colon cancer cells with high fluorescent intensity, referred to as LRCCs, have the greatest cancer stem cell (CSC)-like capacities and that they preferentially express CSC markers and stemness-related genes. Moreover, we found that Lgr5, which has been reported to be a marker of rapid cycling CSCs, is almost negatively expressed in LRCCs but that its expression is gradually increased in the differentiation process of LRCCs. Interestingly, we found that LRCCs are especially sensitive to the pro-apoptotic effect of IFN-γ treatment both in vitro and in vivo because LRCCs possess higher IFN-γR levels compared with non-LRCCs, which results in the upregulation of the apoptosis pathway after IFN-γ treatment. Furthermore, we found that IFN-γ shows synergistic effects with the conventional anticancer drug Oxaliplatin to eliminate both LRCCs and non-LRCCs. In conclusion, this is the first study to suggest that LRCCs, as a distinct tumor-initiating population, can be selectively eradicated by IFN-γ, which may provide a novel therapeutic strategy for colon cancer treatment.