Hiroyoshi Sasaki

Induction of Heat Shock Protein 47 Synthesis by TGF-β and IL-1β Via Enhancement of the Heat Shock Element Binding Activity of Heat Shock Transcription Factor 1

With most immunological reactions, tissue fibrosis, collagen overproduction caused by immune cytokines, is inevitably associated. Among the various immune cytokines, heat shock protein 47 (HSP47) is a procollagen-specific molecular chaperon and is essential for secretion of procollagen from cells. Induction of HSP47 by TGF-β has been previously reported in rat skeletal myoblasts and mouse osteoblasts, but not in human diploid fibroblasts. As for IL-1β, its effect on HSP47 has not been elucidated. In the present study, using human embryonic lung fibroblast cells, we first disclosed that both TGF-β and IL-1β induced HSP47 synthesis. We then revealed that the binding of the heat shock element (HSE) by heat shock transcription factor 1 (HSF1) was enhanced by both cytokines. We further demonstrated that trimer formation of HSF1, which is essential for its binding to HSE, was induced by these cytokines. The enhancement of HSP47 synthesis and their trimer formation of HSF1 were augmented by using a combination of both cytokines. Collectively, TGF- β and IL-1β were found to induce trimer formation of HSF1 which in turn bound to HSE of HSP47, resulting in the enhancement of HSP47 expression. Thus, HSP47 could well be a good candidate for molecular targeting in controlling tissue fibrosis, given that both principal fibrinogenetic cytokines (TGF-β, IL-1β) are commonly involved in its induction through HSF1 trimerization.

Serum tumor necrosis factor activity in inflammatory bowel disease

Serum tumor necrosis factor (TNF) levels in 33 patients with inflammatory bowel disease (IBD) were measured by using a sensitive enzyme immunoassay. Four of five Crohns diseases (CD) and nine of twenty eight ulcerative colitis (UC) had elevated levels of serum TNF. In active CD or UC, a greater fraction of patients studied had significantly increased serum TNF levels (3/3 for CD and 8/11 for UC). Production of TNF by peripheral blood monocytes when stimulated by lipopolysaccharide was also increased in these patients and correlated with their serum TNF levels. These results suggest that TNF may have some pathoetiological meaning in IBD.

Mechanism of Synergistic Cytotoxic Effect between Tumor Necrosis Factor and Hyperthermia

We previously reported that recombinant human tumor necrosis factor (rhTNF) and hyperthermia had a synergistic effect against tumors, in vitro and in vivo. We have now investigated the mechanism of this synergy by measuring the lysosomal enzyme activity and hydroxyl radical production of L‐M cells treated with rhTNF and/or hyperthermia. A synergistic activation of lysosomal enzyme and the induction of hydroxyl radical production in L‐M cells treated with both rhTNF and hyperthermia was observed. A synergistic cytotoxic effect was observed when rhTNF and hyperthermia were combined, and was inhibited by the addition of a reactive oxygen scavenger, dimethyl sulfoxide or bipyridine. The results show that the augmenting effect of hyperthermia on lysosomal enzyme activation and induction of hydroxyl radical production by rhTNF plays an important role in the synergistic cytotoxic effect.

Endogenous Tumor Necrosis Factor Inhibits the Cytotoxicity of Exogenous Tumor Necrosis Factor and Adriamycin in Pancreatic Carcinoma Cells

Pancreatic carcinoma is one of the most devastating neoplasms with regard to its resistance to conventional therapy. In a previous report, we found that endogenous tumor necrosis factor (enTNF) exerts an intracellular protective effect against exogenous TNF- and Adriamycin (ADM)-induced cytotoxicity by scavenging oxygen free radicals (OFR) with induced manganous superoxide dismutase (MnSOD). We also know that glutathione S-transferase pi (GST-pi) and glutathione (GSH) also scavenge OFR. It remains unclear to what extent enTNF and MnSOD induced by enTNF regulate the sensitivity to ADM and exogenous TNF among different carcinoma cells. In this study, we examined the relationship between ADM and exogenous TNF sensitivity and en-TNF expression and MnSOD activity in four pancreatic carcinoma lines. We determined whether ADM and exogenous TNF sensitivity could be predicted by measuring enTNF expression and MnSOD activity in the carcinoma cells. The sensitivity to TNF and ADM varied with the cell lines, and TNF sensitivity correlated well with Adriamycin sensitivity. Moreover, enTNF expression and Mn-SOD activity correlated positively with resistance to ADM and exogenous TNF. When MIAPaCa-2 cells, which had the lowest enTNF expression and the highest sensitivity to exogenous TNF and ADM, were transfected with the nonsecretory-type human TNF gene (pTNF delta pro) to increase enTNF synthesis, their intracellular MnSOD activity and exogenous TNF and ADM resistance were increased. These findings suggest that MnSOD plays a critical role in scavenging OFR induced by ADM and exogenous TNF. enTNF is the most important factor that regulates the production of MnSOD. Therefore, it is plausible that inhibition of enTNF expression or MnSOD activity in pancreatic carcinoma would improve the efficacy of therapies for pancreatic carcinoma.

Recombinant Human Tumor Necrosis Factor Causes Regression in Patients with Advanced Malignancies

Fifteen patients with advanced solid tumors of various types were treated by the intratumoral administration of recombinant human tumor necrosis factor (rH-TNF). The treatment appeared to benefit the 4 cases of superficial tumors: there were 1 complete response, 1 partial response and 2 minor responses. In all 11 patients with deep-seated tumors, including 6 cases of pancreatic cancer, 4 of liver cell cancer and 1 of metastatic liver tumor, no tumor regression was observed, but progression stopped in all these tumors. Seven of the 11 with deep-seated tumors showed a decrease in tumor markers and/or the development of tumor necrosis. Fever, hypotension and fatigue were the main clinical side effects. No significant changes were found in hematologic, renal or liver parameters. These results suggest that administration of rH-TNF to the tumor site has the potential for controlling local tumor growth.

Endogenous Tumor Necrosis Factor Functions as a Resistant Factor against Hyperthermic Cytotoxicity in Pancreatic Carcinoma Cells via Enhancement of the Heat Shock Element-Binding Activity of Heat Shock Factor 1

To elucidate the relationship between two distinct resistant factors, endogenous tumor necrosis factor (enTNF) and heat shock proteins (HSPs), against hyperthermia, we assessed whether enTNF enhances HSP72 expression. Although there was a variability in the sensitivity of pancreatic carcinoma cell lines to heat, enTNF and HSP72 expression as well as MnSOD activity correlated positively with heat resistance. When MIAPaCa-2 pancreatic carcinoma cells, which had the lowest enTNF expression and highest heat sensitivity, were transfected with a nonsecretory-human TNF gene (pTNF delta pro), intracellular manganous superoxide dismutase (MnSOD) activity, HSP72 expression, and heat resistance were significantly increased. Furthermore, in these cells, enTNF expression correlated with the binding activity of heat shock factor 1 (HSF 1) to an oligonucleotide containing the human heat shock element. These results indicate that enTNF participates in the intrinsic resistance against heat via induction of MnSOD, enhances HSF1-binding activity, and augments of HSP72 expression. Therefore, inhibition of enTNF expression in pancreatic carcinoma cells would improve the efficacy of hyperthermia for pancreatic carcinoma.

Protein Kinase C Inhibitors Augment Tumor-Necrosis-Factor-Induced Apoptosis in Normal Human Diploid Cells

In this report we show augmentation of tumor necrosis factor (TNF)-induced apoptosis by protein kinase C (PKC) inhibitors in human embryonic lung fibroblast (HEL) cells. Staurosporine (STS) reportedly potentiates TNF-mediated cytotoxicity in cancer cell lines via inhibition of PKC. We investigated whether STS or another potent PKC inhibitor, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H7), augmented TNF-induced apoptosis in normal human diploid cells and examined the effects of the PKC inhibitors on the expression of endogenous TNF (enTNF) and manganous superoxide dismutase (MnSOD) as possible cell resistance factors opposing TNF-induced apoptosis. Both PKC inhibitors augmented TNF-mediated DNA fragmentation in HEL cells, which are normally TNF resistant and constitutively express high amounts of enTNF and MnSOD activity. Neither the number nor the affinity of TNF receptors were altered by STS and H7. The expression of enTNF and MnSOD was suppressed by the presence of STS or H7 along with TNF. The results indicate that PKC inhibitors augment TNF-induced apoptosis not only in tumor cells but also in normal cells by inhibitory effects on cellular resistance factors such as enTNF and MnSOD.

Differentiation induction by a tumor-necrosis-factor mutant 471 in human myelogenous leukemic cells via tumor-necrosis-factor receptor-p55.

The present study examined differentiation‐inducing activity by various tumor‐necrosis‐factor(TNF) mutants against the human leukemic cell lines HL‐60 and U‐937. Mutant TNF 471, from which 7 N‐terminal amino acids of native TNF were deleted and Pro8, Ser9 and Asp10 were replaced by Arg, Lys and Arg, possessed the highest activity among the TNF mutants, and its activity was 120‐fold that of native TNF. The various biological activities of TNF were signaled through 2 distinct receptors, p55 and p75. Although cytotoxicity was reported to involve mainly p55, this differentiation‐inducing activity was not well understood. The fact that the affinity of TNF 471 was higher to p55 and lower to p75 than that of native TNF by a binding competition assay suggested that the differentiation‐inducing activity was also signaled through p55. To verify this hypothesis, the human myelogenous leukemic cell line, KG‐1, which scarcely expresses either receptor and does not differentiate with TNF, was transduced with the p55 or p75 gene. Subsequently p55 transfectants manifested a greater ability to differentiate; however, p75 transfectants did not differ from parental cells or from mock‐transfectants. Further, the differentiation of p55 transfectants induced by TNF was reduced by the inhibitor of protein‐kinase‐C (PKC), staurosporine. These results indicate that the differentiation‐inducing activity was signaled through the TNF receptor, p55, via PKC and that the excellent ability of TNF 471 to induce differentiation was related to its high affinity for p55. Int. J. Cancer 78:223–232, 1998.© 1998 Wiley‐Liss, Inc.

Reversal of Tumor Necrosis Factor Resistance in Tumor Cells by Adriamycin via Suppression of Intracellular Resistance Factors

Tumor necrosis factor (TNF) and various chemotherapeutic drugs show synergistic antitumor effects in vitro and in vivo, though the mechanism is not clear. Based on our previous finding that endogenous TNF (enTNF) acts as an intracellular resistance factor against exogenous TNF by scavenging oxygen free radicals (OFR) with induced manganous superoxide dismutase (MnSOD), we examined the suppression of these resistance factors by chemotherapeutic drugs and the resulting increase in TNF cytotoxicity. Pretreatment of HeLa cells, which produce an appreciable amount of enTNF and show apparent TNF resistance, with TNF followed by adriamycin (ADM) resulted in an additive effect, whereas pretreatment with ADM followed by TNF resulted in a synergistic effect. After treatment of HeLa cells with ADM, the expression of enTNF was remarkably suppressed and MnSOD activity was decreased by one‐half. These results indicate that suppression of the intracellular resistance factors, i.e., enTNF and MnSOD, by ADM plays an important role in the mechanism of the synergistic antitumor effect of TNF in combination with ADM.

Enhanced Antitumor Effect of Recombinant Human Tumor Necrosis Factor in Combination with Recombinant Human Granulocyte Colony‐stimulating Factor in BALB/c Mice

The synergistic antitumor effect of tumor necrosis factor (TNF) and granulocyte colony‐stimulating factor (G‐CSF) was investigated. G‐CSF was administered subcutaneously to BALB/c mice inoculated with Meth‐A cells at a dose of 2.5 μg/day for 5 consecutive days. When TNF (1 × 103 U) was administered intravenously to mice which had been pretreated with G‐CSF, tumor growth showed a 74.1% inhibition 17 days after the tumor cell inoculation, compared to that of untreated mice. In this experiment, G‐CSF significantly (P<0.025) enhanced the antitumor effect of TNF. The in vitro cytotoxicity of TNF (10 U/ml) towards Meth‐A cells was increased about 5.2‐fold in the presence of neutrophils (E/T=50) as compared to the cytotoxicity obtained with TNF alone. A combination of TNF and G‐CSF (50 ng/ml) in the presence of neutrophils, resulted in a 2.1 times greater cytotoxicity against Meth‐A cells as compared to that obtained without G‐CSF. Significant augmenting effects of G‐CSF on superoxide (O2−) production by TNF‐stimulated neutrophils were observed. These observation suggest that the neutrophil plays an important role in the antitumor action of TNF on Meth‐A cells, and that the antitumor effect of TNF is enhanced by combination with G‐CSF.