Shinichiro Akiyama

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.

Oral Vitamin C Supplementation in Hemodialysis Patients and Its Effect on the Plasma Level of Oxidized Ascorbic Acid and Cu/Zn Superoxide Dismutase, an Oxidative Stress Marker

Background/Aim: Oxidative stress is known to be enhanced in hemodialysis patients, and one of its useful markers is plasma copper/zinc superoxide dismutase (Cu/Zn-SOD). The increase in plasma Cu/Zn-SOD can be inhibited by orally administered lipid-soluble vitamin E. We examined the antioxidative effects of water-soluble vitamin C administered orally on Cu/Zn-SOD levels in hemodialysis patients. Methods: Vitamin C was orally administered to 16 maintenance hemodialysis patients before each dialysis session. Doses were increased from 200 to 1,000 mg over 3 months. The levels of plasma vitamin C and Cu/Zn-SOD and its mRNA expression in leukocytes were determined 1, 2, and 3 months after the start of vitamin C administration. Furthermore, the levels of oxidized and reduced forms of plasma vitamin C were determined before the start of vitamin C administration and before and after dialysis at 1,000-mg vitamin C doses. Results: Following oral administration, the plasma levels of vitamin C and its oxidized form were increased. However, significant changes in plasma Cu/Zn-SOD or its mRNA expression in leukocytes were not observed. Conclusion: In maintenance hemodialysis patients, vitamin C administration resulted in a significant increase in the postdialysis level of the oxidized form of vitamin C, which suggested an increase in antioxidant effect. However, water-soluble vitamin C did not significantly suppress Cu/Zn-SOD expression enhancement.

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.

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.

Pharmacodynamic Study of the Saltz Regimen for Metastatic Colorectal Cancer in a Hemodialyzed Patient

Objective: Combination therapy with irinotecan (CPT-11), 5-fluorouracil (5-FU) and leucovorin is widely used for the treatment of metastatic colorectal cancer. However, little is known about the safety of chemotherapy of malignancies in hemodialysis (HD) patients. We encountered a patient with colorectal carcinoma on HD. We decided to administer combination chemotherapy while monitoring the pharmacodynamics of the patient. Case Report: A 74-year-old male received regular HD 3 times a week because of type 1 diabetes mellitus. He was diagnosed with stage IV colorectal cancer in November 2004. After sigmoidectomy, the patient received chemotherapy: a weekly schedule of CPT-11 (50 mg/m2) was administered followed by l-leucovorin (10 mg/m2) and 5-FU (400 mg/m2) just after HD. During the course, the plasma concentrations of both SN-38, an active metabolite of CPT-11, and 5-FU were not increased compared with those of patients with normal renal function. Our patient presented with grade III hematological toxicity that was easily recovered by granulocyte colony-stimulating factor. Conclusion: These data suggest that the dose-reduced Saltz regimen can be feasible for colorectal cancer patients receiving dialysis as postoperative adjuvant chemotherapy.

Tumor necrosis factor and interferon-gamma augment anticolon antibody- dependent cellular cytotoxicity in ulcerative colitis.

The effect of tumor necrosis factor (TNF) and interferon (IFN)-gamma on antibody-dependent cellular cytotoxicity (ADCC) in patients with ulcerative colitis (UC) was investigated. ADCC activity was measured by the 51Cr release assay, using peripheral blood mononuclear cells of healthy subjects as effector cells and RPMI 4788 cells derived from human colon cancer as target cells. ADCC activity under sera from healty subjects remained low whether or not the effector cells were pretreated with TNF (100 U/ml, 16h). Under sera from UC patients, ADCC activity of 13.9%, compared to 9.6% when pretreatment was deleted. The effect of IFN pretreatment (100 U/ml, 16h) was also examined under sera from UC patients; in that experiment activity rose to 26.8%, in comparison to a 10.7% when IFN-gamma pretreatment was deleted. Finally, when the effector cells were pretreated with both TNF and IFN-gamma (100 U/ml of each, 16h) the ADCC activity under sera from UC patients was higher than when either TNF or IFN-gamma were used alone. These results suggest that TNF and IFN-gamma, by increasing ADCC activity in UC lesions, are involved in cell injury in the colonic epithelium. IFN-gamma appears to increase ADCC activity by increasing the number of high affinity monocyte Fc gamma RI receptors, while TNF increases ADCC activity by a different mechanism.