Masahiro Mise

Membrane-type matrix metalloproteinase-1(MT1-MMP) gene is overexpressed in highly invasive hepatocellular carcinomas

Abstract Background/Aims: The matrix metalloproteinase (MMP) family play important roles in the invasion of cancer cells by degrading the extracellular matrices. The current study was designed to determine the expression pattern of membrane-type matrix metalloproteinase-1 (MT1-MMP) in hepatocellular carcinomas and its participation in invasion potential. Methods: MT1-MMP mRNA expression was examined in 25 human hepatocellular carcinoma specimens using Northern blot, and the correlation to clinico-pathological features was evaluated. In situ hybridization and immunohistochemistry were performed to study the localization and the cells responsible for the production. Results: Northern blot analysis revealed high levels of MT1-MMP mRNA, expression in tumorous portions in all cases, whereas in non-tumorous portions moderate or faint expression was evident in 22/25 cases. In 21/25 cases, the expression levels in tumorous portion were higher than those in non-tumorous portion. In particular, hepatocellular carcinoma with capsule infiltration demonstrated significantly higher expression than those without ( p In situ hybridization and immunohistochemical study revealed MT1-MMP transcripts and proteins in cancer cells and stromal cells, respectively. MT1-MMP positive cells were preferentially observed in the invading border of tumor nests. The MMP-2 transcript showed a similar pattern to that of MT1-MMP by in situ hybridization. Conclusion: The present study showed that the MT1-MMP gene is strongly expressed in hepatocellular carcinoma cells and is involved in the invasion potential of hepatocellular carcinoma, and also that MT1-MMP may be one of the key molecules responsible for the invasion potential of hepatocellular carcinoma. Furthermore, the evidence suggests that MT1-MMP and MMP-2 cooperate in the process of cancer invasion.

Pathogenic role of Kupffer cell activation in the reperfusion injury of cold-preserved liver.

The present study was designed to investigate the possible participation of Kupffer cells in the development of reperfusion injury of the cold-stored liver graft. In the cold preservation of Kupffer cells with Euro-Collins solution, the proportion of asialo-GM1-positive cells was significantly increased at 12 and 24 hr of storage, and the TNF alpha-producing activity in these cells was approximately fivefold greater than control. Northern blot analysis demonstrated that TNF alpha mRNA was remarkably elevated in the reperfusion of the cold-preserved liver, although that of the prereperfused graft was only slightly induced. The reperfusion experiments of the cold-stored liver graft showed that addition of anti-TNF alpha antibody to the perfusate suppressed the elevation of the effluent levels of GOT and LDH significantly, and that pretreatment with a Kupffer cell inhibitor, gadolinium chloride, inhibited the increase of these enzymes in the effluents almost completely. Histological study revealed deposition of a fibrinlike substance in the sinusoid and the central veins extensively in the reperfused liver graft, whereas no apparent deposition was observed in the gadolinium-pretreated liver. Thus, the present study showed that Kupffer cells were primed by cold preservation with Euro-Collins solution, and then activated when the reperfusion was done. It seems likely that the Kupffer cell activation induced by cold preservation/reperfusion plays a major role in reperfusion injury with sinusoidal microcirculatory disturbance, and that TNF alpha is responsible for the impairment of the reperfused liver graft.

Expression of membrane-type matrix metalloproteinase-1 in human pancreatic adenocarcinomas

Abstract The expression of a new type of matrix metalloproteinase, membrane-type matrix metalloproteinase-1 (MT-MMP-1), was examined in 24 cases of primary pancreatic adenocarcinomas and 9 cases of secondary liver tumors derived from pancreatic adenocarcinomas, using a non-radioactive in situ hybridization and immunohistochemical methods. Out of 24 cases of primary pancreatic adenocarcinomas, 18 showed positive expression of MT-MMP-1 transcripts in cancer cells and 20 of 24 showed positive expression in the tumor stromal cells. The immunoreactivity of the gene products for MT-MMP-1 was demonstrated to be almost the same, as shown by in situ hybridization in these 24 cases. In particular, both the staining intensity for MT-MMP-1 transcripts and the immunoreactivity of the gene products in the tumor stromal cells of mucinous cystadenocarcinomas were significantly weaker than those of common-type ductal adenocarcinomas among the 24 cases. All of the 9 cases of secondary liver tumors derived from pancreatic adenocarcinomas showed positive expression for MT-MMP-1 transcripts but less immunoreactivity for the gene products. These results suggest that MT-MMP-1 is transcribed and translated in both cancer cells and the tumor stromal cells in human pancreatic adenocarcinomas. Furthermore, considering that common-type ductal adenocarcinoma of the pancreas usually shows a strong desmoplastic reaction, while mucinous cystadenocarcinoma typically does not, MT-MMP-1 expressed in the tumor stromal cells of common-type adenocarcinomas may be involved in processes leading to the desmoplastic reaction.

Decreased expression and rare somatic mutation of the CIP1/WAF1 gene in human hepatocellular carcinoma

CIP1/WAF1, a critical downstream effector of tumor suppressor p53, encodes a cyclin-dependent kinase inhibitor. By Northern blot analysis, the CIP1/WAF1 mRNA level in the tumor was significantly lower than that in the corresponding normal liver from 19 Japanese patients with hepatocellular carcinoma (P < 0.05). In the tumor from only one out of 19 patients (5%), somatic mutations of the CIP1/WAF1 as well as that of p53 gene were identified by RT-PCR/SSCP analysis. These results suggest that the decreased CIP1/WAF1 expression is involved in the carcinogenesis or the progression of hepatocellular carcinoma.

Hepatic macrophage malfunction in rats with obstructive jaundice and its biological significance

The present study was designed to investigate the pathophysiology of obstructive jaundice by analyzing the function of hepatic macrophages and their role in immune responses and homeostasis in rats. The phagocytic index, determined by the rate of disappearance of 51Cr-endotoxin from the peripheral blood after intravenous injection, was increased in obstructive jaundice 2 weeks after bile duct ligation. The superoxide production of isolated hepatic macrophages and peripheral blood monocytes, measured by the superoxide dismutase inhibitable ferricytochrome c reduction method, was increased. Prostaglandin E2 release, measured by RIA, was markedly increased in rats with obstructive jaundice, but there was no significant difference in interleukin-1 release between jaundiced and control rats. The flow-cytometric analysis of surface molecules of hepatic macrophages showed decreased expression of interleukin-2 receptor in rats with obstructive jaundice. Thus, the functions of hepatic macrophages in rats with obstructive jaundice were impaired. This malfunction may disturb the immunoregulatory network and metabolism, although the exact implications of the altered function of hepatic macrophages have not yet been clarified.

Inhibition of tumor growth and microvascular angiogenesis by the potent angiogenesis inhibitor, TNP-470, in rats

The antiangiogenic effects of TNP-470 on the neovascularization of tumors were studied by examining ultrastructural alterations in the vasculature and interstitial fluid pressure (IFP) of tumors. Wistar rats were first inoculated subcutaneously (s.c.) with the Walker 256 carcinosarcoma cell line, then either vehicle medium or TNP-470, 30 mg/kg, was injected s.c. on day 1. A tumor growth assay, the necrotic area, and the IFP in the tumor were all measured on day 12. The antiangiogenic effects of TNP-470 were studied by scanning electron microscopic images of tumor vascular casts. TNP-470 was observed to inhibit tumor growth and increase the necrotic area significantly. In the TNP-470-treated group, the IFP in the superficial layer, defined as 2–3 mm from the tumor capsule, and in the deep layer, defined as 8–10 mm from the tumor capsule, were significantly higher than the corresponding values in the control. Moreover, vascular casts showed a significant reduction in the budding of sprouts in the superficial layer, and a decrease in the maximum diameter of the tumor vessels in the deep layer. It is possible that the higher IFP in the TNP-470-treated tumors might have prevented tumor vessel dilation. The findings of this study demonstrated that TNP-470 inhibited the budding of tumor vessel sprouts, and increased the IFP. These processes seem to act synergistically to suppress tumor angiogenesis.

Effect of PGE2 on interleukin-1 and superoxide release from primary-cultured human hepatic macrophages

In order to learn more about how human hepatic macrophages function, we analyzed the effect of exogenous PGE2 on the amounts of interleukin-1 (IL-1) and superoxide (O2-) released from primary-cultured human hepatic macrophages (HHM phi). When endogenous PGE2 production was blocked by indomethacin, exogenous PGE2 reduced IL-1 release from HHM phi in a dose-dependent manner, whereas it tended to increase O2- release from HHM phi. These results may suggest the probable contribution of PGE2 in regulating HHM phi mediator release in vivo.

Tumoricidal activity of Kupffer cells augmented by anticancer drugs

The effect of Mitomycin-C (MMC) and Adriamycin (ADM) on the antitumor-associated function of Kupffer cells was examined. MMC and ADM enhanced the production of superoxide by Kupffer cells in cultures at low concentrations likely to occur in clinical use. The expression of interleukin-2 receptor, Ia antigen and asialoGM1 antigen, measured by flowcytometry, was increased by contact with MMC. Growth inhibition of AH130, rat ascites hepatoma, and P815, murine mastocytoma, by Kupffer cells treated with anticancer drugs was greater than that by Kupffer cells alone or anticancer agent alone. These results show that MMC and ADM activate Kupffer cells, leading to synergistic antitumor activity. The results suggest that some anticancer agents act through immunological mechanisms as well as through direct antineoplastic activity.

Augmented local immunity in the liver by a streptococcal preparation, OK432, related to antitumor activity of hepatic macrophages

The aim of this study was to investigate the augmentative effect of a streptococcal preparation, OK432, on the immunological competence of hepatic macrophages. We found that OK432 was distributed predominantly to hepatic macrophages after intravenous injection, and Northern blot analysis revealed that OK432 induced the gene expression of IL-1 alpha, beta, and TNF alpha in the liver. The induction of mRNAs was evident 1 h after the intravenous injection of OK432 and their accumulation reached a maximal level at 3 h. TNF production of hepatic macrophages was also increased by the intravenous injection of OK432. Furthermore, OK432 significantly increased the proportion of IL-2 receptor-positive hepatic macrophages. As for antitumor activity in the liver being augmented by OK432, the cytotoxic and cytostatic activity of hepatic macrophages from OK432-treated rats against tumor cells was significantly increased and OK432 markedly reduced the number of tumor nodules in the liver after the inoculation of tumor cells via the portal vein. These findings, which indicate that OK432 has various immuno-stimulating actions on hepatic macrophages, leading to the augmentation of antitumor activity in the liver, suggest that OK432 may be of some benefit in helping to prevent hepatic metastasis, at least in part, via its activation of hepatic macrophages.

Membrane-type matrix metalloproteinase-1 (MT1-MMP) gene is overexpressed in highly invasive hepatocellular carcinomas

BACKGROUND/AIMS The matrix metalloproteinase (MMP) family play important roles in the invasion of cancer cells by degrading the extracellular matrices. The current study was designed to determine the expression pattern of membrane-type matrix metalloproteinase-1 (MT1-MMP) in hepatocellular carcinomas and its participation in invasion potential. METHODS MT1-MMP mRNA expression was examined in 25 human hepatocellular carcinoma specimens using Northern blot, and the correlation to clinicopathological features was evaluated. In situ hybridization and immunohistochemistry were performed to study the localization and the cells responsible for the production. RESULTS Northern blot analysis revealed high levels of MT1-MMP mRNA expression in tumorous portions in all cases, whereas in non-tumorous portions moderate or faint expression was evident in 22/25 cases. In 21/25 cases, the expression levels in tumorous portion were higher than those in non-tumorous portion. In particular, hepatocellular carcinoma with capsule infiltration demonstrated significantly higher expression than those without (p<0.05). In situ hybridization and immunohistochemical study revealed MT1-MMP transcripts and proteins in cancer cells and stromal cells, respectively. MT1-MMP positive cells were preferentially observed in the invading border of tumor nests. The MMP-2 transcript showed a similar pattern to that of MT1-MMP by in situ hybridization. CONCLUSION The present study showed that the MT1-MMP gene is strongly expressed in hepatocellular carcinoma cells and is involved in the invasion potential of hepatocellular carcinoma, and also that MT1-MMP may be one of the key molecules responsible for the invasion potential of hepatocellular carcinoma. Furthermore, the evidence suggests that MT1-MMP and MMP-2 cooperate in the process of cancer invasion.