Takashi Kurizaki

Effect of Platelet-Derived Growth Factor Receptor-β Inhibition with STI571 on Radioimmunotherapy

Whereas radioimmunotherapy of hematologic malignancies has evolved into a viable treatment option, the responses of solid tumors to radioimmunotherapy are discouraging. The likely cause of this problem is the interstitial hypertension inherent to all solid tumors. Remarkable improvements in tumor responses to radioimmunotherapy were discovered after the inclusion of STI571 in the therapy regimen. A combination of the tumor stroma-reactive STI571, a potent platelet-derived growth factor receptor-beta (PDGFr-beta) antagonist, and the tumor-seeking radiolabeled antibody B72.3 yielded long-lasting growth arrest of the human colorectal adenocarcinoma LS174T grown as s.c. xenografts in athymic mice. The interaction of STI571 with the stromal PDGFr-beta reduced tumor interstitial fluid pressure (P(IF)) by >50% and in so doing improved the uptake of B72.3. The attenuation of P(IF) also had a positive effect on the homogeneity of antibody distribution. These effects were dose-dependent and under optimized dosing conditions allowed for a 2.45 times increase in the tumor uptake of B72.3 as determined in the biodistribution studies. Single-photon emission computed tomography imaging studies substantiated these results and indicated that the homogeneity of the radioisotope distribution was also much improved when compared with the control mice. The increased uptake of radioimmunotherapy into the tumor resulted in >400% increase in the tumor absorbed radiation doses in STI571 + radioimmunotherapy-treated mice compared with PBS + radioimmunotherapy-treated mice. The improved antibody uptake in response to the attenuation of tumor P(IF) was identified as the primary reason for the growth arrest of the STI571 + radioimmunotherapy-treated tumors. Two related causes were also identified: (a) the improved homogeneity of monoclonal antibody distribution in tumor and (b) the increased tumor radiosensitivity resulting from the improved tumor oxygenation.

Characterization of cancer cell dissociation factor in a highly invasive pancreatic cancer cell line

Background. Two pancreatic cancer cell lines, the highly invasive and metastatic cell line PC‐1.0 and the weakly invasive and rarely metastatic cell line PC‐1, were established from a pancreatic ductal carcinoma induced by N‐nitrosobis (2‐oxopropyl) amine in a Syrian golden hamster.

Zonula occludens-1 (ZO-1) redistribution is involved in the regulation of cell dissociation in pancreatic cancer cells.

In our previous study, dissociation factor (DF) and mitogen-activated protein kinase kinase 2 (MEK2) were isolated as factors relating to cancer cell dissociation in pancreatic cancer cells. On the other hand, tight junction protein zonula occludens 1 (ZO-1) has been indicated to be involved in carcinogenesis. In this study, the expression of ZO-1 and a downstream kinase of MEK2, extracellular signal-regulated kinase 2 (ERK2), was analyzed to clarify the regulatory mechanism of cell dissociation in pancreatic cancer cells. Two hamster (PC-1.0 and PC-1) and two human (AsPC-1 and CAPAN-2) pancreatic cancer cell lines were used. Immunocytochemical study was performed using anti-ZO-1, ERK2, and phosphorylated ERK1/2 (p-ERK1/2) antibodies. DF treatment obviously disrupted ZO-1 expression at the sites of cell–cell contact and markedly induced ERK2 and p-ERK1/2 expression, as well as the dissociation of cell clones in PC-1 and CAPAN-2 cells. In contrast, U0126 (a MEK1/2 inhibitor) treatment significantly induced the peripheral distribution of ZO-1 as well as cell aggregation in PC-1.0 and AsPC-1 cells, which usually grew as single cells, but seriously suppressed ERK2 and p-ERK1/2 expression. We conclude that redistribution of ZO-1 is closely correlated with cell dissociation status in pancreatic cancer cells through activation of ERK2.

Analysis of the signal transduction pathway in relation to invasion and metastasis in pancreatic cancer

Abstract The signal transduction pathway related to the potential of invasion and metastasis was analyzed using two pancreatic cancer cell lines, the highly invasive and metastatic cell line, PC-1.0, and weakly invasive and rarely metastatic cell line, PC-1 established from a pancreatic ductal carcinoma induced by N -nitrosobis (2-oxopropyl) amine (BOP) in a Syrian golden hamster. The cell motility of PC-1.0 was found to be six times higher than that of PC-1. The relationship between the cell motility and the c-fos mRNA expression was detected in these two cell lines. The cell motility of PC-1.0 was inhibited by cyclic AMP antagonist, PKC inhibitor (staurosporine) and antisence c-fos oligonucleotide. The difference in mRNA expression between these two cell lines was studied using the representational difference analysis (RDA) method, and MAP kinase 2 (MEK2) was isolated as a factor related to the difference in cell functions. The constitutive activation of MEK2 was observed in PC-1.0 and while only faint expression was found in PC-1. And the MEK2 inhibitor U0126 was found to induce cell aggregation in PC-1.0. The results from this study suggest that the activation of MEK2, cyclic AMP dependent PKC and c-fos are involved in the mechanism of invasion and metastasis through the induction of cell dissociation and cell motility.

Possible relationship between the expression of membrane-associated phospholipase A2 and the proliferation of interstitial tissue in human pancreatic cancer

Abstract The immunohistochemical localization of membrane-associated phospholipase A2 (M-PLA2) in normal human pancreases and 30 cases of pancreatic ductal carcinomas was investigated. In pancreatic ductal carcinomas, the immunoreactivity was observed in 25 cases (83%). Among the clinocopathological factors of pancreatic cancer, the incidence of expression of this enzyme is significantly higher in infiltrative type cancers. Furthermore, the expression of M-PLA2 was significantly increased in tumors, which had a larger amount of interstitial tissue. On the other hand, human M-PLA2 added exogenously to the fibroblast cell lines Swiss 3T3 and BALB/3T3 was found to augment their DNA synthesis. The stimulation of DNA synthesis was not affected by treatment with indomethacin. These results suggest that this enzyme could be involved directly in the proliferation of interstitial tissue through its own function.

Biological significance of the production of membrane-associated phospholipase A2 in human gastric cancer

We examined the immunohistochemical expression of membrane-associated phospholipase A2 (M-PLA2), belonging to group II PLA2, in 44 advanced gastric cancers, using the ABC method and monoclonal antibody anti-human M-PLA2. M-PLA2 mRNA was also examined in the same tumors by Northern blot analysis. In addition, the content of M-PLA2 protein and prostaglandin E2 (PGE2) in the malignant lesion and in the non-malignant gastric mucosa was examined. The expression was detected in cancer cells in 31 out of 44 advanced gastric cancer tissues (70.4%) by the ABC method. M-PLA2 mRNA was detected in 36 out of 44 gastric cancer tissues (81.8%), and the density was observed to be higher in tumor tissues than in the adjacent non-malignant gastric mucosa. The M-PLA2 protein was detected both in malignant tissues and in non-malignant gastric mucosa, and the content of M-PLA2 protein was significantly higher in malignant tissues than in the non-malignant gastric mucosa. There was a significant positive correlation between the expression of M-PLA2 mRNA and the amount of M-PLA2 protein. PGE2 was also detected in the malignant tissues and in the non-malignant mucosa. The content of PGE2 was significantly higher in the former. These results indicate that M-PLA2 is produced both in malignant and non-malignant cells of the stomach, the former producing higher amounts of this enzyme than the latter. M-PLA2 may be involved in cancer progression through its function or through the function of products of this enzymes action such as PGE2 in gastric cancer.

Expression of group II phospholipase A2 in malignant and nonmalignant human hepatocytes

Abstract Using a monoclonal antibody (MoAb) against human group II PLA2, we investigated the expression of human group II PLA2 in hepatocytes from adults, and hepatocellular carcinoma (HCC) and hepatoblastoma cells. The immunoreactivity of group II PLA2 was found only in a limited number of hepatocytes from adults. In HCC, however, immunoreactivity was found in 15 of 25 cases (60.0%) and 14 of 16 moderately differentiated HCC expressed group II PLA2. In contrast, immunoreactivity was observed in only one of eight poorly differentiated HCC, and no immunoreactivity was detected in one undifferentiated HCC and six hepatoblastomas. The incidence of the expression of this enzyme was significantly higher in trabecular-type HCC than in compact-type HCC. Moreover, expression of group II PLA2 was higher in the infiltrative tumor than in an expansive tumor. A high incidence was found in the tumor with larger amounts of interstitial tissue and 2/2 scirrhous-type HCC expressed this enzyme. Our observations suggest that hepatocytes in the adult liver have the potential to produce group II PLA2, and that expression of this enzyme is positively related to differentiation of the hepatocyte. The possibility was discussed that the expression of this enzyme may be involved in the differentiation, cancer cell invasion and proliferation of interstitial tissue of the HCC.

Correlation of extracellular signal-regulated kinase 2 expression and the cell dissociation induced by dissociation factor in pancreatic cancer cells

Abstract In our previous studies, the cancer dissociation factor (DF), which can induce cell dissociation of weakly invasive pancreatic cancer cells (PC-1), was isolated from a highly invasive cell line (PC-10). In further study, mitogen-activated protein kinase 2 (MEK2) was discovered to be an invasion-metastasis related factor in this model. Extracellular signal-regulated kinase 2 (ERK2) is known to be the downstream kinase of MEK2 in the mitogen-activated protein kinase (MAPK) signaling pathway. Demonstration of the role of ERK2 in DF induced cell dissociation may contribute to elucidation of the signaling regulatory mechanism of tumor invasion-metastasis. The two cell lines mentioned above and the immunofluorescent method were used. In untreated cells, both total ERK2 and phosphorylated ERK (p-ERK) presented constitutively strong expressions in PC-1.0 cells, whereas the relevant expressions were quite weak in PC-1 cells. Nevertheless, addition of the conditioned medium containing DF to the PC-1 cells, significantly induced total ERK2 and p-ERK expressions and dissociation of islandlike cell colonies. Furthermore, the total ERK2 and p-ERK expressions, either the induced expressions in PC-1 cells or constitutive expressions in PC-1.0 cells, were seriously suppressed after treatment with the MEK2 phosphorylation inhibitor, U0126. Correspondingly, obvious cell colonies formation was observed in these two cell lines. In conclusion, ERK2 activation is closely correlated with the DF induced cell dissociation in pancreatic cancer cells.

Study of the Mechanism of Invasion and Metastasis in Pancreatic Cancer. Analysis of Cancer Cell Dissociation Factor.

膵臓癌の転移機構を明らかにする目的で, BOP誘導実験膵癌組織から樹立した高転移株 (PC-1・0) 培養上清中に存在する癌細胞解離因子 (DF) 分離精製し, その本態と膵癌の浸潤転移機構との関連について解析を行った. DFの精製を進め構成アミノ酸解析を行った結果, DFは新しいタイプのmetalloproteaseあるいはムチン様の構造をもつFc binding proteinであることが示唆された. 生物学的活性を解析した結果, DFには癌細胞コロニーを濃度依存的に解離し, 細胞運動能を増強させ, さらに, fibronectinに対する接着能を選択的に増強し, MATRIBGELに対する細胞浸潤能を増強する作用が存在した. 本研究の結果から, DFは膵臓癌の浸潤転移機構と密接に関連した生物学的特性を有する新しい因子であることが示唆された.