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Epithelial-mesenchymal transition (EMT), a key process in the tumor metastatic cascade, is characterized by the loss of cell-cell junctions and cell polarity, as well as by the acquisition of migratory and invasive properties. However, the precise molecular events that initiate this complex EMT process are poorly understood. Snail expression induces EMT in Madin-Darby canine kidney (MDCK) cells and the human epidermoid carcinoma cell line, A431. Snail is a zinc finger transcription factor and triggers EMT by suppressing E-cadherin expression. In the present study, to broaden our knowledge of Snail‑induced EMT, we generated stable Snail transfectants using Madin-Darby bovine kidney (MDBK) cells. Contrary to the MDCK or A431 cells examined in our previous studies, the MDBK cells transfected with the Snail construct maintained an epithelial morphology and showed no sign of reduced cell-cell adhesiveness compared to the control cells. Consistent with these observations, the downregulation of epithelial marker proteins, e.g. E-cadherin and desmoglein, and the upregulation of mesenchymal marker proteins, e.g., N-cadherin and fibronectin, were not detected. Furthermore, the E-cadherin promoter was not methylated. Therefore, in the MDBK cells, the ectopic expression of Snail failed to induce EMT. As previously demonstrated, in MDCK cells, Snail expression is accompanied by the increased expression of other EMT-inducing transcription factors, e.g., Slug and zinc finger E-box-binding homeobox 1 (ZEB1). However, the MDBK cells transfected with the Snail construct did not exhibit an increased expression of these factors. Thus, it is possible that the failure to upregulate other EMT-related transcription factors may explain the lack of Snail-mediated induction of EMT in MDBK cells.

Thymidine phosphorylase (TP) is an angiogenic factor that plays a pivotal role in tumor angiogenesis. Various kinds of solid tumors express TP and high TP activity is correlated with microvessel density. We have previously reported that TP enhances interleukin-8 (IL-8) expression in KB human epidermoid carcinoma cells. In this study, TP was shown to be involved in enhanced expression of IL-8 in EJ human bladder cancer cells and Yumoto human cervical cancer cells as well as KB human epidermoid carcinoma cells. The enzymatic activity of TP was required for the enhanced expression of IL-8. A degradation product of thymidine was implicated in the enhanced expression of IL-8. TP augmented reactive oxygen species (ROS) generation in KB and Yumoto cells, and the enzymatic activity of TP was again required for the generation of ROS. An antioxidant, N-acetylcysteine (NAC), attenuated the generation of ROS and IL-8 mRNA expression in KB and Yumoto cells, and H2O2 increased IL-8 mRNA expression in Yumoto cells, suggesting that ROS generated by TP caused the increased expression of IL-8 mRNA. Since TP also reduced cellular glutathione levels and transcription of γ-GCS in KB cells, the TP-induced augmentation of ROS may be partially attributed to the decreased glutathione. Our findings suggest that thymidine-derived sugars enhanced ROS generation and consequently increased IL-8 expression.

Arsenic trioxide (As2O3) has been used with success in the treatment of acute promyelocytic leukemia. However, resistance to arsenite agents reduces their efficacy. We have isolated arsenite-resistant human epidermoid carcinoma KB cells, termed KAS. KAS cells were resistant to sodium arsenite (22-fold) and showed a reduced accumulation of arsenite as a result of an active efflux mechanism. Further analysis indicated that resistance of KAS cells extended to other drugs including cisplatin (17-fold), antimony potassium tartrate (11-fold) and doxorubicin (27-fold). Although increased expression of multidrug resistance protein 1 (MRP1) in KAS cells was confirmed by quantitative RT-PCR and immunoblot analysis, specific inhibitors of MRP1 did not completely eliminate arsenite resistance. The level of glutathione (GSH) in KAS cells was 3-fold higher than that in KB-3-1 cells, and the inhibition of GSH synthesis by buthionine sulfoximine (BSO) considerably increased the cytotoxic effect of arsenite on KAS cells. A pyridine analog, 2-[4-(diphenylmethyl)-1-piperazinyl ethyl 5-(trans-4,6-dimethyl-1,3,2-dioxaphosphorinan-2-yl)-2,6-dimethyl-4-(3-nitrophenyl)-3-pyridine-carboxylate P oxide (PAK-104P), partially reversed the arsenite resistance and increased the arsenite accumulation in KAS cells. We suggest that the increased level of GSH is involved in arsenite resistance and an as yet unidentified arsenite transporter is expressed in the arsenite-resistant KAS cells.

Disseminated carcinimatosis of the bone marrow is accompanied by solid tumors, and gastric cancer accounts for the majority. The prognosis of this condition is poor, however, the pathogenesis for wide-spread bone lesions has yet to be elucidated. In 9 patients with gastric cancer demonstrating disseminated carcinomatosis of the bone marrow, the characteristic clinicopathological features were examined. Immunohistochemistry for receptor activator of NF-kappaB ligand (RANKL) and parathyroid hormone-related protein was also performed on gastric cancer tissue and bone marrow specimens to identify the factors responsible for the occurrence of bone lesions in patients presenting with this condition. The characteristic features of disseminated carcinomatosis of the bone marrow due to gastric cancer include a yonger patient age, an elevation of serum alkaline phosphatase and/or lactate dehydrogenase levels, wide-spread bone metastases with osteolytic bone destruction, a low incidence of hypercalcemia and a histological gastric cancer type of either signet ring cell carcinoma or poorly diffentiated adenocarcinoma. The expression of RANKL, which is one of the master regulators of osteoclastic bone resorption in bone metastasis, was also found in gastric cancer cells obtained from such patients. The RANKL expressed in gastric cancer may therefore play a critical role in the promotion of osteoclast formation, which has been suggested to be involved in the pathogenesis of bone lesions.

Thymidine phosphorylase (TP), also known as platelet-derived endothelial cell growth factor (PD-ECGF), has been implicated in bladder cancer angiogenesis and invasion. However, the molecular basis of its role in invasion remains unclear. We investigated the expression of TP and 10 invasion-related genes in bladder cancers from 72 randomly selected patients by real-time two-step RT-PCR assay. We found that the expression levels of TP, MMP-9, uPA, and MMP-2 were significantly higher in invasive tumors than those in superficial tumors. Also, the expression level of TP significantly correlated with that of uPA, MMP-1, MMP-9, PAI-1 and VEGF. KK47/TP cells, bladder cancer cells that overexpress TP, had a higher expression of MMP-7 and MMP-9 than KK/CV cells that express lower level of TP in hypoxic condition. PC/TP cells, prostate cancer cells that overexpress TP, also had a higher expression of MMP-1 and MMP-7 than PC/CV cells that express no detectable TP. Taken together these data indicate that TP enhances the invasion of tumor cells through the induction of invasion-related genes.

Vaults are barrel-shaped cytoplasmic ribonucleoprotein particles composed of three proteins. One of the components, the major vault protein (MVP) initially named the lung resistance-related protein (LRP), was found to be overexpressed in various multidrug resistant cancer cell lines and clinical samples. In this study, we investigated whether anticancer drugs could directly induce MVP protein or gene expression in the SW-620 human colorectal cancer cell line, in which MVP has been shown to be induced by the differentiation-inducing agent, sodium butyrate (NaB). MVP protein levels were enhanced in SW-620 cells after a 72 h treatment with doxorubicin (Adr), etoposide (VP-16), cis-platinum (II) diammine dichloride (CDDP) or SN-38, but not vincristine (VCR) or paclitaxel (Taxol) at their IC50 concentration. Treatment for 48 h with Adr, VP-16 and SN-38 at their IC50 concentration also enhanced the expression of MVP mRNA. Moreover, Adr could directly enhance the transcriptional activity of MVP promoter regions. On the other hand, the Adr treatment did not affect the stability of MVP mRNA. Furthermore, MVP levels were also elevated after treatment with the DNA-damaging agents, ethidium bromide (EtBr) and ultraviolet light (UV) irradiation. Our findings therefore suggest that DNA damage enhances MVP promoter activity. Since the MVP protein and mRNA have low turnover rates, a slight enhancement of MVP promoter activity could lead to a considerable increase in the level of MVP.

Endoscopic mucosal resection (EMR) is a minimally invasive, standard treatment for intramucosal (early) gastric cancers, but is not standard for submucosal gastric cancers based on existing criteria. We evaluated the possibility of extending EMR as a therapy for submucosal gastric cancers by analyzing nodal micrometastasis through immunohistochemical staining in patients with apparent node-negative submucosal gastric cancer, the patients for whom EMR might be appropriate. We used anti-cytokeratin (AE1/AE3) antibody to immunohistochemically detect nodal micrometastasis that was not identified by routine pathological examination in 162 patients (total, 2048 lymph nodes) with apparent node-negative submucosal gastric cancer. The relationship between the incidence of nodal micrometastasis and clinicopathological factors was analyzed. Micrometastasis was detected in 45 of 2048 nodes (2.2%), representing 31 of 162 patients (19%). A significantly high incidence of nodal micrometastasis was found with submucosal cancers of large size (>2 cm), as well as with tumors that showed lymphatic or venous invasion and deeper submucosal invasion (p<0.0001). Nodal micrometastasis was also recognized in 2 cases of histologically well-differentiated tumors with focal submucosal invasion without venous or lymphatic invasion. Of the 162 patients, only 2 died of recurrent disease regardless of nodal involvement. Based on the present results, risk factors for nodal micrometastasis are tumor size, presence of lymphatic-vascular invasion, and depth of tumor, which are nearly the same as those established in previous pathological studies that used hematoxylin and eosin staining. We conclude that EMR is not recommended for patients with submucosal gastric cancer.

Thymidine phosphorylase (dThdPase) is known to promote the development of new blood vessels. Increased dThdPase expression in solid tumors has been shown to correlate with tumor growth, invasion and metastasis. In the present study, we measured dThdPase levels in the tumor tissue and in the serum from the tumor drainage and peripheral venous blood obtained from patients with resectable colorectal cancer. Serum dThdPase levels, measured by a modified ELISA method, were significantly higher in patients with hematogenous metastasis. In the tumor tissue specimens, no significant difference was observed between patients with or without hematogenous metastasis. These results suggest that the serum dThdPase levels are a novel marker to predict occurrence of hematogenous metastasis in patients with resectable colorectal cancer.

We previously reported that enhanced active efflux of cisplatin and increased GSH level were observed in KCP-4 cells. In the present study, KCP-4 cells were found to be cross-resistant to ultraviolet (UV) compared with parental KB-3-1 cells. Enhanced nucleotide excision repair (NER) was verified by time-dependent repair of UV-induced DNA damage. In addition, the amount of platinum bound to DNA after exposure to cisplatin decreased in a time-dependent manner in KCP-4 cells and this was reversed by aphidicolin, a DNA polymerase inhibitor. In stationary phase cultures, aphidicolin increased the sensitivity of KCP-4 cells to cisplatin. The expression of xeroderma pigmentosum complementation group F (XPF), an endonuclease involved in NER, was upregulated in KCP-4 cells. In KCP-4 cells the expression of hMSH6, one of the mismatch repair (MMR) factors, was decreased compared to parental KB-3-1 and revertant KCP-4R cells. However, KCP-4 cells were cross-resistant to oxaliplatin, and microsatellite instability was not observed in them. These findings suggest that the enhanced NER activity for DNA damage caused by cisplatin may be involved in cisplatin resistance in KCP-4 cells.

To investigate the relationship between tumor angiogenesis and hematogenous metastasis in colorectal cancer, an immunohistochemical analysis using antibody against factor VIII was carried out on archival specimens of 35 primary tumors. In addition, we also evaluated the levels of vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8), by an enzyme-linked immunosorbent assay (ELISA), in tumor specimens and the serum in the drainage venous blood. The levels of VEGF showed no correlation with the microvessel density and also did not increase significantly in patients with hepatic metastasis. On the other hand, the IL-8 levels in the tumor tissue (r=0.45) and the serum IL-8 levels (r=0.49) showed a significant correlation with the microvessel density. The serum IL-8 levels in patients with Dukes' C colorectal cancer and hepatic metastasis were significantly higher than in those without hepatic metastasis (p<0.05). In addition, the serum levels of IL-8 in patients with Dukes' C cancer without hepatic metastasis and those with Dukes' A and B cancer were also closely similar. These results suggest that IL-8 is associated with the microvessel density in primary tumors and thus play an important role in the occurrence of hepatic metastasis in patients with colorectal cancer. As a result, elevated levels of IL-8 in the drainage vein are considered to be a useful predictor for developing hepatic metastasis in patients with resectable colorectal cancer.

Insulin-like growth factor 2 (IGF2) stimulates cell proliferation and development in normal human growth. In several human cancers, the IGF2 gene is overexpressed and is thus considered to be a growth factor for tumors mediated through both the paracrine and autocrine pathways. However, the significance of IGF2 mRNA expression in gastric cancer has yet to be clarified. We semi-quantitatively measured the expression of IGF2 mRNA in 57 Japanese cases of gastric cancer by means of the reverse transcription polymerase chain reaction and also analyzed the relation between the IGF2 expression status and other clinicopathologic factors. We also performed immunohistochemical staining for IGF2. In 41 of 57 cases (72%), the expression of IGF2 mRNA was greater in tumor tissue (T) than in normal tissue (N). The average tumor/normal (T/N) expression ratio of IGF2 mRNA corrected for that of control gene mRNA was 1.42, while ranging from 0.36 to 3.65. The T/N ratio of infiltrative-type cancers was greater than that of expanding-type cancers (p<0.05). The cases with lymphatic permeation showed a greater T/N ratio than those without lymphatic permeation in expanding-type cancers (p<0. 05). Immunohistochemical staining revealed IGF2 to be detected in cancer cells themselves, especially at the margin of the cancer tissue. The IGF2 gene may thus play an important role in lymph vessel permeation especially in expanding-type gastric cancers.

We examined the expression of ornithine decarboxylase (ODC) mRNA in 53 female cases of breast cancer by a reverse transcriptase-polymerase chain reaction (RT-PCR) assay to determine the clinicopathologic significance of its expression. A significantly higher expression of ODC mRNA was, observed in younger patients than in older patients. The patients with a larger sized tumour possessed a significantly higher expression of ODC mRNA. In addition, the cases with a poor prognosis showed significantly higher expression of ODC. Previous studies have reported in vivo and in vitro correlation between the expression of ODC and c-myc genes in human carcinomas. We disclosed a significant correlation between these genes in primary breast carcinomas. We conclude that the expression of ODC may potentially be a new biological marker for breast carcinoma.

Integrin alpha 6 is believed to be involved in malignant biological development. High tumor/normal (T/N) ratio of integrin alpha 6 was observed in intestinal type tumors in gastric carcinoma. In an immunohistochemical study, an intense and continuous staining was seen at the basement layer in the intestinal type tumors. Concerning the variant, there was a significant correlation between the expression ratio of variant B/variant A and the depth of tumor invasion. In conclusion, i) integrin alpha 6 expression was intimately correlated with histologic differentiation, and ii) the overexpression of variant B may be correlated with tumor invasion of gastric carcinoma.

The human gene MAGE encodes tumor specific peptide antigens and consists of at least 12 families. Some antigens coded by the MAGE genes may be potentially useful for cancer specific immunotherapy. There is, however, so far little information on the expression of these gene families in human esophageal carcinomas. We investigated the expression of MAGE-1, -2, -3, -4, -6, -8, -9, -10, -11, and -12 genes in 24 human esophageal carcinoma cell lines, and in 50 pairs of tumor and corresponding normal tissue specimens from the human esophagus by means of reverse transcription polymerase chain reaction. The expression rate varied from 13% of MAGE-6 and 8 to 79% of MAGE-4 in the esophageal carcinoma cell lines, and from 6% of MAGE-6 to 62% of MAGE-4 in clinical tumor samples. The most frequently and the least expressed gene were the MAGE-4 and MAGE-6 genes, respectively, in both the cell lines and the clinical samples. Forty-seven of the 50 clinical tumors expressed at least one MAGE gene. No significant clinicopathologic difference between the tumor cases was observed, regardless of the presence or absence of MAGE gene expression. The findings of this study thus demonstrated that the MAGE gene family is frequently expressed in clinical samples as well as in the cell lines of human esophageal carcinomas. Therefore, to identify the MAGE gene family may be useful, not only for esophageal tumor specific immunotherapy but for molecular diagnostic usage as well.

The expression of pyrimidine nucleoside phosphorylase (PyNPase) mRNA in tumor (T) and normal (N) biopsy specimens obtained from 41 cases of colorectal carcinoma was examined by the reverse transcriptase/polymerase chain reaction (RT-PCR). The higher T/N ratio of the expression of PyNPase mRNA correlated significantly with the presence of lymph vessel invasion (p=0.039), the positive lymph node metastasis (p=0.014) and the advanced stage of the disease (p=0.014). There was a significant con-elation between the results determined by enzyme activity and those determined by RT-PCR (p=0.005). The findings suggested that the determination of PyNPase mRNA by RT-PCR may give useful information on tumor aggressiveness of colorectal carcinoma and this method can be used instead of enzyme activity.

Recent studies have shown that microsatellite instability (MSI) play an important role in the development of various types of cancer. To clarify the clinicopathologic significance of MSI in colorectal carcinoma (CRC), the presence of MSI was examined in 54 Japanese cases of CRC using the polymerase chain reaction-based method. The incidence of MSI in CRC cases was 13 out of 54 cases (24%). CRC with MSI also showed a significant tendency not to have lymph node metastasis (P<0.05), although neither the survival nor the prognosis of the cases examined in this study were available due to the short period of follow-up. The present study showed that the incidence of MSI in Japanese CRC was 24% and suggests that CRC with MSI may behave in a less malignant manner.

Thymidine phosphorylase (TP) is identical to platelet-derived endothelial cell growth factor (PD-ECGF), and has angiogenic activity. We examined the involvement of TP activity in tumor growth and angiogenesis. KB cells were transfected with wild-type or mutant (L148R) PD-ECGF cDNA, and two sublines with high TP activity, KB/wt4 and KB/wt6, and one subline with no TP activity, KB/L148R, were cloned, respectively. The doubling times of these subclones in vitro were similar to that of KB cells. However, the growth of KB/wt4 and KB/wt6 cells was significantly faster when xenografted into nude mice than that of control cells with no TP activity. The tumors with high TP activity (KB/wt4 and KB/wt6) had significantly more microvessels than those with no TP activity (KB/-, KB/CV and KB/L148R) (P<0.01). These results, taken together with previous reports, suggest that the TP enzyme activity itself is involved in angiogenesis and growth of the KB tumors.

Prothymosin-alpha (PT-alpha) is a nuclear protein involved in cell proliferation. c-myc is implicated in the carcinogenesis of many human cancers. PT-alpha gene transcription is reported to be regulated by the c-myc gene in vitro. However, little has been reported on the PT-alpha and c-myc mRNA expressions in gastric cancer. We semi-quantitatively determined the PT-alpha and c-myc mRNA expressions in 60 pairs of gastric cancer tissue (T) and corresponding normal tissue (N) using the reverse transcriptase-polymerase chain reaction method. The average of T/N ratio was 1.20 for PT-alpha and 1.30 for c-myc. Cases demonstrating a T/N ratio of more than 1.0 were seen in 33 (55%) and 30 (50%) cases for PT-alpha and c-myc, respectively. No significant correlation was observed between either of these two mRNA expressions and any of the examined clinicopathologic factors for gastric cancer. However, a significant correlation was seen between the expressions of both genes (p<0.0001). The findings support the hypothesis that, regarding human gastric cancer, the transcription of PT-alpha is considered to be under the control of c-myc gene, however, the value of these gene expressions do not reflect biological behavior.