T Tsuchida

Vascular endothelial growth factor (VEGF) mRNA isoform expression pattern is correlated with liver metastasis and poor prognosis in colon cancer.

Vascular endothelial growth factor (VEGF) is a well known factor that induces angiogenesis. Four isoforms, i.e. VEGF206, 189, 165, and 121, have been identified. We examined the isoform patterns of VEGF mRNA using reverse transcription polymerase chain reaction (RT-PCR) analysis in 61 colon cancers. All the colon cancers examined expressed VEGF121. The isoform patterns were classified into three groups: type 1, VEGF121; type 2, VEGF121 + VEGF165; type 3, VEGF121 + VEGF165 + VEGF189. Three of the 61 colon cancers examined showed type 1 expression, 26 showed type 2 expression and 32 showed the type 3 pattern. The patients with liver metastases showed the type 3 isoform expression pattern at a significantly higher incidence (12 of 16, 75%) than those without liver metastasis (20 of 45, 44%) (P=0.036). The type 3 isoform pattern was significantly associated with M1 stage (P=0.019). The patients with colon cancer and the type 3 isoform pattern showed significantly poor prognosis (P < 0.01, Cox-Mantel). The colon cancers with the type 3 pattern showed a significantly higher involvement of veins (P=0.006). These observations suggest that the aberrant type 3 expression pattern of VEGF189 mRNA isoforms is correlated with liver metastasis, M stage, and poor prognosis in colon cancer.

Expression pattern of vascular endothelial growth factor isoform is closely correlated with tumour stage and vascularisation in renal cell carcinoma.

Vascular endothelial growth factor (VEGF) has five isoforms (VEGF206, 189, 165, 145 and 121). Increased VEGF expression in renal cell carcinoma (RCC) is associated with angiogenesis, but, it is not apparent which isoform is involved in this effect. We examined the isoform patterns of VEGF by reverse transcription-polymerase chain reaction (RT-PCR) in 47 RCCs. All showed increased VEGF expression as compared with extraneoplastic renal tissue. Four of the 47 RCCs showed VEGF121 alone, 10 showed VEGF121 + 165, and 33 showed the VEGF121 + 165 + 189 pattern. Patients with pathological stage pT3-4 RCC showed the VEGF121 + 165 + 189 isoform pattern at a significantly higher incidence (10/10, 100%) than those with pT0-2 (23/37, 62%) (P < 0.022). The VEGF121 + 165 + 189 isoform pattern was also significantly associated with high vessel counts and density (P = 0.0002, Mann-Whitney U test). These observations suggested that the VEGF189 mRNA isoform is closely associated with angiogenesis and results in the growth of RCC.

Thrombospondin 2 expression is correlated with inhibition of angiogenesis and metastasis of colon cancer

SummaryTwo subtypes of thrombospondin (TSP-1 and TSP-2) have inhibitory roles in angiogenesis in vitro, although the biological significance of these TSP isoforms has not been determined in vivo. We examined TSP-1 and TSP-2 gene expression by reverse transcription polymerase chain reaction (RT-PCR) analysis in 61 colon cancers. Thirty-eight of these 61 colon cancers were positive for TSP-2 expression and showed hepatic metastasis at a significantly lower incidence than those without TSP-2 expression (P = 0.02). TSP-2 expression was significantly associated with M0 stage in these colon cancers (P = 0.03), whereas TSP-1 expression showed no apparent correlation with these factors. The colon cancer patients with TSP-2 expression showed a significantly low frequency of liver metastasis correlated with the cell-associated isoform of vascular endothelial growth factor (VEGF-189) (P = 0.0006). Vascularity was estimated by CD34 staining, and TSP-2(–)/VEGF-189(+) colon cancers showed significantly increased vessel counts and density in the stroma (P < 0.0001). TSP-2(–)/VEGF-189(+) colon cancer patients also showed significantly poorer prognosis compared with those with TSP-2(+) / VEGF-189(–) (P = 0.0014). These results suggest that colon cancer metastasis is critically determined by angiogenesis resulting from the balance between the angioinhibitory factor TSP-2 and angiogenic factor VEGF-189.

Clinical implications of interleukin (IL)-10 induced by non-small-cell lung cancer

BACKGROUND The type 2 cytokine interleukin (IL)-10 has been reported to inhibit the antitumour activity of the regional immunity against various neoplasms. Certain lung cancers produce IL-10, but the clinical significance of IL-10 expression is not well understood. PATIENTS AND METHODS We examined IL-10 and IL-10 receptor (IL-10R) mRNA expression in 82 non-small-cell lung cancers (NSCLC) by reverse transcription-polymerase chain reaction (RT-PCR) assay. Immunohistochemistry (IHC) and enzyme immunoassay (EIA) were applied to evaluate the cellular localisation and the serum levels of IL-10. RESULTS RT-PCR assay revealed IL-10 mRNA expression in 68 (83%) of 82 NSCLC surgical specimens (40 of 50 adenocarcinomas, 22 of 26 squamous cell carcinomas, 5 of 5 large-cell carcinomas, 1 of 1 adenosquamous-cell carcinoma). RT-PCR assay also revealed IL-10R mRNA expression in 79 cases of NSCLC (96.1%). IL-10 expression was confirmed within tumour cells by IHC. EIA showed no significant serum IL-10 elevation in the 12 NSCLC positive for IL-10 mRNA expression (0-2.99 pg/ml). The NSCLC patients with IL-10 production showed significantly poorer prognosis than those without IL-10 production (P < 0.05, Kaplan Meier, log-rank test). CONCLUSIONS These results suggested that the cytoplasmic IL-10 correlated to clinical prognosis, and that IL-10 expression is a prognostic factor for NSCLC.

Adenovirus-mediated anti-K-ras ribozyme induces apoptosis and growth suppression of human pancreatic carcinoma.

Human pancreatic cancer is a lethal malignancy, and the lesions show a very high incidence of point mutations of the K-ras oncogene. These alterations can be used as potential targets for specific ribozyme (Rz)-mediated growth suppression of the cancer cells. We designed an anti-K-ras Rz against mutant K-ras gene transcripts (codon 12, GGT to GTT) and generated a recombinant adenovirus (rAd) to express the Rz (rAd/anti-K-ras Rz). More than 95% of Capan-1 human pancreatic cells were infected with rAd/anti-K-ras Rz when treated with the virus at 200 plaque-forming units/cell. The virus, rAd/anti-K-ras Rz, significantly suppressed mutant K-ras gene expression and inhibited the growth of Capan-1 cells. At 3 days postinfection, we observed maximum growth suppression of the cells, characteristic morphological changes of apoptosis such as nuclear condensation and oligonucleosomal DNA fragmentation, and suppression of bcl-2 oncoprotein. These changes were not found in control virus-infected cells. Our results indicated that the virus rAd/anti-K-ras Rz specifically down-regulated the K-ras/bcl-2 pathway and induced apoptotic changes in Capan-1 pancreatic carcinoma cells. High-efficiency adenovirus-mediated delivery of anti-K-ras Rz could become a significant gene therapy strategy against human pancreatic cancer.

Multidrug resistance-associated protein (MRP) expression is correlated with expression of aberrant p53 protein in colorectal cancer

Multidrug resistance-associated protein (MRP) is one of the major factors responsible for non-P-glycoprotein (Pgp)-mediated multidrug resistance of human tumour cells. In this study, we examined MRP and aberrant p53 expression in 54 colorectal cancers (CRC), 35 carcinoma in adenomas (CIA) and 40 adenomatous polyps by immunohistochemical procedures. 38 of 54 (70%) CRCs, 16 of 35 (46%) CIAs and 3 of 40 (8%) adenomatous polyps were MRP positive (chi 2 test, P < 0.0001). 36/54 (67%) CRCs, 10/35 (29%) CIAs and 0/40 adenomatous polyps were p53 positive. 30 of the 36 p53-positive CRCs were also MRP positive and 8/10 CIAs were both p53 and MRP positive. MRP overexpression correlated with aberrant p53 accumulation in CRCs and CIAs (chi 2 test, P < = or 0.01). Coexpression of MRP and p53 in the same cells was confirmed in the CRCs and CIAs by double staining procedures. These results suggested that MRP overexpression is related to aberrant p53 expression in CRC.

Alterations in tumour suppressor gene p53 correlate with inhibition of thrombospondin-1 gene expression in colon cancer cells

Abstract If activation of the p53 gene is involved in the progression or metastasis of colon cancer, it may affect the angiogenic phenotype in vivo. To verify this hypothesis, we studied the correlation between p53 accumulation and expression of thrombospondin-1 (TSP1) in colon cancer specimens. Levels of TSP1 gene expression were estimated by Northern blotting in 65 colon cancers. Accumulation of p53 and the distribution of TSP1 protein were evaluated immunohistochemically. Various levels of TSP1 gene expression were seen in colon cancers, while p53 accumulation was confirmed in 42 of the 65 colon cancers. The level of TSP1 gene expression demonstrated a significant inverse correlation with p53 accumulation in colon cancer. Colon cancer cells expressed TSP1 protein and p53 accumulation reciprocally in the same nests. These results suggest that alterations in the tumour suppressor gene p53 may inhibit TSP1 expression in colon cancer.

Ribozyme-mediated inactivation of mutant K-ras oncogene in a colon cancer cell line

Mutation of c-K-ras oncogene is an important step in progression of colon cancer. We used a hammerhead ribozyme (KrasRz) against mutated K-ras gene transcripts (codon 12, GTT) to inactivate mutant K-ras function in the colon cancer cell line SW480, harbouring a mutant K-ras gene. The β-actin promoter-driven KrasRz sequence (pHβ/KrasRz) was introduced into these cells (SW480/KrasRz), and we evaluated its effects on growth of the colon cancer. The gene expression of angiogenesis-related molecules (vascular endothelial growth factor and thrombospondin) was also estimated in SW480/KrasRz. KrasRz specifically and efficiently cleaved the mutant K-ras mRNA but not wild-type mRNA in vitro. SW480/KrasRz showed decreased growth rate under tissue culture conditions (P< 0.01, Dunnett’s test). The xenotransplantability of SW480/KrasRz (XeSW480/KrasRz) was significantly decreased in nude mice (P< 0.05, Fisher’s exact test). Tumour volume of the xenografts XeSW480/KrasRz was significantly smaller than that of XeSW480/DisKrasRz (P< 0.01, Dunnett’s test). Gene expression of VEGF was suppressed in SW480/KrasRz, while TSP1 gene expression was enhanced. The SW480/KrasRz cells showed apoptosis-related features including nuclear condensation and DNA fragmentation. These results suggested that the hammerhead ribozyme-mediated inactivation of the mutated K-ras mRNA induced growth suppression, apoptosis and alteration of angiogenic factor expression.

Ribozyme approach to downregulate vascular endothelial growth factor (VEGF) 189 expression in non-small cell lung cancer (NSCLC).

The aim of this study was to further clarify the role of the cell-associated isoform of vascular endothelial growth factor (VEGF189) on tumour growth and vascularity. Five isoforms of VEGF have been identified with different biological activities. VEGF121, VEGF145, VEGF165, VEGF189, VEGF206 are generated by alternative splicing. We used a hammerhead-type ribozyme (V189Rz) to suppress VEGF189 mRNA. The V189Rz specifically cleaved exon 6 of VEGF189 mRNA, but showed no activity against the VEGF121 or VEGF165 isoforms. The V189Rz was introduced into the human non-small cell lung cancer (NSCLC) cell line (OZ-6/VR). The expression level of VEGF189 mRNA was decreased in the OZ-6/VR cells, while VEGF121 and 165 expression was unaltered. The OZ-6/VR cells xenotransplanted into nude mice showed markedly reduced vascularisation and growth, whereas the cell line did not show any decreased growth under tissue culture conditions. The OZ-6/VR cells (1 x 10(5) cells/mouse) formed no tumours, whereas the parental OZ-6 cells formed large tumours within 8 weeks. The specific suppression of VEGF189 by the ribozyme decreased vascularity and xenotransplantability of the lung cancer cell line. Thus, the cell-associated isoform of VEGF, VEGF189, might have a key role in stromal vascularisation and the growth of NSCLC xenografts in vivo.

Acinar-islet cell tumor of the pancreas: report of a malignant pancreatic composite tumor.

An unusual case of malignant pancreatic composite tumor with both components of acinar cell tumor (ACT) and islet cell tumor (ICT) was investigated histologically, immunohistochemically, and ultrastructurally. The pancreatic tumor with central cyst formation was found on computerized tomographic examination of a 72-year-old man reporting appetite and weight loss. The ACT component was present in the original pancreatic region and the ICT region was adjacent to the ACT. ACT was immunohistochemically positive for pancreatic amylase, whereas ICT had argyrophil tumor cells immunohistochemically positive for chromogranin A. There were several tumor cell nests positive for both pancreatic amylase (acinar differentiation) and chromogranin A (islet differentiation). We speculated that ICT may have arisen from the de-differentiated tumor cells in the ACT after the occurrence of ACT.