Jan Show Chu

In situ detection of Epstein–Barr virus in breast cancer

Epstein-Barr virus (EBV) is strongly associated with nasopharyngeal carcinoma and some lymphoepithelioma-like carcinomas from other anatomic sites. This study investigates the presence of EBV in breast cancer. Immunohistochemistry for EBV proteins (EBV nuclear antigen-2 and latent membrane protein-1) and in situ hybridization for EBV-encoded small nuclear RNAs (EBER-1 and -2) were performed in 60 invasive breast cancers. None of the 60 breast cancer samples showed detectable EBV. These results suggest that EBV may not play a significant role in the etiology of breast cancers in Taiwan.

Breast cancer vascularity: color Doppler sonography and histopathology study.

SummaryIn this prospective study, the authors examined 50 patients with breast tumors (malignant, n = 32; benign, n = 18) to investigate the correlation between color Doppler flow mapping and histopathological findings and to evaluate the clinical significance of color Doppler mapping. Among the 32 patients with breast cancer, color Doppler signals were detected in 24 patients (75%). The maximum flow velocities varied from 5 to 34 cm/sec, with 16 (67%) of them above 15 cm/sec. Among the 18 patients with benign tumors, color Doppler signals could be detected in 7 (39%). The maximum flow velocity varied from 3 to 30 cm/sec but was over 15 cm/sec in only two patients (28%). Histological studies revealed that color Doppler signals detected by Doppler sonography correlated with disordered neovascularization penetrating the lesion from its periphery, consisting of thin-walled blood vessels and large arteriovenous shunts. Although large tumors tend to have high Doppler flow, there is no significant correlation between the maximum flow velocity and tumor size. There is also no significant correlation between the detection of high flow color Doppler signals and the age, receptor status, tumor size, lymph node metastases, or clinical stage of patients with breast cancer. However, there is a positive association (p < 0.05) between nodal metastases and higher tumor flow velocity in T1 (≤ 2 cm) breast tumors, but not in larger tumors. It is concluded that color Doppler is useful in the assessment of tumor vascularity but is of limited value in the differentiation of benign from malignant lesions. However, the presence of color Doppler signals in Tl breast cancer suggesting early dissemination of the cancer might be of important clinical significance in detecting those small, apparently early, but aggressive tumors with poor prognosis.

Expressions of E-cadherin and exon v6-containing isoforms of CD44 and their prognostic values in human transitional cell carcinoma.

Cell surface adhesion molecules, E-cadherin and exon v6 containing CD44 isoforms (CD44v6), were readily found in well-to-moderately differentiated urothelial cell lines but were down-regulated in poorly differentiated cell lines. One hundred and fifteen tumors of transitional cell carcinoma (TCC) were examined with E-cadherin and CD44v6 specific antibodies. Sixty-five (56.5%) tumors exhibited a preserved type while 50 (43.5%) showed a reduced type for CD44v6. Sixty-seven (58.3%) tumors were classified as the preserved type, and 48 (41.7%) were classified as the reduced type for E-cadherin. The staining pattern of E-cadherin was the same as that of CD44v6 in 87.0% (100 of 115) of tumors. The frequency of the reduced type was higher in poorly differentiated carcinomas (32 of 52 for CD44v6, p = 0.001; 27 of 52 for E-cadherin, p = 0.112) and tumors with an invasive growth pattern (22 of 27 for CD44v6, p < 0.001; 20 of 27 for E-cadherin, p < 0.001) than it was in well-differentiated carcinomas and tumors with expansile growth. However, the association with lymph node involvement or distant metastasis did not reach statistical significance. There was no difference in survival in reference to the expression patterns of CD44v6 and E-cadherin. Furthermore, neither marker was a significant prognostic factor for tumor recurrence and survival according to Coxs multiple variant regression analysis.

In vitro and in vivo studies of the anticancer action of terbinafine in human cancer cell lines: G0/G1 p53‐associated cell cycle arrest

Terbinafine (TB) (Lamisil®), a promising oral antifungal agent used worldwide, has been used in the treatment of superficial mycosis. In our study, we demonstrated that TB dose‐dependently decreased cell number in various cultured human malignant cells. Flow cytometry analysis revealed that TB interrupts the cell cycle at the G0/G1 transition. The TB‐induced cell cycle arrest in colon cancer cell line (COLO 205) occurred when the cyclin‐dependent kinase (cdk) system was inhibited just as the levels of p53, p21/Cip1 and p27/Kip1 proteins were augmented. In the TB‐treated COLO 205, the binding between p53 protein and p53 consensus binding site in p21/Cip1 promoter DNA probe was increased. Pretreatment of COLO 205 with p53‐specific antisense oligodeoxynucleotide decreased the TB‐induced elevations of p53 and p21/Cip1 proteins, which in turn led to arrest in the cell cycle at the G0/G1 phase. Moreover, in the p53 null cells, HL60, TB treatment did not induce cell cycle arrest. Taken together, these results suggest an involvement of the p53‐associated signaling pathway in the TB‐induced antiproliferation in COLO 205. We further examined whether administration of TB could affect the growth of tumors derived from human colon cancer cells in an in vivo setting. COLO 205 cells implanted subcutaneously in nude mice formed solid tumor; subsequent intraperitoneal injections of TB (50 mg/kg) led to obvious decline in tumor size, up to 50–60%. In these tumors, increases in the p21/Cip1, p27/Kip1 and p53 proteins and the occurrence of apoptosis were observed. Combined treatment with TB and nocodazole (ND), a clinically used anticancer agent, potentiated the apoptotic effect in COLO 205. These findings demonstrate for the first time that TB can inhibit the proliferation of tumor cells in vitro and in vivo.

Mucin expression in mucinous carcinoma and other invasive carcinomas of the breast

To investigate mucin expression in breast cancer, immunohistochemical staining was performed on 30 mucinous carcinomas and 95 non-mucinous invasive carcinomas. MUC2 expression was detected in all mucinous carcinomas, but only in 11.1% of invasive ductal carcinomas, and in none of the invasive lobular carcinomas and medullary carcinomas. MUC1 is often expressed in invasive breast carcinoma, but not in medullary carcinoma. Strong cytoplasmic staining was seen in invasive ductal carcinoma, in contrast to surface membrane staining in mucinous carcinoma and intracytoplasmic vacuole staining in invasive lobular carcinoma. CA19-9 and CA50 expression in more than 25% of tumor cells was seen in 17.2 and 16.0% of invasive ductal carcinomas, respectively, but not in mucinous carcinomas. CA125 and human gastric mucin were rarely expressed in breast cancer, irrespective of histologic type.

Correlation of expression of CD44 isoforms and E-cadherin with differentiation in human urothelial cell lines and transitional cell carcinoma.

Using immunostaining, immunoblot, reverse-transcriptase polymerase chain reaction and Southern blot, we found that expressions of CD44 isoforms and E-cadherin were very closely linked and were correlated with the differentiation status in human urothelial cell lines and clinical specimens of transitional cell carcinoma. Normal urothelium, well to moderately differentiated cell lines and surgical samples expressed E-cadherin and large CD44 isoforms containing exon v6, which was pivotal in metastasis of rat pancreatic cell line model. Poorly differentiated cell lines and surgical samples, were E-cadherin-negative and expressed primarily standard form CD44, which did not contain exon v6. We concluded that CD44v6 isoforms and E-cadherin were both down-regulated during the carcinogenesis of urothelium. The large exon v6 containing CD44 isoforms were readily detected in normal urothelium, therefore, were not likely linked to cancer metastasis. E-cadherin and CD44v6 may be used as differentiation markers for human urothelial tumors. Immunohistochemical study solely with antibody against epitopes encoded by exon v6 alone is not informative enough as other alternatively spliced exons may change the function of CD44v6 isoforms.

MCPIP1 Suppresses Hepatitis C Virus Replication and Negatively Regulates Virus-Induced Proinflammatory Cytokine Responses

Human MCP-1–induced protein 1 (MCPIP1, also known as ZC3H12A and Regnase-1) plays important roles in negatively regulating the cellular inflammatory response. Recently, we found that as an RNase, MCPIP1 has broad-spectrum antiviral effects by targeting viral RNA. In this study, we demonstrated that MCPIP1 expression was induced by hepatitis C virus (HCV) infection in Huh7.5 hepatoma cells. MCPIP1 expression was higher in liver tissue from patients with chronic HCV infection compared with those without chronic HCV infection. Knockdown of MCPIP1 increased HCV replication and HCV-mediated expression of proinflammatory cytokines, such as TNF-α, IL-6, and MCP-1. However, overexpression of MCPIP1 significantly inhibited HCV replication and HCV-mediated expression of proinflammatory cytokines. Various mutants of functional domains of MCPIP1 showed disruption of the RNA binding and oligomerization abilities, as well as RNase activity, but not deubiquitinase activity, which impaired the inhibitory activity against HCV replication. On immunocytochemistry, MCPIP1 colocalized with HCV RNA. Use of a replication-defective HCV John Cunningham 1/AAG mutant and in vitro RNA cleavage assay demonstrated that MCPIP1 could directly degrade HCV RNA. MCPIP1 may suppress HCV replication and HCV-mediated proinflammatory responses with infection, which might contribute to the regulation of host defense against the infection and virus-induced inflammation.

Ketoconazole potentiates the antitumor effects of nocodazole: In vivo therapy for human tumor xenografts in nude mice

Our previous studies demonstrated that the oral antifungal agent ketoconazole (KT) induces apoptosis and G0/G1 phase cell cycle arrest in human cancer cell lines. In this study, we first demonstrated that KT (1 μM) potentiated the apoptotic effects of nocodazole (ND, 1 nM) in COLO 205 cancer cells. We further demonstrated the therapeutic efficacy of a combined treatment of KT (50 mg/kg/three times per week) and ND (5 mg/kg/three times per week) in vivo by treating athymic mice bearing COLO 205 tumor xenografts. The antitumor effects of ND were significantly potentiated by KT in mice after 6 wk of treatment. No gross signs of toxicity were observed in mice receiving these treatment regimens. The apoptotic cells were detected in a microscopic view of the terminal deoxynucleotidyl transferase–mediated dUTP nick‐end labeling staining and by observation of DNA fragmentation in KT + ND–treated tumor tissues. The levels of cell cycle regulatory proteins were determined by Western blot analysis. Treatment with KT inhibits tumor growth through elevation of p53, p21/CIP1, and p27/KIP1 as well as inhibition of cyclin D3 and cyclin‐dependent kinase 4 protein expression. Immunohistochemical staining analysis showed that p53, p21/CIP1, and p27/KIP1 immunoreactivity were induced in the tumor tissues. To clarify the roles of the p21/CIP1 and p27/KIP1 protein expression involved in G0/G1 arrest and/or apoptosis induced by a combined treatment with KT and ND, antisense oligodeoxynucleotides (ODNs) specific to p21/CIP1 and p27/KIP1 were used. Our results demonstrated that apoptotic phenomena, including BAX induction and cytochrome C released from mitochondria induced by KT + ND, were significantly attenuated by pretreatment the cells with the p27/KIP1–specific antisense ODNs. These results indicate that p27/KIP1 protein does indeed play a critical role in the KT + ND–induced apoptosis. Our study revealed the molecular mechanism of KT + ND in regression of the tumor growth. The apoptotic effects of KT in a great variety of cancer cells make it a very attractive agent for cancer chemotherapy.

Proliferating cell nuclear antigen (PCNA) immunolabeling as a prognostic factor in invasive ductal carcinoma of the breast in Taiwan

To evaluate the prognostic significance of proliferating cell nuclear antigen (PCNA) in breast cancer, an immunohistochemical assay was performed in 150 patients with invasive ductal carcinomas. The PCNA labeling index (PCNA-LI) was classified into two subgroups at a cut-off point of 45% that gave the best prognostic estimates for PCNA in survival analyses. Seventy-eight tumors had a low PCNA-LI of < or =45% and 72 tumors had a high PCNA-LI of >45%. A high PCNA-LI correlated significantly with p53 overexpression (P<0.03), positive axillary node (P<0.04), short disease-free survival (P<0.001) and overall survival (P<0.0002), but not with other factors. In multivariate analysis, the PCNA-LI predicted the disease-free (P<0.008) and overall survival (P = 0.0007) independently. Our study indicates that the PCNA-LI has independent prognostic value.

p53 point mutation enhanced by hepatic regeneration in aflatoxin B1-induced rat liver tumors and preneoplastic lesions

Aflatoxin B1 (AFB1) is a well-known mutagen and carcinogen which induces human hepatocellular carcinoma (HCC). It has been found to be an important factor in inducing a high frequency of codon 249 mutation in the p53 gene. We characterized p53 mutations in specimens from preneoplastic lesions or tumors from the liver of rats induced by AFB1 with or without regeneration by partial hepatectomy treatment. PCR-SSCP and direct sequencing were used for screening and identification of p53 gene mutations in these samples. In rats treated with AFB1 with or without liver regeneration, 29% (5/17) of rats with hepatoma or neoplastic lesion had p53 mutation. No p53 mutations were found in the tumor samples from the rats without liver regeneration. However, in samples from the rats with liver regeneration, 38% (5/13) of the rats with hepatoma or neoplastic lesion were found to have p53 mutations. In one of these samples, we also observed a transversion mutation G --> T on codon 247, compared to codon 249 in humans. These findings suggest that the process of hepatocarcinogenesis induced by AFB1 does not necessarily involve p53 mutation, but mutation of the p53 gene can be enhanced by liver regeneration and that there is a possibility of a high mutability of the third base of codon 247, even though there is a small probability of detecting such a mutation in rats due to its silent mutation.