Etsuko Negishi

Connexin32 as a tumor suppressor gene in a metastatic renal cell carcinoma cell line

Connexin genes expressing gap junction proteins have tumor-suppressive effects on primary cancers with certain cell specificity, but the suppressive effects on metastatic cancers are still conflicting. In this study, we show that connexin32 (Cx32) has a strong tumor-suppressive effect on a human metastatic renal cell carcinoma cell line (Caki-1 cell). Cx32 expression in Caki-1 cells reduced in vitro malignant phenotypes of the cells such as anchorage independency and invasion capacity. Furthermore, the Cx32 expression drastically reduced the development of Caki-1 cells in nude mice. We also determined that Cx32 reduced the malignant phenotypes in Caki-1 cells mainly through the inactivation of Src signaling. Especially, Cx32-dependent inactivation of Src decreased the production of vascular epithelial growth factor (VEGF) via the suppression of signal transducers and activators of transcription 3 (Stat3) activation, and we confirmed this result using short interfering RNA. In nude mice, Cx32-transfected Caki-1 cells showed lower serum level of VEGF comparing mock transfectant, and the development of the cells in nude mice positively related to the VEGF level. These data suggest that Cx32 acts as a tumor suppressor gene in Caki-1 cells and that the tumor-suppressive effect partly depends on the inhibition of Src-Stat3-VEGF signal pathway.

Association of cytosine-adenine repeat polymorphism of the estrogen receptor-β gene with menopausal symptoms

BACKGROUND The nature and severity of menopausal symptoms are highly variable among women. Polymorphisms of the estrogen receptor-beta (ERbeta) gene, such as cytosine-adenine (CA) dinucleotide repeats in intron 5, have been implicated in various diseases. OBJECTIVE We investigated the possible role of a CA dinucleotide repeat polymorphism in intron 5 of the ERbeta gene in the occurrence of menopausal and premenstrual symptoms. METHODS Fifty-one postmenopausal Japanese women were interviewed about premenstrual symptoms, menopausal symptoms, and use of hormone replacement therapy. Menopausal symptoms were divided into vasomotor, psychological, and musculoskeletal symptoms, and summary scores were created on the basis of severity. CA repeat polymorphism of the ERbeta gene was examined using denaturing high-performance liquid chromatography with the WAVE DNA Fragment Analysis System (Transgenomic Inc., Omaha, Nebraska). RESULTS The number of CA repeats of the ERP gene ranged from 14 to 25, and subjects could be divided into 3 groups: those with < or =17 repeats, or extremely short (E); 18 to 21 repeats, or short (S); and > or =22 repeats, or long (L). Four genotypes of CA repeats (EL, SS, SL, and LL) were found among the subjects, with SL being the most common. Relative to subjects with the SL genotype, women with SS had a 7.0-fold increased risk of vasomotor symptoms (odds ratio [OR] = 7.0; 95% CI, 1.25-39.15; P < 0.05), a 13.0-fold higher risk of psychological symptoms (OR = 13.0; 95% CI, 1.44-117.2; P < 0.01), and a 7.6-fold increased risk of premenstrual symptoms (OR = 7.6; 95% CI, 1.61-35.9; P = 0.01). The EL genotype was associated with an increased risk of vasomotor symptoms (P < 0.05), depression (P < 0.01), and premenstrual symptoms (P < 0.01). CONCLUSIONS CA repeat polymorphism of the ERbeta gene may be associated with menopausal and premenstrual symptoms. Premenstrual symptom scores were significantly related to menopausal symptom scores.

Negative growth control of renal cell carcinoma cell by connexin 32: Possible involvement of Her-2

Connexin (Cx) genes have negative growth effects on tumor cells with certain cell specificity. We have previously reported that Cx32 is specifically downregulated in human renal cell carcinoma cell (RCC) lines as well as cancerous regions of kidneys and that the Cx is expressed in the progenitor cells of the carcinoma. However, the precise role of Cx32 in growth control of RCC cells remains unknown. In this study, we examined whether Cx32 could act in growth control against a human RCC cell, Caki‐2 cell. In order to estimate the cell growth control, we established Caki‐2 cells that have stable expression of Cx32 genes. Cx32 expression in Caki‐2 cells induced contact inhibition of growth and reduced anchorage‐independent growth ability, but did not significantly affect lag phase growth rates. This growth control by Cx32 was dependent on the inhibition of the cell‐cycle transition from G1 to S phase at high cell density, and the inhibition of the cell‐cycle transition related to the suppression of Her‐2 activation. Furthermore, the suppression of Cx32 expression in Caki‐2 cells by short interfering RNA induced the activation of Her‐2. These data suggest that Cx32 has negative growth control of Caki‐2 cells, partly due to the inhibition of the Her‐2 activation.

Connexin 32 potentiates vinblastine-induced cytotoxicity in renal cell carcinoma cells.

We have reported that connexin (Cx) 32 gene, a member of gap junction protein family, acts as a tumor suppressor gene in human renal cell carcinoma (RCC). Of solid tumors, RCC is one of the most chemoresistant cancers, and there is no effective cancer chemotherapy against RCC at present. In this study, we examined if the combination of Cx32‐dependent tumor‐suppressive effect and vinblastine (VBL), a chemotherapeutic agent which has been utilized for clinical RCC treatment, could be effective in enhancing the sensitivity of RCC to VBL treatment. Cx32 expression in a human metastatic RCC cell (Caki‐1 cell) significantly enhanced in vitro and in vivo VBL‐induced cytotoxicity on the cell. Cx32 expression in the RCC cells potentiated VBL‐induced apoptosis compared to the Cx32‐negative RCC cells in vitro as well as in vivo. The enhancing apoptosis in the RCC cells by Cx32 mainly depended on the decrease of P‐glycoprotein (P‐gp), a multidrug resistance gene‐1 (MDR‐1) product responsible for reduction of VBL accumulation into the cells. We also observed that silencing of Cx32 by short interfering RNA (siRNA) treatment elevated the level of P‐gp in Caki‐1 cells and that inhibition of P‐gp function enhanced VBL‐induced apoptosis in the RCC cells. These results suggest that Cx32 is effective to enhance VBL‐induced cytotoxicity in Caki‐1 cells via the reduction of P‐gp. Overall, it seems that the combination of Cx32‐dependent tumor‐suppressive effect and VBL is promising as a new cancer therapy against RCC.

Enhancing effect of connexin 32 gene on vinorelbine-induced cytotoxicity in A549 lung adenocarcinoma cells

PurposeConnexin (Cx) genes exert negative growth effects on tumor cells with certain cell specificity, and tumor-suppressive effects of the Cx genes contribute to enhancement of chemotherapeutical agents-induced cytotoxicity in some cancer cells. Since we and others have been reported that Cx32 acts as a tumor suppressor gene in lung adenocarcinomas, this study was undertaken to estimate if the combination of Cx32-dependent tumor-suppressive effect and vinorelbine (VBN), a chemotherapeutic agent which has been utilized for clinical lung adenocarcinoma treatment, could be effective in enhancing the sensitivity of the lung cancer to VBN treatment.MethodsWe established the A549 cells (a human lung adenocarcinoma cell line) which had stable expression of Cx32 and estimated effect of Cx32 on VBN-induced cytotoxicity in the established cells.ResultsCx32 expression in A549 cells significantly potentiated VBN-induced cytotoxicity on the cells due to enhancement of apoptosis induction. The enhancing cytotoxicity in A549 cells by Cx32 mainly depended on a decrease in expression of multi-drug resistance-1 (MDR-1) gene responsible for reduction of VBN accumulation into the cells. We also observed that silencing of Cx32 by siRNA treatment elevated the expression level of MDR-1 mRNA in A549 cells and that inhibition of MDR-1 gene product-dependent function enhanced VBN-induced cytotoxicity in the cells.ConclusionThese results suggest that Cx32 contributes to the enhancement of VBN-induced cytotoxicity in A549 cells via the reduction of MDR-1 expression.

Evaluation of allele frequencies in the PADI4 gene and anti‐cyclic citrullinated peptide antibodies of patients with rheumatoid arthritis in a Japanese population

A case–control linkage disequilibrium study in a Japanese population showed that the peptidyl arginine deiminase type 4 ( PADI4 ) is a susceptibility locus for rheumatoid arthritis (p<0.05).1 Ikari et al 2 also showed an association of the PADI4 haplotype with rheumatoid arthritis in an independent Japanese study, but the relationship between the level of anti-cyclic citrullinated peptide (anti-CCP) antibodies and the haplotype has not been shown. Barton et al 3,4 and Caponi et al 5 could not validate the association in the UK or Caucasian French populations. The purpose of this study was to ascertain the relationship between levels of anti-CCP antibodies and PADI4 allele frequencies in a …