Shinji Oie

Akt-dependent nuclear localization of Y-box-binding protein 1 in acquisition of malignant characteristics by human ovarian cancer cells

Y-box-binding protein 1 (YB-1), which is a member of the DNA-binding protein family containing a cold-shock domain, has pleiotropic functions in response to various environmental stimuli. As we previously showed that YB-1 is a global marker of multidrug resistance in ovarian cancer and other tumor types. To identify YB-1-regulated genes in ovarian cancers, we investigated the expression profile of YB-1 small-interfering RNA (siRNA)-transfected ovarian cancer cells using a high-density oligonucleotide array. YB-1 knockdown by siRNA upregulated 344 genes, including MDR1, thymidylate synthetase, S100 calcium binding protein and cyclin B, and downregulated 534 genes, including CXCR4, N-myc downstream regulated gene 1, E-cadherin and phospholipase C. Exogenous serum addition stimulated YB-1 translocation from the cytoplasm to the nucleus, and treatment with Akt inhibitors as well as Akt siRNA and integrin-linked kinase (ILK) siRNA specifically blocked YB-1 nuclear localization. Inhibition of Akt activation downregulated CXCR4 and upregulated MDR1 (ABCB1) gene expression. Administration of Akt inhibitor resulted in decrease in nuclear YB-1-positive cancer cells in a xenograft animal model. Akt activation thus regulates the nuclear translocation of YB-1, affecting the expression of drug-resistance genes and other genes associated with the malignant characteristics in ovarian cancer cells. Therefore, the Akt pathway could be a novel target of disrupting the nuclear translocation of YB-1 that has important implications for further development of therapeutic strategy against ovarian cancers.

Overexpression of Class III β-Tubulin Predicts Good Response to Taxane-Based Chemotherapy in Ovarian Clear Cell Adenocarcinoma

Purpose: Of the various microtubule-associated molecules, β-tubulin III has been reported to be closely associated with the therapeutic efficacy of taxane-based chemotherapy against ovarian cancer. Stathmin and microtubule-associated protein 4 (MAP4) have been reported to play an important role in microtubule stabilization. In this study, we investigated whether expression of these microtubule-associated factors affects the therapeutic efficacy of taxane-based chemotherapy in ovarian clear cell adenocarcinoma. Experimental Design: Drug sensitivity of paclitaxel or cisplatin was assessed in ovarian cancer cell lines treated with small interfering RNA of tubulin isoforms, MAP4, and stathmin. We examined 94 surgically resected ovarian clear cell adenocarcinoma specimens from patients treated with taxane-containing regimens (n = 44) and with taxane-free regimens (n = 50), using immunohistochemistry to detect expression of β-tubulin III, stathmin, and MAP4. Results: Knockdown of β-tubulin III and IV specifically conferred drug resistance to paclitaxel in one ovarian cancer cell line, but not to other molecules. Estimated overall survival revealed a significant synergistic effect between taxane and β-tubulin III in patients with ovarian clear cell adenocarcinoma. Of three microtubule-related molecules, among the taxane-based chemotherapy group, cases with higher β-tubulin III expression were associated with a significantly more favorable prognosis compared with those having lower β-tubulin III expression. By contrast, there was no statistical significance in the synergistic relationships between stathmin and taxane or between MAP4 and taxane. Conclusions: Taxane-based chemotherapy was effective for patients with ovarian clear cell adenocarcinomas who were positive for β-tubulin III but not for those who were negative for these proteins.

High expression of insulin-like growth factor binding protein-3 is correlated with lower portal invasion and better prognosis in human hepatocellular carcinoma

Insulin‐like growth factor binding protein‐3 (IGFBP‐3) modulates cell proliferation of various cancer cell types. However, it remains unclear how IGF–IGFBP‐3‐signaling is involved in growth and progression of hepatocellular carcinoma (HCC). The aim of the present study was to evaluate the role of IGFBP‐3 in HCC. Type 1 receptor for IGF (IGF‐1R) was expressed at various levels in the seven lines examined, but IGF‐2R was not expressed. Of the seven lines, the growth of HAK‐1B, KIM‐1, KYN‐2 and HepG2 cells was stimulated in a dose‐dependent manner by the exogenous addition of IGF‐I or IGF‐II, but the HAK‐1A, KYN‐1 and KYN‐3 cell lines showed no growth. Exogenous addition of IGFBP‐3 markedly blocked IGF‐I and IGF‐II‐stimulated cell growth of KYN‐2 and HepG2 cells, and moderately stimulated that of KIM‐1 and HAK‐1B cells, but no growth of the KYN‐1, KYN‐3 and HAK‐1A cell lines was observed. IGF‐I enhanced the phosphorylation of IGF‐1R, Akt and Erk1/2 in KYN‐2 cells, and coadministration of IGFBP‐3 blocked all types of activation by IGF‐I investigated here. In contrast, no such activation by IGF‐I was detected in KYN‐3 cells. IGFBP‐3 also suppressed IGF‐I‐induced cell invasion by KYN‐2 cells. Moreover, we were able to observe the apparent expression of IGFBP‐3 in KYN‐3 cells, but not in the other six cell lines. Furthermore reduced expression of IGFBP‐3, but not that of IGF‐1R, was significantly correlated with tumor size, histological differentiation, capsular invasion and portal venous invasion. Low expression of IGFBP‐3 was independently associated with poor survival. IGFBP‐3 could be a molecular target of intrinsic importance for further development of novel therapeutic strategy against HCC. (Cancer Sci 2006; 97: 1182–1190)

Growth inhibitory effects of pegylated IFN α‐2b on human liver cancer cells in vitro and in vivo

Abstract: Purpose: We investigated the effects of pegylated IFN‐α2b (PEG‐IFN‐α2b) on the growth of human liver cancer cells.

17β-estradiol induces down-regulation of Cap43/NDRG1/Drg-1, a putative differentiation-related and metastasis suppressor gene, in human breast cancer cells

Purpose: Cap43 is known as a nickel- and calcium-inducible gene. In the present study, we examined whether 17β-estradiol (E2) could affect the expression of Cap43 in breast cancer. Experimental Design: Real-time PCR, immunoblotting, and immunocytochemistry were used to examine the expression of Cap43 and estrogen receptor-α (ER-α) in breast cancer cell lines. MDA-MB-231 and SK-BR-3 cell lines were transfected with ER-α cDNA to establish cells overexpressing ER-α. Immunohistochemistry was used to evaluate the expression of the Cap43 protein in breast cancer patients (n = 96), and the relationship between Cap43 expression and clinicopathologic findings was examined. Results: Of the eight cell lines, four expressed higher levels of Cap43 with very low levels of ER-α, whereas the other four expressed lower levels of Cap43 with high ER-α levels. Treatment with E2 decreased the expression of Cap43 dose-dependently in ER-α-positive cell lines but not in ER-α-negative lines. Administration of antiestrogens, tamoxifen and ICI 182780, abrogated the E2-induced down-regulation of Cap43. Overexpression of ER-α in both ER-α-negative cell lines, SK-BR-3 and MDA-MB-231, resulted in down-regulation of Cap43. Immunostaining studies showed a significant correlation between Cap43 expression and the histologic grade of tumors (P = 0.0387). Furthermore, Cap43 expression was inversely correlated with the expression of ER-α (P = 0.0374). Conclusions: E2-induced down-regulation of Cap43 seems to be mediated through ER-α-dependent pathways in breast cancer cells both in culture and in patients. Cap43 has potential as a molecular marker to determine the therapeutic efficacy of antiestrogenic anticancer agents in breast cancer.

Establishment and characterization of the RMG-V cell line from human ovarian clear cell adenocarcinoma

A cell Line, designated as RMG-V, was established from a patient with clear cell adenocarcinoma of the ovary. The cell line has grown without interruption and has been propagated continuously by serial passaging (more than 36 times) over 5 years. The cells are spindle-shaped, display neoplastic and pleomorphic features, and grow in a jigsaw puzzle-like arrangement while forming monolayers without contact inhibition. These cells proliferate rapidly, and the population doubling time is about 15.5 hours. The number of chromosomes ranges between 77 and 85, with a modal number of 83.

Alteration of dihydropyrimidine dehydrogenase expression by IFN-α affects the antiproliferative effects of 5-fluorouracil in human hepatocellular carcinoma cells

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme in the catabolism of 5-fluorouracil (5-FU) and its activity is closely associated with cellular sensitivity to 5-FU. This study examines the role of DPD in the antiproliferative effects of 5-FU combined with IFN-α on hepatocellular carcinoma (HCC) cells in culture and asks whether IFN-α could affect DPD expression. The combined action of IFN-α and 5-FU on three HCC lines was quantified by a combination index method. Coadministration of IFN-α and 5-FU showed synergistic effects against HAK-1A and KYN-2 but antagonistic effects against KYN-3. The cellular expression levels of DPD mRNA and protein were markedly up-regulated in KYN-3 cells by IFN-α but were down-regulated in HAK-1A and KYN-2. The expression of thymidylate synthase mRNA and protein was down-regulated by IFN-α in all three cell lines. Coadministration of a selective DPD inhibitor, 5-chloro-2,4-dihydroxypyridine (CDHP), enhanced the antiproliferative effect of 5-FU and IFN-α on KYN-3 ∼4-fold. However, the synergistic effects of 5-FU and IFN-α on HAK-1A and KYN-2 were not affected by CDHP. The antiproliferative effect of 5-FU could thus be modulated by IFN-α, possibly through DPD expression, in HCC cells. Inhibition of DPD activity by CDHP may enhance the efficacy of IFN-α and 5-FU combination therapy in patients with HCC showing resistance to this therapy. [Mol Cancer Ther 2007;6(8):2310–8]

Loss of integrin α3 expression is associated with acquisition of invasive potential by ovarian clear cell adenocarcinoma cells

Among various types of surface epithelial ovarian carcinoma, clear cell adenocarcinoma often has a particularly poor prognosis even when diagnosed in stage I. It is resistant to existing anticancer drugs and appears to have different biological properties to other histological types of ovarian cancer.The present study was conducted using cell lines derived from ovarian clear cell adenocarcinoma in order to identify genes associated with the acquisition of malignant potential by this type of cancer. Two cell lines derived from ovarian clear cell adenocarcinoma (RMG-I and RMG-V), with different levels of invasive potential in an invasion assay, were used. DNA fragments were extracted from the band showing differences in the level of expression by RT-PCR with fluorescent differential display. A total of 56 different DNA base sequences were determined by direct sequencing. Primers were established using these base sequences and the level of expression in each cancer cell line was determined by real-time PCR Integrin a3, the gene of which is present on chromosome 17q, was identified. It was also detected by a microarray analysis as one of the molecules showing a different level of expression between the two cell lines. Then the pattern of integrin α3 expression was investigated using immunocytochemical staining, and was found to differ between the two cell lines. The findings obtained using these cell lines derived from ovarian clear cell adenocarcinoma indicate that integrin α3 may associated with the acquisition of malignant potential by clear cell adenocarcinoma.

Up-regulation of insulin-like growth factor-binding protein 3 by 5-fluorouracil (5-FU) leads to the potent anti-proliferative effect of androgen deprivation therapy combined with 5-FU in human prostate cancer cell lines

In this study, we investigated the synergistic mechanism of anti-androgen and 5-fluorouracil (5-FU) combination therapy against castration-resistant prostate cancer (CRPC). Four prostate cancer cell lines, LNCaP, 22Rv1, DU145 and PC3, were examined for their growth dependency on androgens and the insulin-like growth factor 1 (IGF1). We assessed the expression changes of certain growth factor receptors and regulating proteins when treated with 5-FU, and found that 5-FU increased the expression of the IGF-binding protein 3 (IGFBP3). Furthermore, 5-FU inhibited the phosphorylation of Akt and p70 S6K, while the knockdown of IGFBP3 reduced the levels of poly (ADP-ribose) polymerase cleaved by 5-FU in PC3 cells. Therefore, the up-regulation of IGFBP3 by 5-FU not only inhibits cell growth by reducing the IGF1 signal but also induces apoptosis in PC3 cells. The synergistic effect of bicalutamide and 5-FU on 22Rv1 cells was reduced by IGFBP3 gene silencing using small-interfering RNA. These results suggest that the up-regulation of IGFBP3 induced by 5-FU plays an important role in the potent anti-tumor effect of 5-FU combined with anti-androgens on CRPC. Androgen-deprivation therapy combined with 5-FU could therefore be an appropriate therapy for CRPC patients.

Hydroxyflutamide enhances cellular sensitivity to 5-fluorouracil by suppressing thymidylate synthase expression in bicalutamide-resistant human prostate cancer cells

We investigated the antitumor effects of combination therapy with anti-androgens and 5-fluorouracil (5-FU), and examined the underlying mechanism of the treatment. Initially, we established the bicalutamide-resistant subline CDX25R from the androgen receptor (AR)-positive human prostate cancer cell line LNCaP through continuous exposure to bicalutamide. CDX25R cells lost the ability to respond to androgens, but still expressed AR. They showed significant resistance to bicalutamide, but had high sensitivity to hydroxyflutamide (OH-flutamide) compared with LNCaP cells. The CDX25R subline was thus considered to be a suitable model for prostate cancer that has developed resistance to first-line hormonal therapy but shows sensitivity to an alternative approach. Combined treatment with 5-FU and OH-flutamide had a synergistic effect on CDX25R cells. OH-flutamide decreased expression of the transcription factor E2F1, and subsequently of thymidylate synthase (TS), in CDX25R cells but not in AR-negative DU145 cells. This suggested that OH-flutamide enhanced the growth-inhibitory activity of 5-FU in CDX25R cells by reducing TS expression through the AR pathway. Combined therapy with 5-FU and OH-flutamide may, therefore, be appropriate for patients with prostate cancer that has acquired resistance to initial hormone therapy including bicalutamide.