Kazushige Kiguchi

Therapeutic strategy targeting the mTOR-HIF-1α-VEGF pathway in ovarian clear cell adenocarcinoma

Malignant tumors usually involve a relatively hypoxic state, which induces overexpression of hypoxia‐inducible factor‐1α (HIF‐1α) to satisfactorily enable the tumor to survive. Thus, inhibition of the mammalian target of rapamycin (mTOR) pathway including HIF‐1α is expected to play a major role in suppression of tumor cell growth, having recently drawn much attention as an anti‐cancer therapeutic strategy for various malignant tumors. In the present study, which compared clear cell adenocarcinoma (CLA) of the ovary with serous adenocarcinoma (SEA), the immunohistochemical expression of mTOR, phosphorylated‐mTOR (p‐mTOR), HIF‐1α, and vascular endothelial growth factor (VEGF) was examined in surgically resected specimens of 29 SEA and 47 CLA. There were no significant differences in expression of mTOR, HIF‐1α and VEGF between SEA and CLA, but it was noted that p‐mTOR expression was more prominent in CLA than SEA. Then, using the cell lines of CLA (RMG‐1 and W3uF), an experimental study was designed to clarify whether tumor suppression due to downregulation of mTOR activity could represent a promising therapeutic strategy for CLA. After treatment of an analogue of rapamycin (everolimus), expression of mTOR, p‐mTOR, HIF‐1α and VEGF was examined on western blot. As a result, although mTOR expression remained unchangeable, expression of p‐mTOR, HIF‐1α and VEGF was shown to be sharply depressed. The same expression alterations were demonstrated in the xenograft model treated with everolimus. In conclusion, mTOR‐targeted therapy through usage of drugs such as everolimus may be more effective for CLA of the ovary because of its significant expression of p‐mTOR.

Alterations in the glycolipid composition and cellular properties of ovarian carcinoma-derived RMG-1 cells on transfection of the α1,2-fucosyltransferase gene

Transfection of the mouse Fut1 and Fut2, and human FUT1 genes into human ovarian carcinoma‐derived RMG‐1 cells resulted in 20–30‐fold increases in cellular α1,2‐fucosyltransferase activity, and in alteration of the glycolipid composition, including not only fucosylated products, but also precursor glycolipids. Although globo‐series glycolipids were not significantly affected by the transfection, the major glycolipids belonging to the lacto‐series type 1 chain family in RMG‐1 cells and the transfectants were the Lc4Cer, Lewis a (Le)a and Leb, and H‐1 glycolipids, respectively, suggesting that fucosylation of Lc4Cer to the H‐1 glycolipid prevents the further modification of Lc4Cer to Lea and Leb in the transfectants. Also, the lacto‐series type 2 chains in RMG‐1 cells were LeX, NeuAc‐nLc4Cer and NeuAc‐LeX, and those in the transfectants were LeX and LeY, indicating that the sialylation of nLc4Cer and LeX is restricted by increased fucosylation of LeX. As a result, the amount of sialic acid released by sialidase from the transfectants decreased to 70% of that from RMG‐1 cells, and several membrane‐mediated phenomena, such as the cell‐to‐cell interaction between cancer cells and mesothelial cells, and the cell viability in the presence of an anticancer drug, 5‐fluorouracil, for the transfectants was found to be increased in comparison to that for RMG‐1 cells. These findings indicate that cell surface carbohydrates are involved in the biological properties, including cell‐to‐cell adhesion and drug resistance, of cancer cells. (Cancer Sci 2005; 96: 26 –31)

A patient-like orthotopic implantation nude mouse model of highly metastatic human ovarian cancer.

Clinically relevant animal models of human cancer are important for studies of cancer biology, invasion and metastasis, and for investigating new forms of prognostic diagnosis and therapy. An ovarian tumor line (RMG-1: human clear cell carcinoma of the ovary) previously grown subcutaneously was implanted ortho-topically as intact tissue into the ovarian capsule of 22 nude mice. The tumors showed progressive growth at the orthotopic site in all animals. Tumor-associated serum galactosyltransferase (GAT) tended to be posi-tive in all nude -mice. The tumors invaded or metastasized to the contralateral ovary, retroperitoneum, mesentery and peritoneum, and omentum, and metastasized to the subcutaneous tissue, lymph nodes and distant organs including the liver, kidney, pancreas, and diaphragm. In striking contrast, subcutaneous trans-plantation of this tumor resulted in growth in only 2 of 5 animals with local lymph node and kidney involve-ment but no retroperitoneal or peritoneal involvement. These findings suggest that orthotopic implantation provides a suitable micro-environment in which ovarian cancer can express its intrinsic clinically-relevant properties. This approach is relevant to the clinical features of ovarian cancer and is thought to be a useful model for studies of therapy for this cancer.© Kluwer Academic Publishers 1998

Melatonin binding sites in estrogen receptor‐positive cells derived from human endometrial cancer

Abstract: Our previous work showed that melatonin (N‐acetyl‐5‐methoxytryptamine) inhibits proliferation of the human endometrial cancer cell line, Ishikawa, which is estrogen receptor‐positive. The aim of the present study was to determine whether Ishikawa cells possess membrane melatonin receptors. Binding of the radioligand 2‐[125I]‐iodomelatonin to membrane preparations obtained from Ishikawa cells was detectable, saturable and stable. Scatchard analysis revealed that the dissociation constant (Kd) of the binding sites was 179.0 pm (similar to that of the MT2 [Mel1b] melatonin receptor subtype), and that the concentration (Bmax) of the binding sites was 12.9 fmol/mg protein. Luzindole, a selective MT2 melatonin receptor antagonist, significantly suppressed binding of 2‐[125I]‐iodomelatonin at all concentrations tested (10−8 to 10−4 m). These results suggest that the MT2 melatonin receptor subtype is present in the membranes of Ishikawa cells, and that the antiproliferative effect of melatonin on Ishikawa cells is mediated via the MT2 receptor. This may have implications for the use of melatonin in endometrial cancer therapy.

Side population is increased in paclitaxel-resistant ovarian cancer cell lines regardless of resistance to cisplatin

OBJECTIVES In recent years, cancer stem cells (CSCs) have been reported to be correlated with chemoresistance and may also be enriched in side populations (SPs). In this study, the relationship between resistance to paclitaxel (PTX) and cisplatin (CDDP) and side populations was examined in three parental PTX- and CDDP-sensitive ovarian cancer cell lines (2008, KF28, and TU-OM-1) and several other cell lines derived from these as well as the additional effects of interferon-alpha (INF-α). METHODS SP of three different parental cell lines and PTX- and/or CDDP-resistant cell lines derived from these was analyzed with flow cytometry. The expression of ABCB1 and ABCG2 in KF28 and its derived cell lines was examined. Additional cell-death effect of INF-α with PTX was also examined. RESULTS In the three parental cell lines and the PTX-sensitive cell lines derived from these lines, SP was very low. Conversely, in PTX-resistant cell lines, regardless of CDDP resistance, SP increased. ABCB1 was strongly expressed in the PTX-resistant cells, but not in their parental lines, which are sensitive to PTX. While INF-α showed only slight enhancement of the cell-death effect of PTX in PTX-sensitive cells, INF-α itself strongly induced apoptosis in PTX-resistant cells regardless of PTX concentration. CONCLUSIONS The SP could be correlated with resistance to PTX. SP could be a target of INF-α, and resistance to PTX might be overcome by INF-α.

Characteristic expression of globotriaosyl ceramide in human ovarian carcinoma‐derived cells with anticancer drug resistance

The transporter protein genes and lipids in human ovarian carcinoma‐derived KF28 cells with anticancer‐drug‐sensitive properties were compared with those in resistant cells, taxol‐resistant KF28TX, cisplatin‐resistant KFr13, and taxol‐ and cisplatin‐resistant KFr13TX, to identify the molecules required for anticancer‐drug resistance. In accordance with previous reports, taxol and cisplatin resistance was closely correlated with expression of the multidrug resistance 1 and bile acid export pump, and multidrug resistance‐associated protein 2 genes, respectively. In addition, we found a distinct difference in glycosphingolipids between the sensitive and resistant cells. Although GlcCer was the major glycolipid (83.0%) in sensitive cells, GalCer, LacCer and, particularly, Gb3Cer were characteristically increased in all resistant cells, irrespective of whether the resistance was to taxol or cisplatin, and comprised 65–84% of total glycosphingolipids. GM3, which was present at 0.04 µg/mg dry weight in the sensitive cells, showed a twofold increase in the taxol‐resistant cells, but was absent in the cisplatin‐resistant cells. The altered glycolipid composition was proven to be due to enhanced or suppressed expression of the respective sugar transferase genes. In addition, the ceramide moiety of ceramide monohexoside in the sensitive cells constituted 83% of non‐hydroxy fatty acids, but that in the resistant cells comprised 67–74% of α‐hydroxy fatty acids. Thus, cells containing Gb3Cer with α‐hydroxy fatty acids were found to survive selectively in the presence of taxol and cisplatin, and modification of the glycolipid structure was revealed to occur in association with anticancer‐drug resistance. (Cancer Sci 2006; 97: 1321–1326)

Establishment and characterization of a human malignant choroids plexus papilloma cell line (HIBCPP)

A cell line designated “HIBSPP” was established from a human malignant choroids plexus papilloma of 29-year-old Japanese woman. This line grew well without interruption for 3 years and was subcultivated over 70 times. The cells were spindle, oval, and polygonal in shape, and neoplastic and pleomorphic features, a jigsaw puzzle-like arrangement, multilayering and forming papillary shuctures without contact inhibition. The cells proliferated slowly, and the population doubling time was about 69 hours. The chromosome number showed a wide distribution of aneuploidy. The mode was in the hypo-tetraploid range, and many marker chromosomes were observed. The culture cells were easily transplanted into the subcutis of nude mice and produced the tumor resembling the original tumor.

Phase II study of neoadjuvant chemotherapy with irinotecan hydrochloride and nedaplatin followed by radical hysterectomy for bulky stage Ib2 to IIb, cervical squamous cell carcinoma: Japanese Gynecologic Oncology Group study (JGOG 1065).

The efficacy and adverse events of neoadjuvant chemotherapy with irinotecan hydrochloride and nedaplatin were evaluated in patients with bulky stage Ib2 to IIb cervical squamous cell carcinoma. Eligibility included patients who received irinotecan (60 mg/m2) on days 1 and 8 and nedaplatin (80 mg/m2) on day 1 of a 21-day cycle. After 1-3 courses of chemotherapy, radical hysterectomy was performed. Sixty-eight patients were enrolled. Sixty-six were included in the full analysis set. Their median age was 47 years (range 22-71), the FIGO stage was Ib2 in 18 patients, IIa in 10, and IIb in 38. Radical hysterectomy was performed after NAC in 63 patients (95.5%). The number of administered courses of NAC was 1 in 13 patients, 2 in 43, and 3 in 10. The response rate, the primary endpoint of this study, was 75.8% (CR in 2 patients, PR in 48, SD in 12, PD in 0, and NE in 4). The mean number of treatment courses required for a response was 1.42 (1 course in 30 patients, 2 courses in 19, and 3 courses in 1). The incidences of grade 3 or 4 hematological toxicities were: neutropenia 72.2%, leukopenia 16.7%, anemia 13.6%, thrombocytopenia 7.6%, febrile neutropenia 1.5%, and elevations of alanine aminotransferase and aspartate aminotransferase 1.5%. Grade 3 or 4 non-hematologic toxicities were as follows: diarrhea 6.1%, nausea 3%, anorexia 1.5%, vomiting 1.5%, fever 1.5%, allergic reactions 1.5%, ileus 1.5% and vesicovaginal fistula 1.5%. Neoadjuvant chemotherapy with irinotecan and nedaplatin was an effective and well-tolerated treatment for patients with bulky stage Ib2 to IIb squamous cell carcinoma of the uterine cervix.

Novel Glycobiomarker for Ovarian Cancer That Detects Clear Cell Carcinoma

Epithelial ovarian cancer (EOC) is often asymptomatic and thus diagnosed at advanced stages with a poor prognosis. False-negative results for the conventional marker CA125 frequently occur in cases of clear cell carcinoma (CCC), a type of EOC; therefore, it is necessary to develop biomarkers with greater sensitivity. We previously reported a strategy to discover glycobiomarker candidates by combined lectin microarray and IGOT-LC/MS analysis. We have now optimized this strategy for discovering EOC biomarkers. Glycopeptides possessing cancerous glycans were enriched from the ascites fluids and culture supernatants of cancer cell lines with a fucose-binding lectin, AAL. IGOT-LC/MS analysis of CCC samples yielded 144 candidate glycoproteins. We selected WFA by lectin microarray as the optimal lectin to distinguish EOC from gastric and colon cancer. The candidates were narrowed by Western analysis of the WFA-bound fraction of ascites fluids. One of the final candidates, WFA-reactive ceruloplasmin, produced higher signals in the ascites fluids of EOC patients, including CCC, in comparison with the benign samples, while CA125 levels were comparable in the sandwich ELISA. Thus, our glycoproteomic strategy featuring efficient enrichment of glycans with disease-related alterations is applicable to various diseases.

Estrogen sulfotransferase and sulfatase: Roles in the regulation of estrogen activity in human uterine endometrial carcinomas

The regulation of estrogen activity through the formation and cleavage of sulfoconjugates of estrogens is known to be related to the progression and metastasis of estrogen‐dependent breast carcinomas, but the involvement of sulfoconjugates in the steroid stimulation of endometrial functions and the progression of endometrial adenocarcinomas is not clearly understood yet. Estrogen sulfotransferase (EST) in the uterine endometria during the follicular phase was more active than during the luteal phase, but estrogen sulfate (ES) sulfatase exhibited lower activity during the follicular phase than during the luteal phase. However, ES sulfatase activities in cancerous tissues were lower than those in normal endometria and endometrial adenocarcinoma‐derived cells, among which the activity was exceedingly high in Ishikawa cells, suggesting that ES sulfatase in Ishikawa cells contributes to the estrogen‐dependent growth of these cells. EST activities higher than that in Ishikawa cells were found in only 3 of 24 cancerous tissues. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis of the EST and ES sulfatase genes in carcinoma‐derived cells demonstrated the extensive expression of both genes in Ishikawa cells. The isolated EST gene was transfected into Ishikawa cells with a mammalian expression vector to establish cell clones with enhanced EST activity, and the estrogen‐dependent cell growth of the resultant cell clones was found to be abolished, due to the enhanced sulfoconjugation of estrogen. Since ES sulfatase activity in cancerous tissues was significantly lower than that in Ishikawa cells, it might be not involved in the enhancement of estrogen activity associated with the pathogenesis of endometrial adenocarcinoma tissues.