Ichiro Nagata

Inhibitory effects of ginsenoside Rh2 on tumor growth in nude mice bearing human ovarian cancer cells

Ginsenoside Rh2 (Rh2), isolated from an ethanol extract of the processed root of Panax ginseng CA Meyer, inhibits the growth of B16 melanoma cells. This study was designed to evaluate the ability of Rh2 to inhibit growth of human ovarian cancer cells (HRA) in vitro and in nude mouse. Rh2 inhibited proliferations of various established human ovarian cancer cell lines in a dose‐dependent manner between 10 and 60 μM in vitro and induced apoptosis at around the IC50 dose. When HRA cells were inoculated s.c. into the right flank of nude mice, all mice formed a palpable tumor within 14 days. Although i.p. administration of Rh2 alone hardly inhibited the tumor growth, when Rh2 was combined with cis‐diamminedichloroplatinum(II) (CDDP) the tumor growth was significantly inhibited, compared to treatment with CDDP alone. When mice were treated p.o. with Rh2 daily (but not weekly), the tumor growth was significantly (P<0.01) inhibited, compared to CDDP treatment alone. When Rh2 was combined with CDDP, the degree of tumor growth retardation was not potentiated. The survival time was significantly (P<, we examined whether p.o. administration of Rh2 has a dose‐dependent inhibitory effect on the tumor growth. I.p. and weekly administration of CDDP had more potent antitumor activity in the order of 1 mg/kg, 2 mg/kg and 4 mg/kg, whereas p.o. and daily administration of Rh2 (0.4 to 1.6 mg/kg) not only had antitumor activity comparable to that of 4 mg/kg CDDP, but also resulted in a significant increase of the survival. Doses of Rh2 used in this study did not result in any adverse side‐effects as confirmed by monitoring hematocrit values and body weight, unlike 4 mg/kg CDDP, which had severe side‐effects. It is noteworthy that p.o. but not i.p. treatment with Rh2 resulted in induction of apoptotic cells in the tumor in addition to augmentation of the natural killer activity in spleen cells from tumor‐bearing nude mice. Thus, particularly in view of the toxicity of CDDP, Rh2 alone would seem to warrant further evaluation for treatment of recurrent or refractory ovarian tumor.

Calcium-regulating hormones and osteocalcin levels during pregnancy: A longitudinal study

We measured serum concentrations of calcium-regulating hormones and osteocalcin in 20 women longitudinally throughout pregnancy, 1,25-Dihydroxyvitamin D levels were high early in pregnancy and increased with advancing gestation. Parathyroid hormone and osteocalcin levels were low in early pregnancy. They declined toward the middle of pregnancy, but increased in late pregnancy. The serum osteocalcin level correlated with the parathyroid hormone level. The synthesis of osteocalcin by osteoblasts is stimulated by the action of 1,25-dihydroxyvitamin D, and serum osteocalcin levels are also related to the levels of parathyroid hormone. During early and mid pregnancy, the stimulatory effect of 1,25-dihydroxyvitamin D on the synthesis of osteocalcin may be overridden by the inhibitory effect of declining parathyroid hormone levels. The increase in osteocalcin level in late pregnancy may be a consequence of increasing levels of both parathyroid hormone and 1,25-dihydroxyvitamin D.

Inhibition of human ovarian cancer cell proliferation in vitro by ginsenoside Rh2 and adjuvant effects to cisplatin in vivo.

In vitro and in vivo effects of ginsenoside Rh2 on human ovarian tumor growth were examined by using a cell line (HRA) derived from ascites of a patient with serous cystadenocarcinoma of the ovary. The HRA cell proliferation in vitro was inhibited in a dose-dependent manner with dosages of 10–100 μM of ginsenoside RH2. DNA, RNA and protein synthesis by the HRA cells was inhibited in a dose-dependent manner at more than 15 μM of ginsenoside RH2. However, the growth of HRA cells transplanted in nude mice was not significantly inhibited by ginsenoside RH2. On the contrary, when cisplatin was administered together with 10 μM (but not 1 μM or 100 μM) ginsenoside RH2, the tumor growth was significantly inhibited 31 days after inoculation and the survival was also significantly prolonged, compared with not only the untreated group but also the groups given cisplatin alone or ginsenoside RH, alone. This indicates synergistic effects between cisplatin and ginsenoside RH2. From monitoring of body weight and hematocrit, concentrations of ginsenoside RH2 used in this study did not seem to cause any adverse effect.

Effect of Korean red ginseng on psychological functions in patients with severe climacteric syndromes

Objective: To evaluate the degree of psychological dysfunction and levels of stress hormones in postmenopausal women with climacteric syndromes and effect of Korean red ginseng (RG) on them. Methods: ACTH, cortisol and DHEA‐S in peripheral blood from 12 postmenopausal women with climacteric syndromes or 8 postmenopausal women without any climacteric syndrome were measured before and 30 days after treatment with daily oral administration of 6 g RG. Blood samples were collected in the early morning on the bed‐rest. In postmenopausal women with climacteric syndromes such as fatigue, insomnia and depression, psychological tests using the Cornell Medical Index (CMI) and the State‐Trait Anxiety Inventory (STAI) were performed before and 30 days after treatment with RG. Results: CMI score as well as anxiety (A)‐state in STAI score in postmenopausal women with climacteric syndromes was significantly higher than that without climacteric syndrome, while DHEA‐S levels in postmenopausal women with climacteric syndromes were about a half of those without climacteric syndrome. Consequently, cortisol/DHEA‐S (C/D) ratio was significantly higher in postmenopausal women with climacteric syndromes than in those without climacteric syndrome. When postmenopausal women with climacteric syndromes were treated with daily oral administration of 6 g RG for 30 days, CMI and STAI A‐state scores decreased within normal range. Although the decreased DHEA‐S levels were not restored to the levels in postmenopausal women without climacteric syndrome, the C/D ratio decreased significantly after treatment with RG. Conclusions: Improvement of CMI and STAI scores in postmenopausal women suffering climacteric syndromes, particularly fatigue, insomnia and depression, by RG seemed to be brought about in part by effects of RG on stress‐related hormones as shown by a decrease in C/D ratio.

Bcl-2 as a Predictor of Chemosensitivity and Prognosis in Primary Epithelial Ovarian Cancer

This retrospective study of ovarian cancer aimed to elucidate whether expression of apoptosis-related proteins, bcl-2, p53 or MDM-2, is associated with resistance to chemotherapy, especially cisplatin (CDDP) based chemotherapy. Expression of bcl-2, p53 and MDM-2 was assessed by immunohistochemical staining of tumour tissues collected at initial surgery prior to treatment with CDDP-based chemotherapy. Among 66 patients with advanced ovarian cancer with measurable tumour following surgery and evaluable for response to chemotherapy, 42, 45 and 56% were positive for bcl-2, p53 and MDM-2, respectively. Significantly fewer tumours of patients who had a complete response to chemotherapy (CR) showed positivity for bcl-2 (2/20) than for p53 (6/20) and MDM-2 (8/20, P < 0.001). There was an inverse correlation between bcl-2 staining and initial response to chemotherapy, especially in serous and endometrial adenocarcinomas. In patients with stage III-IV, serous or endometrioid adenocarcinomas, significantly poorer survival was seen for those with bcl-2 positive tumours than those with negative bcl-2 staining (P = 0.0064). p53 and MDM-2 were not correlated with initial response to chemotherapy. Multivariate analysis revealed that bcl-2, residual tumour size and histology were significant independent prognostic factors. These results suggest that bcl-2 can be a possible predictor of response to chemotherapy and prognosis in patients with advanced ovarian carcinoma.

Inhibitory effects by oral administration of ginsenoside Rh2 on the growth of human ovarian cancer cells in nude mice

Recently two new compounds, ginsenosides Rh1 and Rh2, have been isolated from an ethanol extract of the processed root ofPanax ginseng CA Meyer, and Rh2 (but not Rh1) has been found to cause growth inhibition of cultured B16 melanoma cells. We have also demonstrated that Rh2 caused inhibition of cultured human ovarian cancer cell (HRA) proliferation. The effect of oral administration of Rh2 on tumor growth and survival of nude mice bearing HRA cells was examined. Nude mice were inoculated subcutaneously in the right flank with 106 HRA cells. After 7 days of tumor inoculation 2 mg/kgcis-diamminedichloroplatinum(II) (cisplatin) was administered intraperitoneally once a week for 5 weeks. In Rh2-treated groups. Rh2 was dissolved in absolute ethanol, adjusted with distilled water to 1, 15, and 120 μM, and 0.4 ml of each concentration was administered orally by canula every day for 90 days, from the next day of tumor inoculation. The tumor volume, hematocrit and body weight were measured every week. On days 56 and 63 after tumor inoculation, the tumor volumes in all groups treated with Rh2 were significantly less than those in an ethanol-treated control group and also in cisplatin treated group. After 70 days, the tumor growth in nude mice treated with 15 μM and 120 μM Rh2 was significantly inhibited compared to that in a cisplatin treated group as well as a control group. Consequently, the survival of nude mice treated with 15 μM and 120 μM Rh2 was also significantly prolonged, compared to that of cisplatin treated mice. No toxic effects were observed in any of the mice.

Preoperative determination of several serum tumor markers in patients with primary epithelial ovarian carcinoma.

This study was designed to evaluate the clinical significance of the use of preoperative serum tumor markers in primary epithelial ovarian cancer. Subjects comprised 111 patients with primary epithelial ovarian cancer. Lactate dehydrogenase (LDH), α-hydroxybutyrate dehydrogenase (HBDH), carcinoembryonic antigen (CEA), CA19-9, tissue polypeptide antigen (TPA), CA125 and sialyl TN (STN) serum levels were measured within 7 days before surgery. The tumor marker values were compared with the histopathologic diagnosis. The overall agreement between the test results and the actual outcome was calculated using Student’s t test and analysis of variance (ANOVA). Survival curves were constructed according to the Kaplan-Meier method, and differences in survival were assessed with the log-rank test. The prognostic significance of tumor markers for survival was assessed in a multivariate analysis with the Cox proportional hazards model. Of the tumor markers examined in this study, CA125 showed the highest positive rate (77.6%), followed by 63.2% for STN and 55.9% for CA19-9. When the positive rate was compared according to histologic types, serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioid adenocarcinoma and clear cell carcinoma showed the highest positive rates for CA125 (94.1%), CA19-9 (76.9%), CA125 (91.7%) and STN (75.0%), respectively. Regarding the distribution of tumor marker levels according to the FIGO stage, LDH, HBDH, TPA and CA125 were correlated with the clinical stage while CEA, CA19-9 and STN did not show any correlation. From analyses of tumor marker levels according to histologic types, all patients with a ratio of CA125 to CEA of >1,000 had serous cystadenocarcinoma and a ratio of CA125 to CA19-9 of >50 showed serous cystadenocarcinoma or endometrioid adenocarcinoma. On the other hand, all patients with a ratio of LDH or HBDH to CA19-9 of <1.0 had mucinous cystadenocarcinoma or clear cell carcinoma. From univariate analysis, the survival time of patients with elevated CA125, TPA or STN was significantly shorter than that of patients with normal CA125, TPA or STN levels. When the Cox’s proportional hazard model was used, we identified age, clinical stage, clear cell carcinoma and serum STN as independent prognostic factors. Serum CA125, TPA or STN may give significant prognostic information in epithelial ovarian carcinoma. It is noteworthy that STN has been identified as an independent prognostic factor and has a high rate of positivity in clear cell carcinoma.

Inactivation of p16/CDKN2 and p15/MTS2 is associated with prognosis and response to chemotherapy in ovarian cancer

To define the involvement of p16/CDKN2 and p15/MTS2 tumor‐suppressor genes for response to chemotherapy in primary epithelial ovarian cancer, we analyzed alterations of the gene in 45 patients who were treated with primary cytoreductive surgery followed by 6 courses of cis‐diamminedichloroplatinum (II) (cisplatin)‐based combination chemotherapy. Homozygous deletion of p16/CDKN2 and p15/MTS2 genes was found in 8 (18%) and 15 (33%) cases, respectively. Response to the chemotherapy was confirmed by finding at second surgery after the chemotherapy in 26 patients, resulting in 17 responders and 9 nonresponders. The abundance of gene deletion in nonresponders (56%) was significantly higher (p = 0.0463) when compared to that in responders (18%). Moreover, the deletion of genes was a significant poor prognostic factor (p = 0.0441) in advanced ovarian cancer. These results suggest that deletion of p16/CDKN2 and/or p15/MTS2 is a potential indicator for poor chemotherapy response and adverse prognosis in ovarian cancer patients.

Modulation of cisplatin sensitivity by taxol in cisplatin-sensitive and -resistant human ovarian carcinoma cell lines

Purpose: The aim of this study was to determine whether taxol can circumvent cisplatin resistance, using a KF28 cell line derived from human ovarian carcinoma and a cisplatin-resistant line, KFr13, derived from the parental cell line, KF28, and taxol-resistant cell lines, KF28TX and KFr13TX, derived from the respective parental counterpart. Methods: KF28 is a single-cell clone of the human ovarian carcinoma cell line KF. The cisplatin-resistant KFr13 subline was established by repeated exposure of the parent KF28 cell line to escalating doses of cisplatin. Similarly, KF28TX and KFr13TX were established by repeated exposure of the KF28 and KFr13 cell lines to escalating doses of taxol. A cytotoxicity assay was performed using a crystal violet staining method. Platinum and taxol accumulation were assayed by atomic absorption and reverse-phase high-performance liquid chromatography. The quantitative assay of MDR1 mRNA used polymerase chain reaction. Results: KFr13 cells were about 4.8-fold more resistant to cisplatin and about 1.8-fold more sensitive to taxol than were KF28 cells. When taxol resistance was induced in KF28 and KFr13 cells, sensitivity to cisplatin rose about 1.3- and 1.6-fold respectively. Elevation of sensitivity was correlated with platinum uptake by both KF28TX and KFr13TX cells. Expression of multidrug resistance (MDR1) mRNA, which was not observed in KF28 and KFr13 cells, was observed after induction of taxol resistance. Conclusions: These results may suggest rational therapeutic strategies for patients with cisplatin-resistant or refractory ovarian carcinoma.

Leiomyosarcoma of the Uterus: Ultrasonography and Serum Lactate Dehydrogenase Level

Between January 1, 1979, and September 30, 1990, a total of 1,886 patients in the National Defense Medical College Hospital, a self-referred population, had a hysterectomy because of signs and symptoms presumably resulting from uterine myomas. After hysterectomy with presumed benign disease, a histologic diagnosis of leiomyosarcoma was made in 7 patients (0.37%). Preoperative diagnosis of leiomyosarcoma was not made in any of the 7 patients. However, serum lactate dehydrogenase levels were abnormally elevated in 3 of them, and degenerative changes were found within the tumor by ultrasonography in 5 of them. Furthermore, increased lactate dehydrogenase levels and degenerative changes within the tumor were found in 3 of the patients whose tumors had 10 or more mitoses per 10 high-power fields. The prognosis for the leiomyosarcomas with increased mitotic rates is very poor. Therefore, a degenerative change within the uterine mass and an increased lactate dehydrogenase level, when present, should suggest the diagnosis of leiomyosarcoma.