Takehiko Tode

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.

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.

Endometriotic Tissues Produce Immunosuppressive Factors

In order to study the possible role of the immune functions in the pathogenesis of endometriosis, effects of supernatants from endometrial and endometriotic tissue cultures on the natural killer (NK) activity were examined by determining the lymphocyte cytotoxicity toward an erythroleukemic cell line (K562). Endometrial and endometriotic tissues were obtained at the time of operation from patients with ectopic endometriosis or uterine myoma. The normal peritoneum and myoma tissues were used as controls. The supernatants obtained by culturing the ectopic endometriotic tissues for 24-48 h had significant (p < 0.01) suppressive effects on NK activity of peripheral blood lymphocytes (PBL) from a healthy woman, compared to the supernatants from the normal peritoneum and the myoma tissues. There was no significant difference between NK activities of PBL from patients with ectopic endometriosis and age-matched healthy women. However, the NK activity in patients with low NK status before surgery was significantly elevated after surgery while that in patients with normal NK status before surgery was not changed. Although PBL from the same healthy women could significantly lyse the endometrial cells, the ability was about-one-third the level at which they could lyse K562 cells. These results suggest that the immunosuppressive factors secreted by endometriotic tissues may be involved in the local implantation and development of ectopic endometriosis.

Altered Expression of γ-Glutamylcysteine Synthetase, Metallothionein and Topoisomerase I or II during Acquisition of Drug Resistance to Cisplatin in Human Ovarian Cancer Cells

This study was designed to elucidate the mechanisms of cisplatin (CDDP) resistance using two human ovarian cancer cell lines, KF and TYK, and two CDDP‐resistant lines, KFr and TYK/R, derived from the former lines. KFr and TYK/R showed about 3‐fold higher resistance to the cytotoxic effects of CDDP than their parental lines. They also showed a significant increase in sensitivity to not only etoposide, but also (+)‐(4S)‐4, ll‐diethyl‐4‐hydroxy‐9‐[(4‐piperidino‐piperidino)carbonyloxy]‐lH‐pyrano[3′,4′:6,7]inodolizino[l,2‐b]quinoline‐3,14(4H, 12H)‐dione hydrochloride trihydrate (CPT‐11). Cellular CDDP accumulation levels in KFr and TYK/R were decreased from those of the parental cells. By contrast, the cellular glutathione (GSH) content in KFr cells was 1.7‐fold higher than that in KF, whereas TYK/R cells had a 40% lower content than TYK cells. Cellular mRNA levels of drug‐resistance‐related genes, such as DNA topoisomerase (topo) I and topo II, glutathione S‐transferase‐π (GST‐π;), γ‐glutamylcysteine synthetase (.γ‐GCS), and metallothionein (hMT) genes, were compared between drug‐sensitive KF or TYK and KFr or TYK/R. KFr cells had 8.5‐ and 24.7‐fold higher mRNA levels of γ‐GCS and topo II genes than KF cells while KFr had only a slight increase in GST‐π mRNA level as compared with KF. By contrast, TYK/R cells had 2.9‐ and 1.7‐fold higher hMT and topo I mRNA levels than TYK cells. Acquisition of CDDP resistance in human ovarian cancer cells thus appeared to be related mainly to expression of γ‐GCS, topo II and hMT genes, and partly to that of topo I and GST‐π genes, in addition to a decrease in CDDP accumulation

Clinical Usefulness of Korean Red Ginseng in Postmenopausal Women with Severe Climacteric Disturbance

The aim of this study is to evaluate clinical usefulness of Korean red ginseng (RG) on various postmenopausal syndromes. Total plasminogen inhibitor-1 (tPAI-1) in peripheral blood from 9 postmenopausal women with climacteric syndromes (CS) was measured before and 3 months after treatment with daily oral administrat i on of 6g RG and that from 8 postmenopausal women without any CS was also measured as healthy controls. Blood samples were collected in the early morning on the bed-rest. Psychological conditions of postmenopausal women with CS were measured before and 3 months after treatment with RG using simplified menopausal index (SMI). In addition, OKETSU (blood stagnation) syndrome scores and KI deficiency (generalized energy stagnation) scores proposed by Terasawa et al., were recorded before and 3months after treatment with RG in postmenopausal women with CS and in healthy postmenopausal women. OKETSU syndrome scores and tPAI-1 levels in postmenopausal patients with CS were significantly (P P<0.01) higher than those in healthy postmenopausal women without CS. Similarly, SMI scores and KI deficiency scores in postmenopausal patients with CS were about three-fold higher than those without any CS. When RG was administered for 3 months, KI deficiency scores and OKETSU scores as well as SMI scores declined around the levels of healthy postmenopausal women. Although tPAI-1 levels significantly (P<0.05) decreased after treatment with RG, those did not reach the levels of healthy postmenopausal women. Clinical usefulness of administration of RG to postmenopausal women with CS was confirmed from evaluation not only by modern medicine but also by traditional KAMPO medicine.

Growth-inhibitory effects of N,N-diethyl-2-[4-(phenylmethyl)phenoxy]-ethanamine-HCl combined with cisplatin on human ovarian cancer cells inoculated into nude mice.

In 5‐day incubation of an estrogen receptor‐negative human ovarian cancer cell line (KF) with diethyl‐2‐[4‐(phenylmethyl)phenoxy]ethanamine‐HCl (DPPE), the concentration of DPPE required for 50% inhibition of KF cell proliferation (IC50) was 1.7 μM. The IC50 of DPPE for inhibition of protein kinase C (PKC) activity was 3.0 μM, a similar value to those of other antiestrogens such as tamoxifen and clomiphene. DPPE also inhibited phosphorylation of mitogen‐activated protein kinase in KF cells. When treatment with DPPE was started 7 days after inoculation of KF cells into nude mice, 50 mg/kg DPPE alone resulted in a significant growth retardation in the early stage of tumor growth. Although 25 mg/kg DPPE showed a similar effect to 2 mg/kg cisplatin (CDDP), the combination had the most marked tumor growth‐inhibitory effect. Nude mice treated with combinations of CDDP and DPPE survived significantly longer than not only untreated, but also CDDP‐alone‐treated mice, while 50 mg/kg but not 25 mg/kg DPPE alone had an effect comparable to that of 2 mg/kg CDDP alone. If treatment with DPPE was begun from the day after tumor inoculation, the inhibitory effect of DPPE was further enhanced, especially when combined with CDDP. If treatment with DPPE was started in nude mice with a lower tumor burden, 25 mg/kg as well as 50 mg/kg DPPE had a similar effect to 2 mg/kg CDDP, in terms of survival. When DPPE was combined with CDDP, the effect was significantly enhanced, compared to that of either alone. These treatments could be done without any adverse side effect. Thus, we conclude that DPPE has an antiestrogen action and its tumor growth‐inhibiting activity is enhanced on administration in combination with CDDP.