Adjuvant effects of calmodulin antagonists on antitumor activity of 1-(2-tetrahydrofuryl)-5-fluorouracil (ftorafur) and a mixture of ftorafur and uracil (UFT) in nude mice bearing human ovarian carcinomas.

Effects of N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5) and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on antitumor activity of 1-(2-tetrahydrofuryl)-5-fluorouracil (ftorafur, FT) and a mixture of FT and uracil (UFT) were determined in nude mice bearing human ovarian carcinoma (KF cells). All nude mice developed palpable tumor 17 days after KF cell inoculation unless treated. A combination of UFT and calmodulin antagonists (W-5 and W-7) resulted in a delay in tumor formation in nude mice. One of 10 nude mice treated with a combination of UFT and W-5 and 3 of 10 nude mice treated with UFT and W-7 did not develop tumors during the experimental period. Tumor volumes in groups treated with the combinations of UFT and calmodulin antagonists were significantly smaller than those in the untreated group after 31 days of tumor inoculation. In addition, tumor volumes in a group treated with UFT plus W-7 were significantly smaller than not only those in the untreated group but also those in groups treated with UFT alone, FT alone, W-5 alone, or W-7 alone. The longest median survival time was also observed in the group treated with UFT plus W-7. 5-Fluorouracil (5-FU) content of the tumors of groups treated with FT plus W-5 or W-7 was similar to that of the group treated with FT alone. On the other hand, 5-FU content of a group treated with UFT plus W-7 was about twofold higher than that of a group treated with UFT alone. These results suggest that a synergistic effect of W-7 and UFT on inhibition of tumor growth and prolongation of survival time may result from accumulation of 5-FU in the tumor.

Effects of lymphokine-activated killer cells and interleukin-2 on the ascites formation and the survival time of nude mice bearing human ovarian cancer cells.

The effect of intraperitoneal instillations of interleukin-2 (IL-2) and/or lymphokine-activated killer (LAK) cells on the ascites formation and the survival time was examined using nude mice as a model, with malignant ascites produced by intraperitoneal inoculation of human ovarian cancer cells derived from ascites of a patient with serous cystadenocarcinoma of the ovary. Twenty-eight days after tumor inoculation, all nude mice in the untreated group and in the group treated with spleen cells alone formed ascites. Two of ten nude mice treated with IL-2 alone after tumor inoculation survived without forming ascites during the experimental period. On the other hand, all nude mice treated with LAK cells alone had formed ascites 14 days after tumor inoculation. When LAK cells and IL-2 were combined, five of ten mice survived without forming ascites during the experimental period. The survival time of the group treated with IL-2 alone was significantly prolonged compared to the groups that received medium alone, spleen cells alone and LAK cells alone. When administration of LAK cells was followed by IL-2, the survival time was further prolonged.

Prediction of clinical course and recurrence of human ovarian cancer by several serum tumor markers.

Measurement of serum tumor markers [lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (HBD), LDH-4, erythrocyte sedimentation rate (ESR), carcinoembryonic antigen (CEA), beta 2-microglobulin (beta 2M) and CA 125] was done before and after operation, and during the course of chemotherapy in 43 patients with advanced primary ovarian cancer. Pre-operative positive results for these serum tumor markers were 94.4% for CA 125, 62.2% for beta 2M, 54.8% for HBD, 51.3% for ESR and 46.5% for LDH, respectively. In a group of patients from whom most of the tumor mass had been removed, LDH, LDH-4 and HBD significantly declined after the operation, whereas in a group of patients with a large residual tumor after operation no significant change in any of the serum tumor markers examined was observed after operation. Except for CEA, all serum tumor markers in patients with complete response to chemotherapy significantly decreased after 3 courses of chemotherapy. From the analysis of predictive values for the recurrence of ovarian cancer, the most reliable tumor markers as a single marker appeared to be CA 125 and CEA, followed by ESR, LDH and LDH-4. However, in about 10% of cases, abnormal levels of CA 125 or CEA before treatment returned to the normal range after treatment and an increase in the other tumor markers was observed with a relapse of disease in the absence of an increase in CA 125 or CEA. These results suggest that in addition to such tumor markers as CA 125 or CEA, a combination assay of several tumor markers is necessary for monitoring of treatment of ovarian cancer.

Adjuvant effects of prostaglandin D2 to cisplatin on human ovarian cancer cell growth in nude mice.

Adjuvant effects of prostaglandin D2 to cisplatin on tumor growth were studied by using nude mice bearing HR cells derived from human ovarian carcinoma. Combinations of 0.2 or 0.4 microgram/ml cisplatin and 0.05 or 0.1 microgram/ml prostaglandin D2, which did not affect the HR cell proliferation alone, resulted in a significant inhibition of cell proliferation. In addition, tumor take of HR cells by nude mice in groups treated with a combination of cisplatin and prostaglandin D2 was inhibited. Although there was no significant difference between tumor volumes in mice treated with prostaglandin D2 alone or cisplatin alone and untreated mice, when cisplatin was administered with 1 mg/kg prostaglandin D2 the tumor volume was significantly smaller on days 21 and 35, compared to that of untreated mice. When cisplatin and 2 or 4 mg/kg prostaglandin D2 were combined, the tumor growth was significantly inhibited after day 21, compared not only to that of untreated mice but also of mice treated with cisplatin alone or prostaglandin D2 alone. Such a combination therapy by cisplatin and prostaglandin D2 seemed to result in prevention by prostaglandin D2 of immunological suppression which may be induced by cisplatin. Thus, such a combination therapy brought about a significant prolongation to the survival time.

Effects of cimetidine on interleukin-2 production by peripheral blood lymphocytes in advanced ovarian carcinoma.

The present study was designed to elucidate the effect of cimetidine on CD4 and CD8 positive cells, interleukin-2 (IL-2) production and IL-2 receptor expression in peripheral blood lymphocytes (PBL) from patients with advanced ovarian carcinoma during the course of combination chemotherapy. The absolute number of CD8 (but not CD4) positive cells in PBL from patients with advanced ovarian carcinoma before surgery was significantly higher than that with benign ovarian tumor, while the IL-2 productivity was significantly lower. However, the IL-2 receptor expression was comparable to that with benign ovarian tumor. When a combination chemotherapy consisting of cisplatin, adriamycin and cyclophosphamide was given to these ovarian cancer patients, the IL-2 production was markedly depressed. If cimetidine was given with the combination chemotherapy, the inhibition of IL-2 production by chemotherapy was significantly diminished with a significant increase of CD4 positive cells. On the other hand, the IL-2 receptor expression was not affected by this treatment. When treatment with cimetidine was initiated after completion of chemotherapy, the depressed IL-2 production was restored to the level of control patients with benign ovarian tumor. The restoration seemed to result from an increase in proportion of CD4 positive cells. However, the expression of IL-2 receptor remained unchanged even if cimetidine was given.

The mechanism of acquired resistance to cisplatin by a human ovarian cancer cell line.

The present study was designed to elucidate the mechanism of resistance to cisplatin. A cisplatin-resistant cell line (KFr) was established from KF cells derived from human serous cystadenocarcinoma of the ovary. The DNA histogram revealed an increase of S-phase cells and a decrease of G1-phase cells in cultured KFr cells, compared to that in cultured KF cells. Although the cisplatin content in the KF cells incubated with cisplatin at 10 micrograms/ml increased in a time-dependent manner, that in the KFr cells remained unchanged during the experimental period. When 0.5 mg of cisplatin was administered ip to nude mice with KF or KFr tumor, the cisplatin content in the KFr tumor was significantly lower than that in the KF tumor. The KFr cells showed a cross-resistance to L-phenylalanine mustard, while no cross-resistance to vincristine or 5-fluorouracil was observed. These findings suggest that the mechanism of cisplatin resistance in the KFr cells involves a decrease of cisplatin accumulation in the tumor cells.

[Effect of calmodulin antagonists (W-5 and W-7) on antitumor activity of Tegafur and UFT (Tegafur plus uracil)].

The effect of calmodulin antagonists (W-5 and W-7) on antitumor activity of 1-(2-Tetrahydrofuryl)-5-fluorouracil (Tegafur; FT) and FT plus uracil (UFT) was examined by using nude mice bearing human ovarian carcinoma (KF cells). All nude mice developed a palpable tumor 17 days after KF cell inoculation unless treatment was done. A combination of UFT and calmodulin antagonists (W-5 or W-7) resulted in a delay in tumor formation in nude mice. One of 10 nude mice treated with a combination of UFT and W-5 and 3 of 10 nude mice treated with UFT and W-7 did not develop tumors during the experimental period. Tumor volume in the treatment groups combined with calmodulin antagonists was significantly less than in those in the untreated group after 31 days of tumor inoculation. In addition, tumor volume in a UFT plus W-7 treated group was significantly less than those not only in the untreated group but also in the UFT alone, FT alone, W-5 alone and W-7 alone treated groups, suggesting a synergistic inhibitory effect on tumor growth. The longest median survival time was also observed in a UFT plus W-7 treated group. 5-Fluorouracil (5-FU) content in the tumor of groups treated with FT plus W-5 or W-7 was similar to that in a group treated with FT alone. On the other hand, that of a group treated with UFT plus W-7 was about 2-fold higher than that of a UFT -only treated group.(ABSTRACT TRUNCATED AT 250 WORDS)

[Establishment of a cisplatin-resistant human ovarian cancer cell line and the mechanism of resistance].

A cisplatin-resistant cell line was established by using KF-1 cells derived from human serous cystadenocarcinoma of the ovary. This resistant cell line, designated "KFr", was capable of proliferating in the presence of 1.0 microgram/ml cisplatin. It had doubling times of 24.8 and 27.2 hr in the presence of 0.5 and 1.0 microgram/ml cisplatin, respectively. The morphologic characteristics of the KFr cells were an enlarged nucleus and prominent nucleoli, unlike the nucleus and nucleoli of the parent KF-1 cells. The degree of resistance to cisplatin of the KFr cells was about 20 times as great as that of the KF-1 cells, with regard to the concentrations of cisplatin required for 50% inhibition of cell proliferation. Although the cisplatin content in the KF-1 cells incubated with 10 micrograms/ml cisplatin was increased in a time-dependent manner, that in the KFr cells reached the plateau level after 1.5 hr incubation with cisplatin. After about 4 hr incubation, the cellular content in the KFr cells was about a half of that in the KF-1 cells. When 0.5 mg cisplatin was administered i.p. to nude mice with KF-1 or KFr tumor, the cisplatin content in the KFr tumor was significantly lower than that in the KF-1 tumor. The KFr cells showed a cross-resistance to melphalan, while no cross-resistance to vincristine or 5-fluorouracil was observed. When 5 microM W-5 or W-7 was added in the presence of concentrations of cisplatin that hardly inhibited cell proliferation, the KFr cell proliferation was markedly inhibited. These findings suggest that the cisplatin resistance in the KFr cells may be due to an impaired cisplatin-transport mechanisms and can be overcome by calmodulin antagonists.

Interleukin-2 activity and the response of peripheral blood lymphocytes in patients with gynecologic malignancies.

The interleukin-2 (IL-2) production in peripheral blood lymphocytes (PBL), the response to IL-2, and the IL-2 absorption by PBL was studied in 34 patients with gynecologic malignancies. In addition measurement of the OKT 4/8 cell ratios were made. The OKT 4/8 cell ratio in patients with advanced gynecologic malignancies was significantly lower than that in patients with benign tumor. PBL from advanced cancer patients activated by phytohemagglutinin (PHA) had significantly lower IL-2 productivity than that from patients with benign tumors, while no significant difference was observed in its abilities to respond to IL-2 and to absorb IL-2. The OKT 4/8 cell ratio and IL-2 productivity in PBL of patients with good prognosis were significantly elevated after chemotherapy. On the other hand, PBL of patients with poor prognosis declined to about one-half of the preoperative levels. Although in patients with good prognosis the ability of PBL to respond to IL-2 was not changed before and after treatment, in patients with poor prognosis the response was significantly increased after chemotherapy. However, the ability to absorb IL-2 was not affected by treatment.

[Potentiation of the antitumor effect of cisplatin by calmodulin antagonists (W-7 and W-5) in nude mice bearing human ovarian carcinoma].

The present study was designed to potentiate the antitumor effects of cisplatin by combination with calmodulin antagonists (W-7 and W-5) by using nude mice bearing human ovarian carcinoma. Tumor growth in nude mice treated with W-7 or W-5 combined with cisplatin was significantly inhibited, compared to that in nude mice treated with W-7 alone, W-5 alone or cisplatin alone. Although treatment with cisplatin alone markedly inhibited lytic activity of the spleen cells from tumor bearing nude mice against the tumor cells, the inhibitory effect was eliminated by combination with W-7 or W-5. There was no significant difference in the survival time among untreated, cisplatin-treated, W-7-treated and W-5-treated groups. Only when cisplatin was followed by W-7 or W-5, a significant enhancement by W-7 or W-5 of the antitumor effect of cisplatin was observed with respect to inhibition of the tumor growth as well as prolongation of the survival time.

Enhancement of antineoplastic effects of cisplatin by a calmodulin antagonist (W-7) in nude mice bearing human ovarian carcinoma.

The present study was designed to potentiate antineoplastic effects of cisplatin by combination with a calmodulin antagonist (W-7) using nude mice bearing human ovarian carcinoma. Tumor growth in nude mice treated with W-7 after (but not before) administration of cisplatin was significantly inhibited. Although treatment with cisplatin alone markedly inhibited lytic activity of the spleen cells in tumor-bearing nude mice against the tumor cells, the inhibitory effect was eliminated by subsequent treatment with W-7. There was no significant difference in the survival time between untreated and cisplatin-treated groups. Only when cisplatin was followed by W-7 was a significant enhancement by W-7 of the antitumor effect of cisplatin observed with respect to inhibition of the tumor growth as well as prolongation of the survival time.

Effects of PSK on interleukin-2 production by peripheral lymphocytes of patients with advanced ovarian carcinoma during chemotherapy.

The effects of PSK on OKT 4/OKT 8 cell ratio, interleukin-2 (IL-2) production and expression of IL-2 receptor were examined in peripheral blood lymphocytes (PBL) from patients with advanced ovarian cancer during the course of chemotherapy. Preoperative levels of OKT 4/OKT 8 cell ratio and IL-2 production in PBL from patients with advanced ovarian cancer were significantly lower than those in cases of benign ovarian tumor. However, the expression of IL-2 receptor did not show any significant difference between ovarian cancer and benign ovarian tumor patients. When a combination chemotherapy of cisplatin, adriamycin and cyclophosphamide was given, the OKT 4/OKT 8 cell ratio was significantly increased with a significant decrease of the absolute number of the OKT 8 cell subset, while the expression of IL-2 receptor and the absolute number of the OKT 4 cell subset remained unchanged. In contrast, the IL-2 production was markedly depressed after the first course of chemotherapy. When PSK was combined with combination chemotherapy, the degree of inhibition of IL-2 production was reduced (though the effect was not statistically significant). If treatment with PSK was initiated after completion of combination chemotherapy, in addition to a significant elevation of OKT 4/OKT 8 cell ratio the depressed IL-2 production was restored to benign control levels. On the other hand, the expression of IL-2 receptor remained unchanged even if PSK was given after completion of chemotherapy.

Interleukin 2 activity in peripheral blood mononuclear cells of patients with gynecologic malignancies.

We studied production of, absorption of and response to interleukin 2 (IL-2) by peripheral blood mononuclear cells (PBMC) from 66 patients with gynecologic malignancies, in addition to measurement of the OKT 4/OKT 8 cell ratio. Patients with benign tumor served as controls. The OKT 4/OKT 8 cell ratio in patients with advanced (but not early) gynecologic malignancies was significantly lower than that in patients with benign tumor. PBMC from advanced cancer patients activated with phytohemagglutinin (PHA) had significantly lower IL-2 production compared to that from patients with benign tumors, while significant changes in their ability to respond to IL-2 and to absorb IL-2 were not observed. Absolute numbers of OKT 8 positive cells in PBMC of patients with good prognosis were significantly decreased after surgery and chemotherapy, while those of OKT 4 positive cells remained unchanged. Although IL-2 production in PBMC of patients with good prognosis was significantly elevated after chemotherapy, that in PBMC of patients with poor prognosis declined to about a half of pre-operative levels. The ability of PBMC to respond to IL-2 was significantly elevated not only in patients with good prognosis but also in patients with poor prognosis after termination of chemotherapy. On the other hand, the ability of PBMC to absorb IL-2 remained unchanged during the course of treatment. These findings may contribute to the understanding of tumor-induced immune suppression.

Enhancement of antineoplastic effects of cisplatin by calmodulin antagonists in nude mice bearing human ovarian carcinoma.

The present study was designed to potentiate the antineoplastic effects of cisplatin by combination with calmodulin antagonists [N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5)] in nude mice bearing human ovarian carcinoma. Tumor growth of nude mice treated with W-7 or W-5 combined with cisplatin was significantly inhibited, compared to that of nude mice treated with W-7 alone, W-5 alone, or cisplatin alone. Although treatment with cisplatin alone markedly inhibited lytic activity of spleen cells from tumor-bearing nude mice against the tumor cells, the inhibitory effect was eliminated by combination with W-7 or W-5. There was no significant difference in survival time among untreated, cisplatin-treated, W-7-treated, and W-5-treated groups. Only when cisplatin was followed by W-7 or W-5 was a significant enhancement by W-7 or W-5 of the antitumor effect of cisplatin observed with respect to inhibition of tumor growth as well as prolongation of survival time.

[Effects of cimetidine and PSK on interleukin-2 production by PBL in patients with advanced ovarian carcinoma during the course of chemotherapy].

The present study was designed to elucidate the effect of cimetidine and PSK on T-cell subsets, interleukin-2 (IL-2) production and its receptor in peripheral blood lymphocytes (PBL) from patients with advanced ovarian carcinoma during the course of chemotherapy. Preoperative levels of OKT 4/8 cell ratio and IL-2 production in PBL from patients with advanced ovarian carcinoma were significantly lower than those with benign ovarian tumor. When a combination chemotherapy which consisted of cisplatin, adriamycin and cyclophosphamide was given to these ovarian cancer patients, the OKT 4/8 cell ratio was significantly increased while the IL-2 production was markedly inhibited. When PSK and cimetidine were combined with the combination chemotherapy, the inhibition of IL-2 production was reduced. When treatment with PSK or cimetidine was initiated after completion of the combination chemotherapy, the OKT 4/8 cell ratio was significantly elevated on 30 days and 60 days, compared to 7 days after the completion of chemotherapy. The depressed IL-2 production after completion of the combination chemotherapy was increased to about 8 times by cimetidine and about 2.5 times by PSK on 60 days after completion of chemotherapy. On the other hand, the IL-2 receptor remained unchanged even if PSK or cimetidine was administered.

Effects of naloxone on human ovarian cancer cell growth in vitro and in vivo.

The effects of naloxone on human ovarian cancer cell growth in vitro and in vivo were examined by using an established cell line, designated KF, derived from human ovarian carcinoma. When the KF cells were incubated in the presence of various concentrations of naloxone for 72 hr, the cell proliferation was inhibited in a dose-dependent manner in the concentration range of 30 to 120 microM naloxone. The growth-inhibitory effect of naloxone could not be detected by 51Cr-release assay or colony-forming assay. The inhibition of cell proliferation by naloxone resulted from the inhibitory effect on protein synthesis in the KF cells, as confirmed by a marked decrease of incorporation of [3H]valine. In order to determine the effect of naloxone on tumor growth in vivo, ip injections 3 times a week of 2.5 or 5.0 mg/kg naloxone were initiated either one week prior to tumor inoculation (pretreated groups) or one week after tumor inoculation (post-treated groups). Nude mice in the pretreated groups had significantly smaller tumor volumes than those in the untreated group as long as naloxone was administered. On the other hand, in the post-treated groups a significant growth retardation was observed 2 weeks after initiation of treatment with 2.5 mg/kg naloxone. In comparison to a median survival time of 38 days for untreated controls, the naloxone pre- and post-treated groups showed increases of 26-71% in median survival.

Adjuvant effects of calmodulin antagonists to 5-fluorouracil on tumor cell proliferation and the mechanisms.

The adjuvant effect of calmodulin antagonists (W-5 and W-7) to 5-fluorouracil (5-FU) was studied by using two human cultured cell lines derived from ovarian adenocarcinoma. Enhancement of the uptake of 5-[6-3H]fluorouracil ([3H]FU) by cancer cells was observed only when calmodulin antagonists were added in the presence of [3H]FU. Similarly, the adjuvant effect by W-5 or W-7 on cancer cell proliferation was also observed only when calmodulin antagonists were added in the presence of 5-FU.

[Inhibition of human ovarian cancer cell growth by prostaglandin D2].

The effects of prostaglandin D2 (PGD2) on human ovarian tumor growth were examined in vitro and in vivo by using a cell line, designated HR, derived from patients with serous cystadenocarcinoma of the ovary. The cell proliferation was dose-dependently inhibited by PGD2 between concentrations of 0.1 and 4.0 micrograms/ml after 24 hrs, 48 hrs and 72 hrs of contact time. Concentrations of PGD2 required for 50% inhibition of the cell proliferation were 2.0, 1.1 and 0.55 micrograms/ml with 24, 48 and 72 hrs of contact time, respectively. From the results of 51Cr-release assay, the inhibition of cell proliferation by PGD2 was considered to result from the direct cytotoxic effects. The incorporations of 3H-thymidine, 3H-uridine and 3H-valine were inhibited in a dose-dependent fashion with more than 1.0 micrograms/ml of PGD2. Tumor growth in nude mice treated with 0.3 mg/mouse PGD2 was significantly inhibited, compared to that of untreated nude mice. In untreated nude mice the tumor growth curve was parallel to the changes in the plasma alpha-hydroxybutyrate dehydrogenase (HBD). In both PGD2-treated groups with 0.1 mg/mouse and 0.3 mg/mouse, the HBD activity markedly decreased on the 14th and the 21st day after inoculation. The 50% survival time in untreated mouse, 0.1 mg/mouse and 0.3 mg/mouse PGD2-treated groups was 52 days, 55 days and 67 days, each respectively.