Acquired platinum resistance enhances tumour angiogenesis through angiotensin II type 1 receptor in bladder cancer.

BACKGROUND: We investigated the changes in reactive oxygen species (ROS) and angiogenesis through angiotensin II (Ang II) type 1 receptor (AT1R) after the development of acquired platinum resistance in bladder cancer. METHODS: Four invasive human bladder cancer cell lines, T24, 5637, T24PR, and 5637PR, were used in vitro, whereas in vivo, T24 and T24PR cells were used. T24PR and 5637PR cells were newly established at our institution as acquired platinum-resistant sublines by culturing in cisplatin (CDDP)-containing conditioned medium for 6 months. RESULTS: Ang II induced significantly higher vascular endothelial growth factor (VEGF) production in T24PR and 5637PR cells than in their corresponding parent cells in vitro, whereas Ang II induced a further increase in VEGF production. These platinum-resistant cells also showed significantly higher AT1R expression than their corresponding parent cells. ROS was also significantly upregulated in T24PR and 5637PR cells, whereas increased AT1R expression was significantly downregulated by scavenging free radicals. We also demonstrated the efficacy of AT1R blockade at suppressing the growth of platinum-resistant xenograft model. CONCLUSION: Our findings indicate a new molecular mechanism for upregulated AT1R signalling through increased ROS when tumours progressed after the CDDP-based regimens, and shed light on the importance of AT1R blockade for platinum-resistant bladder cancers.

Cyclophosphamide and 5-fluorouracil act synergistically in ovarian clear cell adenocarcinoma cells.

Chemosensitivity to the drugs plays a crucial role in the treatment of ovarian cancer. In this study, we evaluate the cytotoxicity of chemotherapeutic agents in six ovarian cancer cell lines; four clear cell adenocarcinoma and two serous papillary adenocarcinoma, using seven single drugs and seven sets of drug combinations with tetrazolium-based semiautomated colorimetric (MTT) assay. The drug concentration which produced 50% growth inhibition (IC50) of cisplatin was within clinically achievable range in five cell lines. The area under the curve (AUC) at IC50 of cyclophosphamide was below the clinically achievable AUC in two serous papillary cell lines. Paclitaxel was more effective in clear cells than serous papillary cells. The intensification of cytotoxicity was observed in the combinations of paclitaxel and cisplatin, and cyclophosphamide and cisplatin or 5-fluorouracil irrespective of histopathological characteristics of the original tumor. Our results indicate that ovarian cancer cell lines respond to chemotherapeutic agents heterogeneously depending upon histopathological features, indicating individualized regimens may improve survival in ovarian cancer patients.

[Trial for transdermal administration of sulfonylureas].

Sulfonylureas are widely used as oral hypoglycemic drugs in the treatment of non insulin dependent diabetes mellitus (NIDDM). Since sulfonylureas are usually taken for a long period, the compliance of the patients is very important. Therefore, for the improvement of the compliance of the patients, the development of a transdermal dosage form of sulfonylureas was attempted in this study. Glibenclamide (GLI) or chlorpropamide (CHL) was chosen as a principal agent and ointments were prepared by mixing 5% of GLI or CHL with a FAPG ointment base. Penetration and shearing stresses of the ointments were determined as physical characteristics of the ointments. There was no obvious difference of characteristics between the GLI ointment, or the CHL ointment and the FAPG ointment base. In drug release tests, the CHL ointment showed better release of the drug than the GLI ointment. In both ointments, comparatively rapid release of drug was observed in the initial 1 h, and continuous slow release was observed thereafter. When the ointments were applied on the back of male Wistar rats and the plasma glucose level was measured, both CHL and GLI ointments gave lower blood glucose levels than the control (FAPG base). At the all measuring points, the GLI ointment brought about significantly lower blood glucose levels than the control (p < 0.01). Thus, it was demonstrated that sulfonylureas were absorbed through the skin and lowered the blood glucose levels. The results suggest the possibility of transdermal administration of sulfonylureas for the treatment of NIDDM.

[Preparation and utility of glibenclamide suppository for hospital use].

Glibenclamide (GC) is widely used as an oral hypoglycemic drug in the treatment of non-insulin dependent diabetes mellitus (NIDDM). Since GC is usually taken for a long period, side effects and noncompliance are among the problems. In order to solve those problems, we prepared GC suppositories and examined their usefulness. Suppositories containing 4, 20, and 40 mg of GC were prepared and examined for drug release, drug absorption and blood glucose levels after the rectal administration of suppositories in rabbits. In the release test, GC suppositories released the drug continuously for 6 hours. The areas under the drug release time curve (ADT) of 20 and 40 mg GC suppositories were 3.5 and 6.2 times of 4 mg GC suppositories respectively. The plasma concentrations after administration of 4 and 20 mg GC suppositories showed about the same profiles for 6 hours. After administration of 40 mg GC suppositories, the maximum plasma concentration (Cmax) was observed at 2 hours. All the GC suppositories showed lower blood glucose levels compared with the control. The remainder of the area under the blood glucose concentration time curve between the control (RAUC) in the case of 40 mg GC suppository was 1.3 times larger than that of the 4 mg GC suppository. The GC suppositories sufficiently lowered the blood glucose levels. These results suggest that the GC suppositories should be useful in the hospital preparation for the treatment of NIDDM patients.

[Effect of temperature on drug release and drug absorption in mixed type diclofenac sodium suppositories].

New types of diclofenac sodium suppositories known to control a drug release function for hospital preparations were developed based on a concept of the drug delivery system. Hard fat (Witepsol) used as a base of the suppository consists of a mixture of triglycerides, diglycerides and monoglycerides, and each Witepsol is characterized by its physicochemical properties. Authors disclosed that the amount of drug release measured in the commercially available diclofenac sodium suppositories decreased at a low temperature (36 degrees C). Mixed types of diclofenac sodium suppositories consisting of Witepsol W35 and Witepsol E85 as a base were also prepared and their drug release functions investigated in vitro and in vivo. The in vitro drug release properties changed with the mixing ratios of the two bases and with the temperature of the fluid tested. The amount of released diclofenac sodium increased with increases of both the ratio of Witepsol W35 in the suppository and the temperature of the test fluid. Moreover, several processes causing these phenomena were evidenced by the image analysis. The in vivo absorption of diclofenac sodium was found to be also influenced by these factors. Consequently, it is predicted that such factors as the ratio of Witepsol W35 in the suppository and the temperature will influence the drug absorption and the pharmacological effect of diclofenac sodium suppositories.

[Sustained release double-layered progesterone suppository for luteal support therapy].

A sustained release suppository containing progesterone with a double-layered structure was prepared for the treatment of the luteal phase defect. Hydroxypropylcellulose-H (HPC) and Carbopol-934P (CP) were used as bases of the inner layer and Witepsol W35 was used as a base of the outer layer. The strength of the inner layer (stick) decreased with the increase of the rate of content of HPC component. The strength of the stick which was prepared from a mixture of HPC and CP in a ratio of 1:1, was inverse by proportional to the rate of the addition of crystalline cellulose (CC) and the amount of released drug was proportional to the rate of the addition of CC. The area under the drug release curve of the stick containing 60% of CC in the base was about 12 times of the stick containing no CC (control stick). Furthermore, the mean release time of the stick containing 60% of CC became about a half of the control stick. It was suggested that the drug release of progesterone from the stick could be controlled by changing the rate of the addition of CC. Two types of suppository which containing progesterone in both phases (suppository A) and in the stick alone (suppository B) were prepared. Both suppositories showed a sustained release property and suppository B had a lag time of two hours. When the suppositories were administered in to the vagina of rabbits, they showed a sustained release property and a rapid rise in the serum concentration was more suppressed than an ordinary Witepsol suppository. One hour after the administration of the two layered suppository, some parts of the suppository was identified macroscopically to be remained in the vagina. The usefulness of the double-layered suppository as a hospital preparation should be suggested after the attainment of the optimization of the formulation.

[Studies on enzymeimmunoassay for human chorionic gonadotropin and its clinical application].

Two enzyme immunoassays for hCG were developed and compared with HAR and RIA by using many urine and serum samples. The EIA was characterized by management of trophoblastic disease. 1. An EIA by hCG-beta antibody immobilized on a microplate and hCG antibody conjugated with HRP enzyme quantified hCG from 0.5 to 100ng/ml, while the other by biotinyl hCG antibody and avidine-HRP conjugate quantified hCG from 0.1 to 20ng/ml. Both of them had a good correlation with RIA; y = 1.02x + 2.3, r=0.99 and Y=0.96x-0.38. The C.V.S. were 1.8 and 5.2% (intraassay), or 5.8 and 9.5% (interassay), respectively. 2. The EIA was carried out to test hCG patients whose urinary hCG by HAR was under the LH level. The values in 371 samples were under 2ng/ml, but those in 40 samples were higher than 2ng/ml (Max 15.8 ng/ml). Diagnosis by EIA distinguished recurrence from remission 2 month earlier than by HAR. The EIA was useful in the management of the disease.

[MTX sensitivity and the mechanism of resistance of various choriocarcinoma cells in culture].

The sensitivity to methotrexate (MTX) and mechanisms of the drug resistance of seven kinds of human choriocarcinoma cell lines were studied in vitro. The results were as follows. Each cell line of HCCM-5 , BeWo, IMa, and NUC-1 secreted more than 200ng/10(6) cells/48 hours of human chorionic gonadotropin (hCG), whereas GCH-1, ENAMI-1, and SCH secreted less than 20ng/10(6) cells/48 hours. HCCM-5 cell line was the most sensitive to MTX judging from the inhibition of both 3H-thymidine uptake and cell growth in vitro. Each cell line of BeWo, IMa, and NUC-1 revealed sensitivity to 2 X 10(-8)M MTX, but such cell lines as GCH-1, ENAMI-1, and SCH showed resistance to MTX. The cell lines which revealed sensitivity to MTX incorporated more 3H-MTX and showed less intracellular dihydrofolate reductase activity than resistant cell lines.

[Antitumor effect of interferon against human choriocarcinoma cell lines].

In this report, we describe the inhibitory effect of two kinds of human interferons, IFN-alpha and IFN-beta, on the growth of human choriocarcinoma cell lines in vitro and in vivo on comparing the other various cell lines derived from gynecologic malignancies. As compared to human endometrial (HEC-1B) or ovarian (HOC-21) adenocarcinoma cells which were highly resistant to the inhibitory effect of IFN-beta on the DNA synthesis at any doses up to 10,000U/ml, two human choriocarcinoma cell lines, BeWo and HCCM-5, and yolk sac tumor cell line YS-K were relatively sensitive. However, the inhibition was not dose-dependent. The growth of BeWo and HCCM-5 cells were also inhibited by either IFN-alpha or IFN-beta at concentration of 1,000U/ml and the inhibition became conspicuous when the treatment was prolonged to 4 days or more. When the hamsters transplanted with BeWo or HCCM-5 cells were treated with 10,000U IFN-beta on every day from the first till the 7th day after inoculation of cells, the tumor weights decreased to about one-half of those of non-treated controls. In contrast, the tumor weights were almost the same as non-treated controls when the animals were given with IFN-beta from the 7th day till the 13th day.

[Isolation and properties of methotrexate-resistant choriocarcinoma cells in vitro].

The sensitivity to methotrexate (MTX) of ten kinds of human choriocarcinoma cell lines was studied in vitro. The cell line most sensitive to MTX was HCCM-5 and it was fed with a medium containing increasing concentrations of MTX. A resistant subline (HCCM-5R) which could proliferate in the medium containing 5 X 10(-7)M MTX was obtained after about 80 weeks of feeding. The properties of HCCM-5R cells were compared with those of the parent HCCM-5 cells. i) There were no apparent differences in morphology between the HCCM-5 and HCCM-5R lines. The population doubling time was almost the same for both cell lines. ii) The hCG level in the culture fluid of the HCCM-5R cells was about twice as high as that of the HCCM-5 cells. iii) The concentration of MTX which inhibited 50% of DNA synthesis in HCCM-5R cells was 10,000 times higher than that for HCCM-5 cells. iv) Each X and Y chromosome was identified in 90% of the HCCM-5 cells, while no Y chromosome was detected in the HCCM-5R cells. v) The DNA distribution pattern for HCCM-5R cells consisted of a large fraction of each cell with DNA G1 phase content and distributed in the tetraploid range, whereas that of HCCM-5 showed no particular cell cycle pattern. vi) The incorporation of 3H-MTX in the HCCM-5R cells was one tenth of that in the HCCM-5. vii) The intracellular DHFR activity of the HCCM-5R cells was about 5 times higher than that of HCCM-5. These results suggest that MTX-resistant cells among the HCCM-5 cells were selected in long-term contact with MTX, and sensitivity to MTX was concerned with both the MTX transport and intracellular DHFR levels.

[Effects of various agents on the proliferation and hCG secretion of choriocarcinoma cells in vitro].

Effects of sex steroids, LH-RH, cyclic AMP and differentiation promoters on the proliferation and hCG secretion of two choriocarcinoma cell lines (BeWo and HCCM-5) were examined in vitro. The results were as follows. 1) Physiological concentrations of estradiol or progesterone revealed no effect on proliferation or hCG secretion, but pharmacological ones (more than 10 micrograms/ml) inhibited both the cell proliferation and its hCG secretion. 2) LH-RH and cyclic AMP slightly inhibited the proliferation of cells, and hCG secretion per cell was concomitantly enhanced. Cyclic AMP enhanced hCG secretion more markedly than LH-RH. 3) Retinoic acid had no effect on either cell proliferation or hCG secretion, whereas sodium butyrate inhibited both cell proliferation and hCG secretion. These results suggest that modifiers of hCG secretion may be grouped as follows; agents that inhibited cell proliferation slightly and enhanced hCG secretion markedly (LH-RH, cyclic AMP), agents that inhibited cell proliferation markedly and enhanced hCG secretion slightly (anticancer drugs) and agents that inhibited both cell proliferation and hCG secretion (estradiol, progesterone and sodium butyrate).

Enzyme immunoassay for placental protein 4 (PP4) and its possible diagnostic significance in patients with genital tract cancer.

We have established an enzyme immunoassay for placental protein 4 (PP4), by using avidin-biotin binding reaction, and set its normal range below 10.9 ng/ml (mean + 2 sigma). Throughout the menstrual cycle, the serum PP4 profile was similar to that of serum progesterone. In the follicular and ovulatory phase, PP4 remained relatively low, with the mean levels of 1.5 ng/ml and 1.8 ng/ml, respectively. In the luteal phase, the mean level was 3.2 ng/ml. In normal pregnancy, serum PP4 levels were low irrespective of gestational age, with a mean level of 3.0 ng/ml. There was only one case in which the serum PP4 level over 10.9 ng/ml. Mean serum PP4 levels and the frequencies of elevated serum PP4 levels were respectively 6.3 ng/ml and 11% in patients with benign ovarian neoplasms, 4.7 ng/ml and 6% in patients with endometriosis, and 5.5 ng/ml and 18% in patients with uterine myomata. The frequency of raised PP4 levels was 48% and the mean value was 13.3 ng/ml in patients with endometrial carcinoma, and the values were 44% and 13.4 ng/ml respectively in patients with cervical carcinoma. In patients with ovarian malignancy, the respective values were 15% and 7.0 ng/ml. The results did not relate to clinical stages of disease (FIGO), while the frequencies of elevated serum PP4 in patients with uterine carcinoma was over 40% in stage I diseases. Compared with other tumor markers such as carcino-embryonic antigen (CEA), tissue polypeptide antigen (TPA) and cancer antigen 125 (CA125), PP4 seems to be more promising as a marker of endometrial carcinoma.(ABSTRACT TRUNCATED AT 250 WORDS)

[Synergistic effect of amphotericin B and actinomycin-D against two human choriocarcinoma cell lines].

The sensitivity to actinomycin-D (Act-D) and the changes in survival rate of two human choriocarcinoma cell lines (BeWo and SCH) were studied in vitro and the following results were obtained. BeWo was shown to be more sensitive to Act-D than SCH, when the survival rate was compared in the two cell lines. 3H X Act-D uptake was 39 pmol/10(6) cells in BeWo and 12 pmol/10(6) cells in SCH after a two hours treatment. Those results suggested that the sensitivity to Act-D of choriocarcinoma cells was positively correlated with the intracellular Act-D concentration. The intracellular Act-D concentration was increased depending upon the concentrations of amphotericin B (AMB). After a two hours treatment with Act-D and AMB, the intracellular Act-D concentrations were twice in BeWo, and 2.3 times in SCH comparing with those treated with Act-D alone. The synergistic effects of Act-D and AMB on the survival rate were 1,000 times in BeWo and 100 times in SCH compared with those treated with Act-D alone. From the above, combination therapy with Act-D and AMB was supposed to be one of the trial methods in the treatment of drug resistant choriocarcinoma.

[Fundamental and clinical study of chemotherapy of refractory choriocarcinoma].

Since the introduction of MAC therapy in 1973, choriocarcinoma has become one of the most curable gynecologic malignancies. But in MAC resistant cases, the therapeutic results have been unsatisfactory. To establish the proper therapy for MAC resistant choriocarcinoma, fundamental experiments with MTX resistant HM cell lines were carried out with the aim of their clinical application. The effective combination in the treatment of choriocarcinoma appeared to be 10 to the minus 5th power mol of MTX and 10 to the minus 8th power mol of Act-D, considering its effect in cell proliferation inhibition, deoxyuridine uptake inhibition and dihydrofolate reductase activity suppression. Taking the effects of Oncovin confirmed by the experiments with nude mice into consideration, a combined moderate dose of MTX, Oncovin and Act-D, namely MOA therapy was used. The protocol of MOA therapy is as follows: The first day, MTX 150mg bolus, 300mg drip infusion for 4 hours, Oncovin 2mg bolus and Act-D 0.5mg bolus. From the second to the fifth day, Act-D bolus. MAC was effective in only one of the eight recurrent cases, but on the other hand, MOA was effective in five cases of choriocarcinoma including two MAC resistant cases. Therefore, MOA seemed to be a more effective therapy than MAC.

Immunochemical measurement and immunohistochemical detection of membrane-associated placental tissue protein 1.

Using the avidin-biotin binding system, an enzyme immunoassay procedure was developed to measure the membrane-associated placental tissue protein 1 (MP1) in serum. The standard curve covered the range from 10 to 1000 ng/ml of MP1. The intra- and inter-assay coefficient of variations (C.Vs) were less than 5 and 10%, respectively. Recoveries of MP1 added to serum ranged between about 96 and 101%. The MP1 serum level was over 10 and under 112 ng/ml in non-pathological men, and under 240 ng/ml in non-pathological women. The MP1 level in the ovulatory phase was higher than in other phases of the menstrual cycle. In pregnancies during 6-39 weeks, the MP1 level ranged from 10 to 540 ng/ml, and it increased during the third trimester of gestational age. In benign gynecologic diseases, the MP1 concentration in serum ranged from 10 to 215 ng/ml. The MP1 levels in benign diseases were compared with those in ovarian malignancies, in endometrial carcinoma, and in uterine cervical cancer. The immunohistochemical location of MP1 was detected in the cell membrane of ovarian cystadenocarcinoma.

[Anticancer effect of VP16-213 (etoposide) on three choriocarcinoma cell lines].

A new anticancer drug, VP16-213 was studied in vitro to determine its effect upon 3H-thymidine uptake and the survival rate of choriocarcinoma cells in comparison with the effect of MTX and Act-D. Three human choriocarcinoma cell lines, BeWo, HCCM-5 and SCH were used. The results were as follows; Serum VP16-213 levels were 10.3 +/- 1.19 micrograms/ml and 1.35 +/- 0.38 micrograms/ml at one hour and 12 hours after the administration of 100mg of VP16-213, respectively. In those three cell lines, VP16-213 suppressed cellular 3H-thymidine uptake by 70% as compared with the control. On the sixth day after exposure to VP16-213, the survival rate of choriocarcinoma cells was less than 10% in each of the three cell lines. One of the most prominent pharmacodynamic characteristics of VP16-213 was a rapid influx and efflux mechanism as seen in MTX. From the above, the anticancer effect of VP16-213 upon those three cell lines was supposed to be equal to or more than those of MTX and Act-D.

Clinical significance of a solid-phase immunoradiometrical assay for sialyl SSEA-1 antigen in obstetrics and gynecology.

The clinical usefulness of sialyl SSEA-1 antigen was evaluated in obstetrics and gynecology. Serum levels of sialyl SSEA-1 were measured in patients with benign or malignant gynecologic diseases, and in normal pregnant women. Moreover, in 10 cases of full term delivery, samples of maternal sera immediately prior to delivery, soon after delivery and 5-day-puerperium, cord sera from the umbilical artery and vein, and amniotic fluid were taken to measure its concentration. During the course of pregnancy, serum SSEA-1 levels were within the normal range (below 38 U/ml), showing no significant correlation with gestational weeks. Of patients with gynecologic diseases, those with malignant ovarian neoplasms, uterine cervical carcinoma and benign ovarian neoplasms exhibited elevated (over 38 U/ml) levels in 26%, 15% and 6% of all cases, respectively. In cases of full term delivery, the concentrations of sialyl SSEA-1 in the maternal and cord sera were within the normal range. Concentrations were extremely high, however, in the amniotic fluid.

Effect of methotrexate on the growth and human chorionic gonadotropin secretion of human choriocarcinoma cell lines in vitro.

The effect of methotrexate (MTX) on the growth and human chorionic gonadotropin (hCG) secretion of five gestational and two nongestational human choriocarcinoma cell lines was studied in vitro. A striking heterogeneity in hCG secretion was noted among the cell lines. The growth of cells which secreted large quantities of hCG, such as HCCM-5, BeWo, and IMa, was inhibited by continuous exposure to 2 X 10(-8)M MTX. In contrast, cells which secreted little hCG, such as SCH, ENAMI-1, and GCH-1, showed no response to the growth inhibitory action of 2 X 10(-8)M MTX and the 3H-thymidine uptake was not reduced by treatment with MTX at doses of up to 10(-4)M for 48 hours. The common morphologic alterations observed in the cells which responded to MTX were an increase in the number of multinucleated giant cells, the appearance of vacuoles and granules in the cytoplasm, and enlargement of the nuclei. Increased hCG secretion was observed in accordance with the appearance of such morphologically altered cells. Part of the mechanisms of resistance to MTX appeared to involve both impairment of MTX uptake by the cells and an increase in the level of intracellular dihydrofolate reductase.

[Studies on the recurrent trophoblastic disease].

We studied the remission and the recurrent of trophoblastic disease treated in our clinic, during 1973 to 1980. 1) The remission and recurrent rates of 168 cases were 91.1% (153/168) and 5.2% (8/153) respectively. In these percentages, high risk was 65.9% (29/44) and 20.7% (6/29), low risk was 100% (124/124) and 1.6% (2/124), choriocarcinoma was 58.3% (21/36) and 28.6% (6/21), invasive mole was 100% (44/44) and 0, and undetermined was 100% (88/88) and 2.3% (2/88). 2) The interval to recurrence: 5 cases appeared under a year, 3 cases did over a year. And remission rate of recurrent case was (4/8), high risk was (2/6), and low risk was (2/2). 3) HCG-beta titers when U-hCG decreased under the LH levels was higher until 3 months in the recurrent case of high risk than that unrecurrent, while we did not recognize any differences after 3 months. Therefore criteria of remission in high risk would occurred on continuous LH levels of U-hCG for 3 months. 4) High risk has been treated by 3 courses of additional chemotherapy, but that when hCG-beta titer over 1.0ng/ml require more chemotherapy.

Characterization of human choriocarcinoma cell lines secreting high and low amounts of chorionic gonadotropin.

The secretory potential of human chorionic gonadotropin (hCG) in eight strains of human choriocarcinoma cell lines was examined in vitro and the correlation between the secretory potential and certain cell biological characteristics was studied. Multinucleated giant cells (syncytiotrophoblast-like cells) were occasionally observed only in high hCG-secreting cell lines in accordance with inhibition of growth. Appearance of abundant dilated rough endoplasmic reticulum and aggregated glycogen particles in the cytoplasm seemed to represent ultrastructural characteristics of high hCG-secreting cell lines. A shift of nuclear DNA to hyperploidy, its wide distribution, and S phase accumulation of cells by flow cytometry were also characteristic of them. However, no correlation was noted between the hCG secretory potential and population doubling time of the cell lines in vitro.