Nitrosourea compounds have been widely used in the chemotherapy of malignant brain tumors, because of their blood-brain barrier permeability. However, drug resistance to nitrosoureas has been recently a major concern. Using an in vitro colony formation assay, intrinsic and acquired resistances to an anticancer nitrosourea, 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU), were analyzed in rat 9L and C6 glioma cells. 9L and C6 cells were treated with varying doses of ACNU for 2 hours. Ten days after, the cells were fixed and stained with crystal violet. Colonies consisting more than 50 cells were counted. The survival fraction following treatment is the ratios of colony efficiency of treated cells to the colony efficiency of untreated control cells. The dose-response curve for ACNU indicated the existence of a shoulder (Dq, quasithreshold dose) at doses and an exponential cell-killing at higher doses with D0(37% survival dose). Based on dose-response curves corresponding to multitarget single-hit model, 9L cells showed 7.4 microM, 2.9 microM, and 14 microM at Dq, D0, and SD10 (10% survival dose) values, respectively, whereas C6 cells showed respective values of 6.4 microM, 30 microM, and 75 microM. 9L cells had significantly less intrinsic resistance to ACNU than C6 cells at the p less than 0.005 level by a covariance analysis of the curves. As with changes of drug susceptibility after ACNU treatment, both parent cells were treated every other day (1, 5, and 10 repeated times) with various doses up to approximately 1% survival dose of the parent cells.(ABSTRACT TRUNCATED AT 250 WORDS)

We investigated a new chemosensitivity test, MTT-hybrid assay, which was a hybrid of MTT colorimetric assay and double-layered soft agar colony assay, using human bone and soft tissue tumor cells. MTT formazan crystals produced by viable cells in the soft agar medium were solubilized by SDS at 60 degrees C. The absorbance (560 nm) is directly proportional to the cell number over a wide range. The absorbance increased in proportion to colonial growth of osteosarcoma cells, while it decreased in a human diploid cell strain in a few days. Drug sensitivity of tumor cells is supposed to be assessed without contaminating normal cells by MTT-hybrid assay in primary tumor samples. Good correlation of IC50 was observed between MTT-hybrid assay and colony assay. The MTT-hybrid assay shows potential value as a rapid predictive test for chemotherapeutic agents in an individual patient.

Though radiotherapy combined with chemotherapy is used increasingly more frequently in the recent treatment of malignant tumors, very little is known about what dose of an antitumor agent equals what radiation dose in effect. We tried to determine those radiation doses which are required to produce the same effect as certain specific doses of antitumor agents (isoeffective dose) by studying the early effects of radiation and drugs chiefly on the intestine of dd strain mice. Parameters used were food consumption, body weight change, 3H-TdR radioactivity in the intestine and the labelling index of intestinal crypt cells. The results were as follows: 1) To determine that radiation dose which is equal to a drug dose (1/2 its LD50) in systemic effect, body weight change and food consumption were suggested to be useful parameters. 2) To determine that radiation dose which is equal to a drug dose in the effect on a specific organ, parameters suitable for the target cells must be selected. For evaluating the effect on the intestine, the labelling index of intestinal epithelium was a useful parameter. 3) The results of treatment with combined radiation and slight amounts of drugs suggested that the degree of aggravation of radiation injury due to drugs might vary with the time of drug administration. 4) Graphic representation of the severity of injury as indicated by parameters enabled us to grasp visually the characteristics of drugs.

In 34 patients with primary advanced breast cancer, intra-arterial administration of ADR (50 mg X 3, total dose 150 mg, 10 cases), 4' epi ADR (50 mg X 3, 150 mg, 8 cases; 70 mg X 3, 210 mg, 10 cases) and THP-ADR (50 mg X 3, 150 mg, 6 cases) was performed, and its effects and side effect were analyzed. The clinical and histological response rate were superior in the ADR (150 mg) regimen and 4'-epi-ADR (150 mg) regimen. Signs of systemic toxicity such as gastrointestinal disorders, leukocytopenia and thrombocytopenia were the side effects in patients treated with THP-ADR, but the frequency of alopecia was lower. No cardiotoxicity was recorded in any of the patients. These results indicated that 4'-epi-ADR given the total dose of 150 mg in a single dosage of 50 mg was the most effective agent in intra-arterial infusion chemotherapy for advanced breast cancer.

Five different types of anticancer drugs were individually entrapped into fibrin clots using our own material, "G.T.XIII" to provide an "anticancer drug-fibrin clot" for regional cancer chemotherapy. Anticancer drugs used in the present study were ADM, MMC, MTX, 5-FU and cDDP. The release of drugs from fibrin clots was studied in vitro. Each fibrin clot was intraperitoneally administered to cancer (AH-130)-bearing rats to evaluate the oncolytic effects. The activities of anticancer drugs delivered from the clots were maintained for more than two weeks. Survival terms of cancer bearing rats were remarkably prolonged with the anticancer drug-fibrin clots. Neither recurrence of ascites nor metastases of malignant cells was observed in the rats treated with such clots. Our newly devised anticancer drug-fibrin clots showed a sustained release of oncolytic drugs and favorable antineoplastic effects. This newly devised drug delivery system suggested a clinical potential for regional cancer chemotherapy.

A conventional limulus test can detect not only endotoxin but also beta(1----3) glucan. Therefore, with a quantitative limulus test, the limulus colorimetric test, we studied the pharmacokinetics of lentinan, an antitumor beta(1----3) glucan preparation, in the blood of 10 healthy volunteers and 20 patients with advanced cancers. The calibration curve of lentinan in the human plasma was linear in the range of 0 to 100 ng/ml. Two mg of lentinan was administered once a week and blood samplings for the measurement were done immediately before the next lentinan administration to document trough-levels serially at a week interval. Trough-levels tended to rise gradually and showed a rapid elevation between 4 and 8 weeks after beginning of administration. This phenomenon may correlate closely with the antitumor effect of lentinan and, therefore, deserves further investigations.

We studied whether or not cyclosporin A (CSA) has a usefulness in subrenal capsule assay (SRCA) with normal immunocompetent mice. Sixty mg/kg of CSA was given to BDF1 mice daily subcutaneously, and various dosages of adriamycin (ADR) was given intravenously on day 2. The body weight of BDF1 mice decreased over 20% within ten days when ADR was given at more than 5 mg/kg. MX-1, a human breast carcinoma line is known to be sensitive to ADR. This tumor was implanted subcutaneously in the back of BALB/c nu/nu mice and chemosensitivity was tested against ADR. ADR resulted to be positive at the dose of 8 mg/kg. On the contrary, the dose of 5 mg/kg proved to be negative, and hence the result of SRCA would be false negative, if the dose of ADR is reduced to avoid the toxicity of CSA. The tumor grew slowly when only 60 mg/kg of CSA was given daily for three weeks, and the inhibition rate was 56.2%. The toxicity of CSA was neglected because of the body weight loss was approximately 13%. CSA may have the antitumor effect by itself, and we therefore suggest that the CSA is not useful for SRCA.

Sensitivity of human transitional cancer cells to anticancer agents was evaluated utilizing cultured cell lines. T-24, MGH-U1 and KU-1. Simultaneously, chemosensitivity tests combined with 42 degrees C hyperthermia were performed. Cells inoculated in 96-well multiplates for 48 hours, were exposed to graded concentrations of doxorubicin (DOX), mitomycin C (MMC), bleomycin (BLM), peplomycin (PEP), cis-diamminedichloroplatinum (II) (CDDP) for 2 to 48 hours. After additional culture for 48 hours, viable cell numbers were estimated by the dye exclusion assay (DEA) and tetrazolium-based colorimetric assay (MTT-assay). In 2-hour exposure, most of anti-cancer agents did not significantly suppress the growth of the cell lines. Only DOX suppressed the cell growth. In 6-hour and 48-hour exposure, DOX, MMC and CDDP showed significant growth inhibitory effect on the transitional cancer cell lines. The effect of BLM and PEP was insufficient. The hyperthermia of 42 degrees C enhanced the growth inhibitory effect of MMC and CDDP, but did not influence the effect of DOX. In comparison of DEA and MTT-assay, viable cell numbers measured by DEA well correlated with the optical density in MTT-assay. Since MTT-assay is a semiautomated, rapid and inexpensive assay with good reproducibility, it can be a useful substitute for DEA in chemosensitivity testing of cancer cells.

One of the major problems in cancer chemotherapy is the development of drug resistance during treatment. The nature of drug resistance in cancer patients is complex. Recently, it has been found that tumor cells can acquire resistance to anticancer drugs. It is now generally accepted that drug resistance at the cellular level (cellular resistance) is also an important mechanism of drug resistance in patients. The elucidation of the resistance mechanisms has progressed well recently owing to the application of cellular and molecular techniques in addition to the usual biological and biochemical techniques. In this article, I describe the mechanisms of cellular resistance, especially those of multidrug resistance at the molecular level, and I also discuss possible approaches to overcoming drug resistance.

3'-5'-Dioctanoyl-5-fluoro-2'-deoxyuridine (TT 82), one of the lipophilic prodrugs of 5-fluoro-2'-deoxyuridine mixed with Lipiodol (LPD), was injected into the hepatic artery before hepatic resection in 4 patients with hepatocellular carcinoma. Later, the anti-cancer effects of the drug were evaluated mainly by the examination of resected specimens. The serum alpha-fetoprotein levels of three patients in whom the values were over normal range fell to less than 50% of pretreatment values from 7 to 14 days after the injections. In pathological observation of the specimens, the necrosis rates of main tumors were 100%, 70%, 30% and 0%, respectively. In three patients whose main tumors showed complete or partial necrosis, selective depositions of LPD in the tumors were observed on soft X-ray photographs. The concentrations of TT 82 and its metabolites in the tissues of LPD-depositing areas were markedly higher than those in tissue of the regions without LPD deposits. These findings suggested that TT82-Lipiodol mixture remained selectively in the cancerous tissues and exhibited anticancer effects following injection into the hepatic artery.

In case of an arterial infusion chemoembolization therapy for primary or metastatic liver cancer, gradual release of the anti-cancer drug from lipiodol is a very important factor for a higher drug concentration in the tumor and for longer contact. We studied basic points about what kind of drug form has a gradual drug release. We prepared 3 forms of drugs. (1) Powder form: Powder of ADM, MMC and CDDP was suspended in lipiodol with ultrasonic suspender. (2) URO form: ADM and MMC were dissolved with Urografin and mixed with lipiodol. (3) Surfactant form: ADM and MMC were dissolved with water and then mixed with lipiodol using surfactant. We put lipiodol suspension into physiological saline and then stirred water at 100 rpm with the paddle method, measuring drug release from the suspension or emulsion. Powder form had a lowest drug release. In clinical trials, we administered intra-arterially (1) ADM, MMC dissolved with physiological saline water as usually used (physiological saline water form) (2) Powder form; (3) URO from; (1) CDDP solution as usually used was administered; (2) Powder form. Then we studied the changes of serum concentration of ADM, MMC and CDDP. The results indicated that powder form had the lowest drug release. Thus, the water-soluble anti-cancer drugs ADM, MMC and CDDP should be used in powder form.

Evaluation of "Gianturco-Wallace chemotherapy pulser," which was developed to produce a more homogeneous drug distribution of the tumors in intra-arterial infusion chemotherapy, was assessed by comparative study of pulsed and nonpulsed arterial radionuclide infusion using Tc-99m pertechnetate for 18 cases of hepatic carcinomas (11 cases of hepatocellular carcinomas and 7 cases of metastatic hepatic carcinomas). Tc-99m pertechnetate, 740 MBq (20 mCi) diluted with saline (30 mL) was infused with or without pulse through the catheter into the hepatic artery at a rate of 1mL per minute. The intrahepatic dynamic radionuclide distribution was analyzed by the time activity curves of ROIs in the tumor and nontumor areas. Pulsed infusion interrupted laminar flow and produced more homogeneous radionuclide distribution in the liver, and combination of pulsed and nonpulsed infusion also produced better radionuclide distribution in the areas of the tumors. This method using Tc-99m pertechnetate was very useful as a simulation to determine the dynamic drug distribution of the tumor and non-tumor region in intraarterial infusion methods.

The authors studied the localization of neocarzinostatin (NCS) in cultured cells and in tumor-bearing rats by means of immunofluorescent staining. Anti-NCS antibody was obtained through immunization of rabbits with NCS. Cellular uptake of NCS was dose-dependent (1.0 to 1000 micrograms/ml) in 9L rat gliosarcoma cells in monolayer. In monolayer cells of 9L, KMG-4 (derived from human glioblastoma), and KMS II (human ependymoma) treated with 1 mg/ml of NCS, drug uptake occurred within a few seconds. Accumulation was much higher in the cytoplasm than in the nucleus and, although nuclear uptake increased slightly over time, there appeared to be no increase in total cellular uptake. Mitotic cells, which were spherical in culture, showed greater intracellular accumulation than other cells. There was no significant difference in uptake among non-mitotic cells. Cells surviving 20 hours of treatment retained accumulation as high as that in killed cells. In KMG-4 monolayers, cytoplasmic and nuclear NCS distribution still differed, whereas 9L monolayers exhibited more even intracellular distribution. In 9L spheroid models treated with 1 mg/ml of NCS, the drug permeated almost all layers within 10 minutes, and at 120 minutes had heavily accumulated in the central necrotic areas. In rats with transplanted brain tumors, NCS selectively accumulated in neoplastic tissues following intra-arterial administration. However, NCS uptake by arterial endothelium was also seen, which suggests the potential for vascular toxicity. The therapeutic value of NCS is discussed in terms of its pharmacokinetic characteristics.

The cell growth inhibitory effects of prostaglandin D2 (PGD2) were examined on cultured and implanted malignant tumor cell lines. The results were as follows. 1) PGD2 showed significant cytotoxicity against Sarcoma-180, Ehrlich cancer, Yoshida sarcoma and KATO-III in culture. The cytotoxicity of PGD2 against KATO-III was the lowest. 2) PGD2 at a dose of 40 micrograms/mouse/day was intraperitoneally injected into ddY mice for 10 days starting on the first day after an intraperitoneal implant of 2 X 10(5) Sarcoma-180 cells. As a result, the duration of survival was significantly prolonged. However, intravenous injection was less effective than intraperitoneal injection. 3) Addition of Forskolin to the cultured KATO-III cells with PGD2 resulted in a significant increase of cyclic AMP (cAMP) levels. However, the growth inhibition of PGD2 was independent of elevated levels of cAMP. 4) DNA histograms using flow cytometry showed PGD2 blocked significantly G2 + M phase DNA synthesis in the cultured Sarcoma-180 cells, and S phase DNA synthesis in KATO-III cells was significantly blocked with PGD2 at a concentration of 100 micrograms/ml. The results indicate that PGD2 possesses an antitumor effect on 4 malignant tumor cell lines. Furthermore, it seems that the antitumor mechanism of PGD2 is due to the inhibition of DNA synthesis rather than the action through cAMP-receptor, and the blockade to cell cycle progression is dependent on kinds of tumor cell lines.

FK 973, a new substituted dihydrobenzoxazine, was obtained by chemical modification of a novel antibiotic which was isolated from the fermentation products of streptomyces sandaensis No. 6897. FK 973 had cytotoxic effects against in vitro cultured human and murine glioma cells. The concentration of FK 973 required to inhibit cell growth by 50% was 0.06-5 micrograms/ml, after 2-day exposure of this drug against human glioblastoma (ONS-6, 12, 23, and ONS-12/ACNU), human medulloblastoma (ONS-76, 81), human neuroblastoma (ST), and murine glioblastoma (RSV-M glioma). FK 973 showed antitumor efficacy in the meningeal gliomatosis models by RSV-M glioma cells. The median survival time (MST) of models treated by FK 973 (i.t.) was 30 days. However, the MST of control group was 23 days. In the in vitro neurotoxicity test, FK 973 proved to be slightly more toxic than ACNU and MTX, but it had no crucial problems, compared with ADM.