Experimental studies on biochemical modulation targeting topoisomerase I and II in human tumor xenografts in nude mice.

Topoisomerase (topo) I and II are nuclear enzymes which are novel targets of cancer chemotherapy. A new camptothecin (CPT) analog, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]carbonyl-oxy-CPT (CPT-11), is a topo-I inhibitor with a higher activity and less toxicity than CPT. To investigate topo-I and -II-targeting chemotherapy in an in vivo model, we studied the effect of sequential or co-treatment using CPT-11 and adriamycin (ADR) a topo-II inhibitor, in 6 human tumor xenografts (2 esophageal, 2 gastric and 2 colon tumor lines). In sequential treatment, adriamycin was administered i.v. 24 hr after CPT-11 treatment, and no antagonistic effect of this treatment schedule was observed. ADR cytotoxicity was potentiated significantly by CPT-11 pretreatment in the case of 2 esophageal and 2 gastric tumor lines and 1 colon tumor line. On the other hand, co-treatment abolished the sensitivity to CPT-11 and ADR in all 6 tumor lines. Moreover, CPT-11 did not significantly enhance the cytotoxicity of other agents tested, including mitomycin C (MMC) and cisplatin (CDDP). Flow cytometry and dot-blot analyses showed that CPT-11 pretreatment induced an increase in the S-phase cell population with an increase of topo-II mRNA expression after 24 and 48 hr, respectively, in the esophageal and colon tumor lines. These results suggest that CPT-11 can modulate topo-11 levels to enhance the effect of topo-II inhibitors in some human tumors, and this suggests a new clinical method of topo-I and -II targeting chemotherapy for human solid tumors.

Factors contributing to adriamycin sensitivity in human xenograft tumors: the relationship between expression of the MDR1, GST-pi and topoisomerase II genes and tumor sensitivity to adriamycin.

To study the factors contributing to tumor sensitivity to adriamycin (ADR) in vivo, the relationship between mRNA expression of the MDR1, GST-pi and topoisomerase II genes and tumor response to ADR was examined in six human xenograft tumors derived from two esophageal, two gastric and two colon cancers. A significant tumor response to ADR was observed in two esophageal xenograft tumors of six tumor lines, and one gastric tumor partially responded to ADR. mRNA expression of the MDR1 and GST-pi genes was elevated in five tumor lines including three ADR responsive tumors, whereas mRNA expression of the topoisomerase II gene was detected in all six tested tumor lines. Topoisomerase II mRNA expression levels in ADR responsive tumors were higher compared with those of ADR unresponsive tumors. No significant relationship between mRNA expression of the MDR1 and GST-pi genes and ADR sensitivity was found. In contrast, topoisomerase II mRNA expression was significantly correlated with tumor sensitivity to ADR (p less than 0.01). Moreover, topoisomerase II mRNA expression was significantly correlated with the growth fraction (S-phase fraction) in the cell cycle kinetics (p less than 0.01). These results indicate that topoisomerase II mRNA expression in association with the high growth fraction may be an important in vivo factor to contribute to ADR sensitivity in human tumors.

[Loco-regional cancer therapy for hepatic metastasis].

UNLABELLED: We undertook a clinical evaluation of chemotherapy for hepatic metastasis of gastric, colorectal and breast cancer. Between 1980 and 1989, chemotherapy for hepatic metastasis of gastric cancer was performed in 96 cases. Between 1973 and 1989, chemotherapy for hepatic metastasis of colorectal cancer and breast cancer was performed in 40 and 14 cases. RESULTS: (1) In hepatic metastasis of gastric cancer, the 50% survival period was 149 days in local injection therapy, 132 days in arterial infusion therapy and 117 days with no chemotherapy. There was no significant difference in the survival period in gastric cancer. (2) In hepatic metastasis of colorectal cancer, the 50% survival period was 445 days in arterial infusion therapy, 206 days in local injection therapy and 96 days with no chemotherapy. The survival period with hepatic metastasis of colorectal cancer that had undergone chemotherapy was longer than for no chemotherapy. (3) In hepatic metastasis of breast cancer, arterial infusion therapy was more effective, and the survival period was prolonged significantly.

[Study on the intra-tumor tissue concentration of zinostatin stimalamer in the liver following intra-arterial injection of zinostatin stimalamer (YM881) suspension].

A suspension of zinostatin stimalamer (YM881), an antitumor protein antibiotic in iodized poppy oil-fatty acid ester, was injected into the hepatic artery of 2 patients who received surgical treatment of hepatic tumors. Single doses of 4 mg of zinostatin stimalamer were injected into the hepatic artery by Seldinger's method. Two and 4 weeks after the dose, tissue samples were collected from the tumor and non-tumor regions close to and distant (normal tissues) from the tumor, and zinostatin stimalamer concentrations in these tissues were assayed by RIA. In one of the two patients, plasma concentrations of the drug were also assayed. Concentrations of zinostatin stimalamer in the tumor tissues (381.8 ng/g) were about 55 times greater than those in the normal tissue (6.9 ng/g) in patient No. 1 (week 4), and about 17 times in patients No. 2 (13.9 ng/g in the normal tissue and 229.8 ng/g in the tumor tissues in week 2). Plasma concentrations were assayed in patient No. 2, and were 510.3 ng/ml after one hr., 385.3 ng/ml after 3 hrs., 184.4 ng/ml after 6 hrs., and 45.4 ng/ml after 24 hrs., with a half life of 7.1 hrs. These results indicate that zinostatin stimalamer suspended in iodized poppy oil fatty acid ester persisted in the hepatic tumor tissues at the high concentrations, when injected into the hepatic artery, but was eliminated fairly rapidly from the plasma.

[Clinical studies of in vitro chemosensitivity test evaluated by ATP assay of gastrointestinal cancer].

In order to determine the most effective anticancer agent for individual human tumor, we have performed several chemosensitivity tests, such as human tumor clonogenic assay (HTCA), succinic dehydrogenase inhibition test (SDI-T), nude mouse isotope assay (NM-IA) and subrenal capsule assay (SRCA). In this study, an novel in vitro chemosensitivity test (ATP-assay) measuring ATP amounts of cancer cells was carried out in 69 fresh gastro-intestinal tumors obtained at surgery. As the results, the evaluable rate of ATP assay was 87.0%. The positive rate of ATP assay against all tumors were 13.3% in mitomycin-C (MMC), 11.7% in adriamycin (ADM), 13.3% in 5-fluorouracil (5-FU) and 18.3% in cis-diamminedichloroplatinum (CDDP), respectively. Overall predictive accuracy rate was 82.8%. The comparative study of the survival rates of the patients with stage IV gastric cancer, receiving sensitive anticancer agents assayed by ATP assay, and those receiving negative anticancer agents revealed that the survival rate of the patients treated with sensitive drugs was longer with Kaplan-Meier analysis. From these results, it seems reasonable to conclude that ATP assay is of value in determining the chemosensitivity of gastrointestinal cancer in each patient.

Prediction of the resistance of human tumors to adriamycin by chemosensitivity tests and DNA analysis of the multidrug resistance gene.

In order to predict natural resistance to Adriamycin (ADM), the amplification of multidrug resistance gene 1 (MDR1) was investigated in 50 human cancer specimens using Southern blot analysis. Genomic DNA was extracted from both human solid tumors and adjacent normal tissues for the analysis. MDR1 gene amplification was not observed in any of the patients tested, including 5 patients in whom ADM was not clinically effective. On the other hand, chemosensitivity tests performed on the tumor cells of these 5 patients indicated resistance to ADM. Our results therefore indicate that MDR1 gene amplification is rarely seen among clinical samples and that conventional chemosensitivity tests might be more useful for the prediction of ADM resistance in cancer patients than the analysis of MDR1 gene amplification.

Antitumor effect of KW2149, a new mitomycin derivative, administered by different modalities.

The effectiveness of a new mitomycin derivative, KW2149, against human tumors was evaluated by the 4 days subrenal capsule assay (SRCA) and the nude mice screening assay (NMSA). Evaluation by the SRCA showed a 50% response rate at a maximum dose of 3.8 mg/kg for 3 consecutive days. When evaluated by NMSA, the response rate was 100, 75 and 25% after the intermittent administration of 7.5, 5.6 and 4.5 mg/kg (q4dx3) respectively. Although the efficacy was reduced when mice were administered a single dose equivalent to the intermittent one, the new analog was along more effective than MMC administered by either modality.

The histological assessment and evaluation of a 4 day subrenal capsule assay by the percentage inhibition of DNA/protein.

A four day subrenal capsule assay was investigated in order to determine its ability to clinically predict tumor chemosensitivity. To establish more objective and accurate evaluation criteria, a histological assessment and measurement of the DNA and protein content of excised tumor implants was conducted in ddY mice. The histological studies provided qualitative results concerning the percentage of cancer cells in the xenograft, the number of mitoses, the amount of necrosis, and the extent of lymphocytic infiltration. The DNA content was measured by a modified version of the Schmidt-Thannhauser-Schneider method and the protein content was estimated using the Bio-Rad protein assay. The percentage of cancer cells in the xenograft correlated poorly with the relative increase in tumor size, weight and the percentage inhibition of DNA/protein (per cent DNA/protein), however, the per cent DNA/protein correlated well with the clinical effects in 85.7 per cent of the tumors studied. Moreover, the histological assessment information was only consistent with those results obtained for per cent DNA/protein in the control group.

Chemosensitivity tests in colorectal cancer patients.

In order to assess the usefulness of chemosensitivity tests in the treatment of colorectal cancer, 71 tumor specimens were tested for chemosensitivity in the following assays: nude mouse isotope assay (NMIA), subrenal capsule assay (SRCA), human tumor clonogenic assay (HTCA) and adenosine triphosphate inhibition assay (ATPA). The agents examined were: mitomycin C (MMC), 5-fluorouracil (5-FU), cyclophosphamide (CPM), adriamycin (ADM) and cis-diamminedichloroplatinum (CDDP). The evaluability rates were 90.8, 93.9 and 92.3 per cent in NMIA, SRCA and ATPA, respectively, but only 42.9 per cent in HTCA. The tumor response rates were 50.8, 45.2, 16.7 and 33.3 per cent in NMIA, SRCA, HTCA and ATPA, respectively. Individual drug sensitivity rates differed among all 4 assays, ranging from 0 to 33.3 per cent. In the arbitrary judgment of the 4 assays, the most sensitive agent was CDDP, followed by CPM, ADM, 5-FU and MMC. In the prospective study, predictive accuracy rates of the clinical responses were 81.3, 66.7, 100, 100 and 76.5 per cent in NMIA, SRCA, HTCA, ATPA and the arbitrary judgment, respectively. A significant correlation between the survival time and the results of SRCA was detected retrospectively. These results suggested that colorectal cancer might not be completely resistant to anticancer agents, and that chemosensitivity tests might be useful in the individual therapy of colorectal cancer patients.

[Comparative study on the anticancer activities of KW2149 and mitomycin C against human tumor xenografts using subrenal capsule assay].

The anticancer activity of KW2149, a new derivative of mitomycin C (MMC), was investigated against 5 human tumor xenografts derived from digestive organs using 4-day subrenal capsule assay (SRCA). Normal immunocompetent mice were used in this assay. For the comparative study, KW 2149 and MMC were administered intraperitoneally for 3 days after implantation, and the anticancer activity and the weight loss of mice were evaluated. The total doses were determined as 1/2, 1/3 and 1/4 of LD50 value of each anticancer agent. The anticancer activities of the two drugs were almost the same with no significant difference in 3 xenografts. Thus, it may be suggested the difference of the anticancer spectrum between the two drugs. The anticancer activity of KW2149 indicated higher correlation with the administered doses as compared with MMC. The toxicity of KW2149 was almost the same as MMC according to the weight loss of mice.

[Comparison of succinic dehydrogenase inhibition test with adenosine triphosphate inhibition assay for human solid tumors as in vitro chemosensitivity tests].

In order to determine the most effective anticancer agents for individual human tumor, succinic dehydrogenase inhibition test (SDI-T) and adenosine triphosphate inhibition assay (ATP-A) as in vitro chemosensitivity tests were performed. Fifty tumors and 57 tumors derived from cancer patients surgically methods were examined by SDI-T and ATP-A respectively. As the results, the evaluable rate was 70% by SDI-T and 94.7% by ATP-A, respectively. With SDI-T, the positive rate against all tumors was 51.4% in mitomycin-C (MMC), 42.9% in adriamycin (ADM), 20.0% in 5-fluorouracil (5-FU), 54.3% in cis-diamminedichloroplatinum (CDDP). On the other hand, with ATP-A, that was 20.4% in MMC, 29.5% in ADM, 20.6% in 5-FU, 20.4% in CDDP, respectively. Retrospective and prospective clinical trials were also carried out to determine the usefulness of both assays. With SDI-T, overall predictive accuracy rate was 57.1% while with ATP-A that was 88.9%. Furthermore, the rates of sensitivity for the same tumors using SDI-T and ATP-A were compared. The rate of the same sensitive cases in both assays were 30% with MMC, 70% with 5-FU, 42.1% with ADM, 36.8% with CDDP, respectively. In conclusion, it is suggested that ATP-A was more useful than SDI-T as in vitro chemosensitivity test to determine the most adequate drug for cancer patients.

[Experimental study of normal liver regeneration in hepatic arterial infusion with degradable starch microspheres (DSM)].

Influence of hepatic arterial infusion with degradable starch microspheres (DSM) or with anti-cancer drug or with both for normal liver regeneration in rat was determined by histological and microautoradiographical examination and liquid scintilation before or after 70% hepatectomy. DSM was seen in interlobular artery in early phase after hepatic arterial infusion, but disappeared into the central vein by degrees for 2 hours later. Adriamycin (ADM) in serum was detected for a long time after hepatic arterial infusion with DSM + ADM. In the heart muscle, a significantly lower concentration of ADM was seen in the DSM + ADM group than in the ADM only group. In the case of hepatic arterial infusion with DSM only after hepatectomy, there was no influence on normal liver regeneration. No significant difference was seen between hepatic arterial infusion and intravenous infusion with ADM only. However, in the case of hepatic arterial infusion with DSM + ADM after hepatectomy, a significant difference was seen in the inhibition of liver regeneration from any other groups.

[Combination chemotherapy of CPM-MTX-5-FU in non-resectable and recurrent cancer patients].

Fifty-two non-resectable and recurrent cancer patients with prior treatment, were entered in this study; 1 esophageal, 33 gastric, 1 duodenal, 4 colorectal, 2 pancreatic, 2 bile duct, and 9 breast cancer. The protocol of this therapy was as follows: On day 1, 500 mg/body cyclophosphamide (CPM) was administered by drip infusion, and on day 2, 200 mg/m2 methotrexate (MTX) was infused intravenously for 30 min; immediately after, 500 mg/body 5-fluorouracil (5-FU) was injected by bolus infusion for 5-10 min. On day 3, 24 hours after MTX administration, leucovorin rescue was added. This combination chemotherapy was repeated every two weeks. As a result, 35 of 52 patients were evaluable and the response rate (CR + PR) was investigated; 2/21 (9.5%) for gastric, 2/7 (28.6%) for breast, and 0% for miscellaneous. As complications for side effect, general fatigue, anorexia, nausea, vomiting and stomatitis were observed symptomatically, and leukopenia and thrombocytopenia were recognized in laboratory data as dose limiting factors.

[Evaluation and problems of second-look operations in resectable gastric cancer].

During the period from June 1973 to December 1985, one thousand and ninety-seven patients with primary gastric cancer have been operated on in our Dept. of Surgery. Of the 1097 gastric cancer patients, 59 were reoperated and evaluated for chemotherapeutic effects. The cases were 21/548 (3.83%) for absolute curatively resection, 16/202 (7.92%) for relative curatively resection, 9/64 (14.0%) for relatively noncurative resection and 13/283 (4.59%) for absolutely noncurative resection, respectively, The median survival period from primary gastrectomy to second look operation was less than 2 years, and the prognoses were not very good. The factors of relaparotomy were peritoneal dissemination 44/59 (78.6%), invasion to contiguous structures, lymph node metastases and liver metastasis. Those with local recurrence or remnant stomach cancer could be resected in 5 cases, and one patient was well more than 2 years following surgery. The reoperation procedures were reconstruction of artificial anus, intestinal anastomosis or intestinal fistula. Some 7 of 59 patients were found to be entirely beyond surgical aid at the second look operation and were administered large-dose OK-432 or ADM patch method. These methods are suggested for their usefulness for peritoneal dissemination.

[Inhibition of thymidylate synthetase and antiproliferative effect by 1-hexylcarbamoyl-5-fluorouracil].

In order to estimate tumor chemosensitivity of fluoropyrimidine derivative, inhibition of thymidylate synthetase (TS) was investigated using a nude mouse experimental system. Four human tumors xenografted in nude mice; H-111, SH-8 and SH-10, each established from gastric cancer, and EH-1 from esophageal cancer, were used. When the transplanted tumor volumes reached to approximately 200 mm3, 1-hexylcarbamoyl-5-fluorouracil (HCFU) was given for 5 days. Tumors was removed for the measurement of total and free TS at 0 hr, 6 hrs and 24 hrs after the last administrations. Simultaneously, the anti-proliferative effects were investigated according to the therapeutic protocol of NCI. No positive correlation between the inhibition rate of TS and the anti-proliferative effects was observed, although the absolute values of free TS were similar to the tumor inhibition rates. The measurement of total TS provided a highest concentration in SH-8, while extremely low in EH-1. On the analysis of free TS, a significant increase of the concentration was observed at 24 hrs after the last administration compared with at 6 hrs in SH-8. These results indicate that free TS had a potentiality as a new parameter for predicting tumor chemosensitivity of fluoropyrimidine derivative and the analysis of TS should be affected strongly by the characteristics of enzymic activity of examined tumor.

[Anticancer activities of a new mitomycin derivative KW 2149, against human tumors xenografted into nude mice].

Anticancer activity of KW 2149, a new derivative of mitomycin C (MMC), was investigated using 4 human tumors xenografted into nude mice. The basic methodology was essentially the same with NCI's therapeutic protocol. For the comparative study, KW 2149 or MMC was administered intraperitoneally at a schedule of q4d X 3. Daily doses were determined as a 1/3, 1/4 and 1/5 of LD50 value of each anticancer agent (7.5 mg/kg, 5.6 mg/kg and 4.5 mg/kg for KW 2149, and 2.7 mg/kg, 2.1 mg/kg and 1.7 mg/kg for MMC). Anticancer activity of KW 2149 seemed to be dependent on the doses. Comparing with MMC, KW 2149 produced higher response rates at the doses of 1/3 and 1/4 of LD50 and was less toxic judging from the decrease of the body weight. This study may indicate an utility of KW 2149, as a new anticancer agents, or suggest the difference of anticancer activities between these two agents.

[Combined effects of interferon alpha-A/D with fluoropyrimidine derivatives in the subrenal capsule assay].

The combined effects of interferon alpha-A/D (IFN alpha-A/D) with 5-fluorouracil and fluoropyrimidine derivatives such as 1-(2-tetrahydrofuryl)-5-fluorouracil (tegafur), UFT, 1-hexylcarbamoyl-5-fluorouracil (HCFU) and 5'-deoxy-5-fluorouracil (5'-DFUR), were examined by 4-day subrenal capsule assay. Four human tumor xenografts serially transplanted in athymic mice, H-111, SH-10 established from gastric cancers, CH-4 and CH-5 from colon cancers, were used. In this experiment, the adequate dose of each agent was estimated as 50 mg/kg for 5-FU, 473 mg/kg for tegafur, 433 mg/kg for UFT, 50 mg/kg for HCFU, 185 mg/kg for 5'-DFUR and 1 x 10(5) IU for IFN alpha-A/D, respectively. When synergistic, additive and subadditive effects were defined as positive combined effects, all combinations produced positive combined effects against H-111 and CH-5, while negative ones were observed for all combinations against CH-4. The combinations of 5-FU, HCFU and 5'-DFUR with IFN alpha-A/D produced synergistic effects against SH-10. These results indicate that the combination therapy of 5-FU and fluoropyrimidine derivatives with IFN alpha-A/D would be useful.

[Fundamental and clinical studies of intra-arterial chemotherapy combined with degradable starch microspheres (DSM)].

Degradable starch microspheres (DSM, Pharmacia) are specially formulated cross-linked starch microspheres about 45 microns in diameter. The microspheres are degraded by serum amylase and become progressively smaller with t1/2 for complete dissolution between 20 and 30 min. in vitro (in normal serum). In vivo, DSM-occluded microcirculation was observed in lobular artery of liver and disappeared 60 minutes after administration; then PAS-positive debris, which was suspected to be degraded DSM, was found in intrahepatic tissues. We also carried out comparative studies on regrowth liver in rats as an index of 3H-thymidine incorporation to determine the influence for surgical operation. DSM + adriamycin (ADM) was injected into intrahepatic artery at 2 and 5 days before partial liver resection. In DSM + ADM group, regrowth of liver tissue was inhibited with injection 2 days before, while injection 5 days before had no influence. Since the usefulness of DSM combined with anticancer agents had been indicated by the experimental results mentioned above, we carried out a clinical study (phage II). Chemoembolization therapy was undertaken in combination with DSM in 6 patients each with primary and metastatic liver cancers. Six cases were evaluated as Partial Response (PR). Recently, we performed clinical trials to investigate the combination with noradrenaline, too. Noradrenaline was suggested to enhance the effect of DSM.

Renal allograft dysfunction with acute hemorrhagic cystitis caused by adenovirus in a recipient of a transplanted kidney.

We experienced acute hemorrhagic cystitis with renal dysfunction in a recipient of a transplanted kidney. This acute hemorrhagic cystitis was caused by adenovirus type 11. Either elevation of antibody titers or isolation in urine of adenovirus type 11 was identified in six cases out of 11 in seven reports which we collected. Graft dysfunctions were complicated on seven cases out of 11 in these seven reports. These graft dysfunctions have been considered as an acute rejection, but we suggest that they may be graft nephropathy caused by adenovirus type 11 infection.