Temporary interruption of regional blood flow combined with local hyperthermia for cancer chemotherapy.

A striking chemotherapeutically curative effect on tumor was obtained by means of temporary interruption of regional blood flow combined with local hyperthermia. By analyzing various basic conditions required for this system using Ehrlich tumor implanted in the hind limbs of mice, the following were found to be essentially indispensable to obtain satisfactory chemotherapeutic effects: (a) a time interval of 1 to 3 min after systemic i.v. administration of drug to the mice, (b) use of a tourniquet on the tumor-bearing mouse limb to stop blood flow, and (c) warming at 37-41 degrees (d) for a period of at least 30 to 60 min. Among the chemotherapeutic drugs tested in the present study, Carbazilquinone (NSC 134679) was the most effective because it revealed the strongest antitumor effect despite its relative innocuousness to nontumorous adjacent normal tissues. Applying the present method, a large syngeneic mouse sarcoma transplanted to the limb 7 days before the experiment also completely regressed in 6 of 9 mice.

Neural networks applied to quantitative structure-activity relationship analysis.

An application of the neural network to quantitative structure-activity relationship (QSAR) analysis has been studied. The new method was compared with the linear multiregression analysis in various ways. It was found that the neural network can be a potential tool in the routine work of QSAR analysis. The mathematical relationship of operation between the neural network and the multiregression analysis was described. It was shown that the neural network can exceed the level of the linear multiregression analysis.

Increased therapeutic efficacy of intra-arterial carboquone chemotherapy on a limb tumor in rats, by using an acidic vehicle adjusted with lactate.

We previously reported that the cytotoxicity of carboquone (CQ) was potentiated in vitro and in vivo under acidic conditions. In this study, an acidic vehicle adjusted with lactate at various low pHs was used for CQ intra-arterial (i.a.) injection, in order to enhance the antitumor effects of i.a. CQ chemotherapy. Treatments were evaluated in Wistar/KA rats bearing a limb tumor 5 days after the inoculation of 3 x 10(6) syngeneic RBT-1 tumor cells into the hind limb. In chemotherapy experiments using an intrafemoral injection of CQ at 1.5 mg/kg in phosphate-buffered saline (PBS, pH 7.4) or in an acidic vehicle at pH 5.0 or 6.0, the antitumor effects seen in rats given CQ in acidic vehicles, evaluated by tumor weight 14 days after treatment, were significantly greater than that seen in rats given CQ in PBS. There were no significant differences either in changes of body weight or in the number of leukocytes after treatment between the groups given CQ in PBS and in an acidic vehicle at pH 6.0. Although in the group given CQ at 2.0 mg/kg in PBS, the antitumor effect was the same as that observed in rats given CQ at 1.5 mg/kg in an acidic vehicle at pH 6.0, the side effects observed in the former group were much severer than in the latter group. These data suggest that the antitumor effect of i.a. CQ chemotherapy can be potentiated by using an acidic vehicle.

Generation of hydroxyl radical by anticancer quinone drugs, carbazilquinone, mitomycin C, aclacinomycin A and adriamycin, in the presence of NADPH-cytochrome P-450 reductase.

The generation of hydroxyl free radicals in the system consisting of purified NADPH-cytochrome P-450 reductase and anticancer quinone drugs, such as carbazilquinone, mitomycin C, aclacinomycin A and adriamycin, has been confirmed by two methods. In the spin trapping study, using N-tert-butyl-alpha-phenylnitrone as the spin trapping agent, four drugs generated hydroxyl radical-trapped signals, and the formation of the spin adduct was dependent on time and the enzyme concentration. Among the four drugs, the generation time of signal was in the order of carbazilquinone, aclacinomycin A, adriamycin and mitomycin C, but the magnitude of signal intensity was different. In both aclacinomycin A and adriamycin, the signal disappeared in a few minutes. Catalase completely inhibited the formation of the spin adduct, while superoxide dismutase did not significantly inhibit, but effected in some manner. The generation of hydroxyl radical was also confirmed by the ethylene production from methional. Among the four drugs, the order of the magnitude of ethylene production was different from that of signal intensity by ESR study. Catalase potently inhibited the ethylene production, while superoxide dismutase effected in some manner. From these results, the interactions of anticancer quinone drugs with NADPH-cytochrome P-450 reductase and oxygen, and the possible relations of the enzymes to the radical related actions of these drugs are discussed.

The difference in chemosensitivity to antineoplastic agents of human hepatocellular carcinoma cells under normo-oxygenated or hypoxic conditions.

In order to select more effective drugs under hypoxia for the treatment of hepatocellular carcinoma, the cytotoxicity of antineoplastic agents for two hepatoma cell lines, PLC/PRF/5 and HuH-7, was examined under both oxygenated and hypoxic conditions. Mitomycin C was observed potentially to have enhanced cytotoxicity under hypoxic conditions for both hepatoma cell lines. Carboquone showed enhanced cytotoxicity under hypoxia for PLC/PRF/5 alone. On the other hand, there was no cytotoxic enhancement of adriamycin or cisplatin in either cell line. Thus, the sensitivity of tumour cells to the cytotoxic agents altered according to the conditions to which the tumour was exposed. The selection of the antineoplastic drugs for chemotherapy therefore should depend not only on the sensitivity of individual tumours to various drugs, but the alteration of the cytotoxicity of the drugs under certain conditions should also be carefully taken into account.

Sarcoma-180 cells and human colorectal tumor cells under in vitro hypoxic conditions are more sensitive to mitomycin C and carboquone.

The effects of oxygen concentration on the chemosensitivity of mouse sarcoma-180 (S-180) cells and human colorectal cancer tissues to mitomycin C (MMC) or carboquone (CQ) were determined in vitro, since evidence had been obtained that these drugs are more effective in HeLa cells. The results were as follows: (a) S-180 cells exposed to various concentrations of MMC or CQ for 2 h under conditions of normal aeration (about 20%) or hypoxia (5.0% and 0%) were then maintained under normal conditions of aeration for 3 days. Change of viability was assessed by succinate dehydrogenase (SD) activity. With exposure of the cells to MMC or CQ, under anoxic conditions (O2:0%), SD activity decreased to a greater extent than seen in the control cells. The value for CQ was from 61.5% to about 36.2%. (b) The decrease in the SD activity of 20 colorectal cancer tissues kept under conditions of anoxia was compared with findings under normally aerated conditions, following exposure to 30 microM of MMC or 1.6 microM of CQ. On exposure to MMC or CQ under anoxic conditions, SD activity decreased significantly, compared with normally aerated conditions (P less than 0.001 for MMC; P less than 0.05 for CQ). As colorectal cancer is less sensitive than other tissues to various chemotherapeutic agents, we recommend that MMC and CQ be prescribed to treat patients with these malignant lesions.

Hypoxia and acidity increase the cytotoxicity of mitomycin C and carboquone to human tumor cells in vitro.

To clarify the influence of hypoxic and acidic environments on the cytotoxicity of mitomycin C (MMC) and carboquone (CQ), the cytotoxicity was assessed based on decreases in intracellular ATP levels of HeLa cells exposed to the drugs at various acidic pH (pH 5.8-6.8) and low oxygen tension (5% and 0% O2) in vitro. Hypoxia and acidity individually increased the cytotoxicity of MMC, although hypoxia no longer increased it under acidic conditions. On the other hand, the cytotoxicity of CQ was enhanced in the presence of hypoxic and acidic conditions, and even more so with their combination. We conclude that acidity is a determining factor and excels hypoxia in influencing the cytotoxicity of MMC and that these two factors act synergistically on CQ.

How to see characteristics of structural parameters in QSAR analysis: descriptor mapping using neural networks.

In addition to its outstanding abilities in both classification and fitting, the neural network can also accurately predict the values of the untrained region. To rationalize this ability of prediction, the authors mathematically discussed the valid region of prediction. Based on such a background, the authors proposed "descriptor mapping" in the QSAR analysis, which visualizes the nonlinear dependencies between structural parameters. A variable of the linear multiple regression analysis in the QSAR study is supposed to be linear to the biological intensity and is independent of other variables. Analysis by the descriptor mapping method discloses the reality.

[An in vitro chemosensitivity study using the human tumor clonogenic assay in urological malignancies: a preliminary report].

Surgical tumor specimens from 67 urological malignancy patients were subjected to a human tumor clonogenic assay (HTCA) developed by Hamberger and Salmon. Appreciable growth of colonies was obtained in 20 of the 33 renal cancers, 20 of the 30 urothelial cancers and 1 of the 4 testicular cancers examined. Using HTCA, a plating efficiency ranging from 0.01 to 0.5% was obtained in these urologic malignancies. However, colonial growth adequate for chemosensitivity was obtained in 30 of these 67 patients. According to Von Hoff's definition, more than a 70% decrease in the plating efficiency after anticancer drug exposure was defined as susceptible. Susceptibility to vinblastine (VBL) was seen in 4 of the 11 patients with renal cancer. Susceptibility to cis-dichlorodiamine platinum (CDDP) was seen in 4 of the 15 patients with urothelial cancer, 1 of the 4 patients with renal cancer, and that to adriamycin (ADM) was seen in 3 of the 15 patients with urothelial cancer, 2 of the 10 patients with renal cancer and 1 patient with testicular cancer. For comparison, the ratio of IC90 to the peak plasma concentration of the drug tested was used as the "in vivo-in vitro therapeutic index (TI)". According to TI, susceptibility to VBL was seen in 3 of the 7 patients with renal cancer, and that to CDDP was seen in 2 of the 12 patients with urothelial cancer, and 1 of the 2 patients with renal cancer. Susceptibility to ADM was seen in 3 of the 15 patients with urothelial cancer, and 1 of the 6 patients with renal cancer.(ABSTRACT TRUNCATED AT 250 WORDS)

[An in vitro chemosensitivity study using human tumor clonogenic assay in urologic malignancies].

A soft agar colony formation assay, so called human tumor clonogenic assay (HTC assay) similar to that originally described by Salmon and colleagues, was utilized to measure the sensitivity of a total of 85 urologic malignancies including 36 urothelial cell carcinomas, 41 renal cell carcinomas, 5 testicular tumors, and 3 Wilms' tumors to anticancer drugs. In addition, the results obtained were compared with those of a novel dye exclusion method (NDE assay) described by Weisenthal and colleagues. The NDE assay was utilized to measure the sensitivity of a total of 63 urologic malignancies including 28 urothelial cell carcinomas, 25 renal cell carcinomas, 6 testicular tumors, and 4 Wilms' tumors to anticancer agents. In both assay series, the concentration of anticancer drugs tested was approximately one tenth of the maximum serum level achievable after single bolus injection. The colony forming rate inhibition of 70% or more in the HTC assay and the cell survival rate of 30% or less in the NDE assay were defined as "sensitive." Sixteen of the 36 urothelial cell carcinomas, 11 of the 41 renal cell carcinomas, and 1 of the 5 testicular tumors had both more than 30 colonies grown in control plates and enough cells in the specimens to provide at least one drug sensitivity testing. In urothelial cell carcinomas, 3 out of 13 tumors were "sensitive" to adriamycin, 3 out of 16 to cis-platinum, and 4 out of 15 to carboquone. In renal cell carcinomas, 2 out of 9 tumors were "sensitive" to adriamycin, 4 out of 11 to vinblastine, and none of 4 to Interferon alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

[An anticancer drug--carboquone].

Carboquone (CQ) is an anticancer alkylating agent synthesized and developed by Arakawa et al. (Sankyo Co, Ltd.) in 1970, having chemical structure, 2,5-bis-(1-aziridinyl)-3-(2-carbamoyloxy-1-methoxyethyl)-6-methyl- 1,4- benzoquinone. The antitumor efficacies of CQ were reported as excellent, however, the side effects are considerably strong. For the purpose to increase the effectiveness and to eliminate the side effect, various treatment regimen with CQ have been reported. Combination chemotherapies including CQ and cis-Platinum etc. have been reported to increase the antineoplastic activity and CQ combined with immunopotentiator or prednisone have been reported to diminish the side effects. The regimens of PPQ therapy in our department is as follows. CQ is given 7 mg/m2 iv on day 1; cis-Platinum 20 mg/body, drip infusion on day 1-5. Prednisone 3.0 mg p.o. on day 1-5. The response rate found in this regimen was about 30% so far. Antitumor spectrum of this drug has been reported to become broad.

[Effect of carboquone (esquinone) on the development of urinary bladder tumors in rats induced by N-butyl-N-(4-hydroxybutyl) nitrosamine].

The effect of carboquone (CQ) by intraperitoneal administration on the development of urinary bladder tumors in Wistar strain male rats induced by N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) was studied. Urinary bladder tumors were induced in 9 of 11 rats (81.8%) when they were given 0.05% BBN in drinking water for 8 weeks and then given water without BBN for 12 weeks. When CQ 0.25 mg/kg/day was administered intraperitoneally for 7 days after treatment with 0.05% BBN for 8 weeks, incidence of urinary bladder tumors was 19 of 26 rats (73.1%). When CQ 0.25 mg/kg/weekly was given for 12 weeks after treatment with BBN, incidence of urinary bladder tumors was 17 of 27 rats (63.0%). When CQ 0.5mg/kg. B.W./day was given for 7 days after treatment with BBN, tumors developed in the urinary bladder with low incidence as in 8 of 20 rats (40.0%) (P 0.1). When CQ 0.5mg/kg/weekly was given for 12 weeks after treatment of BBN, urinary bladder tumors were induced in 17 of 22 rats (77.3%). Hematotoxity was not observed in any animals treated with CQ. These results showed that CQ inhibited the development of urinary bladder tumors induced by BBN in rats.

[Effect of antibody to rat alpha-fetoprotein (AFP) on protein and DNA synthesis of rat ascites hepatoma AH66 cells].

Effects of horse antibody (purified IgG) to rat AFP on synthesis with both protein and DNA of rat ascites hepatoma cells were examined. The addition of antibody to the cultures showed a decrease in protein synthesis within several hours; whereas, it showed a transient increase in the incorporation of thymidine into tumor cells approximately in 6-12 hours both in vitro and in vivo. Eventually within 24-48 hours, more than 50% of the cells in culture were killed and inhibited the synthesis of DNA by the antibody treatment. The stimulatory effect of antibody on the synthesis of DNA in the tumor cells was more obviously observed in synchronized cells which were capable to obtain by the treatment with thymidine followed by hydroxyurea. The synchronized tumor cells cultivated with antibody containing medium also exhibited the shortening of the cell cycle from 22 to 12-20 hours. Based on these effects of antibody on tumor cell growth, sequential administration of antibody followed by an anticancer drug (carboquone) was proven to be the most effective immunochemotherapeutic schedule for tumor bearing rats, showing 25-33% long-term survival in comparison with any other controls.

[In vitro sensitivity test of a cultured human ovarian cancer cell line to anticancer agents].

In vitro tests were performed to assess the sensitivity to six anticancer agents-ACT-D, ADM, CDDP, CQ, 5-FU and MMC-of a JOHYL-1 (ascites type) cell line from human dysgerminoma which was used as challenge strain. For comparative assessment, anticancer sensitivity was expressed as the ratio of IC50 and IC90 to LD50 (i.v.) in mice. Drug dose-response and time-response curves were plotted, and the IC50 ratio was calculated, for each test compound in order to investigate the mechanism of anticancer action. The results obtained were as follows. CQ proved to be remarkably active and ADM fairly active against JOHYL-1 (ascites), but 5-FU, CDDP and MMC varied remarkably with the parameter of measurement employed. Analysis of IC50 ratio data and patterns of cell growth inhibition indicated the growth-inhibitory effect of CDDP to be concentration-and time-dependent. The results of the present study are in close accord with the pattern of action reported in the literature.

[Chemosensitivity test for gastric cancer by in vitro MTT assay].

In vitro MTT assay was applied for examining chemosensitivity with 104 samples; 56 primary tumors, 31 lymph node, 9 liver, and 8 peritoneal metastases, obtained from 87 patients with advanced gastric carcinoma. The rate of effectiveness of various anticancer drugs were as follows; etoposide, 87.7%; cisplatin, 55.1%; mitomycin C, 51.5%; pirarubicin, 50.0%; aclarubicin, 48.8%; carboquone, 31.8%; doxorubicin, 20.3%; and 5-fluorouracil, 12.9%. Etoposide was found to be most effective against gastric carcinoma in this test. Concerning with the metastatic lesions, liver metastases were resistant to all tested drugs. On the other hand, peritoneal metastases were sensitive to etoposide, mitomycin C, and pirarubicin. The results indicate heterogeneity of the chemosensitivity between primary and metastatic lesions, and it was supposed that etoposide might be useful against human gastric cancer.

[Flow cytometric DNA analysis and chemosensitivity in squamous cell carcinoma of the head and neck].

Cellular DNA content and succinate dehydrogenase activity of 92 human head and neck (34 laryngeal, 24 pharyngeal, 21 oral cavity, 13 maxillary) squamous cell carcinomas were examined, and DNA ploidy status and chemosensitivity were analyzed and compared. DNA aneuploidy was observed in 54 tumors (58.7%). The aneuploid pattern was most common in tumors of the maxillary sinus (84%), and least common in tumors of the larynx (41.3%). Histologically, aneuploidy was detected in 71.4% of poorly-differentiated, 63.8% of moderately-differentiated and 37.5% of well-differentiated squamous cell carcinomas. There was a statistically significant difference between the survival rates of patients with diploid and aneuploid patterns. Chemosensitivity was determined by exposing fresh tumor material to five antitumor drugs: adriamycin (ADM), cisplatin (CDDP), carboquone (CQ), 5-fluorouracil (5-FU) and mitomycin C (MMC). The average decrease in succinate dehydrogenase (SD) activity was 49.8% with ADM, 33.6% with CDDP, 39.9% with CQ, 68.4% with 5-FU and 45.5% with MMC. Histologically, poorly-differentiated squamous cell carcinomas were most sensitive to these five antitumor drugs. We also compared average SD activity in tumors from different organs and found that pharyngeal tumors tend to be most sensitive to these drugs, except for MMC. The chemosensitivity of a tumor with DNA diploidy tended to be higher among well- and moderately-differentiated squamous cell carcinomas. In contrast, tumors with DNA aneuploidy tended to have higher chemosensitivity in the poorly-differentiated type. The results of this study indicate that simultaneous analysis of DNA ploidy and chemosensitivity will be helpful in understanding the characteristics of tumors as well as in predicting the most effective chemotherapy agents for head and neck cancer patients.

[Detection of cellular DNA strand breaks induced by antitumor drug and heat using in situ nick translation].

Cellular DNA strand break induced by an alkylating agent: Carboquone (CQ), and heat (43 degrees C) was detected in HeLa cells in vitro and mouse sarcoma-180 cells in vivo. The break sites in the DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and [3H]-labeled dTTP and sites in the DNA were visualized by autoradiographic observation of grains in the nuclei. These breaks increased in a dose and time dependent manner, compared to findings in the control cells. Our findings show that the surviving response of cells decreases while the level of DNA strand breaks increases following exposure to CQ or heat. The nick translation method is a rapid in situ assay for determining drug and heat induced DNA damage of tumor cells, under in vitro and in vivo conditions and in a semi-quantitative manner.

Carboquone alters the protein synthesis of Chinese hamster V79 cell.

The effect of carboquone (CQ) on protein synthesis in Chinese hamster V79 cells was determined. While the syntheses of higher molecular weight proteins decreased, the relative level of a 43-kilodalton (kd) protein increased following exposure to CQ at the concentration of 0.5 microgram/ml, in a time-related fashion, determined using one-dimensional gel electrophoresis. Using two-dimensional gel electrophoresis, the 43-kd protein may be actin protein, and the syntheses of another two proteins of 8.5/45 and 4.8/270 (designated isoelectric point/molecular weight, kd) were increased in the CQ-treated V79 cells. These changes of the proteins induced by CQ in V79 cells were noted in HeLa cells. Three CQ-induced proteins are candidates for the elucidation of the antineoplastic effect of CQ.

Detection of DNA strand breaks in HeLa cells in vitro and in mouse sarcoma 180 cells in vivo induced by an alkylating agent, carboquone, using in situ nick translation.

We determined the correlation between DNA strand breaks and the toxicity of carboquone (CQ) in HeLa cells in vitro and in mouse sarcoma 180 (S-180) cells in vivo, using in situ nick translation. The break sites in the DNA were translated artificially in the presence of Escherichia coli DNA polymerase I and [3-H]-labelled dTTP, and sites in the DNA were visualized by autoradiographic observation of grains in the nuclei. These breaks appeared as early as 5 min in the CQ-treated HeLa cells and increased in a dose- and time-dependent manner compared to findings in the control cells, i.e., 10.2-fold at 3 x 10(-6) M in 60 min. Strand breaks in the S-180 cells appeared in a dose- and time-dependent manner, i.e., 6.1-fold after the mice had been exposed to CQ (3.6 mg/kg) for 2 h. This level correlated with the increase in host life span. Our findings show that the survival response of cells decreases, while the level of DNA strand breaks increases following exposure to CQ. The nick translation method is a rapid in situ assay for determining drug-induced DNA damage of tumor cells, under in vitro and in vivo conditions and in a semiquantitative manner.