Harukazu Mashiba

Augmentation of in vitro cytotoxicity and in vivo tumor-inhibition by combined use of lymphotoxin-containing supernatants and antitumor drugs

The cytotoxicity of combined antitumor drugs and lymphotoxin (LT)-containing supernatants was studied on target cells. Marked reduction of L cells was observed after combined use of LT-containing supernatants with actinomycin D, adriamycin or aclacinomycin A but not with mitomycin C or vincristine sulphate. Similar effects were observed in Sarcoma 180 cells and Ehrlich ascites tumor cells in vitro. The survival of mice injected i.p. with Ehrlich ascites tumor cells was prolonged by the combined use of LT-containing supernatants and actinomycin D (0.1 or 0.3 microgram/mouse). The mechanisms of the synergistic effect are discussed in regard to the mode of action of antitumor drugs.

Augmentation of Antiproliferative and Antitumor Effect on Human Cancer Cells in Combined Use of Electroporation with a Plant Toxin, Saporin

Effect of electroporation (EP) in combination with a plant toxin, saporin, was studied using a human lung cancer cell line (PC9) and a pancreatic cancer cell line (ASPC-1). Target cells were electroporated in the presence of saporin and washed, and incubated for 72 hr. Proliferation inhibition in combination of EP and saporin was observed in parallel with the voltages and the saporin concentrations used. Proliferation of PC9 cells was completely inhibited at 1000 ng/ml of saporin in combination with EP (80-90 V, 10 ms, n = 8). High degree of proliferation inhibition was also obtained when ASPC-1 cells were electroporated in the presence of saporin (0.1-1000 ng/ml). PC9 or ASPC-1 tumor-bearing nude mice were treated with electroporation following the intratumoral injection of saporin (1 mg). Tumor necrosis was observed 24-48 hr after the combination therapy with saporin and EP. Six of nine mice with established PC 9 tumors and all the mice with established ASPC-1 tumors regressed completely 14 days and 6 days after the combination therapy, respectively.

Inhibition of Meth-A tumor cell proliferation in combined use of disulfiram with catalase

The effect of combined use of disulfiram with catalase on Meth-A tumor cell proliferation was studied. The simultaneous addition of 5 x 10(-7) or 1 x 10(-6) M disulfiram with catalase (4-40 micrograms/ml) induced marked inhibition of cell proliferation. A moderate degree of the antiproliferative effect was also obtained by pretreatment of the target cells with 5 x 10(-6) or 1 x 10(-5) M disulfiram in the presence of catalase (40 micrograms/ml). These results suggest that compounds or metabolites with cytostatic activity are newly formed following the reaction of disulfiram with catalase.

Augmented inhibition of MethA tumor cell proliferation in combined use of diethyldithiocarbamate with catalase or by a nondialysable fraction from co-incubation.

The antiproliferative effect of diethyldithiocarbamate (DDC), a metal chelator, in combined use with catalase on MethA tumor cells was studied. Marked augmentation of the antiproliferative effect was observed when 1 x 10(-7) M DDC was used in combination with catalase (0.004-40 micrograms/ml). Further augmentation of the cytostatic effect was obtained by the simultaneous addition of 2 x 10(-7) M DDC with catalase and more than 97% inhibition of [3H]thymidine uptake by target cells was observed. A nondialysable fraction from the co-incubation of DDC with catalase was also remarkably cytostatic to the target cells. Serum factor(s), probably metal ions, was suggested to be necessary for the induction of the nondialysable fraction with cytostatic activity. The activity was not nullified by the pretreatment of a nondialysable fraction with anti-catalase antibody. These results suggest that an active substance or compound exhibiting an antiproliferative effect on tumor cells might be newly formed as the result of the interaction of DDC with catalase.

Enhancement of radiosensitizing effect of the nitroimidazole derivative RK28 on the proliferation of MethA tumor cells in combined use with diethyldithiocarbamate.

The radiosensitizing effect of the nitroimidazole derivative RK28 and diethyldithiocarbamate (DDC), which is an inhibitor of superoxide dismutase activity, was examined in vitro by using Meth A tumor cells. The radiosensitizing effect of 0.5 mM RK28 was observed in both of 10 Gy and 15 Gy irradiated groups. The addition of 5 x 10(-7) M DDC also enhanced the radiation-induced proliferation inhibition. Marked enhancement of the antiproliferative effect was observed in combined use of 0.2 mM or 0.5 mM RK28 with 2 x 10(-7) M or 5 x 10(-7) M DDC. These results suggest that enhanced oxygen effect could be expected through combined use of the ionizing irradiation with both of these agents.

Proliferation Inhibition of Human Cancer Cells in Combined Use of Electroporation with Attenuated Diphtheria Toxin

Attempts to cause temporary pore formation of cancer cell membrane and to introduce intracellulary attenuated diphtheria toxin (fDT) following treatment with formaldehyde were performed utilizing high-voltage electric pulses. Human pancreatic cancer cell line (ASPC-1) and lung cancer cell line (PC9) were electroporated in the presence or the absence of fDT. Almost complete inhibition of target cell proliferation was observed when the cells were electroporated (90 V, 10 ms, n = 8) in the presence of fDT, even after washing. Similar marked inhibition of PC9 cell proliferation was obtained when anti-DT antibodies were added after electroporation (EP) instead of washing. These results indicate that the presence of fDT is needed only during the time of EP treatment and the side-effects of the agents can be avoided by specific antibodies.

Augmented anti-proliferative effect in combined use of human lymphotoxin with a nitrosourea derivative, ACNU, and the involvement of glutathione redox cycle

The cytotoxic or cytostatic effect of the combined use of human lymphotoxin (LT) with 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3- nitrosourea hydrochloride (ACNU) on L cells or Meth A tumor cells was studied. Simultaneous addition of LT derived from a human lymphoid cell line with ACNU (200 or 500 micrograms/ml) significantly augmented the cytotoxic effect. Similar augmented inhibition was obtained when LT was added to ACNU-treated L cells. The pre-treatment of Meth A tumor cells with ACNU (25 or 50 micrograms/ml) augmented recombinant human LT-mediated cytostasis. However, the addition of glutathione (1.0 mg/ml) to ACNU-treated Meth A tumor cells significantly nullified the augmented anti-proliferative effect of LT (10 U/ml). These results suggest that augmentation of the anti-proliferative effect on tumor cells could be induced through the combined use of LT with ACNU by lowering the intracellular level of glutathione.

Lymphotoxin production by regional lymph node lymphocytes in patients with uterine cervical cancer

The cytotoxin production by regional lymph node cells was examined in 25 patients with uterine cervical cancer and 10 patients with uterine myoma. The patients in stage I had significantly increased spontaneous release of cytotoxins compared with that in stages II, III, and IV. The spontaneous release in stages III and IV was markedly reduced. There was no difference in the release of cytotoxins from peripheral blood lymphocytes between cancer patients and patients with myoma or healthy controls. The cytotoxin production by lymph node cells was increased in stage III by stimulating with formalin-fixed QG-K cells derived from uterine cervical cancer, but not in stages I and II. Almost all of the cytotoxic activity of cytotoxin was abrogated by antilymphotoxin antibody. However, the cytotoxin activity was partially inhibited by anti-tumor necrosis factor antibody. These results suggest that cytotoxins released from the regional lymph node cells of uterine cancer patients are derived from, most of all, lymphotoxin.

Augmentation of antitumor activities in hamster macrophages by yeast cell walls treated with lymphokines in vitro

The effect of the treatment of yeast cell walls (YCW) with PPD-induced lymphokines (LK) was studied. The rate of macrophage spreading increased 1 h and 24 h after incubation with YCW treated with 16-fold or 64-fold diluted LK. Phagocytosis was slightly enhanced after 1 h incubation. Cytostatic activity was obtained when incubated with YCW treated with 16-fold or 64-fold diluted LK, but not with LK of higher or lower dilution. Tumor-inhibitory effect was observed when lymphoma cells were mixed with LK-treated YCW and inoculated s.c.