Ability of sera from mice treated with Ge-132, an organic germanium compound, to inhibit experimental murine ascites tumours

Sera from C57Bl/6 mice treated orally with Ge-132 exhibited antitumour activity against Ehrlich (allogeneic) and RL male 1 (syngeneic) ascites tumours in BALB/c mice. Sera obtained from mice 24 h after Ge-132 administration displayed the greatest antitumour effect and this was dose dependent. Sera prepared from mice 12, 36, or 48 h after Ge-132 treatment had no protective effect. Circulating interferon (IFN) was induced at 24 h after administration of Ge-132 but was not detected in the sera at 12, 36, or 48 h after administration. The antiviral activity of sera from Ge-132-treated mice was inactivated by treatments with trypsin, low pH, and anti-IFN gamma antiserum. The inactivated preparations of serum IFN induced by Ge-132 did not exhibit antitumour activity when administered to tumour-bearing mice. These results suggest that antitumour activity in the sera of Ge-132-treated mice may be expressed through activities of Ge-132-induced lymphokine(s), such as IFN gamma.     

Antitumor mechanisms of carboxyethyl-germanium sesquioxide (Ge-132) in mice bearing Ehrlich ascites tumors

The administration of IFN-containing sera (Ge-sera) obtained from Ge-132-treated mice (Ge-mice) or the passive transfer of macrophages (M phi) to mice bearing ascites tumors resulted in the inhibition of tumor growth. The cooperative role of Ge-sera and Ge-M phi in the display of Ge-132-antitumor activity was studied. When mice were pretreated with antimouse IFN gamma antiserum, no IFN-inducing or antitumor activities of the compound were detected. Cytotoxic activities were detected in peritoneal M phi of mice treated with Ge-sera, and passive transfer of these M phi to tumor-bearing mice resulted in the inhibition of tumor growth. When tumor-bearing mice were pretreated with substances toxic to M phi, no antitumor activity of Ge-sera was observed. However, Ge-132 antitumor activity was observed in mice depleted of T-cells, even though the antitumor effects of the compound itself were not demonstrable in T-cell-depleted mice. Therefore, a part of the antitumor activity of Ge-132 appears to be expressed as follows: Ge-132 stimulates T-cells to produce circulating lymphokine(s) which are inactivated by anti-IFN gamma treatment; activated M phi are generated from resting M phi by these lymphokine(s); the transplanted tumors are inhibited by these M phi.     

Suppression of tumor growth by peritoneal macrophages isolated from mice treated with carboxyethylgermanium sesquioxide (Ge-132)

In a murine model it has been shown that the antitumor activity of carboxyethylgermanium sesquioxide (Ge-132) can be depleted by administration of macrophage (M phi) blockers. In the present study, the role that M phi play in the antitumor activity of the compound was investigated. Oral administration of Ge-132 in mice was demonstrated to be effective in activating M phi (Ge-132-cytotoxic M phi), and the cytotoxic activity of these M phi appeared in the peritoneal cavity of mice 48 hours after the oral administration of the compound. Co-cultivation of RL male-1 leukemia or Ehrlich carcinoma cells with Ge-132-cytotoxic M phi in vitro resulted in marked suppression of the growth of tumor cells. The transfer of peritoneal exudate cells (PEC), or purified M phi fractions of PEC from Ge-132-treated mice to mice bearing Ehrlich or RL male-1 ascites tumors resulted in significant protection. However, when the cytotoxic M phi were depleted by carbonyl-iron treatment in vitro, no antitumor effect was demonstrated in mice bearing Ehrlich or RL male-1 ascites tumors. Macrophage fractions obtained from PEC of Ge-132-treated mice exhibited an inhibitory effect against certain tumors both in vivo and in vitro suggesting that the antitumor effect of Ge-132 observed in vivo resulted from the activation of M phi.     

Antitumor effect in mice of an organic germanium compound (Ge-132) when different administration methods are used

The antitumor effect of an organic germanium compound, carboxyethylgermanium sesquioxide (Ge-132), was examined in mice using two systems: one, the ascitic form of Ehrlich carcinoma in DDI mice, and the other, the solid form of Meth-A fibrosarcoma in BALB/c mice. In the mice with Ehrlich ascitic tumors, a remarkable prolongation in life span was observed after intraperitoneal (i.p.) or per oral (p.o.) administration of Ge-132 (300 mg/kg), but not after intravenous (i.v.) injection of the same compound. Following i.p. or p.o. administration, cytotoxic macrophages (M?) were induced in the peritoneal cavity after 48 h. although this was not the case after i.v. injections. When the in vivo effect of these in vitro active M? was examined after adoptive transfer to mice bearing Ehrlich ascitic tumor cells, a significant antitumor effect was noted. In the mice bearing solid Meth-A tumors, i.v. injections of Ge-132 (100 mg/kg) were found to inhibit tumor growth remarkably, although i.p. and p.o. administrations did not have the same result. This inhibitory effect of Ge-132 by i.v. administration was explained by the continued augmentation of NK activity in peripheral blood, which was followed by the induction of specific killer cells appearing in the spleen. When the mice which had recovered from Meth-A tumor growth, following i.v. injections of Ge-132, were challenged with the same tumor on day 30, all mice were able to tolerate the challenge, but not a challenge of RL male 1 tumor cells. These observations may indicate that the differing antitumor effects of Ge-132 produced when different administration methods are used can be explained by the variation in effector cells induced by such different administration routes.     

Induction of interferon and activation of NK cells and macrophages in mice by oral administration of Ge-132, an organic germanium compound

After oral administration of an organic germanium compound, Ge-132 (300 mg/kg), a significant level of interferon (IFN) activity was detected in the sera of mice at 20 hr and it reached a maximum of 320 U/ml at 24 hr. This IFN activity was lost after heat- or acid-treatment, suggesting that the induced IFN is of gamma nature. The molecular weight of this IFN was estimated to be 19,000 daltons by gel filtration. After the oral administration of Ge-132, NK activity of spleen cells had increased at 24 hr and cytotoxic macrophages were induced in the peritoneal cavity at 48 hr. In mice, receiving intraperitoneal (i.p.) injections of trypan blue or carrageenan 2 days before oral administration of Ge-132, neither induction of IFN nor augmentation of NK activity followed. X ray irradiation of mice also rendered the mice incapable of producing IFN, all suggesting that both macrophages and lymphocytes are required for this IFN induction. After i.p. administration of induced IFN, both NK and cytotoxic macrophage activities appeared at 48 hr with as little as 20 U/ml titered IFN. These facts may indicate that the augmentation of NK and activation of macrophages in mice after oral administration of Ge-132 is mediated by induced IFN.     

Antitumor activity of Ge-132, a new organogermanium compound, in mice is expressed through the functions of macrophages and T lymphocytes

The antitumor activity of Ge-132 against a variety of allogeneic and syngeneic murine ascites tumors was first evaluated. The antitumor effects of Ge-132 were observed when mice inoculated with Ehrlich carcinoma (allogeneic) or RL male 1 leukemia (syngeneic) cells were treated orally. However, Ge-132 had no activity on a T-cell lymphoma (EL 4, syngeneic) or a methylcholanthrene-induced fibrosarcoma (Meth-A, syngeneic). The antitumor effect of Ge-132 in mice was related to the dose administered as well as the administration schedule. The antitumor activity of Ge-132 was next studied in mice pretreated with some blockers against immunocompetent cells. The antitumor efficacy of Ge-132 was not observed when tumor-bearing mice were treated with trypan blue and carrageenan or monoclonal anti-Thy 1.2 antibody. However, when natural killer cells were eliminated from mice bearing RL male 1 or Ehrlich ascites tumors by treatment with anti-asialo GM 1 antiserum, the antitumor efficacy of the compound was unchanged. These results suggest that Ge-132 is effective against certain ascites tumors regardless of whether the tumor is syngeneic or allogeneic. Further, its effect might be expressed through host defense mechanisms, including macrophages and/or T lymphocytes.     

Effect of combination immunochemotherapy with an organogermanium compound, Ge-132, and antitumor agents on C57BL/6 mice bearing Lewis lung carcinoma (3LL)

The antitumor effect of combination immunochemotherapy with Ge-132 and antitumor agent was studied using C57BL/6 mice bearing Lewis lung carcinoma (3LL). Ge-132 was administered orally at a daily dose of 100 mg/kg. Antitumor agents were administered intraperitoneally once a week. Initially, the effect of combination immunochemotherapy with Ge-132 and 5-fluorouracil (5-FU) was studied on 3LL local tumor growth, pulmonary metastases, survival, delayed type hypersensitivity (DTH) and body weight in tumor-bearing mice, and the following results were obtained: Inhibition of tumor growth in the combined group; Enhanced anti-metastatic effect; Prolonged survival time, and; Recovery of loss of both DTH and body weight as a result of combination therapy. These antitumor effects were also obtained by adoptive transfer of Ge-132-stimulated splenocytes in 5-FU-treated mice bearing 3LL. These results therefore suggest that the effects of Ge-132 were expressed through modification of immunocytes. Furthermore, Ge-132 enhanced the antitumor activity of bleomycin as well as that of 5-FU. These facts suggested that Ge-132 is useful for antitumor combination immunochemotherapy.     

DNA-dependent RNA polymerases of Ehrlich carcinoma, other murine ascites tumors, and murine normal tissues.

DNA-dependent RNA polymerase was solubilized from nuclei of Ehrlich ascites carcinoma (EAC) cells by sonic disruption in the presence of 0.3 M (NH4)2 SO4, and the multiple RNA polymerases were separated by chromatography on DEAE-Sephadex A-25. Elution with a nine-step gradient of (NH4)2 SO4 yielded five peaks of activity designated RNA polymerases Ia, Ib, IIa, IIb, and III, of which IIb was the most prominent. Linear-gradient elusion also yielded five peaks of the same designation, but Ia and Ib, as well as IIa and IIb, were not well separated. IIa and IIb were inhibited completely by 0.1 mug alpha-amanitin/ml, whereas the other forms were not. EAC RNA polymerases Ia, Ib, IIa, and IIb possessed Mg2+ ion, Mn2+ ion, and (NH4)2 SO4 optima, molecular weights, and thermal sensitivities similar to those reported for other mammalian DNA-dependent RNA polymerases. As measured by relative ribonucleoside monophosphate incorporation, with native calf thymus DNA template, EAC RNA polymerases Ia and Ib synthesized ribosomal RNA-like products, whereas forms IIa, IIb, and the parent enzyme mixture synthesized compounds that were more similar to DNA. No species specificity was found for DNA templates, and denatured DNA was consistently preferred to the native template by RNA polymerases IIa and IIb; the two kinds of template were about equally efficient for RNA polymerases Ia and Ib. Although EAC RNA polymerases Ia, IIa, and IIb were inhibited by daunomycin, form IIa was preferentially affected. 3',5'-Cyclic AMP, 3',5'-Cyclic GMP, and gibberellic acid, implicated as RNA polymerase regulators in other systems, were generally ineffective. The levels of nuclear RNA polymerase activities, per mg DNA, of 3 mouse ascites tumors (EAC, 6C3HED lymphosarcoma, and TA3 adenocarcinoma) were compared with those from 3 normal mouse tissues (kidney, liver, and spleen). On the average, the tumor cell nuclei contained (per mg of DNA) 8.9, 1.5, 2.7, 20.0, and 3.8 times the activities of RNA polymerases Ia, Ib, IIa, IIb, and III, respectively, as the normal cells, but the difference was significantly only for IIb.     

重组人血管内皮抑制素对肝癌H22腹水瘤小鼠腹水的抑制作用

目的探讨血管内皮抑制素腹腔内给药对H22小鼠腹水瘤生长及腹水的抑制作用。方法取110只昆明小鼠腹腔内接种H22细胞（2x106细胞／只）,建立小鼠腹水瘤模型,接种后随机分为5组：模型对照组（0．9％NS）;低剂量血管内皮抑制素组（4mg／kg）;中剂量血管内皮抑制素组（8mg／kg）;高剂量血管内皮抑制素组（12mg／kg）;阳性对照组（顺铂0．6mg／kg）。接种24h后连续10d给予各药物腹腔内注射,停药24h后处死各组小鼠,测量腹水量并取腹水行相关检测;解剖小鼠,观察腹腔脏器及肺脏的转移情况;通过Evan蓝的吸光度值反映小鼠的腹膜渗透性;观察各组小鼠的生存时间。结果与模型对照组相比,中、高剂量血管内皮抑制素组可显著抑制荷H22腹水瘤小鼠腹水的生成,并减少腹腔内及肺脏转移,延长生存时间。结论血管内皮抑制素腹腔内用药治疗荷H22腹水瘤小鼠腹水具有显著的抑制作用。     

Capacity of murine IL-12 to inhibit the development of primary melanoma tumors and to prevent lung metastases in the melanoma-challenged mice

Interleukin-12 (IL-12) has the capacity to activate cytotoxic lymphocytes, stimulate natural killer cells, induce the production of INF-gamma, and be synergistic with IL-2. We have evaluated this cytokine in an experimental model for metastatic melanoma that approximates the major clinical stages of metastatic dissemination. To develop primary melanoma tumors, mice were injected subcutaneously with 5 x 10(5) cells in a volume of 25 microliters into the middle of the tail (11). In a month, mice were started to be treated for 4 weeks with recombinant murine IL-12 (R mIL-12) at the following doses: 0, 0.5, 2.5, 5.0, 15.0, and 50 micrograms/kg. Diameters of the primary melanoma tumors were measured at weekly intervals. At the end of 13 weeks (9 weeks from the start of treatment with R mIL-12), all surviving mice were sacrificed. Pathological examination of lung metastases (macroscopy) was done with all dead or sacrificed mice. Treatment of mice bearing melanoma at a dose of 300 ng/mouse (15 micrograms/kg) inhibited development of primary tumors in 40% of mice. The primary tumor diameters were significantly lower in the group treated with 300 ng/mouse (15 micrograms/kg) in comparison to controls. At the end of the observation period, groups treated with 0.5, 2.5, 15.0, and 50 micrograms/kg had mean primary tumor diameters smaller than the control group. Evaluation of IL-12 therapy on primary tumor growth, mean diameters of primary tumors, survival rate, and development of lung metastases showed that the best results were observed using 300 ng/mouse (15 micrograms/kg) R mIL-12.     

Effect of purified glutaminase from human ascites fluid on experimental tumor bearing mice

Glutamine is the major respiratory fuel and energy source of the rapidly proliferating tumor cells and that is why glutamine clearance by glutaminase therapy provides an opportunity to fight against the neoplasm. Glutaminase from bacterial source was tried on experimental models but had to be excluded because of its limited efficacy. Search for a better glutaminase continued exploiting the mammalian sources. In the present study, glutaminase purified from human ovarian cancer ascites fluid was used in experimental solid and ascites mice model alone and in combination with Cu-Sulphate and heparin. Cumulative findings indicate that the enzyme alone is quite effective in lowering tumor burden and reducing not only the tumor induced angiogenesis, but also an angiogenic inducer, heparin mediated angiogenesis. However, the presence of Cu with the enzyme, amplified the antineoplastic response by improving anti-angiogenic potential and hematological status of the tumor bearing host. Therefore, Cu-glutaminase combination strengthened the hypothesis that together they may provide a better therapeutic regimen in experimental mice tumor model.     

In vivo anti-tumor activity of a novel indolocarbazole compound, J-107088, on murine and human tumors transplanted into mice.

J-107088 (6-N-(1-hydroxymethyl-2-hydroxy)ethylamino-12,13-dihydro-2,10-dihydroxy- 13-(beta-D-glucopyranosyl)-5H-indolo[2,3-a]-pyrrolo [3,4-c]carbazole-5,7(6H)-dione) is a derivative of NB-506, an indolocarbazole compound previously reported as an anti-tumor agent targeting topoisomerase I. The optimal administration schedule of J-107088 was found to be intermittent injections. The GID75 (75% growth inhibiting total dose) values of J-107088 against LX-1 lung cancer and PC-3 prostate cancer when given by intermittent injection (twice a week for 2 consecutive weeks) were 200 and 15 mg/m2, respectively, whereas the 10% lethal dose (LD10) values of J-107088 against LX-1- and PC-3-bearing mice were 578 and 1200 mg/m2. The ratio of LD10/GID75 indicates the therapeutic window of an anti-tumor agent. Although the ratios of doxorubicin, paclitaxel and cisplatin against PC-3 were <0.3, <0.5 and <0.2, J-107088 showed the widest therapeutic window among the anti-tumor drugs tested. J-107088 was also effective on cells that had acquired resistance related to P-glycoprotein. Furthermore, J-107088 was found to be highly effective in inhibiting proliferation of micro-metastases of tumors to the liver in mice. Therefore, J-107088 is considered to be a promising candidate as an anti-tumor drug for treatment of solid tumors in humans.     

Accelerated growth of syngeneic tumors in mice treated with methylcholanthrene

BALB/c and C3H- mice were given a single injection of methylcholanthrene (MCA) and were tested at various times thereafter for immunologic reactivity and resistance to syngeneic tumor challenge. MCA treatment did not affect the rejection of H-2-incompatible tumor allografts, the rejection of H-2-compatible skin allografts, or the induction of delayed hypersensitivity to DNCB as measured by footpad challenge. In contrast, mice treated with MCA exhibited an increase susceptibility to challenge with several syngeneic ultraviolet-induced and MCA-induced fibrosarcomas. These studies demonstrate that in the autochthonous host the measurement of host immune status by in vivo cell-mediated immune responses to exogenous antigens does not necessarily predict the host response to tumor growth.     

On the surface structure of murine ascites tumors. I. Interactions with various phytoagglutinins

Phytoagglutinins originating from various plants agglutinated murine tumor cells and erythrocytes to varying degrees; some strongly, some moderately, and some not at all. The agglutinability of Yoshida sarcoma cells (rat ascites tumor) by various phytoagglutinins was essentially similar to that of ascites hepatoma 66 cells (rat ascites tumor), and similarity of agglutinability was also found between Ehrlich ascites carcinoma cells and leukemia 1210 cells, both of which were of murine origin. Since the phytoagglutinins react with the corresponding binding sites of cell surfaces in a similar manner to antibodies, the binding sites could be specified by the inhibition of the bindings after the addition of mono- or disaccharides. The treatment of Yoshida sarcoma cells with chemical reagents, hydrolytic enzymes or heat changed their agglutinability against the agglutinins. The extent to which their agglutinability was changed by these treatments, however, differed according to the kind of agglutinin used.     

Immunologic responses to a murine mammary adenocarcinoma. I. Passive transfer of immunity by sera from tumor-bearing mice.

DBA/2 mice bearing a syngeneic mammary adenocarcioma T1699 produced high levels of tumor-specific antibody, detected by indirect immunofluorescence and subsequently identified as the IgG2a subclass. Tumor-bearer sera passively administered to normal recipients protected the animals from subsequent challenge with T1699 tumor cells butnot from challenge with a non-cross-reacting syngeneic tumor, SaD2 fibrosarcoma. Administration of sera prior to tumor challenge was found to be more effective than treatment after the challenge. The protective effect of sera appeared to parallel both antibody titers and appearance of concomitant immunity; however, sera absorbed with T1699 cells, with the indirect fluorescent antibody titers reduced more than 100-fold, conferred an almost identical level of protection. Immune suppression of serum recipients before serum transfer abolished the effect, suggesting that protection depended on a cellular immune response by the host in addition to the possible protective effect(s) of humoral antibody.