[Assessment of Antitumor Effect of Submerged Culture of Ophiocordyceps sinensis and Cordyceps militaris].

Ophiocordyceps sinensis and Cordyceps militaris metabolites showed a high potential in the treatment of tumors as well as some other diseases. Antitumor properties of O. sinensis and C. militaris submerged mycelium were investigated. It was found that the O. sinensis dry biomass in a dose of 50 mg/kg administered once a day to the mice with subcutaneously inoculated P388 lympholeucosis lowered the tumor growth by 65% vs. 54% for the C. militaris dry biomass. The water extract of O. sinensis submerged culture however accelerated the growth of the P388 lympholeucosis tumor node in the mice almost two times, compared to the control. A greater caution in using this fungus as a source of biologically active substances is required since unwanted tumor-stimulating effects can arise.

[Effect of recipient mouse splenocytes taken orally by donor mouse on GVHD after splenocyte transplantation].

This study was aimed to explore whether the GVHD in mice can be ameliorated and the GVL effect in mice can be reserved by transfusion of lymphocytes of donors fed with recipient splenocytes effect. Male (DBA-2) mice (H-2(d)) as donors were fed with BALB/c splenocytes, DBA-2 splenocytes, bovine serum albumin, or regular chow, every other day. Induction of tolerance was assessed by a mixed lymphocyte reaction (MLR). Female (BALB/c) mice (H-2(d)) as recipients received total body irradiation (TBI) of 6.0 Gy ((60)Cogamma-ray) followed by inoculation of 3 x 10(3) P388 mouse leukemia cells on the same day. Subsequently, tail vein injection of 2 x 10(7) splenocytes supplied by DBA-2 was undertaken. Control groups were fed identically without leukemia cell inoculation. The results showed that GVHD was significantly ameliorated and CD4(+)/CD8(+) ratio increased in recipient-mice transplanted with splenocytes of tolerated donors, compared with control animals. There was no significant difference in survival rate between different groups of recipients inoculated with leukemia cell. It is concluded that the peroral recipient-mouse splenocytes can ameliorate GVHD without hampering effect on GVL.

[Selective elimination of alloreactive donor lymphocytes by using TBI and cyclophosphamide].

This study was aimed to investigate a new method of avoiding graft-vs-host disease (GVHD) through selective elimination of alloreactive donor lymphocytes by using total body irradiation (TBI) and cyclophosphamide (Cy). Female (BALB/c x C57BL/6) F1 mice (H-2(d/b)) as recipients received (60)Co gamma-ray sublethal TBI of 4 Gy on day 0 followed by being inoculated with P388D1 leukemia cell line on day 1, injection of allogeneic splenocytes from C57BL/6 male mice (H-2(b)) was carried out for induction of graft-vs-leukemia (GVL) effect prior to stem cell transplantation (SCT), intraperitoneally injection of cyclophosphamide (Cy) (200 mg/kg) and TBI (9 Gy) was given on day 6. One day later, treated mice were rescued with bone marrow hematopoietic stem cells from (BALB/c x C57BL/6) F1 male mice (H-2(d/b)). The results showed that recipients had no occurrence of leukemia and GVHD through selective elimination of alloreactive donor lymphocytes by Cy and TBI, survived more than 210 days, the complete-donor chimerism occurred on day 21 after transplantation. The ratio of chimerism descended subsequently, but still displayed mixed-chimerism at 90 days. Control mice died of GVHD, leukemia or other death-related-transplantation within 20 to 36 days (P<0.01). It is concluded that to induce GVL effects by MHC mismatched splenocytes given before syngeneic bone marrow transplantation followed by selective elimination of alloreactive donor lymphocytes through TBI and Cy, graft-vs-host disease was thus avoided.

Interactions of 2',2'-diflurodeoxycytidine (gemcitabine) with tumor necrosis factor alpha or its mutein VI in murine leukemias L1210 and P388.

The interactions of 2',2'-difluorodeoxycytidine (gemcitabine) with tumor necrosis factor alpha (TNF-alpha) or its mutein VI on the survival time of mice bearing L1210 and P388 leukemia was investigated. Four hundred eighty male CD2F1 mice were used in the experiments. They were given gemcitabine (20 mg/kg) on days 1, 4, 7 and 10 after i.p. inoculation with leukemic cells (day 0). Cytokines were administered i.p. at a dose of 250 microg/kg as daily injections for 8 days or at a dose of 400 microg/kg given 2, 4, 6 and 8 days after day 0. Drugs were administered alone and in combination. Our study indicates that TNF-alpha and its mutein VI decrease the antileukemic effect of gemcitabine on murine leukemias L1210 and P388.

[Biotherapeutic efficacy of adoptive transfer of CD3AK cells in combination with cyclophosphamide and kappa-selenocarrageenan in P 388 leukemic mice].

OBJECTIVE: To study the antileukemia efficacy of a combination of adoptive transfer of CD3AK cells, cyclophosphamide (CTX), and kappa-selenocarrageenan (KSC) in P 388 leukemic mice. METHODS: CD3AK cells in normal DBA/2 murine splenocytes were induced with anti-CD3 antibody and low dose recombinant interleukin-2 (rIL-2). The P 388 murine leukemia model was induced by i.p. injection of P 388 cells into normal DBA/2 mice. The tumor-bearing mice were administrated with adoptively transferred CD3AK Cells and/or CTX and/or KSC. RESULTS: After tumor inoculation, the cellular immune function of P 388-bearing mice was supressed markedly. Adoptive transfer of CD3AK cells with low dose rIL-2 into the P 388 mice significantly enhanced the splenocyte proliferation (SP) induced by Con A, the NK cell activity and the splenocytic IL-2 production and prolonged their survival (45.19%); CTX (200 mg/kg) alone prolonged the survival of P 388-bearing mice (29.90%), but further decreased the immunodeficiency; combination of CTX and CD3AK passive transfer could prevent the reduction of SP, NK activity and IL-2 production in the leukemic mice and prolonged the survival (59.45%), combination of KSC and adoptively transfected CD2AK cells and/or CTX had a much better therapeutic efficacy for P 388 murine leukemia, 12.50%-75.00% of the leukemic mice were cured. CONCLUSION: KSC is a hopeful biological response modifier in cancer biotherapy, and tumor killing effector cells and chemotherapy plus BRM might be a promising candidate for human leukemia biotherapy.

Therapeutic potency of transduction with herpes simplex virus thymidine kinase gene against multidrug resistant mouse leukemia cells.

In order to examine the therapeutic potency of ganciclovir (GCV) against multidrug resistant tumour cells by transduction with the herpes simplex virus thymidine kinase (HSV-TK) gene, vincristine (VCR)-resistant mouse leukemia P388 cells (P388/VCR) were transduced retrovirally with the HSV-TK gene. In vitro, P388/VCR cells expressing HSV-TK (P388/VCR/TK) were approximately 200-fold more sensitive to GCV than P388/VCR cells. Age-matched CDF1 mice were intraperitoneally inoculated with P388/VCR or P388/VCR/TK cells and were treated with either GCV or VCR. The results showed that VCR had little therapeutic effect against P388/VCR/TK-bearing mice, whereas GCV significantly increased the life span of the mice. These results imply the potential value of HSV-TK gene transduction followed by GCV treatment in killing multidrug-resistant tumor cells.

Effect of peritoneal cells on tumors cells growth in vitro.

The cytotoxic and cytostatic activity of PMA-treated macrophages, obtained from pristane-primed BALB/c mice, was analyzed in vitro. The activated macrophages were cytotoxic and cytostatic for YAC-1 lymphoma, P-388 leukemia and P-815 mastocytoma target cells. However, the RPC-5 plasmacytoma target cells appeared to be resistant to their cytotoxicity. The observed cytotoxic or cytostatic effects of macrophages in vitro were not correlated with their ability to produce the superoxide ion. Cytotoxic activity of NK cells, obtained from pristane-primed mice, was also studied. No differences in cytotoxicity of NK cells obtained from pristane-treated and untreated donors, were found. However, only the effector cells from untreated mice were able to respond to stimulatory effect of polyinosinic acid-polycytidylic acid-poly-L-lysine (poly ICLC).

Antitumor immunity induced by photodynamic therapy with aluminum disulfonated phthalocyanines and laser light.

Photodynamic therapy (PDT) is systemic administration of tumor localizing photosensitizers and subsequent irradiation with light of the appropriate wavelength. The combination of drug uptake in malignant tissues and selective delivery of laser-generated light provides effective therapy, with efficient tumor cytotoxicity and minimal normal tissue damage. There have been few studies of the effects of photoactivated photosensitizers on the host immune response. Since immunity is important in the control of tumor growth and spread, we have examined, in our laboratory, the effects of photoactivated phthalocyanines on the antitumor immune response. Immunosuppressed and normal mice bearing the MS-2 fibrosarcoma treated with 5 mg/kg of aluminum disulfonated phthalocyanine (AIS2Pc) and then the tumor mass exposed to laser light (100 mW/cm2 x 10 min) or treated with surgical excision of the tumor survived indefinitely, with no difference between the different groups. The survivors, tumor-free 100 days after the treatment modalities described above, were rechallenged with the parental MS-2. Some groups of surviving animals were immunosuppressed with cyclophosphamide before the injection of the tumor. Resistance to rechallenge was evidenced only in normal surviving animals cured by PDT, while the immunodepressed surviving animals and animals cured by surgery died of tumor. Finally, mice, cured by PDT and tumor-free, rechallenged with L1210 and P388 murine leukemias did not survive. These results suggest that a potential and specific 'antitumor immunity' is induced by PDT with photoactivated AIS2Pc.

Effect of various microbial preparations on P-388 mouse lymphocytic leukemia.

Four bacteria-derived immunopotentiators were tested for their protective effect on a P-388 mouse lymphocytic leukemia model. The microbial test products were prepared from the following bacterial strains: ATCC 35983 Staphylococcus epidermidis isolated from a patient with IV catheter; ATCC 31874, a patented strain listed as Staphylococcus epidermidis isolated from the urine of a cancer patient; ATCC 25615 Staphylococcus hominis obtained from a child with lymphocytic leukemia, and ATCC 25614 Staphylococcus warneri, an isolate from a patient with adenocarcinoma of the breast. A limited degree of protection and prolongation in survival time was observed in the animal group treated with the bacterial strain ATCC 31874.

Effect of various microbial preparations on P-388 mouse lymphocytic leukemia

Four bacteria-derived immunopotentiators were tested for their protective effect on a P-388 mouse lymphocytic leukemia model. The microbial test products were prepared from the following bacterial strains: ATCC 35983 Staphylococcus epidermidis isolated from a patient with IV catheter; ATCC 31874, a patented strain listed as Staphylococcus epidermidis isolated from the urine of a cancer patient; ATCC 25615 Staphylococcus hominis obtained from a child with lymphocytic leukemia, and ATCC 25614 Staphylococcus warneri, an isolate from a patient with adenocarcinoma of the breast. A limited degree of protection and prolongation in survival time was observed in the animal group treated with the bacterial strain ATCC 31874

The activity of two immunotoxins composed of monoclonal antibody MoAb-16 and A-chain of ricin (MoAb-16-RTA) or A-chain of mistletoe lectin I (MoAb-16-MLIA).

The A-chains of ricin obtained from Ricinus communis or mistletoe lectin I from Viscum album were coupled to the monoclonal, anti-L1210V antibody MoAb-16, using SPDP as a cross linking agent. The cytotoxic activity in vitro of these immunotoxins was compared. Each of two immunotoxins tested, applied in vitro for 1 h in appropriate doses, caused irreversible inhibition of leukemic L1210 cells proliferation. Unexpectedly, MoAb-16-MLIA immunotoxin appeared to be cytotoxic to normal bone marrow progenitor cells, as observed in NCFUS tests. Moreover, this immunotoxin revealed cytotoxic effect to the P388 leukemia cells which do not share the antigen, common within L1210 leukemia cells, detected by MoAb-16 antibody.

Antileukemic effects of recombinant human tumor necrosis factor alpha (rh-TNF alpha) with cyclophosphamide or methotrexate on leukemia L1210 and leukemia P388 in mice.

We investigated the influence of recombinant human tumor necrosis factor alpha (rh-TNF alpha) administered as a single agent or in combination with cyclophosphamide (CY) or methotrexate (MTX) on the survival time of mice inoculated with lymphoblastic leukemia L1210 or lymphatic leukemia P388. The median survival time of leukemia L1210 bearing mice treated with rh-TNF alpha at doses ranging from 200 to 275 g/kg in daily i.p. injections was longer than that of control animals. Groups of mice with leukemia L1210 receiving rh-TNF alpha combined with either MTX or CY lived longer than animals treated with these agents individually. We observed only slight prolongation of life of animals inoculated with this tumor and treated with rh-TNF alpha at dose of 800 micrograms/kg in four injections on 2, 4, 6 and 8 day of experiment, and no effect when rh-TNF alpha was administered at dose of 200 or 400 micrograms/kg at the same treatment regime. In contrast no significant differences in lifetime were obtained from either simultaneous or sequential treatment of mice bearing leukemia P388. Groups of mice with this tumor treated with rh-TNF alpha in conjunction with either MTX or CY lived longer than controls, or rh-TNF alpha singly treated mice, but their survivals were not significantly prolonged compared with mice receiving cytostatics alone.

Antitumor effect of Nocardia rubra cell wall skeleton on syngeneically transplanted P388 tumors.

The antitumor activity of the immunomodulator, Nocardia rubra cell wall skeleton (N-CWS), was investigated using syngeneically transplanted P388 leukemia cells in a solid form. The s.c. growth of P388 tumors in DBA/2 mice was significantly suppressed by systemically administered N-CWS, and the effect was dose dependent. The antitumor effect of N-CWS was partially but significantly abrogated in splenectomized mice but not in T-cell or natural killer cell-deficient mice. Although spleen cells from mice treated with 1600 micrograms N-CWS contained no cytolytic activity, they exerted a significant cytostatic effect on P388 cell growth both in vitro and in vivo. Splenic cytostatic activity did not reside in T- or natural killer cells, but in plastic adherent cell population, macrophages. The response to N-CWS immunotherapy appeared to be associated with the number of macrophages infiltrating into the tumor lesions, and this was confirmed by histological analysis showing that P388 tumors from N-CWS-treated mice were intensively and dominantly infiltrated by macrophages. Furthermore, these were shown to be strongly tumor necrosis factor-positive by immunohistochemical analysis. These findings indicate that macrophages are the main effector cells playing a critical role in the suppression of P388 tumor growth in DBA/2 mice, and that tumor necrosis factor produced by these cells may be involved in the macrophage-mediated cytostatic effect induced by N-CWS. The fact that N-CWS suppressed the growth of weakly immunogenic P388 cells in syngeneic DBA/2 mice even when it was systemically injected would support the clinical potential of this agent.

Circumvention of drug resistance of P388/R cells by the combination of adriamycin and mitoxantrone with hyperthermia (42 degrees C).

P388/R, the adriamycin (ADR) resistant subline of murine P388 lymphocytic leukemia was cross-resistant to the drug mitoxantrone (MTN). One hour hyperthermia at 42 degrees C (HT) was employed along with ADR (10 micrograms/ml) and MTN (10 micrograms/ml) for circumventing the drug resistance of P388/R cells in in vitro-in vivo bioassays. Inhibition in the incorporation of tritiated thymidine into cellular DNA was measured to check the in vitro cytotoxic effect. Hyperthermia, ADR and MTN alone could not bring about significant degree of inhibition of DNA biosynthesis whereas the combination of ADR and MTN along with HT resulted in a synergistic cytotoxic action (p less than 0.001 and 0.01, respectively). The cells were treated with the drugs in vitro and inoculated into BDF1 mice. It was observed that ADR, MTN or HT pretreatment of the cells resulted in an increase of the life-span of the mice by 4.0-25.0%, 10-20% and 44-50%, respectively, whereas the pretreatment of cells with the combination of ADR and MTN with HT resulted in an increase by 104-125% and 212-220% in life-span of the mice, respectively. Studies revealed that the combination ADR-HT and MTN-HT resulted in circumvention of resistance of P388/R cells to ADR and MTN.

Effect of hyperthermia and X-irradiation on survival and occurrence of metastases in mice bearing P388 tumor.

Survival of P388 lymphoid tumor-bearing mice and the occurrence of metastasis was studied after combined modality treatment with hyperthermia and X-irradiation. P388 ascites tumor cells were treated at 42 degrees C or 43.5 degrees C for 1 hr in vitro and transplanted on B6D2F1 mice intraperitoneally (i.p.) or intramuscularly (i.m.). Hyperthermic treatment at 43.5 degrees C increased the median survival time (MST). Increased life-span (ILS) was found after i.p. transplantation (54%) and after i.m. transplantation (30%). During the life-span of tumor-bearing animals, significantly fewer metastases were observed in liver and spleen after hyperthermia and 5-10% metastasis occurred after transplantation of ascites tumor cells treated at 43.5 degrees C in vitro compared with 90% in the untreated control animals. The lower occurrence of metastasis could not be ascribed to the higher cell-killing effect of hyperthermia. When both modalities were combined the best tumor growth retardation effect was obtained when ascites tumor cells were treated at 43.5 degrees C for 1 hr before being transplanted i.m. and 1 day later locally X-irradiated with 6 Gy. In this case, 77% ILS was found demonstrating a synergistic effect of the two modalities. While X-irradiation alone did not change the occurrence of metastasis, after combined modality treatment it was as low as with hyperthermia alone (5-10%). In connection with the significantly lower occurrence of metastasis, the possible alterations of P388 tumor cell membrane and surface proteins induced by in vitro hyperthermic treatment are discussed.

Age-dependent antileukemic action and suppression of immune response by 1,4-benzoquinone-guanylhydrazone-thiosemicarbazone (ambazone) in mice.

The antineoplastic activity of 1,4-benzoquinone-guanylhydrazone-thiosemicarbazone (ambazone) against murine leukemia P388 was found to be markedly reduced in 12- and 18-month-old mice as compared to young animals. The immune response against sheep red blood cells (SRBC), a T cell-dependent antigen, was also strongly diminished in tumor-free old mice and was further suppressed after ambazone treatment. Since the antileukemic effect of ambazone disappeared more or less in congenitally athymic nude mice, in neonatally thymectomized or silica-pretreated animals, it has been concluded that the action of the compound seems to be limited to young adult immunocompetent tumor-bearing hosts. Therefore immunosenescence, primarily of T cell functions of old tumor-bearers, may represent a decisive factor influencing the antileukemic, especially curative effect of ambazone in aged animals. A combined treatment with ambazone and immunomodulators (thymalin or a splenopentin derivative) failed to improve the antileukemic effect in young and old leukemia P388-bearing mice.

[A new antitumor drug with immunomodulating activity, ubenimex (bestatin)].

Ubenimex (Bestatin) was discovered by Umezawa et al. in 1976 from the culture broth of Streptomyces olivoreticuli. Bestatin is a compound of low-molecular weight peptide and inhibits leucine aminopeptidase and aminopeptidase B which localized in cell membrane. Bestatin exhibited antitumor effect against murine syngeneic tumors including mouse colon 26 and C1498 leukemia, and also it was active against MNNG-induced rat tumor by oral administration. Combination treatment of mouse colon 26 with bestatin and mitomycin C, 5-FU or CDDP was effective for the life prolongation of the treated mice compared to mono-therapy alone. Bestatin was found to exhibit the antitumor effect through T lymphocyte stimulation, macrophage activation and bone marrow stem cell stimulation were also observed by bestatin treatment experimentally. Values of T cell subsets in cancer patients recovered to the normal levels by Bestatin treatment. Release of Interleukin-1 and -2 was enhanced by Bestatin treatment in vitro. In the phase I study, clinical optimal daily dose was estimated as 10-100 mg to give 2-3 times weekly or daily continuously. In the comparative clinical trials, Bestatin was found to be effective for the prolongation of survival time of the patients with acute non-lymphocytic leukemia after induction of complete remission in combination with maintenance chemotherapy. Minimal side effects were noted.

OK-432-mediated augmentation of antitumor immunity and generation of cytotoxic T lymphocytes.

Sensitization with mitomycin C-treated L1210 or EL-4 tumor cells followed by intraperitoneal injection of a streptococcal preparation OK-432 rendered histocompatible or syngeneic mice immune to the corresponding tumor cells. The antitumor immunity, which was more potent than that induced by attenuated tumor cells alone, was manifested by transplantation resistance to challenge tumor cells, and by cytotoxic activity of spleen cells from the primed mice. The former activity was closely related to the latter, which was found to be mainly due to tumor-specific cytotoxic T lymphocytes. The in vivo immunoaugmentation by OK-432 was susceptible to macrophage toxins such as trypan blue and carragheenins, and was partly dependent on the activity of noncytotoxic Ia-positive peritoneal macrophages. OK-432-mediated enhancement of Ia-positive macrophage functions was confirmed by concanavalin A-blastogenesis and T cell-dependent antibody formation. Allo-reactive cytotoxicity induced in allogeneic or semiallogeneic mice, which had been primed with clonogenic or attenuated tumor cells, was also augmented by concomitant administration of OK-432. These results suggest that OK-432 augments induction of antitumor immunity and alloreactive cytotoxicity, associated with stimulation of noncytotoxic Ia-positive accessory macrophage activity.