Augmentation of the cytostatic activity of granulocytes by biological response modifiers (BRM)

The effects of various biological response modifiers (BRM) such as Nocardia rubra cell wall skeleton (N-CWS), OK-432, muramyl dipeptide (MDP), Staphylococcal protein A, interferon, lymphokine on the cytostatic activity of granulocytes against cultured tumor cells were examined. The cytostatic activity of untreated granulocytes from 10 normal healthy volunteers ranged from 14% to 30%. Lymphokine obtained from the supernatant of lymphocytes cultured with Con A-Sepharose directly suppressed the DNA synthesis of K 562 cells in high concentration, however, other BRM showed no direct cytostatic activity. The cytostatic activity of granulocytes was augmented by low concentrations (2.5 micrograms/ml) of N-CWS, OK-432 and protein A as well as by high concentration (25 micrograms/ml) of MDP. However, the cytostatic activity was not influenced by alpha-interferon. The meanings and mechanisms of the elevation in cytostatic activity of granulocytes by BRM were discussed with regards to the clinical immunotherapy.     

Radiotherapy combined with BRM (biological response modifiers) in the treatment of cancer

The latest treatment of biological response modifiers (BRM) with or without another cancer treatment modalities has been increased. In this paper, radiotherapy combined with some BRM, which were used in systemic administration and intra-arterial infusion, was demonstrated. 1) In experimental study, radiation and IL-2 indicated the synergic effects on the tumor growth inhibition. 2) Radiotherapy and IFN-beta for advanced head and neck and pelvic tumor were successful in tumor effects and tolerable in side effects. 3) The clinical trial showed that pathological effects and clinical tumor response (CR rate) of radiation and LC9018 for cervical cancer stage IIb, III were significantly better than those of radiotherapy alone. These data suggested that the treatment of radiation and BRM was useful and should be considered in multimodal therapy for cancer.     

A morphological analysis of an in vitro model of cytotoxic antitumor activity

Models of adoptive immunotherapy and cytotoxicity of lymphocytes isolated from tumor tissues were studied. A microcytotoxicity model utilizing serum-free culture conditions was evaluated by light microscopy, scanning electron microscopy, and transmission electron microscopy for antitumor activity. By examining morphologically the relationship between peripheral blood mononuclear cells and tumor-infiltrating lymphocytes, it was demonstrated that the latter and macrophages had morphological features similar to the cytotoxic cells obtained in vitro from peripheral blood. When allowed sufficient time in our culture conditions, the tumor-infiltrating cytotoxic cells seem to kill the tumor cells within a few days to many months. The ultrastructural morphology of the interaction between the cytotoxic cells and the tumor cells was described as well as some proteinaceous secretory granules that seem to be transferred to the tumor cells through these interactions.     

Interactions of radiation and biological response modifiers (BRMs) in the treatment of malignant tumor

This article reports on attempt to characterize the interactions of radiation and BRMs in the treatment of malignant tumor on the basis of our some experimental results. BRMs have a high possibility to enhance radiation effects on tumor cells with in a radiation field and bring some benefits to host tumor immune system with minimizing deleterious effects of irradiation to the immune responses. But it is very difficult to determine the rational design of administration of BRMs to induce the most effective interactions on relevant immune system and other host responses in the combination with radiotherapy. We should make it clear how radiation can be useful to potentiate the tumor antigenecity or antitumor immune responses.     

T1 and T2 Proton Nuclear Magnetic Resonance (N.M.R.) relaxation times in vitro and human intracranial tumours

Summary 137 samples of intracranial tumours have been studied in proton NMR spectroscopy. T1 and T2 relaxation times are above those of normal grey and white matter. Differential diagnosis between benign and malignant brain tumours does not seem feasible upon proton T1 and T2 alone. Histological correlations allowed us to specify secondary changes accounting for T1 and T2 variations (oedema, microcyst, stroma reaction, necrosis).     

In vitro and in vivo enhancement of vincristine antitumor activity on B16 melanoma cells by calcium antagonist flunarizine

Flunarizine, a calcium antagonist commonly employed in therapy for vascular diseases, enhances the in vitro and in vivo antitumor activity of vincristine on B16 melanoma cells. In the presence of flunarizine higher intracellular levels of vincristine were observed in vitro and for a longer time. B16 melanoma bearing mice treated with both the drugs presented a median survival time that was significantly longer than that of the controls. The possible mechanism of the enhancement is herein discussed.     

HA22 (R490A) is a recombinant immunotoxin with increased antitumor activity without an increase in animal toxicity.

PURPOSE: RFB4 (dsFv)-PE38 (BL22) is a recombinant immunotoxin containing an anti-CD22 (Fv) fused to truncated Pseudomonas exotoxin A, which induces a high complete remission rate in patients with purine analogue-resistant hairy cell leukemia. HA22 is a mutant of BL22 with mutations in heavy-chain CDR3 resulting in increased cytotoxic activity. Our goal was to improve the activity of HA22. EXPERIMENTAL DESIGN: Arg(490), which is located in the catalytic domain (III) of the immunotoxin HA22, was mutated to alanine. Purified immunotoxins were produced and tested for cytotoxic activity in cell culture and for antitumor activity and nonspecific toxicity in mice. ADP-ribosylation activity was also measured. RESULTS: HA22 (R490A) is approximately 2-fold more cytotoxic than HA22 on several CD22-positive cell lines. When injected i.v., HA22 (R490A) has more potent antitumor activity than HA22 against CA46 tumors in mice. HA22 and HA22 (R490A) have similar LD(50)s (approximately 1.3 mg/kg) and similar plasma half-lives. The R490A mutation also improved the cytotoxicity of the antimesothelin recombinant immunotoxin SS1 (dsFv)-PE38 (SS1P). In vitro ADP-ribosylation assays show that HA22 R490A has increased activity. Increased cytotoxic activity is probably related to this increase in ADP-ribosylation activity. CONCLUSION: Protein engineering can be used to increase the efficacy of recombinant immunotoxins. Because HA22 (R490A) has increased antitumor activity without increased animal toxicity, immunotoxins with this mutation are candidates for clinical development.     

Potentiation of antimetabolite antitumor activity in vivo by dipyridamole and amphotericin B

Summary Previous studies have shown that dipyridamole (DP), a potent nucleoside transport inhibitor blocking the rescue effect of exogenous nucleosides, markedly potentiates the cytotoxicity of antimetabolites. However, no enhancement of the chemotherapeutic effect of antimetabolites by DP in vivo has yet been reported. This study provided evidence that the combination of DP and amphotericin B (AmB) significantly potentiated the inhibitory effect of 5-fluorouracil (FU) or methotrexate (MTX) against a panel of transplantable tumors including sarcoma 180, cervical carcinoma U14, and Lewis lung carcinoma in mice. No significant increase in toxicity was induced by this combination in treated mice. Our results indicate that the combination of DP and AmB with antimetabolites is potentially useful in cancer chemotherapy.     

Augmentation of anti-tumor immunity in low-responder mice by various biological response modifiers: analysis of effector mechanism

In order to elucidate the role of biological response modifiers (BRMs) in anti-tumor immunotherapy, we examined their effect on the induction of anti-tumor immunity in low-responder mice which hardly exhibit anti-tumor resistance against syngeneic Rous sarcoma virus (RSV)-induced tumors, such as B10 or B10.BR mice. The anti-tumor immunity induction in the low-responder mice was 0% on immunization with mitomycin C-treated syngeneic tumor cells alone. However, if BRMs were used as an adjuvant, BCG cell wall skeleton, OK-432 or lentinan augmented the induction of anti-tumor immunity to 50%, 33% and 33%, respectively. In the low-responder mice treated with BRMs, the anti-tumor immune cells had antigen-specificity at the induction phase of in vitro restimulation but not at the effector phase of target cell lysis by the stimulated cells. When T cells were depleted from immune spleen cells just before in vitro stimulation, cytotoxicity was not induced. Furthermore, cytotoxicity was not induced if accessory cells were removed from immune spleen cells at the induction phase. However, cytotoxicity at the effector phase was not mediated by T-lymphocytes, but by non-T cells. These results suggested that the induced cytotoxicity in low-responder mice was associated with the delayed-typed hypersensitivity-like effector mechanism.     

Local antitumor activity of a primary and an anamnestic response to a syngeneic guinea pig hepatoma.

After intradermal (id) injection, the line-10 hepatoma grew progressively in nonimmune guinea pigs, whereas the line-1 hepatoma grew for approximately 2 weeks, developed central necrosis, ulcerated, and regressed. Growth of the line-10 hepatoma was suppressed when line-10 hepatoma cells were mixed with antigenically distinct line-1 hepatoma cells before id injection into syngeneic strain-2 guinea pigs. Mixture of line-10 with irradiated line-1 or viable strain-2 embryo cells did not inhibit tumor growth. Preimmunization of recipients to line-1 cells abrogated the suppression of tumor growth from mixtures of line-1 and line-10.     

Down-regulation of insulin-like growth factor I receptor activity by NVP-AEW541 has an antitumor effect on neuroblastoma cells in vitro and in vivo.

PURPOSE: Signaling through insulin-like growth factor I receptor (IGF-IR) is important for growth and survival of many tumor types. Neuroblastoma is sensitive to IGF. EXPERIMENTAL DESIGN: We assessed the ability of NVP-AEW541, a recently developed small molecule that selectively inhibits IGF-IR activity, for neuroblastoma growth effects in vitro and in vivo. Our data showed that, in a panel of 10 neuroblastoma cell lines positive for IGF-IR expression, NVP-AEW541 inhibited in vitro proliferation in a submicromolar/micromolar (0.4-6.8) range of concentrations. RESULTS: As expected, NVP-AEW541 inhibited IGF-II-mediated stimulation of IGF-IR and Akt. In addition to growth inhibition, the drug also induced apoptosis in vitro. Oral administration of NVP-AEW541 (50 mg/kg twice daily) inhibited tumor growth of neuroblastoma xenografts in nude mice. Analysis of tumors from the drug-treated animals revealed a marked apoptotic pattern and a decrease in microvascularization compared with controls. Interestingly, quantitative real-time PCR detected both in vitro and in vivo a significant down-regulation of mRNA for vascular endothelial growth factor (VEGF) caused by NVP-AEW541. In addition, in Matrigel-coated chambers and in severe combined immunodeficient mice tail vein injected with neuroblastoma cells, tumor invasiveness was significantly reduced by this agent. Analysis of IGF-IR expression in a series of 43 neuroblastoma primary tumors revealed IGF-IR positivity in 86% of cases. CONCLUSIONS: Taken together, these data indicate that NVP-AEW541 can be considered as a novel promising candidate for treatment of neuroblastoma patients.     

In vivo and in vitro antitumor activity expressed by cells of concomitantly immune mice

Growth of a primary tumor is often accompanied by the development of resistance to subsequent challenge implants of the same tumor, i.e., concomitant immunity. Using the P815 mastocytoma tumor, the kinetics of concomitant immunity was found to be governed by duration of exposure to the tumor and tumor mass. By implanting small "challenges" prior to the immunizing tumor, resistance to the growth of existing tumor foci was demonstrated. Winn-type assays revealed that antitumor activity was present in cell populations from the peritoneal exudate and lymph node draining the tumor. Peritoneal exudate cells, when infused systemically, were also able to confer protection against P815 mastocytoma challenge, suggesting their role as mediators of concomitant immunity. The 51Cr release technique indicated that cytolytic activity in lymph node cells, peritoneal exudate cells, and the spleen was present over a time course parallel to the kinetics of in vivo challenge. The peritoneal resident cell population was only slightly active; thus, effectors accumulated in the inflammatory exudate. Removal of specific subsets of cells from effector populations with antibody to surface markers and complement produced similar effects on both Winn and cytolytic assays. Anti-Thy 1.2 ablated measurable activity. It was substantially but not completely reduced by anti-Lyt 1.1 and only to a small degree by anti-Lyt 2.1.     

Radiation-induced antitumor properties of vitamin B5 (pantothenic acid) and its effect on mitomycin C activity: experiments in vitro

Vitamin B5 (pantothenic acid) shows a strongly pronounced antitumor effect under the influence of ionizing radiation. In the frame of experiments in vitro (model: Escherichia coli bacteria, AB1157) performed under the exact knowledge of concentration and kind of the free radicals acting in the various aqueous media (pH 7.4) the following was established: (i) vitamin B5 possesses a very intense antitumor property, (ii) it exerts a strong synergistic effect on mitomycin C (MMC), (iii) the oxidizing species (OH*, O2*-) appears to be most important in the initiation of the observed effect. The generated radiolytic products from vitamin B5 very likely also play an important role in this respect.     

Calcitriol (1,25-dihydroxycholecalciferol) enhances paclitaxel antitumor activity in vitro and in vivo and accelerates paclitaxel-induced apoptosis.

We demonstrated that calcitriol has antiproliferative activity in squamous cell carcinoma and prostatic adenocarcinoma and enhances the antitumor activity of platinum-based agents. In this study, we examined whether calcitriol also increases paclitaxel cytotoxicity. The effect of treatment on growth of the murine squamous cell carcinoma (SCCVII/SF) and human prostatic adenocarcinoma (PC-3) was determined by clonogenic assay, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and monitoring tumor growth. Treatment of SCC or PC-3 cells in vitro with calcitriol prior to paclitaxel significantly reduced clonogenic survival compared with either agent alone. Median-dose effect analysis revealed that calcitriol and paclitaxel interact synergistically. Treatment of SCC or PC-3 tumor-bearing mice with calcitriol prior to paclitaxel resulted in substantially greater growth inhibition than was achieved with either agent alone, supporting the combined use of calcitriol and paclitaxel in the treatment of solid tumors. To explore the molecular basis for the enhanced antitumor activity of this combination, the effect of treatment on p21(Waf-1) (p21), Bcl-2, and poly(ADP-ribose) polymerase expression was evaluated in PC-3. A 72-h pretreatment with calcitriol reduced p21 expression and increased paclitaxel cytotoxicity (measured after 24 h) without evidence of apoptosis [poly(ADP-ribose) polymerase cleavage]. After 48 h, paclitaxel induced apoptosis, the extent of which was increased similarly by pretreatment or concurrent treatment with calcitriol. We therefore propose a model for calcitriol enhancement of paclitaxel cytotoxicity in which the "early" (24 h) effects are schedule dependent and not attributed to enhancement of paclitaxel-induced apoptosis. In contrast, the "delayed" (48-h) enhancement of paclitaxel activity by calcitriol is schedule independent and associated with acceleration of apoptosis.     

Antimycotic activity of miconazole (R 18134) in vitro and in vivo

The in vitro and in vivo activity of miconazoie against a variety of 93 pathogenic fungi was studied. The in vitro test confirmed the results obtained by other authors. The intravenous injection of miconazole was very effective in animals with experimentally induced systemic mycoses. The in vivo results give the evidence that miconazole is more active and better tolerated than the agents actually in use for the clinical treatment of systemic mycoses. In some cases the results obtained with the systemic administration of miconazole in curing the dermatomycoses have been compared with those obtained using a tincture formulation of miconazole.

Zusammenfassung

Die in vitro und in vivo Wirksamkeit von Miconazol gegen 93 verschiedene pathogene Pilze wurde untersucht. Die in vitro Ergebnisse bestätigten die von anderen Autoren erhaltenen Resultate. Die intravenöse Gabe von Miconazol war bei Tieren sehr wirkungsvoll, bei denen systemische Mykosen experimentell verursacht wurden. Die in vivo Ergebnisse geben den Beweis dafür, daß Miconazol wirkungsvoller und besser verträglich ist als die Mittel, die für die klinische Behandlung von Systemmykosen angewendet werden.
Die in einigen Fällen erhaltenen Ergebnisse einer systemischen Anwendung von Miconazol zur Therapie von Dermatomykosen wurden mit den Ergebnissen, die bei lokaler Behandlung mit einer Miconazoltinktur erzielt wurden, verglichen.     

Molecular evidence for increased antitumor activity of gemcitabine by genistein in vitro and in vivo using an orthotopic model of pancreatic cancer

Soy isoflavone genistein exhibits growth inhibitory activity against human pancreatic cancer cell lines. We previously reported the potential of genistein to augment chemotherapeutic response of pancreatic cancer cells in vitro. In the present study, we investigated whether genistein pretreatment could be used as a novel strategy for gemcitabine-induced killing in vitro and enhanced antitumor activity in vivo using an orthotopic tumor model. We conducted our studies using paired isogenic human pancreatic cancer cell line with differences in metastatic behavior (COLO 357 and L3.6pl). In vitro studies were done to measure growth inhibition and degree of apoptotic cell death induced by either genistein alone, gemcitabine alone, or genistein followed by gemcitabine. Our results show that pretreatment of cells with genistein for 24 hours followed by gemcitabine resulted in 60% to 80% growth inhibition compared with 25% to 30% when gemcitabine was used alone. The overall growth inhibition was directly correlated with apoptotic cell death irrespective of the metastatic potential of cells. Several genes that are known to inhibit apoptosis and contribute to chemoresistance such as nuclear factor-kappaB (NF-kappaB) and Akt were assessed to investigate the basis for the observed chemosensitizing effects of genistein. Genistein potentiated the gemcitabine-induced killing by down-regulation of NF-kappaB and Akt. In contrast, Akt and NF-kappaB were found to be up-regulated when pancreatic cancer cells were exposed to gemcitabine alone, suggesting the potential mechanism of acquired chemoresistance. In addition to in vitro results, we show here for the first time, that genistein in combination with gemcitabine is much more effective as an antitumor agent compared with either agent alone in our orthotopic tumor model. But most importantly, our data also showed that a specific target, such as NF-kappaB, was inactivated in genistein-treated animal tumors and that gemcitabine-induced activation of NF-kappaB was completely inhibited in animal tumors treated with genistein and gemcitabine. These results provide strong molecular in vivo evidence in support of our hypothesis that inactivation of NF-kappaB signaling pathway by genistein could also abrogate gemcitabine-induced activation of NF-kappaB resulting in the chemosensitization of pancreatic tumors to gemcitabine, which is likely to be an important and novel strategy for the treatment of pancreatic cancer.     

Inhibition of invasion activity in vitro by a novel class of antitumor agents: silatrane derivatives

The effect of several silatranes on in vitro invasion of the human amnion basement membrane (BM) by A549 human lung carcinoma cells was examined. Cells treated for two days with the derivatives were examined for invasive activity in the absence of the compounds. From silatrane dose-invasion response curves, an 80% inhibition of invasiveness compared to untreated cells was obtained with 40 micrograms/mg of 1-vinyl silatrane, 50 micrograms/ml of 1-(p-aminophenyl) silatrane, 80 micrograms/mg of 1-(3-phenylthiocarbamidopropyl) silatrane, 66 micrograms/ml of parent silatrane or 171 micrograms/ml of 1-bromosilatrane. Treatment with these doses had no effect on viability, growth or BM attachment of A549 cells.