Metastases development following local tumour treatment.

Transplantable mouse methylcholanthrene- induced fibrosarcoma (CMC4 tumour growing in CBA/HZgr mice), characterized by lung metastases developing shortly after local tumour cell transplantation, was used as an experimental model to investigate the problem of tumour metastases after local tumour treatment. Surgery and/or irradiation were performed on locally growing tumour of particular size. Further, heavily irradiated, viable but not dividing tumour cells, imitating the situation in treated tumour-bearing organism, were injected intraperitoneally in a parallel group of treated tumour-bearing mice. The animals were killed 35 days after tumour transplantation and the number and volume of lung metastases were determined. Depending on the treatment performed, when the tumour mass was reduced or even eliminated, the number of lung metastases and their volume were significantly lower than in control mice, but the addition of tumour mass (injection of heavily irradiated tumour cells) resulted in a significant increase in lung metastases parameters, pointing to a possible role of the host's immune reaction against the tumour. Further, the release of a simple molecule, such as nitric oxide, from tumour mass seems to be detrimental for the survival of tumour cells and subsequently their metastases through the induction of angiogenesis and possible suppression of immune reaction. Thus, complex mechanisms could be involved when a locally growing tumour is exposed to a particular therapeutic approach.

The antitumor activity of thymoquinone and thymohydroquinone in vitro and in vivo.

AIM: The aim of the study was to investigate antitumor activity of thymoquinone (TQ) and thymohydroquinone (THQ) in vitro and in vivo. MATERIALS AND METHODS: In the in vitro experiments, L929 mouse broblasts and two tumor cell lines (squamous cell carcinoma (SCC VII) and fibrosarcoma (FsaR)) were used. The cells were cultured with 0.1 or 0.01 mg/ml TQ or THQ for 24 h, and cytotoxicity assay was performed with the use of crystal violet staining technique. For in vivo antitumor efficiency evaluation of new compounds two murine tumor models (fibrosarcoma (FsaR) and squamous cell carcinoma (SCC VII)) were used. The used dose was equal for both substances. Antitumor effect of 4 intratumoral injections of TQ and THQ at the dose of 5 mg/kg was evaluated by comparison of tumor growth kinetics between treated and control animals. RESULTS: In vitro study showed that TQ and THQ exhibit statistically significant cytotoxic activity (p less, similar 0.01). The cytotoxic activity was dose dependent and more expressed against tumor cells than against L929 fibroblasts. The result of antitumor activities of TQ and THQ in vivo reached TGI = 52% and it was statistically significant (p less, similar 0.05). CONCLUSION: The results indicate that THQ antitumor activity may be improved with further dose increase of the investigated substance.

Natural zeolite clinoptilolite: new adjuvant in anticancer therapy.

Natural silicate materials, including zeolite clinoptilolite, have been shown to exhibit diverse biological activities and have been used successfully as a vaccine adjuvant and for the treatment of diarrhea. We report a novel use of finely ground clinoptilolite as a potential adjuvant in anticancer therapy. Clinoptilolite treatment of mice and dogs suffering from a variety of tumor types led to improvement in the overall health status, prolongation of life-span, and decrease in tumors size. Local application of clinoptilolite to skin cancers of dogs effectively reduced tumor formation and growth. In addition, toxicology studies on mice and rats demonstrated that the treatment does not have negative effects. In vitro tissue culture studies showed that finely ground clinoptilolite inhibits protein kinase B (c-Akt), induces expression of p21WAF1/CIP1 and p27KIP1 tumor suppressor proteins, and blocks cell growth in several cancer cell lines. These data indicate that clinoptilolite treatment might affect cancer growth by attenuating survival signals and inducing tumor suppressor genes in treated cells.

Spleen peptides (Polyerga) inhibit development of artificial lung metastases of murine mammary carcinoma and increase efficiency of chemotherapy in mice.

There are numerous attempts to find novel anticancer drugs or to improve therapeutic protocols based on application of chemotherapeutic agents and immunomodulators (biological response modifiers, cytokines, various plant or bacterial products). Among the preparations that have beneficial effects for the cancer bearing organism are preparations of spleen peptides (Polyerga). Hence, we analyzed if treatment with spleen oligopeptides GP-1 (active substance for the manufacture of Polyerga ampoules' solution injected as 0.5 microgram/kg every second day) if given alone or combined with chemotherapy (Endoxan 50 mg/kg single i.p. dose) of mice bearing artificial lung metastases of mammary carcinoma will have an impact on the metastases count and survival of the animals. The results obtained have shown that chemotherapy reduced metastases count and increased survival of the tumor bearing mice, while the use of GP-1 alone did not affect metastases development. However, combined GP-1 treatment and chemotherapy were more efficient in prevention of the metastases development than chemotherapy alone. Thus, in mice treated with GP-1 and Endoxan, the average metastases count was four times lower than in the mice treated by chemotherapy only, while 2/12 animals were without tumor nodules in the lungs. Finally, all the animals treated by chemotherapy alone died until the 42nd day after tumor transplantation, while at the same time, only 5/10 animals died receiving combined therapy. Thus, these results give an experimental support for the use of the spleen peptides in biotherapy (or combined therapy) of cancer.

Comparison of the effects of Viscum album lectin ML-1 and fresh plant extract (Isorel) on the cell growth in vitro and tumorigenicity of melanoma B16F10.

Numerous findings indicate that specific plant lectins acting against cancer could be major active components of Viscum album extracts, although activity of low molecular weight components (peptides, carbohydrates and alkaloids) might be as essential for the beneficial activity of the plain plant extracts, too. Thus, active principle of Viscum album extracts is still not understood, and is difficult to be analysed because of the complex composition of the extracts and uncertainty of the standardised effectiveness (batch consistency) of the extracts. The aims of this study were to compare the concentration dependent effects of the pure mistletoe lectin (ML-1) with the fresh plant Viscum album extract (Isorel) and its different MW components on the in vitro growth of ConA stimulated lymphocytes, on the growth and tumorigenicity (artificial lung metastases development) of murine melanoma B16F10 cells, and to compare concentration dependent effects of the different types of the Viscum album extracts in vitro (applying novel type of MTT assay). The results obtained indicate that the effects of Isorel used at high dose could be result of toxic activity of the mistletoe lectins ("ML-1 like" activity). Unlike ML-1, if used at low concentrations, Isorel selectively inhibited tumor cells, due the activity of the low MW components. On the other hand, the number of tumor nodules was reduced (in comparison to the control) equally in the lungs of mice injected with B16F10 cells pre-treated in vitro with the plain Viscum album extract or any of its modifications or ML-1. Hence, it is supposed that the beneficial therapeutic effects of Isorel might result from the combined biological activity of the high and the low MW components not lectins only. Similarly, in MTT assay low concentrations of all types of the Viscum album extract showed stronger inhibiting activity for B16F10 and HeLa cells than pure ML-1. According to these results we propose a standardisation of aqueous Viscum album extracts by comparing their and ML-1 concentration dependent activity on the tumor cells in vitro applying MTT bioassay described which should be relevant for further evaluation of their active principle and for improvement of biotherapy of cancer.

The Viscum album preparation Isorel inhibits the growth of melanoma B16F10 by influencing the tumour-host relationship.

The aim of this study was to analyse whether Viscum album (mistletoe; Isorel) modulates the tumour-host relationship and whether this might be a basic mechanism of the antitumorous activity of the drug. The effects of a single intraperitoneal injection of the drug (100 mg/kg single 'planta tota' dose) were analysed for mice-bearing melanoma B16F10 growing in the hind limb. Injection of Isorel reduced the size of the tumour and caused abundant tumour necrosis with inflammatory response, oedema and destruction of the malignant tissue. Furthermore, the lymphocytes of saline-treated tumorous mice were not able to respond to the mitogenic lectin concanavalin A in vitro, while those of mistletoe extract-treated mice showed high reactivity too the mitogen, but only if cultured in the medium supplemented with the plasma of the mistletoe extract-treated mice. Moreover, melanoma cells exposed to the mistletoe extract were more sensitive to the cytotoxic activity of the lymphocytes than the control tumour cells, particularly in the presence of the plasma of mistletoe extract-treated mice. The plasma itself, however, did not show any cytotoxic activity. These results indicate that the antitumour activity of the mistletoe drug is due to a modulation of the tumour-host relationship, mediated by direct cytotoxicity of the drug to tumour cells and/or through a potentiation of immune response by certain, as yet unidentified, growth modifying humoral factors of the host.

Viscum album L. preparation Isorel modifies the immune response in normal and in tumour-bearing mice.

The Viscum album (mistletoe) preparation Isorel is able to destroy tumour cells and to modify immune reactivity against a particular antigen in normal and in tumour-bearing animals. CBA/HZgr mice and methylcholanthrene-induced fibrosarcoma were used in these studies. A single dose of Isorel M (140 mg/kg or 1400 mg/kg body weight) significantly increased the number of plaque forming cells if applied at the time of injection of sheep red blood cells or 1 day earlier. The application of Isorel 1 day after sheep red blood cells did not modify the number of plaque forming cells in comparison to the controls. The higher the dose of Isorel the stronger is the immune response to sheep red blood cells. Furthermore, one dose of Isorel (140 mg/kg body weight) restored the suppressed immune response of fibrosarcoma-bearing mice to a significant extent. Besides modification of the humoral immune response, the survival time of C57BI/GoZgr male skin grafts on syngeneic female recipients was significantly shorter if Isorel was applied at a particular time after grafting. However, according to plaque forming cell numbers, a prolonged application of Isorel was significantly immunosuppressive in normal mice and particularly in tumour-bearing mice. It should be mentioned that the doses of Isorel used in this experiment were much higher than generally used in cancer patients. In view of the immunomodulating effects of Isorel, the monitoring of the immune response of the patients treated with mistletoe preparations is to be recommended.

Porcine splenic peptides (Polyerga) decrease the number of experimental lung metastases in mice.

Preparations of splenic peptides under the name of Polyerga are being tested in numerous experimental immunomodulating and antitumorous models and are also used during supportive treatment of tumorous patients. Further, the incidence of experimental lung metastases of melanoma cells in mice was significantly reduced if we used Polyerga preparations. The aim of our investigation was to determine whether Polyerga is active directly against tumor cells or whether its activity is manifested by modulating immune and other possible abilities of the organism. To clarify the problem glycopeptides containing Polyerga were incubated with melanoma B16F10 cells in vitro and the plating efficiency of these cells determined when cultivated in medium, or in medium with different doses of the same Polyerga preparation. The cells preincubated in medium only reacted to the addition of increasing doses of Polyerga, 150 pg or more, by raising colonies number. However, 24-h incubation of melanoma cells in the presence of 150 micrograms of Polyerga per ml significantly reduced the number of tumor cell colonies in comparison to the corresponding cell cultures previously not exposed to Polyerga. These in vitro studies were extended to in vivo application using C57B1/GoZgr mice injected i.v. with melanoma cells pretreated with Polyerga in vitro or previously not treated. A group of the treated mice was further injected i.p. with Polyerga. All the mice were killed at a particular time and the number of lung nodules determined. A significant difference to the control values was noticed in each group that used Polyerga, regardless of the exposure of melanoma cells to Polyerga in vitro, in vivo or to combined treatment. The efficiency of Polyerga application 7 days following i.v. injection of control melanoma cells (cultivated in medium only) when the nodules already exist, was further evaluated in a combined treatment using DTIC, a drug of choice in melanomas. The smallest incidence of experimental lung metastases was observed in the group exposed to the combination of DTIC and Polyerga. Polyerga preparation is thus active against melanoma cells, particularly in vivo and if combined with chemotherapy.

Analysis of the in vitro secretory activity of human pituitary adenomas: modification of corticotropin release from adenoma tissue explant cultures by addition of a human plasma ultrafiltrate bioactive fraction.

The lack of control of tumour behaviour is manifested in different ways, depending primarily on the type of tumour. This results in numerous problems of tumour diagnosis and therapy. In the case of "benign" tumours, like pituitary adenomas, in vitro studies are often used for evaluation of the tumour. The use of tissue explant cultures of human pituitary adenomas and the comparison of the feature of cultured tumours with their behaviour in vivo showed that corticotropin is released not only from the tumours associated with Cushing's disease, but also from clinically non-functioning tumours. Hence, it was supposed that the release of corticotropin in vivo from non-secreting tumours is probably under the influence of certain neuroendocrine and/or systemic humoral factors. To test this possibility, samples of 22 tumours were cultured in plain culture medium or in the presence of the "human plasma ultrafiltrate bioactive fraction" (tentatively termed as TBP) prepared by anion-exchange chromatography. In the presence of TBP the release of corticotropin was strongly inhibited in adenomas showing relatively high spontaneous secreting activity in vitro (> 200 ng/l in 24 hours), while immunohistochemistry of these tumours indicated accumulation of corticotropin inside the cells. In contrast, TBP stimulated corticotropin release from tumours that showed relatively low basic corticotropin release (< 200 ng/l in 24 hours), with no obvious change in cellular corticotropin immunoreactivity. Such a dual activity of TBP was not observed for 8 samples of adenomas cultured in the presence of surrounding pituitary tissue, probably because TBP did not affect corticotropin secretion by the normal pituitary cells (as indicated by immunohistochemistry). From these results, it appears that TBP could be one of the humoral factors involved in the regulation of corticotropin release from pituitary adenoma tissue. Its possible involvement in the regulation of corticotropin release from normal pituitary tissue, however, is uncertain.

A mathematical model for the effect of red light penetration depth on tumor growth.

The effect of different local temperature increase in normal and tumorous tissue, influenced by red light commonly used in photodynamic therapy, on tumor growth rate was reanalyzed, taking into account the tumor tissue penetration depth of the light used. The rear part of the tumor is definitely not receiving the same light dose, and consequently the same amount of heat energy, as the front part. The effect is taken into account by using a mathematical model of tumor growth for the rear part of the tumor. Positive experimental results of hyperthermia due to the energy deposited by the red light, are more encouraging after such a correction than previously reported.

Inhibition of melanoma B16-F10 growth by lipid peroxidation product 4-hydroxynonenal.

Since a gradual benign-to-malignant progression of murine melanoma B16 after exposure in vitro to hypoxia was described recently, the aim of this study was to test if exposing melanoma B16-F10 cells to aldehyde 4-hydroxynonenal (HNE), which is considered not only as one of the major "second toxic messengers" of oxygen free radicals (or oxidative stress), but as a normal constituent of many cells and tissues, might have opposite effects. Treatment of the tumor cells with 50 microM HNE in vitro or in vivo did not prevent development of the tumors, but inhibited their growth. Tumor growth inhibition was equal for in vitro and in vivo treatment, but appeared after a delay of almost one week, since there was no difference of the tumor volume to the control observed during the initial period of the tumor growth. Similarly, both HNE treatment of the tumor cells before transplantation and HNE treatment of the melanoma bearing mice resulted in equally prolonged survival time. Thus, the results obtained suggest that while hypoxia could increase the malignancy of the murine melanoma cells, exposing these cells to one of the major "second toxic messengers" of oxygen free radicals, HNE, has almost opposite effects and further indicate the possible use of the aldehyde in vivo.

Mutual dependence of growth modifying effects of 4-hydroxynonenal and fetal calf serum in vitro.

Recently, the hypothesis has been put forward that 4-hydroxynonenal (HNE), an aldehydic product of lipid peroxidation, contributes to the mechanisms of oxygen toxicity and to the selective pressure exerted by exposure to hyperoxia. Here it has been studied whether HNE itself is involved in mechanisms that convey increased resistance of the cells to the toxicity of HNE. The following four cell lines, different in their basic biological features, were used: nonmalignant Chinese hamster lung fibroblasts V79 (established cell line), human carcinoma HeLa (established cell line), pigmented murine melanoma B16f10 (primary culture), and amelanotic murine melanoma B16BL6 (primary culture). The cells were pretreated in vitro with a toxic dose of HNE (50 microM), and afterwards the effect of a second exposure to the same dose of HNE on 3H-thymidine incorporation was examined. Cells were cultured in the absence and in the presence of fetal calf serum (FCS), because it had been shown that a growth modifying effect of HNE depends on an unknown serum factor. The results showed that, regardless of the type of cells, preculturing them with 50 microM HNE in the presence of serum changed the reactivity of the cells to added serum as well as to additional HNE treatment. Thus, HNE precultured cells incorporated less 3H-thymidine in the presence of serum than if cultured under serum-free conditions. On the other hand, HNE precultured cells became less sensitive to further HNE treatment, but only if cultured in the presence of serum.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of HeLa cell growth by physiological concentrations of 4-hydroxynonenal.

The aim of this study was to analyze the growth response of HeLa cells over a prolonged period of time to a single exposure of physiological and supraphysiological concentrations of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-polyunsaturated fatty acids. Furthermore, the growth modulating effect of serum factors, particularly albumin, on the growth pattern was examined. The effects of HNE on the growth rate and viability of the cells, as well as on the incorporation of labelled amino acids were monitored daily over a period of four days. Fetal calf serum not only had a growth stimulating effect but also modulated the action of HNE. In neither respect was albumin able to substitute for serum indicating that the influence of serum was not exerted via an albumin-HNE conjugate. HNE had a clear dose-dependent effect and a distinction could be made between a supraphysiological concentration (100 microM), which was primarily cytotoxic and a physiological range (below 10 microM) which showed growth modulatory effects. These effects consisted of a transient inhibition in the initial phase of the cell growth, which under optimal conditions (in presence of serum) was followed by a period of increased proliferation, compared to untreated control cultures, until confluence was attained. It is suggested that HNE is not only a toxic product of lipid peroxidation, but a physiological growth regulating factor as well.

Antitumorous and immunomodulatory effects of the Viscum album L. preparation Isorel.

There are numerous data on the immunostimulative and antitumorous activity of various Viscum album tissue extracts. Isorel (Novipharm, Austria) is one of these compounds. We found that in mice an increased number of plaque-forming cells to sheep red blood cells (SRBC) followed the injection of Isorel together with SRBC. Further, survival time of a foreign skin graft was shortened if Isorel was applied at the correct time. Finally, suppressed immune reactivity in tumorous mice recovered following Isorel injection. Isorel was further shown to be cytotoxic to tumor cells in vitro. Its application to tumor-bearing mice could prolong their life but without any therapeutic effect. However, a combination of local irradiation and Isorel was very effective: following 43 Gy of local irradiation to a transplanted methylcholanthrene-induced fibrosarcoma (volume about 240 mm3) growing in syngeneic CBA/HZgr mice, the tumor disappeared in about 25% of the animals; the addition of Isorel increased the incidence of cured animals to over 65%. The combined action of Isorel, influencing tumor viability on the one hand and the host's immune reactivity on the other, seems to be favorable for its antitumor action in vivo.

Mitogenicity of the earthworm's (Eisenia foetida) insulin-like proteins.

1. Biologically active glycolipoprotein complex (G-90), isolated from whole earthworm tissue extract (Eisenia foetida), was separated into seven fractions by gel-filtration. 2. It has been shown by radioimmunoassay that each of the fractions, except the lightest one, is cross-reactive with porcine anti-insulin antibodies. Molecules that possess such activity were detected by immunoblotting. 3. All fractions, except the heaviest and the lightest one, stimulate mammalian normal and transformed cell proliferation in serum-free conditions in vitro. The intensity of stimulation depends on cell type. Stimulation is completely abolished if the medium is supplemented with fetal calf serum.

The influence of mouse sera, regenerating liver extracts and bacterial products on the abilities of different cells in vitro.

In the complexity of host tumor relations, the regeneration of the tissue in which the tumor is growing, or in some other tissue in the organism, could influence the maturation of tumor cells, i.e. tumor reversion. Clinical observations and experiments on plants, lower animals, or animal embryos, performed by several authors, and our results on the influence of regenerating mouse liver on the abilities of tumor transplanted there or elsewhere in the organism led us to study the in vitro growth of different cells or bacteria exposed to the extracts of normal or regenerating liver and/or sera from these animals. Further, sterile used bacterial media were added to bacterial or cell cultures, respectively. Depending on the model, liver extracts-particularly extracts and sera from mice with regenerating liver-were shown to inhibit radioactive thymidine incorporation in the cells. In these experiments, the number of bacteria or cells per culture was lower than in otherwise treated corresponding cultures. Further, used sterile media of bacterial cultures stimulated the growth of bacteria but inhibited thymidine incorporation into fibrosarcoma cells in vitro. Whether this means that one or several common regulators exist in nature appears as an intriguing, but still completely open question. The idea of controlling tumor growth by using such regulatory growth factors seems very provocative.

[Inhibition of growth of B16 melanoma caused by liver regeneration]. / Inhibicija rasta melanoma B16 izazvana regeneracijom jetara.

Phenomenon of spontaneous regression of cancer indicates that recovery from malignant disease can happen without the application of any so far known therapeutic treatment. The most of the self-cured patients have undergone surgical operation which did not eliminate entire tumor or did not affect malignant tissue at all. Furthermore, regression or reversion of tumors (transformation of tumor cells into normal, nonmalignant cells) can be achieved in plants or amphibia after exposure of tumor cells to the influence of normal, regenerating tissue. Thus, it seems that during tissue regeneration certain local changes in tissue happen (synthesis of some regulatory growth factors) which induce dying of tumor cells or modify main features of malignant cells. Previously we have studied growth of murine malignant tumors in regenerating tissue of liver and skin. Obtained results indicated that under the influence of regenerating tissue anaplasia of fibrosarcoma decreases, as well as do pigmentation and the incidence of live cells in melanoma B16 tissue. Thus, it is obvious that mechanism of regenerating tissue growth control, which can also change characteristics of tumor cells, exists in mammalia, too. In order to analyse whether the same homeostatic mechanism is responsible for the phenomenon of spontaneous regression of human cancer, we have analysed the growth of melanoma B16 in back limb of nonoperated and sham or partially hepatectomized mice. Regeneration of skin and abdominal wall tissue in sham hepatectomized animals slowed down tumor growth, while liver regeneration completely inhibited tumor progression. Tumor growth inhibition was result of tumor tissue necrosis which developed around blood vessels. However, the structure and integrity of blood vessels themselves was normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of semiconductor GaAs laser irradiation on pain perception in mice.

The influence of subacute exposure (11 exposures within 16 days) of mice to the low power (GaAs) semiconductive laser-stimulated irradiation on pain perception was investigated. The pain perception was determined by the latency of foot-licking or jumping from the surface of a 53 degrees C hot plate. Repeated hot-plate testing resulted in shortening of latencies in both sham- and laser-irradiated mice. Laser treatment (wavelength, 905 nm; frequency, 256 Hz; irradiation time, 50 sec; pulse duration, 100 nsec; distance, 3 cm; peak irradiance, 50 W/cm2 in irradiated area; and total exposure, 0.41 mJ/cm2) induced further shortening of latencies, suggesting its stimulatory influence on pain perception. Administration of morphine (20 mg/kg) prolonged the latency of response to the hot plate in both sham- and laser-irradiated mice. This prolongation tended to be lesser in laser-irradiated animals. Further investigations are required to elucidate the mechanism of the observed effect of laser.