Elevated neutrophil elastase and acrolein-protein adducts are associated with W256 regression.

The involvement of granulocytes in immune response against cancer is not well understood. Depending on the cytokine milieu in which they act and on their oxidative burst, granulocytes may play either an inhibitory or stimulatory role in tumour growth. Unsaturated fatty acids, essential components of cellular membranes and storage lipids, are susceptible to granulocyte-derived reactive oxygen species (ROS). ROS can induce lipid peroxidation (LPO) resulting in the destruction of biomembranes. Thus, murine W256 tumour progressing and tumour regressing animal models were used to study the involvement of plasma inflammatory mediators and oxidative burst of circulating granulocytes in malignant destruction and detrimental tumour growth. The involvement of LPO-derived aldehydes (i.e. acrolein, 4-hydroxy-2-nonenal and malondialdehyde) and myeloperoxidase (MPO) appearance in the granulocyte anti-cancer response were further evaluated. The results obtained revealed a significant increase in neutrophil elastase in animals with regressing tumour. Furthermore, the presence of MPO in tumour microenvironment was accompanied by the formation of acrolein only 5 h after tumour transplantation and its presence increased during tumour regression. Later, at an early stage of tumour regression, the presence of other LPO-derived aldehydes were also observed. The results obtained suggest that elevated neutrophil elastase and initiation of LPO may play an important role in the tumour development leading to tumour regression.

Distribution and time-course of 4-hydroxynonenal, heat shock protein 110/105 family members and cyclooxygenase-2 expression in the hippocampus of rat during trimethyltin-induced neurodegeneration.

Trimethyltin (TMT), an organotin compound considered a useful tool to obtain an experimental model of neurodegeneration, exhibits neurotoxicant effects selectively localised in the limbic system and especially in the hippocampus, which are different in the rat and in mice. In the rat hippocampus, we investigated the expression of aldehyde 4-hydroxynonenal, a major bioactive marker of membrane lipid peroxidation, heat shock protein (HSP) 110/105 family members, markers of oxidative stress, and the neuroinflammatory marker cyclooxygenase-2 after TMT-intoxication at various time points after treatment. Our data show that TMT-induced neurodegeneration in the rat hippocampus is associated specifically with oxidative stress and lipid peroxidation, but not with HSP expression, indicating species-specific differences in the neurotoxicity of TMT between rats and mice.

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.

Overview on major lipid peroxidation bioactive factor 4-hydroxynonenal as pluripotent growth-regulating factor.

The reactive aldehyde 4-hydroxynonenal (HNE) is major bioactive marker of lipid peroxidation generated under oxidative stress from polyunsaturated fatty acids. Biomedical significance of HNE was first revealed in pathogenesis of various degenerative and malignant diseases. Thus, HNE was considered for decades only as cytotoxic molecule, "second toxic messenger of free radicals" responsible for numerous undesirable consequences of oxidative stress. However, the increase of knowledge on physiology of redox signaling revealed also desirable, physiological roles of HNE, especially in the field of cellular signaling pathways regulating proliferation, differentiation, and apoptosis. These pluripotent effects of HNE can be explained by its concentration-dependent interactions with the cytokine networks and complex cellular antioxidant systems also showing cell and tissue specificities. Therefore, this paper gives a comprehensive, yet short overview on HNE as pluripotent growth-regulating factor.

The pathophysiology of otosclerosis: Review of current research.

Otosclerosis is a complex disease of the human otic capsule with highest incidence in adult Caucasians. So far, many possible etiological factors like genetics, HLA, autoimmunity, viruses, inflammation, and hormones have been investigated but still the development of the disease remains unclear. Currently, the surgical replacement of stapes (stapedotomy) remains the best possible treatment option. In this review, we analyze different etiological factors studied so far in otosclerosis pathophysiology and discuss most recent findings and possible new research pathways.

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)

4-Hydroxynonenal modifies the effects of serum growth factors on the expression of the c-fos proto-oncogene and the proliferation of HeLa carcinoma cells.

In this study, the effect of 4-hydroxynonenal (HNE), a peroxidation product of omega-6-poly-unsaturated fatty acids, on the expression of the c-fos proto-oncogene and growth factor-induced proliferation of HeLa carcinoma cells in vitro was investigated. The Fos protein forms the heterodimer AP-1 with the Jun protein and regulates the cell cycle by inducing cyclin D1. Agents that are able to induce c-fos include serum, platelet-derived growth factor (PDGF), and epidermal growth factor (EGF), all of which were used in this study. The proliferation rate was determined by cell counting (viable and dead cells according to trypan blue exclusion) and the BrdU assay. The c-fos mRNA level was monitored by the reverse transcriptase/polymerase chain reaction. In the absence of HNE, serum-deprived cells responded to serum stimulation with a more than 10-fold increase of the c-fos mRNA level as well as with an increased rate of DNA synthesis and cell multiplication. Both EGF and PDGF (applied in combination with insulin) were able to substitute for FCS and induced rapid growth of the tumor cells preincubated in serum-deprived medium. In the absence of growth factors a negative correlation between the HNE concentration (range: 1-250 microM) and the c-fos mRNA level was observed. We suppose that HNE interferes in this case with the basal activity of the c-fos promoter. EGF, when applied after the HNE treatment, induced rapid growth of the tumor cells preincubated in serum-free medium, if HNE was used in a physiological concentration (1 microM). No difference was observed compared to the HNE-free control. c-fos mRNA level was nearly unchanged. In contrast, a cytotoxic concentration of the aldehyde (100 microM) caused a complete inhibition of proliferation, although a twofold increase of the c-fos mRNA level immediately after the aldehyde treatment was observed. A similar effect of HNE in cytotoxic concentration on c-fos expression was observed when cells were grown in presence of PDGF instead of EGF. Hence, in both cases HNE possibly interferes with the signal transduction pathway, which is initiated by external growth factors. The increased c-fos expression might be part of an abortive attempt to overcome the stressful condition raised by a cytotoxic concentration of HNE.

Treatment of the budding yeast Saccharomyces cerevisiae with the lipid peroxidation product 4-HNE provokes a temporary cell cycle arrest in G1 phase.

The effects of 4-hydroxy-2-nonenal (HNE) on the cell division cycle were investigated in the yeast Saccharomyces cerevisiae. A short treatment with HNE at a concentration in the range of the IC50 value in S. cerevisiae SP-4 cells induced a significant increase in the proportion of G0/G1 cells at the expense of S-phase cells. A similar delay in cell cycle progression upon treatment with HNE has recently been shown for HL-60 neoplastic cells. Long-term exposure in a synchronized yeast culture resulted in a pronounced dose-dependent block between G0G1- and S-phase, probably at or close to a point in the cell cycle that has been designated as "START." Incorporation of radioactively labeled precursors of macromolecules revealed that DNA synthesis was most susceptible to HNE in comparison to RNA and protein synthesis. Production of glutathione appeared to be required for the continuation of the cell cycle. HNE-treated yeast cells reentered the cell cycle when their glutathione content exceeded about twice the level of control cells. The release from the cell division cycle delay was followed by an enhanced growth to an extent that HNE-treated cells exceeded the number of control cells. These results indicate that HNE causes a biphasic modulation of cell proliferation. It was concluded that this effect was conserved during evolution from yeast to mammalian cells, emphasizing once more the usefulness of this unicellular organism as a model system for the investigation of the effects of free radical-derived products on the proliferation of eukaryotes.

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.

Gastric pentadecapeptide BPC 157 accelerates healing of transected rat Achilles tendon and in vitro stimulates tendocytes growth.

In studies intended to improve healing of transected Achilles tendon, effective was a stable gastric pentadecapeptide BPC 157 (GEPPPGKPADDAGLV, M.W. 1419). Currently in clinical trials for inflammatory bowel disease (PLD-116, PL 14736, Pliva), it ameliorates internal and external wound healing. In rats, the right Achilles tendon transected (5 mm proximal to its calcaneal insertion) presents with a large tendon defect between cut ends. Agents (/kg b.w., i.p., once time daily) (BPC 157 (dissolved in saline, with no carrier addition) (10 microg, 10 ng or 10 pg) or saline (5.0 ml)), were firstly applied at 30 min after surgery, the last application at 24 h before autopsy. Achilles functional index (AFI) was assessed once time daily. Biomechanical, microscopical and macroscopical assessment was on day 1, 4, 7, 10 and 14. Controls generally have severely compromised healing. In comparison, pentadecapeptide BPC 157 fully improves recovery: (i) biomechanically, increased load of failure, load of failure per area and Young's modulus of elasticity; (ii) functionally, significantly higher AFI-values; (iii) microscopically, more mononuclears and less granulocytes, superior formation of fibroblasts, reticulin and collagen; (iv) macroscopically, smaller size and depth of tendon defect, and subsequently the reestablishment of full tendon integrity. Likewise, unlike TGF-beta, pentadecapeptide BPC 157, presenting with no effect on the growth of cultured cell of its own, consistently opposed 4-hydroxynonenal (HNE), a negative modulator of the growth. HNE-effect is opposed in both combinations: BPC 157+HNE (HNE growth inhibiting effect reversed into growth stimulation of cultured tendocytes) and HNE+BPC 157(abolished inhibiting activity of the aldehyde), both in the presence of serum and serum deprived conditions. In conclusion, these findings, particularly, Achilles tendon transection fully recovered in rats, peptide stability suitable delivery, usefully favor gastric pentadecapeptide BPC 157 in future Achilles tendon therapy.

4-Hydroxynonenal as a second messenger of free radicals and growth modifying factor.

Immunohistochemical analysis of the distribution of the lipid peroxidation product 4-hydroxynonenal (HNE) in the brain of baboons exposed to experimental hemorrhagic traumatic shock or sepsis showed that systemic oxidative stress and the thereby generated HNE affect the blood:brain barrier and the regulation of cerebral blood flow determining secondary brain damage. Similarly, HNE was determined during ischemia in the brain blood vessels of rats exposed to ischemia/reperfusion injury of the brain. After reperfusion, HNE disappeared from the blood vessels but remained in neurones and in glial cells. Since HNE modulates cell proliferation and differentiation (including proto-oncogene expression), it is postulated that HNE might have prominent local and systemic effects that are not only harmful but beneficial, too, determining the outcome of various pathophysiological conditions based on oxidative stress.

The carcinostatic and proapoptotic potential of 4-hydroxynonenal in HeLa cells is associated with its conjugation to cellular proteins.

BACKGROUND: Previous studies have shown that the lipid peroxidation product 4-hydroxynonenal (HNE) acts as a cell growth modulator if used at low, physiological concentrations being strongly cytotoxic at higher concentrations for a number of cells. These effects of HNE also appeared to be mutually dependent on the effects of serum growth factors. The aim of this investigation was to study the concentration-dependent response of human cervical carcinoma (HeLa) cells in vitro with respect to the intracellular uptake of exogenous HNE, the cellular energy metabolism, DNA synthesis, overall gene expression and susceptibility to apoptosis. MATERIALS AND METHODS: MTT assay was applied as an index of energy metabolism and the replicative activity was quantitated by the 3H-thymidine incorporation assay. The occurence and intracellular distribution was studied with monoclonal antibodies directed against HNE-protein conjugates. Binding of HNE to serum proteins was determined with the same antibodies by Western blotting. Differential gene expression was studied by differential display RT-PCR while a novel photometric assay, denoted Titer-TACS, was used for in situ detection and quantitation of apoptosis in monolayer cell cultures. RESULTS: A physiological concentration of HNE (1 microM) had hardly any effect on the parameters of the replicative activity and the energy metabolism. No morphological changes were observed and the number of HNE-positive cells was not significantly different when compared to the untreated control cells, while most of the aldehyde appeared to be bound to serum proteins (albumin fraction). A ten-fold higher concentration (10 microM) was found to be cytostatic. Spindle-shaped cells with a picnotic nucleus were observed occasionally, as well as membrane blebs, which were HNE-positive. The number of HNE-positive cells was significantly increased compared both to the control cells and cells treated with 1 microM HNE, but in the presence of serum the effects of 10 microM HNE were negated due to its binding to the serum proteins. Finally, 100 microM HNE was cytotoxic for the HeLa cells. Most of the cells were picnotic, together with a few spindle-shaped or oval cells. The staining for HNE was diffuse and strong (90% of the cells were HNE-positive) while even binding of the aldehyde to serum proteins did not prevent its cytotoxic effects. This concentration of HNE caused acute stress response of the cells resulting in the decreased expression of several as yet unidentified genes. The altered pattern of gene expression was followed by programmed cell death, i.e. an increased number of apoptotic cells after treatment with low (1 and 10 microM) concentrations of HNE. A rebound effect was observed, i.e. a decrease of apoptotic cells after 24 hours followed by an overshooting increase after 48 hours. CONCLUSIONS: For HeLa carcinoma cells there appears to be a concentration range of HNE where it does not cause necrosis but preferentially apoptosis. At this concentration range HNE is cytochemically detectable within the cells as a protein conjugate. It is proposed that a possible differential sensitivity of cancer cells and their normal counterparts to the cytostatic activity of HNE should be explored.

Proatrial natriuretic peptide (1-98), but not cystatin C, is predictive for occurrence of acute renal insufficiency in critically ill septic patients.

INTRODUCTION: N-terminal prohormone of atrial natriuretic peptide ((proANP(1-98)) has been extensively analyzed in patients with chronic renal failure. It has been found to be closely related to the renal function and to interdialytic hydration status. The clinical relevance of proANP(1-98) and cystatin C, a novel marker of glomerular filtration, has not been investigated in the subgroup of critically ill septic patients with no history of chronic renal impairment. METHODS: We measured plasma level ofproANP(1-98) and cystatin C in 29 critically ill septic patients on admittance to the surgical intensive care unit and correlated it with the occurrence of acute renal failure. RESULTS: The proANP(1-98) plasma level was significantly higher in the group of patients who developed renal failure (12,722 +/- 12,421 vs. 2,801+/- 2,023 fmol/ml, p < 0.05). Multiple regression analysis shows that proANP(1-98) on the first day in the intensive care unit has a superior predictive value for the occurrence of renal failure to diuresis, calculated creatinine clearance or cystatin C (r = 0.42, p < 0.039). proANP(1-98) is also higher in non-survivors (9,303.8 +/- 11,053 vs. 2,448.5 +/- 1,803 fmol/ml, p < 0.018). CONCLUSION: proANP(1-98) is possibly a better predictor of acute renal failure to calculated creatinine clearance or diuresis among critically ill septic patients. Cystatin C was not correlated with occurrence of acute renal failure in this subgroup of patients.

Impaired proliferation and DNA synthesis of a human tumor cell line (HeLa) caused by short treatment with the anti-anemic drug jectofer (ferric-sorbitol-citrate) and the lipid peroxidation product 4-hydroxynonenal.

Anti-anaemic drug, ferric-sorbitol-citrate complex (FSC), inhibit tumour cell growth through the mechanisms which are complex and not entirely understood. The probable mechanisms of described effects of iron is iron-induced oxidative stress of the treated cells. Hence, the effects of FSC on HeLa cell growth in vitro were compared with the biological activity of one of the major mediators of the oxygen free radicals--aldehyde 4-hydroxinonenal (HNE), to see if the effects of FSC and of HNE resemble each other. Impaired proliferative ability and DNA synthesis of HeLa cells was observed after treatment with anti-anaemic drug FSC for 24 hours. After treatment with FSC and culturing of HeLa cells in fresh medium for 24 or 96 hours the cells did not proliferate at all, DNA synthesis was transiently recovered and then diminished again. HNE blocked cell proliferation during the time the aldehyde was present in culture and 24 h later. Afterwards, the cells proliferated as control non-treated cells. HNE did not inhibit DNA synthesis during treatment, but intensity of 3H-thymidine incorporation was lower after preincubation. Thus, both FSC and HNE interfere with the basic mechanisms of the cell growth regulation, while antitumour activity of FSC resembles, but does not necessarily include iron induced lipid peroxidation.

Inhibition of HeLa cell proliferation by 4-hydroxynonenal is associated with enhanced expression of the c-fos oncogene.

Previous studies have shown that the highly reactive aldehyde 4-hydroxynonenal (HNE), a mediator of oxidative stress, can either stimulate or inhibit cell proliferation, depending on the concentration of the aldehyde and the presence of serum. HNE can also induce differentiation of tumour cells in vitro and inhibit the tumour development in vivo. The aim of the study presented was to find out more details about the basic mechanisms by which HNE influences cell growth behaviour. Therefore we analysed the effect of HNE on the transcription of the c-fos gene in HeLa cells, to clarify the pathway by which the aldehyde modulates gene transcription and growth behaviour of the cells. At a supraphysiological concentration (50 microM) the aldehyde caused an enhanced c-fos transcription (as measured by the reverse transcriptase/polymerase chain reaction assay), while it inhibited cell proliferation markedly. Therefore, we assume that among the "early" effects of HNE on cellular growth regulation might be an altered expression of the "early response" genes (c-fos), while a "late" effect might be an altered autocrine/paracrine growth regulation of the cells. This finding on the possible basic mechanisms of the biological effects of HNE together with the already described high toxicity of the aldehyde for cancer cells give support for the further evaluation of the possible use of HNE in cancer biotherapy.

Involvement of lipid peroxidation, oncogene expression and induction of apoptosis in the antitumorous activity of ferric-sorbitol-citrate.

We described before that iron-containing, anti-anaemic drug, ferric-sorbitol-citrate complex (FSC) inhibited proliferation of various murine cancer cells in vitro and caused tumour regression in vivo, but did not affect proliferation of the non-malignant cells. The aim of this study was to evaluate further the anticancer activity mechanism of FSC using human colon cancer cell line CaCo2. After treatment with FSC for 72 hours impaired proliferative ability and viability of CaCo2 cells as observed. Growth modification caused by FSC involved diminished expression of Bcl-2, and over-expression of mp53 proto-oncogenes, accompanied by increased incidence of apoptosis. Immunostaining the cells applying monoclonal antibodies for lipid peroxidation product 4-hydroxynonenal (HNE) showed that FSC-iron increased intracellular HNE, but did not induce severe HNE-mediated oxidative stress. Thus, antitumorous mechanism of FSC involves modulation of oncogene expression and induction of apoptosis apparently not triggered by lipid peroxidation-mediated oxidative stress, although FSC might restore endogenous HNE production in the CaCo2 cells to level resembling physiological for various non-malignant cells and tissues. Higher dose of FSC increased also number of intracellular ferritin positive CaCo2 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.

An overview on anticancer activities of the Viscum album extract Isorel.

The activity principle of the mistletoe (Viscum album L.) phytotherapeutics could be considered as combined cytotoxic and "biological response modifying" activities (increasing host defense against cancer) that result from the activities of the plant lectins and the other biologically relevant substances. We found before that the aqueous extract Isorel, produced by Novipharm GmbH (Pörtschach, Austria) from the entire plant (planta tota) of fresh mistletoe under standardized conditions with bioassay validated batch consistency, can be valuable in experimental adjuvant cancer therapy increasing efficiency of cyclophosphamide chemotherapy. In current study we found that Isorel increases the reactivity of the tumor-bearing mice lymphocytes to the mitogens (ConA and LPS) in vitro, thus indicating its immune stimulating effects for the cancer-immunosuppressed lymphocytes. Moreover, Isorel inhibited the incorporation of 3H-labelled amino acids (protein synthesis) in various malignant cell lines. For the growth inhibition mostly higher MW components were responsible, although even less than 500 Da components were also active. We further analyzed the effects of drug application in vicinity of tumor (murine mammary carcinoma) and compared it with systemic effects. The animals carried mammary carcinoma in both hind limbs and were also injected with tumor cells i.v. to develop artificial lung metastases. Isorel was applied only at the right side (in the limb distal from the tumor) and caused persistent and almost complete inhibition of the tumor growth for 2/7 animals. Anticancer effects were less pronounced on the contralateral side tumors, although tumor growth rate was transiently reduced for some mice. Histology revealed that Isorel treatment, both at the side of tumor and systemically, increased the incidence of apoptosis and necrosis in the tumors, while reduction of mitosis was noticed only for the tumors in vicinity of the tumor exposed to Isorel. Finally, animals treated with Isorel had, on the average, three times less lung metastases than the controls. Thus, we conclude that both local and systemic effects of the application of Isorel could be of benefit for the tumor-bearing organism resulting in immunomodulation combined with tumor growth inhibition and reduction of metastases. According to the in vitro results, antitumorous effects could be the result not only of the mistletoe lectins and the other high MW factors, but also of the very low MW (< 500 Da) substances that deserve further analyses.

Corticosteroid-impairment of healing and gastric pentadecapeptide BPC-157 creams in burned mice.

The amelioration of corticosteroid-impairment of healing by a stable gastric pentadecapeptide BPC-157 (GEPPPGKPADDAGLV, M(w) 1419, currently in early clinical trials for inflammatory bowel disease) was studied in thermally injured mice. Its effects on corticosteroid impaired healing of deep partial skin thickness burns, and burn-gastric lesions were investigated. Male NMRI-Hannover mice (sacrificed at 1-3,7,14 and 21 days following burning 20% of total burn area at the back (open flame for 7s) received intraperitoneally (per kg bw) 6alpha-methylprednisolone (Depo-medrol, 1.0 or 10.0mg), or an equal volume of saline (5.0 ml), once daily, first application 30 min after injury, last 24h before sacrifice. The injury was subsequently treated by topical application of a thin layer of pentadecapeptide BPC-157 cream at three different levels a neutral cream of no treatment. Pentadecapeptide BPC-157 consistently improved given burn healing (both microscopical and tensionmetry assessment), and counteracted corticosteroid-impairment of burn healing. In burn-gastric lesions investigation of the effects of BPC showed an anti-ulcer effect of its own in burned non-corticosteroid-treated mice and potentiated the anti-ulcer effect observed in 6alpha-methylprednisolone-treated mice. Pentadecapeptide BPC-157 inhibited corticosteroid immunosuppression. In vitro, in spleenic cells assessment, animals (sacrificed at day 21) treated with 6alpha-methylprednisolone 1mg showed decreased reactivity to nitrogen in comparison with control, healthy animals, while the addition of BPC-157 (1 microg/g cream) returned cell reactivity to values noted in control healthy animals.