cytochalasin-b and Melanoma

Background Despite inherent differences between the cytoskeletal networks of malignant and normal cells, and the clinical antineoplastic activity of microtubule-directed agents, there has yet to be a microfilament-directed agent approved for clinical use. One of the most studied microfilament-directed agents has been cytochalasin B, a mycogenic toxin known to disrupt the formation of actin polymers. Therefore, this study sought to expand on our previous work with the microfilament-directed agent, along with other less studied cytochalasin congeners. Materials and Methods We determined whether cytochalasin B exerted significant cytotoxic effects in vitro on adherent M109 lung carcinoma and B16BL6 and B16F10 murine melanomas, or on suspension P388/ADR murine leukemia cells. We also examined whether cytochalasin B, its reduced congener 21, 22-dihydrocytochalasin B (DiHCB), or cytochalasin D could synergize with doxorubicin (ADR) against ADR-resistant P388/ADR leukemia cells, and produce significant cytotoxicity in vitro. For in vivo characterization, cytochalasins B and D were administered intraperitoneally (i.p.) to Balb/c mice challenged with drug sensitive P388-S or multidrug resistant P388/ADR leukemias. Results Cytochalasin B demonstrated higher cytotoxicity against adherent lung carcinoma and melanoma cells than against suspension P388/ADR leukemia cells, as assessed by comparative effects on cell growth, and IC₅₀ and IC₈₀ values. Isobolographic analysis indicated that both cytochalasin B and DiHCB demonstrate considerable drug synergy with ADR against ADR-resistant P388/ADR leukemia, while cytochalasin D exhibits only additivity with ADR against the same cell line. In vivo, cytochalasins B and D substantially increased the life expectancy of mice challenged with P388/S and P388/ADR leukemias, and in some cases, produced long-term survival. Conclusion Taken together, it appears that cytochalasins have unique antineoplastic activity that could potentiate a novel class of chemotherapeutic agents.

Topics: Animals; Antineoplastic Agents; Cell Survival; Cytochalasin B; Cytochalasin D; Cytochalasins; Doxorubicin; Drug Synergism; Leukemia P388; Lung Neoplasms; Melanoma; Mice; Mice, Inbred BALB C; Neoplasms; Tumor Cells, Cultured

It was previously found that L-tyrosine oxidation product(s) are cytotoxic, genotoxic and increase the sister chromatid exchange (SCE) levels in human melanoma cells. In this work, the micronucleus assay has been performed on human melanotic and amelanotic melanoma cell lines (Carl-1 MEL and AMEL) in the presence of 1.0, 0.5 and 0.1 mM L-tyrosine concentrations to investigate if melanin synthesis intermediate(s) increase micronuclei production. L-Tyrosine oxidation product(s) increased the frequency of micronuclei in melanoma cells; 0.1 mM phenylthiourea (PTU), an inhibitor of L-tyrosine oxidation by tyrosinase, lowered the micronucleus production to the control levels. The culture of melanoma cells with high L-tyrosine in the culture medium resulted in a positive response to an ELISA-based apoptotic test. For comparison the effect of L-tyrosine on micronuclei production in human amelanotic melanoma cells was also investigated; the micronucleus production in the presence of 1 mM L-tyrosine in the culture medium was lower than that found with melanotic melanoma cells of the same cell line. The data suggest that melanin synthesis intermediates arising from L-tyrosine oxidation may cause micronuclei production in Carl-1 human melanoma cells; the addition of PTU in the presence of L-tyrosine decreased the frequency of micronuclei to about the control values thus the inhibition of melanogenesis may have some clinical implication in melanotic melanoma.

Topics: Apoptosis; Cell Nucleus; Cytochalasin B; Dose-Response Relationship, Drug; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Humans; Melanoma; Melanoma, Amelanotic; Micronucleus Tests; Monophenol Monooxygenase; Phenylthiourea; Skin Neoplasms; Tumor Cells, Cultured; Tyrosine

Cytokinesis-block micronucleus assay (CB-MNA) was applied for comparison of radiation sensitivity of 25 human malignant melanomas in primary culture. Cells obtained from tumor specimens were irradiated (0-4.Gy) on dishes, incubated with cytochalasin B (2 micrograms/ml) to block cytokinesis, stained in situ and micronuclei (MN) scored in binucleate cells (BNC). Proportions of BNC in nonirradiated controls after fixed time of incubation (96 h) ranged from 2.3 to 38% indicating great differences (C.V. = 74%) in proliferative activity among tumors evaluated. No correlation was observed between proliferative activity and susceptibility of cells to induction of MN by radiation. The great inter-tumor heterogeneity was observed in respect of radiation sensitivity expressed either as normalized (Net) frequency (Fq) of BNC with MN or as number of MN per BNC. Both endpoints differed widely at 2 Gy and 4 Gy as well (Net FqBNC with MN = 0.28-25.4% or 1.5-45% and MN/BNC = 0.004-0.309 or 0.013-0.593 respectively at 2 Gy and 4 Gy) with coefficients of variation ranging from 44 to 57%. Extreme difference in MN frequency was also observed between one primary tumor and its metastasis indicating intra-tumor heterogeneity. Our results suggest that CB-MNA may contribute some clinically useful information for discriminating tumors that will eventually respond to radiotherapy and those that will probably not. However, studies aimed at comparison of MN induction in vitro with clinical radioresponsiveness of malignant melanomas are urgently required.

Topics: Antibodies, Monoclonal; Cell Division; Cell Nucleus; Cytochalasin B; Female; Flow Cytometry; Humans; Immunohistochemistry; Male; Melanoma; Micronucleus Tests; Ploidies; Radiation Tolerance; Skin Neoplasms; Tumor Cells, Cultured

Consumption of soy products has been linked to a reduced mortality and morbidity from a number of cancers. Genistein, one of the principal soy isoflavones, has been shown to inhibit the growth of a number of tumour cell lines in vitro; however, a role of genistein in retarding tumour growth in vivo is less well documented. In this study, in addition to examining the effects of genistein on the growth of murine B16 melanoma cells in vitro, we have examined the effects of feeding a genistein-rich diet on s.c. growth of these tumour cells in mice. In vitro, the melanoma cells showed an increase in sensitivity to genistein with increasing time of exposure, culminating in a 50% growth inhibition (IC50) at 12.5 microM after 7 days. Genistein at 25 microM induced micronucleus formation after 24 hr and at concentrations as low as 2.5 microM induced morphological changes indicative of differentiation. Growth of solid tumours implanted into female C57BL/6J mice was inhibited by 50% when mice were fed genistein for 1 week before and for 1 week after inoculation with B16 melanoma cells. Plasma genistein concentrations at the time of tumour removal were 1.1 microM, which is similar to levels reported in humans consuming diets high in soybeans or soybean products, while control animals had no detectable genistein in plasma. Our results provide additional in vivo evidence suggesting that genistein retards the growth of implanted tumours, adding further to studies suggesting that this isoflavonoid is a biologically active component of soy foods.

Topics: Animals; Antineoplastic Agents; Cell Differentiation; Cell Division; Cytochalasin B; Dose-Response Relationship, Drug; Etoposide; Female; Genistein; Growth Inhibitors; Melanoma; Mice; Mice, Inbred C57BL; Neoplasm Transplantation; Time Factors; Tumor Cells, Cultured

While the primary targets for granulocyte-macrophage colony-stimulating factor (GM-CSF) are hematopoietic precursors and mature myeloid cells, GM-CSF receptors (GMR) are also found on normal tissues including placenta, endothelium, and oligodendrocytes as well as certain malignant cells. The function of GMR in these nonhematopoietic cells is unknown. We studied the function of GMR in human melanoma cell lines. Six of seven cell lines tested (clones 1-5 and 3.44 of SK-MEL-131, SK-MEL-188, SK-MEL-23, SK-MEL-22, and SK-MEL-22A) expressed mRNA encoding the membrane-bound and soluble isoforms of the alpha subunit of the GMR. Melanoma cell lines in early stages of differentiation expressed the largest quantities of alpha-subunit mRNA. Although five of these lines expressed trace levels of mRNA encoding the beta subunit of the GMR, Scatchard analysis of equilibrium binding data derived from three of the cell lines showed that they expressed only low-affinity GMR. Clones 3.44 and 1-5 of SK-MEL-131, and SK-MEL-188 cells expressed receptors with a dissociation constant (kd) for GM-CSF in the following ranges: 0.7 to 0.8, 1.2 to 1.8, and 0.4 to 0.8 nmol/L, respectively. GM-CSF stimulated glucose uptake in four of the melanoma cell lines expressing the alpha subunit, presumably through facilitative glucose transporters, as uptake was blocked by cytochalasin B but not cytochalasin E. Stimulation of glucose uptake was transient, with maximum stimulation occurring at approximately 30 minutes in the presence of 1 nmol/L GM-CSF. GM-CSF stimulated glucose uptake 1.4- to 2.0-fold but did not stimulate cell proliferation. These results suggest a metabolic role for the low-affinity GMR in melanoma cell lines and indicate that the alpha subunit of the GMR can signal for increased glucose uptake in nonhematopoietic tumor cells.

Topics: Base Sequence; Biological Transport, Active; Cell Line; Cytochalasin B; Cytochalasins; Deoxyglucose; DNA Primers; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Kinetics; Leukemia, Myeloid; Macromolecular Substances; Melanoma; Molecular Sequence Data; Monosaccharide Transport Proteins; Polymerase Chain Reaction; Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; RNA, Messenger; Signal Transduction; Tumor Cells, Cultured

The radiation-induced genotoxic damage in three established cell lines and 15 primary cultures of human malignant melanoma and ovarian carcinoma showing different radiosensitivity was tested by the cytokinesis-block micronucleus assay. A dose-related increase in micronucleus frequency was observed in all the cell systems. The mean number of micronuclei per Gy of ionising radiation per binucleated cell was respectively 0.44 +/- 0.0075 and 0.43 +/- 0.04 for M14 and JR8 malignant melanoma cell lines and 0.19 +/- 0.013 for the A2780 ovarian cancer cell line. The number of micronuclei did not rank the cell lines in the same order of radiosensitivity as clonogenic cell survival, which showed a surviving fraction at 2 Gy of 0.38 +/- 0.02 for JR8, 0.34 +/- 0.05 for M14 and 0.22 +/- 0.007 for A2780. As regards primary tumour cultures, no correlation was observed between micronucleus induction and surviving fraction at 2 Gy. In conclusion, the discrepancy we observed between micronucleus formation and cell death raises doubts about the potential of the micronucleus assay as a preclinical means to predict radiosensitivity.

Topics: Carcinoma; Cell Survival; Clone Cells; Cytochalasin B; DNA Damage; DNA, Neoplasm; Dose-Response Relationship, Radiation; Female; Humans; In Vitro Techniques; Melanoma; Micronucleus Tests; Ovarian Neoplasms; Radiation, Ionizing; Tumor Cells, Cultured

Effects of cytochalasins on actin polymerization state in living cells were measured using fluorimetry of TRITC-phalloidin bound to F-actin. Normal (3T3) and tumour (SV-3T3, B16 melanoma, and Ehrlich ascites) cells were treated with cytochalasin B and cytochalasin D (1 microgram/ml). Three effects of cytochalasins were demonstrated--depolymerization of F-actin, promotion of polymerization, and redistribution of actin without change in polymerization state. Occurrence of a given effect was dependent on cell type, cell density, cytochalasin concentration and type. This indicates that cells from different lines, and even the same cells in different culture conditions may differ significantly in their state of actin polymerization, which we suppose is the cause of their different reactions to cytochalasins. Accordingly, caution should be taken in generalizing the results concerning the effect of cytochalasis on the polymerization state of actin.

Topics: 3T3 Cells; Actins; Animals; Carcinoma, Ehrlich Tumor; Cell Line, Transformed; Cytochalasin B; Cytochalasin D; Melanoma; Mice; Microscopy, Fluorescence; Polymers; Simian virus 40; Tumor Cells, Cultured

Improvement in the predictive assertion of the micronucleus assay was achieved by treating human malignant melanoma cells (Mewo) with cytochalasin B (CB), generating binucleated cells (BNC) representing cells after a single karyokinesis. Optimal cell binucleation was determined by testing several cytochalasin B concentrations and different incubation times. On average, 56% binucleated cells were found after incubation with 2 to 3 micrograms/ml cytochalasin B for 48 h. Cells with at least one micronucleus (Mn) were defined as fraction of cells with micronuclei and describes the degree of damaged cells. We found in binucleated cells 2.2 fold the fraction of cells with micronuclei than in mononucleated cells (MNC), as expected assuming that an induced micronucleus is associated with only one single daughter cell after mitosis. The mean of micronuclei per binucleated cells, however, was enhanced about 2.9 fold in relation to that of micronuclei per mononucleated cells and is related to the nuclear damage per cell. The application of cytochalasin B did not enhance the fraction of damaged cells although the degree of the injury per cell is intensified. A micronuclei promoting or inhibiting effect of the experimental design due to changes in cell proliferation was excluded by cytofluorometric investigations of DNA content and synthesis after cytochalasin B application. A comparison of the modified with the conventional micronucleus assay shows the superiority of the former.

Topics: Cell Line; Cell Nucleus; Cytochalasin B; DNA, Neoplasm; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Melanoma; Micronucleus Tests; Prognosis; Time Factors; Tumor Cells, Cultured

Four B16 melanoma cell variants were investigated to determine if there exists a correlation between their deformability and their metastatic potential. Cell deformability was measured as the percentage of cells traversing 10-mum diameter Nuclepore filter membranes at constant pressure as a function of time. A method was devised to circumvent common problems encountered in cell filtration experiments, i.e., cell aggregation and adhesion to the filter and failure to recover the input. F1a cells with the lowest spontaneous metastatic rate required 44 s for 50% of the cell input to traverse the filter, whereas No. 4 cells, featuring the highest metastatic rate, needed 12 s despite the fact that the cells had identical dimensions. Other variants tested showed intermediate filterability which also correlated with their metastatic potential. Cells, when pretreated with cytochalasin B at a final concentration of 21 microM exhibited increased filterability (75% and 42% greater than control for F1a and No. 4 cells, respectively). Somewhat smaller increases were observed after colchicine treatment. The findings imply major involvement of the cytoskeleton in the filterability and thus deformability of these B16 variants. Such physiochemical factors may play an important role in the metastasis of this and possibly other tumor types.

Topics: Cell Nucleus; Colchicine; Cytochalasin B; Humans; Melanoma; Neoplasm Metastasis

The effect of insulin on glucose metabolism through different pathways and the glucose transporters in Harding-Passey melanoma cells have been studied. Glucose was utilized at a rate of 6.9 +/- 2.3 (SD) mumol X g-1 X h-1 with 86% transformed into lactate and pyruvate and only 0.43 and 3% metabolized through the tricarboxylic acid cycle and the pentose phosphate pathway, respectively. Of the total glucose consumed 2% was used in protein synthesis and 2% was used for lipid synthesis. Hexokinase isoenzyme was type I and enolase was present mainly in the alpha gamma hybrid form. The glucose transporters were cytochalasin B sensitive. The number of high affinity cytochalasin B binding sites was 175,000 receptors/cell (about 0.6 pmol/mg protein) and Kd = 1 X 10(-7) M. Insulin increased glucose utilization and lactate production by about 70% and caused a 56% increase in transport without alterations in the Kd of the site. Insulin receptors were quantified by binding assay using 125I-insulin. Kd was 11 X 10(-9) M with the number of receptors calculated as 11,500/cell. Harding-Passey melanoma cells could thus be a useful model to study basic metabolic events and their modulation by hormones or other effectors.

Topics: Animals; Biological Transport; Carbon Dioxide; Carbon Radioisotopes; Cytochalasin B; Glucose; Hexokinase; Insulin; Melanoma; Mice; Mice, Inbred C57BL; Monosaccharide Transport Proteins; Phosphopyruvate Hydratase; Receptor, Insulin

The mechanism of autodegradation of cell-surface macromolecules shed by human melanoma cells was studied by incubating radio-iodinated shed macromolecules with unlabeled sister cells and measuring the appearance of acid-soluble radioactivity. After a preliminary latent period of 1-3 h, degradation continually increased up to 24 h and was concentration-dependent. By contrast, binding to cells was very rapid reaching half-maximal value within 15 min. Autodegradation was markedly reduced (44-82%) by pharmacological agents which interfere with endocytosis or lysosomal enzyme activity, including drugs which inhibit receptor migration into coated pits (dansylcadaverine), endocytosis and intracellular transport (colchicine, cytochalasin B, and monensin), and the activity of lysosomal enzymes (chloroquine, ammonium chloride, leupeptin). Degradation was almost totally suppressed (95%) at 4 degrees C. These data suggest that surface macromolecules shed by melanoma cells are autodegraded in part by re-uptake into melanoma cells followed by degradation in lysosomes.

Topics: Ammonium Chloride; Biological Transport; Cadaverine; Cell Line; Chloroquine; Coated Pits, Cell-Membrane; Colchicine; Cytochalasin B; Endocytosis; Humans; Leupeptins; Lysosomes; Melanoma; Membrane Proteins; Monensin; Neoplasm Proteins; Time Factors; Tunicamycin

A permanent cell line has been established from a human intracranial secondary melanoma. During 3 years of continuous growth in vitro the cells have maintained their characteristic phenotypic properties including melanin production. The cultured cells are highly tumorigenic in the athymic mouse and the tumours produced are histologically identical to the human tumour of origin.

Topics: Animals; Brain Neoplasms; Cell Division; Cell Line; Cell Nucleus; Chromosomes; Cytochalasin B; Humans; Melanins; Melanoma; Mice; Mice, Nude; Neoplasm Transplantation; Transplantation, Heterologous

Human tumor cell lines derived from melanoma, glioblastoma, and carcinoma of the prostate, bladder, and kidney multinucleated in response to growth in cytochalasin B-supplemented medium, whereas cell lines derived from normal prostate, kidney, skin, lung, and other nonmalignant diseases remained predominantly binucleate under comparable conditions. The multinucleate cytochalasin B phenotype was dissociable from the anchorage-independent phenotype of tumor cells, suggesting that these markers of cellular transformation are under separate control. These results suggest that uncontrolled nuclear division by tumor cells may be a general marker of abnormal growth or regulation.

Topics: Cell Line; Cell Nucleus; Cytochalasin B; Glioma; Humans; Male; Melanoma; Neoplasms; Prostate; Prostatic Hyperplasia; Prostatic Neoplasms; Urinary Bladder Neoplasms

The fate of cell surface macromolecules released by human melanoma cells in vitro was studied. Labeled surface macromolecules released by lactoperoxidase-radioiodinated melanoma cells were incubated cells. It was found that some of these macromolecules were autocatabolized to acid-soluble fragments by the cells which had released them. Degradation did not occur in the absence of cells, was almost completely inhibited at 4 degrees, and was partially suppressed by cytochalasin B (10 micrograms/ml) and by some inhibitors of energy production, i.e., iodoacetamide (10(-4) M) and a combination of 2-deoxyglucose (18 mg/ml) and 2,4-dinitrophenol (10(-4) M). Radioiodinated surface macromolecules were degraded much more rapidly than radioiodinated serum proteins. Thus, degradation required the presence of cells, was in part an active process, and was selective. These results suggest that one of the pathways for the turnover of surface macromolecules on tumor cells is shedding followed by autocatabolism of the shed material by the cells which they have released.

Topics: 2,4-Dinitrophenol; Cell Membrane; Cells, Cultured; Cytochalasin B; Deoxyglucose; Dinitrophenols; Humans; Iodoacetamide; Kinetics; Melanoma; Membrane Proteins

The effects of treatment with colchicine, cytochalasin B, and low temperature (4 degrees C) on the metastatic behavior of the B16-F10 melanoma cell line were examined. The growth of metastases and the distribution of radiolabeled tumor cells were monitored in inbred C57/BL6 mice given iv injections of B16-F10 cells. Cells treated previously with both drugs, but not with low temperature, produced fewer lung nodules than did control cells and displayed alterations in tumor dissemination patterns. In vitro studies revealed that both drugs reduced the rate of adhesion of the tumor cells to bovine endothelial cell monolayers, the rate of migration from agarose droplets, the formation of homotypic aggregates, and agglutination by wheat germ agglutinin. The drugs also induced morphologic alterations of the cells grown in monolayer culture but had little effect on cell volume.

Topics: Animals; Cell Adhesion; Cell Aggregation; Cell Movement; Cell Survival; Colchicine; Cold Temperature; Cytochalasin B; Cytoskeleton; Melanoma; Mice; Microtubules; Neoplasm Metastasis; Neoplasms, Experimental

Mouse B16 melanoma cells in cultures were treated with 2 microgram/ml cytochalasin B. Melanosomes, localized in dendrites as well as those in the peripheral cell body, formed aggregates which then showed centripetal migration. After 24 hr of treatment, melanosomes were found as large aggregates near the nucleus. However, cytochalasim B showed no effect on the distribution of mitochondria and endoplasmic reticulum. When cells were released from the 24 hr-treatment with cytochalasin B, each melanosome began centrifugal migration. In this process, melanosomes were situated in the periphery, and were in close association with microtubules. Side arm-like structures were observed between microtubules and melanosomes, or mitochondria. These results seem to indicate the presence of mechanism(s) specific to melanosome migration, and possible participation of microtubules in this melanosome migration.

Topics: Animals; Cells, Cultured; Cytochalasin B; Dendrites; Endoplasmic Reticulum; Melanocytes; Melanoma; Mice; Microtubules; Mitochondria; Neoplasms, Experimental

Topics: Carcinoma, Squamous Cell; Cell Line; Cytochalasin B; Humans; In Vitro Techniques; Melanoma; Skin Neoplasms

Cloudmann S 91 mouse melanoma cells treated for 5 days with melanocyte-stimulating hormone (MSH), cytochalasin B (CB), or both, exhibited changes in cell volume, population, nucleation, and pigment production. Cells treated with CB or CB in combination with MSH were eight to nine times larger, rounder, multinucleated, and heavily pigmented. CB alone increased melanin and DNA per nucleus threefold. CB in combination with MSH increased melanin per nucleus 30-fold. Data on DNA per nucleus suggest that CB-treated cells remained in the G phase longer than did control cells. MSH alone caused a reduction in cell population and a fourfold increase in melanin per nucleus.

Topics: Animals; Cell Division; Cell Nucleus; Cytochalasin B; DNA, Neoplasm; Drug Synergism; Melanins; Melanocyte-Stimulating Hormones; Melanoma; Mice; Neoplasms, Experimental

Topics: Acridines; Alkaloids; Animals; Antineoplastic Agents; Cell Adhesion; Cell Division; Cell Line; Cell Nucleus; Cells, Cultured; Cyclic AMP; Cytochalasin B; Female; Golgi Apparatus; Humans; Leukemia, Experimental; Leukemia, Lymphoid; Melanoma; Mice; Microscopy, Electron; Mitochondria; Mitosis; Simian virus 40; Time Factors; Tumor Virus Infections; Uterine Cervical Neoplasms