cytochalasin-b and Neoplasm-Metastasis

Rho GTPases control cytoskeletal dynamics through cytoplasmic effectors and regulate transcriptional activation through myocardin-related transcription factors (MRTFs), which are co-activators for serum response factor (SRF). We used RNA interference to investigate the contribution of the MRTF-SRF pathway to cytoskeletal dynamics in MDA-MB-231 breast carcinoma and B16F2 melanoma cells, in which basal MRTF-SRF activity is Rho-dependent. Depletion of MRTFs or SRF reduced cell adhesion, spreading, invasion and motility in culture, without affecting proliferation or inducing apoptosis. MRTF-depleted tumour cell xenografts showed reduced cell motility but proliferated normally. Tumour cells depleted of MRTF or SRF failed to colonize the lung from the bloodstream, being unable to persist after their arrival in the lung. Only a few genes show MRTF-dependent expression in both cell lines. Two of these, MYH9 (NMHCIIa) and MYL9 (MLC2), are also required for invasion and lung colonization. Conversely, expression of activated MAL/MRTF-A increases lung colonization by poorly metastatic B16F0 cells. Actin-based cell behaviour and experimental metastasis thus require Rho-dependent nuclear signalling through the MRTF-SRF network.

Topics: Actins; Animals; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cytochalasin B; Cytoskeleton; Gene Expression Regulation, Neoplastic; Genes, Reporter; Humans; Mice; Mutation; Neoplasm Invasiveness; Neoplasm Metastasis; Rats; rho GTP-Binding Proteins; RNA, Messenger; Serum Response Factor; Signal Transduction; Transcription Factors

Using porous cell culture chambers, we have simultaneously assessed growth and locomotion of cancer cells to investigate whether certain agents affect cell motility in addition to cell division. First, cells from a murine fibrosarcoma cell line, 1.0/L1, were grown in ordinary flask cultures to determine appropriate cell inocula. Doses of agents were selected to reduce the final 4 day culture cellularity to about 50%, when present during the last two days of culturing. Secondly, the effects of these agents on cell numbers in the porous chambers and on cell migration out of the chambers ('emigration fraction') were recorded. We also examined, using a similar type of porous chamber, whether the agents could affect leucocyte chemotaxis. Hydroxyurea (an inhibitor of DNA synthesis) reduced cancer cell emigration as well as cell growth, without interfering with leucocyte chemotaxis. Cytochalasin B (a microfilament disrupting agent) inhibited cancer cell motility and growth, as well as leucocyte chemotaxis. Vinblastine (a microtubule disrupting agent), at the very low dose chosen, reduced cancer cell growth, but did not consistently affect the migration of either cell type. The experimental anti-metastasis agent Razoxane reduced growth, but had no detectable effects on motility. High doses of natural murine interferon-alpha/beta weakly inhibited both cancer cell growth and locomotion. This motivates for further studies of these and other cytokines, as treatment with agents inhibiting cancer cell locomotion might possibly prevent peri-operative spread of cancer in patients.

Topics: Animals; Antineoplastic Agents; Cell Division; Cell Movement; Chemotaxis; Cytochalasin B; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Humans; Hydroxyurea; Interferons; Leukocytes; Mice; Neoplasm Metastasis; Razoxane; Tumor Cells, Cultured; Vinblastine

Cytochalasin B (CB), at 100 or at 10 mg/kg single dose s.c. in carboxymethyl-cellulose (2%)/Tween-20 (1%) 24 h after s.c. challenge of B6D2F1 mice with trocar implants of B16F10 tumor s.c., delayed the appearance of measurable tumor nodules by 157 and 93%, respectively, and extended host survival by 65 and 26%. Tumor growth was also delayed when CB treatment was given 1 day after the appearance of palpable tumor nodules. By in vivo bioassay, in vitro cloning, and dye exclusion measurements, solid tumor nodules treated in vivo with CB at either 100 or 10 mg/kg showed the same viability and tumorigenicity as did vehicle-treated nodules 4 and 6 days after drug treatment, at which time growth inhibition was still apparent. This indicates that growth inhibition by CB is not dependent on a gross cytotoxic effect. CD2F1 mice challenged s.c. with Madison 109 lung carcinoma cells and treated with CB s.c. at 100 or 150 mg/kg 24 h later showed a 66% delay in the median day of tumor nodule appearance. When administered under these conditions or at the time of nodule appearance. CB markedly inhibited the rate of tumor growth, prevented tumor invasion at day 23, extended life span by 23%, and significantly inhibited spontaneous lung metastases measured 28 days after tumor challenge. Maximum tolerated doses of CB administered i.p., s.c., or i.v. in suspension or in solution are defined. These results delineate the conditions under which CB can be tested for in vivo biological activities and establish that this microfilament-active natural product in a single-agent protocol inhibits local tumor growth and extends survival in B16F10 melanoma and Madison 109 lung carcinoma, and, in the latter model, inhibits invasion and spontaneous lung metastases by mechanisms that do not appear to depend on cytotoxicity. This work on formulation, tolerated doses in vivo, and localized peritumoral effects of CB now permits evaluation of systemic antitumor effects of cytochalasin B as a single agent. It also permits chemotherapy studies using CB as a potential amplifier of the activity of other antitumor agents in vivo.

Topics: Animals; Cell Survival; Chromatography, Thin Layer; Cytochalasin B; Dose-Response Relationship, Drug; Drug Evaluation, Preclinical; Lung Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Neoplasm Invasiveness; Neoplasm Metastasis; 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

Cloned metastatic Lewis lung carcinoma cells, C3, were more resistant to natural killer (NK) lysis than were nonmetastatic variant cells, C8. This was influenced by the tumor cell adhesiveness and morphology. When the nonadherent round C3 cells were cultured with dimethylsulfoxide, they became adherent and spread, sensitive to NK lysis and less metastatic. When the adherent and spread C8 cells were made nonadherent and round with cytochalasin B, they became more resistant to NK lysis and more metastatic. These metastatic differences were not observed in 3-week-old NK-deficient mice.

Topics: Animals; Cell Adhesion; Clone Cells; Cytochalasin B; Cytotoxicity, Immunologic; Dimethyl Sulfoxide; Female; Killer Cells, Natural; Lung Neoplasms; Mice; Mice, Inbred C57BL; Neoplasm Metastasis

Cytochalasin B pretreatment of tumor cells alters the distribution pattern of metastases after i.v. injection. We now have studied Cytochalasin B cell pretreatment affects the i.v. distribution and elimination of 125IUDR-labeled TA3-Ha tumor cells. We found no differences between control and CB-treated cells in their initial distribution. but 4-8 hours after the injection, in the redistribution phase, there were more CB cells than control cells extrapulmonally. Rather than a consequence of CB paralysis, we interpret the results in terms of altered surface properties of CB cells during their recovery.

Topics: Animals; Cell Movement; Cytochalasin B; Female; Male; Mice; Neoplasm Metastasis; Neoplasm Seeding; Neoplasms, Experimental; Surface Properties; Time Factors

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

The influence of Cytochalasin B (CB) on TA3 ascites tumor cells was studied in vivo in order to assess whether CB-induced cell paralysis would affect the transplanation behavior of the cells and in particular tumor distribution after IV cell infusion ("experimental metastases"). Tumor cell pre-treatment with CB (1 mug/ml) did not alter the SC or IV transplantability of TA3 cells. Pre-treatment with 10 mug/ml CB, in contrast, consistently increased the incidence and number of extra-pulmonary tumor takes from IV transfused cells. The amount of pulmonary tumors was not significantly altered. SC transplantability was not affected by 10 mug/ml CB. The importance of cell mobility and cell surface topography for tumor cell nidation in vessels is discussed.

Topics: Animals; Carcinoma, Ehrlich Tumor; Cytochalasin B; Mice; Mice, Inbred Strains; Neoplasm Metastasis; Neoplasm Transplantation; Neoplastic Cells, Circulating; Transplantation, Isogeneic

The evidence is reviewed that two types of cell attachment occur, depending upon the presence or absence of serum in the medium. In the absence of serum, attachment has many characteristics of a nonphysiological process. In the presence of serum, attachment occurs as a series of steps: adsorption of serum components onto the substratum, contact between the cell and substratum, initial attachment, and progressive attachments leading to cell spreading. Although there is a close interdependence of these events, they could be experimentally distinguished. Studies are reported indicating that cell spreading requires the adsorption of a specific serum glycoprotein onto the substratum surface. The relationship between cell adhesiveness and the altered behavior of malignant cells is discussed.

Topics: Animals; Cations, Divalent; Cell Adhesion; Cell Aggregation; Cell Line; Culture Media; Cytochalasin B; Ethylmaleimide; Neoplasm Metastasis; Osmolar Concentration; Serum Albumin, Bovine; Temperature; Trypsin