cytochalasin-b and Leukemia

Cytochalasin B is a mycogenic toxin that preferentially damages malignant cells through multiple mechanisms. The microfilament-disrupting agent inhibits cytokinesis, producing enlarged and multinucleated neoplastic cells without enlarging or producing multinucleated normal cells. In addition, cytochalasin B has been shown to induce apoptosis and to increase the mitochondrial activity of malignant cells. In spite of these pharmacological properties potentially exploitable in cancer chemotherapy, no cytochalasin congener or derivative and indeed no microfilament-directed agent has yet been examined in the clinic. Nevertheless, it will likely be necessary to combine microfilament-directed agents with other chemotherapeutic agents, and potentially with other anti-neoplastic modalities to amplify the mechanisms by which microfilament-directed agents inflict damage. These combinations could increase the likelihood of obtaining clinically useful activities with microfilament-directed agents and decrease the often inevitable emergence of drug resistance. Therefore, this study intends to determine appropriate chemotherapeutic agents to use concurrently with cytochalasin B and with other microfilament-directed agents.. Since cytochalasin B has shown in vitro efficacy against anchorage-independent growth, as well as against attached malignancies, both U937 human monocytic leukemia and MCF7 human breast carcinoma cells were evaluated. These cell lines were assessed for their sensitivity to a comprehensive array of chemotherapeutic agents that could amplify the cytoskeletal effects of microfilament-directed agents or that could themselves be potentiated by the cellular effects of such agents. In addition, clinically-approved microtubule-directed agents, as well as clinically-active anti-neoplastic agents not specifically cytoskeletal-directed, were examined for their ability to potentiate cell enlargement, one of the hallmark features of microfilament-directed agents. Conditions for inducing optimal enlargement and multinucleation of neoplastic cells with cytochalasin B were also defined.. U937 and MCF7 cells have differing sensitivities to chemotherapeutic agents indicating that different regimens will likely be needed for various cell types in concomitant cytochalasin B-mediated chemotherapy. It was noted that microtubule-directed agents (paclitaxel and vincristine) would likely have a synergistic effect with cytochalasin B as they produced a substantial enlargement in viable cells at their 50% inhibitory (IC50) values. Interestingly, doxorubicin and mitomycin C also produced considerable cell enlargement, suggesting that nucleic acid-directed agents may be used to further enhance the cell-enlargement and multinucleation effects of microfilament-directed agents if appropriate sequences and concentrations can be found for the combination of agents. A subsequent publication in this series will examine the optimal combinations of chemotherapeutic agents with microfilament-directed agents in regards to drug concentrations and sequential timing. U937 cells exposed to cytochalasin B exhibited substantial cell enlargement and multinucleation that was still prevalent 8 days post-administration depending on the concentration used.. Taken together, it appears that cytochalasin B has substantial synergistic potential with microtubule- and nucleic acid-directed agents.

Topics: Antineoplastic Agents; Cell Enlargement; Cell Nucleus; Cytarabine; Cytochalasin B; Doxorubicin; Drug Screening Assays, Antitumor; Humans; Inhibitory Concentration 50; Leukemia; MCF-7 Cells; Methotrexate; Mitomycin; Radiation-Sensitizing Agents; Sound

Sonodynamic therapy (SDT) is a form of ultrasound therapy that has been shown to preferentially damage malignant cells based on the relatively enlarged size and altered cytology of neoplastic cells in comparison to normal cells. This study sought to determine whether cytoskeletal-directed agents that either disrupt (cytochalasin B and vincristine) or rigidify (jasplakinolide and paclitaxel) microfilaments and microtubules, respectively, affect ultrasonic sensitivity. U937 human monocytic leukemia cell populations were treated with each cytoskeletal-directed agent alone, and then sonicated at 23.5 kHz under relatively low power and intensity (20-40 W; 10-20 W/cm(2)), or at 20 kHz using moderate power and intensity (60 W; 80 W/cm(2)). In addition, human leukemia lines U937, THP1, K562, and Molt-4, and the murine leukemia line L1210 were sonicated using pulsed 20 kHz ultrasound (80.6 W; 107.5 W/cm(2)) both with and without the addition of cytoskeletal-directed agents to assess whether cytoskeletal-directed agents can potentiate ultrasonic sensitivity in different leukemia lines. Human hematopoietic stem cells (hHSCs) and leukocytes were sonicated with continuous 23.5 kHz ultrasound (20 W; 10 W/cm(2)) to determine whether this approach elicited the preferential damage of neoplastic cells over normal blood components. To determine whether ultrasonic sensitivity is exclusively dependent on cell size, leukemia cells were also enlarged via alteration of cell growth parameters including serum deprivation and re-addition, and plateau-phase subculturing. Results indicated that cytochalasin B/ultrasound treatments had the highest rates of initial U937 cell damage. The cells enlarged and partially synchronized, either by serum deprivation and re-addition or by plateau-phase subculturing and synchronous release, were not comparably sensitive to ultrasonic destruction based solely on their cell size. In addition, cytochalasin B significantly potentiated the ultrasonic sensitivity of all neoplastic cell lines, but not in normal blood cells, suggesting that preferential damage is attainable with this treatment protocol. Therefore, it is likely that ultrasonic cell lysis depends not only on cell size and type, but also on the specific molecular mechanisms used to induce cell enlargement and their effects on cell integrity. This is supported by the fact that either the microfilament-or microtubule-disrupting agent produced a higher rate of lysis for cells of a given size th

Topics: Animals; Antineoplastic Agents; Cell Cycle; Cell Death; Cell Growth Processes; Culture Media, Serum-Free; Cytochalasin B; Cytoskeleton; Depsipeptides; Humans; Leukemia; Leukemia L1210; Mice; Microtubules; Paclitaxel; U937 Cells; Ultrasonic Therapy; Ultrasonography; Vincristine

Sonodynamic therapy (SDT) is a form of ultrasound therapy in which chemotherapeutic agents known as sonosensitizers are administered to increase the efficacy of ultrasound's preferential damage to neoplastic cells. Perhaps one of the most intriguing capabilities of ultrasound is its ability to preferentially lyse cells based on size. Cytochalasin B is a cytokinesis inhibitor that preferentially enlarges and multinucleates malignant cells, making them much more sensitive to ultrasonic irradiation.. The present study investigated the extent of preferential damage inflicted by cytochalasin B on U937 leukemia/human blood cell populations. Cell mixtures were treated with cytochalasin B and then sonicated under a relatively low intensity (3W/cm(2)).. Cytochalasin B preferentially damages U937 cells both before and after sonication. This agent also reduces rapid proliferation as the clonogenicity of U937 cells was considerably reduced following treatment.. Cytochalasin B may have profound therapeutic applications when combined with SDT.

Topics: Antineoplastic Agents; Cytochalasin B; Humans; Leukemia; U937 Cells; Ultrasonic Therapy

The advanced technique of DNA microarray makes it possible to monitor the expression of thousands of genes simultaneously in one hybridization experiment. This technique accelerates demonstration of anti-tumor drug mechanisms and discovery of new drug targets. This study was designed to investigate the differential gene expression of K562 cells after cytochalasin B treatment using cDNA microarray.. Restriction display polymerase chain reaction (RD-PCR) products of 277 human genes were spotted on a glass slide in microarray. K562 cells grew in RPMI 1640 medium with 10 microg/ml cytochalasin B. After 24 hours, the total RNA was isolated from K562 cells, and mRNA was purified. Both mRNA from the treated K562 cells and the controlled K562 cells were reversely transcribed into cDNA and labeled with two different fluorescence dyes: Cy5 or Cy3, using a method of restriction digestion and PCR labeling (RD-PCR). The probes were hybridized to the cDNA microarrays. After high-stringent washing,the cDNA microarray was scanned for the fluorescent signals and showed difference between the two cells.. Among the 277 target genes, 18 down-regulated genes were identified after cytochalasin B treatment.. There is a consistent tendency toward lower-expressed genes in partial K562 cells after cytochalasin B treatment. Most down-regulated genes were correlated with cell proliferation, signal transduction, and transcription factor.

Topics: Cytochalasin B; Down-Regulation; Gene Expression; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; K562 Cells; Leukemia; Neoplasm Proteins; Oligonucleotide Array Sequence Analysis

Using the DNase I inhibition assay, fluorimetric measurements, and immunoblot analysis, we studied quantitatively changes in the actin polymerization dynamics in primary cultures of normal and malignant human lymphocytes, normal human endometrial cells, and in various leukemic and endometrial adenocarcinoma cell lines. The G/total-actin ratio of malignant cells was found to be 1.37 to 1.81-fold higher compared to normal cells, indicating that malignant cells express reduced amounts of polymerized actin. The above findings were corroborated by fluorescence measurements of the amounts of rhodamine-phalloidin-labeled F-actin in normal and neoplastic cells, which showed significantly lower F-actin content in malignant cell preparations. Moreover, the total actin content, as quantitated by the DNase I inhibition assay and by immunoblot analysis, was found to be significantly decreased in the primary cultures of malignant human lymphocytes and endometrial cells when compared to the total actin levels in corresponding normal cells. Proliferation and viability measurements of normal and neoplastic cells in culture, treated equally with cytochalasin B (CB), revealed an increased susceptibility of malignant cells to this anticytoskeletal agent. This was not due to increased CB incorporation in neoplastic cells, as indicated by 3H-CB uptake experiments. In addition, fluorescence microscopy, in the presence of graded concentrations of CB, showed destabilization of microfilaments in the poorly differentiated endometrial adenocarcinoma HEC-50 cells, compared to the well-differentiated Ishikawa cells. In conclusion, all investigated malignant cells are characterized by: (a) higher G/total-actin ratio; (b) decreased F- and total-actin content; and (c) lower resistance to CB treatment. These quantitatively determined parameters may represent potential biochemical indicators reflecting malignant transformation. Moreover, it seems worthwhile to explore whether or not the differential sensitivity of malignant cells to anticytoskeletal drugs may provide a valuable approach to the manipulation of malignant cells.

Topics: Actins; Cell Division; Cell Survival; Cells, Cultured; Cytochalasin B; Dose-Response Relationship, Drug; Endometrial Neoplasms; Endometrium; Female; Humans; Leukemia; Lymphocytes; Microscopy, Fluorescence; Polymers; Tumor Cells, Cultured

Apoptosis is a form of cell death in which the cell "participates," such that metabolic energy and often protein synthesis are required for the death to occur. Once begun, the process of apoptosis proceeds in an ordered fashion. In the earliest phase DNA fragmentation occurs, accompanied by cell shrinkage and dilation of the endoplasmic reticulum. This is followed by cell fragmentation with the formation of sealed membrane vesicles, termed apoptotic bodies. In the present study we have demonstrated that the fungal metabolite cytochalasin B inhibits cell fragmentation and the formation of apoptotic bodies, probably by its ability to interfere with actin polymerization. This effect was seen when HL-60 cells were pretreated with cytochalasin B and then exposed to one of a number of apoptosis-inducing agents, including UV irradiation, camptothecin, aphidocholin, or PMA plus ionomycin. The observed effect was not peculiar to HL-60 cells, inasmuch as it was also seen for both Molt-4 and U-937 cell lines. Cytochalasin B had no effect on DNA fragmentation occurring in the earliest stage of apoptosis, and it appeared to have no inhibitory effects on nuclear fragmentation. Staurosporin had an effect similar to that seen with cytochalasin B, probably due to its ability to inhibit protein kinase C, which is a known potentiator of microfilament assembly. These data demonstrate that microfilament assembly is necessary for the formation of apoptotic bodies in the later stages of the apoptotic process.

Topics: Actin Cytoskeleton; Aphidicolin; Camptothecin; Cell Death; Cell Line; Cell Survival; Cytochalasin B; Humans; Ionomycin; Kinetics; Leukemia; Tetradecanoylphorbol Acetate; Ultraviolet Rays

Venous blood from healthy donors or from patients with various lympho- and myeloproliferative diseases was incubated in vitro in the presence of cytochalasin B for the induction of binucleated lymphocytes. The time at which cytochalasin B was added depended on the proliferation rate of the lymphocytes. Proliferation was monitored using a semiautomatic microscope photometer/computer system. The background level of micronuclei in binucleated lymphocytes of the patients before radiotherapy was statistically indistinguishable from that of healthy persons. Blood from both groups was irradiated in vitro for the study of the dose-response relationship. The dose-response curves were very similar up to 3.75 Gy, and a somewhat lower micronucleus frequency was found in lymphocytes of patients after a 5-Gy exposure. These in vitro results were compared with in vivo exposure after total-body irradiation of leukemic patients. Due to heavy medication that accompanied radiation therapy, only two doses (1.25 and 2.5 Gy) could be checked after in vivo exposure. There was no statistically significant difference between in vitro and in vivo results after 1.25 Gy, but a slightly lower number of micronuclei was observed after in vivo exposure to 2.5 Gy.

Topics: Adult; Cell Division; Cytochalasin B; Dose-Response Relationship, Radiation; Gamma Rays; Humans; In Vitro Techniques; Leukemia; Lymphocytes; Micronuclei, Chromosome-Defective; Radiation Dosage

Undifferentiated and differentiated HL-60 leukemic cells possess nucleotide receptors which functionally couple to phospholipase C via pertussis toxin-sensitive guanine nucleotide-binding proteins (G-proteins). We investigated the role of extracellular nucleotides in the regulation of beta-glucuronidase release in HL-60 cells. In dibutyryl cyclic AMP (Bt2cAMP)-differentiated HL-60 cells, the chemotactic peptide, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe), the phosphorothioate analogue of ATP, adenosine 5'-O-[3-thio]triphosphate (ATP[gamma S]), and UTP increased cytosolic Ca2+ from 100 nM up to 1.2 microM with EC50 values of 4 nM, 1 microM and 100 nM, respectively. In these cells, ATP[gamma S] induced exocytosis with an EC50 of 4 microM and an effectiveness amounting to 50-70% of that of fMet-Leu-Phe. ATP, ITP, UTP, CTP, and uridine 5'-O-[2-thio]diphosphate activated exocytosis as well. Phorbol myristate acetate (PMA) induced exocytosis with an EC50 of 115 ng/ml and an effectiveness similar to that of ATP[gamma S]. Cytochalasin B (CB) differently potentiated exocytosis induced by ATP[gamma S], fMet-Leu-Phe and PMA. Treatment of Bt2cAMP-differentiated HL-60 cells with pertussis toxin (500 ng/ml) for 24 h resulted in ADP-ribosylation of more than 97.5% of the G-proteins. Under these conditions, pertussis toxin almost completely inhibited the increase in cytosolic Ca2+ and beta-glucuronidase release induced by fMet-Leu-Phe but only partially inhibited the effects of ATP[gamma S] and UTP. fMet-Leu-Phe at a non-stimulatory concentration (1 nM) potentiated ATP[gamma S]-induced beta-glucuronidase release in the presence but not in the absence of CB. In contrast, ATP[gamma S] and fMet-Leu-Phe synergistically activated superoxide formation in the absence of CB. PMA potentiated superoxide formation induced by ATP[gamma S] or fMet-Leu-Phe and did not affect exocytosis induced by ATP[gamma S] or fMet-Leu-Phe. In undifferentiated HL-60 cells, fMet-Leu-Phe, ATP[gamma S], UTP and PMA did not induce beta-glucuronidase release. fMet-Leu-Phe did not increase cytosolic Ca2+ in undifferentiated HL-60 cells, whereas ATP[gamma S] and UTP were similarly potent and effective as in Bt2cAMP-differentiated cells. In differentiated HL-60 cells, fMet-Leu-Phe induced aggregation, and ATP[gamma S] induced a transient shape change. Our results show (I) that exocytosis in HL-60 cells does not obligatorily depend on CB. (II) Purine and pyrimidine nucleotides activate exoc

Topics: Bucladesine; Calcium; Cell Differentiation; Cytochalasin B; Exocytosis; Glucuronidase; Humans; Leukemia; N-Formylmethionine Leucyl-Phenylalanine; Nucleotides; Phorbol Esters; Superoxides; Tumor Cells, Cultured

Cytochalasin B and D enhanced vincristine (VCR) and daunomycin (DAU) accumulation in tumor cells, especially in VCR- and DAU-resistant cell lines. The effect of cytochalasin B, and to a lesser extent cytochalasin D, was almost equivalent to that observed for verapamil, a calcium channel blocker which has been reported to enhance drug accumulation in tumor cells. Cytochalasin B was most effective in VCR- and DAU-sensitive cells; however, the effect in resistant cells was less than that observed for verapamil, suggesting a different mode of action between these drugs in sensitive and resistant cells. Enhanced accumulation of VCR and DAU by cytochalasins was mediated by the inhibition of outward transport of VCR and DAU from tumor cells. Colchicine had no effect on VCR and DAU accumulation. Cytochalasins, especially cytochalasin D is a specific inhibitor of microfilament assembly in cells. These results indicate that the cellular microfilament system plays a prominent role in drug transport of tumor cells, and that an intact microtubular system is less involved.

Topics: Animals; Cell Line; Cell Survival; Colchicine; Cytochalasin B; Cytochalasin D; Cytochalasins; Daunorubicin; Drug Resistance; Humans; Leukemia; Leukemia P388; Leukemia, Myeloid, Acute; Mice; Verapamil; Vincristine