Variation in the activity of distinct cytochalasins as autophagy inhibitiors in human lung A549 cells.

Autophagy is a cell survival process that represents a therapeutic target in cancer treatment. Many types of cytochalasins have been identified and some of them have been reported to interfere with the formation of the autophagosome, although only limited data are available to assess their potential effects. Therefore, in this study, we examined the effects of cytochalasins and structurally related compounds on cell survival and the regulation of autophagy in human lung A549 adenocarcinoma cells. Cytochalasin D (CD) and cytochalasin E (CE) prominently inhibited the growth of A549 cells in a dose-dependent manner. Following treatment with CE, F-actin filaments were disrupted, and the proportion of binucleated cells increased, whereas no such effects were observed with the seven other cytochalasins tested. We found that cytochalasin H (CH), CD, and especially CE could induce the up-regulation of autophagy-related protein (LC3-II) and SQSTM1/p62. Using bafilomycin A1, we demonstrated that CD, CE, and CH inhibited autophagosome turnover, resulting in a dysfunctional autophagic process. The results of this study reveal that CE is the most potent cytochalasin in terms of its ability to induce cell death and inhibit autophagy. CE may therefore be an effective therapeutic agent against lung cancer.

Chemotherapy with cytochalasin congeners in vitro and in vivo against murine models.

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.

Tolerated doses in zebrafish of cytochalasins and jasplakinolide for comparison with tolerated doses in mice in the evaluation of pre-clinical activity of microfilament-directed agents in tumor model systems in vivo.

BACKGROUND/AIM: Chemotherapeutic approaches involving microtubule-directed agents such as the vinca alkaloids and taxanes are used extensively and effectively in clinical cancer therapy. There is abundant evidence of critical cytoskeletal differences involving microfilaments between normal and neoplastic cells, and a variety of natural products and semi-synthetic derivatives are available to exploit these differences in vitro. In spite of the availability of such potential anti-neoplastic agents, there has yet to be an effective microfilament-directed agent approved for clinical use. Cytochalasins are mycogenic toxins derived from a variety of fungal sources that have shown promising in vitro efficacy in disrupting microfilaments and producing remarkable cell enlargement and multi-nucleation in cancer cells without producing enlargement and multi-nucleation in normal blood cells. Jasplakinolide is a sponge toxin that stabilizes and rigidifies microfilaments. Insufficient in vivo data has been acquired to determine whether any of the microfilament-directed agents have valuable preferential anticancer activity in pre-clinical tumor model systems. This is partly because the limited availability of these agents precludes their initial use in large-scale mammalian pre-clinical studies. Therefore, the present study sought to determine the tolerated in vivo doses of cytochalasins and jasplakinolide in zebrafish (Danio rerio), a well-studied fish cancer model that is 1.5% the size of mice. We also determined the tolerated levels of a variety of clinically active anti-neoplastic agents in zebrafish for comparison with tolerated murine doses as a means to allow comparison of toxicities in zebrafish expressed as µM concentrations with toxicities in mice expressed in mg/kg. MATERIALS AND METHODS: Tolerated doses in zebrafish with various cytochalasins or jasplakinolide were determined by adding the solubilized test agent to water in which the fish were maintained for 24 h, then restored to their normal tanks and monitored for a total of 96 h. RESULTS: Cytochalasin D at 0.2 µM gave an approximate LD50 in zebrafish, while cytochalasin B was fully-tolerated at 5 µM, and gave an LD50 of 10 µM. 21,22-dihydrocytochalasin B was fully-tolerated at 10 µM. Cytochalasin C was tolerated fully at 1 µM, ten-fold higher than the level for cytochalasin D that was tolerated. Jasplakinolide at 0.5 µM did not exhibit any apparent acute toxicity or affect fish behavior for four days, but delayed toxicity was evident at days 4 and 6 when the fish died. Further, the addition of 5 µM glutathione (GSH) at the time of treatment substantially decreased the toxicity of 10 µM cytochalasin B, a level of cytochalasin B that not otherwise tolerated in vivo. Such observations were likely due to GSH-mediated alkylation of C-20 in cytochalasin B, thereby reducing the rate of oxidation to the highly toxic congener, cytochalasin A, and reacting with any cytochalazin A formed. The protective effects of GSH are further supported by its ability to react with α, ß-unsaturated ketone moieties, as is found in cytochalasin A. GSH at 0.8 uM was able to reduce the toxicity of 0.8 µM cytochalasin D, but it took 20 µM GSH to fully protect against the toxicity of 0.8 µM cytochalasin D. CONCLUSION: Pre-clinical evaluation of rare natural products such as microfilamented-directed agents for efficacy in vivo in tumor-bearing zebrafish is a feasible prospect. Dose-limiting toxicities in zebrafish expressed as µM concentrations in water can be used to estimate in vivo toxicities in mice expressed as mg/kg.

Role of microfilaments and microtubules in the invasion of EPC cells by Aeromonas hydrophila.

The influence of the cytoskeleton on the invasion of Aeromonas hydrophila strain AhJ-1, isolated from diseased fish, in the monolayer cell of epithelioma papillosum cells of carp (EPC) was evaluated by the recovery of gentamicin-resistant bacteria from Triton X-100 cell lysates. The depolymerization of microfilaments (MF) by cytochalasin B and D inhibited the uptake of A. hydrophila in a dose-dependent manner and that of microtubules (MT) by colchicines and nocodazole did not affect the invasion of A. hydrophila in EPC cells significantly. The invasion frequency decreased approximately 62% with the addition of 0.1 microg/ml cytochalasin D and nearly 86% by the addition of 5.0 microg/ml. Invasion decreased approximately 49% and 83% by addition of cytochalasin B in a concentration of 2.5 microg/ml and 10.0 microg/ml. Colchicine and nocodazole, inhibitors of MT formation appears to have little effect on the invasion of EPC cells by strain Ah J-1. Thus MF formation, but not MT formation seems to play an important role in the internalization of A. hydrophila J-1.

Effects of small doses of cytochalasins on fibroblasts: preferential changes of active edges and focal contacts.

The effects of low doses of cytochalasin B (2 micrograms/ml) and cytochalasin D (0.2 microgram/ml) on the spreading of normal mouse fibroblasts in culture were investigated to find out which components of cell-substrate interactions are most sensitive to alterations of the state of actin cytoskeleton. Cytochalasin B disorganized the cortical layer of actin microfilaments and caused partial or complete disappearance of microfilament bundles; focal contacts with the substrate seen by interference-reflection microscopy also disappeared. Diffuse close contacts were apparently insensitive to cytochalasin B. Low doses of cytochalasin B did not inhibit the outgrowth and maintenance of lamellas at the cell periphery. However, in contrast to controls, these lamellas had no distal zones with convex outer edges and ruffles at the upper surfaces. The disappearance of these ruffling active edges was accompanied by loss of the ability to clear the surface of the lamellas from the concanavalin A receptors crosslinked by the corresponding ligand. The effects of cytochalasin D were similar to those of cytochalasin B. Thus, ruffling active edges and focal contacts can be regarded as specialized parts of lamellas with increased sensitivity to cytochalasins; the presence of ruffling active edges is essential for the initiation of centripetal movement of the patches of crosslinked surface receptors.

Acute toxic effects of chaetoglobosin A, a new cytochalasan compound produced by Chaetomium globosum, on mice and rats.

A new cytochalasan compound, chaetoglobosin A produced by Chaetomium globosum, was studied for its toxicity to animals. By subcutaneous injection, 2 mg/kg body weight of chaetoglobosin A killed young Wistar rats acutely, and in the mouse of DDD strain, LD50 values of chaetoglobosin A were estimated 6.5 and 17.8 mg/kg for male and female, respectively. By oral administration, 400 mg/kg of chaetoglobosin A caused little adverse effect on mice and rats. Pathological examination of the mice, injected subcutaneously with 5 mg/kg of the toxin, revealed marked edema at the injection site which appeared in several hours after injection and subsided in a week. In other tissues necrosis of the thymus and spleen and degeneration of the spermatocytes in the testicles were noticeable. With characteristic cytotoxic effects like cytochalasin B, chaetoglobosin A is an interesting mycotoxin, although it may be of little significance as a food- or feed-borne toxin.

Teratogenicity of cytochalasin D in the mouse.

Cytochalasin D, a mold metabolite identified in food, was injected intraperitoneally in doses of 0.4 to 0.9 mg per kilogram on gestational days 7 through 11 and produced evidence of teratogenicity in two out of three strains of mice. Exencephaly, hypognathia and axial skeletal defects were found in strains C57BL/6J and BALB/c while no increase in defects was observed in the Swiss Webster strain. In all three strains, a significantly increased resorption rate was found. Oral doses of approximately 7.0 mg per kg on days 7 through 11 in the BALB/c produced exencephaly in the offspring. Autoclaved cytochalasin D retained its teratogenic potential.