F-actin (Kd = 2.2 nM, with Mg2+); F-actin (Kd = 1.4 nM, with Mg2+/K+)[1]

Cytochalasin B is a cell-penetrating mycotoxin that binds to the barbed ends of actin filaments and inhibits the lengthening and shortening of actin filaments. In the presence of MgCl2 (2 mM) or MgCl2, F-actin The Kds were 2.2 nM and 1.4 nM (2 mM) correspondingly with KCl[1]. Cytochalasin B (0.1-10 μM) shows inhibitory effects on multiple mouse cancer cell lines with IC50 of 2.56 μM (M109c), 10.46 μM (B16BL6), 105.5 μM (P388/ADR), 51.9 μM (P388/S), and IC80s after 3 hours of treatment were 12.23 μM (M109c), 44.86 μM (B16BL6), 188.4 μM (P388/ADR), 84.1 μM (P388/S), and IC50 were 0.25 μM (M109c), 0.37 μM (B16F10), 0.87 μM (B16BL6), the IC80 after 4 days of treatment were 0.75 μM (M109c), 1.21 μM (B16F10), and 10.41 μM (B16BL6) [2]. Cytochalasin B (6 μM) raises the myofibrillar fragmentation index (MFI), which is caused by the strong fragmentation of myofibrillar proteins into small fragments. Cytochalasin B also increases the breakdown of actin filaments. In addition, Cytochalasin B can also speed the conversion of F-actin to G-actin, lower the F-actin concentration, and considerably enhance the G-actin band during postmortem processing [3].

Balb/c mice with P388/ADR leukemia have a dose-dependent improvement in life expectancy when treated with cytochalasin B (10, 25, 50 mg/kg, ip). Long-term survival rates with cytochalasin B at 50 mg/kg were 10% in the drug-resistant P388/ADR cohort and 40% in the drug-sensitive P388/S cohort [2].

---

cytochalasin B appeared to increase the life expectancy of Balb/c mice challenged with either P388/ADR or P388/S leukemias (Fig. 6). It was discerned from the P388/ADR protocol that Balb/c mice could take up to 50 mg/kg/day i.p. for eight consecutive days (Days 1–8). Therefore, only this dose was examined for P388/S challenged mice, as the antitumor activity of cytochalasin B appeared to be dose dependent. Interestingly, 50 mg/kg cytochalasin B was able to produce 10 % long-term survival in the multidrug resistant P388/ADR cohort, and 40 % long-term survival in the drug sensitive P388/S cohort[2].
The antitumor effects of cytochalasin B were mirrored by cytochalasin D at much lower concentrations (Fig. 6b). It only took 2 mg/kg/day cytochalasin D administered for eight consecutive days (Days 1–8) to produce marked prolongation in the life expectancy of mice challenged with P388/S, as well as a 20 % long-term survival rate. Whether or not cytochalasin D would exhibit the same antitumor effect against P388/ADR at these lower concentrations remains unclear, as not enough mice remained to establish another treatment group of sufficient quantity. Nevertheless, it is likely that at least some prolongation in life expectancy would be observed. The cytochalasin vehicle CMC/Tw did not affect the life span of mice challenged with either leukemia, demonstrating that there is no effect of the lipophilic detergent vehicle on the leukemia challenges in the absence of cytochalasins.[2]

It is generally accepted that cytochalasin B (CB), as well as other cytochalasins, shorten actin filaments by blocking monomer addition at the fast-growing ("barbed") end of these polymers. Despite the predominance of this mechanism, recent evidence suggests that other interactions may also occur between CB and F-actin. To investigate this possibility further we have employed an actin derivative, prepared by substitution at Cys374 by a glutathionyl residue. We demonstrate here that CB did not significantly bind to glutathionyl F-actin under several ionic conditions. We further show that in the presence of CB the glutathionyl-F-actin exhibits a significantly higher ATPase activity than the non-modified F-actin. These data argue that the incorporation of glutathionyl groups prevents the high-affinity binding of CB to the barbed end of actin filaments, probably due to a decreased hydrophobicity of the CB binding site by the introduction of the hydrophilic glutathionyl residue. Despite the lack of substantial binding at equilibrium, we have found that the addition of CB to glutathionyl-F-actin results in extensive fragmentation of the filaments, as demonstrated by electron microscopy and by a significant reduction of the relative viscosity of actin solutions. These results are consistent with the idea that CB shortens glutathionyl-actin filaments by a mechanism distinct from barbed end capping. Glutathionyl F-actin offers an interesting model to study the complex mechanism of interaction of actin filaments with cytochalasins and with the physiologically important actin capping/severing proteins.[1]

---

Breast muscles of twenty-four Eastern Zhejiang White Geese were randomly divided into three groups: control, cytochalasin B (Cyt B) and Jasplakinolide (Jasp) treatments during postmortem conditioning. The myofibrillar fraction index (MFI), actin filaments and the levels of F-actin, G-actin and actin associated proteins (cofilins and tropomodulins) during conditioning were investigated. In control, the degraded tropomodulins, increased G-actin and disrupted actin filaments were observed at 4 and 7days; the increase of MFI and decrease of F-actin content were shown during conditioning. Cyt B treatments accelerated the transformation from F-actin to G-actin, weakened actin filaments and increased MFI compared to the control, while Jasp gained the opposite effect against Cyt B. We concluded that depolymerization of actin filaments regulated by tropomodulins contributed to myofibrillar fraction during conditioning. This work provided a new pathway of tenderization by the depolymerization of actin filaments.[3]

Effect of cytochalasin B on cancer cell lines in vitro[2]
The attached cell lines M109c, B16BL6, and B16F10 were seeded at 1 to 4 × 104 cells/ml in 2 ml volumes in 24-well culture plates 1 day prior to treatment with cytochalasin B. Conditions for treatment of the attached cell lines were as detailed earlier for B16BL6 and B16F10 cells. The suspension culture of P388/ADR cells was seeded at 5 × 104 cells/ml and allowed to grow overnight before cytochalasin B treatment. Cells were treated with cytochalasin B for 3 h, as well as 2, 3, or 4 days. In the case of continuous exposure for 2, 3, or 4 days, attached cells were trypsinized and counted with a hemacytometer. Leukemia cell suspensions were counted with a Coulter Counter. In the case of short-term exposure, cells were washed twice with fresh medium, then trypsinized (except for P388/ADR cells), reseeded, and allowed to regrow for 3 days, at which time they were counted. Growth results were calculated as the number of cells generated above the seeding density compared to the untreated control cells and graphically presented as percent of control increase.[2]
M109c clonogenic cells were determined by seeding aliquots containing 400–2000 trypsinized cells from each well into wells in another 24-well plate, culturing for 7 days, fixing in methanol (5 min), and staining with 0.1 % methylene blue (5 min). Colonies of greater than 10 cells were counted with a dissecting microscope.

---

Determining the extent of drug synergy between cytochalasins and doxorubicin[2]
To assess whether cytochalasin B, D or DiHCB synergizes with ADR, cells were treated with a cytochalasin congener for 2.5 h over a concentration range of 0 to 150 μM, followed by ADR over a concentration range of 0 to 9 μM for 3 h. IC50 and IC80 values were taken at a series of concentrations for each chemotherapeutic agent in order to construct an isobologram. IC50 and IC80 values for the single agents and for combinations were determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays.[2]
In addition, drug synergy was assessed by clonogenic assays in which P388/ADR cells were seeded in 24-well plates at early log phase. Cytochalasin B or DiHCB were administered for 2.5 h, followed by ADR for 3 h at a series of concentrations. Aliquots of the treated cells were then removed and cloned in soft agar in additional 24-well plates. Results from the assays were plotted as log surviving fractions at a given cytochalasin concentration as a function of ADR concentration. Fold-synergism was then calculated at relatively low concentrations of cyotchalasin where cytochalasin B or DiHCB-alone had minimal influence on cloning efficiency.

P388 leukemias in vivo[2]
For chemotherapy testing, Balb/c mice under isoflurane anesthesia were challenged with 2 × 105 trypan blue negative P388/S or P388/ADR cells subcutaneously (s.c.) in a volume of 200 μl. Untreated mice were kept in order to determine the lethality of the challenge without chemotherapeutic intervention. Long-term survival was defined as challenged mice that survived the duration of the observation period.

---

cytochalasin B and D intraperitoneal administration[2]
cytochalasin B and D were prepared in suspension form in 2 % carboxymethyl cellulose 1 % tween 20 (CMC/Tw) for intraperitoneal (i.p.) administration, as previously described. The congeners or the vehicle were administered to leukemia-challenged mice on Days 1–8 following the initial challenge.

Metabolism / Metabolites
...In the synthesis of cytochalasin B (PHOMIN) by Phominis and cytochalasin D by Zygosporium masonii...various (14)C and (3)H precursors were provided to Phominis, and precise degradation reactions were carried out to reveal the process by which cytochalasin B is formed from one phenylalanine unit, nine acetate-malonic acid units, and two methionine units. The labeling pattern of cytochalasin D was also confirmed. The direct conversion of labeled deoxyphosphoproteins by Phominis demonstrated that cytochalasin B (phosphoprotein) is formed through enzymatic Bayer-Villiger type oxygen insertion.

Intraperitoneal LD50 in rats: 11 mg/kg

---

Interactions
Treatment of wild-type S49 lymphoma cells with the microfilament disruptor cytochalasin B reversibly and in a highly dose-dependent manner enhanced the cell response to subsequent addition of the β-adrenergic agonist (-)-isoproterenol, prostaglandin E1, or cholera toxin to accumulate cyclic adenosine monophosphate (cAMP). INSEL PA, KOACHMAN AM; Cytochalasin B enhances cyclic adenosine monophosphate (cAMP) accumulation in hormone- and cholera toxin-stimulated S49 lymphoma cells; J BIO CHEM 257(16) 9717 (1982)
Cytochalasin B cannot transform 3T3-like tumor cells, but it can increase the cell transformation frequency of polyomavirus by 8-40 times. SEIF R; Factors that disrupt microtubules or microfilaments can increase the frequency of polyomavirus cell transformation; J VIROL 36(2) 421 (1980)
Cytochalasin B significantly enhances pinocytosis induced by concanavalin A in amoeba. PRUSCH RD; Effects of concanavalin A and cytochalasin B on pinocytosis in amoeba; Protoplasm 106(3-4) 223 (1981)
Cytochalasin B inhibited elongation of wheat coleoptile segments and maize roots in indole-3-acetic acid, with the only ultrastructural change being the accumulation of secretory vesicles. Cytochalasin B apparently blocks cell elongation growth by inhibiting vesicle transport and the secretion of cell wall components.

---

Interactions
Treatment of wild-type S49 lymphoma cells with the microfilament disruptor cytochalasin B reversibly and in a highly dose-dependent manner enhanced the cell's cyclic adenosine monophosphate (cAMP) accumulation response to the β-adrenergic agonist (-)-isoproterenol, prostaglandin E1, or cholera toxin.
Cytochalasin B did not transform 3T3-like tumor cells, but it did increase cell proliferation by 8–40-fold. Cell transformation frequency of polyomaviruses.
In amoebas, pinocytosis induced by concanavalin A was significantly enhanced by cytochalasin B.
Cytochalasin B inhibited elongation of wheat coleoptile segments and maize roots in indole-3-acetic acid, with ultrastructural changes limited to the accumulation of secretory vesicles. Cytochalasin B apparently blocks elongation growth by inhibiting vesicle transport and the secretion of cell wall components.

[1]. Cytochalasin B may shorten actin filaments by a mechanism independent of barbed end capping. Biochem Pharmacol. 1994 May 18;47(10):1875-81.

[2]. Chemotherapy with cytochalasin congeners in vitro and in vivo against murine models. Invest New Drugs. 2015 Apr;33(2):290-9.

[3]. The effect of Cytochalasin B and Jasplakinolide on depolymerization of actin filaments in goose muscles during postmortem conditioning. Food Res Int. 2016 Dec;90:1-7.

[4]. Discovery of the migrasome, an organelle mediating release of cytoplasmic contents during cell migration. Cell Res. 2015 Jan;25(1):24-38.

Cytochalasin B is an organic heterocyclic tricyclic compound belonging to the fungal toxin class. It penetrates cell membranes and inhibits cytokinesis by blocking the formation of contractile microfilaments. It has multiple functions, including as a metabolite, platelet aggregation inhibitor, fungal toxin, and actin polymerization inhibitor. It is a cytochalasin, an organic heterocyclic tricyclic compound belonging to the lactam and lactone classes. Cytochalasin B has been reported in Boeremia exigua, Sonchus mauritanicus, and other organisms with relevant data. It is a cytotoxic member of the cytochalasin family. Mechanism of Action: Major biological effects include blocking cytoplasmic lysis, leading to multinucleated cell formation; inhibiting cell motility; and inducing nuclear ejection… Other reported effects include inhibition of glucose transport, thyroid secretion, growth hormone release, phagocytosis, platelet aggregation, and thrombus contraction. Cytochalasin
The precise binding sites and subcellular localization of cytochalasin B and D, labeled with (3)H, are currently being investigated… The experimental results obtained to date support the view that its main function is membrane-friendly, possibly involving the binding of microfilaments to the plasma membrane. Notably, cytochalasin B has different effects on normal cells and transformed cells; the latter have a higher degree of multinucleation than normal cells.
The effects of cytochalasin B and E on the intestinal digestion of maltose and sucrose and their digestion products (glucose and fructose) were investigated in an in vitro mouse model. After incubation for 60 minutes at concentrations of 5.0 and 10.0 μg/ml, cytochalasin B or E had no effect on the digestion of maltose or sucrose in the mouse jejunal fossa or on the activity of maltase or sucrase. However, cytochalasin E (5.0 μg/ml) inhibited the absorption of glucose from maltose or sucrose digestion by 68.5% and 65.9%, respectively, while the same concentration of cytochalasin B inhibited glucose absorption by 29.5% and 13.1%, respectively. Cytochalasin B and E appear to stimulate the absorption of fructose produced from sucrose digestion in the mouse jejunum. The inhibitory effect of cytochalasin E on galactose absorption in the mouse jejunal eversion pouch was investigated. Cytochalasin B showed the highest inhibitory efficacy against galactose absorption when added to the mucosal solution, followed by cytochalasin E, A, C, and D. For more complete data on the mechanisms of action of cytochalasin B (14 in total), please visit the HSDB record page.

---

Therapeutic Use
Experimental Use: The effects of vincristine, colchicine, lidocaine, and cytochalasin B on the killing of tumor cells by BCG-activated macrophages were investigated. Cytochalasin B can disrupt microfilaments and, at a concentration of 10⁻⁷ mol, enhances the lysis and inhibition of tumor cells by activated macrophages. Vincristine and lidocaine appear to act on the macrophages themselves, while cytochalasin B primarily acts on tumor cells. Microtubules and microfilaments may play a role in activating macrophages to destroy tumor cells. Experimental applications: When cytochalasin B and colchicine were added to Yoshida sarcoma cells at concentrations of 5 μg/ml and 0.4 μg/ml, respectively, the number of motile cells was reduced. Cytochalasin B also reduced cell motility and inhibited cell growth in a dose-dependent manner. Experimental applications: In cultured IRC 741 rat leukemia cells, cytochalasin B (1.5–4 μg/ml) induced dose-dependent binucleation and inhibited the normal increase in cell number. In cells that entered the mitotic phase after exposure to cytochalasin B for up to 24 hours, the formation of mitotic grooves was completely inhibited in a certain proportion of cells, and the degree of inhibition was dose-dependent.

C29H37NO5

479.6078

479.267

C, 72.62; H, 7.78; N, 2.92; O, 16.68

14930-96-2

5311281

White to off-white solid powder

1.2±0.1 g/cm3

740.6±60.0 °C at 760 mmHg

218-223ºC

401.7±32.9 °C

0.0±2.6 mmHg at 25°C

1.596

3.71

3

5

2

35

859

8

C[C@@H]1CCC[C@H](/C=C/C(=O)O[C@]23[C@@H](/C=C/C1)[C@@H](C(=C)[C@H]([C@H]2[C@@H](NC3=O)CC4=CC=CC=C4)C)O)O

GBOGMAARMMDZGR-TYHYBEHESA-N

InChI=1S/C29H37NO5/c1-18-9-7-13-22(31)15-16-25(32)35-29-23(14-8-10-18)27(33)20(3)19(2)26(29)24(30-28(29)34)17-21-11-5-4-6-12-21/h4-6,8,11-12,14-16,18-19,22-24,26-27,31,33H,3,7,9-10,13,17H2,1-2H3,(H,30,34)/b14-8+,16-15+/t18-,19-,22-,23+,24+,26+,27-,29-/m1/s1

(1S,4E,6R,10R,12E,14S,15S,17S,18S,19S)-19-benzyl-6,15-dihydroxy-10,17-dimethyl-16-methylidene-2-oxa-20-azatricyclo[12.7.0.01,18]henicosa-4,12-diene-3,21-dione

CYTOCHALASIN B; Phomin; 14930-96-2; cytochalasin-B; CHEBI:23527; Cytochalasin B (Phomin); MFCD00077704; MLS000028816;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~83.33 mg/mL (~173.75 mM)
Ethanol : ~25 mg/mL (~52.13 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.21 mM) (saturation unknown) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (5.21 mM) (saturation unknown) in 10% EtOH + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (5.21 mM) (saturation unknown) in 10% EtOH + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 4: ≥ 2.08 mg/mL (4.34 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

Solubility in Formulation 5: ≥ 2.08 mg/mL (4.34 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

---

Solubility in Formulation 6: ≥ 2.08 mg/mL (4.34 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

Solubility in Formulation 7: 5 mg/mL (10.43 mM) in 0.5% CMC-Na 0.5% Tween-80 (add these co-solvents sequentially from left to right, and one by one), Suspened solution; with ultrasonication.

---

 (Please use freshly prepared in vivo formulations for optimal results.)