Actin polymerization

Cytochalasin D (3 and 10 μM; 30 min) induces F-actin to change from long fibers to punctate structures, as well as the contraction and branching of COS-7 cells [1]. In COS-7 cells, cytochalasin D (0.3, 1, 3, and 10 μM; 30 min) significantly lowers the rate of actin depolymerization [1]. Yap phosphorylation and fibrosis are both disrupted when cytokinin D (1 μM) is shortened in NIH3T3 cells, although the cells maintain their previous state.

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.[Invest New Drugs. 2015 Apr;33(2):290-9.]

Cofilin, a key regulator of actin filament dynamics, binds to G- and F-actin and promotes actin filament turnover by stimulating depolymerization and severance of actin filaments. In this study, cytochalasin D (CytoD), a widely used inhibitor of actin dynamics, was found to act as an inhibitor of the G-actin-cofilin interaction by binding to G-actin. CytoD also inhibited the binding of cofilin to F-actin and decreased the rate of both actin polymerization and depolymerization in living cells. CytoD altered cellular F-actin organization but did not induce net actin polymerization or depolymerization. These results suggest that CytoD inhibits actin filament dynamics in cells via multiple mechanisms, including the well-known barbed-end capping mechanism and as shown in this study, the inhibition of G- and F-actin binding to cofilin[1].

Western Blot Analysis
Cell Types: COS-7 cells[1]
Tested Concentrations: 3 and 10 μM
Incubation Duration: 30 min
Experimental Results: Causes the retraction and arborization of COS-7 cells and the transformation of F-. Actin changes from long fibers to punctate structures.

---

Western Blot Analysis
Cell Types: COS-7 cells expressing YFP-actin [1]
Tested Concentrations: 0.3, 1, 3 and 10 μM
Incubation Duration: 30 minutes
Experimental Results: Concentration-dependent decrease in actin lysis in COS-7 cells polymerization rate.

P388 leukemias in vivo[Invest New Drugs. 2015 Apr;33(2):290-9.]
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 D intraperitoneal administration[Invest New Drugs. 2015 Apr;33(2):290-9.]
Cytochalasin D was 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 studies using stereospecifically labeled precursors, L-phenylalanine was shown to be the major precursor of cytochalasin D, and the rapid equilibration of the D- and L-isomers via phenylpyruvic acid was shown to explain the equally efficient incorporation of both enantiomers. In the biosynthesis of cytochalasin D in Zygosporium masonii, [1,2-(13)C]-acetate was added to the fungal growth culture. The arrangement of the complete acetate units was determined…cytochalasin D consists of phenylalanine, methionine, and nine complete acetate units. Eight of these acetate units are linked head-to-tail to form a C16 polyketide moiety. Addition of saturated acids with higher even carbon numbers, such as C1-labeled butyric acid, myristic acid, and palmitic acid, did not show direct incorporation. However, they undergo β-oxidation to generate C1-labeled acetate, which can be incorporated into cytochalasin D.

Intraperitoneal LD50 in rats: 900 ug/kg.
Interactions
Pretreatment of rabbit kidney cells with cytochalasin D increased herpes simplex virus type 2 infectivity by 3 to 6 times compared to values obtained using standard calcium chloride techniques.
Treatment of permissive or semi-restrictive cell lines with cytochalasin D enhanced their susceptibility to poliovirus infection.

[1]. Cytochalasin D acts as an inhibitor of the actin-cofilin interaction. Biochem Biophys Res Commun. 2012 Jul 20;424(1):52-7.

[2]. Cytochalasins block actin filament elongation by binding to high affinity sites associated with F-actin. J Biol Chem. 1980 Feb 10;255(3):835-8.

[3]. Inhibiting extracellular vesicles formation and release: a review of EV inhibitors. J Extracell Vesicles. 2019 Dec 19;9(1):1703244.

[4]. Hippo pathway regulation by cell morphology and stress fibers. Development. 2011 Sep;138(18):3907-14.

Cytochalasin D is needle-like or fluffy white powder. (NTP, 1992)
Cytochalasin D has been reported in Ascochyta rabiei, Zygosporium masonii, and Metarhizium anisopliae, with relevant data available.
It is a fungal metabolite that inhibits cytokinesis by blocking the formation of contractile microfilaments, leading to multinucleated cell formation, reversible inhibition of cell motility, and cell extrusion. Other reported effects include inhibition of actin polymerization, DNA synthesis, sperm motility, glucose transport, thyroid secretion, and growth hormone release.
Mechanism of Action

The main biological effects are the blocking of cytokinesis leading to multinucleated cell formation, inhibition of cell motility, and induction of nuclear extrusion… Other reported effects include inhibition of glucose transport, thyroid secretion, growth hormone release, phagocytosis, platelet aggregation, and thrombus contraction. In the early stages of cell biology research, the mechanism of action of cytochalasin was explained as directly disrupting microfilaments, thereby interfering with the contractile movement of mammalian cells. Further research, particularly observing its effects on the entry of various substances into cells, revealed a more complex mechanism of action. Due to the rapid and reversible nature of its action, it is clear that the binding between cytochalasin and its active site is not covalent. Currently, (3)H-labeled cytochalasin B and D are being used to study their precise binding sites and subcellular localization... The experimental results obtained to date support the following view: its main function is membrane-friendly, possibly involving the binding of microfilaments to the plasma membrane. Although cytochalasin D cannot prevent the attachment of Toxoplasma gondii, it can prevent its entry into peritoneal macrophages and bladder tumor 4934 cells. For more complete data on the mechanisms of action of cytochalasin D (9 types in total), please visit the HSDB record page.

---

Therapeutic Use
Nucleic Acid Synthesis Inhibitor
Experimental Use: ...Researchers investigated the application of .../Zygosporin A (cytochalasin D), E, F, G and more than 30 derivatives/as antitumor drugs, but the results were not ideal.
Experimental Use: Cytochalasin D (0.322-5.0 mg/kg/day, intraperitoneal injection) has antitumor activity against ascites, AH-130 hepatoma, and Murphy-Sturm lymphosarcoma in rats.

C30H37NO6

507.62

507.262

C, 70.98; H, 7.35; N, 2.76; O, 18.91

22144-77-0

5458428

White to off-white solid powder

1.2±0.1 g/cm3

712.1±60.0 °C at 760 mmHg

255-260ºC

384.5±32.9 °C

0.0±2.4 mmHg at 25°C

1.597

2.64

3

6

4

37

996

9

C[C@H]1C/C=C/[C@H]2[C@@H](C(=C)[C@H]([C@@H]3[C@@]2([C@@H](/C=C/[C@@](C1=O)(C)O)OC(=O)C)C(=O)N[C@H]3CC4=CC=CC=C4)C)O

SDZRWUKZFQQKKV-JHADDHBZSA-N

InChI=1S/C30H37NO6/c1-17-10-9-13-22-26(33)19(3)18(2)25-23(16-21-11-7-6-8-12-21)31-28(35)30(22,25)24(37-20(4)32)14-15-29(5,36)27(17)34/h6-9,11-15,17-18,22-26,33,36H,3,10,16H2,1-2,4-5H3,(H,31,35)/b13-9+,15-14+/t17-,18+,22-,23-,24+,25-,26+,29+,30+/m0/s1

[(1R,2R,3E,5R,7S,9E,11R,12S,14S,15R,16S)-16-benzyl-5,12-dihydroxy-5,7,14-trimethyl-13-methylidene-6,18-dioxo-17-azatricyclo[9.7.0.01,15]octadeca-3,9-dien-2-yl] acetate

Lygosporin A; Zygosporin A; 22144-77-0; Zygosporin A; Cytohalasin D; Lygosporin A; MFCD00077706; SY9F0FZ3TO; NSC209835; Cytochalasin D

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 : ~25 mg/mL (~49.25 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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