Cell experiment:

LX-2 cells or L02 cells are plated at a density of 5 × 103 cells per well in a 96-well plate and treated with Casticin (0-40 μM) for 48 h in growth medium containing serum. Cell proliferation is determined using a CCK-8 assay kit[2].

Animal experiment:

Mice[2]Male mice 6-8 weeks of age weighing 20-30 g are kept in a temperature-controlled room with an alternating 12 h dark and light cycle. A total of 32 mice are divided randomly into four groups of 8 animals each: control, Casticin, CCl4, and CCl4 + Casticin. To induce liver fibrosis, CCl4 dissolved in olive oil (20%) is injected intraperitoneally into mice (1.0 mL/kg body weight) in the CCl4 and CCl4 + Casticin groups twice a week for six weeks. Mice in the control group and Casticin group are injected with an equivalent volume of olive oil. Casticin is dissolved in 0.25% Tween-80. After treatment with CCl4 or olive oil for six weeks, mice in the Casticin group and CCl4 + Casticin group receives Casticin (20 mg/kg) by gastric gavage daily for two weeks, and the other two groups are given the equivalent volume of 0.25% Tween-80. After the eight week intervention period, mice are euthanatized under 3% pentobarbital sodium anesthesia (40 mg/kg ip), and the livers and blood from all animals are collected. Serum is obtained by centrifugation (1600 g, 15 min) and stored at –20℃ for further examination[2].

Casticin is a methyoxylated flavonol isolated from Viticis Fructus, with antimitotic and anti-inflammatory effect. Casticin inhibits the activation of STAT3.

Casticin (0.2-1.0 μM) dose-dependently inhibits the proliferation of KB cells, with an IC50 of 0.23 μM on day 3, while shows no significant inhibition on 3T3 Swiss Albino and TIG-103 cells. Casticin (0.6 μM) alters spindle morphology with partial mitotic spindle breakdown or with disordered spindles[1]. Casticin (0-40 μM) dose-dependently inhibits the proliferation of LX2 cells. Casticin (40 μM) suppresses L02 cells proliferation and induces apoptosis. Casticin inhibits fibrotic effects of TGF-β1 on ECM deposition in LX2 cells by evaluating the mRNA levels of TGF-β, collagen α1(I), MMP-2, MMP-9, TIMP-1 and TIMP-2[2]. Casticin (0-8 μM) reduces the viability of 786-O, YD-8, and HN-9 cells, but shows no significant effect on that of the normal HEL 299 cells. Casticin (5 μM) increases cleavage caspase-3 and PPAR, diminishes the levels of B-cell lymphoma-extra large (Bcl-xl), Bcl-2, IAP-1/-2, vascular endothelial growth factor (VEGF), matrix metallopeptidase 9 (MMP-9), and cyclooxygenase 2 (COX-2) proteins in 786-O, YD-8, and HN-9 cells. Casticin (5 μM) also promotes apoptotic cell death, inhibits constitutively active STAT3 in tumor cells, modulates STAT3 activation by altering the activity of upstream STAT3 regulators, and abrogates IL-6-induced STAT3 activation. In addition, Casticin (2.5 μM) enhances the effect of ionizing radiation in 786-O cells and potentiates the therapeutic effect of radiotherapy[3].

Casticin (20 mg/kg, p.o.) has toxic effect on the liver in mice with CCl4-and BDL-induced hepatic injury. Casticin attenuates liver fibrosis induced by CCl4 or BDL in vivo. Casticin inhibits HSC activation and collagen matrix expression by blocking TGF-β/Smad signaling in vivo[2].

References:
[1]. Kobayakawa J, et al. G2-M arrest and antimitotic activity mediated by casticin, a flavonoid isolated from Viticis Fructus (Vitex rotundifolia Linne fil.). Cancer Lett. 2004 May 10;208(1):59-64.
[2]. Zhou L, et al. Casticin attenuates liver fibrosis and hepatic stellate cell activation by blocking TGF-β/Smad signaling pathway. Oncotarget. 2017 Apr 27;8(34):56267-56280.
[3]. Lee JH, et al. Casticin inhibits growth and enhances ionizing radiation-induced apoptosis through the suppression of STAT3 signaling cascade. J Cell Biochem. 2018 Dec 5.