Cryptotanshinone (Cryptotanshinon), a STAT3 inhibitor (IC50: 4.6 μM) in a cell-free assay, strongly inhibits phosphorylation of STAT3 Tyr705, with a little effect on STAT3 Ser727, but no inhibition for STAT1 nor STAT5.

STAT3:4.6 μM

In ob/ob mice (C57BL/6J-Lepob), db/db mice (C57BL/KsJ-Leprdb), and ZDF rats, treatment with Cryptotanshinone (600 mg/kg/day) for 3 days significantly reduced blood glucose levels, maintaining this reduction throughout the monitoring period. In ob/ob mice (C57BL/6J-Lepob) and diet-induced obese mice, Cryptotanshinone significantly reduced body weight and food intake in a dose-dependent manner. It also notably decreased fat in adipose tissues and significantly lowered serum cholesterol and triglyceride levels, with AMPK activity in skeletal muscle being 2.5-3 times higher than that in the control mice.

In DU145 cells, Cryptotanshinone treatment (7 μM) for 30 minutes significantly inhibits the phosphorylation of STAT3 at Tyr705 without affecting the phosphorylation at Ser727. After 4 hours, it markedly inhibits the phosphorylation of JAK2 (IC50: 5 μM) but does not affect the phosphorylation of downstream kinases c-Src and EGFR. This suggests that the inhibition of STAT3 phosphorylation at Tyr705 is due to binding to the STAT3 SH2 domain. Cryptotanshinone, a natural product isolated from the root of Salvia miltiorrhiza, significantly inhibits STAT3-dependent luciferase activity, blocks phosphorylation at Tyr705, and dimerization of STAT3. It notably suppresses the proliferation of the DU145 cell line, which possesses constitutively active STAT3 (GI50: 7 μM), by inhibiting STAT3 activity, leading to the downregulation of cyclin D1, Bcl-xL, and anti-apoptotic genes, and subsequently causing cell accumulation in the G0-G1 phase. Cryptotanshinone exhibits mild inhibitory effects on the growth of PC3, LNCaP, and MDA-MB-468 cells.

STAT3-dependent dual-luciferase assay: HCT-116 cells are transiently transfected with reporter plasmid having the STAT3-binding element for regulating luciferase assay. Cells are treated with Cryptotanshinone for 24 hours at a concentration range of 0.2 to 50 μM. After treatment, cells are harvested in 20 μL of passive lysis buffer and luciferase activity is evaluated by the Dual Luciferase Reporter Assay kit on Wallac Victor2. The concentration of Cryptotanshinone that inhibits the luciferase activity by 50% represents IC50 value.

Cells are exposed to Cryptotanshinone for 24 or 48 hours. For the determination of cell proliferation, the cell proliferation reagent WST-1 is added and WST-1 formazan is quantitatively measured at 450 nm using an ELISA reader.(Only for Reference)