In vitro activity: Cryptotanshinone, a natural compound isolated from the roots of Salvia miltiorrhiza Bunge (Danshen), significantly inhibits STAT3-dependent luciferase activity, the STAT3 Tyr705 phosphorylation and the dimerization of STAT3, compared to tanshinone IIA which exhibits no activity. Cryptotanshinone (7 μM) dramatically blocks STAT3 Tyr705 phosphorylation but not STAT3 Ser727 phosphorylation in DU145 cells, and significantly inhibits JAK2 phosphorylation with IC50 of ~5 μM without affecting the phosphorylation of upstream kinases c-Src and EGFR, suggesting the inhibition of STAT3 Tyr705 phosphorylation might due to a direct mechanism probably by binding to the SH2 domain of STAT3. Cryptotanshinone significantly inhibits the proliferation of DU145 prostate cancer cells harboring constitutively active STAT3 with GI50 of 7 μM by blocking STAT3 activity, which leads to the down-regulation of cyclin D1, Bcl-xL, and survivin, subsequently the accumulation in the G0-G1 phase. Cryptotanshinone exhibits less growth inhibitory effect on PC3, LNCaP and MDA-MB-468 cells.

Kinase 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.

Cell Assay: Cells (KATO III, DU145, PC3, LNCaP, MDA-MB-231, MDA-MB-468, MDA-MB-453, MCF-7, MCF-10A, HeLa and HCT-116) 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.

Cancer Res. 2009 Jan 1;69(1):193-202; Mol Pharmacol. 2007 Jul;72(1):62-72.