Kinase experiment:

The caspase-3 activity is measured using a caspase-3 activity assay kit. Briefly, the cells treated by different concentrations of Sanguinarine (0.5 μM, 1 μM, 2 μM, 4 μM) or control DMSO are collected, washed and lysed in a lysis buffer for 30 min on ice. The supernatants are then collected by centrifuging at 1,2000 rpm for 10 min. The Ac-DEVD-pNA (2 mM) is added to each sample and incubated at 37℃ for 1 h. The optical density (OD) of each sample is finally quantified at a wavelength of 405 nm using a spectrophotometer. The p-NA standard is used to calibrate the caspase-3 activity of each sample[1].

Cell experiment:

The cell viability of Sanguinarine is determined by CCK-8 assay using a cell counting kit-8. Briefly, 22B-cFluc cells are seeded in a 96-well plate (5×103 cells/well) and treated with different concentrations of Sanguinarine (0.5 μM, 1 μM, 2 μM, 4 μM) for 24 h. Then 10 mL CKK-8 is added to each well for 4 h and the absorbance at 450 nm is measured with a microplate reader. The optical density (OD) values are determined to reflect the viable cell populations from each well[1].

Animal experiment:

Mice[1]Xenografted tumor models are prepared by injection of 1×107 22B-cFluc cells suspended in PBS into nude mouse (n=6). After tumors reach a volume of approximately 100 mm3, Sanguinarine (10 mg/kg) is i.v. injected into mice. After injection for 24 h, 48 h and 72 h, mice are given a single i.p. dose of 150 mg/kg D-luciferin and bioluminescence imaging are performed using a Xenogen Lumina II system. The signal intensity in the region of interest is expressed using the Living Image software 4.1. For the anti-tumor therapy studies, one group of tumor-bearing mice (n=6) receive intravenously 10 mg/kg of Sanguinarine every other day throughout the experimental period, while the control group of mice (n=6) receive DMSO only. Tumor growth measurement is calculated[1].

Sanguinarine chloride is a potent and specific inhibitor of PP2C with Ki value of 0.68 μM, and is also a selective and cell-active inhibitor of MKP-1 with IC50 value of 10 μM. Sanguinarine is also an allosteric activator of AMPK [1][2][3].

Protein phosphatase 2C (PP2C) is a serine/threonine-specific phosphatase, the activity of which is dependent on Mg2+ or Mn2+. PP2C dephosphorylates a number of substrates such as cyclin-dependent kinase, mitogen-activated kinase (MAPK) and Bad. Mitogen-activated protein kinase phosphatase-1 (MKP-1) is a dual specificity phosphatase. AMP-activated protein kinase (AMPK) plays an important role in the regulation of cellular metabolism [1][2][3].

Sanguinarine chloride is an inhibitor of PP2C and MKP-1, and also an allosteric activator of AMPK with antibiotic and antitumor activity. Sanguinarine competed with α-casein to inhibit PP2C and exhibited selectivity for PP2C as compared with PP1, PP2A and PP2B. In human promyelocytic leukemia cell line HL60, sanguinarine exhibited cytotoxicity with IC50 value of 0.37 μM and induced apoptosis via a caspase-3/7-dependent mechanism involving the phosphorylation of p38, a PP2C substrate [1]. Sanguinarine inhibited MKP-1 and MKP-L with IC50 values of 17.3 and 12.5 μ
M. In PANC-1 human pancreatic cancer cells, sanguinarine increased ERK and JNK/SAPK phosphorylation [2]. In the MDA MB-231 cell line, sanguinarine caused AMPK and the downstream acetyl-CoA carboxylase (ACC) phosphorylation [3]. In LNCaP and DU145 cells, sanguinarine inhibited cell growth, induced G0/G1 phase arrest and apoptosis [4].

References:
[1].  Aburai N, Yoshida M, Ohnishi M, et al. Sanguinarine as a potent and specific inhibitor of protein phosphatase 2C in vitro and induces apoptosis via phosphorylation of p38 in HL60 cells. Biosci Biotechnol Biochem, 2010, 74(3): 548-552.
[2].  Vogt A, Tamewitz A, Skoko J, et al. The benzo[c]phenanthridine alkaloid, sanguinarine, is a selective, cell-active inhibitor of mitogen-activated protein kinase phosphatase-1. J Biol Chem, 2005, 280(19): 19078-19086.
[3].  Choi J, He N, Sung MK, et al. Sanguinarine is an allosteric activator of AMP-activated protein kinase. Biochem Biophys Res Commun, 2011, 413(2): 259-263.
[4].  Adhami VM, Aziz MH, Reagan-Shaw SR, et al. Sanguinarine causes cell cycle blockade and apoptosis of human prostate carcinoma cells via modulation of cyclin kinase inhibitor-cyclin-cyclin-dependent kinase machinery. Mol Cancer Ther, 2004, 3(8): 933-940.