Standardized herbal extracts of Japanese Kampo medicine and their effects on human and murine pancreatic cancer cells

Traditional Japanese Kampo prescriptions have anti-inflammatory and anti-cachexia effects. Epigenetic effects have also been described for herbal remedies. Here, we analyze the effect of standardized single and composite prescriptions of Japanese Kampo medicine in human and murine pancreatic cancer cells. PANC1, NKCII and KPCbl6 cells were cultivated in DMEM (+ 10% FCS, + 1% NEAA) until confluence. Standardized methanolic, ethanolic (25%) and traditional aqueous extracts of Glycyrrhiza uralensis root and Scutellaria baicalensis root were used. The concentration and time-dependent effect on cell viability was tested with MTT-at 24, 48, 72 and 96h. Protein expression of proliferation markers (CDK6, Cyclin D1, CyclinD3), transcription factors (NFATc1), tumor suppressor genes (p15, p21, p27, p53), and histone modifications were analyzed by Western Blot in whole protein lysates at 24 and 48h. Furthermore, gene/transcript expression of proliferation markers CDK6, Cyclin D1 and CyclinD3, and the tumor suppressor gene p21 were analyzed by qPCR. FACS analysis was used to study cell cycle arrest after treatment with G. uralensis root and S. baicalensis root. The different extracts of G. uralensis root and S. baicalensis root inhibited tumor growth in vitro. This effect was concentration dependent in all three tested cell lines. Cyclines and cell-cycle inhibitors were regulated accordingly. These results could be verified on both protein and transcript level. FACS analysis showed a G1 arrest. G. uralensis also induced histone H3K27 acetylation, indicating that specific genomic areas, e.g. enhancer regions, were activated upon treatment. The presented experimental data demonstrate that Kampo extracts have anti-proliferative effects in vitro.