Planta Medica International Open 2017; 4(S 01): S1-S202
DOI: 10.1055/s-0037-1608053
Poster Session
Georg Thieme Verlag KG Stuttgart · New York

How natural products act on molecular level: understanding the effect of cryptotanshinone on keratinocyte differentiation using qPCR, DARTS and proteomics

S Esch
1   University of Münster, Institute of Pharmaceutical Biology and Phytochemistry, Münster, Germany
,
A Hensel
1   University of Münster, Institute of Pharmaceutical Biology and Phytochemistry, Münster, Germany
,
S Brandt
1   University of Münster, Institute of Pharmaceutical Biology and Phytochemistry, Münster, Germany
,
S König
2   University of Münster, Interdisciplinary Centre for Clinical Research, Core Unit Proteomics, Münster, Germany
› Author Affiliations
Further Information

Publication History

Publication Date:
24 October 2017 (online)

 

Cryptotanshinone (CTS), one of the main compounds from the roots of Salvia miltiorrhiza Bunge was investigated on its potential influence on the differentiation process of human keratinocytes (NHEK) in order to rationalize the traditional use of this plant for psoriasis treatment.

In vitro studies indicated strong influence of CTS (1µM) on keratinocyte differentiation: CTS suppresses the differentiation-specific keratins KRT1/10 nearly completely on protein level. Gene expression analysis by qPCR proved > 90% reduction of CK1/10 after 60h.

At higher concentrations CTS influenced cell vitality negatively (vitality assay IC50= 14.4µM, proliferation assay IC50= 8.2µM).

For pinpointing the signaling pathways induced by CTS (1µM) qPCR screening plates (BioRad) and cDNA from NHEK, treated with CTS (1µM) for 60h, were used. As expected distinct pathways towards keratin expression were reduced, indicating that this kind of qPCR screening plates can be a powerful tool for identifying signaling pathways.

To determine possible target structures in the differentiation-specific signaling pathways, Drug Affinity Responsive Target Stability (DARTS) in combination with proteomic approach was used. Stability of proteins, obtained from a total cell lysate from HaCaT keratinocytes with and without CTS treatment, versus the proteases thermolysin and pronase, was determined by using colloidal Coomassie staining. This method allows a distinguished and sensitive protein staining with low background and no interference with the subsequent protein sequencing. The full proteome analysis leads to the identification of Heat shock protein 90 as a possible major molecular target for CTS. An inhibition of this chaperon is known to result in reduced KRT1/10 expression [1]. From these data the use of CTS for skin diseases like lamellar ichthyosis seems to be rationalized.

[1] Sadanori Miyoshi et al. FEBS openbio