Compound Astragalus and Salvia miltiorrhiza extracts modulate MAPK-regulated TGF-β/Smad signaling in hepatocellular carcinoma by multi-target mechanism.
BACKGROUND
Astragalus membranaceus Bunge (Leguminosae) and Salvia miltiorrhiza Bunge (Lamiaceae) are two important Chinese herbs with a long history of extensive ethnobotanical usage in the treatment of liver-related diseases over many centuries. Presently, these two herbs are being used either as a single herbal formulation or a composite formula for the treatment of liver related conditions. In response, recent studies on these two herbs have focused on elucidating their mechanisms of action, particularly with regards to their anti-hepatocarcinogenic effects. Previously, we have reported that Compound Astragalus and Salvia miltiorrhiza extract (CASE), a synergized composite extract from Astragalus membranaceus and Salvia miltiorrhiza ameliorates liver fibrosis and hepatocellular carcinoma (HCC) by modulating the TGF-β/Smad pathway. Meanwhile, MAPK activation and MAPK-dependent linker phosphorylation of Smad2/3 and their preferential nuclear import are crucial for overall oncogenic role of TGF-β/Smad signaling in HCC. To elucidate further, we studied the effect of CASE on the MAPK pathway and how it affects MAPK-dependent regulation of TGF-β/Smad signaling using both cell and animal models of HCC.


METHODS
We used immunofluorescence and western blot techniques to monitor effect of CASE on the activation of the MAPKs (pERK, pJNK and pp38) in TGF-β1-stimulated hepatic stellate cells (HSCs), HepG2 cells and also diethylnitrosamine (DEN)-induced HCC in rats. Also phosphorylation and subcellular distribution of pSmad2/3, Smad4 and Imp7/8 in TGF-β1-stimulated HSC and HepG2 cells were monitored. The expression of pERK, pJNK, pp38 and PAI-1 gene were monitored by using western blot technique. The effect of CASE on domain-specific phosphorylation of Smad2/3 and their subcellular distribution, and the expression of Smad4 and its subcellular distribution in TGF-β1-stimulated HSCs and HepG2 cells were evaluated by using immunofluorescence technique. And the expression of Imp7/8 and their subcellular distribution were assessed by both immunofluorescence and western blot techniques, while PAI-1 gene expression was assessed by western blot


RESULTS
In vitro, CASE in a concentration-dependent manner increased the expression of pp38 but decreased the expression of pERK and pJNK; however, in vivo, CASE in a dose dependent manner decreased the expression of pERK, pJNK as well as pp38. Also, CASE concentration dependently inhibited pSmad2C/L, pSmad3L, Smad4, Imp7/8 and their nuclear import; it had no effect on pSmad3C in HepG2 cells; significantly decreased PAI-1 gene expression in both in vitro and in vivo.


CONCLUSIONS
CASE blocked MAPK activation, MAPK-dependent linker phosphorylation of Smad2/3, Smad4 expression, Imp7 expression and their nuclear import leading to significant down-regulation of PAI-1 gene expression; further highlighting the multi-target anti-HCC effect of CASE and its potential drug candidature.
Department of Pharmacology and Institute of Natural Medicine, Anhui Medical University, Hefei 230032, China.
Alex
A
Boye
Department of Pharmacology and Institute of Natural Medicine, Anhui Medical University, Hefei 230032, China.
Chao
C
Wu
Department of Pharmacology and Institute of Natural Medicine, Anhui Medical University, Hefei 230032, China.
Yufeng
Y
Jiang
Department of Pharmacology and Institute of Natural Medicine, Anhui Medical University, Hefei 230032, China.
Jiyu
J
Wang
Department of Pharmacology and Institute of Natural Medicine, Anhui Medical University, Hefei 230032, China.
Jiajun
J
Wu
Department of Pharmacology and Institute of Natural Medicine, Anhui Medical University, Hefei 230032, China.
Xiaochuan
X
Yang
Department of Pharmacology and Institute of Natural Medicine, Anhui Medical University, Hefei 230032, China. Electronic address: yangyan@ahmu.edu.cn.
Yan
Y
Yang
D000475:Alkenes / Q000302:isolation & purification / Q000494:pharmacology; D000818:Animals; D027885:Astragalus propinquus / Q000737:chemistry*; D006528:Carcinoma, Hepatocellular / Q000139:chemically induced / Q000378:metabolism*; D004052:Diethylnitrosamine; D015536:Down-Regulation; D015870:Gene Expression / Q000187:drug effects; D056945:Hep G2 Cells; D055166:Hepatic Stellate Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D028884:Karyopherins / Q000378:metabolism; D008113:Liver Neoplasms / Q000139:chemically induced / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D008297:Male; D010936:Plant Extracts / Q000737:chemistry / Q000494:pharmacology*; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D059808:Polyphenols / Q000302:isolation & purification / Q000494:pharmacology; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D027561:Salvia miltiorrhiza / Q000737:chemistry*; D012503:Saponins / Q000302:isolation & purification / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects; D051899:Smad2 Protein / Q000378:metabolism*; D053773:Transforming Growth Factor beta1 / Q000378:metabolism* / Q000494:pharmacology; D014315:Triterpenes / Q000302:isolation & purification / Q000494:pharmacology; D015854:Up-Regulation
D000475:Alkenes; C466996:IPO7 protein, human; D028884:Karyopherins; D010936:Plant Extracts; D017395:Plasminogen Activator Inhibitor 1; D059808:Polyphenols; D011134:Polysaccharides; D018160:Receptors, Cytoplasmic and Nuclear; C497782:SMAD2 protein, human; D012503:Saponins; D051899:Smad2 Protein; D053773:Transforming Growth Factor beta1; D014315:Triterpenes; C568740:salvianolic acid; C052064:astragaloside A; D004052:Diethylnitrosamine
CASE; HSC; HepG2; Hepatocellular carcinoma; MAPK; TGF-β(1)