Compound Astragalus and Salvia miltiorrhiza extract inhibits hepatocellular carcinoma progression via miR-145/miR-21 mediated Smad3 phosphorylation
Graphical abstract
Introduction
Hepatocellular carcinoma (HCC), one of the most frequent solid organ malignancies, is very prevalent in Asia and Africa, where many people are infected with hepatitis B-or-C virus (HBV or HCV), which is the foremost etiology to generate HCC (Llovet et al., 2016). Currently, therapeutic strategies against HCC on clinic, surgical resection is preferred for patients with early HCC and/or non-metastatic tumors of extrahepatic tissues; chemotherapy and radiotherapy are commonly applied for patients with intermediate and advanced HCC and/or complementary therapies of surgical resection (Trojan et al., 2016). Nevertheless, the high mortality of HCC still exists on account of the high incidence and mobility of HCC, additional deficiencies of present therapeutic approaches, for instance, tumor recurrence after surgical resection, low sensitivity in HCC but high cytotoxicity in normal tissues to chemotherapeutics and/or radiotherapy (Mitra et al., 2015). Traditional Chinese medicine (TCM) is emerging as a potential candidate for treating HCC because of its effectiveness, hypotoxicity and inexpensiveness (Gao et al., 2016). Two important Chinese medical herbs, Astragalus membranaceus (Fisch.) Bge. and Salvia miltiorhiza Bge., have been empirically used for treatment of these patients with various hepatic diseases including hepatitis, cirrhosis and HCC on TCM clinic spanning several centuries (Du et al., 2008, Zhu et al., 2013), however, scientific evidences with respect to their mechanisms of action, especially molecular mechanisms against HCC, remain unclear.
Widely known, the pathogenesis of HCC at the cellular and molecular level, is generally considered to be derived from the trans-differentiation of hepatocytes owing to proto-oncogene activation and anti-oncogene inactivation under carcinogens (e.g. HBV, HCV and aflatoxin), which characterizes dysregulated proliferation and migration/invasion of cells (Kirstein and Vogel, 2014). Persistent inflammation mediated by cytokines is direct motivity of cell phenotype trans-differentiation and maintenance in tumor micro-environment (Martin et al., 2012, Matsuzaki et al., 2007). Transforming growth factor beta (TGF-β), especially TGF-β1 isoform, one of the most pivotal cytokines, is highly implicated in the occurrence and progression of HCC (Matsuzaki et al., 2007). TGF-β1 combining its receptors (TβRII and TβRI) located in the target cell membrane activates intracellular canonical Smad signaling and non-canonical signaling such as mitogen-activated protein kinase (MAPK) pathways (Derynck and Zhang, 2003), these dysregulated signaling pathways contribute to the trans-differentiation of cell phenotypes, and then induce HCC occurrence and progression. MicroRNAs, small conserved non-coding RNAs of 19–25 nucleotides in length, are key endogenous post-transcriptional regulators of gene expression through their interaction with the 3′ untranslated region (3′UTR) of target mRNA resulting in the degradation or translational suppression. Accumulating evidences manifest that microRNAs function via the transcriptional regulation of some specific oncogenes and/or tumor suppressors in oncogenesis and progression (Boye and Yang, 2014, McManus, 2003). MicroRNA-145 (miR-145) was found to be down-regulated in HCC with HBV or HCV infection (Gao et al., 2011, Varnholt et al., 2008), and in vitro experiments indicated that restoring miR-145 level markedly suppressed cell proliferation, invasion and migration of Hep3B and HepG2 cell lines (Gao et al., 2011). On the contrary, another microRNA, namely miR-21 was reported to be up-regulated in patients with HCC (Karakatsanis et al., 2013), and one in vitro study demonstrated that up-regulated miR-21 promoted HepG2 cell proliferation (Xu et al., 2013). Hence, theoretically, targeted regulation of these pivotal molecules to rectify aberrant cell signaling transduction may be underlying molecular mechanisms of TCM and its extracts exerting curative efficacy against HCC.
Compound Astragalus and Salvia miltiorrhiza extract, abbreviated as CASE, a formula is made up of astragalosides, astragalus polysaccharide regarded as active compounds of Astragalus membranaceus (Fisch.) Bge. and salvianolic acids regarded as active compounds of Salvia miltiorhiza Bge. Several previous studies have shown that CASE suppressed hepatocarcinogenesis induced by diethylnitrosamine (DEN) in rats, which was associated with repressive TGF-β/Smad signaling and MAPK pathways (Boye et al., 2015, Hu et al., 2014, Rui et al., 2014); CASE inhibited HepG2 cell proliferation and invasion by regulating the activation of Smad proteins and MAPKs (Boye et al., 2015, Liu et al., 2010). Quite recently, the crucial interaction between miR-145/miR-21 and domain-specific phosphorylation of Smad3 was implicated in the pathogenesis and progression of HCC (Wang et al., 2017). On the basis of this initial finding, it was hypothesized that CASE might regulate inverse expression of miR-145/miR-21 and their interaction with Smad3 phosphorylation in order to truncate HCC progression. This study investigated the effects of CASE on phenotypic hallmarks (Cell proliferation, migration, and apoptosis) of HCC and the expression patterns of miR-145/miR-21, domain-specific Smad3 phosphorylation, and the activation of MAPKs using cell (HepG2 cells) and xenografted nude mice models of HCC.
Section snippets
Preparation of CASE
The two herbs Astragalus membranaceus (Fisch.) Bge. and Salvia miltiorhiza Bge. were purchased from Bozhou Huqiao Pharmaceutical Co., Ltd. (Bozhou, China) and authenticated by Prof. Xiaoxiang Zhang (Department of Pharmaceutical Engineering, Hefei University of Technology, Hefei, China), a specialist in traditional Chinese herbal medicine. Voucher specimens were deposited in the specimen room of traditional Chinese medicine (Anhui University of Chinese Traditional Medicine, Hefei, China). The
CASE up-regulated miR-145 but down-regulated miR-21 expression in rats with DEN-induced HCC and TGF-β1-stimulated HepG2 cells
To observe the effects of CASE on miR-145/miR-21 expression in the development of HCC, in vivo (DEN-inducted HCC in rats) and in vitro (TGF-β1-stimulated HepG2 cells) models of HCC were established and administrated with CASE, miR-145 and miR-21 were measured. Our results showed that miR-145 was markedly decreased in rat liver tissues with HCC induced by DEN and TGF-β1-stimulated HepG2 cells, while observably increased by CASE treatment (Fig. 1A, C); synchronously, miR-21 was slightly decreased
Discussion
Recently, increasing evidences indicate that some active compounds from TCM are excellent potential candidates used for the treatment of cancers, especially HCC, in view of their reported effectiveness such as inhibited cell proliferation, migration and invasion as well as induced apoptosis in hepatoma cell lines, which function via the adjustment of multi-target and multi-level (Boye et al., 2015, Ma et al., 2013, Meng et al., 2013, Zhu et al., 2013). Hereby, we reported that CASE, a formula
Conclusion
CASE exhibits well anti-HCC effects on in vivo and in vitro models of human liver cancer via regulating the crosstalk of miR-145/miR-21 and pSmad3C/pSmad3L, especially miR-145/miR-21 mediated Smad3 phosphorylation, which maybe provides an important theoretical foundation for CASE's anti-HCC therapy used for patients in a near future.
Acknowledgments
We thank Prof. Koichi Matsuzaki (Department of Gastroenterology and Hepatology, Kansai Medical University, Osaka, Japan) for providing us with some specific antibodies (Rabbit anti-pSmad3L antibody) and plasmids (Smad3 WT, Smad3 EPSM and Smad3 3S-A plasmids). Also, we appreciate the support from the National Natural Science Foundation of China (No. 81573652 and 81374012).
Conflicts of interest
All authors declare no potential conflicts of interest.
Author contribution
Yan Yang conceived the project and acquired funds. Yan Yang designed and administrated the research. Chao Wu, Weiyang Chen, Meng Fang, Xiangming Tao, Yuanyuan Xu and Shu Hou performed these experiments. Chao Wu, Weiyang Chen and Meng Fang arranged, analyzed the data and interpreted the results. Chao Wu and Alex Boye drafted the manuscript. Yan Yang reviewed the manuscript. Yan Yang, Chao Wu, Meng Fang and Alex Boye participated in revising the manuscript.
References (49)
- et al.
Transforming growth factor-beta in the gastrointestinal and hepatic tumor microenvironment
Gastroenterology
(2011) - et al.
Smad-mediated regulation of microRNA biosynthesis
FEBS Lett.
(2012) - et al.
Compound Astragalus and Salvia miltiorrhiza extracts modulate MAPK-regulated TGF-beta/Smad signaling in hepatocellular carcinoma by multi-target mechanism
J. Ethnopharmacol.
(2015) - et al.
Deregulation of microRNA expression occurs early and accumulates in early stages of HBV-associated multistep hepatocarcinogenesis
J. Hepatol.
(2011) - et al.
Modulation of K-Ras-dependent lung tumorigenesis by MicroRNA-21
Cancer Cell
(2010) - et al.
A novel reciprocal loop between microrna-21 and TGFbetaRIII is involved in cardiac fibrosis
Int. J. Biochem. Cell Biol.
(2012) - et al.
Controversies on the role of Th17 in cancer: a TGF-beta-dependent immunosuppressive activity?
Trends Mol. Med.
(2012) Smad phospho-isoforms direct context-dependent TGF-beta signaling
Cytokine Growth Factor Rev.
(2013)MicroRNAs and cancer
Semin. Cancer Biol.
(2003)- et al.
Ethnopharmacological and bioactivity guided investigation of five TCM anticancer herbs
J. Ethnopharmacol.
(2013)
Novel delivery approaches for cancer therapeutics
J. Control. Release.: Off. J. Control. Release Soc.
Compound Astragalus and Salvia miltiorrhiza extract suppresses hepatocellular carcinoma progression by inhibiting fibrosis and PAI-1 mRNA transcription
J. Ethnopharmacol.
MiR-145 regulates PAK4 via the MAPK pathway and exhibits an antitumor effect in human colon cells
Biochem. Biophys. Res. Commun.
Compound Astragalus and Salvia miltiorrhiza extract exerts anti-fibrosis by mediating TGF-beta/Smad signaling in myofibroblasts
J. Ethnopharmacol.
miR-21 and miR-145 cooperation in regulation of colon cancer stem cells
Mol. Cancer
Hepatic microRNA orchestra: a new diagnostic, prognostic and theranostic tool for hepatocarcinogenesis
Mini Rev. Med. Chem.
miR-145-5p inhibits epithelial-mesenchymal transition via the JNK signaling pathway by targeting MAP3K1 in non-small cell lung cancer cells
Oncol. Lett.
SMAD proteins control DROSHA-mediated microRNA maturation
Nature
Smad-dependent and Smad-independent pathways in TGF-beta family signalling
Nature
The clinic research of huang qi injection combined with transcatheter arterial chemoembolization to treat primary hepatocellular carcinoma
Gansu Med. J.
Molecular targets of Chinese herbs: a clinical study of hepatoma based on network pharmacology
Sci. Rep.
TGF-beta signaling from receptors to smads
Cold Spring Harb. Perspect. Biol.
Compound Astragalus and Salvia miltiorrhiza extracts suppress hepatocarcinogenesis by modulating transforming growth factor-beta/Smad signaling
J. Gastroenterol. Hepatol.
miR-145 inhibits invasion and metastasis by directly targeting Smad3 in nasopharyngeal cancer
Tumour Biol.: J. Int. Soc. Oncodev. Biol. Med.
Cited by (0)
- 1
Also affiliated to Department of Pharmacology, School of Pharmaceutical Sciences and Drug Discovery, University of Cape Coast, Ghana.