Elsevier

Journal of Ethnopharmacology

Volume 231, 1 March 2019, Pages 98-112
Journal of Ethnopharmacology

Compound Astragalus and Salvia miltiorrhiza extract inhibits hepatocellular carcinoma progression via miR-145/miR-21 mediated Smad3 phosphorylation

https://doi.org/10.1016/j.jep.2018.11.007Get rights and content

Abstract

Ethnopharmacological relevance

Compound Astragalus and Salvia miltiorrhiza extract (CASE), containing astragalosides, astragalus polysaccharide extracted from Astragalus membranaceus (Fisch.) Bge. and salvianolic acids from Salvia miltiorhiza Bge., has been found to inhibit hepatocarcinogenesis via mediating transforming growth factor-β (TGF-β)/Smad signaling, especially Smad3 phosphorylation. The crucial interaction between microRNA-145/microRNA-21 (miR-145/miR-21) and Smad3 phosphorylation is implicated in the pathogenesis and progression of hepatocellular carcinoma (HCC). However, effects of CASE on HCC progression involved in the expression of miR-145/miR-21 and their interaction with Smad3 phosphorylation downstream of TGF-β/MAPK/Smad pathway remain unclear. This study addressed above questions using in vitro (HepG2 cells) and in vivo (Xenografts of nude mice) models of HCC.

Materials and methods

In vivo [Diethylnitrosamine (DEN)-induced HCC in rats] and in vitro [TGF-β1-stimulated HepG2 cells] models of HCC were established and co-administrated using graded doses/concentrations CASE (60, 120, 240 mg/kg used in rats; 20, 40, 80 µg/ml used in HepG2 cells), miR-145 and miR-21 were measured. HepG2 cells were transfected with miR-145 antagomir, miR-21 agomir and Smad3C/L plasmids (Smad3 EPSM, Smad3 3S-A and Smad3 WT related to up-regulated expression of pSmad3C, pSmad3L and pSmad3C/3L respectively) and then treated by CASE (80 µg/ml). Similarly, HepG2 cell xenografted nude mice were administered with miR-145 antagomir, miR-21 agomir and CASE (310 mg/kg); Smad3 WT, Smad3 EPSM and Smad3 3S-A plasmids stably transfected HepG2 cell lines were constructed respectively and their xenografted nude mice were established, and then treated by CASE (310 mg/kg). Cell proliferation, migration, apoptosis, tumor growth and histopathologic characteristics of xenografts were assessed; also, domain-specific Smad3 phosphorylation isoforms (pSmad3C/pSmad3L), activated MAPKs (pERK1/2, pJNK1/2, pp38) and miR-145, miR-21 were measured.

Results

CASE up-regulated miR-145 while down-regulated miR-21 expression in both rats with DEN-induced HCC and TGF-β1-stimulated HepG2 cells; CASE inhibited cell migration, proliferation and tumor growth while facilitated cell apoptosis in TGF-β1-stimulated HepG2 cells and xenografts of nude mice with miR-145 antagomir/miR-21 agomir treatment via increasing miR-145 and facilitating miR-145 modulated pSmad3L→pSmad3C signaling switch while decreasing miR-21 and inhibiting miR-21 modulated MAPK-dependent Smad3L phosphorylation. Also, up-regulated pSmad3C enhanced inhibited effect of CASE on tumor growth and facilitated effect of CASE on cell apoptosis involved in increased miR-145 while decreased miR-21 expression, however, inverse phenomena were observed when up-regulated pSmad3L.

Conclusion

Our results suggest that CASE inhibits HCC progression via mediating the interaction of miR-145/miR-21 and Smad3 phosphorylation, 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.

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.

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    Also affiliated to Department of Pharmacology, School of Pharmaceutical Sciences and Drug Discovery, University of Cape Coast, Ghana.

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