The extract of huanglian, a medicinal herb, induces cell growth arrest and apoptosis by upregulation of interferon-β and TNF-α in human breast cancer cells
Jing X. Kang*,Jing Liu,Jingdong Wang,Chengwei He and Frederick P. Li1
Abstract Huanglian (Coptidis rhizoma), a widely used herb in traditional Chinese medicine, has been shown recently to possess anticancer activities. However, the molecular mechanism underlying the anticancer effect of the herb is poorly understood. Specifically, whether huanglian extract affects the expression of cancer-related genes has not been defined. This study used DNA microarray technology to examine the effect of the herbal extract on expression of the common genes involved in carcinogenesis in two human breast cancer cell lines, the ER-positive MCF-7 and ER-negative MDA-MB-231 cells. Treatment of the cancer cells with huanglian extract markedly inhibited their proliferation in a dose- and time-dependent manner. The growth inhibitory effect was much more profound in MCF-7 cell line than that in MDA-MB-231 cells. DNA microarray assay revealed that treatment with huanglian dramatically increased the mRNA expression of interferon-β (IFN-β) and tumor necrosis factor-α in MCF-7 cells. Quantitative analysis by real-time PCR or western blotting confirmed the upregulation of the two genes (especially IFN-β) in MCF-7 cells, but not in MDA-MB-231 cells. Addition of neutralizing antibody against IFN-β to culture medium markedly inhibited the huanglian-induced antiproliferative effect, confirming the involvement of IFN-β in the huanglian's effect and also suggesting an autocrine pathway for the action of IFN-β in this setting. Given that IFN-β is among the most important anticancer cytokines, the upregulation of this gene by huanglian is, at least in part, responsible for its antiproliferative effect. The results of this study implicate huanglian as a promising herb for chemoprevention and chemotherapy of certain cancers.
Discussion The data presented here demonstrate that huanglian extract is highly effective in inhibiting cell proliferation and inducing apoptotic cell death in MCF-7 breast cancer cells. The observed anticancer effects of the herbal extract result mainly from its ability to enhance the expression of two anticancer cytokines, IFN-β and TNF-α (especially IFN-β), as evidenced by the increased levels of mRNA and protein of these cytokines in the cells treated with the herbal extract. The upregulated cytokines act through an autocrine pathway to induce cell growth arrest and apoptosis, as indicated by the fact that addition of a neutralizing antibody against IFN-β to culture medium could significantly attenuate the growth inhibitory effect of huanglian extract. Thus, the results of this study provide evidence for the anticancer activity of huanglian and, more importantly, the molecular basis for its effect.
Our results showed that the two breast cancer cell lines MCF-7 (estrogen receptor positive) and MDA-MB-231 (estrogen receptor negative) responded to the herbal treatment differently. The MDA-MB-231 cells did not increase the synthesis of IFN-β and TNF-α in response to huanglian treatment and, accordingly, exhibited a much smaller degree of growth arrest and apoptosis when compared with the MCF-7 cells. This phenomenon supports the notion that the upregulation of IFN-β and TNF-α is probably the mechanism underlying the growth inhibitory effect of huanglian in this particular cell type. The differential responses of the two cell lines also raise a question as to whether estrogen receptor is involved in or essential for the action of huanglian. Future study on this subject matter and experiments to elucidate the whole signal transduction pathway for huanglian are warranted.
The extract of huanglian contains several components (15). Berberine is known to be the dominant one (7,15). In this study, we found that purified berberine was significantly less effective than the whole huanglian extract. Similar results have been reported previously by others (7,8). This indicates that there are constituents in the herb other than berberine that are critical for its growth inhibitory effect. In this context, it seems better to develop the whole herbal extract, rather than its dominant components, for cancer therapy. Nevertheless, identification and characterization of the active components present in the whole extract of huanglian is needed.
Both INF-β and TNF-α are the important cytokines that regulate cell growth and death (16–19). The biological effects of these cytokines have been investigated extensively over the last decades. The anticancer activity of INF-β has been well recognized (16–19), whereas TNF-α plays a paradoxical role in carcinogenesis (20). It is well known that IFN-β can inhibit cell growth and kill cancer cells. Thus, enhancing the production of IFN-β in cancer cells seems to be an effective anticancer mechanism and the identification of compounds that have such a property should be a new direction of cancer drug development. Our finding that huanglian is highly effective in enhancing synthesis of IFN-β in MCF-7 cancer cells provides a molecular basis for huanglian as a promising anticancer agent. Although huanglian has been previously shown to alter expression of some genes in several different cancer cell lines (7,8), the present study is the first to show upregulation by huanglian of the two anticancer cytokines, IFN-β and TNF-α, in human cancer cells. Previously, Iizuka et al. (8) reported that use of oligonucleotide microarray identified 13 various genes whose levels of expression were correlated with the ID50 values of both berberine and C.rhizoma in pancreatic cancer cell lines. Li et al. (7) found that huanglian extract suppressed the expression of cyclin B1 in a human gastric cancer cell line (MKN-74). It is possible that the genetic effects of huanglian are different in different cell types. Whether the effect of huanglian on cytokine expression also occurs in non-cancer cells are now under investigation in our laboratory.
Since IFN and TNF are also potent immunomodulators and play critical roles in treatment of certain infections, the finding of the present study that huanglian can enhance the expression of these two cytokines (especially IFN) might provide an explanation for the use of huanglian as a key herb to treat infectious conditions in Chinese medicine.
A full understanding of molecular effect (i.e. gene expression) of a herb is very important for evaluation of its efficacy as well as safety (side effect). Microarray technology seems to be quite helpful in this regard. DNA chip studies may help identifying compounds with novel therapeutic effect and clarify the roles of various compounds of herbs in the physiological activity. Our strategy, as used in the present study, could serve as a framework to study medicinal herbs.
Huanglian has been widely used in China for several thousand years, mainly, for the treatment of infectious conditions. This herb has been shown to be quite safe for human consumption (21). This advantage plus the emerging evidence of its anticancer effects make huanglian a very promising candidate for being an effective and safe anticancer agent.
Source Carcinogenesis (November 2005) 26 (11): 1934-1939. doi: 10.1093/carcin/bgi154 LINK TO FULL ARTICLE
Hepatoprotective effects of berberine on carbon tetrachloride-induced acute hepatotoxicity in rats Yibin Feng1, Ka-Yu Siu1, Xingshen Ye1, Ning Wang1, Man-Fung Yuen2 , Chung-Hang Leung3, Yao Tong1 and Seiichi Kobayashi4
1 School of Chinese Medicine, The University of Hong Kong, 10 Sassoon Road, Pokfulam, Hong Kong SAR, China 2 Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulam Road, Hong Kong SAR, China 3 Department of Chemistry and Open Laboratory of Chemical Biology of the Institute of Molecular Technology for Drug Discovery and Synthesis, Faculty of Science, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, China 4 Department of Medical Laboratory Science, Faculty of Health Sciences, Hokkaido University, Kita-12, Nishi-5, Kita-ku, Sapporo, Japan
Background Berberine is an active compound in Coptidis Rhizoma (Huanglian) with multiple pharmacological activities including antimicrobial, antiviral, anti-inflammatory, cholesterol-lowering and anticancer effects. The present study aims to determine the hepatoprotective effects of berberine on serum and tissue superoxide dismutase (SOD) levels, the histology in tetrachloride (CCl4)-induced liver injury.
Methods Sprague-Dawley rats aged seven weeks were injected intraperitoneally with 50% CCl4 in olive oil. Berberine was orally administered before or after CCl4 treatment in various groups. Twenty-four hours after CCl4 injection, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, serum and liver superoxide dismutase (SOD) activities were measured. Histological changes of liver were examined with microscopy.
Results Serum ALT and AST activities significantly decreased in a dose-dependent manner in both pre-treatment and post-treatment groups with berberine. Berberine increased the SOD activity in liver. Histological examination showed lowered liver damage in berberine-treated groups.
Conclusion The present study demonstrates that berberine possesses hepatoprotective effects against CCl4-induced hepatotoxicity and that the effects are both preventive and curative. Berberine should have potential for developing a new drug to treat liver toxicity.