Elsevier

Journal of Ethnopharmacology

Volume 176, 24 December 2015, Pages 55-68
Journal of Ethnopharmacology

Molecular mechanisms of rosmarinic acid from Salvia miltiorrhiza in acute lymphoblastic leukemia cells

https://doi.org/10.1016/j.jep.2015.10.020Get rights and content

Abstract

Ethnopharmacological relevance

Rosmarinic acid (RA), a major hydrosoluble bioactive compound found in the Chinese medicinal herb, Salvia miltiorrhiza Bunge, which has been used in traditional Chinese medicine to treat various diseases, including cancer. However, the mechanisms have not been fully elucidated.

Aim of the study

Guided by microarray hybridization and Ingenuity Pathway Analysis, we identified modes of action of rosmarinic acid (RA) isolated from S. miltiorrhiza on acute lymphoblastic leukemia cells.

Materials and methods

Microarray data were verified by independent methods: Real-time RT-PCR (mRNA expression), resazurin assay (cytotoxicity of RA towards parental CCRF-CEM, multidrug-resistant CEM/ADR5000 cells and normal lymphocytes), flow cytometry (cell cycle arrest, apoptosis, necroptosis, generation of reactive oxygen species (ROS), disruption of mitochondrial membrane potential (MMP)), single cell gel electrophoresis (DNA damage), molecular docking and gene promoter binding motif analysis (NFκB), Western blotting (nuclear NFκB translocation, PARP cleavage, caspase 3/7/9 expression), and fibronectin-based cell adhesion assay.

Results

RA dose-dependently inhibited CCRF-CEM and CEM/ADR5000 cells, but caused less cytotoxicity towards normal lymphocytes. RA simultaneously induced apoptosis and necrosis, as shown by cell morphology and annexin V-PI assay. DNA damage was dose-dependently induced without ROS generation, which subsequently led to cell cycle arrest. RA-stimulated MMP dysfunction activated PARP-cleavage and caspase-independent apoptosis. In accordance with molecular docking and gene promoter binding motif analyses, p65 translocation from the cytosol to the nucleus was blocked by RA, indicating a mechanistic role of the NFκB pathway to explain RA's action. RA affected cellular movement as evaluated by ameliorating cell adhesion to fibronectin.

Conclusions

RA induced apoptosis and necrosis in a ROS-independent DNA damage and caspase-independent manner. These results may contribute to the rationale use of S. miltiorrhiza and RA in traditional medicine of leukemia.

Introduction

Cancer belongs to the leading public health issues and represents a major cause of death worldwide (Hanahan and Weinberg, 2000). Conventional antineoplastic drugs are used to eradicate malignant cells, however, reoccurrence due to incomplete removal of cancer cells and severe side effects in normal organs are often accompanied with chemotherapy. For this reason, new approaches to improve efficiency and tolerability of chemotherapeutic agents are urgently needed for anti-cancer treatment. Natural herbal medicine has been considered as promising option to combat cancer, since approximately 60% of approved anti-cancer drugs are originated from nature (Newman and Cragg, 2012). In traditional Chinese medicine, cancer was described as the results of blood stasis or qi stagnation (Zhang et al., 2012, Hu et al., 2015). Herbal plants which are able to promoting circulation and removing blood stasis (a Chinese medicinal term named huoxue huayu) are traditionally used for patients with cancers (Chen et al., 2014). Salvia miltiorrhiza Bunge (Lamiaceae, also named danshen in Chinese) is a well-known Chinese medicinal herb and is classified as huoxue huayu. Therefore, besides the clinical use as a hemorrheologic agent, S. miltiorrhiza has also traditionally used in the treatment of cancer (Du and Zhang, 2014).

In modern pharmacological investigations, S. miltiorrhiza exerts multiple therapeutical potentials against cancer, Alzheimer and cardiovascular diseases etc. (Cheng, 2007, Chen et al., 2014, Zhang et al., 2015). The root of the plant contains the hydrophilic phenolic acids and the hydrophobic tanshinones (Zhou et al., 2005). Rosmarinic acid (RA), classified as a polyphenolic compound, is an ester derivative of caffeic acid and one of the main hydrophilic phenolic acids in S. miltiorrhiza. RA modulates the immune system (Hur et al., 2007, Mesaik et al., 2012) and acts anti-microbial (Abedini et al., 2013, Chung et al., 2015) as well as neuroprotective (Hamaguchi et al., 2009, Hasanein and Mahtaj, 2015). This compound also inhibited colon cancer cell proliferation by repressing 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced binding of AP-1, c-Jun and c-Fos to cyclooxygenase-2 (COX-2) and reducing COX-2 activity (Scheckel et al., 2008, Tao et al., 2014). Furthermore, RA induced cancer cell apoptosis and inhibited invasion by modulating phosphorylation of ERK (Xavier et al., 2009, Xu et al., 2010). Moreover, RA inhibited DNA methyltransferase activity, which in turn promoted transcriptional expression of tumor suppressor genes (Paluszczak et al., 2010). In addition, RA sensitized tumor necrosis factor (TNF)α-induced apoptosis by down-regulating ROS regeneration and NFκB activation in U937 leukemia cells (Moon et al., 2010).

Despite its anticancer and chemopreventive properties, the molecular modes of action of RA in cancer cells have not been analyzed in detail, especially on acute lymphoblastic leukemia (ALL). Here, we investigated the inhibitory effects of RA towards sensitive (CCRF-CEM) and multidrug-resistant cell lines (CEM/ADR5000) derived from ALL, which represent an aggressive and malignant form of hematopoietic tumors characterized by uncontrolled growth of abnormal progenitor cells unable to differentiate into T-cells and B-cells. To the best of our knowledge, the detailed mechanisms of RA treatment towards CCRF-CEM and CEM/ADR5000 have not been explored.

Section snippets

Substances

RA (purity:≥97%, Fig. 1A) and necrostatin-1 were purchased from Enzo Life Science GmbH (Lörrach, Germany) and were dissolved in DMSO. z-VAD-fmk was obtained from Selleckchem (München, Germany). Doxorubicin was provided by the University Hospital of the Johannes Gutenberg University (Mainz, Germany). TNFα was purchased from Sino Biological Inc. (Beijing, People's Republic of China).

Cancer cell lines

CCRF-CEM and P-glycoprotein (P-gp) overexpressing, multidrug-resistant CEM/ADR5000 leukemia cells were cultured in

Cytotoxicity of RA towards sensitive and multidrug-resistant leukemia cell lines.

Multidrug resistance (MDR) is mediated predominately by energy-dependent ATP-binding cassette (ABC) transporters and represents a major reason for the failure of cancer chemotherapy. P-gp, ABCB1/MDR1 is the best known ABC transporter, which pumps drugs out of cells leading to survival of cancer cells (Ferté, 2000, Ambudkar et al., 2003). We firstly investigated, whether RA suppresses the growth of sensitive CCRF-CEM cells and the P-gp-overexpressing subline CEM/ADR5000. RA induced cytotoxicity

Discussion

Multidrug resistance (MDR) is a major obstacle in clinical oncology and new therapeutic options are urgently needed. This is also illustrated in the present investigation. P-glycoprotein overexpressing CEM/ADR500 cells were more than 3320-fold more resistant to doxorubicin than their parental wild-type CCRF-CEM cells. As previously reported, CEM/ADR5000 cells also reveal high degrees of cross-resistance to other established anticancer drugs such as epirubicin, vincristine, vinblastine,

Funding

We are thankful to the National Science Council (Taiwan) and German Academic Exchange Service (DAAD) for providing a scholarship to Ching-fen Wu.

Conflict of interest

The authors declare that they have no conflict of interest.

Acknowledgment

We thank the microarray unit of the DKFZ Genomics and Proteomics Core Facility for providing the Illumina Whole-Genome Expression Beadchips and related services, and technical support from the Cytometry Core Facility in IMB (Mainz, Germany).

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