Dong-Mei Ren

Brusatol enhances the efficacy of chemotherapy by inhibiting the Nrf2-mediated defense mechanism

The major obstacle in cancer treatment is the resistance of cancer cells to therapies. Nrf2 is a transcription factor that regulates a cellular defense response and is ubiquitously expressed at low basal levels in normal tissues due to Keap1-dependent ubiquitination and proteasomal degradation. Recently, Nrf2 has emerged as an important contributor to chemoresistance. High constitutive expression of Nrf2 was found in many types of cancers, creating an environment conducive for cancer cell survival. Here, we report the identification of brusatol as a unique inhibitor of the Nrf2 pathway that sensitizes a broad spectrum of cancer cells and A549 xenografts to cisplatin and other chemotherapeutic drugs. Mechanistically, brusatol selectively reduces the protein level of Nrf2 through enhanced ubiquitination and degradation of Nrf2. Consequently, expression of Nrf2-downstream genes is reduced and the Nrf2-dependent protective response is suppressed. In A549 xenografts, brusatol and cisplatin cotreatment induced apoptosis, reduced cell proliferation, and inhibited tumor growth more substantially when compared with cisplatin treatment alone. Additionally, A549-K xenografts, in which Nrf2 is expressed at very low levels due to ectopic expression of Keap1, do not respond to brusatol treatment, demonstrating that brusatol-mediated sensitization to cisplatin is Nrf2 dependent. Moreover, a decrease in drug detoxification and impairment in drug removal may be the primary mechanisms by which brusatol enhances the efficacy of chemotherapeutic drugs. Taken together, these results clearly demonstrate the effectiveness of using brusatol to combat chemoresistance and suggest that brusatol can be developed into an adjuvant chemotherapeutic drug.

Naringenin-7-O-glucoside protects against doxorubicin-induced toxicity in H9c2 cardiomyocytes by induction of endogenous antioxidant enzymes.

Doxorubicin, a widely used chemotherapeutic agent, can give rise to severe cardiotoxicity that limits its clinical use by generation of reactive oxygen species (ROS) and apoptosis. Protection or alleviation of doxorubicin cardiotoxicity can be achieved by administration of natural phenolic compounds via activating endogenous defense systems and antiapoptosis. Naringenin-7-O-glucoside (NARG), isolated from Dracocephalum rupestre Hance, has been demonstrated to protect against cardiomyocyte apoptosis. In the present study, we investigated the effects of NARG on endogenous antioxidant enzymes against doxorubicin toxicity and the potential role of extracellular signal-regulated kinase (ERK) in regulation of NARG-induced Nrf2-dependent gene expression in H9c2 cardiomyocytes. The mRNA expression of glutamate-cysteine ligase modifier subunit (GCLM) and glutamate-cysteine ligase catalytic subunit (GCLC) was upregulated by NARG as detected by RT-PCR. NARG (10, 20, and 40microM) pretreatment increased NAD (P) H: quinone oxidoreductase (NQO1), ERK, and Nrf2 protein levels in cardiomyocytes as detected by Western blotting. These results suggest that NARG could prevent cardiomyocytes from doxorubicin-induced toxicity by induction of endogenous antioxidant enzymes via phosphorylation of ERK1/2 and nuclear translocation of Nrf2.

Protective effect of naringenin-7-O-glucoside against oxidative stress induced by doxorubicin in H9c2 cardiomyocytes.

Doxorubicin (DOX) is one of the most effective chemotherapeutic agents, but cardiotoxicity limits its clinical use. Although the mechanisms are not entirely understood, reactive oxygen species (ROS) and cardiomyocyte apoptosis appear to be involved in DOX cardiotoxicity. Protection or alleviation of DOX cardiotoxicity can be achieved by administration of natural phenolic compounds via activating endogenous defense systems and antiapoptosis. Naringenin-7-O-glucoside (NARG), isolated from Dracocephalum rupestre Hance, could protect from cardiomyocyte apoptosis and induce endogenous antioxidant enzymes against DOX toxicity, but the effects on intracellular ROS generation and cell membrane stability were not demonstrated. In the present study, we investigated the effects of NARG on H9c2 cell morphology, viability, lactate dehydrogenase (LDH) and creatine kinase (CK) leakage, glutathine peroxidase (GSH-Px) activity, intracellular Ca2+ concentration, and ROS generation. Compared with DOX alone treatment group, the morphological injury of the cells in groups treated by DOX plus NARG was alleviated, cell viability was increased, the amount of released LDH and CK was significantly decreased, the activity of GSH-Px was increased, the content of intracellular Ca2+ and ROS generation was lowered remarkably. These results suggest that NARG could prevent cardiomyocytes from DOX-induced toxicity by their property of stabilizing the cell membrane and reducing ROS generation.

Stereochemistry of flavonoidal alkaloids from Dracocephalum rupestre

Phytochemical studies on the aerial parts of Dracocephalum rupestre led to the isolation of four groups of flavonoidal alkaloids, dracocephins A-D. They were elucidated as conjugates of flavanone with pyrrolidin-2-one on the basis of extensive spectroscopic analysis. The two stereogenic centers rendered each group of the dracocephins as two pairs of enantiomers simultaneously. All of the sixteen isomers were separated successfully by chiral HPLC and their stereochemical features were determined by their CD data and single-crystal X-ray diffraction analysis of one stereoisomer. The additive relation of the chiroptical contributions resulting from the two stereogenic centers was generalized. The CD contribution of the chiral carbon in the pyrrolidin-2-one ring was proposed by subtraction of their respective contributions.

Eriodictyol-7-O-glucoside, a novel Nrf2 activator, confers protection against cisplatin-induced toxicity

Eriodictyol-7-O-glucoside, a flavonoid isolated from Dracocephalum rupestre, is among the most potent free radical scavenger. In the present study, we identified eriodictyol-7-O-glucoside as a novel nuclear factor E2-related factor 2 (Nrf2) activator using a high-throughput cellular screening method. This compound activated Nrf2 signaling pathway and was able to stabilize Nrf2 by delaying Nrf2 degradation, resulting in accumulation of Nrf2 protein and activation of the Nrf2-dependent protective response. Recent studies have suggested that activation of Nrf2 pathway would confer protection against cisplatin-induced toxicity. The protective role of eriodictyol-7-O-glucoside in cisplatin-induced toxicity was investigated in a human renal mesangial cell line, HRMC. Cotreatment of HRMC cells with eriodictyol-7-O-glucoside significantly improved cell survival under cisplatin exposure. These findings demonstrated the feasibility of using natural compounds targeting Nrf2 as a therapeutic approach to subvert the side effects of cisplatin in normal cells.

Dracotanosides A-D, spermidine glycosides from Dracocephalum tanguticum: structure and amide rotational barrier.

Four new spermidine glycosides, dracotanosides A-D (1-4), have been isolated from Dracocephalum tanguticum. These molecules represent the first spermidine glycosides from this plant genus. The structures, including absolute configurations, were determined by spectroscopic and chemical methods. The amide bond rotational barrier of aglycone 1a was calculated by density functional theory (DFT) computation.

The genus Litsea in traditional Chinese medicine: An ethnomedical, phytochemical and pharmacological review

ETHNOPHARMACOLOGICAL RELEVANCE The genus Litsea, mainly distributed in the tropical and subtropical regions, has been used in traditional and indigenous Chinese medicines for the treatment of diarrhea, stomachache, dyspepsia, gastroenteritis, diabetes, edema, cold, arthritis, asthma, pain, traumatic injury, etc. for a long history. The present review aims to provide a comprehensive summary on the ethnomedical uses, phytochemistry, and pharmacology of the Litsea species used in traditional Chinese medicine (TCM). Based on these data, evidences supporting their ethnopharmacological effectiveness are illustrated, and opportunities for the future research and development as well as the therapeutic potential of this genus are analyzed to highlight the gaps in our knowledge that deserves further investigation. MATERIAL AND METHODS Information on the Litsea species was collected via electronic search (using Pubmed, SciFinder, Google Scholar, Web of Science and CNKI) and a library search for articles published in peer-reviewed journals. Furthermore, information was also obtained from some local books on ethnopharmacology. RESULTS Twenty plants of the genus Litsea are found to be important traditional medicines in China, and have a long medicinal application for diarrhea, stomachache, dyspepsia, gastroenteritis, diabetes, edema, cold, arthritis, asthma, pain, traumatic injury, etc. Over 200 ingredients have been identified from these 20 Litsea species used in TCM, and flavonoids, terpenoids and alkaloids are considered as the characteristic and bioactive constituents. The crude extracts and the isolated metabolites of these medicinal plants have exhibited some in vitro and in vivo pharmacological effects, including antimicrobial, hepatoprotection, anti-inflammatory, antiasthmatic, immunomodulation, anti-diabetic, anticholelithogenic, as well as function on central nervous system, etc. CONCLUSIONS The extensive literature survey reveals Litsea species to be a group of important medicinal plants used for the ethnomedical treatment of gastrointestinal diseases, diabetes, inflammatory disorders, and microbial infection in TCM. Pharmacological investigations have supported the use of some Litsea species in the traditional medicines. In addition, further researches targeting individual ingredients responsible for the pharmacological effects, as well as their mechanisms of action are necessary. The outcome of these studies will further support the therapeutic potential of the genus Litsea, and provide convincing evidences to its future clinical applications in modern medicine.

Plant Extracts of the Family Lauraceae: A Potential Resource for Chemopreventive Agents that Activate the Nuclear Factor-Erythroid 2-Related Factor 2/Antioxidant Response Element Pathway

Cells and tissues counteract insults from exogenous or endogenous carcinogens through the expression of genes encoding antioxidants and phase II detoxifying enzymes regulated by antioxidant response element promoter regions. Nuclear factor-erythroid 2-related factor 2 plays a key role in regulating the antioxidant response elements-target gene expression. Hence, the Nrf2/ARE pathway represents a vital cellular defense mechanism against damage caused by oxidative stress and xenobiotics, and is recognized as a potential molecular target for discovering chemopreventive agents. Using a stable antioxidant response element luciferase reporter cell line derived from human breast cancer MDA-MB-231 cells combined with a 96-well high-throughput screening system, we have identified a series of plant extracts from the family Lauraceae that harbor Nrf2-inducing effects. These extracts, including Litsea garrettii (ZK-08), Cinnamomum chartophyllum (ZK-02), C. mollifolium (ZK-04), C. camphora var. linaloolifera (ZK-05), and C. burmannii (ZK-10), promoted nuclear translocation of Nrf2, enhanced protein expression of Nrf2 and its target genes, and augmented intracellular glutathione levels. Cytoprotective activity of these extracts against two electrophilic toxicants, sodium arsenite and H2O2, was investigated. Treatment of human bronchial epithelial cells with extracts of ZK-02, ZK-05, and ZK-10 significantly improved cell survival in response to sodium arsenite and H2O2, while ZK-08 showed a protective effect against only H2O2. Importantly, their protective effects against insults from both sodium arsenite and H2O2 were Nrf2-dependent. Therefore, our data provide evidence that the selected plants from the family Lauraceae are potential sources for chemopreventive agents targeting the Nrf2/ARE pathway.

Simultaneous determination of nine major active compounds in Dracocephalum rupestre by HPLC

A new method was developed for the simultaneous determination of nine major constituents in Dracocephalum rupestre, including 5,7-dihydroxychromone (1), eriodictyol-7-O-beta-d-glucoside (2), luteolin-7-O-beta-d-glucoside (3), naringenin-7-O-beta-d-glucoside (4), apigenin-7-O-beta-d-glucoside (5), eriodictyol (6), luteolin (7), naringenin (8) and apigenin (9). The quantitative determination was conducted by reversed phase high-performance liquid chromatography with photodiode array detector (LC-PDA). Separation was performed on an Agilent Eclipse XDB-C(18) column (150 mm x 4.6 mm i.d., 5 microm) with gradient elution of acetonitrile and 0.5% aqueous acetic acid. The components were identified by retention time, ultraviolet (UV) spectra and quantified by LC-PDA at 260 nm. All calibration curves showed good linearity (r(2)>0.999) within test ranges. The reproducibility was evaluated by intra- and inter-day assays and R.S.D. values were less than 3.0%. The recoveries were between 95.15 and 104.45%. The limits of detection (LOD) ranged from 0.002 to 0.422 microg/ml and limits of quantification (LOQ) ranged from 0.005 to 1.208 microg/ml, respectively. The identity of the peaks was further confirmed by high-performance liquid chromatography with triple-quadrupole mass spectrometry system coupled with electrospray ionization (ESI) interface. The developed method was applied to the determination of nine constituents in 14 samples of D. rupestre collected at various harvesting times. Most compounds accumulated at much higher amounts in about June-July. The satisfactory results indicated that the developed method was readily utilized as a quality control method for D. rupestre.

Apoptosis induction of dehydrobruceine B on two kinds of human lung cancer cell lines through mitochondrial-dependent pathway.

BACKGROUND Brucea javanica is an effective traditional medicine listed in Chinese Pharmacopoeia. In China, the seed oil of B. javanica has long been used as commercially available drug for the treatment of tumor in clinic. Dehydrobruceine B (DHB) is a quassinoid isolated from B. javanica. PURPOSE The aim of the present study is to investigate the apoptotic effects induced by DHB in human lung cancer A549 and NCI-H292 cells. The involvement of a mitochondria-mediated intrinsic pathway in the pro-apoptotic action of DHB was also investigated. MATERIAL AND METHODS Cell viability was determined by MTT assay. Cell cycle and apoptosis were assessed by flow cytometry analysis. Mitochondrial membrane potential (MMP) was examined through JC-1 staining. The protein translocation in cells was examined by immunostaining. The expression levels of proteins which are closely related to mitochondria-mediated apoptosis pathway were measured by immunoblot analysis. RESULTS Treatment with DHB decreased cell viability, induced apoptosis and blocked cell cycle at S phase. DHB-induced apoptosis was found to be mediated through mitochondrial intrinsic pathway, evidenced by the loss of MMP, the release of cytochrome c into cytosol, and the cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP). CONCLUSION DHB triggers apoptosis in A549 and NCI-H292 cells via mitochondrial pathway, making it a promising candidate as a therapeutic agent for lung carcinoma.