Yu-Chiang Hung

Functional proteomic and structural insights into molecular targets related to the growth inhibitory effect of tanshinone IIA on HeLa cells.

Certain antitumor agents have recently been extracted from the roots of Salvia miltiorrhiza Bunge. The diterpene derivative, tanshinone IIA, possesses cytotoxic activity against several human carcinoma cell lines. It also inhibits invasion and metastasis of cancer cells. In the present study, we isolated tanshinone IIA from S. miltiorrhiza, and it exhibited strong growth inhibition against human cervical cancer cells in dose‐ and time‐dependent manners with a 50% cell growth inhibition value of 2.5u2009μg/mL (8.49u2009μM). Flow cytometric analysis of cell cycle progression revealed that G2/M arrest was initiated after a 24u2009h exposure to the drug. It also resulted in DNA fragmentation and degradation of poly (ADP‐ribose) polymerase indicating that tanshinone IIA may be a potential antitumor agent. Furthermore, we performed a comprehensive proteomic analysis to survey global protein changes induced by tanshinone IIA treatment on HeLa cells. Significant changes in the levels of cytoskeleton proteins as well as stress‐associated proteins were observed. Immunoblot analysis and immunofluorescence staining were used to confirm the levels of protein expression. Overexpression of the vimentin rescued these tanshinone IIA‐induced events. Computational docking methods indicated that tanshinone IIA could stably bind to the β‐subunit of the microtubule protein. An interaction network analysis of these 12 proteins using MetaCore™ software suggested that tanshinone IIA treatment regulated the expressions of proteins involved in apoptotic processes, spindle assembly, and p53 activation, including vimentin, Maspin, α‐ and β‐tubulin, and GRP75. Taken together, our results suggest that tanshinone IIA strongly inhibited the growth of cervical cancer cells through interfering in the process of microtubule assembly, leading to G2/M phase arrest and sequent apoptosis. The success of this large‐scale effort was assessed by a bioinformatics analysis of proteins through predictions of protein domains and possible functional roles. The possible contributions of these proteins to the cytotoxicity of tanshinone IIA provide potential opportunities for the development of cancer therapeutics.

Functional proteomics reveal the effect of Salvia miltiorrhiza aqueous extract against vascular atherosclerotic lesions.

Salvia miltiorrhiza is a Chinese herb widely used for cardiovascular disorder regimens, yet little is known about the cellular mechanisms that contribute to attenuated growth of smooth muscle cells (SMCs) under oxidative stress such as homocysteine (Hcy) treatment. As anticipated, a low dose (0.015 mg/mL) of S.miltiorrhiza aqueous extract (SMAE) significantly inhibited (>60%) the growth of a rat smooth muscle cell line (A10) under Hcy stimulation and the intracellular reactive oxygen species (ROS) concentration obviously decreased after SMAE treatment in terms of reducing p47(phox) translocation and increasing catalase activity. Signaling profile suggests that SMAE inhibited Hcy-induced A10 cell growth via the PKC/MAPK-dependent pathway. Two-dimensional electrophoresis (2-DE) coupled with mass spectrometry revealed statistically significant changes in the intensity of 14 proteins in response to Hcy and Hcy/SMAE. Meanwhile, SMAE attenuated carbonyl-modification of specific cytoskeleton and chaperone proteins leading to cell type transformation. Moreover, a network analysis using MetaCore shed more light on the molecular basis associated with SMAE efficacy. SMAE exerts its protective effect through the scavenging of ROS and subsequent modulation of protein carbonylation to inhibit cell proliferation. These signature networks and functional proteomics highlighted herein may facilitate the evaluation of potential therapeutic targets and elucidate novel mechanisms through which protein functions can be regulated by the redox status.

Proteomic screening of antioxidant effects exhibited by Radix Salvia miltiorrhiza aqueous extract in cultured rat aortic smooth muscle cells under homocysteine treatment.

AIM OF THE STUDYnStill little is known about the cellular mechanisms that contribute to the attenuated proliferation of aortic smooth muscle cells under the influence of the oxidative stress factors such as homocysteine (Hcy). Thus, we aimed to evaluate whether Salvia miltiorrhiza Bunge (Labiatae), a Chinese medicinal herb widely used in folk medicine for therapy of variety of human cardiovascular disorders would modulate this Hcy promoted growth effect in model animal aortic cells system.nnnMATERIALS AND METHODSnThe Salvia miltiorrhiza roots aqueous extract (SMAE) containing 3,4-dihydroxybenzoic acid, 3,4-dihydroxyphenyl lactic acid and salvianolic acid B, as confirmed by narrow-bore HPLC analyses with binary gradient elution was used in variable concentrations for the treatment of the rat aortic smooth muscle A10 cells under Hcy stimulation. Two-dimensional electrophoresis (2-DE) coupled with MALDI-TOF mass spectrometry was applied for the elucidation of protein changes characterizing the response of the rat A10 cells into the Hcy-induced oxidative stress.nnnRESULTSnThis study showed that a low dose (0.015 mg/mL) of the SMAE significantly inhibited growth (>60%, p<0.05) of the Hcy stimulated rat A10 cells. In addition, concentration of intracellular reactive oxygen species (ROS) obviously decreased in the rat A10 cells after its incubation with SMAE in terms of catalase increasing activity. Next, marked down-regulation of protein kinase C beta-1 (PKC beta-1) and phosphorylated mitogen-activated protein kinase (p-MAPK) expression suggest that observed inhibitory effect of the polyphenol-rich SMAE on the Hcy-induced growth of rat A10 cells was realized via the PKC/p44/42 MAPK-dependent pathway. The intensity changes of 10 protein spots in response of the rat A10 cells to the Hcy-induced oxidative damage as alpha-4-tropomyosin, vimentin, F1F0-ATP synthase (beta subunit), glucose regulated protein 75 (GRP75), actin (fragment), prohibitin, capping protein, plakoglobin, endoplasmic reticulum protein (ERp29), and peptidylprolyl isomerase A (PPIase A), were detected with statistical significance (p<0.05). Meanwhile, it was showed that used here SMAE resist carbonylation of specific cytoskeleton and chaperone proteins as vimentin, alpha-4-tropomyosin and GRP75, respectively, leading to phenotype transformations in the rat A10 cells.nnnCONCLUSIONnThese data suggest that applied here SMAE exerts its protective effect through circulating ROS suppression and subsequent modulation of protein carbonylation in rat aortic smooth muscle A10 cells. Redox-proteomics protocol highlighted in this study may be applicable in facilitating the assessing potential novel molecular therapeutic targets to reduce cardiovascular risk related with elevated Hcy levels in various human populations and elucidating new mechanisms through which protein functions can be regulated by the redox status with the use of naturally occurring antioxidants.

Proteomic analysis reveals tanshinone IIA enhances apoptosis of advanced cervix carcinoma CaSki cells through mitochondria intrinsic and endoplasmic reticulum stress pathways

Cervix cancer is the second most common cancer among women worldwide, whereas paclitaxel, the first line chemotherapeutic drug used to treat cervical cancer, shows low chemosensitivity on the advanced cervical cancer cell line. Tanshinone IIA (Tan IIA) exhibited strong growth inhibitory effect on CaSki cells (IC50 = 5.51 μM) through promoting caspase cascades with concomitant upregulating the phosphorylation of p38 and JNK signaling. Comprehensive proteomics revealed the global protein changes and the network analysis implied that Tan IIA treatment would activate ER stress pathways that finally lead to apoptotic cell death. Moreover, ER stress inhibitor could alleviate Tan IIA caused cell growth inhibition and ameliorate C/EBP‐homologous protein as well as apoptosis signal‐regulating kinase 1 mediated cell death. The therapeutic interventions targeting the mitochondrial‐related apoptosis and ER stress responses might be promising strategies to conquer paclitaxel resistance.

Adjuvant Chinese Herbal Products for Preventing Ischemic Stroke in Patients with Atrial Fibrillation.

Objective Chinese herbal products (CHPs) are widely used for atrial fibrillation (AF) in Taiwan. We investigated the effect of adjuvant CHPs in preventing ischemic stroke in patients with AF. Methods Taiwanese patients in the Health Insurance Database newly diagnosed with AF during 2000–2011 were enrolled. Medication treatment with/without CHPs was administered within 7 days after the AF diagnosis. The clinical endpoint was an ischemic stroke. The Chi-square test, Fisher’s exact test, and Student t test were used to examine differences between the traditional Chinese medicine (TCM) and non-TCM cohorts. Cox proportional hazard regression was used to assess the risk for ischemic stroke between two cohorts. Results Three hundred and eleven patients underwent TCM treatment and 1715 patients did not. Compared to non-TCM users, TCM users had a lower incidence of stroke (12.59% vs. 1.93%, respectively) and lower risk of stroke [CHA2DS2-VASc score = 0–2 (hazard ratio = 0.20; 95% confidence interval = 0.06–0.65)]. Compared to non-TCM users, the stroke risk was significantly lower in TCM users with AF who were female or younger than 65 years, but not in males, people more than 65 years old, or people with comorbidities. Compared to TCM users, non-TCM users who received conventional treatment had a higher ischemic stroke risk. The risk for AF-related hospitalization was significantly lower in TCM users (0.64%) than in non-TCM users (38.1%). Conclusions Users of TCM with AF have a lower risk of new-onset ischemic stroke. Therefore, adjuvant CHP therapy may have a protective effect and may be used in AF patients to prevent ischemic stroke.

Polyphenols and Oxidative Stress in Atherosclerosis-Related Ischemic Heart Disease and Stroke

Good nutrition could maintain health and life. Polyphenols are common nutrient mainly derived from fruits, vegetables, tea, coffee, cocoa, mushrooms, beverages, and traditional medicinal herbs. They are potential substances against oxidative-related diseases, for example, cardiovascular disease, specifically, atherosclerosis-related ischemic heart disease and stroke, which are health and economic problems recognized worldwide. In this study, we reviewed the risk factors for atherosclerosis, including hypertension, diabetes mellitus, hyperlipidemia, obesity, and cigarette smoking as well as the antioxidative activity of polyphenols, which could prevent the pathology of atherosclerosis, including endothelial dysfunction, low-density lipoprotein oxidation, vascular smooth muscle cell proliferation, inflammatory process by monocytes, macrophages or T lymphocytes, and platelet aggregation. The strong radical-scavenging properties of polyphenols would exhibit antioxidative and anti-inflammation effects. Polyphenols reduce ROS production by inhibiting oxidases, reducing the production of superoxide, inhibiting OxLDL formation, suppressing VSMC proliferation and migration, reducing platelet aggregation, and improving mitochondrial oxidative stress. Polyphenol consumption also inhibits the development of hypertension, diabetes mellitus, hyperlipidemia, and obesity. Despite the numerous in vivo and in vitro studies, more advanced clinical trials are necessary to confirm the efficacy of polyphenols in the treatment of atherosclerosis-related vascular diseases.

Roles of Reactive Oxygen Species in Anticancer Therapy with Salvia miltiorrhiza Bunge.

Cancer is a leading cause of death worldwide. We aim to provide a systematic review about the roles of reactive oxygen species (ROS) in anticancer therapy with Salvia miltiorrhiza Bunge (Danshen). Danshen, including its lipophilic and hydrophilic constituents, is potentially beneficial for treating various cancers. The mechanisms of ROS-related anticancer effects of Danshen vary depending on the specific type of cancer cells involved. Danshen may enhance TNF-α-induced apoptosis, upregulate caspase-3, caspase-8, caspase-9, endoplasmic reticulum stress, P21, P53, Bax/Bcl-2, DR5, and AMP-activated protein kinase, or activate the p38/JNK, mitogen-activated protein kinase, and FasL signaling pathways. Conversely, Danshen may downregulate human telomerase reverse transcriptase mRNA, telomerase, survivin, vascular endothelial growth factor/vascular endothelial growth factor receptor 2, CD31, NF-κB, Erk1/2, matrix metalloproteinases, microtubule assembly, and receptor tyrosine kinases including epidermal growth factor receptors, HER2, and P-glycoprotein and inhibit the PI3K/Akt/mTOR or estrogen receptor signaling pathways. Therefore, Danshen may inhibit cancer cells proliferation through antioxidation on tumor initiation and induce apoptosis or autophagy through ROS generation on tumor progression, tumor promotion, and tumor metastasis. Based on the available evidence regarding its anticancer properties, this review provides new insights for further anticancer research or clinical trials with Danshen.

A preliminary randomised controlled study of short-term Antrodia cinnamomea treatment combined with chemotherapy for patients with advanced cancer

BackgroundAntrodia cinnamomea (AC) is a popular medicinal mushroom in Taiwan that has been widely used for treatment of various cancers. Few clinical studies have reported its application and efficiency in therapeutic chemotherapy strategies. We performed a double-blind, randomized clinical study to investigate whether AC given for 30xa0days had acceptable safety and efficacy in advanced cancer patients receiving chemotherapy.MethodsPatients with advanced and/or metastatic adenocarcinoma, performance status (PS) 0–2, and adequate organ function who had previously been treated with standard chemotherapy were randomly assigned to receive routine chemotherapy regimens with AC (20xa0ml twice daily) orally for 30xa0days or placebo. The primary endpoint was 6-month overall survival (OS); the secondary endpoints were disease control rate (DCR), quality of life (QoL), adverse event (AE), and biochemical features within 30xa0days of treatment.ResultsFrom August 2010 to July 2012, 37 subjects with gastric, lung, liver, breast, and colorectal cancer (17 in the AC group, 20 in the placebo group) were enrolled in the study. Disease progression was the primary cause of death in 4 (33.3xa0%) AC and 8 (66.7xa0%) placebo recipients. Mean OSs were 5.4xa0months for the AC group and 5.0xa0months for the placebo group (pu2009=u20090.340), and the DCRs were 41.2 and 55xa0%, respectively (pu2009=u20090.33). Most hematologic, liver, or kidney functions did not differ significantly between the two groups, but platelet counts were lower in the AC group than in the placebo group (pu2009=u20090.02). QoL assessments were similar in the two groups, except that the AC group showed significant improvements in quality of sleep (pu2009=u20090.04).ConclusionsAlthough we found a lower mortality rate and longer mean OS in the AC group than in the control group, A. cinnamomea combined with chemotherapy was not shown to improve the outcome of advanced cancer patients, possibly due to the small sample size. In fact, the combination may present a potential risk of lowered platelet counts. Adequately powered clinical trials will be necessary to address this question.Trial registrationClinicalTrials.gov NCT01287286.

Prescription pattern of Chinese herbal products for heart failure in Taiwan: A population-based study

BACKGROUNDnCertain Chinese herbal products (CHPs) may protect against the progression of heart failure (HF). However, there is a lack of research regarding the use of CHPs in patients with HF. The aims of this study were to analyze CHPs usage patterns in patients with HF and to identify the frequency and combination of CHPs most commonly used for HF.nnnMETHODSnThis retrospective, nationwide, population-based cohort study was conducted using a randomly sampled cohort of one million patients selected from the National Health Insurance Research Database (NHIRD) for the years 2000-2010 in Taiwan. CHP use and the top ten most frequently prescribed formulae and single herbs for treating HF were assessed, including total formulae number and average and frequency of prescriptions. Demographic characteristics, including sex and age at diagnosis of HF, were examined, together with existing comorbidities.nnnRESULTSnThe cohort included 19,988 newly diagnosed AD patients, who were given CHP treatment for HF between 2000 and 2010. Among them, female patients (53.3%) and those over 65years old (63.9%) were more likely to use CM. After adjusting for demographic factors, HF patients suffering from coronary artery disease (CAD) were more likely to seek traditional Chinese medicine (TCM) treatment than those with non-TCM users (57.6% vs. 52.6%). Zhi-Gan-Cao-Tang (4.07%) and Danshen (5.13%) were the most frequent formula CHP and single CHP prescribed by TCM practitioners for treating HF, respectively.nnnCONCLUSIONnMost people with HF who consumed CHPs used CHPs to supplement Yang-Qi, nourish the Ying-blood, and strengthen the heart spirit as complementary medicines to relieve HF-related symptoms, in addition to using standard anti-HF treatments. Further large-scale, randomized clinical trials are warranted in order to determine the effectiveness and safety of these herbal medicines.

Improved medical expenditure and survival with integration of traditional Chinese medicine treatment in patients with heart failure: A nationwide population-based cohort study

Background No previous studies have evaluated the effects of traditional Chinese medicine (TCM) treatment on patients with heart failure (HF). Hence, in this study, we determined whether TCM treatment affects the healthcare burden and survival of HF patients. Methods Samples were retrieved from the registry of catastrophic illness patients of the Taiwan National Health Insurance Research Database (NHIRD). Based on a frequency (1:1) matched case-control design, patients with HF between 2000 and 2010 were designated as cases (TCM users) and controls (non-TCM users). TCM treatment for patients with HF was analyzed. Results Among these patients, 312 used TCM for HF treatment and exhibited significantly increased 5-year survival (p < .0001), with multivariate adjustment, compared with those without TCM use. Mean outpatient clinic visits at 1 year and 5 years after HF diagnosis were higher in TCM users, and accumulated medical costs were lower than in non-TCM users at 1 year. The hospitalization cost at 1-year follow-up was lower for TCM users than for non-TCM users. We found that, compared with non-TCM users, TCM users had an 86% reduction in risk of mortality in the compensated group, and a 68% reduction in the decompensated group receiving TCM treatment (aHR 0.32, 95% CI 0.20–0.52). The hazard ratio (HR) of Chinese herbal medicine (CHM) users with HF was significantly lower than that of non-users (aHR 0.24, 95% CI 0.16–0.35). We also analyzed the most commonly used herbal products as well as the HRs associated with their use, thus providing future research avenues. Conclusions This nationwide retrospective cohort study finds that combined therapy with TCM may improve survival in HF patients. This study also suggests that TCM may be used as an integral element of HF interventions on health care costs.