Targeting Nrf2 to treat thyroid cancer.

Oxidative stress (OS) is recognized as a contributing factor in the development and progression of thyroid cancer. Nuclear factor erythroid 2-related factor 2 (Nrf2) is a pivotal transcription factor involved in against OS generated by excessive reactive oxygen species (ROS). It governs the expression of a wide array of genes implicated in detoxification and antioxidant pathways. However, studies have demonstrated that the sustained activation of Nrf2 can contribute to tumor progression and drug resistance in cancers. The expression of Nrf2 was notably elevated in papillary thyroid cancer tissues compared to normal tissues, indicating that Nrf2 may play an oncogenic role in the development of papillary thyroid cancer. Nrf2 and its downstream targets are involved in the progression of thyroid cancer by impacting the prognosis and ferroptosis. Furthermore, the inhibition of Nrf2 can increase the sensitivity of target therapy in thyroid cancer. Therefore, Nrf2 appears to be a potential therapeutic target for the treatment of thyroid cancer. This review summarized current data on Nrf2 expression in thyroid cancer, discussed the function of Nrf2 in thyroid cancer, and analyzed various strategies to inhibit Nrf2.

Pharmacological effects and mechanisms of YiYiFuZi powder in chronic heart disease revealed by metabolomics and network pharmacology.

Introduction: YiYiFuZi powder (YYFZ) is a classical formula in Chinese medicine, which is commonly used clinically for the treatment of Chronic Heart Disease (CHD), but it's pharmacological effects and mechanism of action are currently unclear. Methods: An adriamycin-induced CHD model rat was established to evaluate the pharmacological effects of YYFZ on CHD by the results of inflammatory factor level, histopathology and echocardiography. Metabolomic studies were performed on rat plasma using UPLC-Q-TOF/MS to screen biomarkers and enrich metabolic pathways; network pharmacology analysis was also performed to obtain the potential targets and pathways of YYFZ for the treatment of CHD. Results: The results showed that YYFZ significantly reduced the levels of TNF-α and BNP in the serum of rats, alleviated the disorder of cardiomyocyte arrangement and inflammatory cell infiltration, and improved the cardiac function of rats with CHD. The metabolomic analysis identified a total of 19 metabolites, related to amino acid metabolism, fatty acid metabolism, and other metabolic pathways. Network pharmacology showed that YYFZ acts through PI3K/Akt signaling pathway, MAPK signaling pathway and Ras signaling pathway. Discussion: YYFZ treatment of CHD modulates blood metabolic pattern and several protein phosphorylation cascades but importance specific changes for therapeutic effect require further studies.

Integrating Metabolomics and Network Pharmacology to Explore the Mechanism of Tongmai Yangxin Pills in Ameliorating Doxorubicin-Induced Cardiotoxicity.

Doxorubicin (DOX) is a broad-spectrum chemotherapeutic drug used in clinical treatment of malignant tumors. It has a high anticancer activity but also high cardiotoxicity. The aim of this study was to explore the mechanism of Tongmai Yangxin pills (TMYXPs) in ameliorating DOX-induced cardiotoxicity through integrated metabolomics and network pharmacology. In this study, first, an ultrahigh-performance liquid chromatography-quadrupole-time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) metabonomics strategy was established to obtain metabolite information and potential biomarkers were determined after data processing. Second, network pharmacological analysis was used to evaluate the active components, drug-disease targets, and key pathways of TMYXPs to alleviate DOX-induced cardiotoxicity. Targets from the network pharmacology analysis and metabolites from plasma metabolomics were jointly analyzed to select crucial metabolic pathways. Finally, the related proteins were verified by integrating the above results and the possible mechanism of TMYXPs to alleviate DOX-induced cardiotoxicity was studied. After metabolomics data processing, 17 different metabolites were screened, and it was found that TMYXPs played a role in myocardial protection mainly by affecting the tricarboxylic acid (TCA) cycle of myocardial cells. A total of 71 targets and 20 related pathways were screened out with network pharmacological analysis. Based on the combined analysis of 71 targets and different metabolites, TMYXPs probably played a role in myocardial protection through regulating upstream proteins of the insulin signaling pathway, MAPK signaling pathway, and p53 signaling pathway, as well as the regulation of metabolites related to energy metabolism. They then further affected the downstream Bax/Bcl-2-Cyt c-caspase-9 axis, inhibiting the myocardial cell apoptosis signaling pathway. The results of this study may contribute to the clinical application of TMYXPs in DOX-induced cardiotoxicity.

A new strategy for the rapid identification and validation of direct toxicity targets of psoralen-induced hepatotoxicity.

The interaction between small-molecule compounds of traditional Chinese medicine and their direct targets is the molecular initiation event, which is the key factor for toxicity efficacy. Psoralen, an active component of Fructus Psoraleae, is toxic to the liver and has various pharmacological properties. Although the mechanism of psoralen-induced hepatotoxicity has been studied, the direct target of psoralen remains unclear. Thus, the aim of this study was to discover direct targets of psoralen. To this end, we initially used proteomics based on drug affinity responsive target stability (DARTS) technology to identify the direct targets of psoralen. Next, we used surface plasmon resonance (SPR) analysis and verified the affinity effect of the 'component-target protein'. This method combines molecular docking technology to explore binding sites between small molecules and proteins. SPR and molecular docking confirmed that psoralen and tyrosine-protein kinase ABL1 could be stably combined. Based on the above experimental results, ABL1 is a potential direct target of psoralen-induced hepatotoxicity. Finally, the targets Nrf2 and mTOR, which are closely related to the hepatotoxicity caused by psoralen, were predicted by integrating proteomics and network pharmacology. The direct target ABL1 is located upstream of Nrf2 and mTOR, Nrf2 can influence the expression of mTOR by affecting the level of reactive oxygen species. Immunofluorescence experiments and western blot results showed that psoralen could affect ROS levels and downstream Nrf2 and mTOR protein changes, whereas the ABL1 inhibitor imatinib and ABL1 agonist DPH could enhance or inhibit this effect. In summary, we speculated that when psoralen causes hepatotoxicity, it acts on the direct target ABL1, resulting in a decrease in Nrf2 expression, an increase in ROS levels and a reduction in mTOR expression, which may cause cell death. We developed a new strategy for predicting and validating the direct targets of psoralen. This strategy identified the toxic target, ABL1, and the potential toxic mechanism of psoralen.

Study on endocrine disruption effect of paclobutrazol and uniconazole on the thyroid of male and female rats based on lipidomics.

The present study investigated the effects of paclobutrazol and uniconazole on thyroid endocrine system in rats. Lipidomic analysis was performed to obtain the biomarkers of thyroid endocrine disruption induced by paclobutrazol and uniconazole. Network pharmacology was further used to discover potential targets of biomarkers related to drugs and diseases. After paclobutrazol and uniconazole administration, seven and four common biomarkers related to thyroid endocrine disruption for female and male rats were obtained, respectively. Paclobutrazol and uniconazole significantly increased the biomarker levels of PG (12:0/15:0), PS (14:0/16:0), PA (20:1/15:0) and PG (13:0/17:0) in both sexes of rats. Exposure to paclobutrazol additionally caused a significant decrease of PG (22:6/20:2), PE (24:1/18:1) and PE (24:0/18:0) in female rats, while an increase in male rats. Changes of the common biomarkers for paclobutrazol and uniconazole revealed similar endocrine disruption effect, which was higher in the females. Network pharmacology and KEGG pathway analysis indicated that the thyroid endocrine disrupting effects of paclobutrazol and uniconazole may be related to V-akt murine thymoma viral oncogene homolog (Akts), mitogen-activated protein kinase (MAPKs), epidermal growth factor receptor (EGFR), Insulin-like growth factor (IGF-1), IGF-IR and V-Raf murine sarcoma viral oncogene homolog B1 (BRAF). The results demonstrated that paclobutrazol and uniconazole could cause thyroid endocrine disorders in male and female rats, which were sex-specific, thus highlighting the importance of safe and effective application of these plant growth regulators.

A New Strategy for the Rapid Identification and Validation of the Direct Targets of Aconitine-Induced Cardiotoxicity.

BACKGROUND: The interaction of small molecules with direct targets constitutes the molecular initiation events of drug efficacy and toxicity. Aconitine, an active compound of the Aconitum species, has various pharmacological effects but is strongly toxic to the heart. The direct targets of aconitine-induced cardiotoxicity remain unclear. METHODS: We predicted the toxic targets of aconitine based on network pharmacology and followed a novel proteomic approach based on the "drug affinity responsive target stability" technology combined with LC-MS/MS to identify the direct targets of aconitine. The identified targets were analysed from the perspective of multilevel and multidimensional bioinformatics through a network integration method. The binding sites were investigated via molecular docking to explore the toxicity mechanism and predict the direct targets of aconitine. Finally, atomic force microscopy (AFM) imaging was performed to verify the affinity of aconitine to the direct targets. RESULTS: PTGS2, predicted by network pharmacology as a toxic target, encodes cyclooxygenase 2 (COX-2), which is closely related to myocardial injury. Furthermore, cytosolic phospholipase A2 (cPLA2) is the upstream signal protein of PTGS2, and it is a key enzyme in the metabolism of arachidonic acid during an inflammatory response. We determined cPLA2 as a direct target, and AFM imaging verified that aconitine could bind to cPLA2 well; thus, aconitine may cause the expression of PTGS2/COX-2 and release inflammatory factors, thereby promoting myocardial injury and dysfunction. CONCLUSION: We developed a complete set of methods to predict and verify the direct targets of aconitine, and cPLA2 was identified as one. Overall, the novel strategy provides new insights into the discovery of direct targets and the molecular mechanism of toxic components that are found in traditional Chinese medicine.

Exploration the Mechanism of Doxorubicin-Induced Heart Failure in Rats by Integration of Proteomics and Metabolomics Data.

Heart failure is a common systemic disease with high morbidity and mortality worldwide. Doxorubicin (DOX) is a commonly used anthracycline broad-spectrum antitumor antibiotic with strong antitumor effect and definite curative effect. However, cardiotoxicity is the adverse reaction of drug dose cumulative toxicity, but the mechanism is still unclear. In this study, proteomics and metabonomics techniques were used to analyze the tissue and plasma of DOX-induced heart failure (HF) in rats and to clarify the molecular mechanism of the harmful effects of DOX on cardiac metabolism and function in rats from a new point of view. The results showed that a total of 278 proteins with significant changes were identified by quantitative proteomic analysis, of which 118 proteins were significantly upregulated and 160 proteins were significantly downregulated in myocardial tissue. In the metabonomic analysis, 21 biomarkers such as L-octanoylcarnitine, alpha-ketoglutarate, glutamine, creatine, and sphingosine were detected. Correlation analysis showed that DOX-induced HF mainly affected phenylalanine, tyrosine, and tryptophan biosynthesis, D-glutamine and D-glutamate metabolism, phenylalanine metabolism, biosynthesis of unsaturated fatty acids, and other metabolic pathways, suggesting abnormal amino acid metabolism, fatty acid metabolism, and glycerol phospholipid metabolism. It is worth noting that we have found the key upstream target of DOX-induced HF, PTP1B, which inhibits the expression of HIF-1α by inhibiting the phosphorylation of IRS, leading to disorders of fatty acid metabolism and glycolysis, which together with the decrease of Nrf2, SOD, Cytc, and AK4 proteins lead to oxidative stress. Therefore, we think that PTP1B may play an important role in the development of heart failure induced by doxorubicin and can be used as a potential target for the treatment of heart failure.

A Proteomics- and Metabolomics-Based Study Revealed That Disorder of Palmitic Acid Metabolism by Aconitine Induces Cardiac Injury.

Currently, research on cardiac injury by aconitine focuses on its effect to directly interfere with the function of cardiac ion channels. Further, abnormal lipid metabolism could cause cardiac injury via inflammatory signaling pathway. In our preliminary study, we discovered that aconitine could alter the metabolism processes of various substances, including palmitic acid. Inspired by these studies, we investigated how elevation of palmitic acid by aconitine causes cardiac injury. Aconitine induced cardiac injury in rats (0.32 mg/kg, d = 7), and the cardiac injury was confirmed by electrocardiogram and serum biochemical study. The proteomic and metabolomic results showed that the palmitic acid level increases in heart tissue, and the NOD-like receptor (NLR) signaling pathway showed a strong effect of cardiac injury. The palmitic acid results in cell viability decline and activates NLR signaling in vitro. The shRNA-mediated knockdown of NLRP3 and NOD1/2 attenuates palmitic acid-induced inhibitory effect on cells and inhibited activation of the NLR signaling pathway. Collectively, this study reveals that aconitine provoked palmitic acid elevation could aggravate cardiac injury via the NLR signaling pathway. This study suggests that drug triggered disorder of the metabolism process could evoke cardiac injury and could propose a new strategy to study drug cardiac injury.

ASBT(SLC10A2): A promising target for treatment of diseases and drug discovery.

Bile acids has gradually become a new focus in various diseases, and ASBT as a transporter responsible for the reabsorption of ileal bile acids, is a key hinge associated to the bile acids-cholesterol balance and bile acids of enterohepatic circulation. The cumulative studies have also shown that ASBT is a promising target for treatment of liver, gallbladder, intestinal and metabolic diseases. This article briefly reviewed the process of bile acids enterohepatic circulation, as well as the regulations of ASBT expression, covering transcription factors, nuclear receptors and gut microbiota. In addition, the relationship between ASBT and various diseases were discussed in this paper. According to the structural classification of ASBT inhibitors, the research status of ASBT inhibitors and potential ASBT inhibitors of traditional Chinese medicine (such resveratrol, jatrorrhizine in Coptis chinensis) were summarized. This review provides a basis for the development of ASBT inhibitors and the treatment strategy of related diseases.

Analysis of biomarkers and metabolic pathways in patients with unstable angina based on ultra­high­performance liquid chromatography­quadrupole time­of­flight mass spectrometry.

Unstable angina (UA) is a coronary disease with a high mortality and morbidity worldwide. The present study aimed to use non­invasive techniques to identify urine biomarkers in patients with UA, so as to provide more information for the early diagnosis and treatment of the disease. Based on metabolomics, urine samples from 28 patients with UA and 28 healthy controls (HCs) were analyzed using ultra­high­performance liquid chromatography­quadrupole time­of­flight mass spectrometry (UPLC­Q­TOF/MS). A total of 16 significant biomarkers that could distinguish between patients with UA and HCs, including D­glucuronic acid, creatinine, succinic acid and N­acetylneuraminic acid, were identified. The major metabolic pathways associated with UA were subsequently analyzed by non­targeted metabolomics. The results demonstrated that amino acid and energy metabolism, fatty acid metabolism, purine metabolism and steroid hormone biosynthetic metabolism may serve important roles in UA. The results of the current study may provide a theoretical basis for the early diagnosis of UA and novel treatment strategies for clinicians. The trial was registered with the Chinese Clinical Trial Registration Center (registration no. ChiCTR­ROC­17013957) at Tianjin University of Traditional Chinese Medicine.

Isoproterenol Triggers ROS/P53/S100-A9 Positive Feedback to Aggravate Myocardial Damage Associated with Complement Activation.

Negative feelings caused by external stress can continually agonize adrenergic receptors via promoting catecholamine secretion, causing cardiovascular disease. This study examines the mechanism by which persistent ß-adrenergic receptor agonism induces myocardial injury. A rat model of cardiac injury was herein established using isoproterenol (5 mg/kg, continuous intraperitoneal injection for 3 days), and multiomics technology combined with metabolomics and proteomics was used to explore the mechanism by which persistent ß-adrenergic receptor agonism induces myocardial injury. The mechanism underlying this phenomenon was further verified at the cellular level. Isoproterenol-induced persistent ß-adrenergic receptor agonism promoted the release of reactive oxygen species, and P53, S100-A9, and complement 3 were shown to be involved in complement system activation pathways. Our data have demonstrated that isoproterenol could trigger ROS/P53/S100-A9 positive feedback to aggravate myocardial damage associated with complement activation.

Integrated Proteomics and Metabolomics Reveal the Mechanism of Nephrotoxicity Induced by Triptolide.

Triptolide (TP), the main active ingredient of Tripterygium wilfordii Hook F., has great potential in the treatment of autoimmune diseases. However, it has been found that the side effects of TP involve multiple organs and systems, of which the most serious side effects relate to the kidney. The mechanism of nephrotoxicity caused by TP requires further investigation. In the present study, we integrated proteomic and metabolomic methods to identify proteins and small molecule metabolites associated with TP-induced nephrotoxicity. There was a significant difference (p value <0.05) in the expression changes of 357 proteins for quantitative proteomics. In addition, high resolution metabolomic data showed significant changes in the levels of 9 metabolites, including hypoxanthine, PC(22:0/18:4), sphingosine, phenylalanine, etc. Finally, based on the Kyoto Encyclopaedia of Genes and Genomes (KEGG) database for network analysis, it was determined that the 7 differentially expressed proteins were highly correlated with these 9 metabolites. Enrichment analysis revealed that the metabolic pathways involved purine and pyrimidine metabolism, glycerol and phospholipid metabolism, sphingolipid metabolism, and amino acid metabolism. The key target proteins were verified by Western blot technology, and the mechanism of TP-induced nephrotoxicity was further elucidated to provide a basis for safe and rational application.

Integrative proteomics-metabolomics strategy reveals the mechanism of hepatotoxicity induced by Fructus Psoraleae.

Fructus Psoraleae (FP), one of the significant traditional Chinese medicines, has been reported to cause hepatotoxicity. However, the mechanism remains undetermined and the reported research is limited. In this study, a tandem mass tag (TMT)-based quantitative proteomics and metabolomics were used to reveal a more comprehensive effect caused by FP. The results showed that aqueous extract of FP can induce liver injury in rats. In total, 575 significantly changed proteins were identified by quantitative proteomics analysis, among which 352 proteins were significantly up-regulated and 223 proteins were significantly down-regulated in liver tissues. And we detected 14 biomarkers such as succinic acid, hypoxanthine, l-carnitine, phenylalanine, glutathione, and glycoursodeoxycholic acid. Correlation analysis of altered metabolites and proteins exhibited the aberrant regulation of metabolic pathways including bile secretion, glutathione metabolism, purine metabolism, glycerophospholipid metabolism, TCA cycle and pyruvate metabolism, which indicated the disorder of bile acid metabolism, oxidative stress, energy metabolism and immune system. Notably, the changed proteins including Cyp7a1, FXR, SHP, BSEP, Sult2a1, Nceh1 in bile acid metabolism may play an essential role in the hepatotoxicity induced by aqueous extract of FP. In conclusion, integrative proteomics and metabolomics provide the potential mechanism of hepatotoxicity induced by FP. SIGNIFICANCE: Fructus Psoraleae, a traditional Chinese medicine, is widely used in Asia for the treatment of osteoporosis and vitiligo. Recently, clinical and experimental reports reveal that FP can induce liver injury. However the mechanism of injury induced by FP is still unclear. In this study, we detected 352 significantly up-regulated proteins and 223 significantly down-regulated proteins in liver tissues by TMT-based quantitative proteomics. And 14 important metabolites were identified by metabolomics analysis. Through integrative analysis of the key metabolites and proteins, several metabolism pathways were selected, which implicated in bile acid metabolism, oxidative stress, energy metabolism, immune system. This is the first integrative study of proteomics and metabolomics for FP exposure, the finding clarified the potential mechanism of hepatotoxicity caused by FP and will promote rational use of FP in clinical application.

iTRAQ-based quantitative proteomics and target-fishing strategies reveal molecular signatures on vasodilation of Compound Danshen Dripping Pills.

Angina pectoris can be used as an early warning for coronary artery disease. Vasodilation is an important mechanism of angina pectoris. Traditional Chinese medicine - Compound Danshen Dripping Pill (CDDP) is widely used to improve the symptoms of cardiovascular diseases (CVDs). To investigate the influence of vasodilation effect and underlying mechanisms of CDDP, we determined the vasodilation effect of thoracic aorta ring on rat induced by norepinephrine (NE). Then targets-fishing method was used to predict the potential mechanism of CDDP on vasodilation, based on the structures of the main components. Then, iTRAQ-based quantitative proteomics analysis was used for verification of the candidate target proteins and pathways to illustrate the underlying mechanisms. Furthermore, the differentially expressed proteins in the enriched pathways were validated by western blotting. In this study, we found that CDDP could significantly inhibit NE induced aortic contraction tension, and the mechanism may be related to platelet activation, cGMP - PKG signaling pathway and vascular smooth muscle contraction. The method provides a new way to uncover the vasodilation mechanism of CDDP, as well as other multi-component herbal medicines.

Liquiritinapioside - A mineralocorticoid-like substance from liquorice.

Surface plasmon resonance (SPR) analysis of the main components of liquorice was performed and a novel strong mineralocorticoid receptor (MR) agonist, namely liquiritinapioside (LA), whose the binding constant was 1.093 × 10-5 M, was reported. As a supplement, LA has been further demonstrated to have a strong MR binding capacity through competitive binding experiments (the enrichment factor of LA was 10.22%). This study also validated the activity of LA on H9c2 cells. The expression of collagen I and the results of Masson staining were used to determine the ability of this substance to cause H9c2 cell fibrosis. Moreover, western blotting was used to verify the mechanism of compound-induced myocardial fibrosis. Overall, the attained results showed that LA could induce the activation of the TGF-ß1/p38 MAPK signalling pathway through the MR to induce H9c2 cell fibrosis.

Ginseng Compatibility Environment Attenuates Toxicity and Keeps Efficacy in Cor Pulmonale Treated by Fuzi Beimu Incompatibility Through the Coordinated Crosstalk of PKA and Epac Signaling Pathways.

Cor pulmonale is characterized by severe right ventricular dysfunction caused by lung disease, particularly chronic obstructive pulmonary disease, which can lead to pulmonary hypertension. Our previous study has demonstrated that Fuzi and Beimu compatibility (FBC), a traditional TCM compatibility taboo, improves lung function in early-stage of pulmonary hypertension through the synergistic action of ß-ARs signals. However, FBC increases cardiotoxicity with prolonged treatment and disease progression. Considering that the compatibility environment influences the exertion of the medicine, we selected ginseng for coordinating the compatibility environment to improve the security and extend the therapeutic time window of FBC. Monocrotaline-induced cor pulmonale rats were treated with FBC, ginseng, or ginseng combined with FBC (G/FBC). Then, the pulmonary and cardiac functions of the rats were examined to evaluate the toxicity and efficacy of the treatments. The crosstalk between PKA and Epac pathways was also studied. Results showed that G/FBC ameliorated lung function similar to or even better than FBC treatment did. Furthermore, G/FBC treatment attenuated FBC-induced cardiotoxicity, which significantly restored cardiac dysfunction and clearly decreased myocardial enzymes and apoptosis. The ßAR-Gs-PKA/CaMKII pathway was inhibited and the Epac1/ERK1/2 axis was activated in G/FBC group. These findings indicate that ginseng compatibility environment could improve pulmonary function and attenuate cardiotoxicity in cor pulmonale via the coordinated crosstalk of PKA and Epac pathways, implying that ginseng could be used to prevent detrimental cardiotoxicity in cor pulmonale treatment.

Quality markers based on biological activity: A new strategy for the quality control of traditional Chinese medicine.

BACKGROUND: The quality and quality evaluation system of traditional Chinese medicine (TCM) are crucial in the safety and effectiveness assessment of TCM. However, they are also the major bottlenecks that restrict the quality control of TCM. Given the nature of Chinese medicine and the limitations of science and technology, the quality evaluation of TCM involves a few difficulties. Therefore, scholars have conducted considerable amount of research on this topic and obtained promising results. Biological potency and biomarkers have been used to evaluate the quality of TCM. Previous studies provided new strategies and methods to establish a system on quality evaluation. PURPOSE: This review aims to provide a new strategy for the quality control of Chinese herbal medicine by combining biological potency and biomarkers based on biological effects. METHODS: We reviewed the quality evaluation system of Chinese herbal medicine, focusing on quality markers (Q-markers) based on biological effects and the application of these markers in the quality evaluation of Chinese herbal medicine. We also reviewed the factors affecting quality, the difficulties related to the quality evaluation system and the attempt of researchers to improve the quality control of TCM. RESULTS: We propose Q-biomarkers by integration of biological potency and biomarkers to evaluate the quality of TCM. The quality markers provided us significant insights in the process of definition. We further optimised the concept of Q-markers and summarised their definition and properties (including quantification, specificity and related to biological response) in accordance with the requirement of the quality evaluation of TCM. CONCLUSION: We propose the use of Q-biomarkers in vivo related to specific diseases as a new strategy for the quality evaluation of Chinese herbal medicine. The quality evaluation system of Q-biomarkers would provide a new perspective to standardise and globalise TCM.

Activation of p53 contributes to pseudolaric acid B-induced senescence in human lung cancer cells in vitro.

AIM: Pseudolaric acid B (PAB), a diterpene acid isolated from the root bark of Pseudolarix kaempferi Gordon, has shown to exert anti-tumor effects via inducing cell cycle arrest followed by apoptosis in several cancer cell lines. Here we reported that PAB induced a mitotic catastrophe in human lung cancer A549 cells, which resulted in senescence without apoptosis or necrosis. METHODS: Three human lung cancer cell lines (A549, H460 and H1299 cells) were examined. Cell growth inhibition was assessed with MTT assay. Cell cycle distribution was determined using a flow cytometer. Cell nuclear morphology was observed under a fluorescence microscope. Senescent cells were detected using SA-ß-Gal staining. Apoptotic and senescent protein expression was examined using Western blot analysis. The expression of p53 and p21 in the cells was downregulated by siRNAs. RESULTS: Treatment with PAB (5-80 µmol/L) inhibited the growth of A549 cells in dose- and time-dependent manners. Prolonged treatment with PAB (20 µmol/L) caused G2/M arrest at day 1 followed by mitotic catastrophe from day 2, which eventually resulted in cell senescence between days 3 and 4 without cell death (apoptosis or necrosis). Knockdown of p53 expression with siRNA significantly suppressed PAB-induced senescence in A549 cells (p53 wild). Furthermore, PAB-induced senescence was also observed in human lung cancer H460 cells (p53 wild), but not in human lung cancer H1299 cells (p53 null). CONCLUSION: The anti-tumor action of PAB against human lung cancer A549 cells in vitro involves the induction of senescence through activation of the p53 pathway.

ATM-p53 pathway causes G2/M arrest, but represses apoptosis in pseudolaric acid B-treated HeLa cells.

Pseudolaric acid B (PAB) is a diterpene acid, isolated from the root and trunk bark of Pseudolarix kaempferi Gordon (Pinaceae). Previous studies demonstrated that PAB induced G2/M arrest and apoptosis in several cancer cell lines, but the relationship between G2/M arrest and apoptosis is still unclear. We examined the relevant signaling pathways for human cervical carcinoma HeLa cells treated with 1 µM PAB. Intriguingly, we found that activation of ATM-p53 signaling pathway by the treatment with 1 µM PAB played a protective role for the subsequent apoptosis. Although the treatment with 1 µM PAB up-regulated the expression of cyclin B1 and p-Histone 3 (mitotic markers) at 12 h, the expression decreased at 24 and 36 h along with the up-down expression of mitotic markers. The expressions of p-ATM and p-p53 that were involved in G2/M arrest increased at 12h after treatment with PAB. However, a prolonged treatment with PAB (longer than 24 h) caused cell apoptosis. When the cells were arrested in G1 or S phase by the treatment with serum starvation, cytosine ß-D-arabinofuranoside (Ara-C) or hydroxyurea (Hu), the apoptotic ratio induced by PAB decreased.

Plasma metabolic profiling analysis of nephrotoxicity induced by acyclovir using metabonomics coupled with multivariate data analysis.

Acyclovir (ACV) is an antiviral agent. However, its use is limited by adverse side effect, particularly by its nephrotoxicity. Metabonomics technology can provide essential information on the metabolic profiles of biofluids and organs upon drug administration. Therefore, in this study, mass spectrometry-based metabonomics coupled with multivariate data analysis was used to identify the plasma metabolites and metabolic pathways related to nephrotoxicity caused by intraperitoneal injection of low (50mg/kg) and high (100mg/kg) doses of acyclovir. Sixteen biomarkers were identified by metabonomics and nephrotoxicity results revealed the dose-dependent effect of acyclovir on kidney tissues. The present study showed that the top four metabolic pathways interrupted by acyclovir included the metabolisms of arachidonic acid, tryptophan, arginine and proline, and glycerophospholipid. This research proves the established metabonomic approach can provide information on changes in metabolites and metabolic pathways, which can be applied to in-depth research on the mechanism of acyclovir-induced kidney injury.