Boningmycin induces AMPK-mediated endoplasmic reticulum-associated degradation of PD-L1 protein in human cancer cells.

Anti-PD-1/PD-L1 monoclonal antibodies have displayed remarkable clinical benefits and revolutionized the treatment of multiple tumor types, but the low response rates and immune-related adverse events limit their application, which promoting the development of small molecule agents to improve the efficacy of PD-1/PD-L1 blockade therapy. Boningmycin (BON), a new small molecule belonging to bleomycin (BLM) family, exhibits potent anticancer activity in vitro and in vivo, as well as negligible lung toxicity, thereby can be an alternative of BLM. However, understandings about the anticancer mechanism of BLM-related compounds are extremely rare, it remains unclear if they affect PD-L1 level in a manner similar to that of other antitumor drugs. In this study, we discover that BON significantly reduces PD-L1 protein level in NCI-H460 and HT-1080 cells. Meanwhile, BON decreases the protein level of PD-L1 in a tumor xenograft model of NCI-H460 cells. Nevertheless, the mRNA level is not influenced after BON exposure. Furthermore, BON-induced PD-L1 reduction is proteasome- dependent. By using specific inhibitors and RNA interference technology, we confirm that the decline of PD-L1 protein by BON is mediated by AMPK-activated endoplasmic reticulum-associated degradation pathway, which is like to the action of metformin. Last but not the least, BON has synergism on gefitinib in vitro and in vivo. In conclusion, it is the first report demonstrating that BON decreases PD-L1 protein level through AMPK-mediated endoplasmic reticulum-associated degradation pathway. These findings will benefit the clinical transformation of BON and aid in the elucidation of molecular mechanism of BLM-related compounds.

Novel patient-derived xenograft and cell line models for therapeutic screening in NF2-associated schwannoma.

Treatment of schwannomas in patients with neurofibromatosis type 2 (NF2) is extremely unsatisfactory, and innovative therapeutic approaches are urgently needed. However, the lack of clinically relevant NF2-associated schwannoma models has severely hampered drug discovery in this rare disease. Here we report the first establishment and characterization of patient-derived xenograft (PDX) and cell line models of NF2-associated schwannoma, which recapitulates the morphological and histopathological features of patient tumors, retain patient NF2 mutations, and maintain gene expression profiles resembling patient tumor profiles with the preservation of multiple key signaling pathways commonly dysregulated in human schwannomas. Using gene expression profiling, we identified elevated PI3K/AKT/mTOR networks in human NF2-associated vestibular schwannomas. Using high-throughput screening of 157 inhibitors targeting the PI3K/AKT/mTOR pathways in vitro, we identified a dozen inhibitors (such as BEZ235, LY2090314, and AZD8055) with significant growth-suppressive effects. Interestingly, we observed that three cell lines displayed differential therapeutic responses to PI3K/AKT/mTOR inhibitors. Furthermore, we demonstrated that two orally bioavailable inhibitors, AZD8055 and PQR309, suppressed NF2-associated schwannoma growth both in vitro and in vivo. In conclusion, our novel patient-derived models of NF2-associated schwannoma closely mimic the phenotypes and genotypes of patient tumors, making them reliable preclinical tools for testing novel personalized therapies. © 2022 The Pathological Society of Great Britain and Ireland.

Decrease of ABCB1 protein expression and increase of G1 phase arrest induced by oleanolic acid in human multidrug-resistant cancer cells.

Oleanolic acid (OA) is a natural compound that can be found in a number of edible and medicinal plants and confers diverse biological actions. However, the direct target of OA in human tumor cells remains poorly understood, preventing its application in clinical and health settings. A previous study revealed that overexpression of caveolin-1 in human leukemia HL-60 cells can increase its sensitivity to OA. The present study aimed to investigate the effects of OA on the doxorubicin-resistant human breast cancer MCF-7 cell line (MCF-7/DOX), harringtonine-resistant human leukemia HL-60 cells (HL-60/HAR) and their corresponding parental cell lines. Western blotting was performed to measure protein expression levels, whilst Cell Counting Kit-8 (CCK-8) assays, cell cycle analysis (by flow cytometry) and apoptosis assays (with Annexin V/PI staining) were used to assess drug sensitivity. CCK-8 assay results suggested that MCF-7/DOX cells, which overexpress the caveolin-1 protein, have similar OA susceptibility to their parent line. In addition, sensitivity of MCF-7/DOX cells to OA was not augmented by knocking down caveolin-1 using RNA interference. HL-60/HAR cells exhibited a four-fold increased sensitivity to OA compared with that in their parental HL-60 cells according to CCK-8 assay. Both of the resistant cell lines exhibited higher numbers of cells at G1 phase arrest compared with those in their parent lines, as measured via flow cytometry. Treatment of both MCF-7 cell lines with 100 µM OA for 48 h induced apoptosis, with increased effects observed in resistant cells. However, no PARP-1 or caspase-3 cleavage was observed, with some positive Annexin V staining found after HL-60/HAR cells were treated with OA, suggesting that cell death occurred via non-classical apoptosis or through other cell death pathways. It was found that OA was not a substrate of ATP-binding cassette subfamily B member 1 (ABCB1) in drug-resistant cells, as indicated by the accumulation of rhodamine 123 assessed using flow cytometry. However, protein expression of ABCB1 in both of the resistant cell lines was significantly decreased after treatment with OA in a concentration-dependent manner. Collectively, these results suggest that OA could reduce ABCB1 protein expression and induce G1 phase arrest in multidrug-resistant cancer cells. These findings highlight the potential of OA for cancer therapy.

Distinct characteristics of dasatinib-induced pyroptosis in gasdermin E-expressing human lung cancer A549 cells and neuroblastoma SH-SY5Y cells.

Dasatinib, a multikinase inhibitor, is used in the treatment of chronic myeloid leukemia and was developed to overcome imatinib resistance. Its mechanism of action involves the induction of apoptosis, autophagy and necroptosis. However, it remains unclear whether dasatinib can induce pyroptosis. In the present study, gasdermin E (GSDME)-expressing SH-SY5Y and A549 cells were chosen for investigation. Typical pyroptotic features, such as cleavage of GSDME protein, leakage of lactate dehydrogenase and large bubbled morphology, were observed in both cell lines after exposure to dasatinib. The generation of GSDME fragments was inhibited by specific caspase-3 inhibitor zDEVD in SH-SY5Y cells and pan-caspase inhibitor zVAD in A549 cells. Moreover, distinct characteristics of pyroptosis were observed in A549 cells, which occurred only with a high percentage of Annexin V/propidium iodide double-stained cells and low level of GSDME protein cleavage. The sensitivity of A549 cells to dasatinib is significantly reduced by increasing cell numbers. The elevation of GSDMD and GSDME protein levels was induced by low concentrations of dasatinib, which was not influenced by the reduction of p53 protein with RNA interference. In conclusion, to the best of our knowledge, this is the first study to report that dasatinib can induce pyroptosis in tumor cells and increase the protein levels of GSDMD and GSDME in a p53-independent manner.

Design, Synthesis, and Bioactivity of Cyclic Lipopeptide Antibiotics with Varied Polarity, Hydrophobicity, and Positive Charge Distribution.

Twenty-three polymyxin analogs with variations at nine amino acid positions were synthesized and assessed for antimicrobial activity and renal cytotoxicity. Compounds M2, 14, S2, and 16 (MIC = 0.125-4 µg/mL) had similar or stronger activities against susceptible and drug-resistant strains of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii compared to polymyxin B (MIC = 1-2 µg/mL). Most synthesized compounds (50% cytotoxic concentration, CC50 ≥ 200 µg/mL) exhibited lower cytotoxicity than polymyxin B (CC50 = 99 ± 6 µg/mL). Polymyxin S2 showed high plasma stability in vitro and strong efficacy in a mouse systemic infection model (ED50 = 0.9 mg/kg) against NDM-1-producing Klebsiella pneumoniae, suggesting that it is a potential candidate for drug development. The activity and cytotoxicity results indicated that the amino acids at positions 2, 3, 6, and 7 might be replaced. Effects on activity and cytotoxicity linked to changes in the number of positively charged amino acids varied among different cyclopeptide skeletons, but the underlying mechanisms are unknown.

Comparison of Clinical, Histopathological, and Genomic Features Between Malignant Peripheral Nerve Sheath Tumors and Cellular Schwannomas of the Eighth Cranial Nerve: A Case Series.

BACKGROUND: Malignant peripheral nerve sheath tumors (MPNSTs) and cellular schwannomas (CSs) of the eighth cranial nerve are exceedingly rare. The purpose of the present study was to evaluate clinical and genetic characterization of these rare tumors. METHODS: The clinical and radiological features were analyzed retrospectively. The histopathological characteristics were assessed by hematoxylin and eosin staining and immunohistochemistry. Genomic abnormalities were evaluated using array comparative genomic hybridization. RESULTS: Of the 1287 surgeries for vestibular schwannomas from 2014 to 2017, 2 were for MPNSTs and 5 were for CSs. The mean age at diagnosis was older for patients with MPNSTs (57.0 ± 4.2 years) than that of patients with CS (35.8 ± 9.4 years; P = 0.03). Two patients with MPNST died of tumor recurrence. None of the patients with CS died. The 2-year overall and progression-free survival of patients with MPNSTs were worse than those for patients with CSs (overall survival, 50.0% ± 35.4% vs. 100%, P = 0.027; progression-free survival, 0% vs. 100%; P = 0.012). The Ki-67 index for the MPNSTs (29.0% ± 3.5%) was greater than that for the CSs (10.3% ± 3.1%; P = 0.001). The common alterations in MPNSTs mainly included gains of chromosomes 7p, 8p, 9q, 12, and 17 and loss of heterozygosity of 1p, 6 and 9p. The common alterations in CSs included gain of 4p16.3, loss of heterozygosity of 2p15-14, and 22q11.1-13.3. CONCLUSIONS: To the best of our knowledge, the present study is the first high-resolution genomic analysis of MPNSTs and CSs of the eighth cranial nerve and has shown a significant difference that might be more accurate to distinguish between these 2 types of rare tumors.

Nrf2 mediates the resistance of human A549 and HepG2 cancer cells to boningmycin, a new antitumor antibiotic, in vitro through regulation of glutathione levels.

NF-E2-related factor 2 (Nrf2) is a transcription factor and a pivotal factor in the induction of the cell defense system. Recent reports show that Nrf2 plays critical roles in tumor heterogeneity and drug resistance. In the present study we investigated whether and how Nrf2 mediated the resistance of human cancer cells to boningmycin (BON), a new antitumor antibiotic of the bleomycin family. We showed that in the expression levels of Nrf2 in human non-small lung cancer A549 cells were much higher than those in human hepatoblastoma HepG2 cells, and their resistance to BON was opposite to Nrf2 expression (the IC50values of BON in A549 cells and HepG2 cells were 5.97 and 0.61 µmol/L, respectively). Similar results were observed with the anticancer agent cisdiamminedichloroplatinum (DDP), which was used as a positive control. In A549 cells, Nrf2 mRNA knockdown significantly increased their susceptibilities to BON and DDP. An enhanced resistance to BON and DDP was observed in HepG2 cells after overexpression of the wild-type Nrf2 protein. Treatment with a specific Nrf2 inhibitor, luteolin, significantly sensitized A549 cells to BON and DDP and increased BON- or DDP-induced apoptosis. The total levels of glutathione (GSH), the final product of the Nrf2 signaling pathway, were much higher in A549 cells than those in HepG2 cells. Supplementation of GSH in HepG2 cells significantly decreased their susceptibility to BON and DDP, wheras depleting GSH with the specific inhibitor L-buthionine sulfoximine in A549 cells significantly increased their susceptibility to BON and DDP. Our results demonstrate that Nrf2 mediates the resistance to BON through regulating glutathione levels in A549 cells and HepG2 cells.

Deregulation of the Hippo Pathway Promotes Tumor Cell Proliferation Through YAP Activity in Human Sporadic Vestibular Schwannoma.

OBJECTIVE: Vestibular schwannomas (VSs) can cause serious neurological defects including hearing loss and facial paralysis. The aim of this study is to identify whether Hippo signaling could be a potential targetable pathway for clinical treatment in VSs. METHODS: Gene expression profiling was performed in 10 sporadic VSs and 4 normal nerves to identify aberrant genes expression of the Hippo pathway. Western blotting and immunohistochemical staining were used to examine the expression of Hippo core components in 20 VS samples. Neurofibromatosis type 2 (NF2) gene sequencing was also performed in all tumors using sanger sequencing. Verteporfin, inhibitor of yes-associated protein (YAP)-TEA domain family member, was used to assess the effect of proliferation inhibition in human primary VS cells and RT4-D6P2T cell line. RESULTS: We found 51 differentially expressed genes of the Hippo pathway between VSs and healthy controls. Unsupervised analysis identified the 2 molecular variants that significantly related with distinct NF2 mutation status. The phosphorylation levels of large tumor suppressor 1 and YAP were significantly decreased in NF2-mutated VSs compared with wild-type VSs and normal nerves. Immunohistochemical staining showed that increased nuclear YAP expression in VSs was positively correlated with high Ki-67 index and low Merlin expression. Verteporfin reduced viability of primary VS cells and RT4-D6P2T cells. CONCLUSIONS: Our findings implicate that deregulation of the Hippo pathway as a molecular mechanism of pathogenesis in human VSs, and suggest inhibition of this pathway as a potential treatment strategy.

Lower endogenous p53 levels and degradation of AKT protein contribute to potent suppression of the new antibiotic Xiakemycin A on tumor cells.

Xiakemycin A (XKA), a new pyranonaphthoquinone antibiotic, is isolated from the fermentation broth of Streptomyces sp. CC8-201. It exerts potent suppression of cell proliferation on some types of tumor cells. In the present study, its underlying mechanism on tumor cells has been investigated. In contrast to the specific AKT inhibitor triciribine hydrate, XKA demonstrated a weak inhibition of the AKT kinase activity in vitro. Knockdown of AKT protein levels reduced XKA-inhibitory action on prostate carcinoma PC-3 cells. Degradation of AKT protein was markedly observed in the XKA-treated PC-3 cells in comparison with triciribine hydrate treatment. There was no typical apoptosis induced by XKA in PC-3 cells. The propidium iodide-stained cells increased concentration-dependently when the cells were treated with XKA. Degradation of apoptosis-related proteins, such as p53 and PARP-1, was also detected in the XKA-treated PC-3 cells. Knockdown of p53 protein levels potentiated XKA action on non-small lung cancer A549 cells. Collectively, the mechanism of XKA potent inhibition was due to degradation of AKT protein and low endogenous p53 levels. As a leading compound, new derivatives based on XKA will be developed to precisely treat tumor cells which have high AKT and low p53 protein levels.

Synthesis and Bioactivity Investigation of the Individual Components of Cyclic Lipopeptide Antibiotics.

In this paper, 26 natural polymyxin components and a new derivative S2 were synthesized, and their differences in efficacy and toxicity have been investigated. Almost all of the synthesized components showed strong activity against both susceptible and resistant strains of E. coli, K. pneumoniae, P. aeruginosa, and A. baumannii. The toxicities were obviously different between the components. Only some of the components were tested for toxicity in vivo. Compounds E2, E2-Val, A2, M2, D2, and S2 showed obviously lower renal cytotoxicity and acute toxicity than polymyxins B and E. The in vivo nephrotoxicity of E2, M2, and S2 was similar to that of polymyxin E. Compound S2, with four positive charges, was especially interesting as it possessed both increased efficacy and decreased toxicity. The SAR and toxicity studies indicated that further structural modification could concentrate on polymyxin S. The results also indicated that S2 could be a new drug candidate.

Neuroprotective effects of chloroform and aqueous fractions of noni juice against t-Butyl hydroperoxide-induced oxidative damage in SH-SY5Y cells.

Oxidative stress is more likely to cause damage to neuronal cells and mediates some neurodegenerative disorders. It is well known that natural antioxidants can prevent oxidative stress damage and become a potential therapeutic strategy. Noni juice obtained from the fruit of the tree Morinda citrifolia, as a folk medicine, has been used for over two thousand years. In the current study, the neuroprotective effect and mechanism of noni juice extracts against tert-Butyl hydroperoxide (TBHP)-induced SH-SY5Y cell damage were investigated. The results demonstrated that chloroform fraction (CF) and aqueous fraction (AF) of noni juice protected SH-SY5Y cells against TBHP-induced oxidative stress and the associated apoptosis effectively. CF and AF treatment significantly weakened the TBHP-induced cytotoxicity, reactive oxygen species generation, mitochondrial membrane depolarization, and apoptotic features. CF and AF restored cellular antioxidant enzyme activity; upregulated expression of heme oxygenase-1, catalase, and superoxide dismutase-1; and increased the nuclear accumulation of nuclear factor-erythroid 2 related factor 2 (Nrf2). The antioxidant and neuroprotection potential of CF may account for its high total phenolic and flavonoid content, while AF may be rich in polysaccharides. These results suggest that CF and AF exhibit antioxidant defense through the upregulation of Nrf2 along with endogenous antioxidants and reduce apoptosis via inhibiting the mitochondrial pathway to protect SH-SY5Y cells damaged by TBHP. CF and AF might be developed as agents for neurodegeneration prevention or therapy.

Involvement of bleomycin hydrolase and poly(ADP-ribose) polymerase-1 in Ubc9-mediated resistance to chemotherapy agents.

Ubiquitin-conjugating protein 9 (Ubc9), the sole enzyme for sumoylation, plays critical roles in many physiological functions, such as DNA damage repair and genome integrity. Its overexpression led to poor prognosis and drug resistance in tumor chemotherapy. However, the underlying mechanism by which Ubc9 promotes tumor progress and influences the susceptibility to antitumor agents remains elusive. In this study, we used nine antitumor agents with distinct actions to explore Ubc9-mediated resistance in human breast carcinoma MCF-7 cells. Increase of susceptibility, respectively, to boningmycin, hydroxycamptothecine, cis-dichlorodiamineplatinum, 5-fluorouracil, vepeside and gemcitabine, but not for doxorubicin, vincristine and norcantharidin, was observed after the knockdown of Ubc9 protein level with RNA interference. Reduction of bleomycin hydrolase and poly(ADP-ribose) polymerase-1 levels after knockdown of Ubc9 suggests their contribution to Ubc9-mediated drug resistance. This is the first report on the sensitivity to hydroxycamptothecine, cis-dichlorodiamineplatinum and gemcitabine that increased after knockdown of bleomycin hydrolase at protein level. In conclusion, Ubc9 plays different roles of action in antitumor agents in chemotherapy. The process requires bleomycin hydrolase and poly(ADP-ribose) polymerase-1. The results are beneficial to deeply understanding of Ubc9 functions and for precise prediction of chemotherapy outcomes in tumors.

Biodiesel Production: Utilization of Loofah Sponge to Immobilize Rhizopus chinensis CGMCC #3.0232 Cells as a Whole-Cell Biocatalyst.

Rhizopus chinensis cells immobilized on loofah (Luffa cylindrica) sponges were used to produce biodiesel via the transesterification of soybean oil. In whole-cell immobilization, loofah sponge is considered to be a superior alternative to conventional biomass carriers because of its biodegradable and renewable properties. During cell cultivation, Rhizopus chinensis mycelia can spontaneously and firmly adhere to the surface of loofah sponge particles. The optimal conditions for processing 9.65 g soybean oil at 40°C and 180 rpm using a 3:1 methanol-to-oil molar ratio were found to be 8% cell addition and 3-10% water content (depending on the oil's weight). Under optimal conditions, an over 90% methyl ester yield was achieved after the first reaction batch. The operational stability of immobilized Rhizopus chinensis cells was assayed utilizing a 1:1 methanol-to-oil molar ratio, thus resulting in a 16.5-fold increase in half-life when compared with immobilized cells of the widely studied Rhizopus oryzae. These results suggest that transesterification of vegetable oil using Rhizopus chinensis whole cells immobilized onto loofah sponge is an effective approach for biodiesel production.

[Tumor heterogeneity and drug resistance of targeted antitumor agents].

The popular application of targeted antitumor agents has greatly improved the efficacies of tumor therapy. However, some patients develop drug resistance after the administration with them for six to twelve months, leading to the failure of treatment. The cause of it is mainly due to tumor heterogeneity. The genesis of tumor heterogeneity is closely associated with tumor stem cells, genetic instability, cell competition and stochastic events. There are a lot of mechanisms involved in the drug resistance to the targeted agents. Tumor heterogeneity and drug resistance are great challenges for precision oncology and are taken account for the process of research and development of new antitumor agents.

Potentiation of resveratrol-induced apoptosis by matrine in human hepatoma HepG2 cells.

Resveratrol, a natural polyphenolic phytochemical, has received considerable attention due to its potential chemopreventive and chemotherapeutic properties. In the present study, we first evaluated the growth-inhibitory effect of resveratrol on HepG2 cells and explored the underlying molecular mechanisms. Resveratrol inhibited proliferation and induced apoptosis in HepG2 cells via activation of caspase-9 and caspase-3, upregulation of the Bax/Bcl-2 ratio and induction of p53 expression. Cell cycle analysis demonstrated that resveratrol arrested cell cycle progression in the G1 and S phase. We further focused on the combination of matrine, a natural component extracted from the traditional Chinese medical herb Sophora flavescens Ait., as a mechanism to potentiate the growth-inhibitory effect of resveratrol on HepG2 cells. Both MTT and colony formation assay results indicated that the combined treatment of resveratrol and matrine exhibited a synergistic antiproliferative effect. In addition, resveratrol-induced apoptosis was significantly enhanced by matrine, which could be attributed to activation of caspase-3 and caspase-9, downregulation of survivin, induction of reactive oxygen species (ROS) generation and disruption of mitochondria membrane potential (Δψm). Our findings suggest that the combination treatment of resveratrol and matrine is a promising novel anticancer strategy for liver cancer; it also provides new insights into the mechanisms of combined therapy.

[Mechanisms of yanshu injection for overcoming multidrug resistance in breast carcinoma MCF-7 cells: an experimental research].

OBJECTIVE: To investigate the mechanism of Yanshu Injection (YI) for overcoming multidrug resistance in breast carcinoma MCF-7 cells. METHODS: Human breast carcinoma MCF-7 cells and doxorubicin-resistant MCF-7/DOX cells were treated with YI. Its inhibition on the cell proliferation was detected by MTT assay. The fluorescence intensity of doxorubicin was detected by flow cytometry. The expression of apoptosis related protein and P-glycoprotein were examined by immunoblotting after treated by YI. RESULTS: The inhibitory action of YI on MCF-7/DOX cells was similar to that of MCF-7 cells, indicating no cross resistance (P > 0.05). 1/16 YI could obviously induce the apoptosis of MCF-7 cells and DOX cells. 1/256 YI +5 nmol/L doxorubicin and 1/128 YI +5 nmol/L doxorubicin could reduce the survival rate of MCF-7/ DOX resistant cells from 86.8% to 74.6% (P < 0.05) and 71.6% (P < 0.01) respectively, showing obvious synergistic effect. Besides, the accumulation of doxorubicin was increased after treated by YI in the MCF-7/ DOX cells. Results of immunoblotting indicated that reduction of P-glycoprotein expression was detected in MCF-7/DOX cells after exposure to YI. CONCLUSION: YI could overcome the multidrug resistance in breast carcinoma MCF-7 cells possibly through reducing the expression of P-glycoprotein.

Puerarin inhibits angiotensin II-induced cardiac hypertrophy via the redox-sensitive ERK1/2, p38 and NF-κB pathways.

AIM: To investigate the effects of puerarin (Pue), an isoflavone derived from Kudzu roots, on angiotensin II (Ang II)-induced hypertrophy of cardiomyocytes in vivo and in vitro. METHODS: C57BL/6J mice were infused with Ang II and treated with Pue (100 mg·kg(-1)·d(-1), po) for 15 d. After the treatment, systolic blood pressure (SBP) and left ventricular wall thickness were assessed. The ratios of heart weight to body weight (HW/BW) and left ventricular weight to body weight (LVW/BW) were determined, and heart morphometry was assessed. Expression of fetal-type genes (ANP, BNP and ß-MHC) in left ventricles was measured using semi-quantitative RT-PCR. Mouse primary cardiomyocytes were treated with Pue (50, 100, 200 µmol/L), then exposed to Ang II (1 µmol/L). ROS level was examined with flow cytometry, the binding activity of NF-κB was determined using EMSA. Western blot was used to measure the levels of ERK1/2, p38 and NF-κB pathway proteins. [(3)H]leucine incorporation was used to measure the rate of protein synthesis. RESULTS: Oral administration of Pue significantly suppressed Ang II-induced increases in the myocyte surface area, HW/BW, LVW/BW, SBP and left ventricular wall thickness. Furthermore, Pue significantly suppressed Ang II-induced increases in ANP, BNP and ß-MHC expression in the left ventricles in vivo. Treatment of cardiomyocytes with Pue (50-500 µmol/L) did not affect the viability of cardiomyocytes in vitro. Pretreatment of cardiomyocytes with Pue dose-dependently inhibited Ang II-induced increases in ROS production, NF-κB binding activity, protein synthesis and cell breadth. Furthermore, pretreatment with Pue significantly suppressed Ang II-induced activation of ERK1/2, p38 and the NF-κB pathway proteins and the expression of ANP and ß-MHC in cardiomyocytes. The positive drug valsartan exerted similar effects on Ang II-induced cardiac hypertrophy in vivo and in vitro. CONCLUSION: Pue attenuates Ang II-induced cardiac hypertrophy by inhibiting activation of the redox-sensitive ERK1/2, p38 and the NF-κB pathways.