Increased Phosphorylation Of P38 Research Articles

Sera from women with different metabolic and menopause states differentially regulate cell viability and Akt activation in a breast cancer in-vitro model.

Obesity is associated with an increased incidence and aggressiveness of breast cancer and is estimated to increment the development of this tumor by 50 to 86%. These associations are driven, in part, by changes in the serum molecules. Epidemiological studies have reported that Metformin reduces the incidence of obesity-associated cancer, probably by regulating the metabolic state. In this study, we evaluated in a breast cancer in-vitro model the activation of the IR-β/Akt/p70S6K pathway by exposure to human sera with different metabolic and hormonal characteristics. Furthermore, we evaluated the effect of brief Metformin treatment on sera of obese postmenopausal women and its impact on Akt and NF-κB activation. We demonstrated that MCF-7 cells represent a robust cellular model to differentiate Akt pathway activation influenced by the stimulation with sera from obese women, resulting in increased cell viability rates compared to cells stimulated with sera from normal-weight women. In particular, stimulation with sera from postmenopausal obese women showed an increase in the phosphorylation of IR-β and Akt proteins. These effects were reversed after exposure of MCF-7 cells to sera from postmenopausal obese women with insulin resistance with Metformin treatment. Whereas sera from women without insulin resistance affected NF-κB regulation. We further demonstrated that sera from post-Metformin obese women induced an increase in p38 phosphorylation, independent of insulin resistance. Our results suggest a possible mechanism in which obesity-mediated serum molecules could enhance the development of luminal A-breast cancer by increasing Akt activation. Further, we provided evidence that the phenomenon was reversed by Metformin treatment in a subgroup of women.

Abstract P164: Identification of the p53 negative feedback loop as a target for enhancing selinexor activity in neuroblastoma

Abstract Neuroblastoma (NB) is the most common extra-cranial pediatric solid tumor and accounts for 15% of cancer-related deaths in children, highlighting an unmet need for novel therapies. Selinexor is a small molecule inhibitor of XPO1. XPO1 shuffles cargo proteins with a nuclear export sequence from the nucleus to the cytosol, many of which are essential for cancer growth and cell maintenance. XPO1 also has a prognostic significance in patients with NB since those with high XPO1 levels have an inferior event-free (P = 1.2e-5, Bonferroni P = 5.4e-3) and overall survival (P = 4.5e-7, Bonferroni P = 2.1e-4) compared to patients with low XPO1 expression. To characterize the global transcriptomic consequences of XPO1 inhibition, we performed RNA-sequencing in the NB cell line IMR-5 where we performed a knockdown of XPO1 or treated cells with selinexor. Conducting a gene set enrichment analysis, we found genes encoding MYCN targets were significantly downregulated by selinexor (normalized enrichment score [NES] = -2.7; nominal P = <0.001) or XPO1 knockdown (NES = 2.2; nominal P = <0.001). Genes associated with the p53 pathway were significantly enriched (NES = 2.7; nominal P = <0.001 [selinexor treatment]; NES = 2.2; nominal P = <0.001 [XPO1knockdown]). Given the large number of XPO1-targets, we applied an advanced tandem mass tag (TMT)-liquid chromatography (LC)/LC-MS/MS based pipeline for deep whole-cell proteomic and phospho-proteomic analyses to interrogate unknown mechanisms of selinexor’s action. We found that selinexor induced its cytotoxic effects in NB through the nuclear accumulation of p53. Drug sensitivity assays with selinexor against seven human and one murine NB cell lines yielded good (median EC50=28.7nM) and intermediate responses (median EC50=133.5nM) with all intermediate responders harboring a TP53 mutation while good responders had TP53 wild type status. Further, phosphoproteomic analysis showed that nuclear accumulation of p53 led to an increase in p53 phosphorylation at site S315 marking p53 for MDM2-mediated ubiquitination. Since this phosphorylation step is undertaken predominantly by aurora kinase A (AURKA), we used a clinically available AURKA inhibitor, alisertib, in combination with selinexor and found synergistic killing activity in vitro and enhanced growth inhibition in two orthotopic xenograft mouse models. These findings suggest a potential therapeutic benefit using selinexor and alisertib to synergistically increase p53-mediated cytotoxicity of high-risk NB. Given the known toxicity profiles and potential therapeutic benefits, these findings support a clinical trial of selinexor and alisertib in children with high-risk NB. Citation Format: Rosa Nguyen, Hong Wang, Ming Sun, Dong Geun Lee, Junmin Peng, Carol J. Thiele. Identification of the p53 negative feedback loop as a target for enhancing selinexor activity in neuroblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P164.

Harmine Hydrochloride Mediates the Induction of G2/M Cell Cycle Arrest in Breast Cancer Cells by Regulating the MAPKs and AKT/FOXO3a Signaling Pathways.

Breast cancer (BC) is one of the most common causes of death among women worldwide. Recently, interest in novel approaches for BC has increased by developing new drugs derived from natural products with reduced side effects. This study aimed to treat BC cells with harmine hydrochloride (HMH) to identify its anticancer effects and mechanisms. HMH treatment suppressed cell growth, migration, invasion, and colony formation in MCF-7 and MDA-MB-231 cells, regardless of the hormone signaling. It also reduced the phosphorylation of PI3K, AKT, and mTOR and increased FOXO3a expression. Additionally, HMH treatment increased p38 phosphorylation in MCF-7 cells and activated c-Jun N-terminal kinase (JNK) phosphorylation in MDA-MB-231 cells in a dose-dependent manner, where activated p38 and JNK increased FOXO3a expression. Activated FOXO3a increased the expression of p53, p21, and their downstream proteins, including p-cdc25, p-cdc2, and cyclin B1, to induce G2/M cell cycle arrest. Furthermore, HMH inhibited the PI3K/AKT/mTOR pathway by significantly reducing p-AKT expression in combination with LY294002, an AKT inhibitor. These results indicate that mitogen-activated protein kinases (MAPKs) and AKT/FOXO3a signaling pathways mediate the induction of cell cycle arrest following HMH treatment. Therefore, HMH could be a potential active compound for anticancer bioactivity in BC cells.

CircCORO1C promotes the proliferation and metastasis of hepatocellular carcinoma by enhancing the expression of PD-L1 through NF-κB pathway.

BackgroundCircular RNA (circRNA) affects the occurrence and development of human cancers, but the specific mechanism of hepatocellular carcinoma (HCC) has not yet been fully understood.MethodsCircRNAs were determined by human circRNA array analysis and quantitative reverse transcription polymerase chain reaction (qRT‐PCR). Cell viability, migration, invasion, and other indicators were used for cell function analysis. Knockdown and overexpression techniques were used to explore the mechanism of circCORO1C in the occurrence and development of HCC by RNA sequencing, qRT‐PCR, western blot, and other methods.ResultsAmong the thousands of circRNAs, 1238 circRNAs were significantly changed. As for the top 10 upregulated circRNAs, the expression of circRNAs, hsa_circ_0036412, hsa_circ_0036411, hsa_circ_0028071, hsa_circ_0036409, hsa_circ_0000437, hsa_circ_0021427, hsa_circ_0097182, hsa_circ_0028067, hsa_circ_0006852, and hsa_circ_0003620 were significantly increased. In regard to the top 10 downregulated circRNAs, the expression of hsa_circ_0123629, hsa_circ_0096121, hsa_circ_0038932, hsa‐circRNA3310‐44, hsa_circ_0045746, hsa_circ_0016836, hsa‐circRNA10899‐9, hsa_circ_0050116, hsa_circ_0035543, and hsa_circ_0092118 decreased significantly. About these circRNAs, the downregulation of hsa_circ_0006852 (circCORO1C) can inhibit the tumorigenesis of HCC cells in vivo and in vitro, and the overexpression of circCORO1C can enhance the proliferation and metastasis ability of HCC cells. Mechanistically, circCORO1C activated the NF‐κB signaling pathway, increased P65 phosphorylation and upregulation of c‐Myc and COX‐2, leading to increased PD‐L1 expression.ConclusionCircCORO1C upregulates c‐Myc and COX‐2 through NF‐κB signaling pathway, leading to the upregulation of PD‐L1, which jointly promotes the development of HCC, suggesting that circCORO1C is a promising biomarker and therapeutic target for HCC.

Protein kinases regulate hyperactivated motility of human sperm.

Protein kinases regulate hyperactivated motility of human sperm.

WIP1 Inhibition by GSK2830371 Potentiates HDM201 through Enhanced p53 Phosphorylation and Activation in Liver Adenocarcinoma Cells.

Simple SummaryPatients with advanced intrahepatic cholangiocarcinoma (iCCA) have a very poor prognosis, and no targeted therapy is approved for advanced iCCA. A therapeutic strategy for wild-type p53 cancers is the reactivation of p53 by inhibition of its the negative regulators, MDM2, and WIP1. In the present study, we used HDM201 (an MDM2-p53 binding antagonist) to increase p53 stabilization and upregulate the expression of downstream targets (p21 and MDM2) in RBE and SK-Hep-1 liver adenocarcinoma cell lines. The survival rate and clonogenicity decreased after HDM201 treatment in a dose-dependent manner. Combined treatment with HDM201 and GSK2830371 (WIP1 inhibitor) increased p53 phosphorylation, leading to sustained p53 activation. This combination treatment resulted in G2/M phase arrest and promoted cytotoxicity compared with MDM2 inhibitor monotherapy. Furthermore, increased expression of p53 signaling pathway target genes were identified following combination treatment with HDM201 and GSK2830371, suggesting potential roles for this combination strategy in iCCA therapy.Background: Intrahepatic cholangiocarcinoma (iCCA) is an adenocarcinoma arising from the intrahepatic bile duct. It is the second most common primary liver cancer and has a poor prognosis. Activation of p53 by targeting its negative regulators, MDM2 and WIP1, is a potential therapy for wild-type p53 cancers, but few reports for iCCA or liver adenocarcinoma exist. Methods: Both RBE and SK-Hep-1 liver adenocarcinoma cell lines were treated with the HDM201 (Siremadlin) MDM2-p53 binding antagonist alone or in combination with the GSK2830371 WIP1 phosphatase inhibitor. Cell proliferation, clonogenicity, protein and mRNA expression, cell cycle distribution, and RNA sequencing were performed to investigate the effect and mechanism of this combination. Results: GSK2830371 alone demonstrated minimal activity on proliferation and colony formation, but potentiated growth inhibition (two-fold decrease in GI50) and cytotoxicity (four-fold decrease in IC50) by HDM201 on RBE and SK-Hep-1 cells. HDM201 increased p53 protein expression, leading to transactivation of downstream targets (p21 and MDM2). Combination with GSK2830371 increased p53 phosphorylation, resulting in an increase in both p53 accumulation and p53-dependent trans-activation. G2/M arrest was observed by flow cytometry after this treatment combination. RNA sequencing identified 21 significantly up-regulated genes and five downregulated genes following p53 reactivation by HDM201 in combination with GSK2830371 at 6 h and 24 h time points compared with untreated controls. These genes were predominantly known transcriptional targets regulated by the p53 signaling pathway, indicating enhanced p53 activation as the predominant effect of this combination. Conclusion: The current study demonstrated that GSK2830371 enhanced the p53-dependent antiproliferative and cytotoxic effect of HDM201 on RBE and SK-Hep-1 cells, providing a novel strategy for potentiating the efficacy of targeting the p53 pathway in iCCA.

Camptothecin enhances 131I-rituximab-induced G1-arrest and apoptosis in Burkitt lymphoma cells.

Rituximab is a chimeric monoclonal antibody against CD20. It is an established immunotherapeutic agent for non-Hodgkin's lymphoma. Even though rituximab has been used in clinics for decades, only 50% of the patients respond to rituximab therapy. To enhance the in vitro effect of rituximab, it was labeled with Iodine-131 (131I) and combined effect of 131I-rituximab and camptothecin (CPT) was studied on a tumor cell line expressing CD20. The aim is to study the magnitude of cell killing and the underlying mechanism responsible for enhancing in vitro therapeutic efficacy. Rituximab was labeled with 131I by the iodogen method. Raji cells were pretreated with CPT (250 nM) for an hour followed by 131I-rituximab (0.37 and 3.7 MBq) and incubated for 24 h in a humidified atmosphere of CO2 incubator at 37°C. Subsequently, Raji cells were harvested and thoroughly washed to carry out studies of cellular toxicity, apoptosis, cell cycle, and mitogen-activated protein kinase (MAPK) pathways. Maximal inhibition of cell proliferation and enhancement of apoptotic cell death was observed in the cells treated with the combination of CPT and 131I-rituximab, compared to controls of CPT-treated and 131I-rituximab-treated cells. Raji cells undergo G1 arrest after 131I-rituximab treatment, which leads to apoptosis and was confirmed by the downregulation of bclxl protein. Expression of p38 was decreased while an increase in phosphorylation of p38 was observed in the combination treatment of CPT and 131I-rituximab. It was concluded from the findings that CPT enhanced 131I-rituximab-induced apoptosis, G1 cell cycle arrest and p38 MAPK phosphorylation in Raji cells.

Hydrostatic pressure promotes migration and filamin-A activation in fibroblasts with increased p38 phosphorylation and TGF-β production

Fibroblast migration is closely regulated by the mechanical characteristics in surrounding microenvironment. While increased interstitial hydrostatic pressure (HP) is a hallmark in many pathological and physiological conditions, little is known about how the HP affects fibroblast motility. Using cell-culture chips with elevated HP conditions, we showed that 20 cmH2O HP significantly accelerated fibroblast migration. The HP-induced migration acceleration was dependent on the augmentation of transforming growth factor-β1, and correlated with the activation of filamin A via the phosphorylation of p38 mitogen-activated protein kinase. Our results suggest that interstitial HP elevation associated with various pathological states could significantly regulate fibroblast migration.

Intracellular Factor H Drives Tumor Progression Independently of the Complement Cascade.

The complement system is a powerful and druggable innate immune component of the tumor microenvironment. Nevertheless, it is challenging to elucidate the exact mechanisms by which complement affects tumor growth. In this study, we examined the processes by which the master complement regulator factor H (FH) affects clear cell renal cell carcinoma (ccRCC) and lung cancer, two cancers in which complement overactivation predicts poor prognosis. FH was present in two distinct cellular compartments: the membranous (mb-FH) and intracellular (int-FH) compartments. Int-FH resided in lysosomes and colocalized with C3. In ccRCC and lung adenocarcinoma, FH exerted protumoral action through an intracellular, noncanonical mechanism. FH silencing in ccRCC cell lines resulted in decreased proliferation, due to cell-cycle arrest and increased mortality, and this was associated with increased p53 phosphorylation and NFκB translocation to the nucleus. Moreover, the migration of the FH-silenced cells was reduced, likely due to altered morphology. These effects were cell type-specific because no modifications occurred upon CFH silencing in other FH-expressing cells tested: tubular cells (from which ccRCC originates), endothelial cells (human umbilical vein endothelial cells), and squamous cell lung cancer cells. Consistent with this, in ccRCC and lung adenocarcinoma, but not in lung squamous cell carcinoma, int-FH conferred poor prognosis in patient cohorts. Mb-FH performed its canonical function of complement regulation but had no impact on tumor cell phenotype or patient survival. The discovery of intracellular functions for FH redefines the role of the protein in tumor progression and its use as a prognostic biomarker or potential therapeutic target.See article by Daugan et al., p. 891 (36).

Nuclear Factor-κB Signaling Mediates Antimony-induced Astrocyte Activation

ObjectiveAntimony (Sb) has recently been identified as a novel nerve poison, although the cellular and molecular mechanisms underlying its neurotoxicity remain unclear. This study aimed to assess the effects of the nuclear factor kappa B (NF-κB) signaling pathway on antimony-induced astrocyte activation. MethodsProtein expression levels were detected by Western blotting. Immunofluorescence, cytoplasmic and nuclear fractions separation were used to assess the distribution of p65. The expression of protein in brain tissue sections was detected by immunohistochemistry. The levels of mRNAs were detected by Quantitative real-time polymerase chain reaction (qRT-PCR) and reverse transcription-polymerase chain reaction (RT-PCR). ResultsAntimony exposure triggered astrocyte proliferation and increased the expression of two critical protein markers of reactive astrogliosis, inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP), indicating that antimony induced astrocyte activation in vivo and in vitro. Antimony exposure consistently upregulated the expression of inflammatory factors. Moreover, it induced the NF-κB signaling, indicated by increased p65 phosphorylation and translocation to the nucleus. NF-κB inhibition effectively attenuated antimony-induced astrocyte activation. Furthermore, antimony phosphorylated TGF-β-activated kinase 1 (TAK1), while TAK1 inhibition alleviated antimony-induced p65 phosphorylation and subsequent astrocyte activation. ConclusionAntimony activated astrocytes by activating the NF-κB signaling pathway.

11-O-Galloyl Bergenin from Corylopsis coreanas Leaves Induces Autophagy and Apoptosis in Human Osteosarcoma.

Osteosarcoma is the most common malignant bone-forming tumor, wherein most patients with high grade osteosarcomas are treated with chemotherapy. Despite this, survival for metastatic or relapsed osteosarcoma patients has remained at an overall 5-year survival rate of 20%. In particular, the extracts of Corylopsis coreana (Korean winter hazel), a cultivated woody plant in South Korea, have shown beneficial anti-inflammatory, anti-oxidative, anti-osteoclastic, and antihyperuricemic properties. Therefore, this study aimed to demonstrate the antitumor activities and underlying mechanism of 11-O-Galloyl bergenin (OGAL) isolated from Corylopsis coreanas leaves in human osteosarcoma cells. Herein, we found that OGAL inhibited MG63 cell proliferation and induced cellular apoptosis as evidenced by cleaved-PARP, cleaved-caspase 3, TUNEL-positive cells, and Annexin V-positive cells. Specifically, OGAL-induced apoptosis was accompanied by p53 and p21 upregulation, BAX expression, and decreased Bcl-2 and cdk2. Moreover, OGAL induced autophagy via AKT inactivation, LC3II upregulation, and MG63 cell autophagosome formation. OGAL-induced autophagy was also accompanied by increased p38 phosphorylation, whereas JNK and ERK1/2 activities were found to be unaffected upon examining the MAPK signaling pathway. Furthermore, wound healing and Boyden chamber assays showed that OGAL suppressed MG63 cell migration and invasion. Given these findings, this study provided evidence that OGAL has antitumor effects by apoptosis and autophagy enhancement through increased p53, AKT, and p38 signaling, suggesting that OGAL may be a potential therapeutic strategy for osteosarcoma treatment.

MgCl2 promotes mouse mesenchymal stem cell osteogenic differentiation by activating the p38/Osx/Runx2 signaling pathway.

Magnesium, an important inorganic mineral component in bones, enhances osteoblast adhesion and osteogenic gene expression. Mg2+-containing hydroxyapatite promotes mouse mesenchymal stem cell (MMSC) osteogenic differentiation. In the present study, MMSCs were cultured in media containing different concentrations of MgCl2 (0 and 20 mM) for different time periods. Western blotting and reverse transcription-quantitative PCR were performed to determine the expression levels of phosphorylated (p)-p38 mitogen-activated protein kinase (MAPK), the osteoblast-specific transcription factor Osterix (Osx), runt-related transcription factor 2 (Runx2), and p38 downstream genes, such as 27 kDa heat shock protein (hsp27), activating transcription factor 4 (Atf4), myocyte enhancer factor 2C (Mef2c) and CCAAT/enhancer-binding protein homologous protein (Ddit3). The facilitatory effect of MgCl2 on MMSC osteogenic differentiation was assessed via Alizarin Red staining. The results suggested that MgCl2 increased p38 phosphorylation compared with the control group. Downstream genes of the p38 signaling pathway, including Osx and Runx2, as well as several osteogenesis-associated downstream target genes, including Hsp27, Atf4, Ddit3 and Mef2c, were significantly upregulated in the Mg2+-treated group compared with the control group. The increased osteogenic differentiation in the Mg2+-treated group was significantly attenuated in MMSCs treated with SB203580, a specific inhibitor of the p38 signaling pathway. The results suggested that appropriate concentrations of MgCl2 promoted MMSC osteogenic differentiation via regulation of the p38/Osx/Runx2 signaling pathway.

Combined Treatment of Heteronemin and Tetrac Induces Antiproliferation in Oral Cancer Cells.

Background: Heteronemin, a marine sesterterpenoid-type natural product, possesses an antiproliferative effect in cancer cells. In addition, heteronemin has been shown to inhibit p53 expression. Our laboratory has demonstrated that the thyroid hormone deaminated analogue, tetrac, activates p53 and induces antiproliferation in colorectal cancer. However, such drug mechanisms are still to be studied in oral cancer cells. Methods: We investigated the antiproliferative effects by Cell Counting Kit-8 and flow cytometry. The signal transduction pathway was measured by Western blotting analyses. Quantitative PCR was used to evaluate gene expression regulated by heteronemin, 3,3’,5,5’-tetraiodothyroacetic acid (tetrac), or their combined treatment in oral cancer cells. Results: Heteronemin inhibited not only expression of proliferative genes and Homo Sapiens Thrombospondin 1 (THBS-1) but also cell proliferation in both OEC-M1 and SCC-25 cells. Remarkably, heteronemin increased TGF-β1 expression in SCC-25 cells. Tetrac suppressed expression of THBS-1 but not p53 expression in both cancer cell lines. Furthermore, the synergistic effect of tetrac and heteronemin inhibited ERK1/2 activation and heteronemin also blocked STAT3 signaling. Combined treatment increased p53 protein and p53 activation accumulation although heteronemin inhibited p53 expression in both cancer cell lines. The combined treatment induced antiproliferation synergistically more than a single agent. Conclusions: Both heteronemin and tetrac inhibited ERK1/2 activation and increased p53 phosphorylation. They also inhibited THBS-1 expression. Moreover, tetrac suppressed TGF-β expression combined with heteronemin to further enhance antiproliferation and anti-metastasis in oral cancer cells.

PERK/NRF2 and autophagy form a resistance mechanism against G9a inhibition in leukemia stem cells

BackgroundThe histone methyltransferase G9a has recently been identified as a potential target for epigenetic therapy of acute myeloid leukemia (AML). However, the effect of G9a inhibition on leukemia stem cells (LSCs), which are responsible for AML drug resistance and recurrence, is unclear. In this study, we investigated the underlying mechanisms of the LSC resistance to G9a inhibition.MethodsWe evaluated the effects of G9a inhibition on the unfolded protein response and autophagy in AML and LSC-like cell lines and in primary CD34+CD38− leukemic blasts from patients with AML and investigated the underlying mechanisms. The effects of treatment on cells were evaluated by flow cytometry, western blotting, confocal microscopy, reactive oxygen species (ROS) production assay.ResultsThe G9a inhibitor BIX-01294 effectively induced apoptosis in AML cell lines; however, the effect was limited in KG1 LSC-like cells. BIX-01294 treatment or siRNA-mediated G9a knockdown led to the activation of the PERK/NRF2 pathway and HO-1 upregulation in KG1 cells. Phosphorylation of p38 and intracellular generation of reactive oxygen species (ROS) were suppressed. Pharmacological or siRNA-mediated inhibition of the PERK/NRF2 pathway synergistically enhanced BIX-01294-induced apoptosis, with suppressed HO-1 expression, increased p38 phosphorylation, and elevated ROS generation, indicating that activated PERK/NRF2 signaling suppressed ROS-induced apoptosis in KG1 cells. By contrast, cotreatment of normal hematopoietic stem cells with BIX-01294 and a PERK inhibitor had no significant proapoptotic effect. Additionally, G9a inhibition induced autophagy flux in KG1 cells, while autophagy inhibitors significantly increased the BIX-01294-induced apoptosis. This prosurvival autophagy was not abrogated by PERK/NRF2 inhibition.ConclusionsPERK/NRF2 signaling plays a key role in protecting LSCs against ROS-induced apoptosis, thus conferring resistance to G9a inhibitors. Treatment with PERK/NRF2 or autophagy inhibitors could overcome resistance to G9a inhibition and eliminate LSCs, suggesting the potential clinical utility of these unique targeted therapies against AML.

Involvement of Angiotensin II Type 1 Receptor and Calcium Channel in Vascular Remodeling and Endothelial Dysfunction in Rats with Pressure Overload.

Vascular remodeling is an adaptive response to various stimuli, including mechanical forces, inflammatory cytokines and hormones. In the present study, we investigated the role of angiotensin II type 1 receptor (AT1R) and calcium channel in carotid artery remodeling in response to increased biomechanical forces by using the transverse aortic constriction (TAC) rat model. TAC was induced on ten-week-old male Sprague-Dawley rats and these models were treated with AT1R blocker olmesartan (1 mg/kg/day) or/and calcium channel blocker (CCB) amlodipine (0.5 mg/kg/day) for 14 days. After the treatment, the right common carotid artery proximal to the band (RCCA-B) was collected for further assay. Results showed that olmesartan, but not amlodipine, significantly prevented TAC-induced adventitial hyperplasia. Similarly, olmesartan, but not amlodipine, signifcantly prevented vascular infammation, as indicated by increased tumor necrosis factor α (TNF-α) and increased p65 phosphorylation, an indicator of nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) activation in RCCA-B. In contrast, both olmesartan and amlodipine reversed the decreased expression of endothelial nitric oxidase synthase (eNOS) and improved endothelium-dependent vasodilation, whereas combination of olmesartan and amlodipine showed no further synergistic protective effects. These results suggest that AT1R was involved in vascular remodeling and inflammation in response to pressure overload, whereas AT1R and subsequent calcium channel were involved in endothelial dysfunction.

Cyclophilin a knokdown inhibits cell migration and invasion through the suppression of epithelial–mesenchymal transition in colorectal cancer cells

Enhanced expression of cyclophilin A (CypA) in colorectal cancer (CRC) was reported; however, how CypA influences CRC progression is not clear. Therefore, we examine the effects of CypA on CRC cell progression. Knockdown of CypA in SW480 cells significantly inhibited cell migration and invasion but had no effect on cell proliferation. In addition, upregulation of E-cadherin and downregulation of N-cadherin and Snail expression were observed by CypA knockdown. These results suggested that CypA knockdown inhibited cell migration and invasion by suppressing epithelial-mesenchymal transition. CypA knockdown was also associated with increased p38 phosphorylation, and the p38 inhibitor treatment led to increase in the number of invasive CypA-knockdown SW480 cells. Therefore, CypA may be a potential therapeutic target in preventing CRC metastasis.

Sarcopenia is attenuated by TRB3 knockout in aging mice via the alleviation of atrophy and fibrosis of skeletal muscles

BackgroundSarcopenia causes several adverse events in elderly people. Muscle fibre atrophy and interstitial fibrosis are the main histopathological changes in sarcopenia and account for decreased muscle function. Tribbles homologue 3 (TRB3) was previously reported to exhibit age‐related expression and play a vital role in cell proliferation, differentiation, and fibrosis. We aimed to investigate how TRB3 affects sarcopenia.MethodsWild‐type and TRB3 knockout C57/BL6J mice were randomly divided into young and old groups. Exercise capacity was evaluated, and single‐muscle function was detected by electrophysiological techniques, after which the mice were sacrificed to collect their gastrocnemius muscles for assessment of atrophy and fibrosis by histopathological and molecular biological methods. TRB3 expression, autophagy level, and MAPK signalling pathway activity were evaluated through western blotting. The interaction of TRB3 with P62 and the association between TRB3 and the MAPK signalling pathway were detected by co‐immunoprecipitation.ResultsIn aged mice, exercise capacity and cross‐sectional area of skeletal muscle fibres were decreased significantly, whereas TRB3, atrophy‐related markers atrogin 1 and MuRF 1, and interstitial fibrosis, including collagen volume fraction, contents of collagens I and III, and ratio of collagens I to III, were increased significantly (P < 0.05 for all). Following TRB3 knockout, the cross‐sectional area of muscle fibres, mainly fast fibres, was elevated (P < 0.05 for both), the atrogin 1 expression was decreased (P = 0.0163), and the corresponding tetanic force of fast muscles was increased (P = 0.0398). Conversely, interstitial fibrosis was substantially decreased and exercise capacity was significantly increased in the knockout mice. In terms of the underlying mechanisms, the autophagy receptor p62 was markedly increased and the MAPK signalling pathway was activated in aged skeletal muscles, which might be attributed to the interaction of TRB3 with p62 and MAPKKs, including MEK1/MEK2, MEK3/MEK6, and MEK4/MKK4. Notably, TRB3 knockout reduced the accumulation of p62 and LC3 (P < 0.05 for both), decreased the phosphorylation of JNK (P = 0.0015), and increased p38 phosphorylation (P = 0.0021).ConclusionsTRB3 knockout in mice attenuated muscle fibre atrophy and reduced skeletal muscle fibrosis by increasing autophagy and inhibiting the MAPK signalling pathway. Correspondingly, in aged knockout mice, exercise capacity was improved. Interfering with TRB3 expression in aged skeletal muscles may serve as a target for the prevention and treatment of age‐related sarcopenia.

The δ-Opioid Receptor Differentially Regulates MAPKs and Anti-inflammatory Cytokines in Rat Kidney Epithelial Cells Under Hypoxia.

Hypoxic injury is one of the most important factors in progressive kidney disorders. Since we have found that δ-opioid receptor (DOR) is neuroprotective against hypoxic stress through a differential regulation of mitogen-activated protein kinases (MAPKs) and anti-inflammatory cytokines, we asked if DOR that is highly expressed in the kidney can modulate renal MAPKs and anti-inflammatory cytokines under hypoxia. We exposed cultured rat kidney epithelial cells (NRK-52E) to prolonged hypoxia (1% O2) with applications of specific DOR agonist or/and antagonist to examine if DOR affects hypoxia-induced changes in MAPKs and anti-inflammatory cytokines. The results showed that endogenous DOR expression remained unchanged under hypoxia, while DOR activation with UFP-512 (a specific DOR agonist) reversed the hypoxia-induced up-regulation of ERK1/2 and p38 phosphorylation. DOR inhibition with naltrindole had no appreciable effect on the hypoxia-induced changes in ERK1/2 phosphorylation, but increased p38 phosphorylation. DOR inhibition with naltrindole attenuated the effects of DOR activation on the changes in ERK1/2 and p38 phosphorylation in hypoxia. Moreover, DOR activation/inhibition differentially affected the expression of transcriptional repressor B-cell lymphoma 6 (Bcl-6), anti-inflammatory cytokines tristetraprolin (TTP), and interleukin-10 (IL-10). Taken together, our novel data suggest that DOR activation differentially regulates ERK1/2, p38, Bcl-6, TTP, and IL-10 in the renal cells under hypoxia.

Naringin Attenuated Acute Lung Injury in Rat Model with Acute Pancreatitis in Pregnancy through Inactivation of p38 MAPK Pathway

Acute pancreatitis in pregnancy can cause fetal lung injury. Naringin exhibits ant-inflammation effect against pulmonary injuries. However, whether naringin can alleviate lung injury associated with acute pancreatitis in pregnancy has not been elucidated. Establishment of acute pancreatitis in pregnancy model by sodium taurocholate showed that sodium taurocholate induced obvious pathological changes in pancreas. Histopathological changes in lungs were also aggravated post sodium taurocholate treatment with enhanced inflammation of neutrophils, thickened alveolar walls and alveolus collapse. Diagnostic blood tests for acute pancreatitis in pregnancy indicated that serum amylase and lipase were increased post sodium taurocholate treatment. Sodium taurocholate-induced rats were intragastrically administered with naringin to investigate the protective effect of naringin on acute lung injury induced by acute pancreatitis in pregnancy. Results revealed that administration of naringin could attenuate pathological changes in pancreas and lungs and decrease amylase and lipase. Moreover, naringin treatment attenuated sodium taurocholate-induced increase in inflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6/1β in lungs. Sodium taurocholate-induced increase in phosphorylation of p38 was also reduced post naringin treatment. In conclusion, naringin could ameliorate acute lung injury and inflammation induced by acute pancreatitis in pregnancy by inhibiting the p38 pathway.

Virus-induced p38 MAPK activation facilitates viral infection.

Rationale: Many viral infections are known to activate the p38 mitogen-activated protein kinase (MAPK) signaling pathway. However, the role of p38 activation in viral infection and the underlying mechanism remain unclear. The role of virus-hijacked p38 MAPK activation in viral infection was investigated in this study.Methods: The correlation of hepatitis C virus (HCV) infection and p38 activation was studied in patient tissues and primary human hepatocytes (PHHs) by immunohistochemistry and western blotting. Coimmunoprecipitation, GST pulldown and confocal microscopy were used to investigate the interaction of p38α and the HCV core protein. In vitro kinase assays and mass spectrometry were used to analyze the phosphorylation of the HCV core protein. Plaque assays, quantitative real time PCR (qRT-PCR), western blotting, siRNA and CRISPR/Cas9 were used to determine the effect of p38 activation on viral replication.Results: HCV infection was associated with p38 activation in clinical samples. HCV infection increased p38 phosphorylation by triggering the interaction of p38α and TGF-β activated kinase 1 (MAP3K7) binding protein 1 (TAB1). TAB1-mediated p38α activation facilitated HCV replication, and pharmaceutical inhibition of p38α activation by SB203580 suppressed HCV infection at the viral assembly step. Activated p38α interacted with the N-terminal region of the HCV core protein and subsequently phosphorylated the HCV core protein, which promoted HCV core protein oligomerization, an essential step for viral assembly. As expected, SB203580 or the HCV core protein N-terminal peptide (CN-peptide) disrupted the p38α-HCV core protein interaction, efficiently impaired HCV assembly and impeded normal HCV replication in both cultured cells and primary human hepatocytes. Similarly, severe fever with thrombocytopenia syndrome virus (SFTSV), herpes simplex virus type 1 (HSV-1) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection also activated p38 MAPK. Most importantly, pharmacological blockage of p38 activation by SB203580 effectively inhibited SFTSV, HSV-1 and SARS-CoV-2.Conclusion: Our study shows that virus-hijacked p38 activation is a key event for viral replication and that pharmacological blockage of p38 activation is an antiviral strategy.