Antiproliferative and apoptotic effects of telmisartan in human glioma cells.

Glioblastoma is the most common primary central nervous system tumor in adults. Angiotensin II receptor blockers (ARBs) are broadly applied to treat hypertension. Moreover, research has revealed that ARBs have the capacity to suppress the growth of several cancer types. In this study, we assessed the effects of three ARBs with the ability to cross the blood brain barrier (telmisartan, valsartan and fimasartan) on cell proliferation in three glioblastoma multiforme (GBM) cell lines. Telmisartan markedly suppressed the proliferation, migration, and invasion of these three GBM cell lines. Microarray data analysis revealed that telmisartan regulates DNA replication, mismatch repair, and the cell cycle pathway in GBM cells. Furthermore, telmisartan induced G0/G1 phase arrest and apoptosis. The bioinformatic analysis and western blotting results provide evidence that SOX9 is a downstream target of telmisartan. Telmisartan also suppressed tumor growth in vivo in an orthotopic transplant mouse model. Therefore, telmisartan is a potential treatment for human GBM.

Targeting human mitochondrial NAD(P)+-dependent malic enzyme (ME2) impairs energy metabolism and redox state and exhibits antileukemic activity in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a fast-growing and highly fatal blood cancer, and recent research has shown that targeting metabolism may be a promising therapeutic approach for treating AML. One promising target is the human mitochondrial NAD(P)+-dependent malic enzyme (ME2), which is involved in the production of pyruvate and NAD(P)H and the regulation of the NAD+/NADH redox balance. Inhibition of ME2 via silencing ME2 or utilizing its allosteric inhibitor disodium embonate (Na2EA) causes a decrease in pyruvate and NADH, leading to a decrease in producing ATP via cellular respiration and oxidative phosphorylation. ME2 inhibition also decreases NADPH levels, resulting in an increase in reactive oxygen species (ROS) and oxidative stress, which ultimately leads to cellular apoptosis. Additionally, ME2 inhibition reduces pyruvate metabolism and the biosynthetic pathway. ME2 silencing inhibits the growth of xenotransplanted human AML cells, and the allosteric ME2 inhibitor Na2EA demonstrates antileukemic activity against immune-deficient mice with disseminated AML. Both of these effects are a result of impaired energy metabolism in mitochondria. These findings suggest that the targeting ME2 may be an effective strategy for treating AML. Overall, ME2 plays an essential role in energy metabolism of AML cells, and its inhibition may offer a promising approach for AML treatment.

Identification of Agents That Ameliorate Hyperphosphatemia-Suppressed Myogenin Expression Involved in the Nrf2/p62 Pathway in C2C12 Skeletal Muscle Cells.

Hyperphosphatemia can occur as a result of reduced phosphate (Pi) excretion in cases of kidney dysfunction, which can induce muscle wasting and suppress myogenic differentiation. Higher Pi suppresses myogenic differentiation and promotes muscle atrophy through canonical (oxidative stress-mediated) and noncanonical (p62-mediated) activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling. However, the crosstalk between myogenin and Nrf2/p62 and potential drug(s) for the regulation of myogenin expression needed to be addressed. In this study, we further identified that myogenin may negatively regulate Nrf2 and p62 protein levels in the mouse C2C12 muscle cell line. In the drug screening analysis, we identified N-acetylcysteine, metformin, phenformin, berberine, 4-chloro-3-ethylphenol, cilostazol, and cilomilast as ameliorating the induction of Nrf2 and p62 expression and reduction in myogenin expression that occur due to high Pi. We further elucidated that doxorubicin and hydrogen peroxide reduced the amount of myogenin protein mediated through the Kelch-like ECH-associated protein 1/Nrf2 pathway, differently from the mechanism of high Pi. The dual functional roles of L-ascorbic acid (L-AA) were found to be dependent on the working concentration, where concentrations below 1 mM L-AA reversed the effect of high Pi on myogenin and those above 1 mM L-AA had a similar effect of high Pi on myogenin when used alone. L-AA exacerbated the effect of hydrogen peroxide on myogenin protein and had no further effect of doxorubicin on myogenin protein. In summary, our results further our understanding of the crosstalk between myogenin and Nrf2, with the identification and verification of several potential drugs that can be applied in rescuing the decline of myogenin due to high Pi in muscle cells.

Dipeptidyl Peptidase-4 Inhibitor-Related Bullous Pemphigoid: Clinical, Laboratory, and Histological Features, and Possible Pathogenesis.

Dipeptidyl peptidase-4 inhibitor (DPP4i) is a widely used antidiabetic agent. Emerging cases of DPP4i-associated bullous pemphigoid (DBP), whose pathogenesis remains unclear, have been reported. Thus, a retrospective study was conducted from January 2016 to June 2021 to determine the clinical, laboratory, and histopathological features of DBP and idiopathic bullous pemphigoid (IBP). We set up in vitro experiments using vildagliptin-treated HaCaT keratinocytes to validate what we found by analyzing published RNA sequencing data about the genes related to the dermal-epidermal junction. We also observed IL-6 expression by HaCaT cells treated with vildagliptin. We enrolled 20 patients with DBP and 40 patients with IBP. The total Bullous Pemphigoid Disease Area Index (BPDAI) score was similar in both groups. However, the BPDAI score of erosions and blisters in DBP was significantly higher than that in IBP (24.6 vs. 16.68, p = 0.0189), and the score for urticaria and erythema was lower in DBP (12 vs. 19.05, p = 0.0183). The pathological features showed that the mean infiltrating eosinophil number per high-power field was significantly lower in DBP than in IBP (16.7 vs. 27.08, p = 0.023). The expression of LAMA3, LAMB3, LAMC2, DST, and COL17A1 decreased significantly in vildagliptin-treated human keratinocytes. On the other hand, IL-6, the hallmark cytokine of bullous pemphigoid (BP) severity, was found to be upregulated in HaCaT cells by vildagliptin. These experimental findings imply less of a requirement for eosinophil infiltration to drive the inflammatory cascades in DBP blistering. Both immunologic and non-immunologic pathways could be employed for the development of DBP. Our findings may help explain the higher incidence of non-inflammatory BP that was observed in DBP.

In Vitro Cell Density Determines the Sensitivity of Hepatocarcinoma Cells to Ascorbate.

Hepatocellular carcinoma (HCC) is the primary histological subtype of liver cancer, and its incidence rates increase with age. Recently, systemic therapies, such as immune checkpoint inhibitors, monoclonal antibodies, and tyrosine kinase inhibitors (TKIs), have been more beneficial than conventional therapies for treating HCC. Nonetheless, the prognosis of late-stage HCC remains dismal because of its high recurrence rates, even with substantial advances in current therapeutic strategies. A new treatment, such as a combination of current systemic therapies, is urgently required. Therefore, we adopted a repurposing strategy and tried to combine ascorbate with TKIs, including lenvatinib and regorafenib, in HepG2 and Hep3B cells. We investigated the potential functional impact of pharmacological concentrations of ascorbate on the cell-cycle profiles, mitochondrial membrane potential, oxidative response, synergistic effects of lenvatinib or regorafenib, and differential responsiveness between HepG2 and Hep3B cells. Our data suggest that the relative level of cell density is an important determinant for ascorbate cytotoxicity in HCC. Furthermore, the data also revealed that the cytotoxic effect of pharmacological concentrations of ascorbate might not be mediated via our proposed elevation of ROS generation. Ascorbate might be involved in redox homeostasis to enhance the efficacy of TKIs in HepG2 and Hep3B cells. The synergistic effects of ascorbate with TKIs (lenvatinib and regorafenib) support their potential as an adjuvant for HCC targeted TKI therapy. This research provides a cheap and new combinatory therapy for HCC treatment.

The Synergistic Cytotoxic Effects of GW5074 and Sorafenib by Impacting Mitochondrial Functions in Human Colorectal Cancer Cell Lines.

Colorectal cancer (CRC) ranks third in the United States for incidence or mortality. Surgical resection is the primary treatment for patients at an early stage, while patients with advanced and metastatic CRC receive combined treatment with chemotherapy, radiotherapy, or targeted therapy. C-RAF plays a key role in maintaining clonogenic and tumorigenic capacity in CRC cells and it might be a potential therapeutic target for CRC. Sorafenib is a popular oral multi-kinase inhibitor, including a B-RAF inhibitor that targets the RAF-MEK-ERK pathway. Sorafenib, as a single agent, has tumor-suppressing efficacy, but its clinical application is limited due to many complex drug resistance mechanisms and side effects. GW5074 is one of the C-RAF inhibitors and has the potential to enhance the efficacy of existing cancer chemotherapies. In this study, we investigated whether the combination of sorafenib with GW5074 could reduce the dosage of sorafenib and enhance its tumor-suppressive effect in two CRC cell lines, HCT116 and LoVo cells. Our findings demonstrate that GW5074 can potentiate the cytotoxicity of sorafenib and dramatically reduce the half-maximal inhibitory concentration (IC50) dose of sorafenib from 17 and 31 µM to 0.14 and 0.01 µM in HCT116 and LoVo cells, respectively. GW5074, similar to sorafenib, suppressed the cellular proliferation and induced cellular apoptosis and cytosolic ROS, but had no further enhancement on the above-mentioned effects when combined with sorafenib. The synergistic effects of GW5074 and sorafenib were mainly found in mitochondrial functions, including ROS generation, membrane potential disruption, and fission-fusion dynamics, which were examined by using the flow cytometry analysis. In summary, the C-RAF inhibitor GW5074 might potentiate the cytotoxicity of the B-RAF inhibitor sorafenib mediated through mitochondrial dysfunctions, suggesting that GW5074 potentially serves as a sensitizer for sorafenib application to reduce the risk of drug resistance of CRC treatment. Our findings also provide novel insights on using C-RAF inhibitors combined with sorafenib, the current CRC therapeutic drug choice, in CRC treatment.

Benzo[a]pyrene exposure in muscle triggers sarcopenia through aryl hydrocarbon receptor-mediated reactive oxygen species production.

BACKGROUND: Benzo[a]pyrene (BaP), a toxic carcinogen, is associated with various adverse effects but is rarely discussed in muscle-related disorders. This study investigated in vitro and in vivo effects triggered by BaP exposure in muscles and hypothesized that exposure might induce conditions similar to sarcopenia due to the shared mechanism of oxidative stress. In vitro experiments used C2C12 mouse myoblasts to examine effects induced by BaP exposure in control (untreated) and BaP-treated (10 µM/ml) muscle cells. An established TNF-α-treated sarcopenia model was utilized to verify our results. In vivo experiments compared immunohistochemical staining of sarcopenia-related markers in rats exposed to clean air and polluted air. RESULTS: In C2C12 cells, after 2-72 h of BaP exposure, elevated mRNA and protein expressions were observed in aryl hydrocarbon receptor (AhR) and cytochrome P450 1A1, subsequently in ROS (NOX2 and NOX4) production, inflammatory cytokines (IL-6, TNF-α, and NF-kB), and proteins mediating apoptotic cell death (caspase-3 and PARP). Two myokines also altered mRNA and protein expressions akin to changes in sarcopenia, namely decreased irisin levels and increased myostatin levels. In addition, N-acetylcysteine, a well-known antioxidant, led to decrease in oxidative markers induced by BaP. The validation by TNF-α-treated sarcopenia model revealed comparable biological responses in either TNF-α or BaP treated C2C12 cells. In vivo experiments with rats exposed to air pollution showed increased expression of BaP, AhR, 8-hydroxydeoxyguanosine, and myostatin and decreased irisin expression in immunohistochemical staining. CONCLUSIONS: Our results suggest that BaP exerts deleterious effects on the muscle, leading to conditions indicative of sarcopenia. Antioxidant supplementation may be a treatment option for BaP-induced sarcopenia, but further validation studies are needed.

Diosmin Inhibits Glioblastoma Growth through Inhibition of Autophagic Flux.

Diosmin, a natural flavone glycoside acquired through dehydrogenation of the analogous flavanone glycoside hesperidin, is plentiful in many citrus fruits. Glioblastoma multiforme (GBM) is the most malignant primary brain tumor; the average survival time of GBM patients is less than 18 months after standard treatment. The present study demonstrated that diosmin, which is able to cross the blood-brain barrier, inhibited GBM cell growth in vitro and in vivo. Diosmin also impeded migration and invasion by GBM8401and LN229 GBM cells by suppressing epithelial-mesenchymal transition, as indicated by increased expression of E-cadherin and decreased expression of Snail and Twist. Diosmin also suppressed autophagic flux, as indicated by increased expression of LC3-II and p62, and induced cell cycle arrest at G1 phase. Importantly, diosmin did not exert serious cytotoxic effects toward control SVG-p12 astrocytes, though it did reduce astrocyte viability at high concentrations. These findings provide potentially helpful support to the development of new therapies for the treatment of GBM.

Functional role of residues involved in substrate binding of human 4-hydroxyphenylpyruvate dioxygenase.

4-Hydroxylphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxylphenylpyruvate (HPP) to homogentisate, the important step for tyrosine catabolism. Comparison of the structure of human HPPD with the substrate-bound structure of A. thaliana HPPD revealed notably different orientations of the C-terminal helix. This helix performed as a closed conformation in human enzyme. Simulation revealed a different substrate-binding mode in which the carboxyl group of HPP interacted by a H-bond network formed by Gln334, Glu349 (the metal-binding ligand), and Asn363 (in the C-terminal helix). The 4-hydroxyl group of HPP interacted with Gln251 and Gln265. The relative activity and substrate-binding affinity were preserved for the Q334A mutant, implying the alternative role of Asn363 for HPP binding and catalysis. The reduction in kcat/Km of the Asn363 mutants confirmed the critical role in catalysis. Compared to the N363A mutant, the dramatic reduction in the Kd and thermal stability of the N363D mutant implies the side-chain effect in the hinge region rotation of the C-terminal helix. The activity and binding affinity were not recovered by double mutation; however, the 4-hydroxyphenylacetate intermediate formation by the uncoupled reaction of Q334N/N363Q and Q334A/N363D mutants indicated the importance of the H-bond network in the electrophilic reaction. These results highlight the functional role of the H-bond network in a closed conformation of the C-terminal helix to stabilize the bound substrate. The extremely low activity and reduction in Q251E's Kd suggest that interaction coupled with the H-bond network is crucial to locate the substrate for nucleophilic reaction.

Differential Cytotoxicity Mechanisms of Copper Complexed with Disulfiram in Oral Cancer Cells.

Disulfiram (DSF), an irreversible aldehyde dehydrogenase inhibitor, is being used in anticancer therapy, as its effects in humans are known and less adverse than conventional chemotherapy. We explored the potential mechanism behind the cytotoxicity of DSF-Cu+/Cu2+ complexes in oral epidermoid carcinoma meng-1 (OECM-1) and human gingival epithelial Smulow-Glickman (SG) cells. Exposure to CuCl2 or CuCl slightly but concentration-dependently decreased cell viability, while DSF-Cu+/Cu2+ induced cell death in OECM-1 cells, but not SG cells. DSF-Cu+/Cu2+ also increased the subG1 population and decreased the G1, S, and G2/M populations in OECM-1 cells, but not SG cells, and suppressed cell proliferation in both OECM-1 and SG cells. ALDH enzyme activity was inhibited by CuCl and DSF-Cu+/Cu2+ in SG cells, but not OECM-1 cells. ROS levels and cellular senescence were increased in DSF-Cu+/Cu2+-treated OECM-1 cells, whereas they were suppressed in SG cells. DSF-Cu+/Cu2+ induced mitochondrial fission in OECM-1 cells and reduced mitochondrial membrane potential. CuCl2 increased but DSF- Cu2+ impaired oxygen consumption rates and extracellular acidification rates in OECM-1 cells. CuCl2 stabilized HIF-1α expression under normoxia in OECM-1 cells, and complex with DSF enhanced that effect. Levels of c-Myc protein and its phosphorylation at Tyr58 and Ser62 were increased, while levels of the N-terminal truncated form (Myc-nick) were decreased in DSF-Cu+/Cu2-treated OECM-1 cells. These effects were all suppressed by pretreatment with the ROS scavenger NAC. Overexpression of c-Myc failed to induce HIF-1α expression. These findings provide novel insight into the potential application of DSF-CuCl2 complex as a repurposed agent for OSCC cancer therapy.

Cardiac calcium dysregulation in mice with chronic kidney disease.

Cardiovascular complications are leading causes of morbidity and mortality in patients with chronic kidney disease (CKD). CKD significantly affects cardiac calcium (Ca2+ ) regulation, but the underlying mechanisms are not clear. The present study investigated the modulation of Ca2+ homeostasis in CKD mice. Echocardiography revealed impaired fractional shortening (FS) and stroke volume (SV) in CKD mice. Electrocardiography showed that CKD mice exhibited longer QT interval, corrected QT (QTc) prolongation, faster spontaneous activities, shorter action potential duration (APD) and increased ventricle arrhythmogenesis, and ranolazine (10 µmol/L) blocked these effects. Conventional microelectrodes and the Fluo-3 fluorometric ratio techniques indicated that CKD ventricular cardiomyocytes exhibited higher Ca2+ decay time, Ca2+ sparks, and Ca2+ leakage but lower [Ca2+ ]i transients and sarcoplasmic reticulum Ca2+ contents. The CaMKII inhibitor KN93 and ranolazine (RAN; late sodium current inhibitor) reversed the deterioration in Ca2+ handling. Western blots revealed that CKD ventricles exhibited higher phosphorylated RyR2 and CaMKII and reduced phosphorylated SERCA2 and SERCA2 and the ratio of PLB-Thr17 to PLB. In conclusions, the modulation of CaMKII, PLB and late Na+ current in CKD significantly altered cardiac Ca2+ regulation and electrophysiological characteristics. These findings may apply on future clinical therapies.

Role of the N-terminus in human 4-hydroxyphenylpyruvate dioxygenase activity.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in tyrosine catabolism, catalysing the oxidation of 4-hydroxyphenylpyruvate to homogentisate. Genetic deficiency of this enzyme causes type III tyrosinaemia. The enzyme comprises two barrel-shaped domains formed by the N- and C-termini, with the active site located in the C-terminus. This study investigated the role of the N-terminus, located at the domain interface, in HPPD activity. We observed that the kcat/Km decreased ∼8-fold compared with wild type upon removal of the 12 N-terminal residues (ΔR13). Interestingly, the wild-type level of activity was retained in a mutant missing the 17 N-terminal residues, with a kcat/Km 11-fold higher than that of the ΔR13 mutant; however, the structural stability of this mutant was lower than that of wild type. A 2-fold decrease in catalytic efficiency was observed for the K10A and E12A mutants, indicating synergism between these residues in the enzyme catalytic function. A molecular dynamics simulation showed large RMS fluctuations in ΔR13 suggesting that conformational flexibility at the domain interface leads to lower activity in this mutant. These results demonstrate that the N-terminus maintains the stability of the domain interface to allow for catalysis at the active site of HPPD.

Ovatodiolide suppresses inflammatory response in BEAS-2B cells by regulating the CREB/AQP5 pathway, and sensitizes nasopharyngeal carcinoma cells to radiation therapy.

Due to the radiosensitivity of the airway epithelium, radiation-induced sinusitis or bronchitis is not uncommon, and makes mitigation of resulting inflammatory airway diseases a principal goal of many investigations. This study examined whether Ovatodiolide (Ova) sensitizes the human metastatic nasopharyngeal cancer (NPC) cell line, NPC-BM2, to irradiation using viability, clonogenicity and Western blot assays. Concurrently, we used varying concentrations of histamine and/or Ova to determine the anti-inflammatory potential of Ovatodiolide on normal bronchus epithelial BEAS-2B cells, as well as on the subcellular distribution of Aquaporin 5 (AQP5) and expression levels of p-CREB, AQP5, p38 MAPK, NF-κB, PI3K, Akt and ERK proteins. We demonstrated that Ova in synergism with irradiation inhibited NPC-BM2 cell viability and suppressed their clonogenicity. Immunofluorescence analysis revealed low-dose (≤ 2.5 µM) Ova reversed histamine-induced suppression of AQP5 expression, and abrogated histamine-enhanced NF-κB nuclear translocation, indicating Ova modulates the p38 MAPK/NF-κB signaling pathway and elicits p-CREB/AQP5-mediated antihistamine effects. Similarly, Ova deregulates the PI3K/Akt/ERK signaling in BEAS-2B cells, suggesting its cytoprotective potential. In conclusion, this study highlights the radio-sensitizing anticancer efficacy of Ova in human metastatic NPC cells, as well as its putative cytoprotective role in normal bronchial cells, for airway surface liquid maintenance and homeostasis during or after radiotherapy.

Metformin causes cancer cell death through downregulation of p53-dependent differentiated embryo chondrocyte 1.

BACKGROUND: Metformin is the most commonly used first-line medicine for type II diabetes mellitus. Acting via AMP-activated protein kinase, it has been used for more than 60 years and has an outstanding safety record. Metformin also offers protection against cancer, but its precise mechanisms remain unclear. METHODS: We first examined the cytotoxic effects of metformin in the HeLa human cervical carcinoma and ZR-75-1 breast cancer cell lines using assays of cell viability, cleaved poly-ADP-ribose polymerase, and Annexin V-fluorescein isothiocyanate apoptosis, as well as flow cytometric analyses of the cell cycle profile and reactive oxygen species (ROS). We later clarified the effect of metformin on p53 protein stability using transient transfection and cycloheximide chase analyses. RESULTS: We observed that metformin represses cell cycle progression, thereby inducing subG1 populations, and had induced apoptosis through downregulation of p53 protein and a target gene, differentiated embryo chondrocyte 1 (DEC1). In addition, metformin increased intracellular ROS levels, but N-acetyl cysteine, a ROS scavenger, failed to suppress metformin-induced apoptosis. Further results showed that metformin disrupted the electron transport chain and collapsed the mitochondrial membrane potential, which may be the cause of the elevated ROS levels. Examination of the mechanisms underlying metformin-induced HeLa cell death revealed that reduced stability of p53 in metformin-treated cells leads to decreases in DEC1 and induction of apoptosis. CONCLUSION: The involvement of DEC1 provides new insight into the positive or negative functional roles of p53 in the metformin-induced cytotoxicity in tumor cells.

Inhibition of NKCC1 Modulates Alveolar Fluid Clearance and Inflammation in Ischemia-Reperfusion Lung Injury via TRAF6-Mediated Pathways.

Background: The expression of Na-K-2Cl cotransporter 1 (NKCC1) in the alveolar epithelium is responsible for fluid homeostasis in acute lung injury (ALI). Increasing evidence suggests that NKCC1 is associated with inflammation in ALI. We hypothesized that inhibiting NKCC1 would attenuate ALI after ischemia-reperfusion (IR) by modulating pathways that are mediated by tumor necrosis-associated factor 6 (TRAF6). Methods: IR-ALI was induced by producing 30 min of ischemia followed by 90 min of reperfusion in situ in an isolated and perfused rat lung model. The rats were randomly allotted into four groups comprising two control groups and two IR groups with and without bumetanide. Alveolar fluid clearance (AFC) was measured for each group. Mouse alveolar MLE-12 cells were cultured in control and hypoxia-reoxygenation (HR) conditions with or without bumetanide. Flow cytometry and transwell monolayer permeability assay were carried out for each group. Results: Bumetanide attenuated the activation of p-NKCC1 and lung edema after IR. In the HR model, bumetanide decreased the cellular volume and increased the transwell permeability. In contrast, bumetanide increased the expression of epithelial sodium channel (ENaC) via p38 mitogen-activated protein kinase (p38 MAPK), which attenuated the reduction of AFC after IR. Bumetanide also modulated lung inflammation via nuclear factor-κB (NF-κB). TRAF6, which is upstream of p38 MAPK and NF-κB, was attenuated by bumetanide after IR and HR. Conclusions: Inhibition of NKCC1 by bumetanide reciprocally modulated epithelial p38 MAPK and NF-κB via TRAF6 in IR-ALI. This interaction attenuated the reduction of AFC via upregulating ENaC expression and reduced lung inflammation.

Zac1 regulates IL-11 expression in osteoarthritis.

Interleukin (IL)-11, a member of the IL-6 family of cytokines, exerts pleiotropic effects under normal and various disease conditions. We assessed IL-11 expression regulation and the IL-11/IL-6 ratio in osteoarthritis (OA) to better guide clinical therapeutic decision-making. Our findings suggest that Zac1, a zinc finger protein that regulates apoptosis and cell cycle arrest, is a transcription factor regulating IL-11 expression. Zac1 overexpression or knockdown respectively induced or suppressed IL-11 expression in HeLa cells. Zac1 acted synergistically with AP-1, human papillomavirus E2, and hypoxia inducible factor 1 alpha (HIF1α). IL-11 expression under various conditions, including hypoxia or treatment with phorbol 12-myristate 13-acetate or copper sulfate. Recombinant IL-11-induced phosphorylation of signal transducer and activator of transcription 3 at tyrosine 705 was reduced in a dose-dependent manner in HeLa cells. Cross-talk between Zac1, IL-11, p53, and suppressor of cytokine signaling 3 was differentially affected by copper sulfate, digoxin, and caffeine. Finally, aggressive vs. conventional treatment of OA patients was primarily determined by IL-6 levels. However, we suggest that OA patients with higher IL-11 levels may respond well to conventional treatments, even in the presence of high IL-6.

Amiodarone promotes cancer cell death through elevated truncated SRSF3 and downregulation of miR-224.

Amiodarone is a widely used class III antiarrhythmic agent which prolongs the action potential and refractory period by blockage of several types of myocardial potassium channels. Emerging evidence suggests that amiodarone sensitize tumor cells in response to chemotherapy. Nevertheless, little is known about the underlying molecular mechanism. To gain further insight, we demonstrated that amiodarone accumulated the population of a premature termination codon-containing isoform of serine and arginine rich splicing factor 3 (SRSF3-PTC) without increasing alternative spliced p53 beta isoform. Amiodarone enhanced reactive oxygen species production and increased cell apoptosis, whereas reduced DNA damage. Moreover, amiodarone suppressed miR-224 and increased its target COX-2 expression. Taken together, our results suggested amiodarone caused cancer cell death might be through increased SRSF3-PTC and miR-224 reduction in a p53-independent manner.

Indoxyl sulfate upregulates the cannabinoid type 1 receptor gene via an ATF3/c-Jun complex-mediated signaling pathway in the model of uremic cardiomyopathy.

BACKGROUND: The risk of cardiovascular disease is notably increased in patients with chronic kidney disease (CKD) and cannabinoid receptor type 1 (CB1R) plays an important role in the development of uremic cardiomyopathy. However, the molecular mechanism underlying the uremic toxin-induced upregulation of CB1R remains elusive. METHODS: The expression of the ATF3/c-Jun complex and CB1R in both in vivo and in vitro models of CKD were measured. We also determined the impact of the ATF3/c-Jun complex on CB1R expression by transfecting H9c2 cells with dominant negative mutants of ATF3 or c-Jun. Inhibitors of organic anion transport, specific MAPK pathways and oxidative DNA damage were also used to assess the pathways mediating the effects of indoxyl sulfate (IS). RESULTS: CB1R upregulation was associated with increased ATF3 expression and c-Jun phosphorylation in CKD both in vivo and in vitro. Expression of dominant-negative ATF3 or c-Jun mutants in IS-treated cells significantly reduced CB1R mRNA levels. Moreover, Co-IP revealed that the ATF3/c-Jun complex is formed and ChIP confirmed its binding to the CB1R promoter, suggesting that this complex directly stimulates CB1R transcription in CKD. Blocking the cellular entry of IS using an organic anion transport inhibitor, as well as inhibiting the ERK1/2 and/or JNK pathways, abrogated the effects of IS on CB1R, ATF3, and c-Jun expression. The IS-induced reactive oxygen species (ROS) was observed in the mitochondria. CONCLUSIONS: We demonstrate that uremic toxins induce ATF3/c-Jun complex-mediated CB1R expression both in vivo and in vitro, possibly by modulating the ERK1/2 and JNK signaling pathways and ROS.

Dexamethasone attenuates methacholine-mediated aquaporin 5 downregulation in human nasal epithelial cells via suppression of NF-κB activation.

BACKGROUND: Cholinergic stimulation plays a major role in inflammatory airway diseases. However, its role in airway surface liquid homeostasis and aquaporin 5 (AQP5) regulation remains unclear. In this study we sought to determine the effects of methacholine and dexamethasone on AQP5 expression in human nasal epithelial cells (HNEpC). METHODS: HNEpC were cultured with methacholine or dexamethasone at 4 concentrations in vitro. The subcellular distribution of AQP5 was explored using immunocytochemistry. The pharmacologic effects of methacholine and dexamethasone on the expression of the phosphorylation of cyclic adenosine monophosphate-responsive element binding protein (p-CREB), AQP5, and nuclear factor-kappaB (NF-κB) were examined using Western blotting. RESULTS: AQP5 was found to be located in cell membrane and cytoplasm and present in every group without a statistically significant difference. Methacholine inhibited expression of AQP5 and p-CREB in HNEpC, whereas dexamethasone increased these protein levels dose-dependently in a statistically significant manner. In turn, HNEpC treated with methacholine and dexamethasone showed the same trends as those intervened separately with these 2 drugs. Moreover, dexamethasone had the ability to reverse the inhibitory effect of methacholine. Western blotting revealed that, after incubation with 10-4 mol/L methacholine, NF-κB increased significantly, by 186.67%, compared with the untreated control group. Again, such an increase could be significantly reversed after dexamethasone treatment. CONCLUSION: NF-κB activation is important for inhibition of p-CREB/AQP5 expression after methacholine intervention, and dexamethasone adjusts it to the opposite side. This observation could provide additional insight into the anti-inflammatory effects of glucocorticoids that contribute to maintaining airway surface liquid and mucosal defense.

Theophylline exhibits anti-cancer activity via suppressing SRSF3 in cervical and breast cancer cell lines.

Caffeine, theophylline, and theobromine are the most well-known members of methylxanthines. Caffeine-induced serine/arginine-rich splicing factor 2, SRSF2, and SRSF3 are required for the alternative splicing of a subset of cancer-associated genes. However, it remains to be investigated whether and how theophylline and theobromine as well as caffeine exert their antitumor effects through mediating the alternative splicing process. Here, we reveal that theophylline down-regulated SRSF3 expression and switched p53 from alpha into a beta isoform as caffeine did in HeLa and MCF-7 cells via the reverse-transcriptase polymerase chain reaction and Western blot analysis. Further functional studies show that theophylline induced cellular apoptosis, senescence, and decreased colony formation. Interestingly, theophylline had a suppressive effect on cellular proliferation, whereas caffeine enhanced cellular proliferation rates via the 5-bromo-2-deoxyuridine analysis. Theophylline and caffeine had no effect on MCF-10A cells, which is a normal breast cell line. Our results provide an insight that theophylline as well as caffeine could be repurposed as antitumor leading compounds via the downregulation of splicing factor SRSF3 and its target genes.