Tat-SynGAP improves angiogenesis and post-stroke recovery by inhibiting MST1/JNK signaling.

Small G protein Ras induces the activation of apoptosis-related molecule mammalian Ste20-like kinase1 (MST1)/JNK signal pathway, which is involved in the regulation of tissue damage under pathological conditions such as ischemic stroke. Our previous study indicated that GTPase-activating protein for Ras (SynGAP), a negative regulator of Ras, could bind with postsynaptic density protein-93 (PSD-93) and Tat-SynGAP (670-685aa) small peptide to exhibit neuroprotective role. Here, we report that Tat-SynGAP (670-685aa) reduced cerebral edema at acute cerebral ischemia/reperfusion (I/R), improved integrity of blood-brain barrier, and decreased cortical and striatum neuronal injury. Mechanistically, Tat-SynGAP (670-685aa) not only inhibited the phosphorylation of MST1 and JNK and the cleavage of caspase-3, but also facilitated the expression of angiogenesis related molecules VEGF and Ang-1. In conclusion, Tat-SynGAP (670-685aa) reduces neuronal apoptosis and cerebral infarction volume and maintains vascular stability and blood-brain barrier integrity by inhibiting MST1/JNK signaling pathway.

PARP10 Multi-Site Auto- and Histone MARylation Visualized by Acid-Urea Gel Electrophoresis.

Poly-ADP-ribose polymerase (PARP)-family ADP-ribosyltransferases function in various signaling pathways, predominantly in the nucleus and cytosol. Although PARP inhibitors are in clinical practice for cancer therapy, the enzymatic activities of individual PARP family members are yet insufficiently understood. We studied PARP10, a mono-ADP-ribosyltransferase and potential drug target. Using acid-urea gel electrophoresis, we found that the isolated catalytic domain of PARP10 auto-ADP-ribosylates (MARylates) at eight or more acceptor residues. We isolated individual species with either singular or several modifications and then analyzed them by mass spectrometry. The results confirmed multi-site MARylation in a random order and identified four acceptor residues. The mutagenesis of singular acceptor residues had a minor impact on the overall auto-MARylation level and no effect on the MARylation of histone H3.1. Together, our results suggest that PARP10 automodification may have functions in the regulation of intramolecular or partner binding events, rather than of its enzymatic catalysis. This contributes to a better understanding of PARP10 functions, and, in the long run, to gauging the consequences of PARP inhibitor actions.

Mas receptor activation attenuates allergic airway inflammation via inhibiting JNK/CCL2-induced macrophage recruitment.

BACKGROUND: Defective absorption of acute allergic airway inflammation is involved in the initiation and development of chronic asthma. After allergen exposure, there is a rapid recruitment of macrophages around the airways, which promote acute inflammatory responses. The Ang-(1-7)/Mas receptor axis reportedly plays protective roles in various tissue inflammation and remodeling processes in vivo. However, the exact role of Mas receptor and their underlying mechanisms during the pathology of acute allergic airway inflammation remains unclear. OBJECTIVE: We investigated the role of Mas receptor in acute allergic asthma and explored its underlying mechanisms in vitro, aiming to find critical molecules and signal pathways. METHODS: Mas receptor expression was assessed in ovalbumin (OVA)-induced acute asthmatic murine model. Then we estimated the anti-inflammatory role of Mas receptor in vivo and explored expressions of several known inflammatory cytokines as well as phosphorylation levels of MAPK pathways. Mas receptor functions and underlying mechanisms were studied further in the human bronchial epithelial cell line (16HBE). RESULTS: Mas receptor expression decreased in acute allergic airway inflammation. Multiplex immunofluorescence co-localized Mas receptor and EpCAM, indicated that Mas receptor may function in the bronchial epithelium. Activating Mas receptor through AVE0991 significantly alleviated macrophage infiltration in airway inflammation, accompanied with down-regulation of CCL2 and phosphorylation levels of MAPK pathways. Further studies in 16HBE showed that AVE0991 pre-treatment inhibited LPS-induced or anisomycin-induced CCL2 increase and THP-1 macrophages migration via JNK pathways. CONCLUSION: Our findings suggested that Mas receptor activation significantly attenuated CCL2 dependent macrophage recruitments in acute allergic airway inflammation through JNK pathways, which indicated that Mas receptor, CCL2 and phospho-JNK could be potential targets against allergic airway inflammation.

LIF maintains mouse embryonic stem cells pluripotency by modulating TET1 and JMJD2 activity in a JAK2-dependent manner.

The LIF-JAK2-STAT3 pathway is the central signal transducer that maintains undifferentiated mouse embryonic stem cells (mESCs), which is achieved by the recruitment of activated STAT3 to the master pluripotency genes and activation of the gene transcriptions. It remains unclear, however, how the epigenetic status required for the master gene transcriptions is built into LIF-treated mESC cultures. In this study, Jak2, but not Stat3, in the LIF canonical pathway, establishes an open epigenetic status in the pluripotency gene promoter regions. Upon LIF activation, cytosolic JAK2 was translocalized into the nucleus of mESCs, and reduced DNA methylation (5mC levels) along with increasing DNA hydroxymethylation (5hmC) in the pluripotent gene (Nanog/Pou5f1) promoter regions. In addition, the repressive histone codes H3K9m3/H3K27m3 were reduced by JAK2. Activated JAK2 directly interacted with the core epigenetic enzymes TET1 and JMJD2, modulating its activity and promotes the DNA and histone demethylation, respectively. The JAK2 effects were attained by tyrosine phosphorylation on the epigenetic enzymes. The effects of JAK2 phosphorylation on the enzymes were diverse, but all were merged to the epigenetic signatures associated with open DNA/chromatin structures. Taken together, these results reveal a previously unrecognized epigenetic regulatory role of JAK2 as an important mediator of mESC maintenance.

A screening study of high affinity peptide as molecular binder for AXL, tyrosine kinase receptor involving in Zika virus entry.

The recent extensive spread of Zika virus has led to increased interest in the development of early diagnostic tests. To the best of our knowledge, this is the first study to demonstrate the successful use of phage display to identify affinity peptides for quantitative analysis of AXL, a tyrosine kinase receptor involved in Zika virus entry. Biopanning of M13 phage library successfully identified a high affinity peptide, with the sequence AHNHTPIKQKYL. To study the feasibility of using free peptides for molecular recognition, we synthesized a series of amino acid-substituted peptides and examined their binding affinity for AXL using electrochemical impedance spectroscopy and square wave voltammetry. Most synthetic peptides had non-identical random coil structures based on circular dichroism spectroscopy. Of the peptides tested, AXL BP1 exhibited nanomolar binding affinity for AXL. To verify whether AXL BP1 could be used as a peptide inhibitor at the cellular level, two functional tests were carried out: a WST assay for cell viability and qRT-PCR for quantification of RNA levels in Zika virus-infected Huh7 cells. The results showed that AXL BP1 had low cytotoxicity and could block Zika virus entry. These results indicate that newly identified affinity peptides could potentially be used for the development of Zika virus entry inhibitors.

Downregulation of PUMA underlies resistance to FGFR1 inhibitors in the stem cell leukemia/lymphoma syndrome.

Resistance to molecular therapies frequently occur due to genetic changes affecting the targeted pathway. In myeloid and lymphoid leukemias/lymphomas resulting from constitutive activation of FGFR1 kinases, resistance has been shown to be due either to mutations in FGFR1 or deletions of PTEN. RNA-Seq analysis of the resistant clones demonstrates expression changes in cell death pathways centering on the p53 upregulated modulator of apoptosis (Puma) protein. Treatment with different tyrosine kinase inhibitors (TKIs) revealed that, in both FGFR1 mutation and Pten deletion-mediated resistance, sustained Akt activation in resistant cells leads to compromised Puma activation, resulting in suppression of TKI-induced apoptosis. This suppression of Puma is achieved as a result of sequestration of inactivated p-Foxo3a in the cytoplasm. CRISPR/Cas9 mediated knockout of Puma in leukemic cells led to an increased drug resistance in the knockout cells demonstrating a direct role in TKI resistance. Since Puma promotes cell death by targeting Bcl2, TKI-resistant cells showed high Bcl2 levels and targeting Bcl2 with Venetoclax (ABT199) led to increased apoptosis in these cells. In vivo treatment of mice xenografted with resistant cells using ABT199 suppressed leukemogenesis and led to prolonged survival. This in-depth survey of the underlying genetic mechanisms of resistance has identified a potential means of treating FGFR1-driven malignancies that are resistant to FGFR1 inhibitors.

Simvastatin ameliorates altered mechanotransduction in uterine leiomyoma cells.

BACKGROUND: Uterine leiomyomas, the most common tumors of the female reproductive system, are characterized by excessive deposition of disordered stiff extracellular matrix and fundamental alteration in the mechanical signaling pathways. Specifically, these alterations affect the normal dynamic state of responsiveness to mechanical cues in the extracellular environment. These mechanical cues are converted through integrins, cell membrane receptors, to biochemical signals including cytoskeletal signaling pathways to maintain mechanical homeostasis. Leiomyoma cells overexpress ß1 integrin and other downstream mechanical signaling proteins. We previously reported that simvastatin, an antihyperlipidemic drug, has antileiomyoma effects through cellular, animal model, and epidemiologic studies. OBJECTIVE: This study aimed to examine the hypothesis that simvastatin might influence altered mechanotransduction in leiomyoma cells. STUDY DESIGN: This is a laboratory-based experimental study. Primary leiomyoma cells were isolated from 5 patients who underwent hysterectomy at the Department of Gynecology and Obstetrics of the Johns Hopkins University Hospital. Primary and immortalized human uterine leiomyoma cells were treated with simvastatin at increasing concentrations (0.001, 0.01, 0.1, and 1 µM, or control) for 48 hours. Protein and mRNA levels of ß1 integrin and extracellular matrix components involved in mechanical signaling were quantified by quantitative real-time polymerase chain reaction, western blotting, and immunofluorescence. In addition, we examined the effect of simvastatin on the activity of Ras homolog family member A using pull-down assay and gel contraction. RESULTS: We found that simvastatin significantly reduced the protein expression of ß1 integrin by 44% and type I collagen by 60% compared with untreated leiomyoma cells. Simvastatin-treated cells reduced phosphorylation of focal adhesion kinase down to 26%-60% of control, whereas it increased total focal adhesion kinase protein expression. Using a Ras homolog family member A pull-down activation assay, we observed reduced levels of active Ras homolog family member A in simvastatin-treated cells by 45%-85% compared with control. Consistent with impaired Ras homolog family member A activation, simvastatin treatment reduced tumor gel contraction where gel area was 122%-153% larger than control. Furthermore, simvastatin treatment led to reduced levels of mechanical signaling proteins involved in ß1 integrin downstream signaling, such as A-kinase anchor protein 13, Rho-associated protein kinase 1, myosin light-chain kinase, and cyclin D1. CONCLUSION: The results of this study suggest a possible therapeutic role of simvastatin in restoring the altered state of mechanotransduction signaling in leiomyoma. Collectively, these findings are aligned with previous epidemiologic studies and other reports and support the need for clinical trials.

Evaluation of VTP-50469, a menin-MLL1 inhibitor, against Ewing sarcoma xenograft models by the pediatric preclinical testing consortium.

BACKGROUND: VTP-50469 is a potent inhibitor of the menin-MLL1 interaction and is implicated in signaling downstream of EWSR1-FLI1. PROCEDURE: VTP-50469 was evaluated against seven Ewing sarcoma (EwS) xenograft models and in vitro against EwS cell lines. RESULTS: VTP-50469 showed limited antitumor activity, statistically significantly slowing tumor progression in four tumor models but with no evidence of tumor regression. In vitro, the IC50 concentration was 10 nM for the mixed lineage leukemia (MLL)-rearranged leukemia cell line MV4;11, but > 3 µM for EwS cell lines. CONCLUSIONS: In contrast to its high level of activity against MLL1-rearranged leukemia xenografts, VTP-50469 shows little activity against EwS models.

miR-543 regulates the epigenetic landscape of myelofibrosis by targeting TET1 and TET2.

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAKV617F mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK2V617F inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-543 was significantly upregulated in nonresponders. We validated these findings by reverse transcription-quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2V617F mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options.

Compound C induces autophagy and apoptosis in parental and hydroquinone-selected malignant leukemia cells through the ROS/p38 MAPK/AMPK/TET2/FOXP3 axis.

Hydroquinone (HQ), a major metabolic product of benzene, causes acute myeloid leukemia (AML) elicited by benzene exposure. Past studies found that continuous exposure of human AML U937 cells to HQ selectively produces malignant U937/HQ cells in which FOXP3 upregulation modulates malignant progression. Other studies revealed that AMPK promotes TET2 activity on DNA demethylation and that TET2 activity is crucial for upregulating FOXP3 expression. This study was conducted to elucidate whether compound C, an AMPK inhibitor, blocked the AMPK-TET2-FOXP3 axis in AML and in HQ-selected malignant cells. We found higher levels of AMPKα, TET2, and FOXP3 expression in U937/HQ cells compared to U937 cells. Treatment of parental Original Article and HQ-selected malignant U937 cells with compound C induced ROS-mediated p38 MAPK activation, leading to a suppression of AMPKα, TET2, and FOXP3 expression. Moreover, compound C induced apoptosis and mTOR-independent autophagy. The suppression of the autophagic flux inhibited the apoptosis of compound C-treated U937 and U937/HQ cells, whereas co-treatment with rapamycin, a mTOR inhibitor, sensitized the two cell lines to compound C cytotoxicity. Overexpression of AMPKα1 or pretreatment with autophagic inhibitors abrogated compound C-induced autophagy and suppression of TET2 and FOXP3 expression. Restoration of AMPKα1 or FOXP3 expression increased cell survival after treatment with compound C. In conclusion, our results show that compound C suppresses AMPK/TET2 axis-mediated FOXP3 expression and induces autophagy-dependent apoptosis in parental and HQ-selected malignant U937 cells, suggesting that the AMPK/TET2/FOXP3 axis is a promising target for improving AML therapy and attenuating benzene exposure-induced AML progression.

A Menin-MLL Inhibitor Induces Specific Chromatin Changes and Eradicates Disease in Models of MLL-Rearranged Leukemia.

Inhibition of the Menin (MEN1) and MLL (MLL1, KMT2A) interaction is a potential therapeutic strategy for MLL-rearranged (MLL-r) leukemia. Structure-based design yielded the potent, highly selective, and orally bioavailable small-molecule inhibitor VTP50469. Cell lines carrying MLL rearrangements were selectively responsive to VTP50469. VTP50469 displaced Menin from protein complexes and inhibited chromatin occupancy of MLL at select genes. Loss of MLL binding led to changes in gene expression, differentiation, and apoptosis. Patient-derived xenograft (PDX) models derived from patients with either MLL-r acute myeloid leukemia or MLL-r acute lymphoblastic leukemia (ALL) showed dramatic reductions of leukemia burden when treated with VTP50469. Multiple mice engrafted with MLL-r ALL remained disease free for more than 1 year after treatment. These data support rapid translation of this approach to clinical trials.

Regulation of GSK3 cellular location by FRAT modulates mTORC1-dependent cell growth and sensitivity to rapamycin.

The mTORC1 pathway regulates cell growth and proliferation by properly coupling critical processes such as gene expression, protein translation, and metabolism to the availability of growth factors and hormones, nutrients, cellular energetics, oxygen status, and cell stress. Although multiple cytoplasmic substrates of mTORC1 have been identified, how mTORC1 signals within the nucleus remains incompletely understood. Here, we report a mechanism by which mTORC1 modulates the phosphorylation of multiple nuclear events. We observed a significant nuclear enrichment of GSK3 when mTORC1 was suppressed, which promotes phosphorylation of several proteins such as GTF2F1 and FOXK1. Importantly, nuclear localization of GSK3 is sufficient to suppress cell proliferation. Additionally, expression of a nuclear exporter of GSK3, FRAT, restricts the nuclear localization of GSK3, represses nuclear protein phosphorylation, and prevents rapamycin-induced cytostasis. Finally, we observe a correlation between rapamycin resistance and FRAT expression in multiple-cancer cell lines. Resistance to Food and Drug Administration (FDA)-approved rapamycin analogs (rapalogs) is observed in many tumor settings, but the underling mechanisms remain incompletely understood. Given that FRAT expression levels are frequently elevated in various cancers, our observations provide a potential biomarker and strategy for overcoming rapamycin resistance.

Interaction of N-acetyl-seryl-aspartyl-lysyl-proline with the angiotensin-converting enzyme 2-angiotensin-(1-7)-Mas axis attenuates pulmonary fibrosis in silicotic rats.

NEW FINDINGS: What is the central question of this study? What are the effects of the antifibrotic peptide acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) on the angiotensin-converting enzyme 2 (ACE2)-angiotensin-(1-7)-Mas axis during the occurrence and progression of silicosis? What is the main finding and its importance? Ac-SDKP inhibited lung fibrosis in rats exposed to silica by activation of the ACE2-angiotensin-(1-7)-Mas axis. Angiotensin-(1-7) potentially promotes Ac-SDKP by increasing the level of meprin α, the major synthetase of Ac-SDKP. Thus, the interaction Ac-SDKP and angiotesin-(1-7) in silicosis could provide a new therapeutic strategy. ABSTRACT: The central role of angiotensin-converting enzyme (ACE) in the occurrence and progression of silicosis has been established. The antifibrotic peptide acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) can be degraded by ACE. The ACE2-angiotensin-(1-7)-Mas axis is protective and acts to counterbalance the detrimental effects of ACE-angiotensin II (Ang II)-Ang II type 1 receptor and exerts antifibrotic effects. Here, we demonstrate an interaction between Ac-SDKP and Ang-(1-7) in the inhibition of collagen deposition and myofibroblast differentiation in rats exposed to silica. Treatment with Ac-SDKP increased the level of ACE2-Ang-(1-7)-Mas in rats or in cultured fibroblasts and decreased the levels of collagen type I and α-smooth muscle actin. Furthermore, exogenous Ang-(1-7) had similar antifibrotic effects and increased the level of meprin α, a major Ac-SDKP synthetase, both in vivo and in vitro. Compared with non-silicotic patients exposed to silica, the level of serum ACE was increased in patients with silicosis phase III; the levels of Ang II and Ang-(1-7) were high in patients with silicosis phase II; and the level of Ac-SDKP was high in the silicosis phase III group. These data imply that Ac-SDKP and Ang-(1-7) have an interactive effect as regulatory peptides of the renin-angiotensin system and exert antifibrotic effects.

Repression of AXL expression by AP-1/JNK blockage overcomes resistance to PI3Ka therapy.

AXL overexpression is a common resistance mechanism to anti-cancer therapies, including the resistance to BYL719 (Alpelisib) - the p110α isoform specific inhibitor of phosphoinositide 3-kinase (PI3K) - in esophagus and head and neck squamous cell carcinoma (ESCC, HNSCC respectively). However, the mechanisms underlying AXL overexpression in resistance to BYL719 remain elusive. Here we demonstrated that the AP-1 transcription factors, c-JUN and c-FOS, regulate AXL overexpression in HNSCC and ESCC. The expression of AXL was correlated with that of c-JUN both in HNSCC patients and in HNSCC and ESCC cell lines. Silencing of c-JUN and c-FOS expression in tumor cells downregulated AXL expression and enhanced the sensitivity of human papilloma virus positive (HPVPos) and negative (HPVNeg) tumor cells to BYL719 in vitro. Blocking of the c-JUN N-terminal kinase (JNK) using SP600125 in combination with BYL719 showed a synergistic anti-proliferative effect in vitro, which was accompanied by AXL downregulation and potent inhibition of the mTOR pathway. In vivo, the BYL719-SP600125 drug combination led to the arrest of tumor growth in cell line-derived and patient-derived xenograft models, and in syngeneic head and neck murine cancer models. Collectively, our data suggests that JNK inhibition in combination with anti-PI3K therapy is a new therapeutic strategy that should be tested in HPVPos and HPVNeg HNSCC and ESCC patients.

Successful Treatment of ROS1-rearranged Lung Cancer Complicated by Hypertrophic Pulmonary Osteoarthropathy with Crizotinib Therapy.

Hypertrophic pulmonary osteoarthropathy (HPO) is a paraneoplastic syndrome characterized by digital clubbing, arthritis, and periostitis. Tumor removal usually leads to the resolution of these symptoms. We herein report the efficacy of crizotinib treatment for treating the symptoms of HPO associated with c-ros oncogene 1 receptor tyrosine kinase (ROS1)-rearranged lung cancer. A 71-year-old woman presented with a pulmonary tumor and arthritis. She was diagnosed with a ROS1-rearranged lung adenocarcinoma [stage IIIB (cT4N2M0) ] with HPO. Crizotinib dramatically reduced the tumor size and resolved the symptoms. After two months of crizotinib treatment, she underwent lobectomy, and a pathological evaluation revealed ypstage IIIA (ypT3a, ypN1). Crizotinib treatment was effective for reducing the tumor size and improving the symptoms of HPO.

Anti-hypersensitive effect of angiotensin (1-7) on streptozotocin-induced diabetic neuropathic pain in mice.

BACKGROUND: We have recently reported that the spinal angiotensin (Ang) converting enzyme (ACE)/Ang II/AT1 receptor axis and downstream p38 MAPK phosphorylation are activated in streptozotocin (STZ)-induced diabetic mice and lead to tactile hypersensitivity. Moreover, our previous results suggested that the intrathecal (i.t.) administration of Ang (1-7), an N-terminal fragment of Ang II, may attenuate the Ang II-induced nociceptive behaviour through the inhibition of p38 MAPK phosphorylation via Mas receptors. Here, we investigated whether the i.t. administration of Ang (1-7) can attenuate STZ-induced diabetic neuropathic pain. METHODS: Tactile and thermal hypersensitivities were determined using the von Frey filament and Hargreaves tests, respectively. The protein expression of ACE2, Mas receptors and phospho-p38 MAPK was measured by western blotting. Spinal ACE2 activity was determined using ACE2 activity assay kit. RESULTS: The i.t. administration of Ang (1-7) significantly reduced the tactile and thermal hypersensitivities on day 14 after STZ injection, and these effects were significantly prevented by the Mas receptor antagonist A779. The expression of ACE2 and Mas receptors in the plasma membrane fraction of the lumbar dorsal spinal cord was both significantly decreased in STZ mice. Spinal ACE2 activity was also decreased while p38 MAPK phosphorylation was increased in the lumbar dorsal region of these mice. This phosphorylation was attenuated by the injection of Ang (1-7), whose effect was reversed by A779. CONCLUSIONS: Our data demonstrate that Ang (1-7) attenuates STZ-induced diabetic neuropathic pain and that this occurs through a mechanism involving spinal Mas receptors and he inhibition of p38 MAPK phosphorylation. SIGNIFICANCE: The ACE2/Ang (1-7)/Mas receptor axis was down-regulated in the spinal cord of STZ mice and the i.t. administration of Ang (1-7) attenuated the STZ-induced diabetic neuropathic pain via Mas receptors. Therefore, the activation of this axis could be an effective therapeutic target to alleviate the neuropathic pain in diabetic patients.

Elevation of tumor mutation burden in ROS1-fusion lung adenocarcinoma resistant to crizotinib: A case report.

RATIONALE: Although most of non-small cell lung cancer (NSCLC) patients with ROS1-fusions respond to crizotinb, acquired resistance eventually develop. The next-generations of ROS1 inhibitors have made some achievements, but the effects of immunotherapy have not been explored. PATIENT CONCERNS: A 44-year-old Chinese women presented with cough and dyspnea with a history of advanced lung adenocarcinoma. DIAGNOSIS: A PET/CT scan revealed primary tumors in bilateral lung lobes and multiple metastases in lymph nodes and bones. And ultrasound-guided left cervical lymph node biopsy revealed the pathological diagnosis was poor differentiated lung adenocarcinoma. INTERVENTIONS: The patients was started to be treated with 4 cycles of pemetrexed, carboplatin and bevacizumab, followed by one cycle of docetaxel, cisplatin and bevacizumab. As the ROS1-fusion was found by next generation sequencing, the patient received crizotinib treatment about 3 months. OUTCOMES: After 5 cycles of chemotherapy, CT scans revealed increased size of bilateral lobe nodules indicative of progressive disease (PD). Then the patient received treatment of crizotinib and his progression-free survival reached 3 months. Due to uncontrollable disease progression, the patient expired. LESSONS: The genetic profile of NSCLC patients might be altered in various therapeutic processes. Thus, repeated genetic testing might be important at each progression. Moreover, immunotherapy might be a powerful weapon to overcome the resistance to Tyrosine kinase inhibitors (TKIs) in future.

Protective roles of autophagy in retinal pigment epithelium under high glucose condition via regulating PINK1/Parkin pathway and BNIP3L.

BACKGROUND: Our study aimed to investigate the roles of autophagy against high glucose induced response in retinal pigment epithelium (ARPE-19 cells). METHODS: The morphological changes and reactive oxygen species (ROS) generation in ARPE-19 cells under high glucose treatment were respectively detected using the transmission electron microscopy and flow cytometry. The expression levels of Parkin, PINK1, BNIP3L, LC3-I and LC3-II in ARPE-19 cells received high glucose treatment were measured by western blot after pretreatment of carbonyl cyanide m-chlorophenylhydrazone (CCCP), 3-methyladenine (3-MA), N-acetyl cysteine (NAC) or cyclosporin A (CsA) followed by high glucose treatment. RESULTS: ARPE-19 cells subjected to high glucose stress showed an obvious reduction in the LC3-I expression and significant increase in the number of autophagosomes, in the intracellular ROS level, and in the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). Pretreatment with CCCP significantly reduced the LC3-I expression and increased the expression levels of Parkin, PINK1, BNIP3L and LC3-II (p < 0.05). ARPE-19 cells pretreated with CsA under high glucose stress showed markedly down-regulated expressions of Parkin, PINK1 and BNIP3L compared with the cells treated with high glucose (p < 0.05). Pretreatment of ARPE-19 cells with NAC or 3-MA under high glucose stress resulted in a marked reduction in the expression levels of PINK1, BNIP3L and LC3-II (p < 0.05). Meanwhile, the expression level of Parkin in the ARPE-19 cells pretreated with NAC under high glucose stress was comparable with that in the control cells. CONCLUSION: Autophagy might have protective roles against high glucose induced injury in ARPE19 cells via regulating PINK1/Parkin pathway and BNIP3L.

The comparative efficacy of renin-angiotensin system blockers in schistosomal hepatic fibrosis.

Schistosomiasis mansoni is involved in hepatic fibrogenesis and portal hypertension. Previous studies proved that blockade of some components of the renin-angiotensin system (RAS) reduce liver fibrogenesis. However, the effects of inhibition of early stages of RAS pathway in schistosomal fibrosis have not been studied yet. Thus, the aim of this study was to compare the role of different antihypertensive drugs on hepatic fibrosis in murine schistosomiasis. BALB/c mice (n = 50) weighing 20g were subjected to inoculation of 50 cercariae and submitted to different treatments: aliskiren, 50 mg/kg (n = 10); bradykinin, 2 µg/kg (n = 5); losartan, 10 mg/kg (n = 10); lisinopril 10 mg/kg (n = 5) and control, proportional volume vehicle (n = 5); daily for 14 weeks. Six animals were not subjected to cercariae inoculation or any type of treatment. Ultrasound, histological, immunohistochemical and proteomic analyzes were performed to evaluate markers associated with hepatic fibrogenesis. The hepatic areas stained with Sirius red and thenumber of cells marked by α-SMA in animals treated with aliskiren, bradykinin, lisinopril and losartan were diminished when compared to control group, demonstrating reduced hepatic fibrosis after RAS blockade. These results were reinforced by ultrasonography analysis and protein expression of TGFß. These findings demonstrated the effect of RAS inhibition on hepatic fibrosis in murine schistosomiasis, with the most evident results being observed in the losartan and aliskiren treated groups. The main mechanisms underlying this process appear to involve anti-fibrogenic activity through the inhibition of collagen and TGFß synthesis.

Sorafenib induces renal cell carcinoma apoptosis via upregulating activating transcription factor 4.

Previous studies have shown sorafenib to function as a multitargeted tyrosine kinase inhibitor in different tumors. However, whether sorafenib improves renal cell carcinoma (RCC) through activating transcription factor 4 (ATF4) has never been explored. In the current study, we showed that sorafenib could suppress RCC cell viability in a time- and dose-dependent manner. Furthermore, sorafenib is demonstrated to enhance the mRNA and protein levels of ATF4. Meanwhile, overexpression of ATF4 was demonstrated to induce ACHN cell cycle arrest and cell apoptosis. Moreover, treatment with sorafenib could enhance the expression of CCAAT/enhancer-binding protein-homologous protein (CHOP) and p53 upregulated modulator of apoptosis (PUMA), thereby leading to ACHN cell apoptosis. More importantly, silencing of ATF4 could largely abolish sorafenib-induced upregulation of CHOP and PUMA in ACHN cells. Meanwhile, sorafenib-induced cell apoptosis may be dependent on the activation of ATF4 since knockdown of ATF4 partially reversed sorafenib-induced ACHN cell apoptosis. In summary, the present study demonstrates that sorafenib activates ATF4-CHOP-PUMA pathway in RCC cells, resulting in enhanced ER stress-related cell apoptosis.