Dynein Function and Protein Clearance Changes in Tumor Cells Induced by a Kunitz-Type Molecule, Amblyomin-X

Amblyomin-X is a Kunitz-type recombinant protein identified from the transcriptome of the salivary glands of the tick Amblyomma cajennense and has anti-coagulant and antitumoral activity. the supposed primary target of this molecule is the proteasome system. Herein, we elucidated intracellular events that are triggered by Amblyomin-X treatment in an attempt to provide new insight into how this serine protease inhibitor, acting on the proteasome, could be comparable with known proteasome inhibitors. the collective results showed aggresome formation after proteasome inhibition that appeared to occur via the non-exclusive ubiquitin pathway. Additionally, Amblyomin-X increased the expression of various chains of the molecular motor dynein in tumor cells, modulated specific ubiquitin linkage signaling and inhibited autophagy activation by modulating mTOR, LC3 and AMBRA1 with probable dynein involvement. Interestingly, one possible role for dynein in the mechanism of action of Amblyomin-X was in the apoptotic response and its crosstalk with autophagy, which involved the factor Bim; however, we observed no changes in the apoptotic response related to dynein in the experiments performed. the characteristics shared among Amblyomin-X and known proteasome inhibitors included NF-kappa B blockage and nascent polypeptide-dependent aggresome formation. Therefore, our study describes a Kunitz-type protein that acts on the proteasome to trigger distinct intracellular events compared to classic known proteasome inhibitors that are small-cell-permeable molecules. in investigating the experiments and literature on Amblyomin-X and the known proteasome inhibitors, we also found differences in the structures of the molecules, intracellular events, dynein involvement and tumor cell type effects. These findings also reveal a possible new target for Amblyomin-X, i.e., dynein, and may serve as a tool for investigating tumor cell death associated with proteasome inhibition.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Butantan Inst, Biochem & Biophys Lab, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biochem, São Paulo, BrazilFAPESP: 2011/05969-4FAPESP: CAT/CEPID 1998/14307-9FAPESP: CETICs 2013/07467-1Web of Scienc

Amblyomin-X induces ER stress, mitochondrial dysfunction, and caspase activation in human melanoma and pancreatic tumor cell

During the last two decades, new insights into proteasome function and its role in several human diseases made it a potential therapeutic target. In this context, Amblyomin-X is a Kunitz-type FXa inhibitor similar to endogenous tissue factor pathway inhibitor (TFPI) and is a novel proteasome inhibitor. Herein, we have demonstrated Amblyomin-X cytotoxicity to different tumor cells lines such as pancreatic (Panc1, AsPC1BxPC3) and melanoma (SK-MEL-5 and SK-MEL-28). Of note, Amblyomin-X was not cytotoxic to normal human fibroblast cells. In addition, Amblyomin-X promoted accumulation of ER stress markers (GRP78 and GADD153) in sensitive (SK-MEL-28) and bortezomib-resistant (Mia-PaCa-2) tumor cells. The intracellular calcium concentration [Ca2+] (i) was slightly modulated in human tumor cells (SK-MEL-28 and Mia-PaCa-2) after 24 h of Amblyomin-X treatment. Furthermore, Amblyomin-X induced mitochondrial dysfunction, cytochrome-c release, PARP cleavage, and activation of caspase cascade in both human tumor (SK-MEL-28 and Mia-PaCa-2) cells. These investigations might help in further understanding of the antitumor properties of Amblyomin-X.Sao Paulo Research Foundation (FAPESP)National Council of Technological and Scientific Development (CNPq, INCTTox)Coordination of Improvement of Higher Education Personnel (CAPES)Uniao Quimica Farmaceutica NacionalButantan Inst, Biochem & Biophys Lab, Ave Vital Brazil 1500, BR-05503900 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biochem, Sao Paulo, SP, BrazilUniv Sao Paulo, Inst Chem, Dept Biochem, Sao Paulo, BrazilUniv Sao Paulo, Sch Med, Expt Oncol Med Invest Lab, LIM 24, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Biochem, Sao Paulo, SP, BrazilFAPESP: 2010/52669-3FAPESP: 2010/07958-7FAPESP: 2011/05969-4FAPESP: CAT/CEPID 1998/14307-9FAPESP: CETICs 2013/07467-1Web of Scienc

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research

Relationship between autophagy marker expression and dynein in the mechanism of action of Amblyomin-X.

<p>Representative western blots of whole-cell lysates of cultured cells treated with vehicle (PBS), 0.5 µM Ambly for 2 h, 4 h or 24 h, 5 µM MG-132 for 24 h or 0.2 µM rapamycin for 16 h. Images are representative of three independent experiments containing the autophagic markers (mTOR, AMBRA1, LC3-I and LC3-II) in <b>A</b>) SK-MEL-28 cells and (<b>B</b>) MIA PaCa-2 cells and (<b>C</b>) autophagic/apoptosis marker (Bim) in SK-MEL-28 cells and (<b>D</b>) MIA PaCa-2 cells. Confocal microscopy analysis of cultured cells treated with vehicle (PBS) or 0.5 µM Ambly for 24 h. The final overlay image represents five fields of three independent experiments in which (<b>E</b>) the red fluorescence represents HC1, while the green fluorescence represents mTOR and the merging of the two is in yellow; or (<b>F</b>) the red fluorescence represents LC8-1/2, while the green fluorescence represents AMBRA1 (originally, the yellow fluorescence was artificially colored by the microscope software) and the merging of the two is in yellow; or (<b>G</b>) the red fluorescence represents LC8-1/2, while the green fluorescence represents Bim and the merging of the two is in yellow.</p

Gene expression of dynein and targets related to intracellular protein quality control induced by Amblyomin-X.

<p>qPCR analysis of dynein chains and two chains of dynactin (p150Glued and dynamitin) with Ambly induction (<b>A</b>) 2 h, (<b>B</b>) 4 h and (<b>C</b>) 24 h. qPCR analysis of targets related to intracellular protein quality control with Ambly induction (<b>D</b>) for 2 h, 4 h and 24 h. Cultured cells were treated with vehicle (phosphate buffered saline, PBS) or 0.5 µM Ambly for 2 h, 4 h or 24 h. The results were calculated related to the control (vehicle) and are expressed as the means ± standard error of fold increase over control (considered as 1) in arbitrary units. Three independent experiments were performed. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant).</p

Protein expression of dynein, NFKB1 and β-actin induced by Amblyomin-X.

<p>Representative western blots of whole-cell lysates of (<b>A</b>) dynein chains in SK-MEL-28 cells, (<b>B</b>) dynein chains in MIA PaCa-2 cells, (<b>C</b>) NFKB1 and β-actin in SK-MEL-28 cells and (<b>D</b>) NFKB1 and β-actin in MIA PaCa-2 cells. Cultured cells were treated with vehicle (PBS), 0.5 µM Ambly for 2 h, 4 h or 24 h or 5 µM MG-132 (NFKB1 and β-actin). Images are representative of three independent experiments.</p

K-linkage profile and visualization of autophagy steps in the mechanism of action of Amblyomin-X.

<p>(<b>A</b>) Mean fluorescence intensity obtained from histograms of flow cytometry analysis of K48 and K63 linkage. Cultured cells were treated with vehicle (PBS) or 0.5 µM Ambly for 24 h. Results are expressed as the means ± standard error in arbitrary units of three independent experiments. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant). (<b>B</b>) Immunofluorescence analysis of autophagic membrane formation. Cultured cells were treated with vehicle (PBS), 5 µM MG-132 for 24 h, 0.2 µM rapamycin for 16 h or 0.5 µM Ambly for 24 h or 48 h. LC3 was stained with FITC and is represented in diffused green fluorescence in the cytoplasm, while the nucleus was stained with DAPI and is represented in blue. Images are representative of five fields from each experiment (n = 3). (<b>C</b>) Fluorescence microscopy analysis using acridine orange stain. Cultured cells were treated with vehicle (PBS), 5 µM MG-132 for 24 h, 0.2 µM rapamycin for 16 h, 0.5 µM Ambly for 24 h pretreated with 0.2 µM rapamycin for 16 h (rapa/Ambly) or 0.5 µM Ambly for 24 h, 48 h or 72 h. Bright red fluorescence indicates acidic vesicles, while green fluorescence indicates the cytoplasm and nucleus. White arrows indicate the zoomed area located in the upper right position of the image. Images are representative of five fields from each experiment (n = 3). (<b>D</b>) Cell viability assay of tumor cells treated with the compounds used in autophagy visualization. Results are reported as the means ± standard error of three independent experiments. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant).</p

Aggresome formation induced by Amblyomin-X.

<p>Cultured cells were treated with vehicle (PBS), 0.5 µM Ambly for 24 h, 5 µM MG-132 for 24 h, 3.5 µM CHX for 2 h or 0.5 µM Ambly for 24 h after pretreatment with 3.5 µM CHX for 2 h (CHX/Ambly). (<b>A</b>) Fluorescence microscopy analysis of aggresomes. Aggresomes were labeled with a commercial kit in red and nuclei were stained with Hoechst 33342 in blue. Images are representative of five fields from each experiment (n = 3). (<b>B</b>) Mean fluorescence intensity obtained from histograms of flow cytometry analysis of aggresomes. Results are reported as the means ± standard error of agressome propensity factor (APF) in arbitrary units calculated according to the manufacturer's instructions. Three independent experiments were performed. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant). (<b>C</b>) Cell viability assay of tumor cells treated with the compounds used in the aggresome analysis. Results are reported as the means ± standard error of three independent experiments. The criteria and representation of statistical significance were set as *p≤0.05, **p≤0.01, ***p≤0.001 or ns (non-significant).</p