Targeting NOX4 Disrupts the Resistance of Papillary Thyroid Carcinoma to Chemotherapeutic Drugs and Lenvatinib

Background: Advanced differentiated thyroid cancer cells are subjected to extreme nutritional starvation which contributes to develop resistance to treatments; however, the underlying mechanism remains unclear.Methods: We used 0.5% serum to mimic starvation during cell culture. A CCK8 assay, cell death Detection ELISAPLUS kit, PI staining were measured to determine cell viability, cell apoptosis and cell cycle respectively in BCPAP cells and TPC-1 cells expressing shRNA against NOX4. The cells were then treated with etoposide and doxorubicin, two chemotherapeutic drugs, as well as lenvatinib to determine the role of NOX4 in resistance. Lenvatinib-resistant BCPAP cells (LRBCs) were also established to confirm the role. Finally, GLX351322, a chemical inhibitor targeting NOX4, was used to inhibit NOX4-derived ROS and detect the the contribution of NOX4 to resistance in vitro and in vivo. Results: NADPH oxidase 4 (NOX4) is highly expressed under serum starvation in BCPAP or TPC-1 cells. NOX4 knockdown impairs cell viability, increases cell apoptosis, extends G1 phase in cell cycle and modulates the level of energy-associated metabolites in starved cells. When these starved cells or Lenvatinib-resistant BCPAP cells (LRBCs) are treated with chemotherapeutic drugs or Lenvatinib, NOX4 knockdown inhibits cell viability and aggravates cell apoptosis depending on NOX4-derived ROS production. GLX351322, a NOX4-derived ROS inhibitor, has a significantly inhibitory effect on cell growth in vitro and the growth of BPCPA-derived even LRBCs-derived xenografts in vivo.Conclusions: These findings highlight NOX4 and NOX4-derived ROS as a potential therapeutic target in resistance of PTC patients.

Increased Inflammatory Properties of Neutrophils in Patients with Venous Thromboembolism and the Effect of Simvastatin on Abrogating Inflamed Neutrophils Adhesiveness

Neutrophils have a complex migrating process out of the vascular lumen, that consists of chemoattraction and rolling, followed by firm attachment and migration to extravascular tissues. In these sites, neutrophils are capable of promoting phagocytosis, secreting proteases, generating reactive oxygen species (ROS), and probably releasing neutrophil extracellular traps (NETs). Furthermore, neutrophil activation also induces major tissue injury associated with acute and chronic inflammatory disorders, such as the venous thromboembolism (VTE). Recently, animal models and clinical studies in acute VTE have explored the participation of neutrophils in the pathophysiology of VTE. However, VTE has been associated with a chronic inflammatory condition, and it remains unclear whether the activation of neutrophils is persistent after the acute phase of the disease. Furthermore, there are clinical evidences supporting that simvastatin may prevent VTE, since the drug has pleiotropic anti-inflammatory effects. The aim of this study is to evaluate the occurrence of neutrophil activation in patients with VTE compared to health controls and determines the effect of simvastatin in these adhesive properties of the neutrophils. Neutrophils were separated from blood collected over Ficoll-Paque densities. Neutrophils activation was determined by the expression of activated adhesive molecules (LFA-1/CD11a and MAC-1/CD11b) and ROS generation, detected by flow cytometry. Chemotaxis assays (chemoTx, Neuro Probe, Inc) and serum nucleosome, a marker of NETs, were quantified by optical density (OD) (Cell Death Detection ELISAPLUS Kit, Roche). Serum high sensitive CRP (hs-CRP) levels were measured in BN ProSpec System (Siemens) by nephelometry. For CD11a and CD11b integrins expression, cells were evaluated under basal conditions and after TNFα inflammatory stimulus, pretreated, or not, with simvastatin. For the migration assays, neutrophils were treated, or not, with IL-8, a neutrophil chemotactic factor. The results were displayed as mean and standard deviation (±SD). The study group consisted of thirty-seven patients with personal history of VTE, the median time since VTE occurred was 25 months (range 13 - 42 months), the event was spontaneous in 51.35% of the cases and 23 patients presented proximal VTE. Thirty-seven controls, matched with patients according to age, gender and ethnicity, were also included. The mean fluorescence intensity (MFI) of CD11a was higher in VTE patient neutrophils, both in basal conditions (30.84 ± 6.82 vs. 38.72 ± 22.75, P= 0.04) and after TNFα stimulus (34.09 ± 9.64 vs. 45.65 ± 33.06, P= 0.01). Higher MFI of CD11b was observed in patient neutrophils, compared with controls, only after TNFα stimulus (149.10 ± 52.74 vs. 200.0 ± 100.5, P= 0.02), and the stimulus was reverted by pre-treatment with simvastatin (200.8 ± 100.50 vs. 174.60 ± 80.63, P= 0.001). The amount of ROS (MFI) was similar in patients and controls (908.30 ± 423.7 vs. 844.0 ± 312.0, P= 0.83). Neutrophils from VTE patients also presented increased basal chemotaxis (17.55% ± 9.79 vs. 12.64% ± 4.78, P=0.02) and IL-8-stimulated chemotaxis (63.48% ± 29.73 vs. 49.88% ± 19.48%, P=0.06). Serum levels of nucleosomes were similar in patients and controls (1.05 ± 0.81 vs. 0.88 ± 0.62, P= 0.64), however higher levels of circulating nucleosomes were observed in patients with severe post-thrombotic syndrome (PTS), compared to patients with non-severe PTS, without PTS and controls (1.49 ± 0.81 vs. 1.06 ± 0.64 vs. 0.64 ± 0.60 vs. 0.88 ± 0.62, P=0.04). Furthermore, serum levels of hs-CRP were significantly higher in VTE patients when compared with controls (0.59 mg/dl ± 0.58 vs. 0.17 mg/dl ± 0.12, P=0.00). We demonstrated that patients with VTE presented patterns of neutrophil activation long time after the acute thrombotic episode. In particular, the stimuli for neutrophil adhesion and chemotaxis were higher in patients, as detected by the increased activation of adhesive molecules and cell migration. Furthermore, we observed that simvastatin may abrogate the expression of CD11b in inflamed neutrophils. Neutrophil activities associated with ROS generation and the releases of nucleosomes were not increased in these patients. The results may support the hypothesis that increased neutrophils activation is part of the chronic inflammatory condition associated with VTE and may be downregulated by the effects of simvastatin. Disclosures No relevant conflicts of interest to declare.

Apoptosis and Phagocytosis Activity of Macrophages Infected by Mycobacterium tuberculosis Resistant and Sensitive Isoniazid Clinical Isolates

Mycobacterium tuberculosis (M.tb) is the main causative pathogen that cause the pulmonary tuberculosis.Intracellular M.tb was reported able to induce macrophages apoptosis, which may have crucial role in the regulationof immun response against M.tb infection. As an intracellular bacteria, M.tb able to live and replicate withinmacrophages. Phagocytosis is the first step to achieved this condition. The induction of macrophages apoptosis byINH resistant and sensitive M.tb clinical isolates, and H37Rv was studied. The macrophages apoptosis level weremeasured using an Ag-capture ELISA for histone and fragmented DNA (Cell Death Detection ELISAplus, RocheDiagnostic GmBH). Phagocytosis activity also analyzed, after staining using fluorescence dye (AcriFluorTM, ScientificDevice Lab.). The results showed that there was no significantly different between INH resistant and sensitive M.tbclinical isolates in respect their ability to induce apoptosis. The phagocytosis activity among the clinical isolates wasshown to be strain dependent, and undistinguishable between the Mtb clinical isolates. There was no associationbetween macrophages apoptosis level and the phagocytosis activity. These data suggested that among the virulentMtb clinical isolates, the ability to induce macrophages apoptosis and phagocytosis were consistently in comparablelevelKeywords: Mycobacterium tuberculosis, apoptosis, phagocytosis, macrophages, isoniazid

Apoptosis and Phagocytosis Activity of Macrophages Infected by Mycobacterium tuberculosis Resistant and Sensitive Isoniazid Clinical Isolates

Mycobacterium tuberculosis (M.tb) is the main causative pathogen that cause the pulmonary tuberculosis.Intracellular M.tb was reported able to induce macrophages apoptosis, which may have crucial role in the regulationof immun response against M.tb infection. As an intracellular bacteria, M.tb able to live and replicate withinmacrophages. Phagocytosis is the first step to achieved this condition. The induction of macrophages apoptosis byINH resistant and sensitive M.tb clinical isolates, and H37Rv was studied. The macrophages apoptosis level weremeasured using an Ag-capture ELISA for histone and fragmented DNA (Cell Death Detection ELISAplus, RocheDiagnostic GmBH). Phagocytosis activity also analyzed, after staining using fluorescence dye (AcriFluorTM, ScientificDevice Lab.). The results showed that there was no significantly different between INH resistant and sensitive M.tbclinical isolates in respect their ability to induce apoptosis. The phagocytosis activity among the clinical isolates wasshown to be strain dependent, and undistinguishable between the Mtb clinical isolates. There was no associationbetween macrophages apoptosis level and the phagocytosis activity. These data suggested that among the virulentMtb clinical isolates, the ability to induce macrophages apoptosis and phagocytosis were consistently in comparablelevel