Lanosterol synthase loss of function decreases the malignant phenotypes of HepG2 cells by deactivating the Src/MAPK signaling pathway.

Cholesterol is critical for tumor cells to maintain their membrane components, cell morphology and activity functions. The inhibition of the cholesterol pathway may be an efficient strategy with which to limit tumor growth and the metastatic process. In the present study, lanosterol synthase (LSS) was knocked down by transfecting LSS short hairpin RNA into HepG2 cells, and cell growth, apoptosis and migratory potential were then detected by Cell Counting Kit-8 cell proliferation assay, flow cytometric analysis and wound healing assay, respectively. In addition, proteins associated with the regulation of the aforementioned cell biological behaviors were analyzed by western blot analysis. The activity of the Src/MAPK signaling pathway was measured by western blotting to elucidate the possible signal transduction mechanisms. LSS knockdown in the HepG2 liver cancer cell line inhibited cell proliferation, with cell cycle arrest at the S phase; it also decreased cell migratory ability and increased apoptosis. The expression proteins involved in the regulation of cell cycle, cell apoptosis and migration was altered by LSS knockdown in HepG2 cells. Furthermore, a decreased Src/MAPK activity was observed in the HepG2 cells subjected to LSS knockdown. LSS loss of function decreased the malignant phenotypes of HepG2 cells by deactivating the Src/MAPK signaling pathway and regulating expression of genes involved in cell cycle regulation, cell apoptosis and migration.

L-asparaginase activity and anti-L-asparaginase antibody as biomarkers in estimating PEG-asp-related anaphylaxis risk in childhood acute lymphoblastic leukemia patients.

BACKGROUND: L-Asparaginase (L-asp), the unconjugated form of polyethylene glycol-conjugated L-asparaginase (PEG-asp), regulates T cell stimulation, antibody production, and lysosomal protease activity to mediate PEG-asp-related anaphylaxis. This study aimed to investigate the relation of L-asp activity and anti-L-asp antibody with anaphylaxis risk and non-anaphylaxis adverse reaction risk in childhood acute lymphoblastic leukemia (ALL) patients who underwent PEG-asp contained therapy. METHODS: In total, 170 childhood ALL patients underwent PEG-asp-contained treatment and their L-asp activity and anti-L-asp antibody were detected on the 7th day after treatment initiation. RESULTS: There were 27 (15.9%) patients who had PEG-asp-related adverse reaction: 17 (10.0%) patients experienced PEG-asp-related anaphylaxis and 14 (8.2%) patients experienced PEG- asp-related non-anaphylaxis adverse reaction. Moreover, L-asp activity was negatively related to anti-L-asp antibody in childhood ALL patients (P<0.001). Elevated L-asp activity was associated with the absence of PEG-asp-related anaphylaxis (P<0.001), PEG-asp-related non-anaphylaxis adverse reaction (P=0.004), and PEG-asp-related adverse reaction (P<0.001). However, the anti- L-asp antibody displayed opposite trend similar to L-asp activity. Receiver operating characteristic (ROC) curve analyses exhibited L-asp activity and anti-L-asp antibody exhibited superior predictive values in estimating PEG-asp-related anaphylaxis risk with area under curve (AUC) of 0.955 and 0.905, respectively compared to PEG-asp-related non-anaphylaxis adverse reaction risk with AUC of 0.730 and 0.675, respectively. Besides, patients with de novo disease, higher risk stratification, and allergic history showed trends linked with PEG-asp-related anaphylaxis risk. CONCLUSION: The monitoring of L-asp activity and anti-L-asp antibody maybe useful for early estimation and prevention of PEG-asp-related anaphylaxis in childhood ALL management.

MiR-363 suppresses the tumor growth of natural killer/T-cell lymphoma via the SIRT6/PI3K/AKT axis.

Background: Natural killer/T cell lymphoma (NKTCL) is a rare and aggressive tumor of non-Hodgkin's lymphoma. The role of micro ribonucleic acid (RNA) (miR)-363 in NKTCL has not yet been elucidated. The present study aimed to investigate the potential role of miR-363 in NKTCL. Methods: The expression of the top five differentially expressed microRNAs (miRNAs) as well as sirtuin 6 (SIRT6) in NK normal cells and its tumor cell lines were explored. The clinical tissues of NKTCL patients were collected and analyzed for expression of miR-363 and SIRT6. In addition, human NK/T-cell lymphoma cells (SNK-6) were transfected into different groups to detect cell proliferation and apoptosis abilities through cell counting kit 8 (CCK-8) experiment and flow cytometry analysis. Western blot assay was employed to examine protein expression. NKTCL nude mice models were constructed by subcutaneous injection of stably transfected SNK-6 cells to validate the mechanism of miR-363 in NKTCL via SIRT6 in vivo. Results: MiR-363 was down-regulated in NKTCL tissues and cell lines. Overexpression of miR-363 inhibited cell proliferation and promoted cell apoptosis. In contrast, SIRT6 was up-regulated in NKTCL and proved to be a downstream target of miR-363. SIRT6 could activate the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Also, miR-363 mimic could suppress the proliferation and induce the apoptosis of NKTCL via the SIRT6/PI3K/AKT axis both in vitro and in vivo. Conclusions: MiR-363 suppresses the SIRT6/PI3K/AKT pathway to restrain cell proliferation and accelerate cell apoptosis during NKTCL progression.

TWEAK/Fn14 interaction induces proliferation and migration in human airway smooth muscle cells via activating the NF-κB pathway.

Asthma, an increasingly common chronic disease among children, are characterized by airway remodeling, which is partly attributed to the proliferation and migration of airway smooth muscle cell (ASMC). The purpose of the present study was to investigate potential roles and mechanisms of the tumor necrosis factor-like weak inducer of apoptosis (TWEAK)/fibroblast growth factor-inducible molecule 14 (Fn14) axis on cell proliferation and migration in HASMCs. Compared to HASMCs from non-asthmatic patients, those from asthmatic patients showed elevated expression levels of both Fn14 and TWEAK. Additionally, similar to the response triggered by platelet-derived growth factor-BB, stimulation with recombinant TWEAK strongly induced cell proliferation and migration in HASMCs. However, depletion of Fn14 remarkably abrogated the enhancement of TWEAK on the cell proliferation and migration of HASMCs. Furthermore, treatment with TWEAK led to the activation of NF-κB. This effect was eliminated by silencing Fn14, indicating that TWEAK-induced NF-κB signaling was mediated via Fn14. Moreover, the TWEAK/Fn14 interaction promoted cell proliferation and migration. These effects were blocked by NF-κB inhibitor SN50, which suggest that the TWEAK/Fn14 signaling system partially depends on NF-κB activity. Collectively, we demonstrated that the TWEAK/Fn14 axis accelerated HASMC cell proliferation and migration by activating the NF-κB pathway, thereby exacerbating airway remodeling in asthma. Altogether, these findings indicate a novel role for the TWEAK/Fn14/NF-κB pathway as a potent option for limiting airway remodeling in asthma.

Depolymerization of cytokeratin intermediate filaments facilitates intracellular infection of HeLa cells by Bartonella henselae.

Bartonella henselae is capable of invading epithelial and endothelial cells by modulating the function of actin-dependent cytoskeleton proteins. Although understanding of the pathogenesis has been increased by the development of an in vitro infection model involving endothelial cells, little is known about the mechanism of interaction between B. henselae and epithelial cells. This study aims to identify the binding candidates of B. henselae in epithelial cells and explores their effect on B. henselae infection. Pull-down assays and mass spectrometry analysis confirmed that some of the binding proteins (keratin 14, keratin 6, and F-actin) are cytoskeleton associated. B. henselae infection significantly induces the expression of the cytokeratin genes. Chemical disruption of the keratin network by using ethylene glycol tetraacetic acid promotes the intracellular persistence of B. henselae in HeLa cells. However, cytochalasin B and phalloidin treatment inhibits B. henselae invasion. Immunofluorescent staining demonstrates that B. henselae infection induces an F-actin-dependent rearrangement of the cytoskeleton. However, we demonstrated via immunofluorescent staining and whole-mount cell electron microscopy that keratin intermediate filaments are depolymerized by B. henselae. The results indicate that B. henselae achieves an intracellular persistence in epithelial cells through the depolymerization of cytokeratin intermediate filaments that are protective against B. henselae invasion.

Staining of Bartonella henselae with carboxyfluorescein diacetate succinimidyl ester for tracking infection in erythrocytes and epithelial cells.

Bartonella infection (Bartonella henselae in particular) is responsible for a widening spectrum of human diseases. The persistent colonization of erythrocytes is a feature of Bartonella infection. Endothelial and epithelial cells are also widely used to study the pathogenesis of bartonellosis in vitro. Exploring a convenient method for visualizing the bacillus without affecting infectivity would be very interesting. Carboxyfluorescein diacetate succinimidyl ester (CFSE) has been previously used for staining several bacterial species to study their adhesion to host cells. The present study demonstrated the efficiency and safety of using CFSE in staining B. henselae. The staining of bacillus-invaded erythrocytes and epithelial cells in vitro successfully allowed for flow cytometry and confocol microscopy analyses. Parallel tests using untreated bacteria confirmed that CFSE staining did not result in side effects on the infectivity of B. henselae. Labeling Bartonella with CFSE is a valuable method for studying the bacteria-host interaction.