Repurposing the diuretic benzamil as an anti-osteosarcoma agent that acts by suppressing integrin/FAK/STAT3 signalling and compromising mitochondrial function.

Aims: Osteosarcoma is the most common primary bone malignancy among children and adolescents. We investigated whether benzamil, an amiloride analogue and sodium-calcium exchange blocker, may exhibit therapeutic potential for osteosarcoma in vitro. Methods: MG63 and U2OS cells were treated with benzamil for 24 hours. Cell viability was evaluated with the MTS/PMS assay, colony formation assay, and flow cytometry (forward/side scatter). Chromosome condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, cleavage of poly-ADP ribose polymerase (PARP) and caspase-7, and FITC annexin V/PI double staining were monitored as indicators of apoptosis. Intracellular calcium was detected by flow cytometry with Fluo-4 AM. The phosphorylation and activation of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) were measured by western blot. The expression levels of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), SOD1, and SOD2 were also assessed by western blot. Mitochondrial status was assessed with tetramethylrhodamine, ethyl ester (TMRE), and intracellular adenosine triphosphate (ATP) was measured with BioTracker ATP-Red Live Cell Dye. Total cellular integrin levels were evaluated by western blot, and the expression of cell surface integrins was assessed using fluorescent-labelled antibodies and flow cytometry. Results: Benzamil suppressed growth of osteosarcoma cells by inducing apoptosis. Benzamil reduced the expression of cell surface integrins α5, αV, and ß1 in MG63 cells, while it only reduced the expression of αV in U2OS cells. Benzamil suppressed the phosphorylation and activation of FAK and STAT3. In addition, mitochondrial function and ATP production were compromised by benzamil. The levels of anti-apoptotic proteins XIAP, Bcl-2, and Bcl-xL were reduced by benzamil. Correspondingly, benzamil potentiated cisplatin- and methotrexate-induced apoptosis in osteosarcoma cells. Conclusion: Benzamil exerts anti-osteosarcoma activity by inducing apoptosis. In terms of mechanism, benzamil appears to inhibit integrin/FAK/STAT3 signalling, which triggers mitochondrial dysfunction and ATP depletion.

Impacts of hyperthermic chemotherapeutic agent on cytotoxicity, chemoresistance-related proteins and PD-L1 expression in human gastric cancer cells.

Objective: Gastric cancer with peritoneal metastasis is considered to be final stage gastric cancer. One current treatment approach for this condition is combined cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (HIPEC). However, the therapeutic mechanisms of HIPEC remain largely undescribed. Method: In order to assess the cellular effects of HIPEC in vitro, we treated AGS human gastric adenocarcinoma cells with or without 5-fluorouracil (5-Fu) at 37 °C or at 43 °C (hyperthermic temperature) for 1 h followed by incubation at 37 °C for 23 h. The impacts of hyperthermia/5-Fu on apoptosis, cell survival signals, oxidative stress, chemoresistance-related proteins and programmed death-ligand 1 (PD-L1) expression were measured. Results: Our results showed that hyperthermia potentiates 5-Fu-mediated cytotoxicity in AGS cells. Furthermore, the combination of 5-Fu and hyperthermia reduces levels of both phosphorylated STAT3 and STAT3, while increasing the levels of phosphorylated Akt and ERK. In addition, 5-Fu/hyperthermia enhances reactive oxygen species and suppresses superoxide dismutase 1. Chemoresistance-related proteins, such as multidrug resistance 1 and thymidylate synthase, are also suppressed by 5-Fu/hyperthermia. Interestingly, hyperthermia enhances 5-Fu-mediated induction of glycosylated PD-L1, but 5-Fu-mediated upregulation of PD-L1 surface expression is prevented by hyperthermia. Conclusion: Taken together, our findings provide insights that may aid in the development of novel therapeutic strategies and enhanced therapeutic efficacy of HIPEC.

Strategic modification of low-activity natural antimicrobial peptides confers antibacterial potential in vitro and in vivo.

Antimicrobial peptides (AMPs) show great promise for clinical applications, but the utility of naturally occurring AMPs is often limited by their stability. Here, we used a rational design approach to improve the characteristics of a pair of inactive peptides, tilapia piscidin 1 and 2 (TP1 and TP2). From each starting peptide, we generated a series of novel derivatives by substituting residues to adjust cationic charge density, percent hydrophobicity and hydrophilicity/hydrophobicity coefficients. This approach yielded a novel peptide, TP2-5 (KKCIAKAILKKAKKLLKKLVNP), that exhibits significant bactericidal potency, low cytotoxicity and high stability. The designed peptide further showed antibiofilm activity, rapid antibacterial action and a low capacity to induce bacterial resistance. Importantly, we also demonstrated that TP2-5 can protect mice in a Vibrio vulnificus-infected wound model. Therefore, our peptide modification strategy successfully generated a novel AMP with high potential for future clinical application.

Magnetic-Field-Assisted Electric-Field-Induced Domain Switching of a Magnetic Single Domain in a Multiferroic/Magnetoelectric Ni Nanochevron/[Pb(Mg1/3Nb2/3)O3]0.68-[PbTiO3]0.32 (PMN-PT) Layered Structure.

We report the magnetic-field-assisted electric-field-controlled domain switching of a magnetic single domain in a multiferroic/magnetoelectric Ni nanochevrons/[Pb(Mg1/3Nb2/3)O3]0.68-[PbTiO3]0.32 (PMN-PT) layered structure. Initially, a magnetic field was applied in the transverse direction across single-domain Ni nanochevrons to transform each of them into a two-domain state. Subsequently, an electric field was applied to the layered structure, exerting the converse magnetoelectric effect to transform/release the two-domain Ni nanochevrons into one of two possible single-domain states. Finally, the experimental results showed that approximately 50% of the single-domain Ni nanochevrons were switched permanently after applying our approach (i.e., the magnetization direction was permanently rotated by 180 degrees). These results mark important advancements for future nanoelectromagnetic systems.

Dielectrically actuated liquid lens.

A packaged liquid lens driven by the dielectric force was demonstrated. The liquid lens consisted of a low dielectric constant droplet and a high dielectric constant sealing liquid. The two non-conductive liquids were sealed in a chamber under the condition of iso-density. Focal length of a liquid lens with an aperture of 3mm changed from 34mm to 12mm in the range of 0-200V. Hysteresis was observed in the liquid lens, with a maximum value measured of 12.5 degrees at 120 volts in terms of droplet's contact angle. The focal spot size measured approximately 80mum. Rise and fall times were 650ms and 300ms, respectively. The lens consumed 1mW of power when applying a 200 volt, 1 kHz signal. The longitudinal and transverse spherical aberrations were estimated to be nearly invariant when the focal length exceeded 20mm.

Variable focus dielectric liquid droplet lens.

A liquid crystal droplet lens driven by the dielectrophoresis (DEP) force was demonstrated. The liquid crystal droplet lens was deformed by the DEP forces under non-uniform AC electric fields. Focal length, hysteresis and electrode design were studied. The focal length varied from 1.6mm to 2.6mm in the range of 0-200V at 1 kHz for electrode spacing of 5 0mum; that is, the tuning ratio of the focal length was about 60% in maximum. The hysteresis of contact angle was found to be less than 3 degrees and it vanished after 1 minute at the rest state. As the electrode spacing over 200 mum, the tuning ratios of the focal length dropped below 5%. The liquid crystal droplet lens that had numerical aperture of about 0.5 consumed power of about 0.1mW. Its response time was measured to be about 220 ms.