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.

Ago2/CAV1 interaction potentiates metastasis via controlling Ago2 localization and miRNA action.

Ago2 differentially regulates oncogenic and tumor-suppressive miRNAs in cancer cells. This discrepancy suggests a secondary event regulating Ago2/miRNA action in a context-dependent manner. We show here that a positive charge of Ago2 K212, that is preserved by SIR2-mediated Ago2 deacetylation in cancer cells, is responsible for the direct interaction between Ago2 and Caveolin-1 (CAV1). Through this interaction, CAV1 sequesters Ago2 on the plasma membranes and regulates miRNA-mediated translational repression in a compartment-dependent manner. Ago2/CAV1 interaction plays a role in miRNA-mediated mRNA suppression and in miRNA release via extracellular vesicles (EVs) from tumors into the circulation, which can be used as a biomarker of tumor progression. Increased Ago2/CAV1 interaction with tumor progression promotes aggressive cancer behaviors, including metastasis. Ago2/CAV1 interaction acts as a secondary event in miRNA-mediated suppression and increases the complexity of miRNA actions in cancer.

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.

Evaluation of the Antibacterial Activity of Eco-Friendly Hybrid Composites on the Base of Oyster Shell Powder Modified by Metal Ions and LLDPE.

Transforming biological waste into high-value-added materials is currently attracting extensive research interest in the medical and industrial treatment fields. The design and use of new antibacterial systems are urgently needed. In this study, we used discarded oyster shell powder (OSP) to prepare calcium oxide (CaO). CaO was mixed with silver (Ag), zinc (Zn), and copper (Cu) ions as a controlled release and antibacterial system to test the antibacterial activity. The inhibition zones of various modified metals were between 22 and 29 mm for Escherichia coli (E. coli) and between 21 and 24 mm for Staphylococcus aureus (S. aureus). In addition, linear low-density polyethylene (LLDPE) combined with CaO and metal ion forms can be an excellent alternative to a hybrid composite. The strength modulus at 1% LLDPE to LLDPE/CaO Ag increased from 297 to 320 MPa. In addition, the antimicrobial activity of LLDPE/CaO/metal ions against E. coli had an antibacterial effect of about 99.9%. Therefore, this hybrid composite material has good potential as an antibacterial therapy and biomaterial suitable for many applications.

Marine Antimicrobial Peptide TP4 Exerts Anticancer Effects on Human Synovial Sarcoma Cells via Calcium Overload, Reactive Oxygen Species Production and Mitochondrial Hyperpolarization.

Synovial sarcoma is a rare but aggressive soft-tissue sarcoma associated with translocation t(X;18). Metastasis occurs in approximately 50% of all patients, and curative outcomes are difficult to achieve in this group. Since the efficacies of current therapeutic approaches for metastatic synovial sarcoma remain limited, new therapeutic agents are urgently needed. Tilapia piscidin 4 (TP4), a marine antimicrobial peptide, is known to exhibit multiple biological functions, including anti-bacterial, wound-healing, immunomodulatory, and anticancer activities. In the present study, we assessed the anticancer activity of TP4 in human synovial sarcoma cells and determined the underlying mechanisms. We first demonstrated that TP4 can induce necrotic cell death in human synovial sarcoma AsKa-SS and SW982 cells lines. In addition, we saw that TP4 initiates reactive oxygen species (ROS) production and downregulates antioxidant proteins, such as uncoupling protein-2, superoxide dismutase (SOD)-1, and SOD-2. Moreover, TP4-induced mitochondrial hyperpolarization is followed by elevation of mitochondrial ROS. Calcium overload is also triggered by TP4, and cell death can be attenuated by a necrosis inhibitor, ROS scavenger or calcium chelator. In our experiments, TP4 displayed strong anticancer activity in human synovial sarcoma cells by disrupting oxidative status, promoting mitochondrial hyperpolarization and causing calcium overload.

Exosomal 2',3'-CNP from mesenchymal stem cells promotes hippocampus CA1 neurogenesis/neuritogenesis and contributes to rescue of cognition/learning deficiencies of damaged brain.

Mesenchymal stem cells (MSCs) have been used in clinical studies to treat neurological diseases and damage. However, implanted MSCs do not achieve their regenerative effects by differentiating into and replacing neural cells. Instead, MSC secretome components mediate the regenerative effects of MSCs. MSC-derived extracellular vesicles (EVs)/exosomes carry cargo responsible for rescuing brain damage. We previously showed that EP4 antagonist-induced MSC EVs/exosomes have enhanced regenerative potential to rescue hippocampal damage, compared with EVs/exosomes from untreated MSCs. Here we show that EP4 antagonist-induced MSC EVs/exosomes promote neurosphere formation in vitro and increase neurogenesis and neuritogenesis in damaged hippocampi; basal MSC EVs/exosomes do not contribute to these regenerative effects. 2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) levels in EP4 antagonist-induced MSC EVs/exosomes are 20-fold higher than CNP levels in basal MSC EVs/exosomes. Decreasing elevated exosomal CNP levels in EP4 antagonist-induced MSC EVs/exosomes reduced the efficacy of these EVs/exosomes in promoting ß3-tubulin polymerization and in converting toxic 2',3'-cAMP into neuroprotective adenosine. CNP-depleted EP4 antagonist-induced MSC EVs/exosomes lost the ability to promote neurogenesis and neuritogenesis in damaged hippocampi. Systemic administration of EV/exosomes from EP4 -antagonist derived MSC EVs/exosomes repaired cognition, learning, and memory deficiencies in mice caused by hippocampal damage. In contrast, CNP-depleted EP4 antagonist-induced MSC EVs/exosomes failed to repair this damage. Exosomal CNP contributes to the ability of EP4 antagonist-elicited MSC EVs/exosomes to promote neurogenesis and neuritogenesis in damaged hippocampi and recovery of cognition, memory, and learning. This experimental approach should be generally applicable to identifying the role of EV/exosomal components in eliciting a variety of biological responses.

EP4 Antagonist-Elicited Extracellular Vesicles from Mesenchymal Stem Cells Rescue Cognition/Learning Deficiencies by Restoring Brain Cellular Functions.

Adult brains have limited regenerative capacity. Consequently, both brain damage and neurodegenerative diseases often cause functional impairment for patients. Mesenchymal stem cells (MSCs), one type of adult stem cells, can be isolated from various adult tissues. MSCs have been used in clinical trials to treat human diseases and the therapeutic potentials of the MSC-derived secretome and extracellular vesicles (EVs) have been under investigation. We found that blocking the prostaglandin E2 /prostaglandin E2 receptor 4 (PGE2 /EP4 ) signaling pathway in MSCs with EP4 antagonists increased EV release and promoted the sorting of specific proteins, including anti-inflammatory cytokines and factors that modify astrocyte function, blood-brain barrier integrity, and microglial migration into the damaged hippocampus, into the EVs. Systemic administration of EP4 antagonist-elicited MSC EVs repaired deficiencies of cognition, learning and memory, inhibited reactive astrogliosis, attenuated extensive inflammation, reduced microglial infiltration into the damaged hippocampus, and increased blood-brain barrier integrity when administered to mice following hippocampal damage. Stem Cells Translational Medicine 2019.

PGE2 /EP4 antagonism enhances tumor chemosensitivity by inducing extracellular vesicle-mediated clearance of cancer stem cells.

Cells expressing mesenchymal/basal phenotypes in tumors have been associated with stem cell properties. Cancer stem cells (CSCs) are often resistant to conventional chemotherapy. We explored overcoming mesenchymal CSC resistance to chemotherapeutic agents. Our goal was to reduce CSC numbers in vivo, in conjunction with chemotherapy, to reduce tumor burden. Analysis of clinical samples demonstrated that COX-2/PGE2 /EP4 signaling is elevated in basal-like and chemoresistant breast carcinoma and is correlated with survival and relapse of breast cancer. EP4 antagonism elicts a striking shift of breast cancer cells from a mesenchymal/CSC state to a more epithelial non-CSC state. The transition was mediated by EP4 antagonist-induced extracellular vesicles [(EVs)/exosomes] which removed CSC markers, mesenchymal markers, integrins, and drug efflux transporters from the CSCs. In addition, EP4 antagonism-induced CSC EVs/exosomes can convert tumor epithelial/non-CSCs to mesenchymal/CSCs able to give rise to tumors and to promote tumor cell dissemination. Because of its ability to induce a CSC-to-non-CSC transition, EP4 antagonist treatment in vivo reduced the numbers of CSCs within tumors and increased tumor chemosensitivity. EP4 antagonist treatment enhances tumor response to chemotherapy by reducing the numbers of chemotherapy-resistant CSCs available to repopulate the tumor. EP4 antagonism can collaborate with conventional chemotherapy to reduce tumor burden.

Transfer of Mammary Gland-forming Ability Between Mammary Basal Epithelial Cells and Mammary Luminal Cells via Extracellular Vesicles/Exosomes.

Cells can communicate via exosomes, ~100-nm extracellular vesicles (EVs) that contain proteins, lipids, and nucleic acids. Non-adherent/mesenchymal mammary epithelial cell (NAMEC)-derived extracellular vesicles can be isolated from NAMEC medium via differential ultracentrifugation. Based on their density, EVs can be purified via ultracentrifugation at 110,000 x g. The EV preparation from ultracentrifugation can be further separated using a continuous density gradient to prevent contamination with soluble proteins. The purified EVs can then be further evaluated using nanoparticle-tracking analysis, which measures the size and number of vesicles in the preparation. The extracellular vesicles with a size ranging from 50 to 150 nm are exosomes. The NAMEC-derived EVs/exosomes can be ingested by mammary epithelial cells, which can be measured by flow cytometry and confocal microscopy. Some mammary stem cell properties (e.g., mammary gland-forming ability) can be transferred from the stem-like NAMECs to mammary epithelial cells via the NAMEC-derived EVs/exosomes. Isolated primary EpCAMhi/CD49flo luminal mammary epithelial cells cannot form mammary glands after being transplanted into mouse fat pads, while EpCAMlo/CD49fhi basal mammary epithelial cells form mammary glands after transplantation. Uptake of NAMEC-derived EVs/exosomes by EpCAMhi/CD49flo luminal mammary epithelial cells allows them to generate mammary glands after being transplanted into fat pads. The EVs/exosomes derived from stem-like mammary epithelial cells transfer mammary gland-forming ability to EpCAMhi/CD49flo luminal mammary epithelial cells.

PGE2 /EP4 Signaling Controls the Transfer of the Mammary Stem Cell State by Lipid Rafts in Extracellular Vesicles.

Prostaglandin E2 (PGE2 )-initiated signaling contributes to stem cell homeostasis and regeneration. However, it is unclear how PGE2 signaling controls cell stemness. This study identifies a previously unknown mechanism by which PGE2 /prostaglandin E receptor 4 (EP4 ) signaling regulates multiple signaling pathways (e.g., PI3K/Akt signaling, TGFß signaling, Wnt signaling, EGFR signaling) which maintain the basal mammary stem cell phenotype. A shift of basal mammary epithelial stem cells (MaSCs) from a mesenchymal/stem cell state to a non-basal-MaSC state occurs in response to prostaglandin E receptor 4 (EP4 ) antagonism. EP4 antagonists elicit release of signaling components, by controlling their trafficking into extracellular vesicles/exosomes in a lipid raft/caveolae-dependent manner. Consequently, EP4 antagonism indirectly inactivates, through induced extracellular vesicle/exosome release, pathways required for mammary epithelial stem cell homeostasis, e.g. canonical/noncanonical Wnt, TGFß and PI3K/Akt pathways. EP4 antagonism causes signaling receptors and signaling components to shift from non-lipid raft fractions to lipid raft fractions, and to then be released in EP4 antagonist-induced extracellular vesicles/exosomes, resulting in the loss of the stem cell state by mammary epithelial stem cells. In contrast, luminal mammary epithelial cells can acquire basal stem cell properties following ingestion of EP4 antagonist-induced stem cell extracellular vesicles/exosomes, and can then form mammary glands. These findings demonstrate that PGE2 /EP4 signaling controls homeostasis of mammary epithelial stem cells through regulating extracellular vesicle/exosome release. Reprogramming of mammary epithelial cells can result from EP4 -mediated stem cell property transfer by extracellular vesicles/exosomes containing caveolae-associated proteins, between mammary basal and luminal epithelial cells. Stem Cells 2017;35:425-444.

Coordinative modulation of human zinc transporter 2 gene expression through active and suppressive regulators.

Zinc transporter 2 (ZnT2) is one of the cellular factors responsible for Zn homeostasis. Upon Zn overload, ZnT2 reduces cellular Zn by transporting it into excretory vesicles. We investigated the molecular mechanism that regulates human ZnT2 (hZnT2) gene expression. Zn induces hZnT2 expression in dose- and time-dependent manners. Overexpression of metal-responsive transcription factor 1 (MTF-1) increases hZnT2 transcription, whereas depletion of MTF-1 reduces hZnT2 expression. There are five putative metal response elements (MREs) within 1kb upstream of the hZnT2 gene. A serial deletion of the hZnT2 promoter region (from 5' to 3') shows that the two MREs proximal to the gene are essential for Zn-induced promoter activity. Further mutation analysis concludes that the penultimate MRE (MREb) supports the metal-induced promoter activity. The hZnT2 promoter has also a zinc finger E-box binding homeobox (ZEB) binding element. Mutation or deletion of this ZEB binding element elevates the basal and Zn-induced hZnT2 promoter activities. Knockdown of ZEB1 mRNA enhances the hZnT2 transcript level in HEK-293 cells. In MCF-7 (ZEB-deficient) cells, expression of ZEB proteins attenuates the Zn-induced hZnT2 expression. However, expressions of MTF-1 target genes such as human ZnT1 and metallothionein IIA were not affected. Our study shows the expression of the hZnT2 gene is coordinately regulated via active and suppressive modulators.

Expression and characterization of SUMO-conjugated metal-responsive transcription factor 1: SIM-dependent cross-interaction and distinct DNA binding activity.

Metal-responsive transcription factor 1 (MTF-1) regulates a variety of genes involving in metal homeostasis and oxidative stress. We have shown that MTF-1 can be conjugated by small ubiquitin-like modifier (SUMO) and forms complexes with cellular factor(s) in a SUMO-interacting motif (SIM)-dependent manner. To investigate whether the interaction of MTF-1 and its SUMO conjugate occurs, we expressed and isolated MTF-1 and sumoylated MTF-1 (S-MTF-1) for functional studies. Various conditions were examined to optimize the expressions of MTF-1 and S-MTF-1. Results from affinity column chromatography demonstrated that the unmodified MTF-1 consistently co-eluted with the S-MTF-1. Mutations at the SIM did not reduce the level of MTF-1 sumoylation but the sumoylated product can then be purified to homogeneity. The presence of MTF-1 cross-interaction was further supported by in vitro pull-down assays. The ability of the purified proteins in binding metal-responsive element (MRE) was assessed with electrophoretic mobility shift assay. Noticeably, MTF-1 required the presence of cell extracts to render the binding activity. However, S-MTF-1 binds MRE in void of other cellular factors. The same characteristic was found for MTF-1 with SUMO fusion at the carboxyl terminus. These results indicate that the presence of SUMO moiety allows the protein to interact directly with MRE.

PTEN interacts with metal-responsive transcription factor 1 and stimulates its transcriptional activity.

MTF-1 (metal-responsive transcription factor 1) is an essential mammalian protein for embryonic development and modulates the expression of genes involving in zinc homoeostasis and responding to oxidative stress. We report in the present paper that PTEN (phosphatase and tensin homologue deleted on chromosome 10) associates with MTF-1 in the cells. These two proteins interact via the acidic domain of MTF-1 and the phosphatase/C2 domain of PTEN. Depletion of PTEN reduced MT (metallothionein) gene expression and increased cellular sensitivity to cadmium toxicity. PTEN did not alter the nuclear translocation, protein stability or DNA-binding activity of MTF-1. Zinc increased MTF-1-PTEN interaction in a dose-dependent manner. The interaction elevated within 2 h of zinc addition and declined afterwards in the cells. The enhanced binding activity occurred mainly in the cytoplasm and reduced after translocating the MTF-1 into the nucleus. Blocking signalling through the PI3K (phosphoinositide 3-kinase) pathway did not alter the zinc-induced MT expression. Analysis of enzymatically inactive PTEN mutants demonstrated that protein but not lipid phosphatase activity of PTEN was involved in the regulation of MTF-1 activity. The same regulatory role of PTEN was also noted in the regulation of ZnT1 (zinc transporter 1), another target gene of MTF-1.

The role of small ubiquitin-like modifier-interacting motif in the assembly and regulation of metal-responsive transcription factor 1.

Metal-responsive transcription factor 1 (MTF-1) is an essential protein required for mouse embryonic development. We report here the occurrence of sumoylation on MTF-1. Mutational studies demonstrated that sumoylation occurs on the lysine residue at position 627 (Lys(627)) of mouse MTF-1. Small ubiquitin-like modifier (SUMO)-1 was fused to the C terminus of MTF-1 to mimic the sumoylated form of the protein and it suppressed the transcriptional activity of MTF-1. The nuclear translocation activity, DNA-binding activity, and protein stability of SUMO-fused MTF-1 are similar to that of wild type MTF-1. The level of sumoylation was reduced by metal in a dose- and time-dependent manner. The fact that zinc reduces MTF-1 sumoylation makes the suppressive role of sumoylated MTF-1 in transcription physiologically less significant because the SUMO moiety of MTF-1 is removed when MTF-1 translocates into nucleus. We further identified a SUMO-interacting motif (SIM) on MTF-1. Remarkably, MTF-1 binds sumoylated MTF-1 and/or other cellular factors in a SIM-dependent manner. This interaction was disrupted by treating cells with zinc. Gel permeation chromatography demonstrated that MTF-1 forms SIM-dependent complexes. This cross-interaction transpires in the cytoplasm and markedly reduces upon nuclear translocation. It can therefore be concluded that SUMO conjugation and the SIM on MTF-1 do not play a critical role in suppressing transcriptional activity. Instead, MTF-1 forms complexes with cellular factors through SIM and SUMO moiety in the cytoplasm. The result explores a new understanding for the mode of MTF-1 assembly and regulation in cells.

A cooperative effect of bifunctionalized nanoparticles on recognition: sensing alkali ions by crown and carboxylate moieties in aqueous media.

Reported here is a cooperative effect that the sensing efficiency of the active group on gold nanoparticles (GNPs) can be significantly influenced by another proximal functional group. We previously developed a visual sensing scheme for K+ by 15-crown-5-CH2O(CH2)12SH functionalized GNPs in aqueous matrix. Upon adding K+, the GNP solution changes from red to blue. Such a transform is triggered by a 2-to-1 sandwich complexation of crown to K+, resulting in the red shift of surface plasmon absorption due to GNP aggregation. Herein, we discover that introducing a second functionality, thioctic acid (TA), onto GNPs significantly affects the sensing efficiency of crown moieties (15-crown-5-CH2O(CH2)n)SH and 12-crown-4-CH2O(CH2)nSH, where n = 4, 8, and 12). The rate constant of K+ recognition by TA- and 15-crown-5-CH2O(CH2)4S-bifunctionalized GNPs is more than 4 orders of magnitude faster than the others containing longer methylene chains. The same chain-length dependence is also found in the case of Na+ sensing by 12-crown-4 functionalized GNPs. The discrepancy in sensing performance is attributed to a cooperative effect that the negatively charged carboxylate of TA may preorganize the crown moiety for K+ recognition. This method is applied to measure K+ and Na+ in human urine by UV-visible spectrometry. By adjusting the concentrations of GNPs, the dynamic ranges tuned for K+ and Na+ are, respectively, 6.25 microM-1.12 mM and 0.156-4.00 mM, suitable for real samples pretreated simply by 10-fold dilution. The results ([K+] = 20.3 mM, [Na+] = 45.1 mM) agree with those obtained from ICP-AES ([K+] = 19.8 mM, [Na+] = 43.8 mM).