The Bcl2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) is an atypical BH3-only protein that is associated with mitochondrial dysfunction and cell death. Bnip3 is also a potent inducer of ...mitochondrial autophagy, and in this study we have investigated the mechanisms by which Bnip3 induces autophagy in cardiac myocytes. We found that Bnip3 induced mitochondrial translocation of dynamin-related protein 1 (Drp1), a protein involved in mitochondrial fission in adult myocytes. Drp1-mediated mitochondrial fission correlated with increased autophagy, and inhibition of Drp1 reduced Bnip3-mediated autophagy. Overexpression of Drp1K38E, a dominant negative of Drp1, or mitofusin 1 prevented mitochondrial fission and autophagy by Bnip3. Also, inhibition of mitochondrial fission or autophagy resulted in increased death of myocytes overexpressing Bnip3. Moreover, Bnip3 promoted translocation of the E3 ubiquitin ligase Parkin to mitochondria, which was prevented in the presence of a Drp1 inhibitor. Interestingly, induction of autophagy by Bnip3 was reduced in Parkin-deficient myocytes. Thus our data suggest that induction of autophagy in response to Bnip3 is a protective response activated by the cell that involves Drp1-mediated mitochondrial fission and recruitment of Parkin.
Preconditioning with lipopolysaccharide (LPS) is used to improve the secretion of anti-inflammatory agents in B cells. However, there are only a few studies on canine B cells.
This study aimed to ...evaluate the immune regulatory capacity of canine peripheral blood mononuclear cell-derived B cells pretreated with LPS.
Canine B cells were isolated from canine peripheral blood mononuclear cells, which were obtained from three healthy canine donors. The B cells were preconditioned with LPS, and then cell viability and the expression of the regulatory B cell marker were assessed. Finally, RNA extraction and immunofluorescence analysis were performed.
LPS primed B cells expressed the interleukin (IL)-10 surface marker and immunoregulatory gene expression, such as IL-10, programmed death-ligand 1, and transforming growth factor beta. Macrophages in the inflammatory condition cocultured with primed B cells were found to have significantly down-regulated pro-inflammatory cytokine, such as tumor necrosis factor-α, and up-regulated anti-inflammatory cytokines such as IL-10. Additionally, it was revealed that co-culture with primed B cells re-polarized M1 macrophages to M2 macrophages.
This study revealed that LPS-primed B cells have an anti-inflammatory effect and can re-polarize macrophages, suggesting the possibility of using LPS-primed B cells as a therapeutic agent for its anti-inflammatory effects and immune modulation.
Multi-drug resistant efflux transporters found in Blood-Brain Barrier (BBB) acts as a functional barrier, by pumping out most of the drugs into the blood. Previous studies showed focused ultrasound ...(FUS) induced microbubble oscillation can disrupt the BBB by loosening the tight junctions in the brain endothelial cells; however, no study was performed to investigate its impact on the functional barrier of the BBB. In this study, the BBB in rat brains were disrupted using the MRI guided FUS and microbubbles. The immunofluorescence study evaluated the expression of the P-glycoprotein (P-gp), the most dominant multi-drug resistant protein found in the BBB. Intensity of the P-gp expression at the BBB disruption (BBBD) regions was significantly reduced (63.2 ± 18.4%) compared to the control area. The magnitude of the BBBD and the level of the P-gp down-regulation were significantly correlated. Both the immunofluorescence and histologic analysis at the BBBD regions revealed no apparent damage in the brain endothelial cells. The results demonstrate that the FUS and microbubbles can induce a localized down-regulation of P-gp expression in rat brain. The study suggests a clinically translation of this method to treat neural diseases through targeted delivery of the wide ranges of brain disorder related drugs.
TREM2 plays a critical role in the alleviation of Alzheimer's disease by promoting Aβ phagocytosis by microglia, but the detailed molecular mechanism underlying TREM2-induced direct phagocytic ...activity of Aβ remains to be revealed. We found that learning and memory functions were improved in aged TREM2 TG mice, with the opposite effects in KO mice. The amount of phagocytosed Aβ was significantly reduced in the primary microglia of KO mice. CD36 expression in primary microglia was greater in TG than in WT mice but was substantially decreased in KO mice. The expression of C/EBPα, an upstream transcriptional activator of CD36, was also elevated in primary microglia of TG mice but decreased in KO mice. The transcription of CD36 was markedly increased by TREM2 overexpression, and this effect was suppressed by a mutation of the C/EBPα binding site on the CD36 promoter. The TREM2-induced expression of CD36 and C/EBPα was inhibited by treatment with PI3K/AKT signaling blockers, and phosphorylation of AKT was elevated in TREM2-overexpressing BV2 cells. The present study provides evidence that TREM2 is required for preventing loss of memory and learning in Alzheimer's disease by regulating C/EBPα-dependent CD36 expression and the consequent Aβ phagocytosis.
Mesenchymal stem cells (MSCs) are attractive candidates for clinical repair or regeneration of damaged tissues. Oct4 and Sox2, which are essential transcription factors for pluripotency and ...self-renewal, are naturally expressed in MSCs at low levels in early passages, and their levels gradually decrease as the passage number increases. Therefore, to improve MSC proliferation and stemness, we introduced human Oct4 and Sox2 for conferring higher expansion and differentiation capabilities. The Oct4-IRES-Sox2 vector was transfected into human adipose tissue MSCs (ATMSCs) by liposomal transfection and used directly. Oct4 and Sox2 were successfully transfected into ATMSCs, and we confirmed maintenance of MSC surface markers without alterations in both red fluorescent protein (RFP) (control) and Oct4/Sox2-ATMSCs. Enhanced proliferative activity of Oct4/Sox2-ATMSCs was shown by WST-1 assay, and this result was further confirmed by cell counting using trypan blue exclusion for a long period. In addition, FACs cell cycle analysis showed that there was a reduction in the fraction of Oct4/Sox2-ATMSCs in G1 with a concomitant increase in the fraction of cells in S, compared with RFP-ATMSCs. Increased levels of cyclin D1 were also seen in Oct4/Sox2-ATMSCs, indicating acceleration in the transition of cells from G1 to S phase. Furthermore, Oct4/Sox2-overexpressing ATMSCs showed higher differentiation abilities for adipocytes or osteoblasts than controls. The markers of adipogenic or osteogenic differentiation were also upregulated by Oct4/Sox2 overexpression. The improvement in cell proliferation and differentiation using Oct4/Sox2 expression in ATMSCs may be a useful method for expanding the population and increasing the stemness of ATMSCs.
Recent studies have shown that tumour cells express tumour necrosis factor‐inducible gene 6 (TSG‐6) and its protein, which is known to play a key role in regulating excessive immune responses and ...proliferation and growth of mesenchymal stem cells (MSCs). It has not been confirmed whether the inhibition of TSG‐6 for tumour cells can suppress tumour cell growth and regulate the activation of immune cells in the tumour microenvironment (TME). TSG‐6‐specific small interfering RNA was transfected into canine and human breast cancer cells (CIPp, CIPm and BT‐20). TSG‐6‐down‐regulated (siTSG‐6) cells showed decreased cell proliferation, migration, and invasion abilities. Decreased mRNA expressions of NF‐κB, STAT3 and Sox2, confirming that TSG‐6 is an upper factor governing tumour growth and metastasis. Notably, siTSG‐6 cells showed significantly decreased expression levels of CD44 and PD‐L1. Direct and indirect co‐culture of canine peripheral blood mononuclear cells (cPBMCs) and the siTSG‐6 cells showed significant activation in M1 type macrophages and cytotoxic T cells. They also showed a tendency to decrease in the expression of CTLA‐4 and increase in the expression of PD‐1. In conclusion, this study suggests that the down‐regulation of TSG‐6 in breast cancer cells could not only suppress tumour growth and metastasis, and but also regulate TME. Since modulation of immune checkpoint proteins occurs in both tumour cells and immune cells, inhibiting TSG‐6 and its protein within the TME could be novel therapeutic target for anticancer treatment.
Mesenchymal stem cells (MSCs) possess regenerative and immunomodulatory properties and can control the immune dysregulation that leads to β-cell destruction. Stem-cell transplantation could thus ...manage insulin-dependent diabetes mellitus (IDDM) in dogs. In this pilot study, we aimed to assess canine adipose tissue-derived MSCs (cAT-MSCs) transplantation as a treatment for canine diabetes mellitus. This study included four dogs with over a year of insulin treatment for IDDM, following diagnosis at the Veterinary Medicine Teaching Hospital of Seoul National University. Allogenic cAT-MSCs were infused intravenously three or five times monthly to dogs with IDDM. Blood and urine samples were obtained monthly. General clinical symptoms, including changes in body weight, vitality, appetite, and water intake were assessed. Three of the four owners observed improvement of vitality after stem cell treatment. Two of the four dogs showed improvement in appetite and body weight, polyuria, and polydipsia. C-peptide has increased by about 5–15% in three of the cases, and fructosamine and HbA1c levels have improved in two of the cases. Hyperlipidemia was resolved in two of the dogs, and there was no concurrent bacterial cystitis in any of the dogs. C-peptide secretion and lipid metabolism are associated with diabetic complications. Improvement in these parameters following the treatment suggests that cAT-MSC transplantation in dogs with IDDM might help to improve their insulin secretory capacity and prevent diabetic complications.
Mesenchymal stem/stromal cells (MSCs) are effective therapeutic agents that ameliorate inflammation through paracrine effect; in this regard, extracellular vesicles (EVs) have been frequently ...studied. To improve the secretion of anti-inflammatory factors from MSCs, preconditioning with hypoxia or hypoxia-mimetic agents has been attempted and the molecular changes in preconditioned MSC-derived EVs explored. In this study, we aimed to investigate the increase of hypoxia-inducible factor 1-alpha (HIF-1alpha)/cyclooxygenase-2 (COX-2) in deferoxamine (DFO)-preconditioned canine MSC (MSC.sup.DFO) and whether these molecular changes were reflected on EVs. Furthermore, we focused on MSC.sup.DFO derived EVs (EV.sup.DFO) could affect macrophage polarization via the transfer function of EVs. In MSC.sup.DFO, accumulation of HIF-1alpha were increased and production of COX-2 were activated. Also, Inside of EV.sup.DFO were enriched with COX-2 protein. To evaluate the transferring effect of EVs to macrophage, the canine macrophage cell line, DH82, was treated with EVs after lipopolysaccharide (LPS) stimulation. Polarization changes of DH82 were evaluated with quantitative real-time PCR and immunofluorescence analyses. When LPS-induced DH82 was treated with EV.sup.DFO, phosphorylation of signal transducer and transcription3 (p-STAT3), which is one of key factor of inducing M2 phase, expression was increased in DH82. Furthermore, treated with EV.sup.DFO in LPS-induced DH82, the expression of M1 markers were reduced, otherwise, M2 surface markers were enhanced. Comparing with EV.sup.DFO and EV.sup.non. DFO preconditioning in MSCs activated the HIF-1alpha/COX-2 signaling pathway; Transferring COX-2 through EV.sup.DFO could effectively reprogram macrophage into M2 phase by promoting the phosphorylation of STAT3.
Background
Reactive oxygen species (ROS) have been shown to promote tumour growth and metastasis in human cell lines. The superoxide anion (•O2−) is produced during ROS formation and is involved in ...tumour cell signalling.
Objectives
Superoxide dismutase (SOD) has been applied to canine mammary gland tumours to investigate its antitumour effects in vitro.
Methods
Cell proliferation, cell cycle cell migration assays, reverse transcription‐quantitative polymerase chain reaction, and western blot analysis were performed to determine the effects of SOD on canine mammary tumour cell line.
Results
SOD treatment resulted in anti‐proliferative effects and mediated cell cycle arrest in the canine mammary gland tumour cell lines (CIPp and CIPm). It also downregulated the expression of N‐cadherin and Vimentin.
Conclusions
The results confirmed that SOD inhibits tumour cell proliferation and migration, thus supporting the potential applications of SOD as a chemotherapeutic agent for canine mammary gland tumours.
SOD treatment showed anti‐proliferative effects and mediated cell cycle arrest in the canine mammary gland tumour cell line (CIPp and CIPm). Our results show that SOD inhibits the proliferation and migration of tumour cells through downregulating the expression of N‐cadherin and Vimentin activity. These findings support the potential application of SOD as a chemotherapeutic strategy for canine mammary gland tumours.
The purpose of this study was to evaluate the effect of extracellular vesicles derived from canine M1-polarized macrophages (M1EVs) on canine tumor cells, such as D17 (osteosarcoma cells) and LMeC ...(melanoma cells).
Protein expression was determined by western blot analysis. Gene expression was determined by RT-qPCR. In addition, cell apoptosis was analyzed by Annexin V/PI staining.
In the case of M1EV, the levels of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1β were increased, and nitrate/nitrite levels were also increased. M1EV induced apoptosis of tumor cells by increasing caspase-3 and caspase-7 activation. In addition, M1EVs decreased expression of CCR4, Foxp3 and CTLA-4 in canine peripheral mononuclear cells cocultured with tumor cells.
M1EV could be an effective anti-cancer therapeutic approach in melanoma and osteosarcoma and M1EVs can be used as immunomodulators in the tumor microenvironment for cancer treatment.
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