Endothelial cell-derived extracellular vesicles modulate the therapeutic efficacy of mesenchymal stem cells through IDH2/TET pathway in ARDS.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a severe and fatal disease. Although mesenchymal stem cell (MSC)-based therapy has shown remarkable efficacy in treating ARDS in animal experiments, clinical outcomes have been unsatisfactory, which may be attributed to the influence of the lung microenvironment during MSC administration. Extracellular vesicles (EVs) derived from endothelial cells (EC-EVs) are important components of the lung microenvironment and play a crucial role in ARDS. However, the effect of EC-EVs on MSC therapy is still unclear. In this study, we established lipopolysaccharide (LPS) - induced acute lung injury model to evaluate the impact of EC-EVs on the reparative effects of bone marrow-derived MSC (BM-MSC) transplantation on lung injury and to unravel the underlying mechanisms. METHODS: EVs were isolated from bronchoalveolar lavage fluid of mice with LPS - induced acute lung injury and patients with ARDS using ultracentrifugation. and the changes of EC-EVs were analysed using nanoflow cytometry analysis. In vitro assays were performed to establish the impact of EC-EVs on MSC functions, including cell viability and migration, while in vivo studies were performed to validate the therapeutic effect of EC-EVs on MSCs. RNA-Seq analysis, small interfering RNA (siRNA), and a recombinant lentivirus were used to investigate the underlying mechanisms. RESULTS: Compared with that in non-ARDS patients, the quantity of EC-EVs in the lung microenvironment was significantly greater in patients with ARDS. EVs derived from lipopolysaccharide-stimulated endothelial cells (LPS-EVs) significantly decreased the viability and migration of BM-MSCs. Furthermore, engrafting BM-MSCs pretreated with LPS-EVs promoted the release of inflammatory cytokines and increased pulmonary microvascular permeability, aggravating lung injury. Mechanistically, LPS-EVs reduced the expression level of isocitrate dehydrogenase 2 (IDH2), which catalyses the formation of α-ketoglutarate (α-KG), an intermediate product of the tricarboxylic acid (TCA) cycle, in BM-MSCs. α-KG is a cofactor for ten-eleven translocation (TET) enzymes, which catalyse DNA hydroxymethylation in BM-MSCs. CONCLUSIONS: This study revealed that EC-EVs in the lung microenvironment during ARDS can affect the therapeutic efficacy of BM-MSCs through the IDH2/TET pathway, providing potential strategies for improving the therapeutic efficacy of MSC-based therapy in the clinic.

LMTK3 promotes tumorigenesis in bladder cancer via the ERK/MAPK pathway.

Lemur tyrosine kinase 3 (LMTK3) is a key member of the serine-threonine tyrosine kinase family. It plays an important role in breast cancer tumorigenesis and progression. However, its biological role in bladder cancer remains elusive. In this study, we demonstrated that LMTK3 was overexpressed in bladder cancer and was positively correlated with bladder cancer malignancy. High LMTK3 expression predicted poor overall survival. Knockdown of LMTK3 in bladder cancer cells triggered cell-cycle arrest at G2/M phase, suppressed cell growth, and induced cell apoptosis in bladder cancer cells. Furthermore, Transwell assays revealed that reduction of LMTK3 decreased cell migration by regulating the epithelial-to-mesenchymal transition pathway. Conversely, LKTM3 overexpression was shown to promote proliferation and migration of bladder cancer cells. We assessed phosphorylation of MEK and ERK1/2 in bladder cancer cells depleted of LMTK3 and demonstrated a reduced phosphorylation status compared with the control group. Using an MAPK signaling-specific inhibitor, U0126, we could rescue the promotion of proliferation and viability in LMTK3-overexpressing cells. In conclusion, we extend the status of LMTK3 as an oncogene in bladder cancer and provide evidence for its function via the activation of the ERK/MAPK pathway. Thus, targeting LMTK3 may hold potential as a diagnostic and prognostic biomarker and as a possible future treatment for bladder cancer.

Lycopene Inhibits Epithelial-Mesenchymal Transition and Promotes Apoptosis in Oral Cancer via PI3K/AKT/m-TOR Signal Pathway.

BACKGROUND: Oral cancer (OC) is one of the most common cancers around the world. Despite the progress in treatment, the prognosis of OC remains poor, especially for patients with advanced diseases. It urges the development of novel therapeutic options against OC. Lycopene (LYC) is an antioxidant with chemoprotective properties against cancer. However, little is known about the mechanisms underlying the protective role of LYC in OC tumorigenesis. METHODS: In this study, we investigated the anti-cancer effect of LYC on the progression of OC in vitro and in vivo and explored the underlying mechanisms involved in this process. RESULTS: LYC inhibited OC cell proliferation, migration, invasion, apoptosis, and xenograft tumor growth in a dose-dependent manner. Furthermore, we found that LYC might inhibit epithelial-mesenchymal transition and induce apoptosis in OC cells by deactivating the PI3K/AKT/m-TOR signaling through increasing the levels of E-cadherin and Bax and downregulating N-cadherin, p-PI3K, p-AKT, p-m-TOR, and bcl-2. CONCLUSION: We reported for the first time that LYC exhibited anti-cancer effects on OC development both in vitro and in vivo via regulating EMT process and apoptosis. These findings provide support for the potential clinical use of LYC in OC treatment.

lncRNA MALAT1 Promotes EMT Process and Cisplatin Resistance of Oral Squamous Cell Carcinoma via PI3K/AKT/m-TOR Signal Pathway.

BACKGROUND: Cisplatin (DDP) is the first-line chemotherapy agent for the treatment of oral squamous cell carcinoma (OSCC). The emergence of DDP resistance leads to diminished drug efficacy and survival benefit. lncRNA MALAT1 has been considered as one of the most important factors in OSCC. It has also been reported to enhance chemo-resistance in other kinds of carcinomas. However, little is known about the role of lncRNA MALAT1 in DDP resistance of OSCC. MATERIALS AND METHODS: Two kinds of human DDP-resistant cell lines (CAL-27R and SCC-9R) were developed from cisplatin-naïve cell lines (CAL-27 and SCC-9, respectively) as in vitro cell models. Cell transfection was performed to overexpress or knockdown MALAT1 in these cells. Mouse xenograft models were also established. The following measurements were performed: cell proliferation, colony formation, wound healing, transwell, and TUNEL assays, as well as Western blot and immunofluorescence staining. RESULTS: DDP-resistant cells showed higher expression level of MALAT1 compared to cisplatin-naïve cells. The overexpression of MALAT1 in cisplatin-naïve cells enhanced DDP resistance and suppressed apoptosis in OSCC cells. However, the knockdown of MALAT1 in DDP-resistance cells induced apoptotic cell death and restored the sensitivity to DDP. Further analyses suggested that MALAT1 might promote DDP resistance via regulating P-glycoprotein expression, epithelial-mesenchymal transition process, and the activation of PI3K/AKT/m-TOR signaling pathway. CONCLUSION: MALAT1 might be a potential therapeutic target for the treatment of DDP-resistant OSCC.

Quercetin Inhibits Epithelial-to-Mesenchymal Transition (EMT) Process and Promotes Apoptosis in Prostate Cancer via Downregulating lncRNA MALAT1.

BACKGROUND: Prostate cancer (PC) is one of the most common carcinomas in men worldwide. The lack of effective therapies urges the development of novel therapeutic options against PC. Quercetin (Quer) is a flavonoid compound that has been shown to effectively inhibit PC in vitro and in vivo. However, the underlying mechanisms await elucidation. Long non-coding RNA MALAT1 has been reported as an oncogenic target in multiple types of cancers, including PC. Previous data showed that quercetin promoted the apoptosis of fibroblast-like synoviocytes by upregulating MALAT1 in rheumatoid arthritis. However, we speculate that mechanisms are different in PC. MATERIALS AND METHODS: Human PC cell line PC-3 and its xenograft tumor were chosen as in vitro and in vivo models for PC. A series of in vitro and in vivo functional experiments were carried out to elucidate the role of MALAT1 in quercetin treatment against PC. Western blot was performed to measure the expression of related proteins to explore underlying molecular mechanisms. RESULTS: We showed for the first time that MALAT1 expression was significantly downregulated in quercetin-treated PC cells in a dose- and time-dependent manner. Also, quercetin inhibited the proliferation of PC cells and the growth of xenograft tumors. Moreover, quercetin suppressed EMT process, promoted apoptosis and deactivated PI3K/Akt signaling pathway during the progression of PC. MALAT1 overexpression in PC cells resulted in the resistance against quercetin treatment. CONCLUSION: Our study illustrated, for the first time, that MALAT1 played an important role in quercetin treatment against PC by inhibiting EMT process and promoting apoptosis, providing a new molecular basis for the application of quercetin in PC treatment.

Quercetin reverses docetaxel resistance in prostate cancer via androgen receptor and PI3K/Akt signaling pathways.

Docetaxel is the first-line chemotherapy agent for metastatic prostate cancer. However, the emergence of resistance diminishes its efficacy and limits the survival benefit. Quercetin is a dietary flavonoid which has been shown to have multiple anti-cancer effects. Also, quercetin has been reported to reverse chemo-resistance in many other cancers. This study was to determine whether quercetin could reverse docetaxel resistance in prostate cancer cells and xenograft models, thereby exploring the underlying mechanism. Depending on the docetaxel-resistant cells (LNCaP/R, PC-3/R) which were established from docetaxel-sensitive cells (LNCaP, PC-3), it was demonstrated that quercetin could reverse docetaxel resistance in prostate cancer on proliferation, colony formation, migration, invasion and apoptosis. Although single docetaxel application had little effect on docetaxel-resistant cells, combining docetaxel with quercetin was significantly effective. Combination therapy could maximally inhibited PI3K/Akt pathway and promoted apoptosis. As shown by in-vivo study, xenograft tumors treated by docetaxel with quercetin had poorest growth. Then, to investigate the underlying mechanisms, the differences among parental cells, docetaxel-resistant subclones and quercetin treated resistant subclones were evaluated. It was found that docetaxel-resistant subclones had stronger activation of androgen receptor and PI3K/Akt pathway, more remarkable mesenchymal and stem-like cell phenotypes, and more P-gp expression than that of parental cells. Interestingly, quercetin could reverse these transformations. Our data revealed that quercetin had docetaxel-resistance reversal effect both in vitro and in vivo and provided in-depth support for clinical use of quercetin in docetaxel-resistant prostate cancer.

Circular RNA circHIPK3 promotes cell proliferation and invasion of prostate cancer by sponging miR-193a-3p and regulating MCL1 expression.

BACKGROUND: While emerging evidence indicates that circHIPK3 is critically involved in tumorigenesis and the development of several cancers, its role in prostate cancer (PCa) is not clearly understood. MATERIALS AND METHODS: Human PCa samples and their matched normal adjacent tissues were obtained from 26 patients to assess the expression of circHIPK3 and its relationship with PCa prognosis. A series of in vitro and in vivo functional experiments were carried out to elucidate the role of circHIPK3 in PCa progression and its underlying molecular mechanisms. RESULTS: In this study, we found that circHIPK3 was overexpressed in PCa tissues and that higher circHIPK3 expression was associated with tumor stage. Moreover, circHIPK3 knockdown markedly inhibited the proliferation, migration, and invasion of PCa cells in vitro and impaired tumor growth in vivo. Bioinformatics analysis and luciferase reporter assays demonstrated that circHIPK3 could promote MCL1 expression by interacting with miR-193a-3p in PCa. Finally, rescue assays illustrated that circHIPK3 knockdown could partially reverse the effects of MCL1 overexpression. CONCLUSION: In summary, our study illustrated, for the first time, that circHIPK3-mediated miR-193a-3p-MCL1 signaling promotes PCa development and progression, providing a novel therapeutic target for PCa.

Ursolic acid improves podocyte injury caused by high glucose.

BACKGROUND: Autophagy plays an important role in the maintenance of podocyte homeostasis. Reduced autophagy may result in limited renal cell function during exposure to high glucose conditions. In this study we investigated the effects of ursolic acid (UA) on autophagy and podocyte injury, which were induced by high glucose. METHODS: Conditionally immortalized murine podocytes were cultured in media supplemented with high glucose and the effects of the PI3K inhibitor LY294002 and UA on protein expression were determined. miR-21 expression was detected by real-time RT-PCR. Activation of the PTEN-PI3K/Akt/mTOR pathway, expression of autophagy-related proteins and expression of podocyte marker proteins were determined by western blot. Immunofluorescence was used to monitor the accumulation of LC3 puncta. Autophagosomes were also observed by transmission electron microscopy. RESULTS: During exposure to high glucose conditions, the normal level of autophagy was reduced in podocytes, and this defective autophagy induced podocyte injury. Increased miR-21 expression, decreased PTEN expression and abnormal activation of the PI3K/Akt/mTOR pathway were observed in cells that were cultured in high glucose conditions. UA and LY294002 reduced podocyte injury through the restoration of defective autophagy. Our data suggest that UA inhibits miR-21 expression and increases PTEN expression, which in turn inhibits Akt and mTOR and restores normal levels of autophagy. CONCLUSIONS: Our data suggest that podocyte injury is associated with reduced levels of autophagy during exposure to high glucose conditions, UA attenuated podocyte injury via an increase in autophagy through miR-21 inhibition and PTEN expression, which inhibit the abnormal activation of the PI3K/Akt/mTOR pathway.

The effect of manganese-induced toxicity on the cytokine mRNA expression of chicken spleen lymphocytes in vitro.

Manganese (Mn) is essential for life, but excess Mn exposure is harmful. This study investigated the effect of excess Mn on the cytokines of spleen lymphocytes in chicken. Lymphocytes were incubated with or without MnCl2 (2, 4, 6, and 8×10(-4) mmol/L) for 12, 24, 36, and 48 h, respectively. The mRNA expression of interleukin (IL) -2, IL-4, IL-6, IL-12ß, and IL-17 and interferon (INF) -γ was examined using RT-PCR. Excess Mn inhibited IL-2, IL-4, IL-6, IL-12ß, and IL-17 mRNA expression in chicken spleen lymphocytes in a dose-dependent manner. IFN-γ was inhibited by 8×10(-4) mmol/L Mn for 48 h. This study demonstrates that excess Mn affects cytokine mRNA expression and causes immunosuppression in chicken spleen lymphocytes. The relationships between IL-6 and IL-17 and between IL-2 and IL-12ß were strong under immunosuppression caused by excess Mn in lymphocytes.

Ursolic acid attenuates diabetic mesangial cell injury through the up-regulation of autophagy via miRNA-21/PTEN/Akt/mTOR suppression.

OBJECTIVE: To investigate the effect of ursolic acid on autophagy mediated through the miRNA-21-targeted phosphoinositide 3 kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) pathway in rat mesangial cells cultured under high glucose (HG) conditions. METHODS: Rat glomerular mesangial cells were cultured under normal glucose, HG, HG with the PI3K inhibitor LY294002 or HG with ursolic acid conditions. Cell proliferation and hypertrophy were assayed using an MTT assay and the ratio of total protein to cell number, respectively. The miRNA-21 expression was detected using RT-qPCR. The expression of phosphatase and tensin homolog (PTEN)/AKT/mTOR signaling signatures, autophagy-associated protein and collagen I was detected by western blotting and RT-qPCR. Autophagosomes were observed using electron microscopy. RESULTS: Compared with mesangial cells cultured under normal glucose conditions, the cells exposed to HG showed up-regulated miRNA-21 expression, down-regulated PTEN protein and mRNA expression, up-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and down-regulated LC3II expression. Ursolic acid and LY294002 inhibited HG-induced mesangial cell hypertrophy and proliferation, down-regulated p85PI3K, pAkt, pmTOR, p62/SQSTMI, and collagen I expression and up-regulated LC3II expression. However, LY294002 did not affect the expression of miRNA-21 and PTEN. Ursolic acid down-regulated miRNA-21 expression and up-regulated PTEN protein and mRNA expression. CONCLUSIONS: Ursolic acid inhibits the glucose-induced up-regulation of mesangial cell miRNA-21 expression, up-regulates PTEN expression, inhibits the activation of PI3K/Akt/mTOR signaling pathway, and enhances autophagy to reduce the accumulation of the extracellular matrix and ameliorate cell hypertrophy and proliferation.