WTAP contributes to the tumorigenesis of osteosarcoma via modulating ALB in an m6A-dependent manner.

PURPOSE: Osteosarcoma (OS) is a prevalent bone malignancy mainly occurred in adolescents. WTAP/N6-methyladenosine (m6A) modification is confirmed to be involved in OS progression. This study is conducted to bring some novel insights to the action mechanism of WTAP/m6A under the hidden pathogenesis of OS. METHODS: qRT-PCR was executed to evaluate the expression levels of WTAP and ALB. ALB protein level in OS cells was measured by western blotting. The content of m6A in total RNA was assessed by m6A quantification assay. Me-RIP, dual luciferase reporter, and mRNA stability assays confirmed the target relationship of WTAP with ALB. With the use of the wound healing, CCK-8, and transwell invasion assays, the functional relationship between WTAP and ALB in OS cells was confirmed. The influences of WTAP on tumor growth in vivo were performed in the xenograft model of mouse. RESULTS: WTAP was increased but ALB was diminished in OS tissues and/or cell lines. WTAP modulated ALB expression in an m6A-dependent manner. Silencing of WTAP retarded the development of OS via inhibiting cell viability, migration, invasion, and tumor growth. Knockdown of ALB exerted the opposite effects on OS progression. Additionally, ALB deficiency partially eliminated the inhibiting effects of WTAP silencing on cellular processes in OS. CONCLUSIONS: This is the first report to clarify the interaction of WTAP/m6A with ALB in OS progression. These experimental data to some extent broadened the horizons of WTAP/m6A in the development of OS.

WTAP contributes to the tumorigenesis of osteosarcoma via modulating ALB in an m6A-dependent manner.

PURPOSE: Osteosarcoma (OS) is a prevalent bone malignancy mainly occurred in adolescents. WTAP/N6-methyladenosine (m6A) modification is confirmed to be involved in OS progression. This study is conducted to bring some novel insights to the action mechanism of WTAP/m6A under the hidden pathogenesis of OS. METHODS: qRT-PCR was executed to evaluate the expression levels of WTAP and ALB. ALB protein level in OS cells was measured by western blotting. The content of m6A in total RNA was assessed by m6A quantification assay. Me-RIP and dual luciferase reporter assays confirmed the target relationship of WTAP with ALB. With the use of the wound healing, CCK-8, and transwell invasion assays, the functional relationship between WTAP and ALB in OS cells was confirmed. The influences of WTAP on tumor growth in vivo were performed in the xenograft model of mouse. RESULTS: WTAP was increased but ALB was diminished in OS tissues and/or cell lines. WTAP modulated ALB expression in an m6A-dependent manner. Silencing of WTAP retarded the development of OS via inhibiting cell viability, migration, invasion, and tumor growth. Knockdown of ALB exerted the opposite effects on OS progression. Additionally, ALB deficiency partially eliminated the inhibiting effects of WTAP silencing on cellular processes in OS. CONCLUSIONS: This is the first report to clarify the interaction of WTAP/m6A with ALB in OS progression. These experimental data to some extent broadened the horizons of WTAP/m6A in the development of OS.

Chondro-Protective Effects of Shikimic Acid on Osteoarthritis via Restoring Impaired Autophagy and Suppressing the MAPK/NF-κB Signaling Pathway.

Osteoarthritis (OA) is a major cause of cartilage pain and limited mobility in middle-aged and elderly individuals. The degeneration of cartilage induced by inflammation and cartilage anabolic and catabolic disorder plays a key role in OA. Shikimic acid (SA), a natural ingredient extracted from Illicium verum, has been shown to exert notable anti-inflammatory effects in previous studies, suggesting its potential effects in the treatment of OA. In this study, we revealed that the pretreatment of SW1353 human chondrocytes with SA before interleukin 1ß (IL-1ß) stimulation effectively decreased the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase (Cox)-2, matrix metalloproteinases (MMPs; MMP3 and MMP13), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5, type X collagen, and p62; increased the expression of type II collagen, ATG7, Beclin-1, and LC3; and increased the autophagic flux. Mechanistically, we found that SA suppressed the IL-1ß-induced activation of the mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-κB) pathways. Furthermore, the results of safranin O staining and toluidine blue staining of primary rat cartilage chondrocytes and a trauma-induced rat model of OA showed that SA alleviated progression of OA in vivo. Collectively, our research enhances understanding of the mechanism of protective effect of SA against the progression of OA, which involves amelioration of cartilage degeneration, thereby providing new evidence for the use of SA as a therapy to prevent the development of OA.

N-(2-18F-fluoropropionyl)-l-glutamate as a potential oncology tracer for PET imaging of glioma.

N-(2-18F-fluoropropionyl)-l-glutamate (18F-FPGLU), a new N-substituted 18F-labeling l-glutamate, is a potential amino acid tracer for oncology PET imaging with good tumor-to-background contrast in several tumor-bearing mice. Herein, we evaluated the potential value of 18F-FPGLU for PET imaging of glioma in orthotopic glioma-bearing SD rats. A series of competitive inhibition experiments with various types of inhibitors were conducted with C6 cells to investigate the transport mechanism of 18F-FPGLU in glioma. Establishment of orthotopic rat C6 glioma-bearing SD rats models was confirmed by MRI. Then PET imaging of 18F-FPGLU was performed on the orthotopic C6 glioma-bearing SD rats and compared with that of 18F-FDG. After the rats sacrificed, the whole brain was collected and immunofluorescence staining of glial fibrillary acidic protein (GFAP) and matrix metalloproteinase 2 (MMP2) were processed. Na+-dependent system XAG- and Na+-independent system XC- are the mainly transporters of 18F-FPGLU in C6 cells. N-methyl-d-aspartate (NMDA) receptor, which is associated with the invasiveness and proliferation of glioma cells, is also involved in the uptake of 18F-FPGLU. High uptake and retention of 18F-FPGLU was observerd in orthotopic glioma with good visualization and the tumor/background ratio reached 2.35 at 60 min post-injection, which was significantly higher than that of 18F-FDG (1.72) in small-animal PET images. High expression of MMP-2 and GFAP was observed in the immunofluorescence staining of glioma xerography slices. 18F-FPGLU seems to be a better potential PET tracer than 18F-FDG for brain glioma imaging with good visualization and ability to assess the tumor activity.

Excitatory glutamate transporter EAAC1 as an important transporter of N-(2-[18F]fluoropropionyl)-L-glutamate in oncology PET imaging.

INTRODUCTION: We have reported that N-(2-[18F]fluoropropionyl)-L-glutamate ([18F]FPGLU) was a potential amino acid tracer for tumor imaging with positron emission tomography (PET). In this study, the relationship between glutamate transporter excitatory amino acid carrier 1 (EAAC1) expression and [18F]FPGLU uptake in rat C6 glioma cell lines and human SPC-A-1 lung adenocarcinoma cell lines was investigated. METHODS: The uptake of [18F]FPGLU was assessed in ATRA-treated and untreated C6 cell lines, and also in EAAC1 knock-down SPC-A-1(shRNA) cells and SPC-A-1(NT) control cells. PET imaging of [18F]FPGLU was performed on the SPC-A-1 and SPC-A-1 (shRNA)-bearing mice models. RESULTS: The uptake of [18F]FPGLU in C6 cells increased significantly after induced by ATRA for 24, 48, and 72 h, which was closely related to expression of EAAC1 in C6 cells (R2 = 0.939). Compared with the SPC-A-1(NT) control cells, the uptake of [18F]FPGLU on EAAC1 knock-down SPC-A-1(shRNA) cells significantly decreased to 64.0%. Moreover, the uptake of [18F]FPGLU in EAAC1 knock-down SPC-A-1(shRNA) xenografts was significantly lower than that in SPC-A-1 xenografts, with tumor/muscle ratios of 3.01 vs. 1.67 at 60 min post-injection of [18F]FPGLU. CONCLUSION: The transport mechanism of [18F]FPGLU in glioma C6 and lung adenocarcinoma SPC-A-1 cell lines mainly involves in glutamate transporter EAAC1. EAAC1 is an important transporter of N-(2-[18F]fluoropropionyl)-L-glutamate in oncologic PET imaging.

Gastric schwannoma with giant ulcer and lymphadenopathy mimicking gastric cancer: a case report.

BACKGROUND: Gastric schwannomas are rare benign tumors originating from the intramuscular plexus of the stomach and account for just 2.6% of gastric mesenchymal tumors. Gastric schwannoma (GS) with a surface ulcer is very rare. Herein, we report a rare case of an ulcer-bearing GS, which in conjunction with multiple enlarged regional lymph nodes, readily mimicked gastric cancer (GC). CASE PRESENTATION: A 79-year-old female presented with poor appetite and intermittent vomiting of gastric contents during the past month. Gastroscopy revealed a giant crateriform ulcer within the stomach body (at the angular notch). Its raised and indurated border was fragile and bled easily. GC was thus suspected. Contrast-enhanced computer tomography (CT) revealed a mild enhancement of the corresponding irregularly thickened gastric wall, and an annular zone of mucosal discontinuity. Enlarged regional lymph nodes were also found, making GC with metastases of lymph nodes our primary concern. 18F-fluorodeoxyglueose position emission tomography (18F-FDG PET)/CT was then performed for further staging. Obviously increased FDG uptake was shown in the gastric lesion ((maximum standardized uptake value (SUVmax) 14.6), but no FDG uptake was observed in the enlarged regional lymph nodes. Given the strong suspicion of GC, subtotal gastrectomy was performed. GS was revealed by postoperative pathology, with no evidence of metastasis in the 13 resected lymph nodes. CONCLUSIONS: This was a rare case of GS with a giant surface ulcer and multiple enlarged regional lymph nodes. The uptake of 18F-FDG in the tumor was substantially higher than previously published literature reports. Under these circumstances, it is difficult to be differentiated from GC.

Pravastatin alleviates interleukin 1ß-induced cartilage degradation by restoring impaired autophagy associated with MAPK pathway inhibition.

BACKGROUND: Osteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage degradation driven by proinflammatory cytokines; meanwhile, statins display anti-inflammatory effects. Here we assessed the effects of pravastatin on inflammatory rat chondrocytes and explored the underlying mechanism. METHODS: Rat articular chondrocytes were pretreated with pravastatin and subsequently stimulated with IL-1ß. Then, the expression levels of OA- and autophagy-related effectors, at the mRNA and protein levels, were examined by real-time polymerase chain reaction (RT-PCR) and Western blotting, respectively. Autophagic flux in chondrocytes in different treatment groups was monitored via GFP-mRFP-LC3 adenovirus transfection and confocal microscopy. Activation of MAPK, PI3K/Akt, and NF-κB pathways in chondrocytes with or without pravastatin treatment during IL-1ß stimulation was examined by Western blotting. RESULTS: Our results showed that pravastatin downregulated the degradation related genes MMP3, MMP13 and ADAMTS5, as well as extracellular matrix degeneration induced by IL-1ß. In addition, pravastatin upregulated the autophagy related genes atg7, atg12, Beclin1, and LC3 II in IL-1ß stimulated chondrocytes. GFP-mRFP-LC3 adenovirus transfection also indicated that pravastatin restored impaired autophagy in OA chondrocytes. Furthermore, we demonstrated that regulation of the MAPK signaling pathway may be responsible for autophagy regulation in the articular cartilage. CONCLUSIONS: Taken together, these findings suggested that pravastatin restores impaired autophagic flux by inhibiting MAPK activation and protects the cartilage against inflammatory responses, suggesting a potential role for autophagic flux in pravastatin-mediated cartilage protection.

Caffeic acid protects against IL-1ß-induced inflammatory responses and cartilage degradation in articular chondrocytes.

Osteoarthritis (OA) is a common articular disease that features cartilage loss and destruction. It has been confirmed that inflammation plays major roles in the progression of osteoarthritis. Caffeic acid (CA), a key dietary nutrient commonly found in coffee, has shown its anti-inflammatory properties in various inflammation diseases. However, the effects of CA in osteoarthritis remain explored. Here we investigated the effects of CA on IL-1ß induced increased expression of inflammatory factors as well as the degradation of Collagen II and aggrecan in rat chondrocytes. CA prevented the cartilage damage induced by IL-1ß in vivo organ culture of articular cartilage. Besides, the IL-1ß induced increased production of inflammation factors such as iNOS and COX2 could be inhibited by CA. Additionally, CA could also suppress IL-1ß induced expression of cartilage matrix catabolic enzymes such as ADAMTS5 and MMPs. Moreover, CA could prevent IL-1ß induced degradation of Collagen II and aggrecan in chondrocytes. Furthermore, CA inhibited NF-κB activity and the activation of JNK pathway. This study reveals that CA inhibits IL-1ß induced inflammation responses through suppression of NF-κB and MAPK related JNK signaling pathways. These results demonstrate that CA may provide new avenues for osteoarthritis treatment in future.

Kaempferol inhibits interleukin­1ß stimulated matrix metalloproteinases by suppressing the MAPK­associated ERK and P38 signaling pathways.

Osteoarthritis (OA) is a common degenerative joint disease in older adults. A number of previous studies have demonstrated that natural flavonoids can serve as promising therapeutic drugs for OA. Kaempferol, a phytochemical ingredient mainly present in various fruits, has exhibited its prominent anti­inflammatory and antioxidant effects in numerous diseases. However, whether Kaempferol ameliorates the deterioration of arthritis remains to be elucidated. The aim of the present study was to investigate the therapeutic role of Kaempferol on OA in rat chondrocytes. The results revealed that Kaempferol significantly inhibited the interleukin (IL)­1ß­induced protein expression of inflammatory mediators such as inducible nitric oxide synthase and cyclo­oxygenase­2. In addition, the common matrix degrading enzymes [matrix metalloproteinase (MMP)­1, MMP­3, MMP­13 and a disintegrin and metalloproteinase with thrombospondin motif­5] induced by IL­1ß were also suppressed by Kaempferol, and consequently abolished the degradation of collagen II. Furthermore, the anti­inflammatory effect of Kaempferol was mediated by the inhibition of the mitogen activated protein kinase­associated extracellular signal­regulated kinase and P38 signaling pathways. These results collectively indicated that Kaempferol can potentially prevent OA development and serve as a novel pharmacological target in the treatment of OA.

Histone deacetylase-4 and histone deacetylase-8 regulate interleukin-1ß-induced cartilage catabolic degradation through MAPK/JNK and ERK pathways.

Interleukin-1ß (IL-1ß)-induced inflammatory response is associated with osteoarthritis (OA) and its development. Histone deacetylase (HDAC) may be involved in regulating this pathogenesis, but the mechanism has yet to be elucidated. The aim of the present study was to investigate the mechanism underlying the regulation of IL­1ß­stimulated catabolic degradation of cartilage by HDAC. An in vitro model of OA was generated using rat articular chondrocytes (rACs) treated with IL­1ß. The role of HDAC in IL­1ß­induced gene expression was investigated using HDAC inhibitors and specific small interfering RNAs (siRNAs). The association of diverse mitogen­activated protein kinase (MAPK) pathways was examined. The IL­1ß­induced expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)­4 and ADAMTS­5, and the production of collagen X and cyclooxygenase­2 in rACs was accompanied by the expression of HDAC4 and HDAC8, and were significantly downregulated by HDAC inhibitors and specific siRNAs. IL­1ß­induced activation of extracellular signal­regulated kinase was downregulated by the HDAC inhibitor Trichostatin A, but not significantly by PCI­34051. The activation of c­Jun N­terminal kinase was observably downregulated by the latter, but only slightly by the former. These results suggest that HDAC4 and HDAC8 may serve as key upstream mediators of MAPK in regulating the IL­1ß­induced cartilage catabolic and degradation. Therefore, inhibiting HDAC4 or HDAC8 or both may be a promising therapeutic strategy in preventing and treating OA.

Sinapic Acid Inhibits the IL-1ß-Induced Inflammation via MAPK Downregulation in Rat Chondrocytes.

Osteoarthritis (OA) is a degenerative joint disease frequently seen in the elderly population. Sinapic acid (SA), a commonly found phenolic acid, has been pharmacologically evaluated for its anti-inflammation effects in various studies. To explore its potential therapeutic role for OA, rat chondrocytes were treated with IL-1ß (10 ng/ml) with different concentrations of SA in vitro. Our study revealed that SA could inhibit the IL-1ß-induced production of nitric oxide (NO) and prostaglandin E2 (PGE2). Consistent with these findings, the increased protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase (Cox)-2 could also be downregulated by SA. Moreover, SA could also suppress the IL-1ß-induced expression of matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) in chondrocytes. Furthermore, our data found that SA could suppress the IL-1ß-induced mitogen-activated protein kinase (MAPK) pathway activation. In general, this paper elucidates that sinapic acid inhibits the IL-1ß-induced inflammation via MAPK pathways and may be a good agent for the treatment of OA.

Chondrogenic effect of cell-based scaffold of self-assembling peptides/PLGA-PLL loading the hTGFß3 plasmid DNA.

With the application of tissue engineering to tissue regeneration, additional new complexes have been made in response to the challenge of cartilage-injury repair. This study was performed to construct a rat precartilaginous stem cells-based scaffold of self-assembling peptides RADA16-I/PLGA-PLL (poly-L-lysine coated PLGA) as extracellular matrix loading the NLS-TAT as a peptide-based carrier for a plasmid DNA containing hTGFß3. After composites were cultured for 1, 2, 3 and 4 weeks, respectively, the results showed that the levels of chondrogenic-related gene expression were higher in the experimental group with and hTGFß3 gene by reverse transcription-polymerase chain reaction, and with higher histochemical and immunohistochemical expression. hTGFß3 protein expression had increased at 4 weeks based on western blot analysis. The results of this study show that a complex may be a suitable scaffold for cartilage repair and offer a strategy for tissue regeneration through the use of tissue engineering.

Chondrogenic effect of precartilaginous stem cells following NLS-TAT cell penetrating peptide-assisted transfection of eukaryotic hTGFß3.

Cell penetrating peptides (CPPs) are a series of promising carriers for delivering exogenous DNA to living cells. Among them, the combination of the human immunodeficiency virus TAT protein (TAT) with the SV40 large T protein nuclear localization signal (NLS) to form NLS-TAT performs well. In the present study, we took advantage of this new carrier to deliver transforming growth factor-beta 3 (TGFß3) genes. TGFß3 was expressed by the pEGFP-N1 vector following transfection of rat precartilaginous stem cells (PSCs), which promoted hTGFß3 protein self-expression. At 24, 48, 72, and 120 h after transfection, the expression levels of hTGFß3 were found to be elevated as compared with the control. The expression of hTGFß3 was found to mediate the chondrogenic effect of PSCs. Thus, we determined the expression of the chondrogenesis-related genes type II collagen, Sox 9, and aggrecan in PSCs at 24, 48, 72, and 120 h after transfection. We found that their transcription and translation was augmented, which indicated a trend of active chondrogenesis in the PSCs. Our results demonstrated that NLS-TAT had the ability to deliver exogenous DNA into rat PSCs and could be actively expressed. This process successfully promoted PSC chondrogenesis. Additionally, PSC, may represent a new type of stem cells, and thus show great potential in regenerative repair following cartilage injury.