Increased Reactive Oxygen Species and Distinct Oxidative Damage in Resveratrol-suppressed Glioblastoma Cells.

Background and Aim: Glioblastoma multiforme (GBM) is a highly aggressive brain malignancy that lacks reliable treatments. Resveratrol possesses anti-cancer effects, but its activity against glioblastoma cells is variable for unknown reasons. One mechanism through which anti-cancer drugs exert their effects is oxidative damage caused by increased reactive oxygen species (ROS) production. Thus, the present study examined the relationship between oxidative stress and sensitivity to resveratrol in glioblastoma cells. Methods: Two GBM cell lines (U251 and LN428) were exposed to 100 µM resveratrol for 48 h, and proliferation and apoptosis were assessed. ROS generation was evaluated using 2'-7'-dichlorodihydrofluorescein diacetate-based flow cytometry and fluorescent microscopy. Immunocytochemical staining and western blotting were conducted at regular intervals to profile the expression patterns of superoxide dismutase-2 (SOD2), catalase, caspase-9, caspase-3, and sulfotransferases (SULTs) in untreated and resveratrol-treated GBM cells. Results: Resveratrol-treated U251 cells, but not resveratrol-treated LN428 cells, exhibited remarkable growth arrest and extensive apoptosis accompanied by elevated intracellular ROS levels and attenuated SOD2 and catalase expression. Mitochondrial impairment and more distinct increases in the expression of activated caspase-9 and caspase-3 were detected in U251 cells following resveratrol treatment. The levels of resveratrol metabolic enzymes (SULT1A1 and SULT1C2) were lower in U251 cells than in LN428 cells. Conclusions: Resveratrol increased ROS generation and induced oxidation-related cellular lesions in U251 cells by activating an ROS-related mitochondrial signal pathway. The levels of SULTs and ROS may indicate the therapeutic outcomes of resveratrol treatment in GBM.

Intra-articular delivery of flurbiprofen sustained release thermogel: improved therapeutic outcome of collagenase II-induced rat knee osteoarthritis.

Knee osteoarthritis (OA) is a common degenerative disease. Intra-articular administration of flurbiprofen is frequently employed in clinic to treat OA, while repeated injections are required because of the limited effective duration. To improve therapeutic outcome and prolong the treatment interval, a poly(ε-caprolactone-co-lactide)-b-poly(ethylene glycol)-b-poly(ε-caprolactone-co-lactide) (PCLA-PEG-PCLA) triblock copolymer based flurbiprofen thermosensitive gel for the sustained intra-articular drug delivery was designed in this study. The anti-OA effects of this flurbiprofen thermogel were investigated on collagenase II-induced rat knee OA model by multiple approaches and compared with that of conventional sodium hyaluronate and flurbiprofen injecta. In vitro drug release studies indicated that flurbiprofen was sustained released from the thermosensitive gel for more than three weeks. This sustained drug release system exerted comparable short-term analgesic effects and distinctly improved long-term analgesic efficacy in terms of the increased percentage of the total ipsilateral paw print intensity and the reduced Knee-Bend scores of OA rats. The inflammatory response was attenuated in the samples of flurbiprofen gel treated group by showing decreased IL-1, IL-6, and IL-11 levels in the joint fluid and down-regulated IL-1, IL-6, IL-11, COX-2, TNF-α, and NF-κB/p65 expression in the articular cartilages. The results suggest the suitability of thermosensitive copolymer PCLA-PEG-PCLA for sustained intra-articular effects of flurbiprofen and provide in vivo experimental evidence for potential clinical application of this flurbiprofen delivery system to better management of OA cases.

STAT3 signaling statuses determine the fate of resveratrol-treated anaplastic thyroid cancer cells.

BACKGROUNDS: Anaplastic thyroid cancer/ATC is highly lethal malignancy without reliable chemotherapeutic drug. Resveratrol possesses anti-ATC activities but encounters resistance in some cases due to certain unknown reason(s). OBJECTIVE: Because signal transducer and activator of transcription/STAT3 signaling is critical for ATC cell survival and the main molecular target of resveratrol, its roles in determining the fates of resveratrol-treated ATC cells were investigated here. METHODS: Human THJ-11T, THJ-16 and THJ-21T ATC cell lines were treated by 100 µM resveratrol and their growth, statuses of STAT3 signaling and STAT3-related gene expression were examined. The relevance of STAT3 activation with resveratrol resistance was elucidated using STAT selective inhibitor AG490. Leukemia inhibitory factor/LIF expression and phosphorylated-STAT3/p-STAT3 nuclear translocation in ATC tissues were immunohistochemically analyzed. RESULTS: Resveratrol inhibited proliferation, p-STAT3 nuclear translocation as well as LIF and STAT3 expression of THJ-16T and THJ-21T but not THJ-21T cells which showed LIF upregulation and more frequent p-STAT3 nuclear translocation. AG490 significantly prevent p-STAT3 nuclear translocation, and reversed the resveratrol tolerance of THJ-11T cells. Immonohistochemical staining revealed 14.3% (4/28) of LIF and 3.6% (1/28) of p-STAT3 detection in noncancerous ATC-surrounding tissues, which increased to 89.5% (17/19) and 52.6% (10/19) respectively among ATC specimens. The correlative analysis indicated the relevance of LIF expression and STAT3 activation (r= 0.825; P< 0.01). CONCLUSIONS: The status of STAT3 activation and LIF expression are closely correlated with the therapeutic effect of resveratrol on ATCs. Frequent LIF upregulation and STAT3 activation are the unfavorable factors of ATCs and the potential targets of anti-ATC therapy.

Sinomenine Attenuates Cartilage Degeneration by Regulating miR-223-3p/NLRP3 Inflammasome Signaling.

Sinomenine (SIN) has been shown to protect against IL-1ß-induced chondrocyte apoptosis in vitro. However, the role of SIN in the anterior cruciate ligament transection (ACLT)-induced osteoarthritis (OA) mouse model and its underlying molecular mechanisms remain unclear. In the present study, the protective effect of SIN on ACLT-induced articular cartilage degeneration and IL-1ß-induced chondrocyte apoptosis miR-223-3p/NLRP3 signaling regulation was investigated. Safranin O staining was performed to evaluate the pathological changes of articular cartilage. Chondrocyte apoptosis was measured with Annexin V-fluorescein isothiocyanate/polyimide (annexin V-FITC/PI) staining using flow cytometry. Gene and protein expression were detected by RT-qPCR and Western blotting, respectively. SIN administration markedly improved articular cartilage degradation in mice undergoing ACLT surgery. In addition, SIN treatment downregulated the levels of inflammatory cytokines and the protein expression of NLRP3 inflammasome components and upregulated the expression of miR-223-3p in OA mice and IL-1ß-stimulated chondrocytes. In vitro, we found that NLRP3 was a direct target of miR-223-3p, and overexpression of miR-223-3p blocked IL-1ß-induced apoptosis and the inflammatory response in chondrocytes. These findings indicate that miR-223-3p/NLRP3 signaling could be used as a potential target of SIN for the treatment of OA.

Upregulation of BAG3 with apoptotic and autophagic activities in maggot extract­promoted rat skin wound healing.

Maggot extract (ME) accelerates rat skin wound healing, however its effect on cell maintenance in wound tissues remains unclear. B­cell lymphoma (Bcl) 2­associated athanogene (BAG)3 inhibits apoptosis and promotes autophagy by associating with Bcl­2 or Beclin 1. Bcl­2, the downstream effector of signal transducer and activator of transcription 3 signaling, is enhanced in ME­treated wound tissues, which may reinforce the Bcl­2 anti­apoptotic activity and/or cooperate with Beclin 1 to regulate autophagy during wound healing. The present study investigated expression levels of BAG3, Bcl­2, Beclin 1 and light chain (LC)3 levels in rat skin wound tissues in the presence and absence of ME treatment. The results revealed frequent TUNEL­negative cell death in the wound tissues in the early three days following injury, irrespective to ME treatment. TUNEL­positive cells appeared in the wound tissues following 4 days of injury and 150 µg/ml ME efficiently reduced apoptotic rate and enhanced BAG3 and Bcl­2 expression. Elevated Beclin 1 and LC3 levels and an increased LC3 II ratio were revealed in the ME­treated tissues during the wound healing. The results of the present study demonstrate the anti­apoptotic effects of BAG3 and Bcl­2 in ME­promoted wound healing. Beclin 1/LC3 mediated autophagy may be favorable in maintaining cell survival in the damaged tissues and ME­upregulated BAG3 may enhance its activity.

SHP2, SOCS3 and PIAS3 Expression Patterns in Medulloblastomas: Relevance to STAT3 Activation and Resveratrol-Suppressed STAT3 Signaling.

BACKGROUND: Activated STAT3 signaling is critical for human medulloblastoma cells. SHP2, SOCS3 and PIAS3 are known as the negative regulators of STAT3 signaling, while their relevance to frequent STAT3 activation in medulloblastomas remains unknown. METHODS: Tissue microarrays were constructed with 17 tumor-surrounding noncancerous brain tissues and 61 cases of the classic medulloblastomas, 44 the large-cell medulloblastomas, and 15 nodular medulloblastomas, which were used for immunohistochemical profiling of STAT3, SHP2, SOCS3 and PIAS3 expression patterns and the frequencies of STAT3 nuclear translocation. Three human medulloblastoma cell lines (Daoy, UW228-2 and UW228-3) were cultured with and without 100 µM resveratrol supplementation. The influences of resveratrol in SHP2, SOCS3 and PIAS3 expression and SOCS3 knockdown in STAT3 activation were analyzed using multiple experimental approaches. RESULTS: SHP2, SOCS3 and PIAS3 levels are reduced in medulloblastomas in vivo and in vitro, of which PIAS3 downregulation is more reversely correlated with STAT3 activation. In resveratrol-suppressed medulloblastoma cells with STAT3 downregulation and decreased incidence of STAT3 nuclear translocation, PIAS3 is upregulated, the SHP2 level remains unchanged and SOCS3 is downregulated. SOCS3 proteins are accumulated in the distal ends of axon-like processes of resveratrol-differentiated medulloblastoma cells. Knockdown of SOCS3 expression by siRNA neither influences cell proliferation nor STAT3 activation or resveratrol sensitivity but inhibits resveratrol-induced axon-like process formation. CONCLUSION: Our results suggest that (1) the overall reduction of SHP2, SOCS3 and PIAS3 in medulloblastoma tissues and cell lines; (2) the more inverse relevance of PIAS3 expression with STAT3 activation; (3) the favorable prognostic values of PIAS3 for medulloblastomas and (4) the involvement of SOCS3 in resveratrol-promoted axon regeneration of medulloblastoma cells.