Suppression of puerarin on polymethylmethacrylate-induced lesion of peri-implant by inhibiting NF-κB activation in vitro and in vivo

Puerarin (PR), a natural isoflavone isolated from Chinese traditional plant pueraria lobata, has attracted considerable attention due to its important biological and pharmacological activities. However, its effects on lesion of peri-implant and related mechanism of action are still not clear, which require further investigation. In this study, we evaluated the effects of PR on polymethylmethacrylate (PMMA)-induced lesion of peri-implant in vitro and in vivo, and explored its possible mechanism of action. Our results indicated that PR could inhibit PMMA-induced osteoclastogenesis in RAW264.7 cells with a dose-dependent manner in vitro and effectively down-regulate mRNA and protein expressions of matrix metalloprotein 9 (MMP-9), tumor necrosis factor (TNF)-α, interleukin (IL)-6, and receptor activator of nuclear factor (NF)-κB (RANK), primarily via the suppression of NF-κB signaling. Furthermore, we found that PMMA induction could directly cause the phosphorylation of IκB and significantly promote the nuclear translocation of p65 in RAW264.7 cells. In other words, PR was able to dose-dependently attenuate the PMMA-induced nuclear translocation of p65 in RAW264.7 cells. In vivo, PR was observed to attenuate PMMA-induced osteoclastogenesis, osteolysis, mRNA expressions of receptor activator of nuclear factor (NF)-κB ligand (RANKL) and RANK, as well as protein levels of MMP-9, TNF-α, IL-6, and p65 in a murine calvarial osteolysis model. These findings suggested that PR might be a potential therapeutic drug to lesion of peri-implant, and provided new insights for understanding its possible mechanism.     

The NEDD4-1 E3 ubiquitin ligase: A potential molecular target for bortezomib sensitivity in multiple myeloma

E3 ubiquitin ligases primarily determine the substrate specificity of the ubiquitin-proteasome system and play an essential role in the resistance to bortezomib in multiple myeloma (MM). Neural precursor cell-expressed developmentally downregulated gene 4-1 (NEDD4-1, also known as NEDD4) is a founding member of the NEDD4 family of E3 ligases and is involved in the proliferation, migration, invasion and drug sensitivity of cancer cells. In the present study, we investigated the role of NEDD4-1 in MM cells and explored its underlying mechanism. Clinically, low NEDD4-1 expression has been linked to poor prognosis in patients with MM. Functionally, NEDD4-1 knockdown (KD) resulted in bortezomib resistance in MM cells in vitro and in vivo. The overexpression (OE) of NEDD4-1, but not an enzyme-dead NEDD4-1-C867S mutant, had the opposite effect. Furthermore, the overexpression of NEDD4-1 in NEDD4-1 KD cells resensitized the cells to bortezomib in an add-back rescue experiment. Mechanistically, pAkt-Ser473 levels and Akt signaling were elevated and decreased by NEDD4-1 KD and OE, respectively. NEDD4-1 ubiquitinated Akt and targeted pAkt-Ser473 for proteasomal degradation. More importantly, the NEDD4-1 KD-induced upregulation of Akt expression sensitized MM cells to growth inhibition after treatment with an Akt inhibitor. Collectively, our results suggest that high NEDD4-1 levels may be a potential new therapeutic target in MM.     

A Phase I Trial of IGF-1R Inhibitor Cixutumumab and mTOR Inhibitor Temsirolimus in Metastatic Castration-resistant Prostate Cancer

BackgroundDespite frequent PTEN (phosphatase and tensin homologue) loss and Akt/mammalian target of rapamycin (mTOR) signaling in prostate cancer, the disease is insensitive to single-agent mTOR inhibition. Insulin-like growth factor-1 receptor inhibition might mitigate the feedback inhibition by Torc1 inhibitors, suppressing downstream Akt activation and, thus, potentiating the antitumor activity of mTOR inhibition.Patients and MethodsIn the present phase I study, patients with metastatic castration-resistant prostate cancer received 6 mg/kg cixutumumab and 25 mg temsirolimus intravenously each week. The primary objective was safety and tolerability. Temsirolimus was decreased if ≥ 2 dose-limiting toxicities (DLTs) were observed in 6 patients. The correlative analyses included measurement of circulating tumor cells, [18F]-fluoro-2-deoxyglucose positron emission tomography, 16β-[18F]-fluoro-α-dihydrotestosterone positron emission tomography, and tumor biopsy.ResultsA total of 16 patients were enrolled across 3 cohorts (1, −1, −2). Two DLTs (grade 3 oral mucositis) were observed in cohort 1 (temsirolimus, 25 mg), and 1 DLT (grade 3 lipase) in cohort −1 (temsirolimus, 20 mg). The most common adverse events included hyperglycemia (100%; 31% grade 3), oral mucositis (63%; 19% grade 3), and diarrhea (44%; 0 grade 3). Low-grade pneumonitis occurred in 7 of 11 patients (44%; 0 grade 3), prompting the opening of a 3-weekly cohort (temsirolimus, 20 mg/kg), without pneumonitis events. No patient had a >50% decline in prostate-specific antigen from baseline. The best radiographic response was stable disease, with median study duration of 22 weeks (range, 7-63 weeks).ConclusionsDespite a strong scientific rationale for the combination, temsirolimus plus cixutumumab demonstrated limited antitumor activity and a greater than expected incidence of toxicity, including low-grade pneumonitis and hyperglycemia. Hence, the trial was stopped in favor of alternative androgen receptor/phosphatidylinositol 3-kinase–directed combinatorial therapies.     

茯苓多糖对人宫颈癌HeLa细胞增殖、迁移、促凋亡的影响及其机制

目的 探讨茯苓多糖（Pachymaran）对人宫颈癌HeLa细胞增殖、迁移、促凋亡的作用及其相关机制。方法 MTT法检测细胞增殖率并筛选出适宜的茯苓多糖低、中、高浓度用于后续实验；不同浓度的茯苓多糖处理细胞后，倒置相差显微镜观察细胞形态学变化；Hoechst 33342染色观察细胞核的变化；平板克隆实验检测细胞克隆形成能力；细胞划痕实验检测细胞迁移能力；流式细胞术检测细胞凋亡率及细胞周期；Western blot法检测凋亡、迁移及ERK通路相关蛋白的表达。结果 茯苓多糖浓度对HeLa细胞活力的影响实验得出，取30、40、50 mg/ml茯苓多糖作为低、中、高浓度进行后续实验；中、高浓度茯苓多糖处理细胞后，细胞和细胞核发生显著的凋亡形态学变化，低浓度下形态学变化不显著；不同浓度茯苓多糖均能降低细胞克隆形成能力；降低细胞迁移率（P<0.05）；使细胞凋亡率增加（P<0.05）；使S期细胞减少并阻滞于G₂/M期（P<0.05）；Cleaved Caspase 3、Cleaved Caspase 8、Cleaved Caspase 9、Bax表达较对照组明显增多，Bcl-2、MMP-9、VEGFA、p-ERK1/2表达明显减少（P<0.05）。ERK1/2表达无明显变化。结论 茯苓多糖能显著抑制HeLa细胞增殖，并诱导其凋亡。其促凋亡机制可能与下调p-ERK1/2表达，抑制ERK信号通路磷酸化有关。同时，茯苓多糖对抑制HeLa细胞的迁移也有一定的作用。     

围产期PFOS暴露对幼鼠海马BDNF/TrkB/CREB信号通路影响

目的 探讨观察母孕鼠围产期全氟辛烷磺酸盐(perfluorooctane sulphonate,PFOS)暴露对幼鼠海马组织BDNF/TrkB/CREB信号通路关键基因表达的影响。 方法 20只昆明种雌性小鼠随机分为对照组和低、中、高剂量组,从孕鼠怀孕第2 d开始(gestation day2, GD2)到幼鼠出生后21 d(postnatal day21,PND21)分别给予低、中、高剂量组孕鼠PFOS剂量为 0.1、1.0、5.0 mg/(kg·bw)灌胃染毒,对照组给予等体积的0.05% Tween-20水溶液。灌胃量为0.1 ml/10 (g·bw)。PND 21 d处死幼鼠,收集脑组织,分离海马及皮层。HE染色观察脑组织常规病理改变,实时荧光定量PCR(Quantitative Real-time PCR,QPCR)检测海马组织中BDNF、TrkB、CREB、Syn1及Syp的mRNA表达水平。 结果 与对照组相比较,低、中、高三个剂量组对幼鼠的死亡率和体质量的影响差异无统计学意义(P>0.05)。但是在高剂量组幼鼠海马组织出现空泡,海马BDNF、TrkB、CREB mRNA水平显著降低,分别由对照组的(0.98±0.11)、(1.03±0.09)、(1.08±0.12)下降到(0.22±0.21)、(0.71±0.14)、(0.37±0.26),并在中、高剂量组引起了幼鼠突触相关蛋白Syn1和Spy的mRNA水平显著降低,分别由对照组的(1.10±0.09)、(0.97±0.08)下降到中剂量的(0.41±0.23)、(0.71±0.17)和高剂量的(0.39±0.19)、(0.63±0.19),差异均有统计学意义(P<0.05)。 结论 PFOS损伤海马BDNF/TrkB/CREB信号通路可能是PFOS神经发育毒性之一。     

白藜芦醇通过激活PI3K/AKT通路抑制内皮细胞凋亡的研究

目的观察白藜芦醇对脂多糖作用下人脐静脉血管内皮细胞凋亡的影响,并探讨其作用机制。方法体外培养人脐静脉血管内皮细胞,实验分为5组:空白组给予单纯培养基培养;模型组培养基中加入1000 ng/mL脂多糖孵育24 h;5μmol/L白藜芦醇组和10μmol/L白藜芦醇组培养基中分别先加入5μmol/L、10μmol/L白藜芦醇孵育12 h,再加入1000 ng/mL脂多糖孵育24 h;PI3K抑制剂组培养基中先加入10μmol/L白藜芦醇和10μmol/L PI3K抑制剂LY294002共处理12 h,再加入1000 ng/mL脂多糖孵育24 h。使用CCK-8试剂盒检测各组细胞的增殖能力;流式细胞术检测各组细胞凋亡率;DHE荧光探针检测各组细胞中活性氧(ROS)含量;ELISA检测各组细胞培养液中一氧化氮(NO)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)水平;Western blot法检测各组AKT的磷酸化情况。结果与空白组相比,模型组细胞增殖能力明显降低,细胞凋亡率和细胞中ROS含量及TNF-α、IL-6水平均明显升高,NO水平及P-AKT/AKT表达水平明显降低,差异均有统计学意义(P均<0.05)。与模型组和PI3K抑制剂组相比,10μmol/L白藜芦醇组细胞增殖能力明显升高,细胞凋亡率和细胞中ROS含量及TNF-α、IL-6水平明显下降,NO水平及P-AKT/AKT表达水平明显升高,差异均有统计学意义(P均<0.05)。结论白藜芦醇能够提升脂多糖作用下人脐静脉血管内皮细胞的增殖能力,抑制细胞凋亡,减轻细胞的氧化应激和炎性反应,恢复细胞的分泌功能,可能与其激活PI3K/AKT通路而发挥保护作用有关。