毛细管气相色谱法测定药用真菌生物转化液中微量斑蝥素的含量

采用毛细管气相色谱法对药用真菌培养液中斑蝥素的含量进行了测定。色谱条件：色谱柱为SPB-5弹性石英毛细管柱；进样口温度240℃；柱温：120℃（5min）→8℃／min→240℃（2min）；检测器为FID，检测温度240℃。栽气：99．999％高纯氮气，流量1．5mL／min；尾吹气：99．999％高纯氮气，流量25mL／min。以香兰素作为内标，采用内标法定量。斑蝥素在1—200mg／L范围内线性关系良好，检测限为0．01mg／L。     

The Discodermia calyx Toxin Calyculin A Enhances Cyclin D1 Phosphorylation and Degradation, and Arrests Cell Cycle Progression in Human Breast Cancer Cells

Cyclin D1 is a key regulator of the cell cycle that is over expressed in more than half of breast cancer patients. The levels of cyclin D1 are controlled primarily through post-translational mechanisms and phosphorylation of cyclin D1 at T286 induces its proteasomal degradation. To date, no studies have explored the involvement of phosphatases in this process. Here we treated human breast cancer cells with the structurally distinct toxins calyculin A, okadaic acid, and cantharidin, which are known to inhibit Ser/Thr phosphatases of the PPP family. At low nanomolar concentrations calyculin A induced T286 phosphorylation and degradation of cyclin D1 via the proteosome in MDA-MB-468 and MDA-MB-231 cells. Cyclin D1 degradation also was dose-dependently induced by okadaic acid and catharidin, implicating a negative regulatory role for type-2A phosphatases. These effects occurred without increasing phosphorylation of p70S6K, cyclin D3, or myosin light chain that were used as endogenous reporters of cellular PP2A and PP1 activity. A reverse phase phosphoprotein array analysis revealed increased phosphorylation of only 6 out of 33 Ser/Thr phosphosites, indicating selective inhibition of phosphatases by calyculin A. Calyculin A treatment induced cell cycle arrest in MDA-MB-468 and MCF-7 breast cancer cells. These findings suggest that a specific pool of type-2A phosphatase is inhibited by calyculin A leading to the degradation of cyclin D1 in human breast cancer cells. The results highlight the utility of toxins as pharmacological probes and points to the T286 cyclin D1 phosphatase inhibited by calyculin A as a possible target for chemotherapy to treat triple negative breast cancer.     

Validation of the cantharidin-induced skin blister as an in vivo model of inflammation

AIM

Pharmacological profiling techniques, such as the cantharidin-induced skin blister, may be used to assess the anti-inflammatory properties of novel drugs. However, no data are available on the reproducibility of this technique or on the blocking effect of anti-inflammatory drugs, such as anti-TNF and corticosteroids.

METHODS

A group of 30 healthy subjects were randomized into three parallel groups treated with placebo, oral methylprednisolone 20 mg day⁻¹ for 7 days or anti-tumour necrosis factor (TNF) (adalimumab, Humira®, Abbott) 40 mg s.c. single dose. A first blister was induced at baseline and collected, immediately before the start of treatment and a second blister was obtained 7 days after the start of treatment. The total number of cells, the cell viability and the differential cell count were evaluated by two independent observers, who were blind to treatment. anova was used to compare change from baseline among the three groups before pairwise comparisons.

RESULTS

Among the placebo group, there was no significant difference in the total cell count, neutrophils, eosinophils and monocytes between day 1 and day 7. Methylprednisolone inhibited the eosinophil influx in mean % (95% CI) (−1.0 (−1.7, −0.3); P < 0.02) and absolute (P < 0.02) values, while anti-TNF inhibited the neutrophil influx in mean % (95% CI) (−19.3 (−29.5, −9.1); P < 0.01) and absolute (P < 0.05) values.

CONCLUSIONS

The cantharidin-induced skin blister is a safe, well tolerated and reproducible procedure. Pre-treatment with anti-TNF or methylprednisolone inhibited the neutrophilic or eosinophilic trafficking, respectively. It could be useful in profiling anti-inflammatory drugs regarding their effects on the cellular inflammatory response.     

Cantharidin induces DNA damage and inhibits DNA repair‐associated protein levels in NCI‐H460 human lung cancer cells

Cantharidin is one of the major compounds from mylabris and it has cytotoxic effects in many different types of human cancer cells. Previously, we found that cantharidin induced cell death through cell cycle arrest and apoptosis induction in human lung cancer NCI‐H460 cells. However, cantharidin‐affected DNA damage, repair, and associated protein levels in NCI‐H460 cells have not been examined. In this study, we determined whether cantharidin induced DNA damage and condensation and altered levels of proteins in NCI‐H460 cells in vitro. Incubation of NCI‐H460 cells with 0, 2.5, 5, 10, and 15 μM of cantharidin caused a longer DNA migration smear (comet tail). Cantharidin also increased DNA condensation. These effects were dose‐dependent. Cantharidin (5, 10, and 15 μM) treatment of NCI‐H460 cells reduced protein levels of ataxia telangiectasia mutated (ATM), breast cancer 1, early onset (BRCA‐1), 14‐3‐3 proteins sigma (14‐3‐3σ), DNA‐dependent serine/threonine protein kinase (DNA‐PK), O⁶‐methylguanine‐DNA methyltransferase (MGMT), and mediator of DNA damage checkpoint protein 1 (MDC1). Protein translocation of p‐p53, p‐H2A.X (S140), and MDC1 from cytoplasm to nucleus was induced by cantharidin in NCI‐H460 cells. Taken together, this study showed that cantharidin caused DNA damage and inhibited levels of DNA repair‐associated proteins. These effects may contribute to cantharidin‐induced cell death in vitro. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1135–1143, 2015.     

斑蝥素在比格犬体内的药代动力学和口服生物利用度研究

目的:测定斑蝥素单剂量静脉和口服给药后的比格犬体内的药-时曲线,并与斑蝥素静脉单剂量给药相比,计算其体内的药代动力学参数和生物利用度.方法:6只比格犬给药,血样盐酸酸化后,乙酸乙酯萃取,使用GC-MS方法测定血浆中的微量斑蝥素,用WinNonLin程序计算药代动力学参数和生物利用度.结果:比格犬静脉注射斑蝥素(34 μg·kg~(-1))的主要药代学参数:AUC(203.5±23.8)h·μg·L~(-1),CL(168.8±18.6)mL·h~(-1)·kg(-1).t_(1/2)(0.69±0.03)h;比格犬单剂量口服斑蝥素(102μg·kg~(-1))主要药代学参数是:AUC(160.4±26.9)h·μg·L~(-1),CL(649.1±97.7)mL·h~(-1)·kg~(-1),t_(1/2)(0.38±0.1)h.与静脉注射相比,生物利用度为26.7%.结论:比格犬口服斑蝥素后,斑蝥素在犬体内的吸收较少,提示要提高斑蝥素的口服生物利用度,提高临床用药有效性.     

用强化靶向材料修饰斑蟊素多相脂质体的实验研究

本试验合成了一种含氧乙基的半乳糖衍生物Gal β1—(CH2—CH2—O)3—C14H29怍为强化靶向材料,使其与卵磷脂(EYL)制得的斑蟊素脂质体相融合,测定其一系列理化性质。结果表明,此种强化靶向材料的加入并不影响脂质体的常规理化性质,小鼠尾静脉注射35h后,强化斑蟊素脂质体是普通斑蟊素脂质体在肝内浓度的3．6倍(P<0.05),滞留时间亦显著延长,因为半乳糖可识别的动物凝聚素存在于肝内,所以此种半乳糖衍生物修饰的脂质体可充当治疗肝病药物的理想载体。     

斑蝥素酸镁对人星形胶质瘤细胞U-251MG生长、凋亡和侵袭的影响

目的 探讨斑蝥素酸镁对人星形胶质瘤细胞U 251MG生长、凋亡和侵袭的影响及机制。方法 体外培养人星形胶质瘤细胞U 251MG,分为空白组（Control组）、低剂量斑蝥素酸镁组（Canthardin 5μM组）、中剂量斑蝥素酸镁组（Canthardin 10μM组）和高剂量斑蝥素酸镁组（Canthardin 20μM组）。采用克隆形成实验检测细胞增殖情况;Hocest染色检测细胞凋亡情况;Transwell法检测细胞侵袭情况;Western blot 检测相关蛋白表达情况。结果 平板克隆实验结果显示,斑蝥素酸镁可以明显抑制人星形胶质瘤细胞U 251MG的增殖,且呈剂量依赖性（P＜0.05）;Hocest染色结果显示,斑蝥素酸镁可以明显促进人星形胶质瘤细胞的凋亡,且呈剂量依赖性（P＜0.05）;Transwell小室实验显示,斑蝥素酸镁可以显著抑制细胞的侵袭,且呈剂量依赖性（P＜0.05）;Western blot 检测结果显示,斑蝥素酸镁处理后,人星形胶质瘤细胞中PCNA蛋白、VEGF蛋白、Bax蛋白及p-AKT蛋白表达均显著降低（均P＜0.05）,Caspase 3蛋白、Bcl 2蛋白表达显著升高（P＜0.05）,AKT总蛋白表达无显著差异（P＞0.05）。 结论 斑蝥素酸镁可以通过调控Akt信号通路抑制人星形胶质瘤细胞U-251MG的增殖和侵袭,并促进其凋亡,且在一定浓度范围内呈剂量依赖性。     

复方斑蝥联合恩替卡韦治疗乙肝相关性肝癌TACE术后效果及对免疫功能、VEGF的影响

正乙肝相关性肝癌(hepataceilular carcinoma,HCC)是临床常见恶性肿瘤,恶性程度高。肝动脉介入栓塞化疗(transcatheter arterial chemoembolization,TACE)是临床新型抗肿瘤技术,在治疗HCC方面具有重要作用,但TACE术并不能彻底清除肿瘤细胞,且存在乙肝病毒激活、机体免疫力下降的可能,增加了HCC患者术后肝癌转移和复发的风险~([1])。术后抗病毒治疗