Quantitation of sorafenib and its active metabolite sorafenib N-oxide in human plasma by liquid chromatography–tandem mass spectrometry

A simple and rapid method with high performance liquid chromatography/tandem mass spectrometry is described for the quantitation of the kinase inhibitor sorafenib and its active metabolite sorafenib N-oxide in human plasma. A protein precipitation extraction procedure was applied to 50 μL of plasma. Chromatographic separation of the two analytes, and the internal standard [²H₃¹³C]-sorafenib, was achieved on a C₁₈ analytical column and isocratic flow at 0.3 mL/min for 4 min. Mean within-run and between-run precision for all analytes were <6.9% and accuracy was <5.3%. Calibration curves were linear over the concentration range of 50–10,000 ng/mL for sorafenib and 10–2500 ng/mL for sorafenib N-oxide. This method allows a specific, sensitive, and reliable determination of the kinase inhibitor sorafenib and its active metabolite sorafenib N-oxide in human plasma in a single analytical run.     

索拉非尼与重组腺病毒H101对肝癌细胞株HepG2的作用及机制研究

目的:研究索拉非尼与重组腺病毒H101对肝癌细胞株HepG2的作用并分析其具体机制。方法:选择索拉非尼、重组腺病毒H101分别组成重组腺病毒H101组、索拉非尼组、两药联合组以及空白对照组,并分别作用于购自中国科学院上海细胞生物研究所细胞库的肝癌细胞株HepG2。采用流式细胞技术检测HepG2细胞的凋亡情况;采用Western blot法检测细胞外信号调节激酶1/2(ERK1/2)、磷酸化ERK1/2(p-ERK1/2)以及髓样细胞白血病-1(Mcl-1)蛋白相对表达量;采用酶联免疫吸附法检测不同组别细胞培养上清液中的血管内皮生长因子(VEGF)水平。结果:重组腺病毒H101组、索拉非尼组、两药联合组的G0-G1期与G2-M期细胞均明显低于空白对照组,S期细胞均明显高于空白对照组,且两药联合组较重组腺病毒H101组与索拉非尼组更明显,差异均有统计学意义(均P0.05);重组腺病毒H101组、索拉非尼组、两药联合组的HepG2细胞凋亡率明显高于空白对照组,而两药联合组又明显高于重组腺病毒H101组与索拉非尼组,差异均有统计学意义(均P0.05)。重组腺病毒H101组、索拉非尼组、两药联合组的p-ERK1/2、Mcl-1蛋白相对表达量和VEGF表达均明显低于空白对照组,而两药联合组又明显低于重组腺病毒H101组与索拉非尼组,差异均有统计学意义(均P0.05)。结论:索拉非尼、重组腺病毒H101均可抑制肝癌细胞株HepG2的增殖,并诱导其凋亡,控制VEGF表达,联合应用具有更明显的效果,其主要机制可能与二者协同作用有效抑制p-ERK1/2、Mcl-1蛋白表达有关。     

索拉非尼联合化疗栓塞治疗晚期原发性肝癌的疗效分析

目的:原发性肝癌(Primary hepatocellular carcinoma,PHC)是最常见的消化系统恶性肿瘤之一,严重威胁人类的健康。目前,治疗晚期肝癌的首选方法是肝动脉化疗栓塞(Transcatheter arterial chemoembolization,TACE),配合抗癌药物使用,治疗效果明显。索拉非尼因具有抑制肿瘤生长的作用而被越来越广泛的用于治疗肝癌。本研究针对索拉非尼的靶向性,探讨该药与化疗栓塞联合治疗晚期原发性肝癌的疗效,旨在为肝癌的临床治疗提供可参考的依据。方法:选取我院2008年10月-2012年6月收治的晚期原发性肝癌患者96例,随机分为对照组和观察组,每组各48例。对照组患者采用肝动脉化疗栓塞单独治疗,观察组患者采用索拉非尼联合化疗栓塞治疗。比较两组患者的客观有效率、临床获益率、治疗一年生存率、两年以上生存率及甲胎蛋白(AFP)水平的变化情况。结果:观察组患者的客观有效率为52.1%,临床获益率为89.6%,均高于对照组患者的对应值33.3%和47.9%,差异有统计学意义(P0.05);观察组患者一年生存率为89.6%,两年生存率为72.9%,均高于对照组患者的对应值58.3%和35.4%,差异显著(P0.05);两组患者治疗后的AFP水平均比治疗前降低,观察组患者AFP水平的改善情况显著优于对照组,差异具有统计学意义(P0.05)。结论:索拉非尼联合化疗栓塞治疗晚期原发性肝癌具有明显的效果,且安全性高,值得临床推广使用。     

Apoptosis of CD4<Superscript>+</Superscript>CD25<Superscript>high</Superscript> T cells in response to Sirolimus requires activation of T cell receptor and is modulated by IL-2

Targeted molecular therapies inhibit proliferation and survival of cancer cells but may also affect immune cells. We have evaluated the effects of Sirolimus and Sorafenib on proliferation and survival of lymphoid cell subsets. Both drugs were cytotoxic to CD4⁺CD25^high T cells, and were growth inhibitory for CD4⁺ and CD8⁺ T cells. Cytotoxicity depended on CD3/CD28 stimulation and was detectable within 12 h, with 80–90% of CD4⁺CD25^high cells killed by 72 h. Cell death was due to apoptosis, based on Annexin V and 7AAD staining. Addition of IL-2 prevented the apoptotic response to Sirolimus, potentially accounting for reports that Sirolimus can enhance proliferation of CD4⁺CD25^high cells. These results predict that Sirolimus or Sorafenib would reduce CD4⁺CD25^high cells if administered prior to antigenic stimulation in an immunotherapy protocol. However, administration of IL-2 protects CD4⁺CD25^high T cells from cytotoxic effects of Sirolimus, a response that may be considered in design of therapeutic protocols.     

Synthesis and biological evaluation of sorafenib- and regorafenib-like sEH inhibitors

To reduce the pro-angiogenic effects of sEH inhibition, a structure–activity relationship (SAR) study was performed by incorporating structural features of the anti-angiogenic multi-kinase inhibitor sorafenib into soluble epoxide hydrolase (sEH) inhibitors. The structural modifications of this series of molecules enabled the altering of selectivity towards the pro-angiogenic kinases C-RAF and vascular endothelial growth factor receptor-2 (VEGFR-2), while retaining their sEH inhibition. As a result, sEH inhibitors with greater potency against C-RAF and VEGFR-2 were obtained. Compound 4 (t-CUPM) possesses inhibition potency higher than sorafenib towards sEH but similar against C-RAF and VEGFR-2. Compound 7 (t-CUCB) selectively inhibits sEH, while inhibiting HUVEC cell proliferation, a potential anti-angiogenic property, without liver cancer cell cytotoxicity. The data presented suggest a potential rational approach to control the angiogenic responses stemming from sEH inhibition.     

Long non-coding RNA GAS5 sensitizes renal cell carcinoma to sorafenib via miR-21/SOX5 pathway

Although the use of sorafenib appears to increase the survival rate of renal cell carcinoma (RCC) patients, there is also a proportion of patients who exhibit a poor primary response to sorafenib treatment. Therefore, it is critical to elucidate the mechanisms underlying sorafenib resistance and find representative biomarkers for sorafenib treatment in RCC patients. Herein, we identified that a long noncoding RNA GAS5 was downregulated in sorafenib nonresponsive RCCs. GAS5 overexpression conferred sorafenib sensitive to nonresponsive RCC cells, whereas knockdown of GAS5 promoted responsive RCC cells resistant to sorafenib treatment in vitro and in vivo. Mechanistically, GAS5 functioned as competing endogenous RNA to repress miR-21, which controlled its down-stream target SOX5. We proposed that GAS5 was responsible for sorafenib resistance in RCC cells and GAS5 exerted its function through the miR-21/ SOX5 axis. Our findings suggested that GAS5 downregulation may be a new marker of poor response to sorafenib and GAS5 could be a potential therapeutic target for sorafenib treatment in RCC.     

肾透明细胞癌MAPK9的表达与索拉非尼疗效的关系

目的:探讨肾透明细胞癌组织中MAPK9表达及其与肾透明细胞癌主要临床病理特征之间的关系,分析MAPK9对索拉非尼靶向治疗疗效的关系。方法:回顾分析2006年5月-2013年10月经病理确诊的32例肾透明细胞癌转移患者的MAPK9的表达情况。所有患者术后均服用索拉菲尼并随访;同时选取10例距肿瘤3 cm癌旁正常肾组织作为对照。对比分析肾透明细胞癌患者与对照组MAPK9的阳性表达率,分析MAPK9阳性表达与疗效的相关性,并分析MAPK9阳性表达与生存率之间的关系。结果:肾透明细胞癌组MAPK9阳性表达21例(65.6%),较正常对照组2例(20.0%)有显著性差异(P0.01)。TNMⅠ~Ⅱ期MAPK9阳性表达12例(85.7%),较TNMⅢ~Ⅳ期10例(55.6%)有显著性差异(P0.05);核分级1~2级MAPK9阳性表达18例(81.8%),较3~4级4例(40.0%)有显著性差异(P0.05)。MAPK9阳性表达患者与阴性表达患者疗效有显著性差异(P0.05)。MAPK9阳性患者PFS为(646±103)d,较阴性患者PFS为(502±89)d,有显著性差异(P0.05)。MAPK9阳性患者OS为(866±75)d,较阴性患者OS为(657±62)d,有显著性差异(P0.01)。结论:MAPK9在肾透明细胞癌中高表达,与肿瘤的临床TNM分期、病理分级相关。MAPK9阳性表达患者其靶向治疗疗效越好,提示MAPK9可能是一个预测索拉菲尼靶向治疗肾透明细胞癌疗效的潜在因子。     

索拉非尼和沙利度胺对肝癌患者血清中VEGF-C、VEGF-D及微血管密度的影响

目的:探讨索拉非尼和沙利度胺这两种不同的化疗药物,对肝癌患者血清中VEGF-C、VEGF-D及微血管密度的影响。方法:将患者分成3组,每组16例。对照组采用常规治疗并服用安慰剂;索拉非尼和沙利度胺这两个组患者中,前者服用索拉非尼400 mg/次,2次/d,治疗6个月;后者服用沙利度胺每日服200mg,每周增加200mg/d,直至最大剂量每日600mg,至少服用4月。ELISA检测患者血清中VEGF-C、VEGF-D;免疫组织化学检测肝癌组织中微血管密度。结果:对照组患者血清中VEGF-C的水平为210ng/ml,索拉非尼组患者血清中VEGF-C的水平为132ng/ml,而沙利度胺组患者血清中VEGF-C的水平为186ng/ml。与对照组相比,索拉非尼组和沙利度胺组患者血清中VEGF-C的水平均降低。对照组患者血清中VEGF-D的水平为322ng/m1,索拉非尼组患者血清中VEGF-D的水平为217ng/ml,而沙利度胺组患者血清中VEGF-D的水平为256ng/ml。与对照组相比,索拉非尼组和沙利度胺组患者血清中VEGF-D的水平均降低。索拉非尼组患者血清中VEGF-D的水平明显低于沙利度胺高(P<0.05)。对照组肝癌组织MVD为(44.32±5.16)个,索拉非尼组患者肝癌组织MVD为(21.75±1.49)个,而沙利度胺组患者肝癌组织MVD为(34.78±2.31)个。结论:多靶点化疗药物索拉非尼对肝癌患者血清中VEGF-C、VEGF-D及微血管密度的影响最大,深入探讨其作用机制,可为其肝癌患者提供新的化疗方案。     

Pregnane X receptor mediates sorafenib resistance in advanced hepatocellular carcinoma

Background

Kinase inhibitor sorafenib is the most widely used drug for advanced HCC clinical treatment nowadays. However, sorafenib administration is only effective for a small portion of HCC patients, and the majority develop sorafenib-resistance during treatment. Thus, it is urgent to discover the endogenous mechanism and identify new pharmaceutical targets of sorafenib-resistance.