Gene expression profiling of L-540 Hodgkin lymphoma cell line after in vitro and in vivo treatment with perifosine in combination with sorafenib. Homo sapiens

Three HL cell lines (HD-MyZ, L-540 and HDLM-2) were used to investigate the effects of perifosine and sorafenib using in vitro assays analyzing cell growth, cell cycle distribution, gene expression profiling (GEP), and apoptosis. Western blotting (WB) experiments were performed to determine whether the two-drug combination affected MAPK and PI3K/AKT pathways as well as apoptosis. Additionally, the antitumor efficacy and mechanism of action of perifosine/sorafenib combination were investigated in vivo in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice using tumor growth rates and survival as endpoints. RESULTS: While perifosine and sorafenib as single agents exerted a limited activity against HL cells, exposure of HD-MyZ and L-540 cell lines, but not HDLM-2 cells, to perifosine/sorafenib combination resulted in synergistic cell growth inhibition (40% to 80%) and cell cycle arrest. Upon perifosine/sorafenib exposure, L-540 cell line showed significant levels of apoptosis (up to 70%, P ≤.0001) associated with severe mitochondrial dysfunction (cytochrome c, apoptosis-inducing factor release and marked conformational change of Bax accompanied by membrane translocation). Apoptosis induced by perifosine/sorafenib combination did not result in processing of caspase-8, -9, -3, or cleavage of PARP, and was not reversed by the pan-caspase inhibitor Z-VADfmk, supporting a caspase-independent mechanism of cell death. In responsive cell lines, WB analysis showed that antiproliferative and pro-apototic events were associated with dephosphorylation of MAPK and PI3K/Akt pathways. GEP analysis of HD-MyZ and L-540 cell lines, but not HDLM-2 cells indicated that perifosine/sorafenib treatment induced upregulation of genes involved in amino acid metabolism and downregulation of genes regulating cell cycle, DNA replication and cell death. In addition, in responsive cell lines, perifosine/sorafenib combination strikingly induced the expression of tribbles homologues 3 (TRIB3) both in vitro and in vivo. Silencing of TRIB3 prevented cell growth reduction induced by perifosine/sorafenib treatment. In vivo, the combined perifosine/sorafenib treatment significantly increased the median survival of NOD/SCID mice xenografted with HD-MyZ cell line as compared to controls (81 vs 45 days, P ≤.0001) as well as mice receiving perifosine alone (49 days, P ≤.03) or sorafenib alone (54 days, P ≤.007). In mice bearing subcutaneous nodules generated by HD-MyZ and L-540 cell lines but not HDLM-2 cell line, perifosine/sorafenib treatment induced significantly increased levels of apoptosis (2- to 2.5-fold, P ≤.0001) and necrosis (2- to 8-fold, P ≤.0001), as compared to controls or treatment with single agents. In addition, perifosine/sorafenib treatment had no effect on HDLM-2 nodules, but significantly reduced L-540 nodules with 50% tumor growth inhibition, compared to controls. CONCLUSIONS: Perifosine/sorafenib combination resulted in strong anti-HL activity both in vitro and in vivo. These results warrant clinical evaluation of perifosine/sorafenib combined-treatment in HL patients. Overall design: The Hodgkin lymphoma cell line L-540 was obtained from the DSMZ (Braunschweig, Germany, EU). Cells were routinely maintained in RPMI medium 1640 (Lonza, Basel, Switzerland) supplemented with 20% FBS (Lonza) and 2 mM glutamine (Lonza). Cells were maintained at 37°C in a water-saturated atmosphere of 5% CO2 in air. 10x10^6 L-540 cells were seeded in 75 cm2 flask and, after 24 hrs, cells were treated with 10 µM perifosine (Aeterna Zentaris, Frankfurt, Germany, EU) and/or 5 µM sorafenib (Bayer, Berlin, Germany, EU) in culture medium for 24 hours. At the end of treatment, cells were collected and RNA extracted.

本研究采用3株霍奇金淋巴瘤（Hodgkin Lymphoma, HL）细胞系（HD-MyZ、L-540与HDLM-2），通过体外实验探究哌立福辛（perifosine）与索拉非尼（sorafenib）的单用及联用效应，实验内容涵盖细胞增殖、细胞周期分布、基因表达谱（gene expression profiling, GEP）分析以及细胞凋亡检测。为明确两药联用是否影响丝裂原活化蛋白激酶（mitogen-activated protein kinase, MAPK）与磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/AKT）通路及细胞凋亡，本研究开展了蛋白质免疫印迹（Western blotting, WB）实验。此外，本研究以肿瘤生长速率与生存期为观测终点，在非肥胖糖尿病/重症联合免疫缺陷（nonobese diabetic/severe combined immunodeficient, NOD/SCID）小鼠体内探究哌立福辛/索拉非尼联合疗法的抗肿瘤效应与作用机制。

结果：单药使用哌立福辛与索拉非尼时，对霍奇金淋巴瘤细胞的增殖抑制活性有限；将HD-MyZ与L-540细胞系（而非HDLM-2细胞）暴露于哌立福辛/索拉非尼联合方案时，可产生协同性细胞增殖抑制效应（抑制率达40%~80%）并诱导细胞周期阻滞。经哌立福辛/索拉非尼联合处理后，L-540细胞系出现显著细胞凋亡（最高达70%，P≤0.0001），同时伴随严重线粒体功能障碍：包括细胞色素c、凋亡诱导因子的释放，以及Bax蛋白构象显著改变并发生膜转位。哌立福辛/索拉非尼联合诱导的细胞凋亡并未伴随半胱天冬酶（caspase）-8、-9、-3的活化，也未出现多聚ADP核糖聚合酶（PARP）的切割，且不能被泛半胱天冬酶抑制剂Z-VAD-fmk逆转，提示其细胞死亡途径不依赖于caspase。在具有应答性的细胞系中，蛋白质免疫印迹分析显示，抗增殖与促凋亡事件与MAPK及PI3K/AKT通路的去磷酸化相关。对HD-MyZ与L-540细胞系（而非HDLM-2细胞）的基因表达谱分析显示，哌立福辛/索拉非尼处理可诱导参与氨基酸代谢的基因上调，同时下调调控细胞周期、DNA复制与细胞死亡的基因。此外，在具有应答性的细胞系中，哌立福辛/索拉非尼联合方案可在体外与体内显著诱导TRIB3（tribbles homologues 3）的表达。沉默TRIB3可阻断哌立福辛/索拉非尼处理诱导的细胞增殖抑制。体内实验显示，相较于对照组（中位生存期45天）、单药哌立福辛组（49天，P≤0.03）与单药索拉非尼组（54天，P≤0.007），哌立福辛/索拉非尼联合处理可显著提升接种HD-MyZ细胞系的NOD/SCID小鼠的中位生存期（联合组中位生存期达81天，P≤0.0001）。在接种HD-MyZ与L-540细胞系（而非HDLM-2细胞系）形成皮下结节的小鼠中，哌立福辛/索拉非尼联合处理可诱导细胞凋亡水平较对照组与单药处理组升高2~2.5倍（P≤0.0001），坏死水平升高2~8倍（P≤0.0001）。此外，哌立福辛/索拉非尼联合处理对HDLM-2细胞形成的皮下结节无显著影响，但可显著抑制L-540细胞形成的皮下结节，肿瘤生长抑制率达50%，相较于对照组。

结论：哌立福辛/索拉非尼联合方案在体外与体内均展现出强效的抗霍奇金淋巴瘤活性。上述结果支持开展哌立福辛与索拉非尼联合疗法在霍奇金淋巴瘤患者中的临床评估。

总体实验设计：L-540霍奇金淋巴瘤细胞系购自德国不伦瑞克的DSMZ（Braunschweig, Germany, EU）。细胞常规培养于添加20%胎牛血清（FBS）与2mM谷氨酰胺的RPMI 1640培养基（Lonza, Basel, Switzerland），培养条件为37℃、5%CO₂的饱和湿度空气环境。将1×10⁷个L-540细胞接种于75cm²培养瓶中，24小时后用10μM哌立福辛（Aeterna Zentaris, Frankfurt, Germany, EU）和/或5μM索拉非尼（Bayer, Berlin, Germany, EU）处理细胞24小时。处理结束后收集细胞并提取RNA。