Gene expression profiling of HD-MyZ 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 HD-MyZ was obtained from the DSMZ (Braunschweig, Germany, EU). Cells were routinely maintained in RPMI medium 1640 (Lonza, Basel, Switzerland) supplemented with 10% 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 HD-MyZ cells were seeded in 150 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）的作用效果，实验分析指标包括细胞增殖、细胞周期分布、基因表达谱（Gene Expression Profiling, GEP）与细胞凋亡。本研究同时开展蛋白质免疫印迹（Western blotting, WB）实验，以明确两药联合是否影响丝裂原活化蛋白激酶（MAPK）、磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/AKT）通路及细胞凋亡过程。此外，本研究以肿瘤生长速率与生存情况为终点指标，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疗效与作用机制。

【结果】单独使用哌立福辛或索拉非尼时，对HL细胞的增殖抑制活性有限；而将HD-MyZ与L-540细胞系（而非HDLM-2细胞）暴露于哌立福辛/索拉非尼联合疗法时，可产生协同性细胞增殖抑制效果（抑制率40%~80%）并诱导细胞周期阻滞。经哌立福辛/索拉非尼处理后，L-540细胞系出现显著凋亡（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蛋白显著构象改变并发生膜转位。哌立福辛/索拉非尼联合疗法诱导的细胞凋亡并未引发半胱天冬酶（caspase）-8、-9、-3的活化，也未导致多聚ADP核糖聚合酶（PARP）的剪切，且无法被泛半胱天冬酶抑制剂Z-VAD-fmk逆转，提示该细胞死亡过程不依赖半胱天冬酶通路。在敏感细胞系中，WB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GEP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，在敏感细胞系中，哌立福辛/索拉非尼联合疗法在体内外均可显著诱导Tribbles同源蛋白3（TRIB3）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。体内实验中，与对照组（中位生存期81天 vs 45天，P ≤ 0.0001）相比，单独使用哌立福辛组（49天，P ≤ 0.03）、单独使用索拉非尼组（54天，P ≤ 0.007），哌立福辛/索拉非尼联合疗法可显著提升HD-MyZ细胞系异种移植的NOD/SCID小鼠的中位生存期。在接种HD-MyZ与L-540细胞系（而非HDLM-2细胞系）皮下结节的小鼠中，与对照组或单药治疗组相比，哌立福辛/索拉非尼处理可显著提升细胞凋亡水平（2~2.5倍，P ≤ 0.0001）与坏死水平（2~8倍，P ≤ 0.0001）。此外，哌立福辛/索拉非尼治疗对HDLM-2皮下结节无显著影响，但可使L-540皮下结节的肿瘤生长抑制率达50%，较对照组显著缩小瘤体。

【结论】哌立福辛/索拉非尼联合疗法在体内外均展现出强效的抗HL活性。上述结果支持开展哌立福辛/索拉非尼联合疗法在HL患者中的临床评估。

【总体实验设计】本研究使用的霍奇金淋巴瘤细胞系HD-MyZ购自德国不伦瑞克的德国微生物和细胞培养物保藏中心（DSMZ）。细胞常规培养于添加10%胎牛血清（Fetal Bovine Serum, FBS）与2mM谷氨酰胺的RPMI 1640培养液（Lonza，瑞士巴塞尔）中，于37℃、5%CO₂饱和水蒸大气环境下传代培养。将10×10⁶个HD-MyZ细胞接种于150cm²培养瓶中，24小时后用10μM哌立福辛（Aeterna Zentaris，德国法兰克福）、或5μM索拉非尼（Bayer，德国柏林）处理细胞24小时。处理结束后收集细胞并提取RNA。