药学学报  2016, Vol. 51 Issue (8): 1250-1256   PDF    
阿西替尼与多柔比星协同抗肿瘤活性研究
许秀丽, 黄园     
四川大学华西药学院, 四川 成都 610041
摘要: 为了研究阿西替尼与多柔比星的协同抗肿瘤活性,在体外用不同浓度的阿西替尼与多柔比星混合溶液处理人肺腺癌A549细胞株,以MTT法测定药物对肿瘤细胞及肿瘤球的生长抑制率,考察其协同作用。通过细胞摄取实验、细胞周期分布实验及DNA ladder实验对阿西替尼与多柔比星协同作用的机制进行初步探索。以A549实体瘤荷瘤裸鼠为模型,考察阿西替尼与多柔比星联合应用的体内抗肿瘤效果。结果显示,阿西替尼与多柔比星联用具有高度协同的抗肿瘤效果,其体外细胞毒性显著强于两种药物单独使用,且对肿瘤细胞和肿瘤球的生长抑制作用呈现时间-浓度依赖性;体内研究表明,阿西替尼与多柔比星联合使用能够显著抑制荷瘤裸鼠的肿瘤生长,且其抑瘤效果显著优于两药单独使用。
关键词: 阿西替尼     多柔比星     联合用药     协同作用     细胞毒性     抗肿瘤活性     A549细胞株    
Synergistic anti-tumor effects of axitinib and doxorubicin
XU Xiu-li, HUANG Yuan     
West China School of Pharmacy, Sichuan University, Chengdu 610041, China
Abstract: To study the synergistic anti-tumor effects of doxorubicin and axitinib in combination, and investigate the underlying mechanism, we performed the MTT cytotoxic assay and tumor spheroids inhibition to investigate in vitro using A549 cells. The cell internalization, cell cycle distribution and DNA ladder experiments were performed to study the synergistic mechanisms. A549 xenograft was established in nude mice and adopted to study the in vivo anti-tumor effect of doxorubicin and axitinib. Results showed that combination of doxorubicin and axitinib exerted significantly higher cytotoxicity than each single drug, and induced a synergistic effect on tumor spheroids growth inhibition. The combination achieved the highest tumor growth suppression in vivo in the A549 xenograft. The combination of axitinib and doxorubicin exhibited the best anti-tumor effects both in vitro and in vivo than each single drug.
Key words: axitinib     doxorubicin     combination therapy     synergistic effect     cytotoxic effect     anti-tumor activity     A549 cell    

癌症是一种威胁人类健康与生命的常见疾病和多发疾病,死亡率已超过心脑血管疾病跃居所有疾病死亡率的首位,已成为世界各国医学科学领域中的重要科研课题。癌症的致病机制复杂,联合用药已成为获得良好预后和降低毒副作用的常用手段[1, 2]。与单一药物治疗相比,联合用药能够同时调控异常细胞的多个信号通路,将治疗效果最大化,并可避免耐药现象的发生[3, 4]

阿西替尼 (axitinib,AXI) 为多靶点的酪氨酸激酶抑制剂,于2012年被美国FDA批准上市[5, 6]。其分子作用靶点为血管内皮生长因子受体1,2,3 (vascular endothelial growth factor receptor 1,2,3,VEGFR 1,2,3) 酪氨酸激酶深部ATP结合位点[7, 8],通过一氧化氮合酶、蛋白激酶B (protein kinase B,Akt) 及细胞外信号调节激酶等抑制下游信号传导[9],从而阻碍血管内皮生长因子介导的内皮细胞对细胞外基质蛋白的黏附与迁移,诱导血管内皮细胞凋亡,从而起到抑制肿瘤组织新生血管形成和降低肿瘤细胞存活率的作用[10]。已有研究发现阿西替尼与其他小分子抗肿瘤药物联合使用,可产生加和或协同抗癌效果,分别进入了不同的临床试验阶段[6, 11, 12]

多柔比星 (doxorubicin,DOX) 是临床上常用的蒽环类抗肿瘤抗生素,其抗瘤谱广,可以广泛用于肝癌、肺癌、乳腺癌和卵巢癌等恶性肿瘤的化疗[13, 14]。DOX主要通过插入细胞DNA,引发拓扑异构酶Ⅱ破坏DNA的三级结构来发挥药效[15, 16]。迄今为止,DOX单独或与其他抗肿瘤药物联合用药,均被认为是治疗实体瘤的强有力的化疗药物[17, 18]。但由于其对心脏、肾脏及神经等正常组织细胞的毒性、用药量大且到达病灶部位发挥抗肿瘤的比例较低等缺陷[19],使得DOX联合用药应运而生。

临床上通常将小分子抗肿瘤药物联用,但AXI与DOX联合用药尚未见报道,因此,本课题拟将AXI与DOX联用,通过一系列体内外实验考察二者的协同抗肿瘤活性,并对二者相互作用的机制进行初步探索。

材料与方法

仪器与试药 超纯水仪 (PCR-10,成都品成科技有限公司); 精密电子天平 (JJ200,美国双杰兄弟集团有限公司); 紫外可见分光光度计 (美国Varian Cary 100公司); 化学发光仪 (Varioskan flash,美国Thermo Scientific公司); 流式细胞仪 (CYTOMICS FC500); 微量高速离心机 (TG16W,长沙平凡仪器仪表有限公司); 磁力搅拌器 (84-1A,上海司乐仪器有限公司); 超声波清洗机 (SB-5200D,宁波新芝生物科技股份有限公司); 旋涡混合器 (IKA VORTEX GENIUS 3,广州仪科实验室技术有限公司); 酸度计 (pHS-3C+,成都世纪方舟科技有限公司); 集热式恒温加热磁力搅拌器 (DF-101S,巩义市予华仪器有限公司); 激光共聚焦显微镜 (CLSM,Live 5 DUO,Carl Zeiss,Jena,Germany); Transwell小室(美国Conning公司); 阿西替尼 (大连美仑生物技术有限公司); 盐酸多柔比星 (DOX·HCl,大连美仑生物技术有限公司); 低熔点琼脂糖、甲醇 (高效液相色谱纯)、二甲基亚砜 (分析纯)、胎牛血清、matrigel (低生长因子基底膜基质胶) 和4',6-联脒-2-苯基吲哚二盐酸盐 (DAPI) (成都宝科生物科技有限公司); 青霉素-链霉素混合液、多聚甲醛 (成都豪乙生物科技有限公司); 3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐 (MTT,美国Sigma公司)。

实验细胞株与动物 人肺腺癌A549细胞株、人脐静脉内皮细胞株(human umbilical vein endothelial cell,HUVEC) (中国科学院典型培养物保藏委员会细胞库); SPF级4周龄雄性BALB/c裸鼠 (成都达硕生物有限公司,动物合格证号: 0015775)。

MTT细胞毒性实验 DOX·HCl供试品溶液: 精密称取一定量DOX·HCl,以DMEM培养基溶解并稀释为不同浓度。AXI供试品溶液: 精密称取一定量AXI,以二甲基亚砜溶解并定容得到8 g·L-1贮备液,再以DMEM培养基稀释至不同浓度。AXI与DOX联用 (combination) 溶液: 分别取DOX·HCl与AXI供试品溶液,按质量比2∶1混合后以DMEM培养基稀释得到不同浓度溶液。

取对数生长期A549细胞,以8×103个/孔接种于96孔板,于37 ℃、5% CO2孵箱中培养24 h。吸出旧的培养基,每孔分别加入上述各供试品溶液200 μL,每个浓度设置3个复孔,以不加药的细胞孔作为阴性对照,不含细胞的培养基孔作为空白对照,于37 ℃、5% CO2孵箱中分别培养24和48 h。随后吸出含药培养基,加入培养基180 μL和5 g·L-1 MTT溶液20 μL,继续培养4 h,吸出混合液并加入DMSO 150 μL,于恒温摇床振荡15 min,使蓝紫色结晶充分溶解,荧光化学发光仪于570 nm波长处测定吸光度值。采用以下公式计算相对抑制率: RIR (%) = [1 - (Ce - C0) / (Cc - C0)] × 100,其中Ce: 实验组吸光度; C0: 空白组吸光度; Cc: 对照组吸光度。给药后24和48 h的半数抑制浓度 (IC50) 分别使用SPSS软件拟合计算得到,并根据Chou-Talalay等效图解公式[20]计算联合用药的协同作用指数 (combination index,CI)。

肿瘤球生长抑制实验 A549细胞以2×103个/孔接种于预铺2% (W/V) 低熔点琼脂糖的96孔板内,孵育48 h,待肿瘤球成型后,将含有5 μg·mL-1 AXI、10 μg·mL-1 DOX的药物单用、联用溶液分别加入各孔内,继续孵育7天,每天以倒置显微镜拍摄记录各组别肿瘤球生长状况,并绘制肿瘤球生长曲线。

HUVEC细胞迁移侵袭抑制实验 HUVEC细 胞以2×104个/孔接种于预铺低生长因子基质胶的transwell小室内,孵育4 h,待细胞贴壁后,将含有0.5 μg·mL-1 AXI、1 μg·mL-1 DOX的药物单用、联用溶液分别加入各孔内,继续孵育24 h,以倒置显微镜拍摄记录并计数各组别HUVEC侵袭状况 (迁移实验除小室内不涂基质胶外,其他步骤均相同)。

细胞周期分布实验 A549细胞以3×105个/孔接种于6孔板,于37 ℃、5% CO2孵箱中培养24 h。然后将含有5 μg·mL-1 AXI、10 μg·mL-1 DOX的药物单用、联用溶液加入各孔内,每个组别设置3个复孔,继续孵育24 h后,吸出各种供试品溶液,使用冷PBS洗涤细胞,以0.22% 胰蛋白酶溶液消化后,2 000 r·min-1离心3 min,吸出上清液,用PBS 400 μL洗涤细胞。使用预冷70% 乙醇溶液400 μL重悬细胞,于4 ℃固定2 h。4 000 r·min-1离心3 min,并使用PBS洗涤,加入RNA酶100 μL,于37 ℃水浴30 min,最后避光加入PI 400 μL于4 ℃染色30 min,使用CYTOMICS FC500流式细胞仪测定细胞平均荧光强度。

细胞摄取实验 A549细胞以1.25×105个/孔接种于铺有玻璃片和未铺玻璃片的12孔板内,孵育24 h,然后将含有5 μg·mL-1 AXI、10 μg·mL-1 DOX的药 物单用、联用溶液分别加入各孔内,每个组别设置 3个复孔,继续孵育4 h后移除含药培养基,细胞以冰乙醇洗涤3次,以4% 多聚甲醛溶液在室温下固定30 min,再加入4',6-联脒-2-苯基吲哚二盐酸盐 (DAPI) 静置10 min,对细胞核进行染色,用激光共聚焦显微镜拍摄记录各药物的摄取图片。

未铺玻璃片的12孔板于37 ℃、5% CO2孵箱中培养4 h后,吸出各供试品溶液,使用冷PBS洗涤细胞,以0.22% 胰蛋白酶溶液消化后,2 000 r·min-1离心3 min,用300 μL PBS重悬细胞,使用 CYTOMICS FC500流式细胞仪测定细胞平均荧光强度。

DNA ladder实验 A549细胞以1×107个/皿培养,将含有0.5 μg·mL-1 AXI、1 μg·mL-1 DOX的药物单用、联用溶液分别加入各培养皿内,孵育48 h,收集所有细胞,PBS清洗细胞,随后以70% 乙醇固定10 min,再次洗涤后转移到EP管内,弃去残余液体,加入lysis buffer 200 μL,然后加入RNase A混匀后37 ℃水浴90 min,加入蛋白酶K继续水浴90 min,酶解后,2 000 r·min-1离心2 min,取上清液电泳。

荷瘤裸鼠模型建立及体内抑瘤活性研究 取对数生长期人肺腺癌A549细胞,计数后,将细胞悬液调至每毫升1×108个,以每只0.1 mL接种于BALB/c雄性裸鼠右腋皮下。接种7天后,肿瘤长至约100 mm3,将荷瘤裸鼠随机分为4组 (saline、DOX·HCl、AXI、combination),每组4只,在0、3、6、9和12天按DOX 5 mg·kg-1、AXI 2.5 mg·kg-1的剂量尾静脉注射 (iv)。从第1次给药开始,每2天用游标卡尺测量肿瘤的长径和短径,按公式: V = a × b2 / 2计算肿瘤体积,其中a表示与瘤块表面平行的最长直径; b表示与瘤块表面平行且垂直于长径的直径。22天测量后将小鼠脱颈处死,并将皮下移植瘤剖出。记录给药22天内肿瘤生长曲线及剖出后肿瘤大小与形态。

统计学分析 采用SPSS 19.0统计软件进行统计学分析,数据用x± s表示,各组间比较采用单因素方差分析 (One-way ANOVA),P < 0.05为差异有统计学意义。

结果 1 AXI与DOX联合用药对人肺腺癌A549细胞生长抑制的影响

MTT细胞毒性实验结果如图 1所示,AXI与DOX联用对A549肿瘤细胞的杀伤力显著强于两种药物单独使用,其24和48 h的IC50值分别由3.028 μg·mL-1、0.604 μg·mL-1降低至1.312 μg·mL-1、0.330 μg·mL-1 (DOX·HCl当量); 二者24和48 h的CI分别为0.441和0.551,说明AXI与DOX联合使用能够产生高度协同作用。

Figure 1 Cell viability of A549 cells. A549 cells were seeded onto 96-well plates at a density of 8×103 cells per well and incubated for 24 h. After treatment with doxorubicin (DOX∙HCl),combination (DOX eq·dose 10,5,2.5,1.25,0.625 and 0.312 5 μg∙mL-1) and axitinib (AXI) (AXI dose 10,5,2.5,1.25,0.625 and 0.3125 μg∙mL-1) for 24,48 h at 37 ℃,medium was removed and cells were incubated for 4 h with 3-(4,5-dimethyl-2-tetrazolyl)-2,5-diphenyl-2H tetrazoliumbromide (5 g∙L-1 MTT,20 µL per well). Then the supernatant was removed before adding dimethylsulfoxide (DMSO,150 µL per well) into the wells to dissolve the formazane of MTT. Absorption at 570 nm was measured using an ELISA plate reader,and cell viability was calculated. n = 6,x± s
2 AXI与DOX联合用药对人肺腺癌A549肿瘤球生长抑制的影响

具有3D (three dimensional) 结构的肿瘤球常被用作体外模型来更好地模拟实体瘤,以考察药物的体外抗肿瘤活性[21, 22]。肿瘤球生长抑制实验结果与A549细胞生长抑制实验结果一致。未给药的对照组肿瘤球体积在7天的测量时间内显著变大,AXI与DOX联合使用能够显著抑制肿瘤球的生长,其抑制效果明显优于DOX·HCl组、AXI组及对照组 (P < 0.01),表明AXI与DOX联用具有更好的体外抗肿瘤效果 (图 2)。

Figure 2 The growth inhibition of A549 three-dimensional multicellular tumor spheroids. A549 cells (2×103 cells/well) were seeded in a 96-well plate pre-coated with 50 µL low melting point agarose (2%). The spheroids (incubated for 2 days) were exposed to AXI,DOX∙HCl and combination (DOX eq·dose 10 μg∙mL-1,AXI eq·dose 5 μg∙mL-1) with a drug-free medium treated group as the blank control. The length and width of each spheroid was monitored every day for 7 days and the volume was calculated by the following formula: V = π×length×width/6. n = 5,x± s. ΔΔP < 0.01 vs control; ##P < 0.01 vs AXI; **P < 0.01 vs DOX∙HCl. Scale bar,100 µm
3 AXI与DOX联合用药对HUVEC迁移侵袭抑制的影响

实验结果显示,AXI与DOX联用组HUVEC细胞迁移、侵袭到transwell小室聚碳酯膜另一侧的数目最少 (图 3),表明AXI与DOX联用抑制血管内皮细胞迁移侵袭的能力最强,AXI与DOX能够产生协同抑制血管内皮细胞迁移与侵袭的作用。

Figure 3 HUVEC migration (A) and invasion (B) inhibition effects. Matrigel invasion assays were performed at 37 ℃ using 24-well transwell inserts coated with 30 µg of matrigel. HUVEC (2×104 cells) suspended in 200 µL of serum-free medium contains AXI,DOX∙HCl and combination (DOX eq·dose 1 μg∙mL-1,AXI eq·dose 0.5 μg∙mL-1) were seeded into the upper chamber. After 24 h,invading cells on the lower membrane surface were fixed,stained and photographed. Transwell cell migration assays were performed similarly without matrigel. n = 3,x± s. ΔP < 0.05,ΔΔP < 0.01 vs control; #P < 0.05,##P < 0.01 vs AXI; P < 0.05,**P < 0.01 vs DOX∙HCl. Scale bar,50 µm
4 AXI与DOX联合用药对人肺腺癌A549细胞周期分布的影响

有文献[23, 24]报道,处于细胞周期不同阶段的肿瘤细胞对药物的摄取能力存在差异,强弱顺序为: G2期、S期 > G1期。实验结果 (图 4) 显示,AXI给药后,处于G2期的细胞数目显著增加。由此可以推断,AXI能够将肿瘤细胞阻滞于G2期。然而,AXI与DOX联合用药后,肿瘤细胞分布于G2期的比重与DOX·HCl组无显著性差异,推测两药联用时DOX对于细胞周期的影响强于AXI,其对肿瘤细胞周期分布的影响起主导作用。值得注意的是,联用组给药后,细胞处于sub-G1期的比重增加,表明二者联用具有协同的肿瘤细胞杀伤作用,产生更多的细胞碎片。

Figure 4 Influence on cell cycle distribution of A549 cells.A549 cells were seeded onto 6-well plates at a density of 3×105 cells per well and pretreated with AXI,DOX·HCl,combination (DOX eq·dose 10 μg·mL-1,AXI eq·dose 5 μg·mL-1) and drug-free medium for 24 h. Then cells were harvested and rinsed with cold phosphate buffer saline (PBS) three times,and fixed with 70% ice-cold ethanol at 4 ℃ for 2 h. After washed with ice-cold PBS and harvested,stained with PI and evaluated using a FACS Aria Cell Sorter. n = 3,x± s
5 AXI与DOX联合用药对细胞摄取的影响

激光共聚焦显微镜结果 (图 5A) 显示,联合给药组的细胞内红色荧光强度强于DOX·HCl组。以流式细胞仪定量测定摄取结果 (图 5B) 也表明,AXI与DOX联合使用能够显著增加细胞对DOX的摄取量 (P < 0.05)。AXI与DOX联用呈现出更多的细胞摄取量,该结果与联合给药组具有更强的肿瘤细胞杀伤力相一致。

Figure 5 Cellular internalization of doxorubicin by A549 cells. Red fluorescence represents doxorubicin; blue fluorescence represents cell nucleus. (A) A549 cells were incubated with DOX∙HCl and combination (DOX eq·dose 10 μg∙mL-1,AXI eq·dose 5 μg∙mL-1) and drug-free medium for 4 h at 37 ℃. Then cells were washed with ice-cold PBS three times,and fixed in 4% paraformaldehyde solution at room temperature for 30 min. 4',6-Diamidino-2-phenylindole (DAPI) was added and incubated for 10 min before imaging in order to visualize nuclei. Cell fluorescence was analyzed by confocal laser scanning microscopy. (B) A549 cells were incubated with DOX∙HCl and combination (DOX eq·dose 10 μg∙mL-1,AXI eq·dose 5 μg∙mL-1) and drug-free medium for 4 h at 37 ℃. After incubation,the medium was removed,and the cells were washed three times with ice-cold PBS,then trypsinized,centrifuged (2 000 r∙min-1,3 min),and re-suspended in PBS followed immediately by flowcytometry analysis. n = 3,x± s. P < 0.05 vs DOX∙HCl. Scale bar,20 µm
6 AXI与DOX联合用药对A549细胞DNA作用的研究

DNA ladder实验结果 (图 6) 显示,DOX和AXI联用与DOX单药相比,并未表现明显的DNA条带差异,说明AXI可能不是通过增强DOX破坏DNA三级结构的能力来发挥药效,二者产生协同作用的机制或许并不在此。

Figure 6 Influence on DNA of A549 cells. a: Control group; b: DOX∙HCl group; c: Combination group. A549 cells were incubated for 24 h. Then the cells were incubated with DOX∙HCl and combination (DOX eq·dose 1 μg∙mL-1,AXI eq·dose 0.5 μg∙mL-1) and drug-free medium for 48 h at 37 ℃. Cells were washed with ice-cold PBS three times,and fixed with 70% ice-cold ethanol at 4 ℃ for 10 min. After washed with ice-cold PBS and harvested,cell were incubated with RNase A and proteinase K and electrophoresis
7 AXI与DOX联合用药的体内抗肿瘤活性研究

荷瘤裸鼠体内抗肿瘤活性结果 (图 7) 显示,AXI与DOX联合用药对于A549皮下移植瘤具有最显著的抑制效果。给药后,各治疗组肿瘤体积增长的速度均低于saline组瘤体积增长的速度。其中联合给药组的肿瘤体积与AXI组相比具有极显著性差异 (P < 0.01),与DOX·HCl组相比具有显著性差异 (P < 0.05)。以上结果表明,AXI与DOX联合用药表现出更好的体内抗肿瘤活性,其原因可能是DOX引发拓扑异构酶Ⅱ破坏DNA的三级结构,直接产生了杀细胞作用; 同时,AXI通过一氧化氮合酶、Akt及细胞外信号调节激酶等抑制下游信号传导,诱导血管内皮细胞凋亡,从而抑制了肿瘤组织新生血管的生成,间接降低了肿瘤细胞存活率,二者产生了协同抗肿瘤作用。

Figure 7 Invivo anti-tumor effects of DOX and AXI. A549 tumor-bearing nude mice were randomly divided into four groups and intravenously injected with DOX∙HCl,AXI,combination or saline on the 0,3,6,9 and 12 days at a dose of 5 mg∙kg-1 DOX∙HCl and 2.5 mg∙kg-1 AXI. Tumor width and length were measured every 2 days from day 0,and tumor size was calculated following the formula: V = (length×width2)/2. n = 4,x± s. P < 0.05 vs DOX∙HCl; ##P < 0.01 vs AXI; ΔΔP < 0.01 vs saline
讨论

AXI为多靶点的酪氨酸激酶抑制剂,通过作用于VEGFR 1,2,3酪氨酸激酶深部ATP结合位点,阻碍血管内皮生长因子介导的内皮细胞对细胞外基质蛋白的黏附与迁移,诱导血管内皮细胞凋亡,从而起到抑制肿瘤组织新生血管形成和降低肿瘤细胞存活率的作用。DOX是临床上常用的蒽环类抗肿瘤抗生素,通过插入细胞DNA,引发拓扑异构酶Ⅱ破坏DNA的三级结构来发挥药效。本研究结果表明: AXI与DOX联合使用能够产生高度协同作用,其机制可能涉及: ① AXI能够显著增加DOX的细胞摄取量; ② AXI通过一氧化氮合酶、Akt及细胞外信号调节激酶等抑制下游信号传导,阻碍血管内皮生长因子介导的内皮细胞对细胞外基质蛋白的黏附与迁移,诱导血管内皮细胞凋亡,从而抑制了肿瘤组织新生血管形成,间接降低了肿瘤细胞存活率,与DOX破坏肿瘤细胞DNA三级结构,直接杀伤肿瘤细胞的作用相协同[9, 10]。值得注意的是,AXI与DOX联合使用更好地抑制了裸鼠皮下移植瘤的生长,其肿瘤体积显著小于单药组及生理盐水组,具有更好的体内抗肿瘤活性。

目前,基于AXI的联合用药体系多着重于在组织器官水平考察AXI进入体内后与抗肿瘤化学药物的协同作用[12, 25]。有研究表明,AXI与多西紫杉醇联合用于转移性乳腺癌治疗,其客观有效率较多西紫杉醇与安慰剂联用更高 (41.1% vs 23.6%,P = 0.011)[25]。另有文献报道,AXI能够增强托泊替康抑制肿瘤细胞增殖和诱导其凋亡的能力[26]。然而,AXI与DOX联用尚未见报道。本文首次在细胞水平上对AXI与DOX相互作用的机制进行了探索,并对其体内抗肿瘤效果进行了考察。本研究表明,AXI进入肿瘤细胞后,能够显著增加肿瘤细胞对DOX的摄取量。若将两者共同装载于纳米载体转运至肿瘤细胞,将AXI作为DOX增敏剂,与DOX共同被肿瘤细胞摄取,两者能够更好地发挥协同治疗效果,该药物联用组合为双载药递药系统的深入研究提供了依据。

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