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Antitumor Activity of TRAIL-Mu3 Protein in vitro and in vivo and the Mechanisms

  • Objective TRAIL-Mu3 was obtained by mutating the N-terminus of human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene to an eight continuous arginine sequence. The present study was designed to explore the antitumor effect of this soluble mutant protein and the underlying mechanisms. Methods The inhibitory effect of TRAIL-Mu3 on the proliferation of lung cancer cell lines NCI-H460, A549, NCI-H1299 and calu-1 was tested by CCK8 assay. The apoptotic rates of A549 and NCI-H460 treated by TRAIL-Mu3 were detected by flow cytometer (FCM). The expressions of apoptosis related proteins death receptor (DR) 4, DR5, Caspase-3, Caspase-8 and X-linked inhibitor of apoptosis protein (XIAP) were detected by Western blot. Moreover, a subcutaneous xenograft tumor mouse model of NCI-H460 was established and treated with TRAIL-Mu3 daily or every other day or three times a week. The expressions of DR4, DR5, Caspase-3, Caspase-8 and XIAP were detected by immunohistochemical staining. Results The in vitro study demonstrated that as compared to the TRAIL, the TRAIL-Mu3 was more toxic and pro-apoptotic by up-regulation of the expression and activity of DR4, Caspase-3 and Caspase-8. Also, the animal study showed a similar antitumor effect between treatment with TRAIL-Mu3 every other day and three time a week, which was better than daily use. All treatments significantly suppressed the growth of xenograft tumor, increased the expression or activity of DR4 and Caspase-3, and down-regulated the expression of XIAP (P<0.05). Conclusion TRAIL-Mu3 could improve antitumor activity in vivo and in vitro through elevating DR4 expression, activating Caspase-3/-8, and inhibiting XIAP activation.
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    [14] VON KARSTEDT S, WALCZAK H. An unexpected turn of fortune: targeting TRAIL-Rs in KRAS-driven cancer. Cell Death Discov, 2020, 6: 14[2020-03-14]. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078304/. doi: 10.1038/s41420-020-0249-4.
    [15] HYMOWITZ S G, O'CONNELL M P, ULTSCH M H, et al. A unique zinc-binding site revealed by a high-resolution X-ray structure of homotrimeric Apo2L/TRAIL. Biochemistry,2000,39(4): 633–640. doi: 10.1021/bi992242l
    [16] KELLEY R F, TOTPAL K, LINDSTROM S H, et al. Receptor-selective mutants of apoptosis-inducing ligand 2/tumor necrosis factor-related apoptosis-inducing ligand reveal a greater contribution of death receptor (DR) 5 than DR4 to apoptosis signaling. J Biol Chem,2005,280(3): 2205–2212. doi: 10.1074/jbc.M410660200
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    [18] YAGOLOVICH A V, ARTYKOV A A, DOLGIKH D A, et al. A new efficient method for production of recombinant antitumor cytokine TRAIL and its receptor-selective variant DR5-B. Biochemistry (Mosc),2019,86(6): 627–636.
    [19] BOSMAN M C, REIS C R, SCHURINGA J J, et al. Decreased affinity of recombinant human tumor necrosis factor-related apoptosis-inducing ligand (rhTRAIL) D269H/E195R to osteoprotegerin (OPG) overcomes TRAIL resistance mediated by the bone microenvironment. J Biol Chem,2014,289(2): 1071–1078. doi: 10.1074/jbc.M113.491589
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    [25] SARAEI R, SOLEIMANI M, MOVASSAGHPOUR A, et al. The role of XIAP in resistance to TNF-related apoptosis-inducing ligand (TRAIL) in leukemia. Biomed Pharmacother,2018,107: 1010–1019. doi: 10.1016/j.biopha.2018.08.065
    [26] DI PIETRO R, ZAULI G. Emerging non-apoptotic functions of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L. J Cell Physiol,2004,201(3): 331–340. doi: 10.1002/jcp.20099
    [27] WAGNER K W, PUNNOOSE E A, JANUARIO T, et al. Death-receptor O-glycosylation controls tumor-cell sensitivity to the proapoptotic ligand Apo2L/TRAIL. Nat Med,2007,13(9): 1070–1077. doi: 10.1038/nm1627
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Antitumor Activity of TRAIL-Mu3 Protein in vitro and in vivo and the Mechanisms

    Corresponding author: JIN Zhao, dr.jinzhao@163.com
  • 1. Cancer Research Institute, Chengdu University of Traditional Chinese Medicine, Chengdu 610037, China
  • 2. Department of Emergency, West China Hospital, Sichuan University, Chengdu 610041, China
  • 3. Department of Gynaecology and Obstetrics, North Sichuan Medical College, Nanchong 637100, China
  • 4. School of Basic Medical Science, Chengdu University of Traditional Chinese Medicine, Chengdu 610037, China

doi: 10.12182/20200560102

Abstract:  Objective TRAIL-Mu3 was obtained by mutating the N-terminus of human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene to an eight continuous arginine sequence. The present study was designed to explore the antitumor effect of this soluble mutant protein and the underlying mechanisms. Methods The inhibitory effect of TRAIL-Mu3 on the proliferation of lung cancer cell lines NCI-H460, A549, NCI-H1299 and calu-1 was tested by CCK8 assay. The apoptotic rates of A549 and NCI-H460 treated by TRAIL-Mu3 were detected by flow cytometer (FCM). The expressions of apoptosis related proteins death receptor (DR) 4, DR5, Caspase-3, Caspase-8 and X-linked inhibitor of apoptosis protein (XIAP) were detected by Western blot. Moreover, a subcutaneous xenograft tumor mouse model of NCI-H460 was established and treated with TRAIL-Mu3 daily or every other day or three times a week. The expressions of DR4, DR5, Caspase-3, Caspase-8 and XIAP were detected by immunohistochemical staining. Results The in vitro study demonstrated that as compared to the TRAIL, the TRAIL-Mu3 was more toxic and pro-apoptotic by up-regulation of the expression and activity of DR4, Caspase-3 and Caspase-8. Also, the animal study showed a similar antitumor effect between treatment with TRAIL-Mu3 every other day and three time a week, which was better than daily use. All treatments significantly suppressed the growth of xenograft tumor, increased the expression or activity of DR4 and Caspase-3, and down-regulated the expression of XIAP (P<0.05). Conclusion TRAIL-Mu3 could improve antitumor activity in vivo and in vitro through elevating DR4 expression, activating Caspase-3/-8, and inhibiting XIAP activation.

  • 20世纪90年代中期,WILEY等和PITTI等先后发现一种对肿瘤具有良好靶向性杀伤作用的内源性细胞因子和免疫因子,分别命名为肿瘤坏死因子相关凋亡诱导配体(TRAIL)[1]和凋亡素2配体(apoptosis ligand 2, Apo2L)[2],后发现为同一种蛋白因子,其特点为可诱导多种肿瘤细胞凋亡的同时对正常细胞毒性较小。该发现迅速掀起了相关作用机理、药物联用、生物工程等方面的研究热潮,TRAIL作为潜在的抗肿瘤药物被认为具有巨大的开发前景[3]

    TRAIL通过与凋亡受体相结合,激活细胞内凋亡通路,诱导肿瘤细胞凋亡。目前发现的TRAIL受体有TRAIL-R1/死亡受体4(death receptor 4, DR4)、TRAIL-R2/死亡受体5(death receptor 5, DR5)和竞争性抑制DR4、DR5的诱骗受体DcR1、DcR2、OPG。TRAIL与凋亡受体DR4和DR5结合后,死亡受体胞内段的Fas相关死亡结构域(Fas-associated death domain, FADD)募集,形成由DR4、DR5和Caspase前体组成的死亡诱导信号复合物(death-inducing signaling complex, DISC),Caspase-8前体自身激活,引发级联反应,然后既可作用于Caspase-3前体,经外源性凋亡通路诱导细胞凋亡,也可作用于Bid(BH3-interacting domain death agonist),通过内源性凋亡通路诱导凋亡[4]

    但经过20余年的研究和开发发现,TRAIL制备成本高[5-6]、体内的半衰期短[7]、耐药率高(约达50%)、单用疗效有限[3]。TRAIL-Mu3是为解决以上问题而设计的TRAIL突变体之一,它在野生型TRAIL可溶性片段的基础上将N端突变为8个连续的精氨酸(第114~122位氨基酸),优点是发酵的目的蛋白产量增多,且主要以可溶性蛋白的形式表达,提高了纯化效率,有利于提高成药质量,在降低成本的同时也降低临床用药发生毒副作用的风险,更重要的是具备更强的抗肿瘤活性[8-9]

    本实验在前期药物制备已经完成的基础上,分别在体内和体外对TRAIL-Mu3的抗肿瘤活性及作用机理进行研究,并对体内给药方案进行初步的摸索,为将来临床试验提供一定支持。

1.   材料与方法
  • 野生型TRAIL和TRAIL-Mu3由本实验室前期构建。紫杉醇购自百时美施贵宝。人大细胞肺癌细胞NCI-H460、人非小细胞肺癌细胞A549和NCI-H1299、人肺癌细胞calu-1均购自中科院细胞生物研究所。RPMI1640、DMEM、McCoY’s 5A、FBS均购自GIBCO(美国)。Balb/c小鼠购自成都达硕实验动物有限公司。Annexin Ⅴ-FITC/PI Apoptosis Detection Kit购自日本同仁化学研究所。BCA蛋白检测试剂盒、苏木素染液、伊红染液、山羊抗鼠IgG(H+L)二抗-HRP、山羊抗兔IgG(H+L)二抗-HRP、SuperSignal West Pico Chemiluminescent Substrate购自Thermo-Fisher(美国)。TRAIL(H-257)兔抗人多克隆抗体、TRAIL-R2鼠来源单克隆抗体、Caspase-8兔来源多克隆抗体、Caspase-3兔来源多克隆抗体、肿瘤坏死因子受体超家族成员10A(TNFRSF10A)鼠来源单克隆抗体、人/小鼠/大鼠XIAP抗体、驴抗羊IgG-HRP、PV-9003山羊超敏二步法兔免疫组化试剂盒均购自Abcam(美国)。

  • 取对数生长期的4株肺癌细胞株(NCI-H460 5×103/孔、A549 8×103/孔、NCI-H1299 5×103/孔、calu-1 5×103/孔),接种于96孔板,每孔依次加入梯度质量浓度的TRAIL-Mu3和野生型TRAIL的培养液100 μL,每种质量浓度设3复孔。48 h后,利用CCK8法检测TRAIL-Mu3对细胞增殖的抑制作用,用酶标仪测定450 nm处吸光度值。按(Ac-As)/(Ac-Ab)×100%计算药物对肿瘤细胞生长的抑制率(%)。As为样品(细胞+CCK8+待测化合物)的吸光度,Ac为阴性对照(细胞+CCK8)的吸光度,Ab为阳性对照(各细胞对应培养基+CCK8)的吸光度。

    运用软件Originpro9.0公式Growth/Sigmoidal.Logistic进行半数致死量(50% inhibition concentration, IC50)曲线拟合并计算出IC50值。

  • 取对数生长期的A549(7.5×104 mL-1)和NCI-H460(12×104 mL-1)细胞接种至6孔板,每孔加2 mL细胞悬液,培养24 h。根据增殖抑制实验所得IC50值,用细胞相对应的培养基将TRAIL-Mu3稀释至上药质量浓度(A549取0.1、0.02 μg/mL,NCI-H460取0.01、0.025、0.005 μg/mL),以对应同体积培养基作为对照,加入对应的细胞孔中,继续培养5 h。胰酶消化细胞,PBS洗两遍,分别进行Annexin Ⅴ和PI染色后,利用流式细胞仪进行检测,计算早期凋亡(Annexin Ⅴ+ PI)+晚期凋亡(Annexin Ⅴ+ PI+)所占百分比,即得到凋亡率。实验重复3次。

  • 将肺癌细胞株NCI-H460接种至90 mm培养瓶皿,待达到80%密度时上药,上药质量浓度为0.001、0.005 μg/mL,或加入相同体积培养基作为对照。药物作用24 h后,收集提取总蛋白,利用BCA蛋白定量法对其进行定量。然后进行配胶、电泳分离、转膜、封闭、一抗孵育、洗膜、二抗孵育、洗膜、显色等步骤后,利用Tanon凝胶成像系统进行图像采集,并通过image J软件检测各条带灰度值,以内参β-actin灰度值为1,计算其它条带的相对灰度值进行半定量分析。

  • 30只雌性4~6周龄Balb/c裸鼠,每只裸鼠于右侧背部皮下接种0.1 mL 3×106个NCI-H460细胞。在肿瘤体积达到200 mm3时,按随机区组法进行随机分组(生理盐水组、紫杉醇组、TRAIL-Mu3每日给药组、TRAIL-Mu3隔日给药组和TRAIL-Mu3每周3次给药组)并开始尾静脉给药,每次给药体积0.1 mL。具体情况见表1

    GroupNumber of miceTest substancesDose/(mg/kg)Dosing frequency
    Control6SalineNot available (0.1 mL)Day 0, 1, 2, 3, 4
    Paclitaxel6Paclitaxel20Day 0, 2, 4
    TRAIL-Mu3 (every day)6TRAIL-Mu360Day 0, 1, 2, 3, 4, 7, 8, 9, 10, 11
    TRAIL-Mu3 (every other day)6TRAIL-Mu360Day 0, 2, 4, 6, 8, 10, 12, 14, 16, 18
    TRAIL-Mu3 (3 times/week)6TRAIL-Mu360Day 0, 2, 4, 7, 9, 11, 14, 16, 18

    Table 1.  Experimental design for the antitumor effect of TRAIL-Mu3 in vivo

    实验期间每周测定两次动物体质量并用游标卡尺测量肿瘤长径和短径。肿瘤体积(V)=(X2×Y)/2,其中X为短径(单位mm),Y为长径(单位mm)。每日观察记录临床症状。给药后第21天采用颈椎脱臼法处死动物,剥取肿瘤并称重,肿瘤组织经甲醛固定,用于免疫组化检测。

  • 标本经脱蜡、水化、抗原修复、染色、一抗孵育、二抗孵育、复染等步骤进行免疫组化EnVision二步法染色或XIAP山羊超敏二步法染色,其中,各抗体的滴度分别为:Caspase-8(1∶200)、Cleaved Caspase-3(1∶100)、DR4(1∶50)、DR5(1∶50)、XIAP(1∶50)。染色后在光镜下观察,采图。每张异种移植瘤组织切片于光镜下随机选取5个高倍视野,免疫组化评分包括阳性细胞百分率和细胞染色强度两方面。阳性细胞百分率(A):阳性细胞计数<5%为0分,5%~25%为1分,26%~50%为2分,51%~75%为3分,>75%为4分。细胞染色强度(B):无着色为0分,淡黄色为1分,棕黄色为2分,棕褐色为3分。免疫组化评分值=A×B。

  • 实验结果以$\bar x \pm s$表示。两组间比较采用非配对t检验;多组间均数比较采用单因素方差分析ANOVA,组间两两比较采用LSD检验或Tamhane’s T2检验。α=0.05。

2.   结果
  • 结果见图1表2。TRAIL-Mu3对NCI-H460、A549和NCI-H1299肺癌细胞株的增殖抑制作用明显强于野生型TRAIL,但对calu-1而言无明显差异。根据药物体外疗效的判定方法,NCI-H460、A549、NCI-H1299为TRAIL-Mu3敏感株,calu-1为TRAIL-Mu3耐药株。

    Figure 1.  The inhibition curve of TRAIL-Mu3 and wild type TRAIL on NCI-H460 (A), A549 (B), NCI-H1299 (C), and calu-1 (D) (n=3)

    Lung cancer cellIC50/(μg/mL)
    TRAIL-Mu3Wild type TRAIL
    NCI-H4600.000 475±0.000 390*61.399 000±2.559 939
    A5490.007 780±0.029 133*121.300 000±0.098 217
    NCI-H12990.008 890±0.000 169*86.710 000±1.716 572
    calu-125.070 000±3.038 763*>122
    *P<0.05, vs. wild type TRAIL

    Table 2.  50% inhibition concentration (IC50) values of TRAIL-Mu3 and wild type TRAIL in different lung cancer cells

  • 图2图3所示,无论是A549细胞,还是NCI-H460细胞,TRAIL-Mu3不同质量浓度间细胞凋亡率差异均无统计学意义(P>0.05),但均高于各自的对照组(P<0.05)。结果表明,TRAIL-Mu3对于肺癌细胞株A549和NCI-H460具有明显促凋亡作用。

    Figure 2.  FCM analysis of apoptosis of A549 and NCI-H460 after treatment with TRAIL-Mu3

    Figure 3.  The apoptotic rates of A549 and NCI-H460 after treatment with TRAIL-Mu3 (n=3)

  • 结果如图4所示,与对照组相比,TRAIL-Mu3低剂量(0.001 μg/mL)组与高剂量(0.005 μg/mL)组DR4表达量均上调(P<0.05),Caspase-3和Caspase-8的活化水平增加(P<0.05),DR5、XIAP的表达差异无统计学意义(P>0.05)。

    Figure 4.  The expression of apoptotic related proteins in NCI-H460 after treatment with TRAIL-Mu3 (n=3)

  • 结果见图5。除紫杉醇组外,其余各组小鼠体质量均保持稳定。紫杉醇组荷瘤鼠体质量在用药7 d内有明显下降,后期略有所恢复。TRAIL-Mu3各组对NCI-H460裸鼠异种移植瘤的生长抑制作用优于紫杉醇组,差异有统计学意义(P<0.05),且在总剂量基本相同的情况下,TRAI-Mu3隔日给药组和每周3次给药组差异无统计学意义(P>0.05),但均优于每日给药组(P<0.05)。TRAIL-Mu3各组荷瘤鼠的肿瘤质量均低于紫杉醇组(P<0.05),抑瘤率均高于紫杉醇组(P<0.05);且TRAIL-Mu3隔日给药组和每周3次给药组的抑瘤效果优于每日给药组,差异有统计学意义(P<0.05)。

    Figure 5.  The antitumor effect on NCI-H460 subcutaneous xenograft mouse model (n=6)

  • 免疫组化结果显示(图6图10):DR4和DR5阳性表达(黄褐色)定位于胞膜或胞浆,Caspase-3、Caspase-8和XIAP阳性表达(黄褐色)定位于胞浆。相较于生理盐水组与紫杉醇组,TRAIL-Mu3各组DR4表达增加,XIAP蛋白的表达降低,差异均有统计学意义(P<0.05),而DR5和Caspase-8无明显变化(P>0.05)。而在每周3次给药组中,还明显观察到Caspase-3活化的增加,与其余组比较差异均有统计学意义(P<0.05)。

    Figure 6.  The expression of DR4 in tumor tissues of NCI-H460 subcutaneous xenograft mouse model by IHC

    Figure 7.  The expression of DR5 in tumor tissues of NCI-H460 subcutaneous xenograft mouse model by IHC

    Figure 8.  The expression of Caspase-8 intumor tissues of NCI-H460 subcutaneous xenograft mouse model by IHC

    Figure 9.  The expression of Caspase-3 in tumor tissues of NCI-H460 subcutaneous xenograft mouse model by IHC

    Figure 10.  The expression of XIAP in tumor tissues of NCI-H460 subcutaneous xenograft mouse model by IHC

3.   讨论
  • 肺癌的发病率和死亡率均居全球恶性肿瘤的首位,预后不佳,5年生存率约为16.6%[7],寻找更加安全有效的治疗药物和治疗手段是当今面临的重要问题。TRAIL自1995年被发现以来,一直作为潜在的抗肿瘤药物来开发[3, 10-12]。内源性TRAIL在生理状态下起着免疫调控和对肿瘤发生起免疫监视的作用[13-14]。TRAIL-Mu3是一种TRAIL突变体,成功实现了TRAIL的工艺放大,并且初步显示出优于野生型TRAIL的抗肿瘤活性,IC50值在纳克级别[8-9],值得进一步研究和开发。

    根据相关文献报道,TRAIL重组蛋白或突变体通常在N端进行改变,且在噬菌体展示技术(构建随机肽库)[15-16]或自动设计算法FOLD-X模拟设计能够与DR4、DR5特异性结合的TRAIL的相关研究[17-25]中,从未出现TRAIL N端前10位氨基酸密码子对受体配体结合起决定性作用的预测。这均符合在不改变其关键抗原决定簇的的基础上建立TRAIL-Mu3的设想,并在前期实验中得到了证实[8-9]

    实验室前期实验还显示,野生型TRAIL体外对9大系统29株传代肿瘤细胞中的15株较为敏感,生物敏感率51.72%,而对于其中的14株表现为耐药,耐药率为48.28%[10-11]。该比例与DI PIETRO等[26](110株细胞株,敏感率62.73%)、WAGNER等[27](119株细胞株,敏感率50%)报道的数据相近。本实验就TRAIL-Mu3对于肺癌细胞株NCI-H460、A549、NCI-H1299、calu-1的抗肿瘤活性进行了测定,证明其体外药效明显优于野生型TRAIL,可高效地诱导A549和NCI-H460细胞的凋亡。

    基于其良好的体外抗肿瘤效果,本研究又探讨了TRAIL-Mu3不同给药方案对移植瘤治疗的效果。结果表明,隔日给药和每周3次给药优于每日给药方案。对此我们猜想TRAIL-Mu3诱导的凋亡信号可能在胞内持续作用一定时间,该现象类似于抗生素类药物的后遗效应,即使TRAIL-Mu3血药浓度低于最低有效血药浓度,肿瘤抑制作用仍然持续发挥。因此虽然TRAIL-Mu3在小鼠体内半衰期仅短短数分钟,但仍然可以在隔日给药或每周3次给药的间隔期间持续发挥诱导凋亡、抑制肿瘤增长的作用。模拟此条件在体外用细胞株进行实验,上药5 min后洗脱药物,48 h后测IC50值,凋亡作用显著,可以作为支持此假设的初步证据之一(尚未发表)。

    从凋亡通路来看,TRAIL与DR4和DR5结合后,通过DISC将凋亡信号传入胞内启动Caspase的级联放大反应[1-2, 4]。此过程中涉及多种促凋亡因子和抗凋亡因子的参与,如XIAP通过抑制Caspase-3、-7、-9的激活起抑制凋亡的作用,而第二个线粒体来源的胱冬肽酶激活剂(second mitochondria-derived activator of caspases, SMAC)可抑制XIAP,通过二次调节促凋亡[25];细胞型死亡结构域样白介素1β转化酶抑制蛋白(cellular Fas-associated death domain-like interleukin 1β-converting enzyme inhibitory protein, cFLIP)通过抑制Caspase-8的激活抑制细胞凋亡[4, 10];核转录因子kappa B(nuclear factor-kappa B, NF-κB)、促分裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)和蛋白激酶B(protein kinase B, PKB)的激活可抑制细胞凋亡[4, 10, 25]。由此可见,两种因子的相互作用结果决定了细胞的凋亡与存活,通过对凋亡相关蛋白的调控可在一定程度上逆转肿瘤细胞对TRAIL的耐药性。

    而本研究中,体外实验Western blot结果显示,TRAIL-Mu3可诱导DR4、Caspase-3、Caspase-8表达上调;体内实验中免疫组化结果显示,相比生理盐水组,TRAIL-Mu3诱导DR4、Caspase-3表达上调,XIAP表达下调。虽然二者结果不完全相符(Caspase-8、XIAP),但考虑体内外环境差异以及实验误差,或是导致此结果的原因之一,可通过重复实验或扩大样本量来解决。本实验仅就有限的凋亡通路进行了检验,而其是否还通过引发其他凋亡通路影响细胞凋亡,尚需要进一步探索。

    综上所述,本研究评价了TRAIL-Mu3重组蛋白对多株肺癌细胞的体内外抑瘤效果,证实其可通过上调肿瘤细胞DR4的表达、增加Caspase-3和Caspase-8的活化来实现更强的凋亡诱导作用,可一定程度逆转肿瘤细胞对野生型TRAIL的耐药性。并初步探讨了其给药方案,证实TRAIL-Mu3隔日给药和每周3次给药抑制作用相近,均优于每日给药方案。本实验为指导TRAIL-Mu3的抗肿瘤研究和临床用药奠定了基础。

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