冬凌草甲素诱导人多发性骨髓瘤细胞凋亡和增强其被激活的Allo-PBMCs杀伤敏感性
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摘要
研究背景与目的
多发性骨髓瘤(multiple myeloma, MM)是血液系统常见的恶性肿瘤,严重危害人类身体健康。目前MM的治疗主要有化学治疗、自体造血细胞移植和异基因造血干细胞移植等。传统化疗虽然能延长患者的中位生存期,但化疗药物本身对机体造成的副反应,以及患者原发／继发耐药等都给传统化学疗法的使用带来了一定障碍。相对于传统化疗,大剂量化疗联合自体干细胞移植能够延长患者中位生存期,异基因造血干细胞移植能够发挥移植物抗骨髓瘤效应,但同时异基因移植也伴随有更高的移植物相关死亡率。因此,需寻找新的药物和治疗手段提高MM的疗效。
冬凌草甲素(Oridonin, Ori)是一种从植物冬凌草中提取的四环二萜类化合物,具有广泛的生物学、药学和病原学功效,如抗炎、抗菌、抗肿瘤、抗氧化等。国内的应用表明,在食管癌、胃癌或肝癌等疾病中,Ori能够延缓疾病进展,降低肿瘤负荷,减少并发症同时延长患者生存期。近来研究显示,Ori可以诱导包括前列腺癌、乳腺癌、非小细胞肺癌、急性白血病(NB4、HL-60细胞)、多形性恶性胶质瘤以及人类黑素瘤在内的肿瘤细胞发生凋亡。此外,冬凌草甲素尚能延长荷淋巴细胞白血病P388细胞小鼠的生存期。尽管大量的研究均证明caspase3、caspase8、p53、Bcl-2/Bax、细胞色素c和核因子kappa B (NF-κB)等信号分子涉及到了Ori诱导的凋亡过程,但其对MM的抗肿瘤机制仍然未完全明了,此外,Ori的临床应用也仍需更进一步的深入研究探讨。
自然杀伤细胞(NK细胞)是遗传性免疫系统的主要效应细胞,对病源感染和转化了的细胞具有自然细胞毒效应。异基因造血干细胞移植中,同种异体反应性NK细胞(Allo-reactive Natutal Killer cell, Allo-NK)能够提升恶性血液病治愈率。NK细胞杀伤靶细胞受HLA-KIA等抑制性信号和NKG2D配体-NKG2D等活化性信号的共同调节。但Allo-NK细胞对MM的作用尚待研究。
依据MM的起源、病因、发病机理等,认为冬凌草甲素将会诱导ARH-77细胞凋亡,并推断Ori能提升肿瘤细胞对NKG2D+细胞的杀伤敏感性。本研究以人多发性骨髓瘤ARH-77细胞为研究对象,探讨冬凌草甲素的体内、体外抗肿瘤效应。为进一步的多靶干预治疗和临床应用提供理论和实验依据。
实验方法
第一章冬凌草甲素诱导人多发性骨髓瘤ARH-77细胞凋亡
以MTT的方法检测不同浓度冬凌草甲素在不同时间对ARH-77细胞的抑制作用。经不同浓度Ori (0、2.5、5、10μmol/L)作用24 h后观察以下指标：采用相差显微镜和Hoechst 33258染色观察细胞凋亡的形态学改变；流式细胞仪检测细胞凋亡率、细胞周期、线粒体膜电位、细胞表面TRAIL受体/Fas/FasL、细胞内活性氧簇的改变；以甲基纤维素半固体培养法测定ARH-77细胞在药物干预前后的体外克隆形成能力和Ori对人脐血单个核细胞克隆形成的影响；RT-PCR测定ARH-77细胞凋亡通路调节分子Bcl-2、Bcl-x、Bax、Bid、p53、NF-κB和β-actin的mRNA表达；分光光度法检测caspase 8、caspase 9的变化；ELISA法检测细胞色素c含量的变化。
第二章冬凌草甲素作用后ARH-77细胞对NKG2D+细胞杀伤敏感性的研究
ARH-77细胞经5μmol/L Ori处理24h,以LDH释放法测定Ori作用前、后ARH-77细胞对NKG2D+细胞杀伤敏感性的差异；RT-PCR方法检测活化性受体NKG2D的配体MICA、MICB、ULBP1、ULBP2和ULBP3在肿瘤细胞mRNA的表达；流式细胞仪测定ARH-77细胞表面NKG2D配体(MICA.MICB.ULBP1、ULBP2和ULBP3)、Fas/FasL/TRAIL的表达状态。
第三章冬凌草甲素对荷人多发性骨髓瘤细胞裸鼠的体内实验研究
选用Bul B/C nu/nu裸鼠为实验动物,通过尾静脉注射ARH-77细胞建立荷人多发性骨髓瘤ARH-77细胞的实验模型,以一般状况、病理学、免疫组织化学检测小鼠组织形态学和人特异性CD20、CD45抗原的表达,以评价实验模型的成功建立。给予治疗组小鼠腹腔注射冬凌草甲素(15mg/kg), PBS治疗作为对照组,以上述指标和TUNEL法观察药物治疗效果。
统计学方法
应用SPSS16.0软件进行数据处理,数据以均数±标准差(x±s)表示,所有统计方法主要包括单因素方差分析、独立样本t检验、析因设计的方差分析、重复测量的方差分析、Kaplan-Meier生存分析法。方差齐时组间多重比较采用SNK法,方差不齐时组间多重比较采用Dunnett's T3法,P<0.05提示差异有统计学意义。
结果
第一章冬凌草甲素诱导人多发性骨髓瘤ARH-77细胞凋亡
1.1冬凌草甲素对ARH-77细胞的生长抑制作用
MTT法测定细胞活性结果显示：Ori能明显抑制ARH-77细胞生长,在同一时间点,不同质量浓度的Ori对ARH-77细胞的抑制率有显著差异；在同一质量浓度Ori药物时,随着时间的延长,Ori对ARH-77细胞的生长抑制作用明显增强。表明Ori对人多发性骨髓瘤ARH-77细胞的生长抑制作用呈时间和剂量依赖性。同时设立DMSO作为溶剂对照,常用化疗药物阿霉素(ADM)为阳性对照,结果表明用于溶解Ori的DMSO对细胞生长无影响,低浓度(2.5、5、10μM)时ADM对ARH-77细胞即可产生明显的生长抑制作用,随浓度增加两组间抑制率的差异逐渐减小。对正常脐血中分离出的单个核细胞的作用结果显示只有高浓度(80、160μM)的药物才能明显抑制脐血单个核细胞的生长。
1.2冬凌草甲素诱导ARH-77细胞凋亡的研究
(1)冬凌草甲素诱导细胞凋亡
经10μmol/L Ori处理24h后可见细胞胞体变小、细胞碎片增多、胞质中出现空泡,Hoechst 33258染色可见处理后有部分细胞出现了细胞核浓缩、聚集,有些可见到凋亡小体。在0、2.5、5、10μmol/L各浓度药物诱导下,ARH-77细胞发生凋亡,流式结果示各组早期凋亡率分别为(5.27±1.46)%,(15.07±0.78)%,(21.00±1.49)%和(27.60±1.77)%,各时间点凋亡率的差异有统计学意义(F=133.331,P=0.000),提示Ori诱导ARH-77细胞凋亡呈浓度依赖性。
(2)冬凌草甲素对ARH-77细胞周期的影响
ARH-77细胞在Ori的作用下细胞周期发生了变化,主要表现为G0／G1期百分比增多,同时S期百分比逐渐减少,在2.5μmol/L这种趋势无统计学意义,而G2／M期百分比没有明显的变化,表明细胞停滞于G1期。细胞的亚二倍体也有增加的趋势,与对照组相比差异具有统计学意义(F=114.958,P=0.000)。
(3)冬凌草甲素对ARH-77细胞体外克隆形成的影响
体外克隆形成实验显示,第14天时,细胞可以形成克隆。与对照组相比,2.5μmol/L Ori能在某种程度上刺激ARH-77细胞克隆的生长,随着药物浓度增加,细胞克隆数锐减,当Ori浓度增加到10μmol/L的时候,培养基中已基本没有克隆的存在,表明人多发性骨髓瘤ARH-77细胞在体外有形成克隆的能力,而Ori能够抑制ARH-77细胞的这种能力。各组克隆数与对照组相比均具有统计学意义(F=952.968,P=0.000)。在第21天时,克隆仍然存在,且有增大趋势,Ori作用组的克隆数较14天时有增多,提示Ori可能对较为早期的干细胞没有抑制作用。不同浓度Ori处理组不能抑制脐血克隆的生成,克隆数比较没有统计学意义。
1.3冬凌草甲素诱导ARH-77细胞凋亡的机制探讨
(1)内源性凋亡通路的变化
RT-PCR检测结果显示：Ori能够下调Bcl-2、Bcl-xl的表达,轻度上调Bax的表达,对Bid没有明显改变。提示Ori通过调节Bcl-2家族比例诱导ARH-77细胞凋亡。此外Ori尚能上调p53 mRNA、NF-κB mRNA的表达,二者有可能参与了冬凌草甲素诱导ARH-77细胞的凋亡过程。
流式结果显示,ARH-77细胞经0、2.5、5、10μmol/L的Ori作用24 h后,细胞线粒体膜电位有降低的趋势,表明随药物浓度增加,线粒体膜发生去极化,且不同药物浓度作用的各组间均有差异(F=252.847,P=0.000)。
ELISA检测细胞色素c发现,随Ori药物浓度的增加ARH-77细胞中总细胞色素c的浓度逐渐减少(F=4.909,,P=-0.047),在10μmol/L Ori处理组与对照组、2.5μmol/L Ori处理组间相比具有统计学意义。
分光光度法检测caspase 9活性结果表明,经过不同浓度冬凌草甲素处理后,细胞内caspase 9的活性与对照组相比发生明显的改变(F=2275,P=0.000)。各组间比较的结果显示,低浓度组的caspase 9变化无统计学意义,5、10μmol/LOri处理后较对照组显著增高。提示经冬凌草甲素处理后内源性凋亡通路中caspase 9被活化,激活内源凋亡通路。
(2)外源性凋亡通路的变化
对照组ARH-77细胞表面,无论死亡受体(DR4、DR5)还是诱骗受体(DcR1、DcR2)的表达均较低,加入2.5μmol/L Ori作用24h后,死亡受体DR4表达有所增高,但DR5的比例却有所下降,而两种诱骗受体的表达均有小幅的提升；经中等浓度药物处理后,死亡受体的表达增加,以DR5增加为主,而诱骗受体的变化不太明显；但接近IC50浓度的Ori却能够明显升高ARH-77细胞表面诱骗受体DcR1的表达,降低死亡受体DR5的表达。10ng/ml TRAIL蛋白能促进ARH-77细胞的生长,100ng/ml时对细胞的影响和对照组无明显差异,在高浓度1000ng/ml TRAIL蛋白的作用下ARH-77细胞的生长受到了明显的抑制。5μmol/L Ori处理的ARH-77细胞对TRAIL蛋白的反应趋势同未处理时基本相同,但在10ng/ml和100ng/ml的浓度时TRAIL蛋白促进细胞生长的效应更加明显,而1000ng/ml TRAIL蛋白对处理后细胞的抑制也更加显著。综上可看出Ori对ARH-77细胞TRAIL受体表达的影响较为复杂,细胞在表达死亡受体、传导凋亡信号的同时也在进行自身适应性调节表达,对抗药物的促凋亡作用。
ARH-77细胞自身高表达Fas,经2.5μmol/L Ori处理即可引起其表面Fas表达的增加,表达率近100%且各个浓度之间无明显差异。ARH-77细胞FasL表达极低,Ori能够明显提升FasL的表达,最高可达(11.67±1.80)%。
Ori能激活外源性凋亡通路中caspase8 (F=116.93,P=0.000),2.5μmol/L和5μmol/L实验组间的差异无统计学意义。
(3)细胞活性氧簇(ROS)的改变
流式细胞术检测结果显示,经低浓度药物(2.5μmol/L)处理后,可使ARH-77细胞内的ROS水平有所提高,增加冬凌草甲素的浓度(5μmol/L)可迅速提升细胞内ROS的产生,随着药物浓度的继续增加(10μmol/L)细胞内ROS进一步升高,差异具有统计学意义(F=3966,P=0.000),且峰值出现明显左移。提示ARH-77细胞内ROS的产生与冬凌草甲素的药物作用浓度有一定关系。
第二章冬凌草甲素作用后ARH-77细胞对激活的Allo-PBMCs细胞杀伤敏感性的研究
(1) Allo-PBMCs细胞杀伤活性测定
测定经rhIL-15共培养后的Allo-PBMCs细胞对冬凌草甲素5μmol/L作用前后多发性骨髓瘤ARH-77细胞的杀伤活性。随着效靶比的升高,实验组和对照组的杀伤活性均有增强,差异有统计学意义(t=-4.048,P=0.016；t=-7.152,P=0.002)；而在同一效靶比时,Allo-PBMCs细胞对冬凌草甲素(5μmol/L)作用后的肿瘤细胞杀伤活性比作用前更强,差异具有统计学意义(t=-10.642,P=0.009;t=-6.458, P=0.003)。ARH-77细胞本身对Allo-PBMCs细胞的杀伤并不敏感,在效比为10：1的时候,杀伤活性只有(7.45±0.39)。结果提示：ARH-77细胞可以靶认为是被NKG2D+细胞杀伤的低度敏感细胞,中等浓度的冬凌草甲素能够提升NKG2D+细胞对该细胞的杀伤敏感性。
(2)冬凌草甲素处理前后ARH-77细胞NKG2D配体mRNA的表达
RT-PCR检测结果显示：两组细胞均扩增出5个片段,与预期片段大小一致。ARH-77细胞本身表达MICA、ULBP2,而MICB、ULBP1和ULBP3表达较低；药物作用后的实验组细胞NKG2D配体mRNA的表达与对照组相比：MICA、ULBP1和ULBP3的表达变化不大,MICB有增加的趋势,而ULBP2的mRNA水平有所减少。
(3)流式细胞术检测ARH-77细胞表面NKG2D配体的表达
FCM检测显示：ARH-77细胞低表达MICB (8.57±1.35),不表达MICA、ULBP1、ULBP2和ULBP3;经5μmol/L冬凌草甲素处理24h后,MICB的表达增加最为显著,差异具有统计学意义(t=-6.833, P=0.002)。MICA、ULBP2的表达也有小幅上升(t=-2.828, P=0.047;t=-4.243, P=0.013),但ULBP3的表达却有下降(t=2.889, P=0.045), ULBP1没有明显的变化。
(4) Allo-PBMCs表面TRAIL、Fas、FasL的表达
经IL-15诱导后,细胞表面表达Fas (86.40±14.71)、FasL (10.20±5.09)、TRAIL (1.00±0.14),在表面几乎检测不到TRAIL的表达,提示Fas/FasL可能在该杀伤效应中同样发挥促凋亡效应,但TRAIL和TRAIL受体通路没有激活。
第三章冬凌甲素对荷人多发性骨髓瘤细胞裸鼠的体内实验研究
(1)荷人多发草性骨髓瘤裸鼠模型的建立
经尾静脉注射骨髓瘤ARH-77细胞后约5周,部分小鼠开始出现体重下降、消瘦、不喜动,到第6周左右,模型组小鼠出现死亡,正常对照组和环磷酰胺预处理对照组小鼠状态良好,未出现体重下降。为进一步明确ARH-77细胞成功移植到小鼠体内,同时进行了病理学和组织化学分析。模型组小鼠肠道、肺脏结构发生异常改变；肝脏、脾脏、骨髓具有肿瘤细胞浸润,而且免疫组化结果显示浸润的人多发性骨髓瘤ARH-77细胞表达人特异性CD45和CD20抗原,且CD20表达高于CD45。肾脏未见明显异常改变。正常对照组小鼠脏器镜下显示结构整齐、无异常细胞浸润,环磷酰胺预处理组结果同正常对照组。提示浸润的细胞来自移植到小鼠体内的人多发性骨髓瘤ARH-77细胞。
(2)冬凌草甲素体内实验研究
模型组小鼠的情况基本与前述建立的模型相同,使用冬凌草甲素治疗组小鼠在第5周未出现模型组的状况,生长状况良好。大约到第8周小鼠有部分出现消瘦、食欲不振等行为。为对药物治疗小组小鼠进行对照性比较,因此实验在处死模型组小鼠同时,对治疗组小鼠也处死1只,在同期进行病理学和组织化学分析比较。模型组小鼠肝脏、脾脏具有肿瘤细胞浸润,而且免疫组化结果显示浸润的肿瘤细胞表达人特异性CD45和CD20抗原,且CD20表达高于CD45,表明异常浸润的是输注的多发性骨髓瘤ARH-77细胞。冬凌草甲素治疗组小鼠肝脏中仅有少量细胞浸润,肝脏、脾脏结构基本正常,而且生存期较模型组延长。TUNEL细胞凋亡原位检测光镜下可见经Ori治疗后,小鼠的肝脏、脾脏内可观察到部分浸润细胞被诱导产生凋亡。提示药物可能抑制了骨髓瘤ARH-77细胞在小鼠体内的生长,诱导浸润细胞凋亡,延长生存期。
结论
1.冬凌草甲素能够诱导人多发性骨髓瘤ARH-77细胞凋亡,其诱导ARH-77细胞凋亡呈时间剂量依赖性；
2.冬凌草甲素诱导ARH-77细胞凋亡是通过调节Bcl-2家族成员、细胞表面死亡受体的表达、以及改变线粒体膜通透性,激发内／外源凋亡通路引起的；
3.冬凌草甲素能够引起ARH-77细胞内活性氧簇(ROS)的变化；
4.5μmol/L冬凌草甲素能够通过提高ARH-77细胞NKG2D配体、Fas的表达,从而提升对激活的Allo-PBMCs杀伤的敏感性。
5.成功建立人多发性骨髓瘤动物体内模型,冬凌草甲素在体内能减少肿瘤细胞的浸润、延长生存期。
Background and Objectives
Oridonin (Ori), a tetracycline diterpenoid isolated from the plant Rabdosia rubescens, has various biological, pharmaceutical, and physiological functions such as anti-inflammation, anti-bacteria activity and others. It was shown in China to be able to suppress disease progress, reduce tumor burden, alleviate syndrome, and prolong survival in patients with esophageal, gastric carcinoma or liver cancer. Recent studies showed that oridonin induced apoptosis in a variety of cancer cells including those from prostate, breast, non-small cell lung cancers, acute leukemia(NB4, HL-60 cells), gioblastoma multiforme, and human melanoma cells. In addition, oridonin could also increase lifespan of mice bearing P388 lymphocytic leukemia. However, though studies showed that caspase 3(casp 3), casp 8, p53, Bcl-2/Bax, cytochrome c (cyto c), and nuclear factor kappa B(NF-icB)were involved in apoptosis induced by oridonin, mechanisms underlying the anti-MM cells activity of oridonin remain largely unknown, and whether oridonin can find clinical application still needs more investigation.
Natural Killer cells (NK cells) are important effectors of the innate immune system with a critical role in early host defense against invading pathogens and transformed cells. In allogeneic hematopoietic transplantation, donor-derived allo-reactive natural killer (Allo-NK) cells have shown improved cure rate in hematopoietic malignancies. Cytotoxicity of NK ells is regulated both by HLA-Killer Immunoglobulin-like Receptors (KIRs) (HLA-KIR) inhibitory signals and NKG2D ligand-NKG2D (NKG2DL-NKG2D) activating signals. Nevertheless, it is unknown whether NKG2D+ cells have an effect on human multiple myeloma ARH-77 cells.
On the basis of origin, etiology and pathogenesis of multiple myeloma, we hypothesize that oridonin could induce apoptosis on human multiple myeloma ARH-77cells and enhance the sensitivity of ARH-77 cells to NKG2D+ cells cytolytic effects. In this study, we choose human multiple myeloma ARH-77 cells as the research object, and investigate the anti-tumor effects by oridonin in vitro and in vivo.
Methods
PartⅠOridonin induced human multiple myeloma ARH-77 cells apoptosis
The growth inhibitions of ARH-77 cells by various concentrations of oridonin at different time were analyzed by MTT assay. ARH-77 cells were treated with various concentrations of oridonin(0、2.5、5、10μmol/L) for 24h, and then observed following results:the morphological changes of apoptotic cells were observed by phasecontrast microscope and Hoechst 33258 staining; the apoptosis rate, the cell cycle, the changes of mitochondrial membrane potential(Δψm), TRAIL receptor on cell surface and intracellular reactive oxygen species(ROX) were detected by flow cytometry (FCM); mRNA expressions of Bcl-2 family members(Bcl-2, Bcl-xl, Bax, Bid), p53, NF-κB andβ-actin were analyzed by RT-PCR; the changes of caspase 8 and caspase 9 were measured by spectrophotometry.
PartⅡThe sensitivities of human multiple myeloma ARH-77 cells to actived Allo-PBMCs mediated cytotoxicity after oridonin treated
ARH-77 cells were treated with 5μmol/L oridonin for 24h, then the differences between oridonin-treated and no oridonin-treated ARH-77 cells in sensitivity to NKG2D+ cells cytotoxicity were measured by LDH releasing assay; mRNA expressions of NKG2D ligands MICA, MICB, ULBP1, ULBP2 and ULBP3 were measured by RT-PCR; the changes of NKG2D ligands(MICA, MICB, ULBP1, ULBP2 and ULBP3) on cell surface were analyzed by flow cytometry (FCM).
PartⅢThe ability of Oridonin to human multiple myeloma ARH-77 cells in vivo
Selected BulB/C nu/nu nude mice as experimental animals, through the tail vein injection of ARH-77 cells establish bearing human multiple myeloma ARH-77 cells experimental model. The general conditions, pathology, immunohistochemistry were used to analyze mice tissue morphology and human-specific CD20, CD45 antigen expression, and then evaluate the success of the establishment of experimental model. The tumor-bearing mice were treated with an intraperitoneal injection of oridonin 15mg/kg), control mice received injection with PBS, observe of the above-mentioned targets as drug therapy effectiveness.
Statistical analysis The analysis was performed using SPSS 16.0 software package. The data was represented as the mean±standard deviation (x±s).Comparisons of means among groups were performed using one-way analysis of variance (ANOVA), independent-samples t-test, factorial design analysis of variance, repeated measures, Kaplan-Meier. If variance were homogenous among groups, the SNK method was used for multiple comparisons, otherwise Dunnett's T3 method was used, P<0.05 were considered to be statistically significant.
Results
PartⅠOridonin induced human multiple myeloma ARH-77 cells apoptosis
1.1 The inhibition to ARH-77 cells with oridonin
MTT results showed that:oridonin significantly inhibited the growth of ARH-77cells, at the same time point, different concentrations of the oridonin on the ARH-77 cells, the inhibition rates were significantly different; at the same concentration, with time, the growth inhibition of oridonin were markedly enhanced. Oridonin obviously inhibited the growth of ARH-77 cells in a time- and dose-dependent manner.
1.2 To investigate the apoptosis of ARH-77 cells induced by oridonin
(1) Oridonin induced ATH-77 cells apoptosis
After treated with 10μmol/L Ori for 24h, the cells shrank, cell debris increased, with vacuolus in it, Hoechst 33258 staining showed that some cells after treatment appeared in nuclear enrichment, aggregation, and some apoptotic bodies can be seen. After 0,2.5,5,10μmol/L oridonin induced, ARH-77 cells apoptosis, FCM showed that, the apoptosis ratios of each groups are (5.27±1.46)%, (15.07±0.78)%, (21.00±1.49)% and (27.60±1.77)%, respectively, and significantly difference at all the concentration (F=133.331, P=0.000).Oridonin induced ARH-77 cells apoptosis in a dose-dependent manner.
(2) Influence of ARH-77 cell cycle induced by oridonin
After treated with different concentration oridonin, ARH-77 cellcycles were changed,mainly of G0/G1 phase cells gradually increased, while decreasing the S phase cells, at low concentrations (2.5μmol/L) this trend does not maintain statistics significance, while the G2/M phase cells have no obvious change, indicating that ARH-77 cells were arrested in G1 phase by oridonin. cells Sub-diploid were also increased, compared with the control group (F=114.958, P=0.000).
(3) In vitro colony formation of ARH-77 cells treated with oridonin
In vitro colony assays indicated, cells could shape clone at Day 14. Contrast with control group, low concentration(2.5μmol/L) of Ori in some extent, could stimulate ARH-77 cell clones formation, with oridonin concentration increased, the number of cell clones drastically reduced, when the oridonin increased 10μmol/L, no clone exists, that indicated human multiple myeloma ARH-77 cells have the ability to form clones in vitro, while the oridonin can inhibit the clone form ability. The numbers of Cloning for each groups compared with the control group were statistically significant (F=952.968, P=0.000)
1.3 The mechanism of ARH-77 cells aopotosis induced with oridonin
(1) Changes in intrinsic apoptosis pathway
RT-PCR results showed that:oridonin could significantly down-regulated the expressions of Bcl-2, Bcl-xl, mild up-regulated the expressions of Bax, and no effect on Bid. Indicate Oridonin induced ARH-77 cells apoptosis through regulated Bcl-2 family members. In addition, oridonin also have capable of raising the expressions of p53 mRNA and NF-κB mRNA expressions, both moleculars were involved in the ARH-77 cells apoptosis induced by Oridonin.
Flow results manifested that, ARH-77 cells were treated with different concertions of oridonin(0,2.5,5,10μmol/L) for 24 h, then cells mitochondrial membrane potential were degreaded, indicating that with the increase of drug concentrations, mitochondrial membrane depolarization occurred (F=252.847, P= 0.000), and there were were differences among the each groups.
To test cytochrome c by ELISA found, with the increased concentration of Ori, the total concentration of cytochrome c in ARH-77 cells were decreasd (F=4.909, P=0.047), there were no dignificant statistics between 10μmol/L Ori-treated group and control group,2.5μmol/L Ori-treated group.
Spectrophotometric detection of caspase 9 activity results showed that, after treatment with different concentrations of oridonin, compared with the control group, the activities of caspase 9 significant changed (F=2275, P=0.000). Comparison between each group, the activitiy of caspase 9 in low concentrations group was no significant change, after treated with 5,10μmol/L oridonin the activities of caspase 9 were significantly higher than the control group. These findings indicated oridonin could activate caspase 9 in extrinsic apoptosis pathway.
(2) Changes in extrinsic apoptosis pathway
On the ARH-77 cell surface, regardless of death receptors (DR4, DR5) or decoy receptors (DcR1, DcR2) expressions were lower, co-incubated with low concentration of Ori (2.5μmol/L) for 24h, the death receptors DR4 expressions was increased, but the proportion of DR5 was decreased, whereas the expression of two decoy receptors had a slight up-grade. Treated with moderate concentrations of oridonin, the death receptor expressions increased mainly about DR5, while the decoy receptors were less obvious; but close to IC50 concentration, decoy receptors DcR1 on ARH-77 cell surface were significantly higher than control group, meantime the expressions of death receptor DR5 were reduced.10ng/ml TRAIL can promote ARH-77 cells growth, 100ng/ml had no obvious affected to cells, the ATH-77 cells were strongly inhibited by 1000ng/ml TRAIL.5μmol/L Ori-treated group had the same trends with control group, in 10ng/ml and 100ng/ml Ori could make TRAIL more easy to enhance of the cells growth and 1000ng/ml TRAIL also could suppresse the ARH-77cells more significantly. therefore, the effects of Ori to the expressions of TRAIL-R in ARH-77 cells were more complex. the cells expressed the DR, transmitted apoptosis signals,meantime they adaptated accommodation to agonistic drugs.
(3) The changes of Reactive Oxygen Species(ROX) on ARH-77 cells
FCM results showed that, after treated with low concentrations of oridonin (2.5μmol/L), the levels of ROS to ARH-77 cells were up-regulated, then increased the concentration of oridonin (5μmol/L) can quickly enhance the production of intracellular ROS, with the continued increase of oridonin concentration (10μmol/ L), the intracellular ROS further increased, the difference was statistically significant (F=3966, P=0.000), and the peak apparent left shift. So, these indicated the generation of ROS by ARH-77 cells were associated with the oridonin concentration.
PartⅡThe sensitivities of human multiple myeloma ARH-77 cells to Allo-PBMCs-mediated cytotoxicity after oridonin treated
(1) Measurement of Allo-PBMCs cytotocixity
Peripheral blood mononuclear cells co-culture with rhIL-15, then measured the cytocixity of Allo-PBMCs against multiple myeloma and ARH-77 cells, which before and after treated oridonin 5μmol/L in vitro. With the effective target(E:T) ratios increased, the cytocixity of the experimental groups and control groups were enhanced, respectively, the difference was statistically significant (t=-4.048, P= 0.016; t=-7.152, P=0.002); while at the same E:T ratio, the cytocixities of Allo-PBMCs against the treated ARH-77 cells were stronger than the role of the former, the difference was statistically significant (t=-10.642, P=0.009; t=-6.458, P=0.003).
ARH-77 were low sensititive to Allo-PBMCs cells cytolytic effects, at the E:T ratio of 10:1, cytotoxic activity was only (7.45±0.39). The results suggested that: ARH-77 cells could be considered to be the low degree of sensitive to Allo-PBMCs, moderate concentration of oridonin can enhance the cytocixity of Allo-PBMCs against multiple myeloma and ARH-77 cells.
(2) The mRNA expression of NKG2D ligands on ARH-77 cells before and after treatment with oridonin
RT-PCR results showed that:two groups were amplified by five fragments consistent with the expected fragment size. ARH-77 cells themselves expressed MICA, ULBP2, while MICB, ULBP1 and ULBP3 expression lower; compare with control group, the expressions of NKG2D ligands mRNA on experimental group treated with oridonin were:MICA, ULBP1 and ULBP3 expressions changed little, mRNA expressions of MICB were increased, the mRNA level of ULBP2 decreased.
(3) The expressions of NKG2D ligand on ARH-77 cell surface by FCM
FCM tests showed:ARH-77 cells with low expressions of MICB (8.57±1.35), did not express MICA, ULBP1, ULBP2 and ULBP3; after treated with oridonin 5μmol/L for 24h, the expressions of MICB were significant increased (t=-6.833, P =0.002). The expressions of MICA, ULBP2 were also moderately enhanced (t=-2.828, P=0.047; t=-4.243, P=0.013), but The expressions of ULBP3 were decreased (t=2.889, P=0.045), and ULBP1 had no significant change.
(4) The espressions of TRAIL、Fas、FasL in Allo-PBMCs
After treated with IL-15, the expressions of Fas, FasL and TRAIL on cell surface were (86.40±14.71), (10.20±5.09), (1.00±0.14), there were almost no TRAIL on Allo-PBMCs. The findings indicated that Fas/FasL may play a role in induce apoptosis effects, but the TRAIL/TRAIL-R pathyway were not activated.
PartⅢThe ability of Oridonin to human multiple myeloma ARH-77 cells in vivo
(1) Establishment the experiment model of nude mice bearing human multiple myeloma
Via the tail vein injection of ARH-77 myeloma cells for about 5 weeks, some mice started appear weight loss, be unwilling to move. to about 6 weeks or so, the model group mice died, the normal control group and cyclophosphamide pre-dealing group mice were in good conditions, did not appear weight loss. To further clarify the ARH-77 cells successfully transplanted into mice, at the same time the organization of the pathology and immunohistochemistry were analysized. The model mice liver and spleen were both with tumor cells infiltration, and immunohistochemistry results showed that infiltrated cells expressed human-specific CD45 and CD20 antigen, and the expressions of CD20 were higner than those of CD45.
The remaining organs (lungs, kidneys, intestines, etc.) had no significant abnormalities. The normal control group mice showed, the structure of organs in microscope were tidy and no abnormal cell infiltration, cyclophosphamide pretreatment group results were in accordance with the normal control group.The findings suggested the infiltrated cells in mice were derive from human multiple myeloma ARH-77 cells.
(2) The study of oridonin in vivo
The condition of model group of mice were the same as the forementioned model, in the first 5 weeks, using oridonin treated group mice does not appear the status of the model group, the growth were in good condition. To the 8 weeks, some mice appeared emaciated, loss of appetite and other behaviors. In order to compare with model group,1 mouse of the treated groupwas sacrificed at the same time with model group mice, in the concurrent analysis and comparison of pathology and immunohistochemistry. Model group mice liver and spleen had tumor cell infiltration, and immunohistochemistry showed that infiltration cells expressed human-specific CD45 and CD20 antigen, and CD20 expressions were higher than CD45. Oridonin treated group mice's livers had only a small amount of abnormal cells infiltrated, the structures of liver and spleen were basically normal.The apoptosis cells were observed in Ori-treated mouse by TUNEL detected. The studies indicated oridonin may inhibit the ARH-77 myeloma cell growth and infiltration in mice and prolong survival.
Conclusions
1. Oridonin can induce human multiple myeloma ARH-77 cell apoptosis in a time-dose-dependent manner;
2. Oridonin induced ARH-77 cells apoptosis by regulating the Bcl-2 family members, the expression of cell surface death receptors, as well as changes in mitochondrial membrane permeability, stimulate intrinsic/extrinsic apoptosis pathway;
3. Oridonin can lead to the changes of reactive oxygen species (ROS) on ARH-77 cells;
4. Medium concentration Oridonin able to improve the expression of NKG2D ligands on ARH-77 cells surface, and thus enhance the cytotoxity sensitivity to Allo-PBMCs.
5. Successful establishment of human multiple myeloma animal model, Oridonin can reduce the infiltration of tumor cells and prolong survival in vivo.
多发性骨髓瘤(multiple myeloma, MM)是血液系统常见的恶性肿瘤,严重危害人类身体健康。目前MM的治疗主要有化学治疗、自体造血细胞移植和异基因造血干细胞移植等。传统化疗虽然能延长患者的中位生存期,但化疗药物本身对机体造成的副反应,以及患者原发／继发耐药等都给传统化学疗法的使用带来了一定障碍。相对于传统化疗,大剂量化疗联合自体干细胞移植能够延长患者中位生存期,异基因造血干细胞移植能够发挥移植物抗骨髓瘤效应,但同时异基因移植也伴随有更高的移植物相关死亡率。因此,需寻找新的药物和治疗手段提高MM的疗效。
冬凌草甲素(Oridonin, Ori)是一种从植物冬凌草中提取的四环二萜类化合物,具有广泛的生物学、药学和病原学功效,如抗炎、抗菌、抗肿瘤、抗氧化等。国内的应用表明,在食管癌、胃癌或肝癌等疾病中,Ori能够延缓疾病进展,降低肿瘤负荷,减少并发症同时延长患者生存期。近来研究显示,Ori可以诱导包括前列腺癌、乳腺癌、非小细胞肺癌、急性白血病(NB4、HL-60细胞)、多形性恶性胶质瘤以及人类黑素瘤在内的肿瘤细胞发生凋亡。此外,冬凌草甲素尚能延长荷淋巴细胞白血病P388细胞小鼠的生存期。尽管大量的研究均证明caspase3、caspase8、p53、Bcl-2/Bax、细胞色素c和核因子kappa B (NF-κB)等信号分子涉及到了Ori诱导的凋亡过程,但其对MM的抗肿瘤机制仍然未完全明了,此外,Ori的临床应用也仍需更进一步的深入研究探讨。
自然杀伤细胞(NK细胞)是遗传性免疫系统的主要效应细胞,对病源感染和转化了的细胞具有自然细胞毒效应。异基因造血干细胞移植中,同种异体反应性NK细胞(Allo-reactive Natutal Killer cell, Allo-NK)能够提升恶性血液病治愈率。NK细胞杀伤靶细胞受HLA-KIA等抑制性信号和NKG2D配体-NKG2D等活化性信号的共同调节。但Allo-NK细胞对MM的作用尚待研究。
依据MM的起源、病因、发病机理等,认为冬凌草甲素将会诱导ARH-77细胞凋亡,并推断Ori能提升肿瘤细胞对NKG2D+细胞的杀伤敏感性。本研究以人多发性骨髓瘤ARH-77细胞为研究对象,探讨冬凌草甲素的体内、体外抗肿瘤效应。为进一步的多靶干预治疗和临床应用提供理论和实验依据。
实验方法
第一章冬凌草甲素诱导人多发性骨髓瘤ARH-77细胞凋亡
以MTT的方法检测不同浓度冬凌草甲素在不同时间对ARH-77细胞的抑制作用。经不同浓度Ori (0、2.5、5、10μmol/L)作用24 h后观察以下指标：采用相差显微镜和Hoechst 33258染色观察细胞凋亡的形态学改变；流式细胞仪检测细胞凋亡率、细胞周期、线粒体膜电位、细胞表面TRAIL受体/Fas/FasL、细胞内活性氧簇的改变；以甲基纤维素半固体培养法测定ARH-77细胞在药物干预前后的体外克隆形成能力和Ori对人脐血单个核细胞克隆形成的影响；RT-PCR测定ARH-77细胞凋亡通路调节分子Bcl-2、Bcl-x、Bax、Bid、p53、NF-κB和β-actin的mRNA表达；分光光度法检测caspase 8、caspase 9的变化；ELISA法检测细胞色素c含量的变化。
第二章冬凌草甲素作用后ARH-77细胞对NKG2D+细胞杀伤敏感性的研究
ARH-77细胞经5μmol/L Ori处理24h,以LDH释放法测定Ori作用前、后ARH-77细胞对NKG2D+细胞杀伤敏感性的差异；RT-PCR方法检测活化性受体NKG2D的配体MICA、MICB、ULBP1、ULBP2和ULBP3在肿瘤细胞mRNA的表达；流式细胞仪测定ARH-77细胞表面NKG2D配体(MICA.MICB.ULBP1、ULBP2和ULBP3)、Fas/FasL/TRAIL的表达状态。
第三章冬凌草甲素对荷人多发性骨髓瘤细胞裸鼠的体内实验研究
选用Bul B/C nu/nu裸鼠为实验动物,通过尾静脉注射ARH-77细胞建立荷人多发性骨髓瘤ARH-77细胞的实验模型,以一般状况、病理学、免疫组织化学检测小鼠组织形态学和人特异性CD20、CD45抗原的表达,以评价实验模型的成功建立。给予治疗组小鼠腹腔注射冬凌草甲素(15mg/kg), PBS治疗作为对照组,以上述指标和TUNEL法观察药物治疗效果。
统计学方法
应用SPSS16.0软件进行数据处理,数据以均数±标准差(x±s)表示,所有统计方法主要包括单因素方差分析、独立样本t检验、析因设计的方差分析、重复测量的方差分析、Kaplan-Meier生存分析法。方差齐时组间多重比较采用SNK法,方差不齐时组间多重比较采用Dunnett's T3法,P<0.05提示差异有统计学意义。
结果
第一章冬凌草甲素诱导人多发性骨髓瘤ARH-77细胞凋亡
1.1冬凌草甲素对ARH-77细胞的生长抑制作用
MTT法测定细胞活性结果显示：Ori能明显抑制ARH-77细胞生长,在同一时间点,不同质量浓度的Ori对ARH-77细胞的抑制率有显著差异；在同一质量浓度Ori药物时,随着时间的延长,Ori对ARH-77细胞的生长抑制作用明显增强。表明Ori对人多发性骨髓瘤ARH-77细胞的生长抑制作用呈时间和剂量依赖性。同时设立DMSO作为溶剂对照,常用化疗药物阿霉素(ADM)为阳性对照,结果表明用于溶解Ori的DMSO对细胞生长无影响,低浓度(2.5、5、10μM)时ADM对ARH-77细胞即可产生明显的生长抑制作用,随浓度增加两组间抑制率的差异逐渐减小。对正常脐血中分离出的单个核细胞的作用结果显示只有高浓度(80、160μM)的药物才能明显抑制脐血单个核细胞的生长。
1.2冬凌草甲素诱导ARH-77细胞凋亡的研究
(1)冬凌草甲素诱导细胞凋亡
经10μmol/L Ori处理24h后可见细胞胞体变小、细胞碎片增多、胞质中出现空泡,Hoechst 33258染色可见处理后有部分细胞出现了细胞核浓缩、聚集,有些可见到凋亡小体。在0、2.5、5、10μmol/L各浓度药物诱导下,ARH-77细胞发生凋亡,流式结果示各组早期凋亡率分别为(5.27±1.46)%,(15.07±0.78)%,(21.00±1.49)%和(27.60±1.77)%,各时间点凋亡率的差异有统计学意义(F=133.331,P=0.000),提示Ori诱导ARH-77细胞凋亡呈浓度依赖性。
(2)冬凌草甲素对ARH-77细胞周期的影响
ARH-77细胞在Ori的作用下细胞周期发生了变化,主要表现为G0／G1期百分比增多,同时S期百分比逐渐减少,在2.5μmol/L这种趋势无统计学意义,而G2／M期百分比没有明显的变化,表明细胞停滞于G1期。细胞的亚二倍体也有增加的趋势,与对照组相比差异具有统计学意义(F=114.958,P=0.000)。
(3)冬凌草甲素对ARH-77细胞体外克隆形成的影响
体外克隆形成实验显示,第14天时,细胞可以形成克隆。与对照组相比,2.5μmol/L Ori能在某种程度上刺激ARH-77细胞克隆的生长,随着药物浓度增加,细胞克隆数锐减,当Ori浓度增加到10μmol/L的时候,培养基中已基本没有克隆的存在,表明人多发性骨髓瘤ARH-77细胞在体外有形成克隆的能力,而Ori能够抑制ARH-77细胞的这种能力。各组克隆数与对照组相比均具有统计学意义(F=952.968,P=0.000)。在第21天时,克隆仍然存在,且有增大趋势,Ori作用组的克隆数较14天时有增多,提示Ori可能对较为早期的干细胞没有抑制作用。不同浓度Ori处理组不能抑制脐血克隆的生成,克隆数比较没有统计学意义。
1.3冬凌草甲素诱导ARH-77细胞凋亡的机制探讨
(1)内源性凋亡通路的变化
RT-PCR检测结果显示：Ori能够下调Bcl-2、Bcl-xl的表达,轻度上调Bax的表达,对Bid没有明显改变。提示Ori通过调节Bcl-2家族比例诱导ARH-77细胞凋亡。此外Ori尚能上调p53 mRNA、NF-κB mRNA的表达,二者有可能参与了冬凌草甲素诱导ARH-77细胞的凋亡过程。
流式结果显示,ARH-77细胞经0、2.5、5、10μmol/L的Ori作用24 h后,细胞线粒体膜电位有降低的趋势,表明随药物浓度增加,线粒体膜发生去极化,且不同药物浓度作用的各组间均有差异(F=252.847,P=0.000)。
ELISA检测细胞色素c发现,随Ori药物浓度的增加ARH-77细胞中总细胞色素c的浓度逐渐减少(F=4.909,,P=-0.047),在10μmol/L Ori处理组与对照组、2.5μmol/L Ori处理组间相比具有统计学意义。
分光光度法检测caspase 9活性结果表明,经过不同浓度冬凌草甲素处理后,细胞内caspase 9的活性与对照组相比发生明显的改变(F=2275,P=0.000)。各组间比较的结果显示,低浓度组的caspase 9变化无统计学意义,5、10μmol/LOri处理后较对照组显著增高。提示经冬凌草甲素处理后内源性凋亡通路中caspase 9被活化,激活内源凋亡通路。
(2)外源性凋亡通路的变化
对照组ARH-77细胞表面,无论死亡受体(DR4、DR5)还是诱骗受体(DcR1、DcR2)的表达均较低,加入2.5μmol/L Ori作用24h后,死亡受体DR4表达有所增高,但DR5的比例却有所下降,而两种诱骗受体的表达均有小幅的提升；经中等浓度药物处理后,死亡受体的表达增加,以DR5增加为主,而诱骗受体的变化不太明显；但接近IC50浓度的Ori却能够明显升高ARH-77细胞表面诱骗受体DcR1的表达,降低死亡受体DR5的表达。10ng/ml TRAIL蛋白能促进ARH-77细胞的生长,100ng/ml时对细胞的影响和对照组无明显差异,在高浓度1000ng/ml TRAIL蛋白的作用下ARH-77细胞的生长受到了明显的抑制。5μmol/L Ori处理的ARH-77细胞对TRAIL蛋白的反应趋势同未处理时基本相同,但在10ng/ml和100ng/ml的浓度时TRAIL蛋白促进细胞生长的效应更加明显,而1000ng/ml TRAIL蛋白对处理后细胞的抑制也更加显著。综上可看出Ori对ARH-77细胞TRAIL受体表达的影响较为复杂,细胞在表达死亡受体、传导凋亡信号的同时也在进行自身适应性调节表达,对抗药物的促凋亡作用。
ARH-77细胞自身高表达Fas,经2.5μmol/L Ori处理即可引起其表面Fas表达的增加,表达率近100%且各个浓度之间无明显差异。ARH-77细胞FasL表达极低,Ori能够明显提升FasL的表达,最高可达(11.67±1.80)%。
Ori能激活外源性凋亡通路中caspase8 (F=116.93,P=0.000),2.5μmol/L和5μmol/L实验组间的差异无统计学意义。
(3)细胞活性氧簇(ROS)的改变
流式细胞术检测结果显示,经低浓度药物(2.5μmol/L)处理后,可使ARH-77细胞内的ROS水平有所提高,增加冬凌草甲素的浓度(5μmol/L)可迅速提升细胞内ROS的产生,随着药物浓度的继续增加(10μmol/L)细胞内ROS进一步升高,差异具有统计学意义(F=3966,P=0.000),且峰值出现明显左移。提示ARH-77细胞内ROS的产生与冬凌草甲素的药物作用浓度有一定关系。
第二章冬凌草甲素作用后ARH-77细胞对激活的Allo-PBMCs细胞杀伤敏感性的研究
(1) Allo-PBMCs细胞杀伤活性测定
测定经rhIL-15共培养后的Allo-PBMCs细胞对冬凌草甲素5μmol/L作用前后多发性骨髓瘤ARH-77细胞的杀伤活性。随着效靶比的升高,实验组和对照组的杀伤活性均有增强,差异有统计学意义(t=-4.048,P=0.016；t=-7.152,P=0.002)；而在同一效靶比时,Allo-PBMCs细胞对冬凌草甲素(5μmol/L)作用后的肿瘤细胞杀伤活性比作用前更强,差异具有统计学意义(t=-10.642,P=0.009;t=-6.458, P=0.003)。ARH-77细胞本身对Allo-PBMCs细胞的杀伤并不敏感,在效比为10：1的时候,杀伤活性只有(7.45±0.39)。结果提示：ARH-77细胞可以靶认为是被NKG2D+细胞杀伤的低度敏感细胞,中等浓度的冬凌草甲素能够提升NKG2D+细胞对该细胞的杀伤敏感性。
(2)冬凌草甲素处理前后ARH-77细胞NKG2D配体mRNA的表达
RT-PCR检测结果显示：两组细胞均扩增出5个片段,与预期片段大小一致。ARH-77细胞本身表达MICA、ULBP2,而MICB、ULBP1和ULBP3表达较低；药物作用后的实验组细胞NKG2D配体mRNA的表达与对照组相比：MICA、ULBP1和ULBP3的表达变化不大,MICB有增加的趋势,而ULBP2的mRNA水平有所减少。
(3)流式细胞术检测ARH-77细胞表面NKG2D配体的表达
FCM检测显示：ARH-77细胞低表达MICB (8.57±1.35),不表达MICA、ULBP1、ULBP2和ULBP3;经5μmol/L冬凌草甲素处理24h后,MICB的表达增加最为显著,差异具有统计学意义(t=-6.833, P=0.002)。MICA、ULBP2的表达也有小幅上升(t=-2.828, P=0.047;t=-4.243, P=0.013),但ULBP3的表达却有下降(t=2.889, P=0.045), ULBP1没有明显的变化。
(4) Allo-PBMCs表面TRAIL、Fas、FasL的表达
经IL-15诱导后,细胞表面表达Fas (86.40±14.71)、FasL (10.20±5.09)、TRAIL (1.00±0.14),在表面几乎检测不到TRAIL的表达,提示Fas/FasL可能在该杀伤效应中同样发挥促凋亡效应,但TRAIL和TRAIL受体通路没有激活。
第三章冬凌甲素对荷人多发性骨髓瘤细胞裸鼠的体内实验研究
(1)荷人多发草性骨髓瘤裸鼠模型的建立
经尾静脉注射骨髓瘤ARH-77细胞后约5周,部分小鼠开始出现体重下降、消瘦、不喜动,到第6周左右,模型组小鼠出现死亡,正常对照组和环磷酰胺预处理对照组小鼠状态良好,未出现体重下降。为进一步明确ARH-77细胞成功移植到小鼠体内,同时进行了病理学和组织化学分析。模型组小鼠肠道、肺脏结构发生异常改变；肝脏、脾脏、骨髓具有肿瘤细胞浸润,而且免疫组化结果显示浸润的人多发性骨髓瘤ARH-77细胞表达人特异性CD45和CD20抗原,且CD20表达高于CD45。肾脏未见明显异常改变。正常对照组小鼠脏器镜下显示结构整齐、无异常细胞浸润,环磷酰胺预处理组结果同正常对照组。提示浸润的细胞来自移植到小鼠体内的人多发性骨髓瘤ARH-77细胞。
(2)冬凌草甲素体内实验研究
模型组小鼠的情况基本与前述建立的模型相同,使用冬凌草甲素治疗组小鼠在第5周未出现模型组的状况,生长状况良好。大约到第8周小鼠有部分出现消瘦、食欲不振等行为。为对药物治疗小组小鼠进行对照性比较,因此实验在处死模型组小鼠同时,对治疗组小鼠也处死1只,在同期进行病理学和组织化学分析比较。模型组小鼠肝脏、脾脏具有肿瘤细胞浸润,而且免疫组化结果显示浸润的肿瘤细胞表达人特异性CD45和CD20抗原,且CD20表达高于CD45,表明异常浸润的是输注的多发性骨髓瘤ARH-77细胞。冬凌草甲素治疗组小鼠肝脏中仅有少量细胞浸润,肝脏、脾脏结构基本正常,而且生存期较模型组延长。TUNEL细胞凋亡原位检测光镜下可见经Ori治疗后,小鼠的肝脏、脾脏内可观察到部分浸润细胞被诱导产生凋亡。提示药物可能抑制了骨髓瘤ARH-77细胞在小鼠体内的生长,诱导浸润细胞凋亡,延长生存期。
结论
1.冬凌草甲素能够诱导人多发性骨髓瘤ARH-77细胞凋亡,其诱导ARH-77细胞凋亡呈时间剂量依赖性；
2.冬凌草甲素诱导ARH-77细胞凋亡是通过调节Bcl-2家族成员、细胞表面死亡受体的表达、以及改变线粒体膜通透性,激发内／外源凋亡通路引起的；
3.冬凌草甲素能够引起ARH-77细胞内活性氧簇(ROS)的变化；
4.5μmol/L冬凌草甲素能够通过提高ARH-77细胞NKG2D配体、Fas的表达,从而提升对激活的Allo-PBMCs杀伤的敏感性。
5.成功建立人多发性骨髓瘤动物体内模型,冬凌草甲素在体内能减少肿瘤细胞的浸润、延长生存期。
Background and Objectives
Oridonin (Ori), a tetracycline diterpenoid isolated from the plant Rabdosia rubescens, has various biological, pharmaceutical, and physiological functions such as anti-inflammation, anti-bacteria activity and others. It was shown in China to be able to suppress disease progress, reduce tumor burden, alleviate syndrome, and prolong survival in patients with esophageal, gastric carcinoma or liver cancer. Recent studies showed that oridonin induced apoptosis in a variety of cancer cells including those from prostate, breast, non-small cell lung cancers, acute leukemia(NB4, HL-60 cells), gioblastoma multiforme, and human melanoma cells. In addition, oridonin could also increase lifespan of mice bearing P388 lymphocytic leukemia. However, though studies showed that caspase 3(casp 3), casp 8, p53, Bcl-2/Bax, cytochrome c (cyto c), and nuclear factor kappa B(NF-icB)were involved in apoptosis induced by oridonin, mechanisms underlying the anti-MM cells activity of oridonin remain largely unknown, and whether oridonin can find clinical application still needs more investigation.
Natural Killer cells (NK cells) are important effectors of the innate immune system with a critical role in early host defense against invading pathogens and transformed cells. In allogeneic hematopoietic transplantation, donor-derived allo-reactive natural killer (Allo-NK) cells have shown improved cure rate in hematopoietic malignancies. Cytotoxicity of NK ells is regulated both by HLA-Killer Immunoglobulin-like Receptors (KIRs) (HLA-KIR) inhibitory signals and NKG2D ligand-NKG2D (NKG2DL-NKG2D) activating signals. Nevertheless, it is unknown whether NKG2D+ cells have an effect on human multiple myeloma ARH-77 cells.
On the basis of origin, etiology and pathogenesis of multiple myeloma, we hypothesize that oridonin could induce apoptosis on human multiple myeloma ARH-77cells and enhance the sensitivity of ARH-77 cells to NKG2D+ cells cytolytic effects. In this study, we choose human multiple myeloma ARH-77 cells as the research object, and investigate the anti-tumor effects by oridonin in vitro and in vivo.
Methods
PartⅠOridonin induced human multiple myeloma ARH-77 cells apoptosis
The growth inhibitions of ARH-77 cells by various concentrations of oridonin at different time were analyzed by MTT assay. ARH-77 cells were treated with various concentrations of oridonin(0、2.5、5、10μmol/L) for 24h, and then observed following results:the morphological changes of apoptotic cells were observed by phasecontrast microscope and Hoechst 33258 staining; the apoptosis rate, the cell cycle, the changes of mitochondrial membrane potential(Δψm), TRAIL receptor on cell surface and intracellular reactive oxygen species(ROX) were detected by flow cytometry (FCM); mRNA expressions of Bcl-2 family members(Bcl-2, Bcl-xl, Bax, Bid), p53, NF-κB andβ-actin were analyzed by RT-PCR; the changes of caspase 8 and caspase 9 were measured by spectrophotometry.
PartⅡThe sensitivities of human multiple myeloma ARH-77 cells to actived Allo-PBMCs mediated cytotoxicity after oridonin treated
ARH-77 cells were treated with 5μmol/L oridonin for 24h, then the differences between oridonin-treated and no oridonin-treated ARH-77 cells in sensitivity to NKG2D+ cells cytotoxicity were measured by LDH releasing assay; mRNA expressions of NKG2D ligands MICA, MICB, ULBP1, ULBP2 and ULBP3 were measured by RT-PCR; the changes of NKG2D ligands(MICA, MICB, ULBP1, ULBP2 and ULBP3) on cell surface were analyzed by flow cytometry (FCM).
PartⅢThe ability of Oridonin to human multiple myeloma ARH-77 cells in vivo
Selected BulB/C nu/nu nude mice as experimental animals, through the tail vein injection of ARH-77 cells establish bearing human multiple myeloma ARH-77 cells experimental model. The general conditions, pathology, immunohistochemistry were used to analyze mice tissue morphology and human-specific CD20, CD45 antigen expression, and then evaluate the success of the establishment of experimental model. The tumor-bearing mice were treated with an intraperitoneal injection of oridonin 15mg/kg), control mice received injection with PBS, observe of the above-mentioned targets as drug therapy effectiveness.
Statistical analysis The analysis was performed using SPSS 16.0 software package. The data was represented as the mean±standard deviation (x±s).Comparisons of means among groups were performed using one-way analysis of variance (ANOVA), independent-samples t-test, factorial design analysis of variance, repeated measures, Kaplan-Meier. If variance were homogenous among groups, the SNK method was used for multiple comparisons, otherwise Dunnett's T3 method was used, P<0.05 were considered to be statistically significant.
Results
PartⅠOridonin induced human multiple myeloma ARH-77 cells apoptosis
1.1 The inhibition to ARH-77 cells with oridonin
MTT results showed that:oridonin significantly inhibited the growth of ARH-77cells, at the same time point, different concentrations of the oridonin on the ARH-77 cells, the inhibition rates were significantly different; at the same concentration, with time, the growth inhibition of oridonin were markedly enhanced. Oridonin obviously inhibited the growth of ARH-77 cells in a time- and dose-dependent manner.
1.2 To investigate the apoptosis of ARH-77 cells induced by oridonin
(1) Oridonin induced ATH-77 cells apoptosis
After treated with 10μmol/L Ori for 24h, the cells shrank, cell debris increased, with vacuolus in it, Hoechst 33258 staining showed that some cells after treatment appeared in nuclear enrichment, aggregation, and some apoptotic bodies can be seen. After 0,2.5,5,10μmol/L oridonin induced, ARH-77 cells apoptosis, FCM showed that, the apoptosis ratios of each groups are (5.27±1.46)%, (15.07±0.78)%, (21.00±1.49)% and (27.60±1.77)%, respectively, and significantly difference at all the concentration (F=133.331, P=0.000).Oridonin induced ARH-77 cells apoptosis in a dose-dependent manner.
(2) Influence of ARH-77 cell cycle induced by oridonin
After treated with different concentration oridonin, ARH-77 cellcycles were changed,mainly of G0/G1 phase cells gradually increased, while decreasing the S phase cells, at low concentrations (2.5μmol/L) this trend does not maintain statistics significance, while the G2/M phase cells have no obvious change, indicating that ARH-77 cells were arrested in G1 phase by oridonin. cells Sub-diploid were also increased, compared with the control group (F=114.958, P=0.000).
(3) In vitro colony formation of ARH-77 cells treated with oridonin
In vitro colony assays indicated, cells could shape clone at Day 14. Contrast with control group, low concentration(2.5μmol/L) of Ori in some extent, could stimulate ARH-77 cell clones formation, with oridonin concentration increased, the number of cell clones drastically reduced, when the oridonin increased 10μmol/L, no clone exists, that indicated human multiple myeloma ARH-77 cells have the ability to form clones in vitro, while the oridonin can inhibit the clone form ability. The numbers of Cloning for each groups compared with the control group were statistically significant (F=952.968, P=0.000)
1.3 The mechanism of ARH-77 cells aopotosis induced with oridonin
(1) Changes in intrinsic apoptosis pathway
RT-PCR results showed that:oridonin could significantly down-regulated the expressions of Bcl-2, Bcl-xl, mild up-regulated the expressions of Bax, and no effect on Bid. Indicate Oridonin induced ARH-77 cells apoptosis through regulated Bcl-2 family members. In addition, oridonin also have capable of raising the expressions of p53 mRNA and NF-κB mRNA expressions, both moleculars were involved in the ARH-77 cells apoptosis induced by Oridonin.
Flow results manifested that, ARH-77 cells were treated with different concertions of oridonin(0,2.5,5,10μmol/L) for 24 h, then cells mitochondrial membrane potential were degreaded, indicating that with the increase of drug concentrations, mitochondrial membrane depolarization occurred (F=252.847, P= 0.000), and there were were differences among the each groups.
To test cytochrome c by ELISA found, with the increased concentration of Ori, the total concentration of cytochrome c in ARH-77 cells were decreasd (F=4.909, P=0.047), there were no dignificant statistics between 10μmol/L Ori-treated group and control group,2.5μmol/L Ori-treated group.
Spectrophotometric detection of caspase 9 activity results showed that, after treatment with different concentrations of oridonin, compared with the control group, the activities of caspase 9 significant changed (F=2275, P=0.000). Comparison between each group, the activitiy of caspase 9 in low concentrations group was no significant change, after treated with 5,10μmol/L oridonin the activities of caspase 9 were significantly higher than the control group. These findings indicated oridonin could activate caspase 9 in extrinsic apoptosis pathway.
(2) Changes in extrinsic apoptosis pathway
On the ARH-77 cell surface, regardless of death receptors (DR4, DR5) or decoy receptors (DcR1, DcR2) expressions were lower, co-incubated with low concentration of Ori (2.5μmol/L) for 24h, the death receptors DR4 expressions was increased, but the proportion of DR5 was decreased, whereas the expression of two decoy receptors had a slight up-grade. Treated with moderate concentrations of oridonin, the death receptor expressions increased mainly about DR5, while the decoy receptors were less obvious; but close to IC50 concentration, decoy receptors DcR1 on ARH-77 cell surface were significantly higher than control group, meantime the expressions of death receptor DR5 were reduced.10ng/ml TRAIL can promote ARH-77 cells growth, 100ng/ml had no obvious affected to cells, the ATH-77 cells were strongly inhibited by 1000ng/ml TRAIL.5μmol/L Ori-treated group had the same trends with control group, in 10ng/ml and 100ng/ml Ori could make TRAIL more easy to enhance of the cells growth and 1000ng/ml TRAIL also could suppresse the ARH-77cells more significantly. therefore, the effects of Ori to the expressions of TRAIL-R in ARH-77 cells were more complex. the cells expressed the DR, transmitted apoptosis signals,meantime they adaptated accommodation to agonistic drugs.
(3) The changes of Reactive Oxygen Species(ROX) on ARH-77 cells
FCM results showed that, after treated with low concentrations of oridonin (2.5μmol/L), the levels of ROS to ARH-77 cells were up-regulated, then increased the concentration of oridonin (5μmol/L) can quickly enhance the production of intracellular ROS, with the continued increase of oridonin concentration (10μmol/ L), the intracellular ROS further increased, the difference was statistically significant (F=3966, P=0.000), and the peak apparent left shift. So, these indicated the generation of ROS by ARH-77 cells were associated with the oridonin concentration.
PartⅡThe sensitivities of human multiple myeloma ARH-77 cells to Allo-PBMCs-mediated cytotoxicity after oridonin treated
(1) Measurement of Allo-PBMCs cytotocixity
Peripheral blood mononuclear cells co-culture with rhIL-15, then measured the cytocixity of Allo-PBMCs against multiple myeloma and ARH-77 cells, which before and after treated oridonin 5μmol/L in vitro. With the effective target(E:T) ratios increased, the cytocixity of the experimental groups and control groups were enhanced, respectively, the difference was statistically significant (t=-4.048, P= 0.016; t=-7.152, P=0.002); while at the same E:T ratio, the cytocixities of Allo-PBMCs against the treated ARH-77 cells were stronger than the role of the former, the difference was statistically significant (t=-10.642, P=0.009; t=-6.458, P=0.003).
ARH-77 were low sensititive to Allo-PBMCs cells cytolytic effects, at the E:T ratio of 10:1, cytotoxic activity was only (7.45±0.39). The results suggested that: ARH-77 cells could be considered to be the low degree of sensitive to Allo-PBMCs, moderate concentration of oridonin can enhance the cytocixity of Allo-PBMCs against multiple myeloma and ARH-77 cells.
(2) The mRNA expression of NKG2D ligands on ARH-77 cells before and after treatment with oridonin
RT-PCR results showed that:two groups were amplified by five fragments consistent with the expected fragment size. ARH-77 cells themselves expressed MICA, ULBP2, while MICB, ULBP1 and ULBP3 expression lower; compare with control group, the expressions of NKG2D ligands mRNA on experimental group treated with oridonin were:MICA, ULBP1 and ULBP3 expressions changed little, mRNA expressions of MICB were increased, the mRNA level of ULBP2 decreased.
(3) The expressions of NKG2D ligand on ARH-77 cell surface by FCM
FCM tests showed:ARH-77 cells with low expressions of MICB (8.57±1.35), did not express MICA, ULBP1, ULBP2 and ULBP3; after treated with oridonin 5μmol/L for 24h, the expressions of MICB were significant increased (t=-6.833, P =0.002). The expressions of MICA, ULBP2 were also moderately enhanced (t=-2.828, P=0.047; t=-4.243, P=0.013), but The expressions of ULBP3 were decreased (t=2.889, P=0.045), and ULBP1 had no significant change.
(4) The espressions of TRAIL、Fas、FasL in Allo-PBMCs
After treated with IL-15, the expressions of Fas, FasL and TRAIL on cell surface were (86.40±14.71), (10.20±5.09), (1.00±0.14), there were almost no TRAIL on Allo-PBMCs. The findings indicated that Fas/FasL may play a role in induce apoptosis effects, but the TRAIL/TRAIL-R pathyway were not activated.
PartⅢThe ability of Oridonin to human multiple myeloma ARH-77 cells in vivo
(1) Establishment the experiment model of nude mice bearing human multiple myeloma
Via the tail vein injection of ARH-77 myeloma cells for about 5 weeks, some mice started appear weight loss, be unwilling to move. to about 6 weeks or so, the model group mice died, the normal control group and cyclophosphamide pre-dealing group mice were in good conditions, did not appear weight loss. To further clarify the ARH-77 cells successfully transplanted into mice, at the same time the organization of the pathology and immunohistochemistry were analysized. The model mice liver and spleen were both with tumor cells infiltration, and immunohistochemistry results showed that infiltrated cells expressed human-specific CD45 and CD20 antigen, and the expressions of CD20 were higner than those of CD45.
The remaining organs (lungs, kidneys, intestines, etc.) had no significant abnormalities. The normal control group mice showed, the structure of organs in microscope were tidy and no abnormal cell infiltration, cyclophosphamide pretreatment group results were in accordance with the normal control group.The findings suggested the infiltrated cells in mice were derive from human multiple myeloma ARH-77 cells.
(2) The study of oridonin in vivo
The condition of model group of mice were the same as the forementioned model, in the first 5 weeks, using oridonin treated group mice does not appear the status of the model group, the growth were in good condition. To the 8 weeks, some mice appeared emaciated, loss of appetite and other behaviors. In order to compare with model group,1 mouse of the treated groupwas sacrificed at the same time with model group mice, in the concurrent analysis and comparison of pathology and immunohistochemistry. Model group mice liver and spleen had tumor cell infiltration, and immunohistochemistry showed that infiltration cells expressed human-specific CD45 and CD20 antigen, and CD20 expressions were higher than CD45. Oridonin treated group mice's livers had only a small amount of abnormal cells infiltrated, the structures of liver and spleen were basically normal.The apoptosis cells were observed in Ori-treated mouse by TUNEL detected. The studies indicated oridonin may inhibit the ARH-77 myeloma cell growth and infiltration in mice and prolong survival.
Conclusions
1. Oridonin can induce human multiple myeloma ARH-77 cell apoptosis in a time-dose-dependent manner;
2. Oridonin induced ARH-77 cells apoptosis by regulating the Bcl-2 family members, the expression of cell surface death receptors, as well as changes in mitochondrial membrane permeability, stimulate intrinsic/extrinsic apoptosis pathway;
3. Oridonin can lead to the changes of reactive oxygen species (ROS) on ARH-77 cells;
4. Medium concentration Oridonin able to improve the expression of NKG2D ligands on ARH-77 cells surface, and thus enhance the cytotoxity sensitivity to Allo-PBMCs.
5. Successful establishment of human multiple myeloma animal model, Oridonin can reduce the infiltration of tumor cells and prolong survival in vivo.
引文
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