阻断Sonic hedgehog信号传导通路逆转难治性急性髓系白血病细胞耐药及其机制研究
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摘要
背景与目的：
急性髓系白血病(acute myeloid leukemia, AML),在我国占全部白血病的60%,是由于造血干／祖细胞发育过程中调控因子失常、细胞分化受阻、增殖失控而引起,目前的治疗以化疗为主。虽然有60-80%的患者可获得完全缓解(complete remission, CR)并延长生存期,但仍有20%-40%的初诊患者根本不能获得完全缓解,即使获得缓解者,其中还有40%-60%患者复发并演变成难治性白血病(refractory leukemia)。白血病细胞多药耐药(multidrug resistance, MDR)仍为当今血液肿瘤治疗中的一大难题,近来认为基因水平异常而导致信号传导通路激活、细胞信号调控网络失衡,是白血病细胞耐药的重要机制。研究发现在白血病耐药过程中多个细胞信号传导通路激活,其中目前研究较多的通路为IGF-1R/PI3K/Akt通路、RAS/RAF/MEK/ERK通路、泛素-蛋白酶体通路等。
HL-60/ADM细胞是公认的急性髓系白血病多药耐药细胞株,我们前期研究证明蛋白酶体抑制剂Bortezomib联合组蛋白去乙酰化酶抑制剂LBH589作用于HL-60/ADM细胞和难治性AML原代细胞具有协同抑制增殖及诱导凋亡作用,并能明显下调多药耐药蛋白(multidrug resistance-associated protein-1, MRPI)的表达,提高细胞内阿霉素摄取率,逆转阿霉素耐药并降低PI3K/Akt/NF-KB信号通路的活性。随后通过细胞生物信息学深度分析整合发现,难治性与非难治性AML-M2a的原代细胞基因谱表达差异显著,难治性／AML-M2a发生发展受一极其复杂的生物信号传导网络调控,其中Sonci hedgehog通路很可能是其中的上游通路,并对其它信号通路起调节作用。
哺乳动物体内Sonic hedgehog蛋白质家族有三个成员,分别为Shh(Sonic hedgehog、Ihh(Indian hedgehog)、Dhh (Desert hedgehog),其中Shh表达最为广泛,与多种器官的发育形成有关,主要由信号分子Shh、膜受体patched (Ptch)、 smoothened(Smo)、一些中间传递分子和核转录因子Glis组成。该通路在胚胎发育、维持内环境稳定及组织损伤后的修复和再生中起重要作用。其异常激活能导致多种肿瘤如基底细胞癌、髓母细胞瘤、胰腺癌、前列腺癌等的形成。血液系统疾病中已证实该通路和慢性粒细胞白血病(CML)干细胞的增殖、CD34+白血病细胞的耐药及淋巴瘤、多发性骨髓瘤相关。但该通路和难治性AML的关系、是否可以逆转难治性AML耐药及与其它信号通路的关系尚无研究。
本课题拟研究Shh通路在难治性AML原代细胞及细胞株中的表达情况及其与临床预后的关系、通路阻断剂NVP-LDE225作用于HL-60/ADM细胞的抑制增殖、诱导凋亡及逆转耐药作用机制,从而试图剖析Shh通路在难治性AML发生发展中的分子作用机制,为寻求早期诊断难治性AML的指标、新的治疗靶点及有效防治难治性AML的提供依据。
研究对象与方法：
1、Shh通路表达的检测：①选择HL60细胞、对应多药耐药细胞HL60/ADM及35例难治性AML和38例非难治AML患者骨髓的单个核细胞,QRT-PCR检测细胞株和其中19例难治性AML、23例非难治性AML单个核细胞中Shh通路组分Gli-1、Ptch、Smo基因的表达情况。②提取细胞株及原代细胞蛋白,Western blot方法检测细胞株及其余16例难治性AML和15例非难治性AML中Shh通路Ptch. Smo、Gli-1蛋白水平的表达情况,分析它们在难治性与非难治性AML中的差异性表达及与治疗反应、临床预后的关系。
2、将HL60及HL60/ADM细胞培养于含有10%小牛血清的RPMI-1640培养基中,阿霉素(ADM)单药分别作用于HL60、HL60/ADM细胞,应用MTT法计算增殖抑制率,以24h阿霉素IC50值比较HL-60/ADM和HL-60细胞对阿霉素的敏感性,耐药倍数大于30倍定义为细胞耐药。后以不同浓度NVP-LDE225药物处理细胞,选择无明显抑制增殖作用的浓度,将HL-60/ADM细胞分为ADM、 DNR、Ara-C、HHT单药处理组、NVP-LDE225联合化疗药物处理组,HL-60细胞分为ADM处理组、NVP-LDE225联合ADM处理组,均设置空白对照组。各组均用MTT法检测细胞增殖,以48hADM单药IC50值除以ADM联合NVP-LDE225-10μM后的IC50值,即为逆转耐药倍数。
3、NVP-LDE225单药不同浓度作用于HL60/ADM细胞48h, AnnexinV-FITC/PI双染后流式细胞仪检测细胞凋亡,及48h后加入ADM0.5μg/ml处理1h后应用流式细胞仪检测HL-60/ADM细胞内阿霉素摄取率。并提取药物处理HL60/ADM细胞后的总蛋白,Western Blot检测MRP1、IGF-1R、p-IGF-1R、 IRS-1、Akt、p-Akt、Gli-1、Bcl-2,探讨Shh通路对IGF-1R/PI3K/Akt通路的调节作用。
4、选择难治性AML患者新鲜骨髓的单个核细胞,无菌条件下淋巴细胞分离液分离单个核细胞,分为NVP-LDE225单药组、ADM单药组、NVP-LDE225-10μM联合ADM处理组,MTT法检测细胞增殖,计算逆转耐药倍数。并用流式细胞仪检测NVP-LDE225不同浓度处理48h后的原代细胞凋亡和阿霉素摄取率,Western Blot方法检测药物处理后细胞表面MRP1蛋白的变化。
5、选择协同作用最强的浓度21nM LBH589联合12nM Bortezomib(前期研究结果)作用于HL60/ADM细胞48h,Western blot检测Shh通路蛋白Gli-1及IGF-1R蛋白的变化。
6、利用SPSS13.0软件进行统计学分析。两组计量资料比较用两独立样本t检验,非正态分布数据以中位数表示,正态分布数据以均数士标准差描述。两组率的比较采用卡方检验。采用Kaplan-Meier法评估患者无复发生存时间(relapse-free survival, RFS)及总生存时间(overall survival, OS)。多组均数比较用单因素方差分析,Levene检验方差齐性,若方差齐使用LSD方法进行多重比较,若方差不齐则使用采用近似F检验(Welch方法)代替方差分析后采用Dunnett's T3方法进行多重比较。以p<0.05认为具有统计学差异。
结果：
1、与HL60细胞比较,多药耐药细胞HL60/ADM在基因及蛋白水平均高表达Shh通路。在19例难治性AML和23例非难治性AML中,QRT-PCR检测基因mRNA表达水平显示,Gli-1、Smo、Ptch在难治性AML的相对表达量分别为0.050(0.011～1.168)、0.108(0.011～3.589)和0.130(0.009～2.916),而在非难治性AML的相对表达量分别为0.025(0.004～0.099)、0.039(0.002～0.306)和0.036(0.005～0.367)。两样本t检验结果提示：P值分别为0.031、0.018、0.043,难治性AML三种基因的表达量均高于非难治性AML。Western blot检测16例难治复发AML中,共有13例表达Gli-1,而在15例非难治性AML中,仅有4例表达Gli-1,表达率分别为81.3%和26.7%,卡方检验结果提示P=0.002,提示难治性AML中激活率高于非难治性AML。分析31例原代AML细胞Gli-1表达情况对临床治疗反应及长期生存的影响,Gli-1高表达组17例,难治性AML占13例(76.5%),非难治性AML占4例(23.5%),≤2个疗程CR者10例(58.8%),大于2个疗程CR或NR者7例(41.2%),CR后一年内复发或多次复发者7例(41.2%),CR后一年后复发或无复发占10例(58.8%)：低表达14例,难治性AML占3例(21.4%),非难治性AML占11例(78.6%),≤2个疗程CR者11例(78.6%),大于2个疗程CR或NR者3例(21.4%),CR后一年内复发或多次复发者1例(7.1%),CR后一年后复发或无复发占13例(92.9%);卡方检验提示：Gli-1的表达对患者CR率无显著影响(P=0.242),对是否发生难治性AML及一年内复发或多次复发具有显著影响(P=0.002和0.031)。高表达Gli-1的患者的中位RFS分别为4.75、6.5个月；低表达Gli-1的患者的中位RFS、OS分别为14、15个月。高表达Gli-1和低表达Gli-1的RFS和OS均有统计学差异(P<0.05)。
2、MTT法检测阿霉素敏感性显示：阿霉素作用于HL60/ADM细胞的IC50值约为6.833μg/ml,作用于HL60细胞的IC50值仅为0.086μg/ml, HL60/ADM细胞耐药倍数为HL60细胞的79倍,说明HL60/ADM细胞对阿霉素高度耐药,可以作为下一步研究的细胞模型。对HL60及HL60/ADM细胞,NVP-LDE225剂量为0-10μM范围内细胞增殖率均大于80%,无明显抑制细胞增殖作用,浓度大于20gM时细胞存活率明显下降。选择对细胞无明显抑制增殖作用的浓度10μM分别联合不同浓度的ADM、Ara-C、DNR、HHT化疗药物共同作用。阿霉素单独作用于HL60/ADM细胞株的IC50为0.061μg/ml,联合NVP-LDE225-10μM后IC50为0.016μg/ml,逆转倍数为3.75倍；柔红霉素单独作用于HL60/ADM细胞株的IC50为0.213μg/ml,联合NVP-LDE225-10μM后IC50为0.04μg/ml,逆转倍数为5.08倍。高三尖杉酯碱单独作用于HL60/ADM细胞株的IC50为0.024μg/ml,联合NVP-LDE225-10μM后IC50为0.011μg/ml,逆转倍数为2.18倍。阿糖胞苷单独作用于HL60/ADM细胞株的IC50为40.8μg/ml,联合NVP-LDE225-10nM后IC50为0.17μg/ml,逆转倍数为234倍。阿霉素单独作用于HL60细胞株的IC50为0.00331μg/ml,联合NVP-LDE225后IC50为0.0031μg/ml。两样本t检验结果提示：NVP-LDE225-10μM可以逆转HL60/ADM细胞对ADM、DNR、HHT、Ara-C的耐药性(P值均小于0.05)。而在HL60细胞中无此现象(P=0.692)。
3、不同浓度NVP-LDE225处理HL60/ADM细胞48h,流式细胞仪检测发现对照组细胞总凋亡率为4.52±1.29%,5μM、10μM和20μM NVP-LDE225处理HL60/ADM细胞总凋亡率分别为6.17±1.73%、11.82±4.21%、52.41±10.57%。单因素方差分析结果显示,不同药物浓度处理HL60/ADM细胞48h细胞总凋亡率之间存在统计学差异(F=46.010; P=0.007)。 Dunnett T3法多重比较分析显示,和对照组及低浓度的NVP-LDE225处理组(5μM、10μM)相比,随着药物浓度的增加,高浓度的NVP-LDE225(20μm)处理组细胞总凋亡率显著增加,P值均小于0.05。HL-60/ADM细胞加入不同浓度NVP-LDE225处理48h后,再加入阿霉素终浓度为0.5μg/ml,继续培养1h后流式细胞仪检测细胞内阿霉素荧光阳性率,单因素方差分析显示,对照组、不同药物浓度处理组阿霉素阳性率之间有显著性差异(F=306.924, P=0.000)。 LSD法多重比较分析显示,5μM NVP-LDE225处理组和10μM NVP-LDE225组分别为65.89±3.93%、70.58±4.71%,均显著高于单药组3.32±1.93%(P=0.000)。提取NVP-LDE225不同浓度处理后的蛋白行Western blot检测,随着NVP-LDE225浓度的增加,细胞表面MRP1表达量逐渐下降。Western blot检测HL-60/ADM细胞经过不同浓度NVP-LDE225处理后的蛋白变化显示,对照组及药物不同浓度处理组IGF-1R、Akt总蛋白水平保持不变,而不同组p-IGF-1R、IRS-1、p-Akt、Bcl-2、Gli-1表达水平存在显著性差异。随着NVP-LDE225浓度的增加,细胞内上述蛋白表达量逐渐下降。
4、原代细胞实验：阿霉素单独作用于难治性AML原代细胞株的IC50为0.0426±0.0013μg/ml,联合NVP-LDE225-10μM后IC50为0.0119±0.0007μg/ml,逆转倍数为3.58倍。两样本t检验显示,P值为0.000,具有显著差异。不同浓度NVP-LDE225处理难治性AML原代细胞48h,流式细胞仪检测细胞凋亡结果显示：对照组细胞凋亡率为21.67±3.06%,5μM、10μM和20μM NVP-LDE225处理原代细胞总凋亡率分别为33.23±1.08%、48.2±2.03%、66.03±4.00%。单因素方差分析结果显示,不同药物浓度处理原代细胞48h细胞总凋亡率间存在统计学差异(F=144.483；P=0.000)。LSD法多重比较分析显示,随着药物浓度的增加,不同处理组细胞总凋亡率显著增加,P值均小于0.05。原代细胞加入不同浓度NVP-LDE225处理48h后,再加入阿霉素终浓度为0.5μg/ml,继续培养1h后流式细胞仪检测细胞内荧光阳性率,单因素方差分析显示,对照组、不同药物浓度处理组阿霉素阳性率之间有显著性差异(F=174.347,P=0.000)。LSD法多重比较分析显示,5μM、10μM、20μM NVP-LDE225处理组分别为16.2±1.68%、25.05±1.78%、42.91±2.72%,均显著高于单药组(4.697±2.12%)(P=0.000)。LSD法多重比较分析显示,随着药物浓度的增加,不同处理组细胞阿霉素摄取率明显增加,P值均小于0.05。提取NVP-LDE225不同浓度处理后的蛋白行Western blot检测,随着NVP-LDE225浓度的增加,细胞表面MRP1表达量逐渐下降。
5、Bor联合LBH589可以显著下调p-IGF-1R、Gli-1蛋白表达量,验证课题组此前研究结果,提示二者作用靶点在上游Shh通路。
结论：
1、和非耐药细胞株HL60及非难治性AML原代细胞相比,耐药珠HL60/ADM及难治性AML原代细胞中存在Shh信号通路的高表达。且高表达该通路的AML患者难治性AML及短期复发或多次复发发生率高,且RFS及OS均低于低表达者。
2、Shh通路阻断剂NVP-LDE225单药-10μM可以逆转HL60/ADM细胞株对ADM、DNR、HHT、Ara-C的耐药,逆转倍数分别为3.75、5.08、2.18、234倍。随着NVP-LDE225药物浓度的增加,HL60/ADM细胞的凋亡率及阿霉素摄取率均逐渐增加。其机制可能是通过下调细胞内p-IGF-1R、IRS-1、p-Akt、Gli-1、 MRP1蛋白的表达而起到逆转耐药的作用。如此也在难治性原代AML细胞中得到了验证。
3、Bor联合LBH589可通过降低Shh通路表达从而抑制IGF-1R/PI3K/Akt通路起到逆转耐药等作用,可能存在的作用机制如下图。
Background and Objectives:
Acute myeloid leukemia (AML) is one of the most common malignant tumors in hematologic system, the proportion is about60%of all leukemias, is due to regulatory factors disorder, blocked cell differentiation, uncontrolled proliferation, during the process of development of hematological stem or progenitor cells, the treatment of the disease is mainly chemotherapy. Although60-80%of patients achieved complete remission and prolong the survival period, but there are still20%-40%of patients with newly diagnosed cannot achieved complete remission, even obtain remission, of which there are40%-60%patients relapsed and become refractory leukemia. The multidrug resistance of leukemia cells (multidrug resistance, MDR) is still a big problem in the treatment of hematological malignancies, the molecular mechanism of MDR was recently thought that geneic abnormalities resulting in signal transduction pathway, the imbalance of cell signaling network, is an important mechanism of drug resistance of leukemia cells. Study found during the process of multi-drug resistance, multiple signal transduction pathway were activated, involved in the mechanism of drug resistance cells, whereas the current pathway more IGF-1R/PI3K/Akt pathway, RAS/RAF/MEK/ERK pathway, the ubiquitin proteasome pathway.
HL-60/ADM cells are recognized as a multidrug resistant cell line of acute myeloid leukemia, our preliminary study proved that different concentrations of proteasome inhibitor Bortezomib combined with LBH589in HL-60/ADM cells and refractory AML has synergistic inhibition of proliferation and induction of apoptosis, and expression regulation of MRP1can significantly improve adriamycin uptake, intracellular adriamycin resistance, reversal and reduce the activity of the PI3K/AKT/NF-KB pathway, participate in the reverse AML resistance. Early results showed, primary cell gene of refractory and non refractory AML-M2a expression significantly, and through the analysis of integration found depth cell bioinformatics, refractory AML-M2a biological signal transduction network regulation by the development of a very complex, in which Sonci hedgehog pathway may be the upstream pathway and can regulate other signal pathways.
Mammalian Hh protein family has three members, respectively Shh (Sonic hedgehog), Ihh (Indian hedgehog), Dhh (Desert hedgehog), in which Shh expression is the most widely used, and various organs formation, mainly by the signal molecule Shh, membrane receptor patched (PTCH), smoothened (Smo), some intermediate transfer molecules and transcription factor Glis. The pathway plays an important role in embryonic development, in the repair and regeneration of the environment and maintain the stable tissue after injury, and its dysregulation is often lead to the occurrence of tumor and other diseases. The abnormal activation can lead to the generation, many kinds of tumors such as:basal cell cancer, medulloblastoma, pancreatic cancer, prostate cancer and so on. The pathway and chronic myelogenous leukemia stem cells CD34+leukemia cell proliferation, drug resistance and a variety of diseases such as lymphoma, myeloma associated has confirmed the blood system diseases. But the correlation between Shh signal and other signals were not clear.
This paper intends to study the expression of Shh pathway in cell lines and primary AML cells, also the correlation between expression and clinical prognosis, using pathway blocker NVP-LDE225on multidrug resistance of acute myeloid leukemia cell line HL-60/ADM, inhibit the proliferation, induction of apoptosis and reversing drug resistance. In order to analyze the Sonic hedgehog pathway in refractory leukemia molecular mechanism in the development of new ways, new therapeutic targets and effective prevention from the search for the early diagnosis of refractory AML index, refractory AML.
Objects and methods:
1、Detection of Sonic hedeghog pathway level:①acute myeloid leukemia cell line HL60and the corresponding multidrug resistant cell line HL60/ADM as the research object, and mononuclear cells were isolated from35patients with refractory acute myeloid leukemia and38patients with non refractory acute myeloid leukemia patients bone marrow using lymphocyte separation liquid, QRT-PCR detection of cell lines and19cases of refractory AML,23patients with non-refractory AML mononuclear cells in Hedgehog pathway components Gli-1, Ptch, Smo gene expression.②The extraction cell lines and primary cell protein, Western blot method for detection of cell lines and16cases of refractory AML,15patients with non-refractory AML in Gli-1, Ptch, Smo, Shh protein expression, discussion on Shh signaling pathway in the treatment of refractory acute myeloid leukemia in the carcinogenesis and development.
2、Doxorubicin (ADM) monotherapy in different effect on HL60cell and HL60/ADM cell proliferation inhibition rate, calculated using the MTT method, the24h IC50value between HL-60/ADM and adriamycin sensitivity of HL-60cells to adriamycin, the drug resistance is30times larger than the defined as cell resistance. After HL60and HL60/ADM cells were cultured in RPMI-1640containing10%serum culture medium with different concentrations of NVP-LDE225, observe the time-dose effect dependent proliferation. Choose the dose of no obvious proliferation inhibition. The HL-60/ADM cells were divided into ADM, Ara-C, DNR, HHT treated-group, NVP-LDE225treatment combined with ADM, Ara-C, DNR, HHT group, HL-60cells were divided into ADM group, NVP-LDE225combined with ADM treatment group, each group of cell number consistent treatment in each group were set up, the blank control group. Groups were detected by MTT cell proliferation, survival curves, calculating the reversal of drug resistance.
3、NVP-LDE225monotherapy in HL60/ADM cells, AnnexinV-FITC/PI staining was used to detect apoptosis, flow cytometry was used to detect the adriamycin uptake rate of HL-60/ADM cells. Total protein in HL60/ADM cells after treatment were extracted and detect changes by Western Blot to detect the protein level of MRP1. To investigate the molecular mechanism of Sonic hedgehog pathway inhibitors reversed the resistance of refractory leukemia cells.Changes in proteinic levels including downstream protein and apoptosis protein were detected by Western Blot method in HL60/ADM cells after treatment of NVP-LDE225monotherapy, detection of proteins including IGF-1R, p-IGF-1R, IRS-1, Akt, p-Akt, Gli-1, Bcl-2, to investigate the effect of the pathway of IGF-1R/PI3K/Akt channel blockers application.
4、Mononuclear cells in bone marrow of refractory AML were isolated mononuclear cells under sterile conditions,and divided into NVP-LDE225single treatment group, NVP-LDE225and ADM treatment group, cell proliferation was detected by MTT, draw the proliferation curve, calculate the reversal of drug resistance. For the detection of NVP-LDE225and treatment with different concentration of primary cells apoptosis and adriamycin uptake rate of flow cytometry, changes in cell surface MRP1protein by Western Blot method for detection of drug treatment.
5、Choose the strongest synergistic effect of concentration of21nM LBH589combined with12nM Bortezomib(preliminary results) on HL60/ADM cells48h, changes of Western blot detection of Shh channel protein Gli-1and protein IGF-1R.
6、Statistical analysis was performed using SPSS13.0software. Two sets of measurement data were compared by statistical analysis using two independent samples t test, non normal data with median said, the normal distribution with mean and standard deviation of data description. The two groups were compared using the chi square test rate. Single factor analysis of variance with more groups, Levene test of homogeneity of variance, Kamo Sai uses the LSD method for multiple comparison, if the variance not neat is using the approximate F test (Welch) instead of variance analysis using Dunnett's T3method for multiple comparisons. The p<0.05that have the difference of statistics.
Results:
1、The relative sensitivity of HL60cells, multidrug resistance cell line HL60/ADM in gene level and protein level of Sonic showed high expression of hedgehog pathway. QRT-PCR was detected in19patients with refractory AML and23patients with non refractory AML, mRNA expression showed that, relative expression of Gli-1, Smo, Ptch in refractory AML were respectively0.050(0.011～1.168),0.108(0.011～3.589) and0.130(0.009～2.916), and in the relative expression of non refractory AML were respectively0.025(0.004～0.099),0.039(0.002～0.306) and0.036(0.005～0.367). Two sample t test results suggest that:the P values were0.031,0.018,0.043, there were significant differences. Western blot was detected in16cases of refractory and relapsed AML and15patients with non refractory primary AML specimens, there were13cases and4cases expressed Gli-1, the positive rates were81.3%and26.7%, chi square test results suggest that there were significant differences in P=0.002. Analysis of31cases of primary Gli-1cell AML expression to clinical treatment response and long-term survival, Gli-1high expression group17cases, refractory AML accounted for13cases (76.5%), non refractory AML accounted for4cases (23.5%),10cases were≤2courses of CR were(58.8%),more than7cases2courses of CR or NR (41.2%),7cases of recurrence within one year or more after recurrence of CR (41.2%, CR) after a year recurrence or relapse free accounted for10cases (58.8%);14cases low expression, refractory AML accounted for3cases (21.4%), non refractory AMLin11cases (78.6%),11cases were≤2courses of CR were (78.6%), more than2courses of CR or NR in3cases (21.4%),1cases of recurrence within one year or more after recurrence of CR (7.1%, CR) after a year recurrence orrelapse free accounted for13cases (92.9%); chi square test indicated:the expression of Gli-1in CR patients had no significant effect on the rate(P=0.242), on whether the occurrence of refractory AML and recurrence within one year or has a significant effect on relapse (P=0.002and0.031). The high expression of Gli-1in RFS patients were4.75,6.5months; median RFS, OS patients with lower expression of Gli-1were14,15months. High expression of RFS and OS, there were significant differences between the Gli-1and the low expression of Gli-1(P<0.05).
2、MTT method for detection of adriamycin sensitivity:IC50adriamycin to HL60/ADM cell value is about6.833μg/ml, acting on the HL60cells and the value of IC50is0.086μg/ml, HL60/ADM cells resistance multiple of79times HL60, indicating that HL60/ADM cells to adriamycin resistant, can be used as the object of study the next step. NVP-LDE225of low concentration of HL60and HL60/ADM cell inhibitory effect is not significant, dose of cell proliferation rate of0-10μM range is greater than80%, no significant inhibition of cell proliferation. No concentration significantly inhibited the proliferation of cells of10μM combined with different concentrations of ADM, Ara-C, DNR, HHT chemotherapy drug testing whether has the effect of reversing multidrug resistance. IC50doxorubicin alone on HL60/ADM cells is0.061μg/ml, combined with NVP-LDE225-10μM IC500.016μg/ml, reverse multiples of3.75times; IC50daunorubicin alone on HL60/ADM cells is0.213μg/ml, combined with NVP-LDE225-10μM IC500.04μg/ml, reverse a multiple of5.08times. IC50homoharringtonine alone on HL60/ADM cells is0.024μg/ml, combined with NVP-LDE225-10μM IC500.011μg/ml, reverse fold was2.18. IC50cytarabine alone on HL60/ADM cells is40.8μg/ml, combined with NVP-LDE225-10μM IC500.17μg/ml, reverse fold was234. IC50doxorubicin alone on HL60cells is0.00331μg/ml, combined with NVP-LDE225IC500.0031μg/ml. Two sample t test results suggest that:NVP-LDE225-10μM can reverse the multidrug resistance of HL60/ADM cells to ADM, DNR, HHT, Ara-C (P<0.05). Without this phenomenon in HL60cells (P=0.692).
3、different concentrations of NVP-LDE225in HL60/ADM cells treated with48h, cell apoptosis was detected by flow cytometry. The control of apoptosis cells group was4.52±1.29%,5μM, lOμM and20μM NVP-LDE225total HL60/ADM cell apoptosis rate were6.17±1.73%,11.82±4.21%,52.41±10.57%. Single factor analysis of variance results show, different concentrations had significant difference between the total HL60/ADM cell apoptosis rate of48h cells (F=46.010; P=0.007). Dunnett T3multiple comparison analysis method, and the control group and the low concentration NVP-LDE225treatment group (5μM,10μM), with the increase of drug concentration, high concentration of NVP-LDE225(20μM) treatment group total apoptosis cells were increased, P values were less than0.05. HL-60/ADM cells with different concentrations of NVP-LDE225after48h treatment, adding doxorubicin concentration is0.5μg/ml, continue to culture1H flow cytometry positive rate of intracellular fluorescence detection, single factor variance analysis showed, there was significant difference between control group, different concentration of adriamycin treatment group the positive rate (F=306.924, P=0.000). LSD multiple comparison analysis showed,5μM NVP-LDE225treatment group and lOμM NVP-LDE225group were65.89±3.93%,70.58±4.71%, significantly higher than the single drug group was3.32±1.93%(P=0.000). Extraction of NVP-LDE225protein after treatment with different concentrations of Western blot detection, can significantly reduce the cell surface expression of MRP1. Display6different protein changes, HL-60/ADM cells to detect Western concentration of blot after NVP-LDE225treatment, the control group and drug treated with different concentrations of IGF-1R, Akt protein levels remained unchanged, and different groups of p-IGF-1R, IRS-1, p-Akt, Bcl-2, Gli-1levels exist significant differences.
4、primary cell experiment:doxorubicin alone in refractory AML primary cell line IC50was0.0426±0.0013μg/ml, combined with NVP-LDE225-10μM IC50is0.0119±0.0007μg/ml, reverse fold was3.58. Two sample t test, P value is0, with significant difference. Different concentrations of NVP-LDE225treatment of refractory AML in primary48h cells, flow cytometry showed:the control of apoptotic cells group was21.67±3.06%,5μM, lOμM and20μM NVP-LDE225total apoptosis of primary cells were33.23±1.08%,48.2±2.03%,66.03±4%. Single factor analysis of variance results show, different concentrations of total apoptosis of primary48h cell rate had significant difference (F=144.483; P=0.000). LSD multiple comparison analysis shows that, with the increase of drug concentration, different treatment group total cell apoptosis rate increased, P values were less than0.05. Primary cells cultured with different concentrations of NVP-LDE225after48h treatment, adding doxorubicin concentration is0.5μg/ml, continue to culture1H flow cytometry positive rate of intracellular fluorescence detection, single factor variance analysis showed, there was significant difference between control group, different concentration of adriamycin treatment group the positive rate (F=174.347, P=0.000). LSD multiple comparison analysis showed,5μM NVP-LDE225treatment group,10μM NVP-LDE225,20μM NVP-LDE225group were16.2±1.68%,25.05±1.78%,42.91±2.72%, significantly higher than the single drug group was4.697±2.12%(P=0.000). LSD multiple comparison analysis shows that, with the increase of drug concentration, different cells treated with adriamycin uptake rate was increased, P values were less than0.05. Extraction of NVP-LDE225protein after treatment with different concentrations of Western blot detection, cell surface MRP1expression decreased obviously.
5、Bor combined with LBH589could downregulate expression of p-IGF-1R and Gli-1, prove the previous conclusion, indicating the targeted point is Shh pathway.
Conclusion:
1、Compared to the non-resistant cells and non refractory primary AML cells, the expression of Sonic Hedgehog pathway is higer in drug-resistant cell lines and refractory primary cells. Higher expression has the high incidence of refractory AML and relapse, also low RFS and OS.
2、The Sonic hedgehog pathway inhibitor single drug NVP-LDE225-10μM can reverse the drug resistance to ADM, DNR, HHT, Ara-C in HL60/ADM cell line. With the increasing concentration of NVP-LDE225, the apoptosis rate of HL60/ADM cells and adriamycin uptake rate was significantly increased, the cell surface expression of MRP1was significantly lower. NVP-LDE225can decrease the intracellular expression of p-IGF-1R, IRS-1, p-Akt, Gli-1protein, suggesting that Sonic hedgehog pathway can reverse the drug-resistance by regulating IGF-1R/PI3K/Akt pathway. In refractory primary AML cells also confirmed the conclusion.
3、LBH589combined with Bortezomib can downregulate the Shh pathway to inhibit the IGF-1R/PI3K/Akt pathway to invese drug-resistance.
急性髓系白血病(acute myeloid leukemia, AML),在我国占全部白血病的60%,是由于造血干／祖细胞发育过程中调控因子失常、细胞分化受阻、增殖失控而引起,目前的治疗以化疗为主。虽然有60-80%的患者可获得完全缓解(complete remission, CR)并延长生存期,但仍有20%-40%的初诊患者根本不能获得完全缓解,即使获得缓解者,其中还有40%-60%患者复发并演变成难治性白血病(refractory leukemia)。白血病细胞多药耐药(multidrug resistance, MDR)仍为当今血液肿瘤治疗中的一大难题,近来认为基因水平异常而导致信号传导通路激活、细胞信号调控网络失衡,是白血病细胞耐药的重要机制。研究发现在白血病耐药过程中多个细胞信号传导通路激活,其中目前研究较多的通路为IGF-1R/PI3K/Akt通路、RAS/RAF/MEK/ERK通路、泛素-蛋白酶体通路等。
HL-60/ADM细胞是公认的急性髓系白血病多药耐药细胞株,我们前期研究证明蛋白酶体抑制剂Bortezomib联合组蛋白去乙酰化酶抑制剂LBH589作用于HL-60/ADM细胞和难治性AML原代细胞具有协同抑制增殖及诱导凋亡作用,并能明显下调多药耐药蛋白(multidrug resistance-associated protein-1, MRPI)的表达,提高细胞内阿霉素摄取率,逆转阿霉素耐药并降低PI3K/Akt/NF-KB信号通路的活性。随后通过细胞生物信息学深度分析整合发现,难治性与非难治性AML-M2a的原代细胞基因谱表达差异显著,难治性／AML-M2a发生发展受一极其复杂的生物信号传导网络调控,其中Sonci hedgehog通路很可能是其中的上游通路,并对其它信号通路起调节作用。
哺乳动物体内Sonic hedgehog蛋白质家族有三个成员,分别为Shh(Sonic hedgehog、Ihh(Indian hedgehog)、Dhh (Desert hedgehog),其中Shh表达最为广泛,与多种器官的发育形成有关,主要由信号分子Shh、膜受体patched (Ptch)、 smoothened(Smo)、一些中间传递分子和核转录因子Glis组成。该通路在胚胎发育、维持内环境稳定及组织损伤后的修复和再生中起重要作用。其异常激活能导致多种肿瘤如基底细胞癌、髓母细胞瘤、胰腺癌、前列腺癌等的形成。血液系统疾病中已证实该通路和慢性粒细胞白血病(CML)干细胞的增殖、CD34+白血病细胞的耐药及淋巴瘤、多发性骨髓瘤相关。但该通路和难治性AML的关系、是否可以逆转难治性AML耐药及与其它信号通路的关系尚无研究。
本课题拟研究Shh通路在难治性AML原代细胞及细胞株中的表达情况及其与临床预后的关系、通路阻断剂NVP-LDE225作用于HL-60/ADM细胞的抑制增殖、诱导凋亡及逆转耐药作用机制,从而试图剖析Shh通路在难治性AML发生发展中的分子作用机制,为寻求早期诊断难治性AML的指标、新的治疗靶点及有效防治难治性AML的提供依据。
研究对象与方法：
1、Shh通路表达的检测：①选择HL60细胞、对应多药耐药细胞HL60/ADM及35例难治性AML和38例非难治AML患者骨髓的单个核细胞,QRT-PCR检测细胞株和其中19例难治性AML、23例非难治性AML单个核细胞中Shh通路组分Gli-1、Ptch、Smo基因的表达情况。②提取细胞株及原代细胞蛋白,Western blot方法检测细胞株及其余16例难治性AML和15例非难治性AML中Shh通路Ptch. Smo、Gli-1蛋白水平的表达情况,分析它们在难治性与非难治性AML中的差异性表达及与治疗反应、临床预后的关系。
2、将HL60及HL60/ADM细胞培养于含有10%小牛血清的RPMI-1640培养基中,阿霉素(ADM)单药分别作用于HL60、HL60/ADM细胞,应用MTT法计算增殖抑制率,以24h阿霉素IC50值比较HL-60/ADM和HL-60细胞对阿霉素的敏感性,耐药倍数大于30倍定义为细胞耐药。后以不同浓度NVP-LDE225药物处理细胞,选择无明显抑制增殖作用的浓度,将HL-60/ADM细胞分为ADM、 DNR、Ara-C、HHT单药处理组、NVP-LDE225联合化疗药物处理组,HL-60细胞分为ADM处理组、NVP-LDE225联合ADM处理组,均设置空白对照组。各组均用MTT法检测细胞增殖,以48hADM单药IC50值除以ADM联合NVP-LDE225-10μM后的IC50值,即为逆转耐药倍数。
3、NVP-LDE225单药不同浓度作用于HL60/ADM细胞48h, AnnexinV-FITC/PI双染后流式细胞仪检测细胞凋亡,及48h后加入ADM0.5μg/ml处理1h后应用流式细胞仪检测HL-60/ADM细胞内阿霉素摄取率。并提取药物处理HL60/ADM细胞后的总蛋白,Western Blot检测MRP1、IGF-1R、p-IGF-1R、 IRS-1、Akt、p-Akt、Gli-1、Bcl-2,探讨Shh通路对IGF-1R/PI3K/Akt通路的调节作用。
4、选择难治性AML患者新鲜骨髓的单个核细胞,无菌条件下淋巴细胞分离液分离单个核细胞,分为NVP-LDE225单药组、ADM单药组、NVP-LDE225-10μM联合ADM处理组,MTT法检测细胞增殖,计算逆转耐药倍数。并用流式细胞仪检测NVP-LDE225不同浓度处理48h后的原代细胞凋亡和阿霉素摄取率,Western Blot方法检测药物处理后细胞表面MRP1蛋白的变化。
5、选择协同作用最强的浓度21nM LBH589联合12nM Bortezomib(前期研究结果)作用于HL60/ADM细胞48h,Western blot检测Shh通路蛋白Gli-1及IGF-1R蛋白的变化。
6、利用SPSS13.0软件进行统计学分析。两组计量资料比较用两独立样本t检验,非正态分布数据以中位数表示,正态分布数据以均数士标准差描述。两组率的比较采用卡方检验。采用Kaplan-Meier法评估患者无复发生存时间(relapse-free survival, RFS)及总生存时间(overall survival, OS)。多组均数比较用单因素方差分析,Levene检验方差齐性,若方差齐使用LSD方法进行多重比较,若方差不齐则使用采用近似F检验(Welch方法)代替方差分析后采用Dunnett's T3方法进行多重比较。以p<0.05认为具有统计学差异。
结果：
1、与HL60细胞比较,多药耐药细胞HL60/ADM在基因及蛋白水平均高表达Shh通路。在19例难治性AML和23例非难治性AML中,QRT-PCR检测基因mRNA表达水平显示,Gli-1、Smo、Ptch在难治性AML的相对表达量分别为0.050(0.011～1.168)、0.108(0.011～3.589)和0.130(0.009～2.916),而在非难治性AML的相对表达量分别为0.025(0.004～0.099)、0.039(0.002～0.306)和0.036(0.005～0.367)。两样本t检验结果提示：P值分别为0.031、0.018、0.043,难治性AML三种基因的表达量均高于非难治性AML。Western blot检测16例难治复发AML中,共有13例表达Gli-1,而在15例非难治性AML中,仅有4例表达Gli-1,表达率分别为81.3%和26.7%,卡方检验结果提示P=0.002,提示难治性AML中激活率高于非难治性AML。分析31例原代AML细胞Gli-1表达情况对临床治疗反应及长期生存的影响,Gli-1高表达组17例,难治性AML占13例(76.5%),非难治性AML占4例(23.5%),≤2个疗程CR者10例(58.8%),大于2个疗程CR或NR者7例(41.2%),CR后一年内复发或多次复发者7例(41.2%),CR后一年后复发或无复发占10例(58.8%)：低表达14例,难治性AML占3例(21.4%),非难治性AML占11例(78.6%),≤2个疗程CR者11例(78.6%),大于2个疗程CR或NR者3例(21.4%),CR后一年内复发或多次复发者1例(7.1%),CR后一年后复发或无复发占13例(92.9%);卡方检验提示：Gli-1的表达对患者CR率无显著影响(P=0.242),对是否发生难治性AML及一年内复发或多次复发具有显著影响(P=0.002和0.031)。高表达Gli-1的患者的中位RFS分别为4.75、6.5个月；低表达Gli-1的患者的中位RFS、OS分别为14、15个月。高表达Gli-1和低表达Gli-1的RFS和OS均有统计学差异(P<0.05)。
2、MTT法检测阿霉素敏感性显示：阿霉素作用于HL60/ADM细胞的IC50值约为6.833μg/ml,作用于HL60细胞的IC50值仅为0.086μg/ml, HL60/ADM细胞耐药倍数为HL60细胞的79倍,说明HL60/ADM细胞对阿霉素高度耐药,可以作为下一步研究的细胞模型。对HL60及HL60/ADM细胞,NVP-LDE225剂量为0-10μM范围内细胞增殖率均大于80%,无明显抑制细胞增殖作用,浓度大于20gM时细胞存活率明显下降。选择对细胞无明显抑制增殖作用的浓度10μM分别联合不同浓度的ADM、Ara-C、DNR、HHT化疗药物共同作用。阿霉素单独作用于HL60/ADM细胞株的IC50为0.061μg/ml,联合NVP-LDE225-10μM后IC50为0.016μg/ml,逆转倍数为3.75倍；柔红霉素单独作用于HL60/ADM细胞株的IC50为0.213μg/ml,联合NVP-LDE225-10μM后IC50为0.04μg/ml,逆转倍数为5.08倍。高三尖杉酯碱单独作用于HL60/ADM细胞株的IC50为0.024μg/ml,联合NVP-LDE225-10μM后IC50为0.011μg/ml,逆转倍数为2.18倍。阿糖胞苷单独作用于HL60/ADM细胞株的IC50为40.8μg/ml,联合NVP-LDE225-10nM后IC50为0.17μg/ml,逆转倍数为234倍。阿霉素单独作用于HL60细胞株的IC50为0.00331μg/ml,联合NVP-LDE225后IC50为0.0031μg/ml。两样本t检验结果提示：NVP-LDE225-10μM可以逆转HL60/ADM细胞对ADM、DNR、HHT、Ara-C的耐药性(P值均小于0.05)。而在HL60细胞中无此现象(P=0.692)。
3、不同浓度NVP-LDE225处理HL60/ADM细胞48h,流式细胞仪检测发现对照组细胞总凋亡率为4.52±1.29%,5μM、10μM和20μM NVP-LDE225处理HL60/ADM细胞总凋亡率分别为6.17±1.73%、11.82±4.21%、52.41±10.57%。单因素方差分析结果显示,不同药物浓度处理HL60/ADM细胞48h细胞总凋亡率之间存在统计学差异(F=46.010; P=0.007)。 Dunnett T3法多重比较分析显示,和对照组及低浓度的NVP-LDE225处理组(5μM、10μM)相比,随着药物浓度的增加,高浓度的NVP-LDE225(20μm)处理组细胞总凋亡率显著增加,P值均小于0.05。HL-60/ADM细胞加入不同浓度NVP-LDE225处理48h后,再加入阿霉素终浓度为0.5μg/ml,继续培养1h后流式细胞仪检测细胞内阿霉素荧光阳性率,单因素方差分析显示,对照组、不同药物浓度处理组阿霉素阳性率之间有显著性差异(F=306.924, P=0.000)。 LSD法多重比较分析显示,5μM NVP-LDE225处理组和10μM NVP-LDE225组分别为65.89±3.93%、70.58±4.71%,均显著高于单药组3.32±1.93%(P=0.000)。提取NVP-LDE225不同浓度处理后的蛋白行Western blot检测,随着NVP-LDE225浓度的增加,细胞表面MRP1表达量逐渐下降。Western blot检测HL-60/ADM细胞经过不同浓度NVP-LDE225处理后的蛋白变化显示,对照组及药物不同浓度处理组IGF-1R、Akt总蛋白水平保持不变,而不同组p-IGF-1R、IRS-1、p-Akt、Bcl-2、Gli-1表达水平存在显著性差异。随着NVP-LDE225浓度的增加,细胞内上述蛋白表达量逐渐下降。
4、原代细胞实验：阿霉素单独作用于难治性AML原代细胞株的IC50为0.0426±0.0013μg/ml,联合NVP-LDE225-10μM后IC50为0.0119±0.0007μg/ml,逆转倍数为3.58倍。两样本t检验显示,P值为0.000,具有显著差异。不同浓度NVP-LDE225处理难治性AML原代细胞48h,流式细胞仪检测细胞凋亡结果显示：对照组细胞凋亡率为21.67±3.06%,5μM、10μM和20μM NVP-LDE225处理原代细胞总凋亡率分别为33.23±1.08%、48.2±2.03%、66.03±4.00%。单因素方差分析结果显示,不同药物浓度处理原代细胞48h细胞总凋亡率间存在统计学差异(F=144.483；P=0.000)。LSD法多重比较分析显示,随着药物浓度的增加,不同处理组细胞总凋亡率显著增加,P值均小于0.05。原代细胞加入不同浓度NVP-LDE225处理48h后,再加入阿霉素终浓度为0.5μg/ml,继续培养1h后流式细胞仪检测细胞内荧光阳性率,单因素方差分析显示,对照组、不同药物浓度处理组阿霉素阳性率之间有显著性差异(F=174.347,P=0.000)。LSD法多重比较分析显示,5μM、10μM、20μM NVP-LDE225处理组分别为16.2±1.68%、25.05±1.78%、42.91±2.72%,均显著高于单药组(4.697±2.12%)(P=0.000)。LSD法多重比较分析显示,随着药物浓度的增加,不同处理组细胞阿霉素摄取率明显增加,P值均小于0.05。提取NVP-LDE225不同浓度处理后的蛋白行Western blot检测,随着NVP-LDE225浓度的增加,细胞表面MRP1表达量逐渐下降。
5、Bor联合LBH589可以显著下调p-IGF-1R、Gli-1蛋白表达量,验证课题组此前研究结果,提示二者作用靶点在上游Shh通路。
结论：
1、和非耐药细胞株HL60及非难治性AML原代细胞相比,耐药珠HL60/ADM及难治性AML原代细胞中存在Shh信号通路的高表达。且高表达该通路的AML患者难治性AML及短期复发或多次复发发生率高,且RFS及OS均低于低表达者。
2、Shh通路阻断剂NVP-LDE225单药-10μM可以逆转HL60/ADM细胞株对ADM、DNR、HHT、Ara-C的耐药,逆转倍数分别为3.75、5.08、2.18、234倍。随着NVP-LDE225药物浓度的增加,HL60/ADM细胞的凋亡率及阿霉素摄取率均逐渐增加。其机制可能是通过下调细胞内p-IGF-1R、IRS-1、p-Akt、Gli-1、 MRP1蛋白的表达而起到逆转耐药的作用。如此也在难治性原代AML细胞中得到了验证。
3、Bor联合LBH589可通过降低Shh通路表达从而抑制IGF-1R/PI3K/Akt通路起到逆转耐药等作用,可能存在的作用机制如下图。
Background and Objectives:
Acute myeloid leukemia (AML) is one of the most common malignant tumors in hematologic system, the proportion is about60%of all leukemias, is due to regulatory factors disorder, blocked cell differentiation, uncontrolled proliferation, during the process of development of hematological stem or progenitor cells, the treatment of the disease is mainly chemotherapy. Although60-80%of patients achieved complete remission and prolong the survival period, but there are still20%-40%of patients with newly diagnosed cannot achieved complete remission, even obtain remission, of which there are40%-60%patients relapsed and become refractory leukemia. The multidrug resistance of leukemia cells (multidrug resistance, MDR) is still a big problem in the treatment of hematological malignancies, the molecular mechanism of MDR was recently thought that geneic abnormalities resulting in signal transduction pathway, the imbalance of cell signaling network, is an important mechanism of drug resistance of leukemia cells. Study found during the process of multi-drug resistance, multiple signal transduction pathway were activated, involved in the mechanism of drug resistance cells, whereas the current pathway more IGF-1R/PI3K/Akt pathway, RAS/RAF/MEK/ERK pathway, the ubiquitin proteasome pathway.
HL-60/ADM cells are recognized as a multidrug resistant cell line of acute myeloid leukemia, our preliminary study proved that different concentrations of proteasome inhibitor Bortezomib combined with LBH589in HL-60/ADM cells and refractory AML has synergistic inhibition of proliferation and induction of apoptosis, and expression regulation of MRP1can significantly improve adriamycin uptake, intracellular adriamycin resistance, reversal and reduce the activity of the PI3K/AKT/NF-KB pathway, participate in the reverse AML resistance. Early results showed, primary cell gene of refractory and non refractory AML-M2a expression significantly, and through the analysis of integration found depth cell bioinformatics, refractory AML-M2a biological signal transduction network regulation by the development of a very complex, in which Sonci hedgehog pathway may be the upstream pathway and can regulate other signal pathways.
Mammalian Hh protein family has three members, respectively Shh (Sonic hedgehog), Ihh (Indian hedgehog), Dhh (Desert hedgehog), in which Shh expression is the most widely used, and various organs formation, mainly by the signal molecule Shh, membrane receptor patched (PTCH), smoothened (Smo), some intermediate transfer molecules and transcription factor Glis. The pathway plays an important role in embryonic development, in the repair and regeneration of the environment and maintain the stable tissue after injury, and its dysregulation is often lead to the occurrence of tumor and other diseases. The abnormal activation can lead to the generation, many kinds of tumors such as:basal cell cancer, medulloblastoma, pancreatic cancer, prostate cancer and so on. The pathway and chronic myelogenous leukemia stem cells CD34+leukemia cell proliferation, drug resistance and a variety of diseases such as lymphoma, myeloma associated has confirmed the blood system diseases. But the correlation between Shh signal and other signals were not clear.
This paper intends to study the expression of Shh pathway in cell lines and primary AML cells, also the correlation between expression and clinical prognosis, using pathway blocker NVP-LDE225on multidrug resistance of acute myeloid leukemia cell line HL-60/ADM, inhibit the proliferation, induction of apoptosis and reversing drug resistance. In order to analyze the Sonic hedgehog pathway in refractory leukemia molecular mechanism in the development of new ways, new therapeutic targets and effective prevention from the search for the early diagnosis of refractory AML index, refractory AML.
Objects and methods:
1、Detection of Sonic hedeghog pathway level:①acute myeloid leukemia cell line HL60and the corresponding multidrug resistant cell line HL60/ADM as the research object, and mononuclear cells were isolated from35patients with refractory acute myeloid leukemia and38patients with non refractory acute myeloid leukemia patients bone marrow using lymphocyte separation liquid, QRT-PCR detection of cell lines and19cases of refractory AML,23patients with non-refractory AML mononuclear cells in Hedgehog pathway components Gli-1, Ptch, Smo gene expression.②The extraction cell lines and primary cell protein, Western blot method for detection of cell lines and16cases of refractory AML,15patients with non-refractory AML in Gli-1, Ptch, Smo, Shh protein expression, discussion on Shh signaling pathway in the treatment of refractory acute myeloid leukemia in the carcinogenesis and development.
2、Doxorubicin (ADM) monotherapy in different effect on HL60cell and HL60/ADM cell proliferation inhibition rate, calculated using the MTT method, the24h IC50value between HL-60/ADM and adriamycin sensitivity of HL-60cells to adriamycin, the drug resistance is30times larger than the defined as cell resistance. After HL60and HL60/ADM cells were cultured in RPMI-1640containing10%serum culture medium with different concentrations of NVP-LDE225, observe the time-dose effect dependent proliferation. Choose the dose of no obvious proliferation inhibition. The HL-60/ADM cells were divided into ADM, Ara-C, DNR, HHT treated-group, NVP-LDE225treatment combined with ADM, Ara-C, DNR, HHT group, HL-60cells were divided into ADM group, NVP-LDE225combined with ADM treatment group, each group of cell number consistent treatment in each group were set up, the blank control group. Groups were detected by MTT cell proliferation, survival curves, calculating the reversal of drug resistance.
3、NVP-LDE225monotherapy in HL60/ADM cells, AnnexinV-FITC/PI staining was used to detect apoptosis, flow cytometry was used to detect the adriamycin uptake rate of HL-60/ADM cells. Total protein in HL60/ADM cells after treatment were extracted and detect changes by Western Blot to detect the protein level of MRP1. To investigate the molecular mechanism of Sonic hedgehog pathway inhibitors reversed the resistance of refractory leukemia cells.Changes in proteinic levels including downstream protein and apoptosis protein were detected by Western Blot method in HL60/ADM cells after treatment of NVP-LDE225monotherapy, detection of proteins including IGF-1R, p-IGF-1R, IRS-1, Akt, p-Akt, Gli-1, Bcl-2, to investigate the effect of the pathway of IGF-1R/PI3K/Akt channel blockers application.
4、Mononuclear cells in bone marrow of refractory AML were isolated mononuclear cells under sterile conditions,and divided into NVP-LDE225single treatment group, NVP-LDE225and ADM treatment group, cell proliferation was detected by MTT, draw the proliferation curve, calculate the reversal of drug resistance. For the detection of NVP-LDE225and treatment with different concentration of primary cells apoptosis and adriamycin uptake rate of flow cytometry, changes in cell surface MRP1protein by Western Blot method for detection of drug treatment.
5、Choose the strongest synergistic effect of concentration of21nM LBH589combined with12nM Bortezomib(preliminary results) on HL60/ADM cells48h, changes of Western blot detection of Shh channel protein Gli-1and protein IGF-1R.
6、Statistical analysis was performed using SPSS13.0software. Two sets of measurement data were compared by statistical analysis using two independent samples t test, non normal data with median said, the normal distribution with mean and standard deviation of data description. The two groups were compared using the chi square test rate. Single factor analysis of variance with more groups, Levene test of homogeneity of variance, Kamo Sai uses the LSD method for multiple comparison, if the variance not neat is using the approximate F test (Welch) instead of variance analysis using Dunnett's T3method for multiple comparisons. The p<0.05that have the difference of statistics.
Results:
1、The relative sensitivity of HL60cells, multidrug resistance cell line HL60/ADM in gene level and protein level of Sonic showed high expression of hedgehog pathway. QRT-PCR was detected in19patients with refractory AML and23patients with non refractory AML, mRNA expression showed that, relative expression of Gli-1, Smo, Ptch in refractory AML were respectively0.050(0.011～1.168),0.108(0.011～3.589) and0.130(0.009～2.916), and in the relative expression of non refractory AML were respectively0.025(0.004～0.099),0.039(0.002～0.306) and0.036(0.005～0.367). Two sample t test results suggest that:the P values were0.031,0.018,0.043, there were significant differences. Western blot was detected in16cases of refractory and relapsed AML and15patients with non refractory primary AML specimens, there were13cases and4cases expressed Gli-1, the positive rates were81.3%and26.7%, chi square test results suggest that there were significant differences in P=0.002. Analysis of31cases of primary Gli-1cell AML expression to clinical treatment response and long-term survival, Gli-1high expression group17cases, refractory AML accounted for13cases (76.5%), non refractory AML accounted for4cases (23.5%),10cases were≤2courses of CR were(58.8%),more than7cases2courses of CR or NR (41.2%),7cases of recurrence within one year or more after recurrence of CR (41.2%, CR) after a year recurrence or relapse free accounted for10cases (58.8%);14cases low expression, refractory AML accounted for3cases (21.4%), non refractory AMLin11cases (78.6%),11cases were≤2courses of CR were (78.6%), more than2courses of CR or NR in3cases (21.4%),1cases of recurrence within one year or more after recurrence of CR (7.1%, CR) after a year recurrence orrelapse free accounted for13cases (92.9%); chi square test indicated:the expression of Gli-1in CR patients had no significant effect on the rate(P=0.242), on whether the occurrence of refractory AML and recurrence within one year or has a significant effect on relapse (P=0.002and0.031). The high expression of Gli-1in RFS patients were4.75,6.5months; median RFS, OS patients with lower expression of Gli-1were14,15months. High expression of RFS and OS, there were significant differences between the Gli-1and the low expression of Gli-1(P<0.05).
2、MTT method for detection of adriamycin sensitivity:IC50adriamycin to HL60/ADM cell value is about6.833μg/ml, acting on the HL60cells and the value of IC50is0.086μg/ml, HL60/ADM cells resistance multiple of79times HL60, indicating that HL60/ADM cells to adriamycin resistant, can be used as the object of study the next step. NVP-LDE225of low concentration of HL60and HL60/ADM cell inhibitory effect is not significant, dose of cell proliferation rate of0-10μM range is greater than80%, no significant inhibition of cell proliferation. No concentration significantly inhibited the proliferation of cells of10μM combined with different concentrations of ADM, Ara-C, DNR, HHT chemotherapy drug testing whether has the effect of reversing multidrug resistance. IC50doxorubicin alone on HL60/ADM cells is0.061μg/ml, combined with NVP-LDE225-10μM IC500.016μg/ml, reverse multiples of3.75times; IC50daunorubicin alone on HL60/ADM cells is0.213μg/ml, combined with NVP-LDE225-10μM IC500.04μg/ml, reverse a multiple of5.08times. IC50homoharringtonine alone on HL60/ADM cells is0.024μg/ml, combined with NVP-LDE225-10μM IC500.011μg/ml, reverse fold was2.18. IC50cytarabine alone on HL60/ADM cells is40.8μg/ml, combined with NVP-LDE225-10μM IC500.17μg/ml, reverse fold was234. IC50doxorubicin alone on HL60cells is0.00331μg/ml, combined with NVP-LDE225IC500.0031μg/ml. Two sample t test results suggest that:NVP-LDE225-10μM can reverse the multidrug resistance of HL60/ADM cells to ADM, DNR, HHT, Ara-C (P<0.05). Without this phenomenon in HL60cells (P=0.692).
3、different concentrations of NVP-LDE225in HL60/ADM cells treated with48h, cell apoptosis was detected by flow cytometry. The control of apoptosis cells group was4.52±1.29%,5μM, lOμM and20μM NVP-LDE225total HL60/ADM cell apoptosis rate were6.17±1.73%,11.82±4.21%,52.41±10.57%. Single factor analysis of variance results show, different concentrations had significant difference between the total HL60/ADM cell apoptosis rate of48h cells (F=46.010; P=0.007). Dunnett T3multiple comparison analysis method, and the control group and the low concentration NVP-LDE225treatment group (5μM,10μM), with the increase of drug concentration, high concentration of NVP-LDE225(20μM) treatment group total apoptosis cells were increased, P values were less than0.05. HL-60/ADM cells with different concentrations of NVP-LDE225after48h treatment, adding doxorubicin concentration is0.5μg/ml, continue to culture1H flow cytometry positive rate of intracellular fluorescence detection, single factor variance analysis showed, there was significant difference between control group, different concentration of adriamycin treatment group the positive rate (F=306.924, P=0.000). LSD multiple comparison analysis showed,5μM NVP-LDE225treatment group and lOμM NVP-LDE225group were65.89±3.93%,70.58±4.71%, significantly higher than the single drug group was3.32±1.93%(P=0.000). Extraction of NVP-LDE225protein after treatment with different concentrations of Western blot detection, can significantly reduce the cell surface expression of MRP1. Display6different protein changes, HL-60/ADM cells to detect Western concentration of blot after NVP-LDE225treatment, the control group and drug treated with different concentrations of IGF-1R, Akt protein levels remained unchanged, and different groups of p-IGF-1R, IRS-1, p-Akt, Bcl-2, Gli-1levels exist significant differences.
4、primary cell experiment:doxorubicin alone in refractory AML primary cell line IC50was0.0426±0.0013μg/ml, combined with NVP-LDE225-10μM IC50is0.0119±0.0007μg/ml, reverse fold was3.58. Two sample t test, P value is0, with significant difference. Different concentrations of NVP-LDE225treatment of refractory AML in primary48h cells, flow cytometry showed:the control of apoptotic cells group was21.67±3.06%,5μM, lOμM and20μM NVP-LDE225total apoptosis of primary cells were33.23±1.08%,48.2±2.03%,66.03±4%. Single factor analysis of variance results show, different concentrations of total apoptosis of primary48h cell rate had significant difference (F=144.483; P=0.000). LSD multiple comparison analysis shows that, with the increase of drug concentration, different treatment group total cell apoptosis rate increased, P values were less than0.05. Primary cells cultured with different concentrations of NVP-LDE225after48h treatment, adding doxorubicin concentration is0.5μg/ml, continue to culture1H flow cytometry positive rate of intracellular fluorescence detection, single factor variance analysis showed, there was significant difference between control group, different concentration of adriamycin treatment group the positive rate (F=174.347, P=0.000). LSD multiple comparison analysis showed,5μM NVP-LDE225treatment group,10μM NVP-LDE225,20μM NVP-LDE225group were16.2±1.68%,25.05±1.78%,42.91±2.72%, significantly higher than the single drug group was4.697±2.12%(P=0.000). LSD multiple comparison analysis shows that, with the increase of drug concentration, different cells treated with adriamycin uptake rate was increased, P values were less than0.05. Extraction of NVP-LDE225protein after treatment with different concentrations of Western blot detection, cell surface MRP1expression decreased obviously.
5、Bor combined with LBH589could downregulate expression of p-IGF-1R and Gli-1, prove the previous conclusion, indicating the targeted point is Shh pathway.
Conclusion:
1、Compared to the non-resistant cells and non refractory primary AML cells, the expression of Sonic Hedgehog pathway is higer in drug-resistant cell lines and refractory primary cells. Higher expression has the high incidence of refractory AML and relapse, also low RFS and OS.
2、The Sonic hedgehog pathway inhibitor single drug NVP-LDE225-10μM can reverse the drug resistance to ADM, DNR, HHT, Ara-C in HL60/ADM cell line. With the increasing concentration of NVP-LDE225, the apoptosis rate of HL60/ADM cells and adriamycin uptake rate was significantly increased, the cell surface expression of MRP1was significantly lower. NVP-LDE225can decrease the intracellular expression of p-IGF-1R, IRS-1, p-Akt, Gli-1protein, suggesting that Sonic hedgehog pathway can reverse the drug-resistance by regulating IGF-1R/PI3K/Akt pathway. In refractory primary AML cells also confirmed the conclusion.
3、LBH589combined with Bortezomib can downregulate the Shh pathway to inhibit the IGF-1R/PI3K/Akt pathway to invese drug-resistance.
引文
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[2]王治香、孟凡义、梁爽、江雪杰。硼替佐米联合LBH589逆转HL60/ADM细胞耐药的分子机制研究,2012年,中华医学会第十二次全国血液学学术会议。
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[2]王治香、孟凡义、梁爽、江雪杰。硼替佐米联合LBH589逆转HL60/ADM细胞耐药的分子机制研究,2012年,中华医学会第十二次全国血液学学术会议。
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[4]Low, J.A. and F.J. de Sauvage, Clinical experience with Hedgehog pathway inhibitors. J Clin Oncol,2010.28(36):p.5321-6.
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