4-氯苯甲酰小檗胺在血液肿瘤中的作用及其机制研究
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摘要
第一部分BBD9抑制骨髓瘤细胞生存并诱导细胞凋亡
目的：
观察BBD9对多发性骨髓瘤细胞生存的影响及其机制。
方法：
MTT法检测BBD9对多种骨髓瘤细胞株及原代细胞的生长抑制作用，瑞氏染色观察细胞形态学变化，AV-PI法检测细胞凋亡，检测DNA ladder，流式细胞术检测细胞周期，western blot法检测caspase，BCL-2家族蛋白及细胞周期调控蛋白。
结果：
在0～4μg／ml BBD9作用下，BBD9能显著抑制四种骨髓瘤细胞株(RPMI8226、U266、MM．1S、MM．1R)的生长。随着BBD9浓度的升高，各细胞株的细胞存活率逐步降低。此外，BBD9对四种细胞株的生长抑制作用也呈时间依赖性，同一浓度下，药物作用时间越长，细胞存活率也越低。BBD9作用于RPMI8226、U266、MM．1S、MM．1R细胞24h的IC50依次是：2．30μg／ml、1．87μg／ml、2．39μg／ml、1．54μg／ml。BBD9对3例病人原代细胞也呈现剂量依赖性的生长抑制。BBD9处理骨髓瘤原代细胞24h的IC50为1．08μg／ml。用正常骨髓单个核细胞进行MTT检测发现，在0-4μg／ml BBD9也呈剂量依赖性地抑制正常骨髓单个核细胞的生长，但其对药物的敏感性低于原代细胞，24h的IC50为3．24μg／ml。形态学观察可见，BBD9处理24h后的U266细胞和RPMI8226细胞出现了典型的凋亡细胞形态：核碎裂，凋亡小体。DNA片段化检测可见经BBD9处理的RPMI8226细胞出现了典型“DNA梯子”，随着药物浓度的增加，“DNA梯子”也越明显。AV-PI法检测细胞凋亡也发现，BBD9可以诱导骨髓瘤细胞出现凋亡。0、1、2、3μg／ml BBD9处理RPMI8226细胞24h后，早期凋亡细胞比例分别为1．57±0．37％、4．79±2．47％、16．18±5．45％、42．94±13．99％；而在U266细胞，早期凋亡细胞的比例分别为2．69±1．28％、7．36±2．22％、19．67±2．83％、50．26±16．29％。BBD9还可以诱导U266细胞和RPMI8226细胞caspase-3，-8，-9及PARP剪切激活。进一步的检测可见，BBD9处理后，促凋亡蛋白Bak、Bax和Bim表达量在U266和RPMI8226细胞均有明显升高，抑凋亡蛋白Mcl-1明显下调。但是，另外两种重要的抑凋亡蛋白BCL-2和BCL-XL则变化不明显。此外，对细胞周期分析表明，BBD9使骨髓瘤细胞周期阻滞在G2／M期。0、1、2、3μg／ml BBD9处理24h后，处于G2／M期的RPMI8226细胞分别为4．49±3．79％、13．61±2．09％、41．03±2．09％、35．99±9．05％；而处于G2／M期的U266细胞分别为14．14±0．6％、19．48±2．84％、26．54±3．44％、33．62±3．0％。细胞周期调控蛋白cyclinA、cyclinB1在BBD9处理的RPMI8226细胞和U266细胞中均明显下调，而CDK1及P27无明显改变。
结论：
(1)BBD9能抑制多种骨髓瘤细胞株及原代细胞生长。
(2)BBD9诱导骨髓瘤细胞凋亡。
(3)BBD9上调促凋亡蛋白Bak、Bax和Bim，下调抑凋亡蛋白Mcl-1。
(4)BBD9通过下调cyclinA、cyclinB1使细胞周期阻滞于G2／M期。
第二部分BBD9诱导的凋亡伴随着FOXO3a／Bim途径激活
目的：
研究BBD9诱导的Bim蛋白表达上调的机制。
方法：
Real-time PCR检测Bim mRNA及mir-17-5p的表达，western blot分析细胞核内FOXO3a及pFOXO3a表达量，免疫荧光显微镜观察FOXO3在细胞内的分布。
结果：
BBD9处理U266细胞8h后，Bim mRNA拷贝数明显增加。与此同时，对BimmRNA起负调控作用的成熟体小RNA—mir-17-5p明显下调。3μg／ml BBD9处理时，mir-17-5p下降至未处理组的0．35±0．23倍。Westem blot结果显示，BBD9处理的U266细胞核内Bim基因转录因子FOXO3a的表达量较未处理的U266细胞明显升高。与此相反，核内pFOXO3a蛋白量随着BBD9浓度的增加而减少。免疫荧光显微镜下可以看到：在BBD9处理前，U266细胞胞核内FOXO3a表达较低，胞浆内FOXO3a表达相对较高；而在2μg／ml BBD9处理24h后，U266细胞胞核内FOXO3a的荧光较强，而胞浆中FOXO3a则较弱。同样地，在BBD9处理前，RPMI8226细胞胞核内FOXO3a荧光较弱，胞浆内FOXO3a荧光则相对更强；而在2μg／mlBBD9处理24h后，RPMI8226细胞胞核内FOXO3a的荧光较强，而胞浆中FOXO3a则较弱。
结论：
(1)BBD9通过提高胞核内FOXO3a的表达量，促进Bim基因转录，增加BimmRNA拷贝数，从而导致Bim蛋白表达上调。
(2)BBD9还通过下调mir-17-5p，减少Bim mRNA降解或对Bim mRNA翻译的抑制，从而使Bim蛋白表达上调。
第三部分BBD9抑制IL-6信号传导途径的激活
目的：
通过观察IL-6信号传导途径中参与信号转导的各种分子的改变，进一步阐明BBD9诱导的骨髓瘤细胞凋亡的分子机制。
方法：
MTT检测外源性IL-6对BBD9诱导的U266细胞生长抑制的影响，ELISA检测U266细胞培养上清IL-6水平，流式细胞术检测细胞膜上IL-6R的表达量，Real-time PCR检测细胞IL-6mRNA及IL-6R mRNA表达量，western blot检测细胞内各种激酶的磷酸化水平。
结果：
外源性IL-6减弱BBD9诱导的生长抑制。U266细胞在1μg／ml BBD9作用24h后，其生存率为88．83％，加用150ng／ml IL-6后，其生存率显著升高至104．83％；2μg／ml BBD9作用24h后，U266细胞的生存率仅有43．03％，而加用IL-6后，其生存率显著升高至55．93％。ELISA法检测U266细胞培养上清IL-6水平，发现BBD9能减少自分泌的IL-6。0、1、2、3μg／ml BBD9处理24h后，U266细胞培养上清中IL-6水平分别为33．49±7．92pg／ml、10．34±5．83pg／ml、13．59±7．22pg／ml、10．78±6．67pg／ml。此外，细胞膜上IL-6R的表达量也略有下降。1、2、3μg／ml BBD9处理8h，IL-6mRNA表达量分别是未处理组的0．9、3、2．7倍，而IL-6R mRNA无明显变化。BBD9处理后，U266细胞内IL-6信号传导途径中两个重要的激酶STAT3及AKT磷酸化水平均明显下调，而其总STAT3及总AKT均无改变。
结论：
(1)BBD9抑制骨髓瘤细胞自分泌IL-6及细胞膜IL-6R的合成，但并不在mRNA水平抑制二者的表达。
(2)BBD9使骨髓瘤IL-6信号传导途径的STAT3和PI3K／AKT途径受抑。
(3)IL-6信号传导途径受阻是BBD9诱导骨髓瘤细胞凋亡的一个重要机制。
第四部分BBD9增加多药耐药K562／A02细胞对阿霉素的敏感性及其机制
目的：
研究BBD9能否逆转p-gp介导的急性白血病多药耐药及其机制。
方法：
MTT检测细胞对阿霉素的耐药性，流式细胞术检测细胞的凋亡率、测定细胞内阿霉素浓度及检测细胞膜表面p-gp表达量的变化，Real-time PCR检测细胞内mdr-1 mRNA表达。
结果：
阿霉素作用于K562／A02细胞24h的IC50为7．57μg／ml，作用于K562细胞24h的IC50为0．651μg／ml，K562／A02细胞耐药指数为11．65。2μg／ml阿霉素与1μg／mlBBD9单独或联合处理K562／A02细胞24h，结果发现：2μg／ml阿霉素处理组细胞凋亡率为6．04±2．28％，1μg／ml BBD9处理组细胞凋亡率为8．21±5．74％，对照组细胞凋亡率为6．59±2．56％。然而，2μg／ml阿霉素与1μg／ml BBD9联合处理组，细胞凋亡率明显升高至35．93±1．87％，与对照组相比差异具有显著统计学意义。但是，0．4μg／ml阿霉素与1μg／ml BBD9单独或联合处理K562细胞24h，结果显示：对照组凋亡率为1．77±0．58％，单用BBD9组凋亡率为3．52±2．0％，单用阿霉素组为11．77±8．44％，而BBD9联合阿霉素组的凋亡率为2．93±0．48％，各组之间差异无统计学意义。检测细胞内阿霉素浓度显示，BBD9处理1h，能使阿霉素阳性K562／A02细胞由单用阿霉素时的8．88±12．98％升高至87．82±12．03％；BBD9处理24h，能使K562／A02细胞阿霉素的平均荧光强度由单用阿霉素时的39．78±3．64％升高至54．4±5．63％。进一步的结果显示，BBD9并不能减少mdr-1 mRNA的表达，也不能影响细胞膜上p-gp蛋白的表达。
结论：
(1)BBD9能增加K562／A02细胞对阿霉素的敏感性，而不能增加K562细胞对阿霉素的敏感性。
(2)BBD9通过提高p-gp高表达的K562／A02细胞内阿霉素浓度使得细胞对阿霉素的敏感性增加。
(3)BBD9对化疗药物的增敏作用并不涉及p-gP蛋白数量的改变，可能是通过抑制p-gP的泵功能达到增敏作用的。
总结：
BBD9具有抑制多发性骨髓瘤细胞株及原代细胞生长的作用。这种生长抑制作用是与细胞凋亡有关的。在BBD9诱导的骨髓瘤细胞凋亡过程中，促凋亡蛋白Bim、Bax及Bak上调，而抑凋亡蛋白Mcl-1则下调。BBD9能通过下调cyclinA、cyclinB1使细胞周期阻滞于G2／M期。此外，BBD9还能增加骨髓瘤细胞核内FOXO3的表达量，抑制mir-17-5p，最终导致Bim蛋白表达增加。BBD9能抑制U266细胞IL-6和IL-6R合成，并抑制IL-6信号传导途径的两个重要途径：STAT3和PI3K／AKT途径。说明IL-6信号传导途径受阻是BBD9诱导的骨髓瘤细胞凋亡的一个重要机制。此外，我们的研究还发现BBD9能增加K562／A02细胞对阿霉素的敏感性，而不能增加K562细胞对阿霉素的敏感性。进一步研究表明，BBD9通过提高K562／A02细胞内阿霉素浓度实现逆转细胞对阿霉素的耐药。由于BBD9既不能降低mdr-1 mRNA表达量，也不能降低p-gP的蛋白表达量，提示BBD9可能是通过抑制p-gP的泵功能达到逆转耐药的作用的。
Section 1 BBD9 inhibited cell growth and induced apoptosisin myeloma cells
Objective:
To study the effects and the mechanisms of BBD9 on myeloma cell growth.
Methods:
Cell growth was measured by MTT assay in myeloma cell lines and primary cellsfrom patients with MM.Cell morphology was observed by Wright-Giemsa stain.AV-PIstaining was used for detecting apoptosis,PI staining was used for cell cycle analysis,and western blot was used for detecting caspase,BCL-2 family proteins and cell cycleregulating proteins.Results:
The cell viabilities of 4 myeloma cell lines,including RPMI8226,U266,MM.1S,MM.1R,were significantly inhibited by BBD9 at the concentration of 0-4μg/ml.Thecell survival rate was decreased with the increasing concentration of BBD9.
Furthermore,BBD9 inhibited cell growth in all 4 cell lines in a time dependent manner.If treated at the same concentration of BBD9,fewer cells would survive when the cellswere treated for an increasing time.The 24h IC50 of BBD9 on RPMI8226,U266,MM.1S,MM.1R were 2.30μg/ml,1.87μg/ml,2.39μg/ml,1.54μg/ml,respectively.Primary cells from three patients with MM were treated with BBD9 for 24h,andshowed growth inhibition in a dose dependent manner.The IC50 for primary cells was1.08μg/ml.The cell growth inhibition induced by BBD9 was also measured inmononuclear cells form normal bone marrow by MTT assay.The results showed thatthe cell growth of normal mononuclear cells was inhibited,in a dose dependant manner,by BBD9 at the concentration of 0-4μg/ml.However,normal mononuclear cells wereless sensitive to BBD9 treatment than primary MM cells were,and the 24h IC50 was3.24μg/ml.After treated by BBD9 for 24h,U266 and RPMI8226 cells,observed byWright-Giemsa stain,showed a typical apoptotic morphology,nuclear fragmentationand apoptotic body.Typical DNA ladders were also seen in RPMI8226 cells treated byBBD9 for 24h.With the concentration of BBD9 increasing,the DNA ladder becamemore significant.BBD9 could induce apoptosis in MM cells,as measured by AV-PIassay.After treated by 0,1,2,3μg/ml BBD9 for 24h,the RPMI8226 cells in earlyapoptosis were 1.57±0.37%,4.79±2.47%,16.18±5.45%,42.94±13.99%,respectively.Meanwhile,the U266 cells in early apoptosis were 2.69±1.28%,7.36±2.22%,19.67±2.83%,50.26±16.29%,respectively.Furthermore,caspase-3,-8,-9 and PARP werecleaved and activated,as measured by western blot,in U266 cells and RPMI8226 cellstreated by BBD9.In addition,pro-apoptotic protein Bak,Bax and Bim wereup-regulated in U266 cells and RPMI8226 cells treated by BBD9 and anti-apoptoticprotein Mcl-1 were down-regulated.However,another two important anti-apoptoticprotein,BCL-2 and BCL-XL,had not changed in protein expression level,as wasmeasured by western blot assay.In addition,the results of cell cycle analysis showedthat BBD9 induced cell cycle arrest in G2/M phase.After treated by 0,1,2,3μg/ml BBD9 for 24h,the RPMI8226 cells arrested in G2/M phase were 4.49±3.79%,13.61±2.09%,41.03±2.09%,35.99±9.05%,respectively.After being treated by 0,1,2,3μg/ml BBD9 for24h,the U266 cells arrested in G2/M phase were14.14±0.6%、19.48±2.84%、26.54±3.44%、33.62±3.0%,respectively.Cell cycle regulators cyclin A,cyclin B1 were bothdown-regulated significantly,as measured by western blot assay,in U266 cells andRPMI8226 cells treated by BBD9 for 24h,whereas,CDK1 and P27 showed no anydistinct down-or up-regulation.
Conclusions:
(1)The cell growth of 4 MM cell lines and primary cells from 3 patients with MMcould be inhibited by BBD9.
(2)BBD9 induced apoptosis in MM cells.
(3)Pro-apoptotic protein Bak,Bax and Bim were up-regulated and anti-apoptoticprotein Mcl-1 was down-regulated by BBD9 in MM cells.(4)BBD9 induced cell cycle arrested in G2/M phase through down-regulation of
cyclin A and cuclin B1.
Section 2 BBD9 induced apoptosis in MM cells accompaniedby the activation of FOXO3a/Bim
Objective:
To study the mechanisms of the up-regulation of Bim protein in apoptotic MMcells induced by BBD9.
Methords:
Real-time PCR was used for quantifying Bim mRNA and mir-17-5p,western blotused for FOXO3a and pFOXO3a analysis,immunofluorescent staining was used forobserving the distribution of FOXO3a in cells.
Results:
The copies of Bim mRNA significantly increased in U266 cells treated by BBD9for 8h.In addition,mir-17-5p,the negative regulator of Bim mRNA,was down-regulated significantly.Mir-17-5p in U266 cells treated with 3μg/ml BBD9 wasdown-regulated to 0.35±0.23 folds of that in control group.Western blot assay showedthat the expression of FOXO3 factor,one of pivotal transcription factors of Bim gene,was significantly higher in nuclear of U266 cells treated with BBD9 than control.Onthe contrary,phosphor-FOXO3a(pFOXO3a)was decreased with the increasing ofBBD9 concentration.Observations using immunofluorescent microscope showed thatwhen U266 cells were not treated with BBD9,FOXO3a in nuclear was lower than thatin cytoplasm.After U266 cells were treated with 2μg/ml BBD9 for 24h,theimmunofluorescence of FOXO3a in nuclear was higher than that in cytoplasm.Likewise,before RPMI8226 cells were treated with BBD9,FOXO3a in nuclear waslower than that in cytoplasm.After RPMI8226 cells were treated with 2μg/ml BBD9 for24h,the immunofluorescence of FOXO3a in nuclear was higher than that in cytoplasm.
Conclusions:
(1)BBD9 up-regulated Bim protein in MM cells through up-regulation ofFOXO3a factor in nuclear and subquently promotion of the Bim gene transcription,BimmRNA up-regulation.
(2)BBD9 down-regulated mir-17-5p which resulted in decreased degradation ofBim mRNA or in decreased inhibition of Bim mRNA translation.
Section 3 BBD9 inhibited IL-6 signaling in MM
Objective:
To better understand the relationship between the apoptosis induced by BBD9 inMM cells and IL-6 signaling pathway.
Methods:
MTT was used for assaying the effect of extraneous IL-6 on cell growth inhibitioninduced by BBD9 in U266 cells.ELISA was used for measuring IL-6 concentration inU266 culture supematant.Membrane IL-6R was measured by FACS,IL-6mRNA andIL-6R mRNA were measured by Real-time PCR,and protein levels were measured bywestern blot.
Results:
Extraneous IL-6 abrogated the cell growth inhibition induced by BBD9 in U266cells.The cell survival rate,which was 88.83% in U266 cells treated with 1μg/ml BBD9for 24h,significantly increased to 104.83% in U266 cells treated with 150ng/ml IL-6and 1μg/ml BBD9.The U266 cell survival rate,which was only 43.03% when cellswere treated with 2μg/ml BBD9 for 24h,significantly increased to 55.93% in U266cells treated with 150ng/ml IL-6 and 2μg/ml BBD9.IL-6 concentration in U266 cellculture was determined using ELISA assay and a decreased auto-secreted IL-6 wasfound.IL-6 concentrations in culture supernatant of U266 cells treated with 0,1,2,3μg/ml BBD9 for 24h were 33.49±7.92pg/ml,10.34±S.83pg/ml,13.59±7.22pg/ml,10.78±6.67pg/ml,respectively.In addition,membrane IL-6R was also slightlydown-regulated.Compared with the untreated control group,the expressions of IL-6mRNA were 0.9,3,2.7 folds,whereas the expressions of IL-6 mRNA were not changed.Furthermore,the phosphorylation level of two important kinases,STAT3 and AKT,were significantly down regulated in U266 cells treated with BBD9,while theexpression of total STAT3 and total AKT kept intact.
Conclusions:
(1)BBD9 inhibited MM cells auto-secretions of IL-6 and the production ofmembrane IL-6R.But they were not regulated at mRNA level.
(2)Two of IL-6 signaling pathways,STAT3 signaling pathway and AKT signaling pathway were inhibited by BBD9.
(3)The blockage of IL-6 signaling pathway was an important mechanism in BBD9induced MM apoptosis.
Section 4 BBD9 increases sensitivity to adriamycin in multi-drugresistant K562/A02 cells
Objective:
To investigate the role of BBD9 in anti-multi-drug resistant acute leukemia and itsmechanisms.Methods:
MTT was used for detecting multi-drug resistance of leukemia cells,apoptosis,theconcentration of ADM in the cells and membrane p-gp were measured by FACS,mdr-1mRNA was measured by Real-time PCR.Results:
24h IC50 of ADM on K562/A02 was 7.57μg/ml,the IC50 of ADM on K562 was0.651μg/ml,and the resistance index was 11.65.K562/A02 cells were treated with2μg/ml ADM and/or 1μg/ml BBD9 for 24h,and the results showed that the apoptosisrate in 2μg/ml ADM group was 6.04±2.28%,the apoptosis rate in 1μg/ml BBD9 groupwas 8.21±5.74%,the apoptosis rate in control group was 6.59±2.56%.However,theapoptosis rate in 2μg/ml ADM combined with 1μg/ml BBD9 group was 35.93±1.87%,and comparing with control,P＜0.05.In addition,K562 cells were treated with 0.4μg/mlADM and/or 1μg/ml BBD9 for 24h,and the results showed that the apoptosis rate in0.2μg/ml ADM group was 11.77±8.44%,the apoptosis rate in 1μg/ml BBD9 group was3.52±2.0%,the apoptosis rate in control group was 1.77±0.58%,the apoptosis rate in0.4μg/ml ADM combined with 1μg/ml BBD9 group was 2.93±0.48%,the difference between these groups were no statistical significance(P＞0.05).The results of measuringADM concentration in cells showed that after treated with BBD9 for lh,ADM positiveK562/A02 increased from 8.88±12.98% in 2μg/ml ADM group to 87.82±12.03% in2μg/ml ADM combined with 1μg/ml BBD9 group.After treatment with BBD9 for 24h,the mean fluorescence intensity of ADM in K562/A02 cells increased from39.78±3.64% in 2μg/ml ADM group to 54.4±-5.63% in 2μg/ml ADM group combinedwith 1μg/ml BBD9 group.In addition,BBD9 decreased neither the expression of mdr-1mRNA nor the membrane p-gp.
Conclusion:
(1)BBD9 increased the sensitivity to adriamycin in multi-drug resistant K562/A02cells but not in K562 cells.
(2)BBD9 increased the sensitivity adriamycin in K562/A02 cells via raising ADMconcentration in cells.
(3)The mechanisms of increasing sensitivity to adriamycin by BBD9 did notinvolved the alteration in p-gp protein,but the efflux-pumping function of p-gp proteinprobably was interfered by BBD9.
Summary:
BBD9 inhibited cell growth in MM cell lines and primary cells from patients withMM apoptosis was induced.Pro-apoptotic protein Bak,Bax and Bim were up-regulatedand anti-apoptotic protein Mcl-1 was down-regulated in BBD9 treated MM cells.Furthermore,BBD9 induced cell cycle arrested in G2/M phase through down-regulationof cyclin A and cuclin B1.In addition,BBD9 increased the expression of FOXO3a innuclear and decreased the expression of mir-17-5p,resulting in the increasement of Bimprotein in MM cells.Furthermore,BBD9 inhibited the production of IL-6 as well asIL-6R,and inhibited 2 important IL-6 signaling pathways:STAT3 and PI3K/AKT, indicating that the blockade of IL-6 signaling is one of the important mechanisms ofapoptosis induced by BBD9 in MM cells.In addition,our study showed that BBD9increased the sensitivity to adriamycin in multi-drug resistant K562/A02 cells but not inK562 cells.BBD9 increased the sensitivity to adriamycin in K562/A02 cells via raisingADM concentration in cells.As BBD9 decreased neither mdr-1 mRNA nor p-gp protein,we deduced that BBD9 increased sensitivity to adriamycin in K562/A02 cells throughinhibition of p-gp function.
目的：
观察BBD9对多发性骨髓瘤细胞生存的影响及其机制。
方法：
MTT法检测BBD9对多种骨髓瘤细胞株及原代细胞的生长抑制作用，瑞氏染色观察细胞形态学变化，AV-PI法检测细胞凋亡，检测DNA ladder，流式细胞术检测细胞周期，western blot法检测caspase，BCL-2家族蛋白及细胞周期调控蛋白。
结果：
在0～4μg／ml BBD9作用下，BBD9能显著抑制四种骨髓瘤细胞株(RPMI8226、U266、MM．1S、MM．1R)的生长。随着BBD9浓度的升高，各细胞株的细胞存活率逐步降低。此外，BBD9对四种细胞株的生长抑制作用也呈时间依赖性，同一浓度下，药物作用时间越长，细胞存活率也越低。BBD9作用于RPMI8226、U266、MM．1S、MM．1R细胞24h的IC50依次是：2．30μg／ml、1．87μg／ml、2．39μg／ml、1．54μg／ml。BBD9对3例病人原代细胞也呈现剂量依赖性的生长抑制。BBD9处理骨髓瘤原代细胞24h的IC50为1．08μg／ml。用正常骨髓单个核细胞进行MTT检测发现，在0-4μg／ml BBD9也呈剂量依赖性地抑制正常骨髓单个核细胞的生长，但其对药物的敏感性低于原代细胞，24h的IC50为3．24μg／ml。形态学观察可见，BBD9处理24h后的U266细胞和RPMI8226细胞出现了典型的凋亡细胞形态：核碎裂，凋亡小体。DNA片段化检测可见经BBD9处理的RPMI8226细胞出现了典型“DNA梯子”，随着药物浓度的增加，“DNA梯子”也越明显。AV-PI法检测细胞凋亡也发现，BBD9可以诱导骨髓瘤细胞出现凋亡。0、1、2、3μg／ml BBD9处理RPMI8226细胞24h后，早期凋亡细胞比例分别为1．57±0．37％、4．79±2．47％、16．18±5．45％、42．94±13．99％；而在U266细胞，早期凋亡细胞的比例分别为2．69±1．28％、7．36±2．22％、19．67±2．83％、50．26±16．29％。BBD9还可以诱导U266细胞和RPMI8226细胞caspase-3，-8，-9及PARP剪切激活。进一步的检测可见，BBD9处理后，促凋亡蛋白Bak、Bax和Bim表达量在U266和RPMI8226细胞均有明显升高，抑凋亡蛋白Mcl-1明显下调。但是，另外两种重要的抑凋亡蛋白BCL-2和BCL-XL则变化不明显。此外，对细胞周期分析表明，BBD9使骨髓瘤细胞周期阻滞在G2／M期。0、1、2、3μg／ml BBD9处理24h后，处于G2／M期的RPMI8226细胞分别为4．49±3．79％、13．61±2．09％、41．03±2．09％、35．99±9．05％；而处于G2／M期的U266细胞分别为14．14±0．6％、19．48±2．84％、26．54±3．44％、33．62±3．0％。细胞周期调控蛋白cyclinA、cyclinB1在BBD9处理的RPMI8226细胞和U266细胞中均明显下调，而CDK1及P27无明显改变。
结论：
(1)BBD9能抑制多种骨髓瘤细胞株及原代细胞生长。
(2)BBD9诱导骨髓瘤细胞凋亡。
(3)BBD9上调促凋亡蛋白Bak、Bax和Bim，下调抑凋亡蛋白Mcl-1。
(4)BBD9通过下调cyclinA、cyclinB1使细胞周期阻滞于G2／M期。
第二部分BBD9诱导的凋亡伴随着FOXO3a／Bim途径激活
目的：
研究BBD9诱导的Bim蛋白表达上调的机制。
方法：
Real-time PCR检测Bim mRNA及mir-17-5p的表达，western blot分析细胞核内FOXO3a及pFOXO3a表达量，免疫荧光显微镜观察FOXO3在细胞内的分布。
结果：
BBD9处理U266细胞8h后，Bim mRNA拷贝数明显增加。与此同时，对BimmRNA起负调控作用的成熟体小RNA—mir-17-5p明显下调。3μg／ml BBD9处理时，mir-17-5p下降至未处理组的0．35±0．23倍。Westem blot结果显示，BBD9处理的U266细胞核内Bim基因转录因子FOXO3a的表达量较未处理的U266细胞明显升高。与此相反，核内pFOXO3a蛋白量随着BBD9浓度的增加而减少。免疫荧光显微镜下可以看到：在BBD9处理前，U266细胞胞核内FOXO3a表达较低，胞浆内FOXO3a表达相对较高；而在2μg／ml BBD9处理24h后，U266细胞胞核内FOXO3a的荧光较强，而胞浆中FOXO3a则较弱。同样地，在BBD9处理前，RPMI8226细胞胞核内FOXO3a荧光较弱，胞浆内FOXO3a荧光则相对更强；而在2μg／mlBBD9处理24h后，RPMI8226细胞胞核内FOXO3a的荧光较强，而胞浆中FOXO3a则较弱。
结论：
(1)BBD9通过提高胞核内FOXO3a的表达量，促进Bim基因转录，增加BimmRNA拷贝数，从而导致Bim蛋白表达上调。
(2)BBD9还通过下调mir-17-5p，减少Bim mRNA降解或对Bim mRNA翻译的抑制，从而使Bim蛋白表达上调。
第三部分BBD9抑制IL-6信号传导途径的激活
目的：
通过观察IL-6信号传导途径中参与信号转导的各种分子的改变，进一步阐明BBD9诱导的骨髓瘤细胞凋亡的分子机制。
方法：
MTT检测外源性IL-6对BBD9诱导的U266细胞生长抑制的影响，ELISA检测U266细胞培养上清IL-6水平，流式细胞术检测细胞膜上IL-6R的表达量，Real-time PCR检测细胞IL-6mRNA及IL-6R mRNA表达量，western blot检测细胞内各种激酶的磷酸化水平。
结果：
外源性IL-6减弱BBD9诱导的生长抑制。U266细胞在1μg／ml BBD9作用24h后，其生存率为88．83％，加用150ng／ml IL-6后，其生存率显著升高至104．83％；2μg／ml BBD9作用24h后，U266细胞的生存率仅有43．03％，而加用IL-6后，其生存率显著升高至55．93％。ELISA法检测U266细胞培养上清IL-6水平，发现BBD9能减少自分泌的IL-6。0、1、2、3μg／ml BBD9处理24h后，U266细胞培养上清中IL-6水平分别为33．49±7．92pg／ml、10．34±5．83pg／ml、13．59±7．22pg／ml、10．78±6．67pg／ml。此外，细胞膜上IL-6R的表达量也略有下降。1、2、3μg／ml BBD9处理8h，IL-6mRNA表达量分别是未处理组的0．9、3、2．7倍，而IL-6R mRNA无明显变化。BBD9处理后，U266细胞内IL-6信号传导途径中两个重要的激酶STAT3及AKT磷酸化水平均明显下调，而其总STAT3及总AKT均无改变。
结论：
(1)BBD9抑制骨髓瘤细胞自分泌IL-6及细胞膜IL-6R的合成，但并不在mRNA水平抑制二者的表达。
(2)BBD9使骨髓瘤IL-6信号传导途径的STAT3和PI3K／AKT途径受抑。
(3)IL-6信号传导途径受阻是BBD9诱导骨髓瘤细胞凋亡的一个重要机制。
第四部分BBD9增加多药耐药K562／A02细胞对阿霉素的敏感性及其机制
目的：
研究BBD9能否逆转p-gp介导的急性白血病多药耐药及其机制。
方法：
MTT检测细胞对阿霉素的耐药性，流式细胞术检测细胞的凋亡率、测定细胞内阿霉素浓度及检测细胞膜表面p-gp表达量的变化，Real-time PCR检测细胞内mdr-1 mRNA表达。
结果：
阿霉素作用于K562／A02细胞24h的IC50为7．57μg／ml，作用于K562细胞24h的IC50为0．651μg／ml，K562／A02细胞耐药指数为11．65。2μg／ml阿霉素与1μg／mlBBD9单独或联合处理K562／A02细胞24h，结果发现：2μg／ml阿霉素处理组细胞凋亡率为6．04±2．28％，1μg／ml BBD9处理组细胞凋亡率为8．21±5．74％，对照组细胞凋亡率为6．59±2．56％。然而，2μg／ml阿霉素与1μg／ml BBD9联合处理组，细胞凋亡率明显升高至35．93±1．87％，与对照组相比差异具有显著统计学意义。但是，0．4μg／ml阿霉素与1μg／ml BBD9单独或联合处理K562细胞24h，结果显示：对照组凋亡率为1．77±0．58％，单用BBD9组凋亡率为3．52±2．0％，单用阿霉素组为11．77±8．44％，而BBD9联合阿霉素组的凋亡率为2．93±0．48％，各组之间差异无统计学意义。检测细胞内阿霉素浓度显示，BBD9处理1h，能使阿霉素阳性K562／A02细胞由单用阿霉素时的8．88±12．98％升高至87．82±12．03％；BBD9处理24h，能使K562／A02细胞阿霉素的平均荧光强度由单用阿霉素时的39．78±3．64％升高至54．4±5．63％。进一步的结果显示，BBD9并不能减少mdr-1 mRNA的表达，也不能影响细胞膜上p-gp蛋白的表达。
结论：
(1)BBD9能增加K562／A02细胞对阿霉素的敏感性，而不能增加K562细胞对阿霉素的敏感性。
(2)BBD9通过提高p-gp高表达的K562／A02细胞内阿霉素浓度使得细胞对阿霉素的敏感性增加。
(3)BBD9对化疗药物的增敏作用并不涉及p-gP蛋白数量的改变，可能是通过抑制p-gP的泵功能达到增敏作用的。
总结：
BBD9具有抑制多发性骨髓瘤细胞株及原代细胞生长的作用。这种生长抑制作用是与细胞凋亡有关的。在BBD9诱导的骨髓瘤细胞凋亡过程中，促凋亡蛋白Bim、Bax及Bak上调，而抑凋亡蛋白Mcl-1则下调。BBD9能通过下调cyclinA、cyclinB1使细胞周期阻滞于G2／M期。此外，BBD9还能增加骨髓瘤细胞核内FOXO3的表达量，抑制mir-17-5p，最终导致Bim蛋白表达增加。BBD9能抑制U266细胞IL-6和IL-6R合成，并抑制IL-6信号传导途径的两个重要途径：STAT3和PI3K／AKT途径。说明IL-6信号传导途径受阻是BBD9诱导的骨髓瘤细胞凋亡的一个重要机制。此外，我们的研究还发现BBD9能增加K562／A02细胞对阿霉素的敏感性，而不能增加K562细胞对阿霉素的敏感性。进一步研究表明，BBD9通过提高K562／A02细胞内阿霉素浓度实现逆转细胞对阿霉素的耐药。由于BBD9既不能降低mdr-1 mRNA表达量，也不能降低p-gP的蛋白表达量，提示BBD9可能是通过抑制p-gP的泵功能达到逆转耐药的作用的。
Section 1 BBD9 inhibited cell growth and induced apoptosisin myeloma cells
Objective:
To study the effects and the mechanisms of BBD9 on myeloma cell growth.
Methods:
Cell growth was measured by MTT assay in myeloma cell lines and primary cellsfrom patients with MM.Cell morphology was observed by Wright-Giemsa stain.AV-PIstaining was used for detecting apoptosis,PI staining was used for cell cycle analysis,and western blot was used for detecting caspase,BCL-2 family proteins and cell cycleregulating proteins.Results:
The cell viabilities of 4 myeloma cell lines,including RPMI8226,U266,MM.1S,MM.1R,were significantly inhibited by BBD9 at the concentration of 0-4μg/ml.Thecell survival rate was decreased with the increasing concentration of BBD9.
Furthermore,BBD9 inhibited cell growth in all 4 cell lines in a time dependent manner.If treated at the same concentration of BBD9,fewer cells would survive when the cellswere treated for an increasing time.The 24h IC50 of BBD9 on RPMI8226,U266,MM.1S,MM.1R were 2.30μg/ml,1.87μg/ml,2.39μg/ml,1.54μg/ml,respectively.Primary cells from three patients with MM were treated with BBD9 for 24h,andshowed growth inhibition in a dose dependent manner.The IC50 for primary cells was1.08μg/ml.The cell growth inhibition induced by BBD9 was also measured inmononuclear cells form normal bone marrow by MTT assay.The results showed thatthe cell growth of normal mononuclear cells was inhibited,in a dose dependant manner,by BBD9 at the concentration of 0-4μg/ml.However,normal mononuclear cells wereless sensitive to BBD9 treatment than primary MM cells were,and the 24h IC50 was3.24μg/ml.After treated by BBD9 for 24h,U266 and RPMI8226 cells,observed byWright-Giemsa stain,showed a typical apoptotic morphology,nuclear fragmentationand apoptotic body.Typical DNA ladders were also seen in RPMI8226 cells treated byBBD9 for 24h.With the concentration of BBD9 increasing,the DNA ladder becamemore significant.BBD9 could induce apoptosis in MM cells,as measured by AV-PIassay.After treated by 0,1,2,3μg/ml BBD9 for 24h,the RPMI8226 cells in earlyapoptosis were 1.57±0.37%,4.79±2.47%,16.18±5.45%,42.94±13.99%,respectively.Meanwhile,the U266 cells in early apoptosis were 2.69±1.28%,7.36±2.22%,19.67±2.83%,50.26±16.29%,respectively.Furthermore,caspase-3,-8,-9 and PARP werecleaved and activated,as measured by western blot,in U266 cells and RPMI8226 cellstreated by BBD9.In addition,pro-apoptotic protein Bak,Bax and Bim wereup-regulated in U266 cells and RPMI8226 cells treated by BBD9 and anti-apoptoticprotein Mcl-1 were down-regulated.However,another two important anti-apoptoticprotein,BCL-2 and BCL-XL,had not changed in protein expression level,as wasmeasured by western blot assay.In addition,the results of cell cycle analysis showedthat BBD9 induced cell cycle arrest in G2/M phase.After treated by 0,1,2,3μg/ml BBD9 for 24h,the RPMI8226 cells arrested in G2/M phase were 4.49±3.79%,13.61±2.09%,41.03±2.09%,35.99±9.05%,respectively.After being treated by 0,1,2,3μg/ml BBD9 for24h,the U266 cells arrested in G2/M phase were14.14±0.6%、19.48±2.84%、26.54±3.44%、33.62±3.0%,respectively.Cell cycle regulators cyclin A,cyclin B1 were bothdown-regulated significantly,as measured by western blot assay,in U266 cells andRPMI8226 cells treated by BBD9 for 24h,whereas,CDK1 and P27 showed no anydistinct down-or up-regulation.
Conclusions:
(1)The cell growth of 4 MM cell lines and primary cells from 3 patients with MMcould be inhibited by BBD9.
(2)BBD9 induced apoptosis in MM cells.
(3)Pro-apoptotic protein Bak,Bax and Bim were up-regulated and anti-apoptoticprotein Mcl-1 was down-regulated by BBD9 in MM cells.(4)BBD9 induced cell cycle arrested in G2/M phase through down-regulation of
cyclin A and cuclin B1.
Section 2 BBD9 induced apoptosis in MM cells accompaniedby the activation of FOXO3a/Bim
Objective:
To study the mechanisms of the up-regulation of Bim protein in apoptotic MMcells induced by BBD9.
Methords:
Real-time PCR was used for quantifying Bim mRNA and mir-17-5p,western blotused for FOXO3a and pFOXO3a analysis,immunofluorescent staining was used forobserving the distribution of FOXO3a in cells.
Results:
The copies of Bim mRNA significantly increased in U266 cells treated by BBD9for 8h.In addition,mir-17-5p,the negative regulator of Bim mRNA,was down-regulated significantly.Mir-17-5p in U266 cells treated with 3μg/ml BBD9 wasdown-regulated to 0.35±0.23 folds of that in control group.Western blot assay showedthat the expression of FOXO3 factor,one of pivotal transcription factors of Bim gene,was significantly higher in nuclear of U266 cells treated with BBD9 than control.Onthe contrary,phosphor-FOXO3a(pFOXO3a)was decreased with the increasing ofBBD9 concentration.Observations using immunofluorescent microscope showed thatwhen U266 cells were not treated with BBD9,FOXO3a in nuclear was lower than thatin cytoplasm.After U266 cells were treated with 2μg/ml BBD9 for 24h,theimmunofluorescence of FOXO3a in nuclear was higher than that in cytoplasm.Likewise,before RPMI8226 cells were treated with BBD9,FOXO3a in nuclear waslower than that in cytoplasm.After RPMI8226 cells were treated with 2μg/ml BBD9 for24h,the immunofluorescence of FOXO3a in nuclear was higher than that in cytoplasm.
Conclusions:
(1)BBD9 up-regulated Bim protein in MM cells through up-regulation ofFOXO3a factor in nuclear and subquently promotion of the Bim gene transcription,BimmRNA up-regulation.
(2)BBD9 down-regulated mir-17-5p which resulted in decreased degradation ofBim mRNA or in decreased inhibition of Bim mRNA translation.
Section 3 BBD9 inhibited IL-6 signaling in MM
Objective:
To better understand the relationship between the apoptosis induced by BBD9 inMM cells and IL-6 signaling pathway.
Methods:
MTT was used for assaying the effect of extraneous IL-6 on cell growth inhibitioninduced by BBD9 in U266 cells.ELISA was used for measuring IL-6 concentration inU266 culture supematant.Membrane IL-6R was measured by FACS,IL-6mRNA andIL-6R mRNA were measured by Real-time PCR,and protein levels were measured bywestern blot.
Results:
Extraneous IL-6 abrogated the cell growth inhibition induced by BBD9 in U266cells.The cell survival rate,which was 88.83% in U266 cells treated with 1μg/ml BBD9for 24h,significantly increased to 104.83% in U266 cells treated with 150ng/ml IL-6and 1μg/ml BBD9.The U266 cell survival rate,which was only 43.03% when cellswere treated with 2μg/ml BBD9 for 24h,significantly increased to 55.93% in U266cells treated with 150ng/ml IL-6 and 2μg/ml BBD9.IL-6 concentration in U266 cellculture was determined using ELISA assay and a decreased auto-secreted IL-6 wasfound.IL-6 concentrations in culture supernatant of U266 cells treated with 0,1,2,3μg/ml BBD9 for 24h were 33.49±7.92pg/ml,10.34±S.83pg/ml,13.59±7.22pg/ml,10.78±6.67pg/ml,respectively.In addition,membrane IL-6R was also slightlydown-regulated.Compared with the untreated control group,the expressions of IL-6mRNA were 0.9,3,2.7 folds,whereas the expressions of IL-6 mRNA were not changed.Furthermore,the phosphorylation level of two important kinases,STAT3 and AKT,were significantly down regulated in U266 cells treated with BBD9,while theexpression of total STAT3 and total AKT kept intact.
Conclusions:
(1)BBD9 inhibited MM cells auto-secretions of IL-6 and the production ofmembrane IL-6R.But they were not regulated at mRNA level.
(2)Two of IL-6 signaling pathways,STAT3 signaling pathway and AKT signaling pathway were inhibited by BBD9.
(3)The blockage of IL-6 signaling pathway was an important mechanism in BBD9induced MM apoptosis.
Section 4 BBD9 increases sensitivity to adriamycin in multi-drugresistant K562/A02 cells
Objective:
To investigate the role of BBD9 in anti-multi-drug resistant acute leukemia and itsmechanisms.Methods:
MTT was used for detecting multi-drug resistance of leukemia cells,apoptosis,theconcentration of ADM in the cells and membrane p-gp were measured by FACS,mdr-1mRNA was measured by Real-time PCR.Results:
24h IC50 of ADM on K562/A02 was 7.57μg/ml,the IC50 of ADM on K562 was0.651μg/ml,and the resistance index was 11.65.K562/A02 cells were treated with2μg/ml ADM and/or 1μg/ml BBD9 for 24h,and the results showed that the apoptosisrate in 2μg/ml ADM group was 6.04±2.28%,the apoptosis rate in 1μg/ml BBD9 groupwas 8.21±5.74%,the apoptosis rate in control group was 6.59±2.56%.However,theapoptosis rate in 2μg/ml ADM combined with 1μg/ml BBD9 group was 35.93±1.87%,and comparing with control,P＜0.05.In addition,K562 cells were treated with 0.4μg/mlADM and/or 1μg/ml BBD9 for 24h,and the results showed that the apoptosis rate in0.2μg/ml ADM group was 11.77±8.44%,the apoptosis rate in 1μg/ml BBD9 group was3.52±2.0%,the apoptosis rate in control group was 1.77±0.58%,the apoptosis rate in0.4μg/ml ADM combined with 1μg/ml BBD9 group was 2.93±0.48%,the difference between these groups were no statistical significance(P＞0.05).The results of measuringADM concentration in cells showed that after treated with BBD9 for lh,ADM positiveK562/A02 increased from 8.88±12.98% in 2μg/ml ADM group to 87.82±12.03% in2μg/ml ADM combined with 1μg/ml BBD9 group.After treatment with BBD9 for 24h,the mean fluorescence intensity of ADM in K562/A02 cells increased from39.78±3.64% in 2μg/ml ADM group to 54.4±-5.63% in 2μg/ml ADM group combinedwith 1μg/ml BBD9 group.In addition,BBD9 decreased neither the expression of mdr-1mRNA nor the membrane p-gp.
Conclusion:
(1)BBD9 increased the sensitivity to adriamycin in multi-drug resistant K562/A02cells but not in K562 cells.
(2)BBD9 increased the sensitivity adriamycin in K562/A02 cells via raising ADMconcentration in cells.
(3)The mechanisms of increasing sensitivity to adriamycin by BBD9 did notinvolved the alteration in p-gp protein,but the efflux-pumping function of p-gp proteinprobably was interfered by BBD9.
Summary:
BBD9 inhibited cell growth in MM cell lines and primary cells from patients withMM apoptosis was induced.Pro-apoptotic protein Bak,Bax and Bim were up-regulatedand anti-apoptotic protein Mcl-1 was down-regulated in BBD9 treated MM cells.Furthermore,BBD9 induced cell cycle arrested in G2/M phase through down-regulationof cyclin A and cuclin B1.In addition,BBD9 increased the expression of FOXO3a innuclear and decreased the expression of mir-17-5p,resulting in the increasement of Bimprotein in MM cells.Furthermore,BBD9 inhibited the production of IL-6 as well asIL-6R,and inhibited 2 important IL-6 signaling pathways:STAT3 and PI3K/AKT, indicating that the blockade of IL-6 signaling is one of the important mechanisms ofapoptosis induced by BBD9 in MM cells.In addition,our study showed that BBD9increased the sensitivity to adriamycin in multi-drug resistant K562/A02 cells but not inK562 cells.BBD9 increased the sensitivity to adriamycin in K562/A02 cells via raisingADM concentration in cells.As BBD9 decreased neither mdr-1 mRNA nor p-gp protein,we deduced that BBD9 increased sensitivity to adriamycin in K562/A02 cells throughinhibition of p-gp function.
引文
[1]Parkin,D.M.,Bray,F.,Ferlay,J.and Pisani,P.Global cancer statistics,2002.CA Cancer J Clin,2005,55:74-108.
[2]Teh,B.S.,Seow,W.K.,Li,S.Y.and Thong,Y.H.Inhibition of prostaglandin and leukotriene generation by the plant alkaloids tetrandrine and berbamine.Int J Immunopharmacol,1990,12:321-6.
[3]Zhang,W.,Chen,S.G.,Ju,H.S.,Zhao,S.H.,Zou,CM.,Hao,J.M.and Liu,Y.Mechanisms of protective effects of berbamine on ischemia/reperfusion injury in isolated rat heart.Methods Find Exp Clin Pharmacol,1992,14:677-84.
[4]Wang,G.Y.,Zhang,J.W.,Lu,Q.H.,Xu,R.Z.and Dong,Q.H.Berbamine induces apoptosis in human hepatoma cell line SMMC7721 by loss in mitochondrial transmembrane potential and caspase activation.J Zhejiang Unix 50/5,2007,8:248-55.
[5]Blade,J.,Kyle,R.A.and Greipp,P.R.Multiple myeloma in patients younger than 30 years.Report of 10 cases and review of the literature.Arch Intern Med,1996,156:1463-8.
[6]Blade,J.and Kyle,R.A.Multiple myeloma in young patients:clinical presentation and treatment approach.Leuk Lymphoma,1998,30:493-501.
[7]Kumar,S.K.,Rajkumar,S.V.,Dispenzieri,A.,Lacy,M.Q.,Hayman,S.R.,Buadi,F.K.,Zeldenrust,S.R.,Dingli,D.,Russell,S.J.,Lust,J.A.,Greipp,P.R.,Kyle,R.A.and Gertz,M.A.Improved survival in multiple myeloma and the impact of novel therapies.Blood,2008,111:2516-20.
[8]Edinger,A.L.and Thompson,C.B.Death by design:apoptosis,necrosis and autophagy.Curr Opin Cell Biol,2004,16:663-9.
[9]Elmore,S.Apoptosis:a review of programmed cell death.Toxicol Pathol,2007,35:495-516.
[10]Ghobrial,I.M.,Witzig,T.E.and Adjei,A.A.Targeting apoptosis pathways in cancer therapy.CA Cancer J Clin,2005,55:178-94.
[11]Youle,R.J.and Strasser,A.The BCL-2 protein family:opposing activities that mediate cell death.Nat Rev Mol Cell Biol,2008,9:47-59.
[12]Ruvolo,P.P.,Deng,X.and May,W.S.Phosphorylation of Bcl2 and regulation of apoptosis.Leukemia,2001,15:515-22.
[13]Haldar,S.,Jena,N.and Croce,CM.Inactivation of Bcl-2 by phosphorylation.Proc Natl Acad Sci USA,1995,92:4507-11.
[14]Thomas,A.,Giesler,T.and White,E.p53 mediates bcl-2 phosphorylation and apoptosis via activation of the Cdc42/JNK1 pathway.Oncogene,2000,19:5259-69.
[15]Ezhevsky,S.A.,Nagahara,H.,Vocero-Akbani,A.M.,Gius,D.R.,Wei,M.C.and Dowdy,S.F.Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb.Proc Natl Acad Sci USA,1997,94:10699-704.
[16]Morris,L.,Allen,K.E.and La Thangue,N.B.Regulation of E2F transcription by cyclin E-Cdk2 kinase mediated through p300/CBP co-activators.Nat Cell Biol,2000,2:232-9.
[17]Bashir,T.,Horlein,R.,Rommelaere,J.and Willwand,K.Cyclin A activates the DNA polymerase delta-dependent elongation machinery in vitro:A parvovirus DNA replication model.Proc Natl Acad Sci USA,2000,97:5522-7.
[18]Mailer,J.L.Mitotic control.Curr Opin Cell Biol,1991,3:269-75.
[19]Mitra,J.and Enders,G.H.Cyclin A/Cdk2 complexes regulate activation of Cdk1 and Cdc25 phosphatases in human cells.Oncogene,2004,23:3361-7.
[20]Pagano,M.,Pepperkok,R.,Verde,F.,Ansorge,W.and Draetta,G.Cyclin A is required at two points in the human cell cycle.Embo J,1992,11:961-71.
[21]Furuno,N.,den Elzen,N.and Pines,J.Human cyclin A is required for mitosis until mid prophase.J Cell Biol,1999,147:295-306.
[22]Miyazaki,T.and Arai,S.Two distinct controls of mitotic cdkl/cyclin Bl activity requisite for cell growth prior to cell division.Cell Cycle,2007,6:1419-25.
[23]Ohi,R.and Gould,K.L.Regulating the onset of mitosis.Curr Opin Cell Biol,1999,11:267-73.
[24]Fletcher,J.I.and Huang,D.C.Controlling the cell death mediators Bax and Bak:puzzles and conundrums.Cell Cycle,2008,7:39-44.
[25]Gomez-Bougie,P.,Oliver,L.,Le Gouill,S.,Bataille,R.and Amiot,M.Melphalan-induced apoptosis in multiple myeloma cells is associated with a cleavage of Mcl-1 and Bim and a decrease in the Mcl-1/Bim complex.Oncogene,2005,24:8076-9.
[26]Gomez-Benito,M.,Balsas,P.,Carvajal-Vergara,X.,Pandiella,A.,Anel,A.,Marzo,I.and Naval,J.Mechanism of apoptosis induced by IFN-alpha in human myeloma cells:role of Jakl and Bim and potentiation by rapamycin.Cell Signal,2007,19:844-54.
[27]Morales,A.A.,Gutman,D.,Lee,K.P.and Boise,L.H.BH3-only proteins Noxa,Bmf,and Bim are necessary for arsenic trioxide-induced cell death in myeloma.Blood,2008,111:5152-62.
[28]Han,J.,Goldstein,L.A.,Gastman,B.R.,Rabinovitz,A.and Rabinowich,H.Disruption of Mcl-1.Bim complex in granzyme B-mediated mitochondrial apoptosis.J Biol Chem,2005,280:16383-92.
[29]Ley,R.,Balmanno,K.,Hadfield,K.,Weston,C.and Cook,S.J.Activation of the ERK1/2 signaling pathway promotes phosphorylation and proteasome-dependent degradation of the BH3-only protein,Bim.J Biol Chem,2003,278:18811-6.
[30]Sunters,A.,Fernandez de Mattos,S.,Stahl,M.,Brosens,J.J.,Zoumpoulidou,G.,Saunders,C.A.,Coffer,P.J.,Medema,R.H.,Coombes,R.C.and Lam,E.W.Fox03a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines.J Biol Chem,2003,278:49795-805.
[31]Stan,S.D.,Hahm,E.R.,Warin,R.and Singh,S.V.Withaferin A causes FOX03a-and Bim-dependent apoptosis and inhibits growth of human breast cancer cells in vivo.Cancer Res,2008,68:7661-9.
[32]Yang,J.Y.,Zong,C.S.,Xia,W.,Yamaguchi,H.,Ding,Q.,Xie,X.,Lang,J.Y.,Lai,C.C.,Chang,C.J.,Huang,W.C.,Huang,H.,Kuo,H.P.,Lee,D.F.,Li,L.Y.,Lien,H.C.,Cheng,X.,Chang,K.J.,Hsiao,CD.,Tsai,F.J.,Tsai,C.H.,Sahin,A.A.,Muller,W.J.,Mills,G.B.,Yu,D.,Hortobagyi,G.N.and Hung,M.C.ERK promotes tumorigenesis by inhibiting FOX03a via MDM2-mediated degradation.Nat Cell Biol,2008,10:138-48.
[33]Hussain,A.R.,Al-Rasheed,M.,Manogaran,P.S.,Al-Hussein,K.A.,Platanias,L.C.,Al Kuraya,K.and Uddin,S.Curcumin induces apoptosis via inhibition of PI3'-kinase/AKT pathway in acute T cell leukemias.Apoptosis,2006,11:245-54.
[34]Yamamura,Y.,Lee,W.L.,Inoue,K.,Ida,H.and Ito,Y.RUNX3 cooperates with Fox03a to induce apoptosis in gastric cancer cells.J Biol Chem,2006,281:5267-76.
[35]Wu,W.,Sun,M.,Zou,G.M.and Chen,J.MicroRNA and cancer:Current status and prospective.Int J Cancer,2007,120:953-60.
[36]Nelson,K.M.and Weiss,G.J.MicroRNAs and cancer:past,present,and potential future.Mol Cancer Ther,2008,7:3655-60.
[37]Fontana,L.,Fiori,M.E.,Albini,S.,Cifaldi,L.,Giovinazzi,S.,Forloni,M.,Boldrini,R.,Donfrancesco,A.,Federici,V.,Giacomini,P.,Peschle,C.and Fruci,D.Antagomir-17-5p abolishes the growth of therapy-resistant neuroblastoma through p21 and BIM.PLoS ONE,2008,3:e2236.
[38]Nilsson,M.B.,Langley,R.R.and Fidler,I.J.Interleukin-6,secreted by human ovarian carcinoma cells,is a potent proangiogenic cytokine.Cancer Res,2005,65:10794-800.
[39]Hirano,T.Interleukin 6 and its receptor:ten years later.Int Rev Immunol,1998,16:249-84.
[40]Klein,B.,Zhang,X.G.,Jourdan,M.,Boiron,J.M.,Portier,M.,Lu,Z.Y.,Wijdenes,J.,Brochier,J.and Bataille,R.Interleukin-6 is the central tumor growth factor in vitro and in vivo in multiple myeloma.Eur Cytokine Netw,1990,1:193-201.
[41]Bataille,R.,Barlogie,B.,Lu,Z.Y.,Rossi,J.F.,Lavabre-Bertrand,T.,Beck,T.,Wijdenes,J.,Brochier,J.and Klein,B.Biologic effects of anti-interleukin-6 murine monoclonal antibody in advanced multiple myeloma.Blood,1995,86:685-91.
[42]Nilsson,K.,Bennich,H.,Johansson,S.G.and Ponten,J.Established immunoglobulin producing myeloma (IgE) and lymphoblastoid (IgG) cell lines from an IgE myeloma patient.Clin Exp Immunol,1970,7:477-89.
[43]Gerlo,S.,Haegeman,G.and Vanden Berghe,W.Transcriptional regulation of autocrine IL-6 expression in multiple myeloma cells.Cell Signal,2008,20:1489-96.
[44]Jourdan,M.,Mahtouk,K.,Veyrune,J.L.,Couderc,G.,Fiol,G.,Redal,N.,Duperray,C,De Vos,J.and Klein,B.Delineation of the roles of paracrine and autocrine interleukin-6 (IL-6) in myeloma cell lines in survival versus cell cycle.A possible model for the cooperation of myeloma cell growth factors.Eur Cytokine Netw,2005,16:57-64.
[45]Bharti,A.C.,Shishodia,S.,Reuben,J.M.,Weber,D.,Alexanian,R.,Raj-Vadhan,S.,Estrov,Z.,Talpaz,M.and Aggarwal,B.B.Nuclear factor-kappaB and STAT3 are constitutively active in CD 13 8+ cells derived from multiple myeloma patients,and suppression of these transcription factors leads to apoptosis.Blood,2004,103:3175-84.
[46]Chatterjee,M.,Stuhmer,T.,Herrmann,P.,Bommert,K.,Dorken,B.and Bargou, R.C.Combined disruption of both the MEK/ERK and the IL-6R/STAT3 pathways is required to induce apoptosis of multiple myeloma cells in the presence of bone marrow stromal cells.Blood,2004,104:3712-21.
[47]Osaki,M.,Oshimura,M.and Ito,H.PI3K-Akt pathway:its functions and alterations in human cancer.Apoptosis,2004,9,:667-76.
[48]Jiang,B.H.and Liu,L.Z.PI3K/PTEN signaling in tumorigenesis and angiogenesis.Biochim Biophys Acta,2008,1784:150-8.
[49]Dillon,R.L.,White,D.E.and Muller,W.J.The phosphatidyl inositol 3-kinase signaling network:implications for human breast cancer.Oncogene,2007,26:1338-45.
[50]West,K.A.,Castillo,S.S.and Dennis,P.A.Activation of the PI3K/Akt pathway and chemotherapeutic resistance.Drug Resist Updat,2002,5:234-48.
[51]Pene,F.,Claessens,Y.E.,Muller,O.,Viguie,F.,Mayeux,P.,Dreyfus,F.,Lacombe,C.and Bouscary,D.Role of the phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma.Oncogene,2002,21,6587-97.
[52]Hideshima,T.,Nakamura,N.,Chauhan,D.and Anderson,K.C.Biologic sequelae of interleukin-6 induced PI3-K/Akt signaling in multiple myeloma.Oncogene,2001,20:5991-6000.
[53]Younes,H.,Leleu,X.,Hatjiharissi,E.,Moreau,A.S.,Hideshima,T.,Richardson,P.,Anderson,K.C.and Ghobrial,I.M.Targeting the phosphatidylinositol 3-kinase pathway in multiple myeloma.Clin Cancer Res,2007,13:3771-5.
[54]Ross,D.D.Novel mechanisms of drug resistance in leukemia.Leukemia,2000,14:467-73.
[55]Legrand,O.,Zittoun,R.and Marie,J.P.Role of MRPl in multidrug resistance in acute myeloid leukemia.Leukemia,1999,13:578-84.
[56]Legrand,O.,Simonin,G.,Zittoun,R.and Marie,J.P.Lung resistance protein (LRP) gene expression in adult acute myeloid leukemia:a critical evaluation by three techniques.Leukemia,1998?12:1367-74.
[57]Meschini,S.,Marra,M,Calcabrini,A.,Monti,E.,Gariboldi,M.,Dolfini,E.and Arancia,G.Role of the lung resistance-related protein (LRP) in the drug sensitivity of cultured tumor cells.Toxicol In Vitro,2002,16:389-98.
[58]Ye,Z.and Song,H.Glutathione s-transferase polymorphisms (GSTM1,GSTP1 and GSTT1) and the risk of acute leukaemia:a systematic review and meta-analysis.Eur J Cancer,2005,41:980-9.
[59]Baines,P.,Cumber,P.and Padua,R.A.Multidrug resistance in leukaemia.Baillieres Clin Haematol,1992,5:943-60.
[60]Nuessler,V.,Stotzer,O.,Gullis,E.,Pelka-Fleischer,R.,Pogrebniak,A.,Gieseler,F.and Wilmanns,W.Bcl-2,bax and bcl-xL expression in human sensitive and resistant leukemia cell lines.Leukemia,1999,13:1864-72.
[61]Gieseler,F.,Glasmacher,A.,Kampfe,D.,Wandt,H.,Nuessler,V.,Valsamas,S.,Kunze,J.and Wilms,K.Topoisomerase Ⅱ activities in AML blasts and their correlation with cellular sensitivity to anthracyclines and epipodophyllotoxines.Leukemia,1996,10 Suppl 3:S46-S49.
[62]Margison,G.P.and Santibanez-Koref,M.F.O6-alkylguanine-DNA alkyltransferase:role in carcinogenesis and chemotherapy.Bioessays,2002,24:255-66.
[63]Dong,Q.H.,Zheng,S.,Xu,R.Z.,Lu,Q.and He,L.[Study on effect of berbamine on multidrug resistance leukemia K562/Adr cells].Zhongguo Zhong Xi Yi Jie He Za Zhi,2004,24:820-2.
[64]Luan,F.J.[Establishment of the multidrug-resistant cell line K562/A02 and its drug-resistant properties].Zhonghua Zhong Liu Za Zhi,1993,15:101-3.
[65]Yang,C.Z.,Luan,F.J.,Xiong,D.S.,Liu,B.R.,Xu,Y.F.and Gu,K.S.Multidrug resistance in leukemic cell line K562/A02 induced by doxorubicin.Zhongguo Yao Li Xue Bao,1995,16:333-7.
[66]Juliano,R.L.and Ling,V.A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants.Biochim Biophys Acta,1976,455:152-62.
[67]Han,Y.Q.,Yuan,J.Y.,Shi,Y.J.,Zhu,Y.and Wu,S.L.[Reversal effect of berbamine on multidrug resistance of K562/A02 cells and its mechanism].Zhongguo Shi YanXue YeXue Za Zhi,2003,11:604-8.
[68]Li,B.Y.,Fu,B.,Zhao,Y.L.and Li,W.H.Effects of berbamine on intracellular calcium concentration in cultured HeLa cells.Zhongguo Yao Li Xue Bao,1999,20:1011-4.
[69]Li,B.Y.,Qiao,G.F.,Zhao,Y.L.,Zhou,H.and Li,W.H.Effects of berbamine on ATP-induced [Ca2+]i mobilization in cultured vascular smooth muscle cells and cardiomyocytes.Zhongguo Yao Li Xue Bao,1999,20:705-8.
[70]Tsuruo,T.,Iida,H.,Tsukagoshi,S.and Sakurai,Y.Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil.Cancer Res,1981,41:1967-72.
[71]Viale,M.,Cordazzo,C,Cosimelli,B.,de Totero,D.,Castagnola,P.,Aiello,C,Severi,E.,Petrillo,G.,Cianfriglia,M.and Spinelli,D.Inhibition of MDR1 activity in vitro by a novel class of diltiazem analogues:toward new candidates.J MedChem,2009,52:259-66.
[72]Xu,Y.H.and Zhang,S.P.A derivative of bisbenzylisoquinoline alkaloid is a new and potential calmodulin antagonist.Biochem Biophys Res Commun,1986,140:461-7.
[73]Johnson,C.K.Calmodulin,conformational states,and calcium signaling.A single-molecule perspective.Biochemistry,2006,45:14233-46.
[74]Ren,Y.,Lu,L.,Guo,T.B.,Qiu,J.,Yang,Y.,Liu,A.and Zhang,J.Z.Novel immunomodulatory properties of berbamine through selective down-regulation of STAT4 and action of IFN-gamma in experimental autoimmune encephalomyelitis．JImmunol， 2008，181：1491-8．
[75]罗崇念，林新，王立为，赵扬，谢蜀生，肖培根．小檗胺对小鼠脾细胞毒性T 淋巴细胞活性的抑制作用．中国药理学与毒理学杂志， 1995，9： 159-160．
[76]Kupeli， E．， Kosar， M．， Yesilada， E．， Husnu， K．and Baser， C．A comparative study on the anti-inflammatory， antinociceptive and antipyretic effects of isoquinoline alkaloids from the roots of Turkish Berberis species．Life Sci， 2002，72： 645-57．
[77]Cao， L．， Luo， C．， Bian， Q．and Xiao， P．Anti-inflammatory activity of berbamine．Jounal of Cinese pharmaceutical sciences， 1999，8： 93-94．
[78]Zhao， X．Y．， He， Z．W．， Wu， D．and Xu， R．Z．Berbamine selectively induces apoptosis of human acute promyelocytic leukemia cells via survivin-mediated pathway．Chin Med J (Engl)， 2007，120： 802-6．
[79]Wu， D．， Lin， M．F．and Zhao， X．Y．[Effects of berbamine on K562 cells and its mechanisms in vitro and in vivo]．Zhongguo Shi Yan Xue Ye Xue Za Zhi， 2005，13： 373-8．
[80]黄志煌，董庆华．小檗胺诱导人白血病Jurkat细胞凋亡的实验研究．中国肿瘤， 2007，16： 722-724．
[81]Xu， R．， Dong， Q．， Yu， Y．， Zhao， X．， Gan， X．， Wu， D．， Lu， Q．， Xu， X．and Yu， X．F Berbamine： a novel inhibitor of bcr／abl fusion gene with potent anti-leukemia activity．Leuk Res， 2006，30： 17-23．
[82]Sun， J．R．， Zhang， X．H．， He， Z．W．， Gu， Y．， Yu， Y．Z．， Fang， Y．M．， Lu， Q．H．， Dong，Q．H．and Xu， R．Z．[The mechanism of apoptosis of chronic myeloid leukemia cells induced by the novel p210 bcr／abl inhibitor berbamine]．Zhonghua Yi Xue Za Zhi， 2006，86： 2246-51．
[83]Wei， Y．L．， Xu， L．， Liang， Y．， Xu， X．H．and Zhao， X．Y．Berbamine exhibits potent antitumor effects on imatinib-resistant CML cells in vitro and in vivo．Acta Pharmacol Sin．2009
[84]孙鹏，吕家驹，赵勇．小檗胺诱导前列腺癌PC-3细胞凋亡及其机制．中华实验外科杂志，2007，24： 957-959．
[85]郭治彬，曹宏宇，徐智，李青．小檗胺对室性心律失常的防治作用．中华心血管杂志，1993，21： 110-111．
[86]Zhao， Y．， Li， X．and Li， B．Effect of Berbamine on Calcium Dynamics in Cultured Vascular Smooth Muscle Cells．Journal of Chinese Pharmaceutical Sciences， 2000，9： 158-160．
[87]He， H．， He， H．-Y．， Zhang， J．-P．and Zhang， C．-Y．Effect of berbamine on DNA damage／repa it and cataractogenes is of diabetic cataract induced by STZ．Chinese Journal of Biochemistry and Molecular Biology， 1998，14： 300-303．
[88]He， H．， Sun， X．， Zhang， J．and Zhang， C．The effect of berbamine eye-drop on prevention and therapeutic of diabetic cataract．Journal of Beijing Medical University，1997，29： 326-328．
[89]周虹，王玲，高云瑞，姜玉泉，王建柱．盐酸小檗胺对实验性脑缺血保护作用的研究．中医药学报，1998，1： 60-61．
[90]周虹，王玲，郝晓敏，高云瑞，郝晓峰．小檗胺对大鼠脑缺血后自由基的影响中国中药杂志，1998，23： 307．
[91]Bao， L．H．， Luo， D．L．， Yang， B．F．， He， S．Z．and Li， W．H．Effects of Berbamine on PAF Production in Human Neutrophils and on Platelet Aggregation．Journal of Chinese Pharmaceutical Sciences， 1997，6： 37-40．
[92]He， Q．Y．， Meng， F．H．and Zhang， H．Q．Reduction of doxorubicin resistance by tetrandrine and dauricine in harringtonine-resistant human leukemia (HL60， cells．Zhongguo Yao Li Xue Bao， 1996，17： 179-81．
[93]Lai， Y．L．， Chen， Y．J．， Wu， T．Y．， Wang， S．Y．， Chang， K．H．， Chung， C．H．and Chen， M．L．Induction of apoptosis in human leukemic U937 cells by tetrandrine．Anticancer Drugs， 1998，9： 77-81．
[94]Chen， B．A．， Qian， X．J．， Cheng， J．and Gao， F．[Effect of tetrandrine combined with Droloxifen on the expression of bcr/abl of K562 at both mRNA and protein levels].Zhongguo Shi YanXue YeXue Za Zhi,2005?13:95-9.
[95]Wang,J.Q.,Chen,B.A.,Cheng,J.,Xu,W.L.and Sun,X.C.[Comparison of reversal effects of 5-bromotetrandrine and tetrandrine on P-glycoproteindependent Resistance to adriamycin in human lukemia cell line K562/A02].Ai Zheng,2008,27:491-5.
[96]Liu,Z.L.,Hirano,T.,Tanaka,S.,Onda,K.and Oka,K.Persistent reversal of P-glycoprotein-mediated daunorubicin resistance by tetrandrine in multidrug-resistant human T lymphoblastoid leukemia MOLT-4 cells.J Pharm Pharmacol,2003,55:1531-7.
[97]Xu,W.L.,Shen,H.L.,Ao,Z.F.,Chen,B.A.,Xia,W.,Gao,F.and Zhang,Y.N.Combination of tetrandrine as a potential-reversing agent with daunorubicin,etoposide and cytarabine for the treatment of refractory and relapsed acute myelogenous leukemia.LeukRes,2006,30:407-13.
[98]Jang,B.C.,Lim,K.J.,Paik,J.H.,Cho,J.W.,Baek,W.K.,Suh,M.H.,Park,J.B.,Kwon,T.K.,Park,J.W.,Kim,S.P.,Shin,D.H.,Song,D.K.,Bae,J.H.,Mun,K.C.and Suh,S.I.Tetrandrine-induced apoptosis is mediated by activation of caspases and PKC-delta in U937 cells.Biochem Pharmacol,2004,67:1819-29.
[99]Chen,B.A.,Su,A.L.,Cheng,J.,Zhao,H.H.,Li,G.H.and Wang,X.M.[Effects of tetrandrine on nuclear factor-kappaB expression in leukemia multidrugresistant cell line K562/A02].ZhongXi Yi Jie He Xue Bao,2008,6:956-9.
[100]Ng,L.T.,Chiang,L.C.,Lin,Y.T.and Lin,C.C.Antiproliferative and apoptotic effects of tetrandrine on different human hepatoma cell lines.Am J Chin Med,2006,34:125-35.
[101]Liu,B.,Wang,T.,Qian,X.,Liu,G.,Yu,L.and Ding,Y.Anticancer effect of tetrandrine on primary cancer cells isolated from ascites and pleural fluids.Cancer Lett,2008,268:166-75.
[102]Chen， Y．， Chen， J．C．and Tseng， S．H．Tetrandrine suppresses tumor growth and angiogenesis of gliomas in rats．Int J Cancer．2008．
[103]Zhu， X．， Sui， M．and Fan， W．In vitro and in vivo characterizations of tetrandrine on the reversal of P-glycoprotein-mediated drug resistance to paclitaxel．Anticancer Res， 2005， 25： 1953-62．
[104]McCubrey， J．A．， Basecke， J．， Cervello， M．， Martelli， A．M．and Franklin， R．A．GSK-3beta is a critical mediator of tetrandrine induced cell cycle arrest and cytotoxicity．Cancer Biol Ther， 2008，7： 1079．
[105]Cho， H．S．， Chang， S．H．， Chung， Y．S．， Shin， J．Y．， Park， S．J．， Lee， E．S．， Hwang， S．K．， Kwon， J．T．， Tehrani， A．M．， Woo， M．， Noh， M．S．， Hanifah， H．， Jin， H．， Xu， C．X．and Cho， M．H．Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells．J Vet Sci， 2009，10： 23-8．
[106]韩冬云，聂宏光，陈磊，宋志国，李金鸣．N-甲基小檗胺对大鼠肠系膜动脉平滑肌细胞钙激活钾电流的作用．中国药理学通报， 2006，22： 488-491．
[107]Zheng， G．H．， Chen， Z．L．and Jiang， J．H．[Effects of O-(4-ethoxyl-butyl)-berbamine on isolated frog hearts]．Zhongguo Yao Li Xue Bao， 1998，19： 497-500．
[108]Liu， J．， Qi， S．， Zhu， H．， Zhang， J．， Li， Z．and Wang， T．The effect of calmodulin antagonist berbaminederivative-EBB on hepatoma in vitro and in vivo．Chin Med J (Engl)， 2002，115： 759-62．
[109]张金红，许乃寒，徐畅，陈家童，刘惠，段江燕．小檗胺衍生物(EBB)体外抑制肺癌细胞增殖机制的初探．细胞生物学杂志， 2001， 23： 218-222．
[110]Fang， B．J．， Yu， M．L．， Yang， S．G．， Liao， L．M．， Liu， J．W．and Zhao， R．C．Effect of O-4-ethoxyl-butyl-berbamine in combination with pegylated liposomal doxorubicin on advanced hepatoma in mice．World J Gastroenterol， 2004，10：950-3．
[111]Cheng， Y．H．， Qi， J．， Xiong， D．S．， Liu， J．W．， Qi， S．L．， Pan， B．， Yang， C．Z．and Zhu， H．F．[Reversal of multidrug resistance in drug-resistant human breast cancer cell line MCF-7／ADR by calmodulin antagonist O-(4-ethoxyl-butyl)-berbamine]．Zhongguo Yi Xue Ke Xue Yuan Xue Bao， 2006，28： 164-8．
[112]Pan， B．， Zhou， Y．， Qi， J．， Xiong， D．S．， Liu， J．W．， Qi， S．L．， Cheng， Y．H．， Yang， C．Z．and Zhu， H．F．[Effect of calmodulin antagonist EBB on invasion of human fibrosarcoma cell HT1080]．Zhongguo Yi Xue Ke Xue Yuan Xue Bao， 2005，27：311-4．
[113]贡岳松，胡卓逸，沈子龙．四种小檗胺衍生物对钙调素诱导的肌球蛋白轻链激酶的抑制作用．中国药科大学学报， 1991，22：93-96．
[2]Teh,B.S.,Seow,W.K.,Li,S.Y.and Thong,Y.H.Inhibition of prostaglandin and leukotriene generation by the plant alkaloids tetrandrine and berbamine.Int J Immunopharmacol,1990,12:321-6.
[3]Zhang,W.,Chen,S.G.,Ju,H.S.,Zhao,S.H.,Zou,CM.,Hao,J.M.and Liu,Y.Mechanisms of protective effects of berbamine on ischemia/reperfusion injury in isolated rat heart.Methods Find Exp Clin Pharmacol,1992,14:677-84.
[4]Wang,G.Y.,Zhang,J.W.,Lu,Q.H.,Xu,R.Z.and Dong,Q.H.Berbamine induces apoptosis in human hepatoma cell line SMMC7721 by loss in mitochondrial transmembrane potential and caspase activation.J Zhejiang Unix 50/5,2007,8:248-55.
[5]Blade,J.,Kyle,R.A.and Greipp,P.R.Multiple myeloma in patients younger than 30 years.Report of 10 cases and review of the literature.Arch Intern Med,1996,156:1463-8.
[6]Blade,J.and Kyle,R.A.Multiple myeloma in young patients:clinical presentation and treatment approach.Leuk Lymphoma,1998,30:493-501.
[7]Kumar,S.K.,Rajkumar,S.V.,Dispenzieri,A.,Lacy,M.Q.,Hayman,S.R.,Buadi,F.K.,Zeldenrust,S.R.,Dingli,D.,Russell,S.J.,Lust,J.A.,Greipp,P.R.,Kyle,R.A.and Gertz,M.A.Improved survival in multiple myeloma and the impact of novel therapies.Blood,2008,111:2516-20.
[8]Edinger,A.L.and Thompson,C.B.Death by design:apoptosis,necrosis and autophagy.Curr Opin Cell Biol,2004,16:663-9.
[9]Elmore,S.Apoptosis:a review of programmed cell death.Toxicol Pathol,2007,35:495-516.
[10]Ghobrial,I.M.,Witzig,T.E.and Adjei,A.A.Targeting apoptosis pathways in cancer therapy.CA Cancer J Clin,2005,55:178-94.
[11]Youle,R.J.and Strasser,A.The BCL-2 protein family:opposing activities that mediate cell death.Nat Rev Mol Cell Biol,2008,9:47-59.
[12]Ruvolo,P.P.,Deng,X.and May,W.S.Phosphorylation of Bcl2 and regulation of apoptosis.Leukemia,2001,15:515-22.
[13]Haldar,S.,Jena,N.and Croce,CM.Inactivation of Bcl-2 by phosphorylation.Proc Natl Acad Sci USA,1995,92:4507-11.
[14]Thomas,A.,Giesler,T.and White,E.p53 mediates bcl-2 phosphorylation and apoptosis via activation of the Cdc42/JNK1 pathway.Oncogene,2000,19:5259-69.
[15]Ezhevsky,S.A.,Nagahara,H.,Vocero-Akbani,A.M.,Gius,D.R.,Wei,M.C.and Dowdy,S.F.Hypo-phosphorylation of the retinoblastoma protein (pRb) by cyclin D:Cdk4/6 complexes results in active pRb.Proc Natl Acad Sci USA,1997,94:10699-704.
[16]Morris,L.,Allen,K.E.and La Thangue,N.B.Regulation of E2F transcription by cyclin E-Cdk2 kinase mediated through p300/CBP co-activators.Nat Cell Biol,2000,2:232-9.
[17]Bashir,T.,Horlein,R.,Rommelaere,J.and Willwand,K.Cyclin A activates the DNA polymerase delta-dependent elongation machinery in vitro:A parvovirus DNA replication model.Proc Natl Acad Sci USA,2000,97:5522-7.
[18]Mailer,J.L.Mitotic control.Curr Opin Cell Biol,1991,3:269-75.
[19]Mitra,J.and Enders,G.H.Cyclin A/Cdk2 complexes regulate activation of Cdk1 and Cdc25 phosphatases in human cells.Oncogene,2004,23:3361-7.
[20]Pagano,M.,Pepperkok,R.,Verde,F.,Ansorge,W.and Draetta,G.Cyclin A is required at two points in the human cell cycle.Embo J,1992,11:961-71.
[21]Furuno,N.,den Elzen,N.and Pines,J.Human cyclin A is required for mitosis until mid prophase.J Cell Biol,1999,147:295-306.
[22]Miyazaki,T.and Arai,S.Two distinct controls of mitotic cdkl/cyclin Bl activity requisite for cell growth prior to cell division.Cell Cycle,2007,6:1419-25.
[23]Ohi,R.and Gould,K.L.Regulating the onset of mitosis.Curr Opin Cell Biol,1999,11:267-73.
[24]Fletcher,J.I.and Huang,D.C.Controlling the cell death mediators Bax and Bak:puzzles and conundrums.Cell Cycle,2008,7:39-44.
[25]Gomez-Bougie,P.,Oliver,L.,Le Gouill,S.,Bataille,R.and Amiot,M.Melphalan-induced apoptosis in multiple myeloma cells is associated with a cleavage of Mcl-1 and Bim and a decrease in the Mcl-1/Bim complex.Oncogene,2005,24:8076-9.
[26]Gomez-Benito,M.,Balsas,P.,Carvajal-Vergara,X.,Pandiella,A.,Anel,A.,Marzo,I.and Naval,J.Mechanism of apoptosis induced by IFN-alpha in human myeloma cells:role of Jakl and Bim and potentiation by rapamycin.Cell Signal,2007,19:844-54.
[27]Morales,A.A.,Gutman,D.,Lee,K.P.and Boise,L.H.BH3-only proteins Noxa,Bmf,and Bim are necessary for arsenic trioxide-induced cell death in myeloma.Blood,2008,111:5152-62.
[28]Han,J.,Goldstein,L.A.,Gastman,B.R.,Rabinovitz,A.and Rabinowich,H.Disruption of Mcl-1.Bim complex in granzyme B-mediated mitochondrial apoptosis.J Biol Chem,2005,280:16383-92.
[29]Ley,R.,Balmanno,K.,Hadfield,K.,Weston,C.and Cook,S.J.Activation of the ERK1/2 signaling pathway promotes phosphorylation and proteasome-dependent degradation of the BH3-only protein,Bim.J Biol Chem,2003,278:18811-6.
[30]Sunters,A.,Fernandez de Mattos,S.,Stahl,M.,Brosens,J.J.,Zoumpoulidou,G.,Saunders,C.A.,Coffer,P.J.,Medema,R.H.,Coombes,R.C.and Lam,E.W.Fox03a transcriptional regulation of Bim controls apoptosis in paclitaxel-treated breast cancer cell lines.J Biol Chem,2003,278:49795-805.
[31]Stan,S.D.,Hahm,E.R.,Warin,R.and Singh,S.V.Withaferin A causes FOX03a-and Bim-dependent apoptosis and inhibits growth of human breast cancer cells in vivo.Cancer Res,2008,68:7661-9.
[32]Yang,J.Y.,Zong,C.S.,Xia,W.,Yamaguchi,H.,Ding,Q.,Xie,X.,Lang,J.Y.,Lai,C.C.,Chang,C.J.,Huang,W.C.,Huang,H.,Kuo,H.P.,Lee,D.F.,Li,L.Y.,Lien,H.C.,Cheng,X.,Chang,K.J.,Hsiao,CD.,Tsai,F.J.,Tsai,C.H.,Sahin,A.A.,Muller,W.J.,Mills,G.B.,Yu,D.,Hortobagyi,G.N.and Hung,M.C.ERK promotes tumorigenesis by inhibiting FOX03a via MDM2-mediated degradation.Nat Cell Biol,2008,10:138-48.
[33]Hussain,A.R.,Al-Rasheed,M.,Manogaran,P.S.,Al-Hussein,K.A.,Platanias,L.C.,Al Kuraya,K.and Uddin,S.Curcumin induces apoptosis via inhibition of PI3'-kinase/AKT pathway in acute T cell leukemias.Apoptosis,2006,11:245-54.
[34]Yamamura,Y.,Lee,W.L.,Inoue,K.,Ida,H.and Ito,Y.RUNX3 cooperates with Fox03a to induce apoptosis in gastric cancer cells.J Biol Chem,2006,281:5267-76.
[35]Wu,W.,Sun,M.,Zou,G.M.and Chen,J.MicroRNA and cancer:Current status and prospective.Int J Cancer,2007,120:953-60.
[36]Nelson,K.M.and Weiss,G.J.MicroRNAs and cancer:past,present,and potential future.Mol Cancer Ther,2008,7:3655-60.
[37]Fontana,L.,Fiori,M.E.,Albini,S.,Cifaldi,L.,Giovinazzi,S.,Forloni,M.,Boldrini,R.,Donfrancesco,A.,Federici,V.,Giacomini,P.,Peschle,C.and Fruci,D.Antagomir-17-5p abolishes the growth of therapy-resistant neuroblastoma through p21 and BIM.PLoS ONE,2008,3:e2236.
[38]Nilsson,M.B.,Langley,R.R.and Fidler,I.J.Interleukin-6,secreted by human ovarian carcinoma cells,is a potent proangiogenic cytokine.Cancer Res,2005,65:10794-800.
[39]Hirano,T.Interleukin 6 and its receptor:ten years later.Int Rev Immunol,1998,16:249-84.
[40]Klein,B.,Zhang,X.G.,Jourdan,M.,Boiron,J.M.,Portier,M.,Lu,Z.Y.,Wijdenes,J.,Brochier,J.and Bataille,R.Interleukin-6 is the central tumor growth factor in vitro and in vivo in multiple myeloma.Eur Cytokine Netw,1990,1:193-201.
[41]Bataille,R.,Barlogie,B.,Lu,Z.Y.,Rossi,J.F.,Lavabre-Bertrand,T.,Beck,T.,Wijdenes,J.,Brochier,J.and Klein,B.Biologic effects of anti-interleukin-6 murine monoclonal antibody in advanced multiple myeloma.Blood,1995,86:685-91.
[42]Nilsson,K.,Bennich,H.,Johansson,S.G.and Ponten,J.Established immunoglobulin producing myeloma (IgE) and lymphoblastoid (IgG) cell lines from an IgE myeloma patient.Clin Exp Immunol,1970,7:477-89.
[43]Gerlo,S.,Haegeman,G.and Vanden Berghe,W.Transcriptional regulation of autocrine IL-6 expression in multiple myeloma cells.Cell Signal,2008,20:1489-96.
[44]Jourdan,M.,Mahtouk,K.,Veyrune,J.L.,Couderc,G.,Fiol,G.,Redal,N.,Duperray,C,De Vos,J.and Klein,B.Delineation of the roles of paracrine and autocrine interleukin-6 (IL-6) in myeloma cell lines in survival versus cell cycle.A possible model for the cooperation of myeloma cell growth factors.Eur Cytokine Netw,2005,16:57-64.
[45]Bharti,A.C.,Shishodia,S.,Reuben,J.M.,Weber,D.,Alexanian,R.,Raj-Vadhan,S.,Estrov,Z.,Talpaz,M.and Aggarwal,B.B.Nuclear factor-kappaB and STAT3 are constitutively active in CD 13 8+ cells derived from multiple myeloma patients,and suppression of these transcription factors leads to apoptosis.Blood,2004,103:3175-84.
[46]Chatterjee,M.,Stuhmer,T.,Herrmann,P.,Bommert,K.,Dorken,B.and Bargou, R.C.Combined disruption of both the MEK/ERK and the IL-6R/STAT3 pathways is required to induce apoptosis of multiple myeloma cells in the presence of bone marrow stromal cells.Blood,2004,104:3712-21.
[47]Osaki,M.,Oshimura,M.and Ito,H.PI3K-Akt pathway:its functions and alterations in human cancer.Apoptosis,2004,9,:667-76.
[48]Jiang,B.H.and Liu,L.Z.PI3K/PTEN signaling in tumorigenesis and angiogenesis.Biochim Biophys Acta,2008,1784:150-8.
[49]Dillon,R.L.,White,D.E.and Muller,W.J.The phosphatidyl inositol 3-kinase signaling network:implications for human breast cancer.Oncogene,2007,26:1338-45.
[50]West,K.A.,Castillo,S.S.and Dennis,P.A.Activation of the PI3K/Akt pathway and chemotherapeutic resistance.Drug Resist Updat,2002,5:234-48.
[51]Pene,F.,Claessens,Y.E.,Muller,O.,Viguie,F.,Mayeux,P.,Dreyfus,F.,Lacombe,C.and Bouscary,D.Role of the phosphatidylinositol 3-kinase/Akt and mTOR/P70S6-kinase pathways in the proliferation and apoptosis in multiple myeloma.Oncogene,2002,21,6587-97.
[52]Hideshima,T.,Nakamura,N.,Chauhan,D.and Anderson,K.C.Biologic sequelae of interleukin-6 induced PI3-K/Akt signaling in multiple myeloma.Oncogene,2001,20:5991-6000.
[53]Younes,H.,Leleu,X.,Hatjiharissi,E.,Moreau,A.S.,Hideshima,T.,Richardson,P.,Anderson,K.C.and Ghobrial,I.M.Targeting the phosphatidylinositol 3-kinase pathway in multiple myeloma.Clin Cancer Res,2007,13:3771-5.
[54]Ross,D.D.Novel mechanisms of drug resistance in leukemia.Leukemia,2000,14:467-73.
[55]Legrand,O.,Zittoun,R.and Marie,J.P.Role of MRPl in multidrug resistance in acute myeloid leukemia.Leukemia,1999,13:578-84.
[56]Legrand,O.,Simonin,G.,Zittoun,R.and Marie,J.P.Lung resistance protein (LRP) gene expression in adult acute myeloid leukemia:a critical evaluation by three techniques.Leukemia,1998?12:1367-74.
[57]Meschini,S.,Marra,M,Calcabrini,A.,Monti,E.,Gariboldi,M.,Dolfini,E.and Arancia,G.Role of the lung resistance-related protein (LRP) in the drug sensitivity of cultured tumor cells.Toxicol In Vitro,2002,16:389-98.
[58]Ye,Z.and Song,H.Glutathione s-transferase polymorphisms (GSTM1,GSTP1 and GSTT1) and the risk of acute leukaemia:a systematic review and meta-analysis.Eur J Cancer,2005,41:980-9.
[59]Baines,P.,Cumber,P.and Padua,R.A.Multidrug resistance in leukaemia.Baillieres Clin Haematol,1992,5:943-60.
[60]Nuessler,V.,Stotzer,O.,Gullis,E.,Pelka-Fleischer,R.,Pogrebniak,A.,Gieseler,F.and Wilmanns,W.Bcl-2,bax and bcl-xL expression in human sensitive and resistant leukemia cell lines.Leukemia,1999,13:1864-72.
[61]Gieseler,F.,Glasmacher,A.,Kampfe,D.,Wandt,H.,Nuessler,V.,Valsamas,S.,Kunze,J.and Wilms,K.Topoisomerase Ⅱ activities in AML blasts and their correlation with cellular sensitivity to anthracyclines and epipodophyllotoxines.Leukemia,1996,10 Suppl 3:S46-S49.
[62]Margison,G.P.and Santibanez-Koref,M.F.O6-alkylguanine-DNA alkyltransferase:role in carcinogenesis and chemotherapy.Bioessays,2002,24:255-66.
[63]Dong,Q.H.,Zheng,S.,Xu,R.Z.,Lu,Q.and He,L.[Study on effect of berbamine on multidrug resistance leukemia K562/Adr cells].Zhongguo Zhong Xi Yi Jie He Za Zhi,2004,24:820-2.
[64]Luan,F.J.[Establishment of the multidrug-resistant cell line K562/A02 and its drug-resistant properties].Zhonghua Zhong Liu Za Zhi,1993,15:101-3.
[65]Yang,C.Z.,Luan,F.J.,Xiong,D.S.,Liu,B.R.,Xu,Y.F.and Gu,K.S.Multidrug resistance in leukemic cell line K562/A02 induced by doxorubicin.Zhongguo Yao Li Xue Bao,1995,16:333-7.
[66]Juliano,R.L.and Ling,V.A surface glycoprotein modulating drug permeability in Chinese hamster ovary cell mutants.Biochim Biophys Acta,1976,455:152-62.
[67]Han,Y.Q.,Yuan,J.Y.,Shi,Y.J.,Zhu,Y.and Wu,S.L.[Reversal effect of berbamine on multidrug resistance of K562/A02 cells and its mechanism].Zhongguo Shi YanXue YeXue Za Zhi,2003,11:604-8.
[68]Li,B.Y.,Fu,B.,Zhao,Y.L.and Li,W.H.Effects of berbamine on intracellular calcium concentration in cultured HeLa cells.Zhongguo Yao Li Xue Bao,1999,20:1011-4.
[69]Li,B.Y.,Qiao,G.F.,Zhao,Y.L.,Zhou,H.and Li,W.H.Effects of berbamine on ATP-induced [Ca2+]i mobilization in cultured vascular smooth muscle cells and cardiomyocytes.Zhongguo Yao Li Xue Bao,1999,20:705-8.
[70]Tsuruo,T.,Iida,H.,Tsukagoshi,S.and Sakurai,Y.Overcoming of vincristine resistance in P388 leukemia in vivo and in vitro through enhanced cytotoxicity of vincristine and vinblastine by verapamil.Cancer Res,1981,41:1967-72.
[71]Viale,M.,Cordazzo,C,Cosimelli,B.,de Totero,D.,Castagnola,P.,Aiello,C,Severi,E.,Petrillo,G.,Cianfriglia,M.and Spinelli,D.Inhibition of MDR1 activity in vitro by a novel class of diltiazem analogues:toward new candidates.J MedChem,2009,52:259-66.
[72]Xu,Y.H.and Zhang,S.P.A derivative of bisbenzylisoquinoline alkaloid is a new and potential calmodulin antagonist.Biochem Biophys Res Commun,1986,140:461-7.
[73]Johnson,C.K.Calmodulin,conformational states,and calcium signaling.A single-molecule perspective.Biochemistry,2006,45:14233-46.
[74]Ren,Y.,Lu,L.,Guo,T.B.,Qiu,J.,Yang,Y.,Liu,A.and Zhang,J.Z.Novel immunomodulatory properties of berbamine through selective down-regulation of STAT4 and action of IFN-gamma in experimental autoimmune encephalomyelitis．JImmunol， 2008，181：1491-8．
[75]罗崇念，林新，王立为，赵扬，谢蜀生，肖培根．小檗胺对小鼠脾细胞毒性T 淋巴细胞活性的抑制作用．中国药理学与毒理学杂志， 1995，9： 159-160．
[76]Kupeli， E．， Kosar， M．， Yesilada， E．， Husnu， K．and Baser， C．A comparative study on the anti-inflammatory， antinociceptive and antipyretic effects of isoquinoline alkaloids from the roots of Turkish Berberis species．Life Sci， 2002，72： 645-57．
[77]Cao， L．， Luo， C．， Bian， Q．and Xiao， P．Anti-inflammatory activity of berbamine．Jounal of Cinese pharmaceutical sciences， 1999，8： 93-94．
[78]Zhao， X．Y．， He， Z．W．， Wu， D．and Xu， R．Z．Berbamine selectively induces apoptosis of human acute promyelocytic leukemia cells via survivin-mediated pathway．Chin Med J (Engl)， 2007，120： 802-6．
[79]Wu， D．， Lin， M．F．and Zhao， X．Y．[Effects of berbamine on K562 cells and its mechanisms in vitro and in vivo]．Zhongguo Shi Yan Xue Ye Xue Za Zhi， 2005，13： 373-8．
[80]黄志煌，董庆华．小檗胺诱导人白血病Jurkat细胞凋亡的实验研究．中国肿瘤， 2007，16： 722-724．
[81]Xu， R．， Dong， Q．， Yu， Y．， Zhao， X．， Gan， X．， Wu， D．， Lu， Q．， Xu， X．and Yu， X．F Berbamine： a novel inhibitor of bcr／abl fusion gene with potent anti-leukemia activity．Leuk Res， 2006，30： 17-23．
[82]Sun， J．R．， Zhang， X．H．， He， Z．W．， Gu， Y．， Yu， Y．Z．， Fang， Y．M．， Lu， Q．H．， Dong，Q．H．and Xu， R．Z．[The mechanism of apoptosis of chronic myeloid leukemia cells induced by the novel p210 bcr／abl inhibitor berbamine]．Zhonghua Yi Xue Za Zhi， 2006，86： 2246-51．
[83]Wei， Y．L．， Xu， L．， Liang， Y．， Xu， X．H．and Zhao， X．Y．Berbamine exhibits potent antitumor effects on imatinib-resistant CML cells in vitro and in vivo．Acta Pharmacol Sin．2009
[84]孙鹏，吕家驹，赵勇．小檗胺诱导前列腺癌PC-3细胞凋亡及其机制．中华实验外科杂志，2007，24： 957-959．
[85]郭治彬，曹宏宇，徐智，李青．小檗胺对室性心律失常的防治作用．中华心血管杂志，1993，21： 110-111．
[86]Zhao， Y．， Li， X．and Li， B．Effect of Berbamine on Calcium Dynamics in Cultured Vascular Smooth Muscle Cells．Journal of Chinese Pharmaceutical Sciences， 2000，9： 158-160．
[87]He， H．， He， H．-Y．， Zhang， J．-P．and Zhang， C．-Y．Effect of berbamine on DNA damage／repa it and cataractogenes is of diabetic cataract induced by STZ．Chinese Journal of Biochemistry and Molecular Biology， 1998，14： 300-303．
[88]He， H．， Sun， X．， Zhang， J．and Zhang， C．The effect of berbamine eye-drop on prevention and therapeutic of diabetic cataract．Journal of Beijing Medical University，1997，29： 326-328．
[89]周虹，王玲，高云瑞，姜玉泉，王建柱．盐酸小檗胺对实验性脑缺血保护作用的研究．中医药学报，1998，1： 60-61．
[90]周虹，王玲，郝晓敏，高云瑞，郝晓峰．小檗胺对大鼠脑缺血后自由基的影响中国中药杂志，1998，23： 307．
[91]Bao， L．H．， Luo， D．L．， Yang， B．F．， He， S．Z．and Li， W．H．Effects of Berbamine on PAF Production in Human Neutrophils and on Platelet Aggregation．Journal of Chinese Pharmaceutical Sciences， 1997，6： 37-40．
[92]He， Q．Y．， Meng， F．H．and Zhang， H．Q．Reduction of doxorubicin resistance by tetrandrine and dauricine in harringtonine-resistant human leukemia (HL60， cells．Zhongguo Yao Li Xue Bao， 1996，17： 179-81．
[93]Lai， Y．L．， Chen， Y．J．， Wu， T．Y．， Wang， S．Y．， Chang， K．H．， Chung， C．H．and Chen， M．L．Induction of apoptosis in human leukemic U937 cells by tetrandrine．Anticancer Drugs， 1998，9： 77-81．
[94]Chen， B．A．， Qian， X．J．， Cheng， J．and Gao， F．[Effect of tetrandrine combined with Droloxifen on the expression of bcr/abl of K562 at both mRNA and protein levels].Zhongguo Shi YanXue YeXue Za Zhi,2005?13:95-9.
[95]Wang,J.Q.,Chen,B.A.,Cheng,J.,Xu,W.L.and Sun,X.C.[Comparison of reversal effects of 5-bromotetrandrine and tetrandrine on P-glycoproteindependent Resistance to adriamycin in human lukemia cell line K562/A02].Ai Zheng,2008,27:491-5.
[96]Liu,Z.L.,Hirano,T.,Tanaka,S.,Onda,K.and Oka,K.Persistent reversal of P-glycoprotein-mediated daunorubicin resistance by tetrandrine in multidrug-resistant human T lymphoblastoid leukemia MOLT-4 cells.J Pharm Pharmacol,2003,55:1531-7.
[97]Xu,W.L.,Shen,H.L.,Ao,Z.F.,Chen,B.A.,Xia,W.,Gao,F.and Zhang,Y.N.Combination of tetrandrine as a potential-reversing agent with daunorubicin,etoposide and cytarabine for the treatment of refractory and relapsed acute myelogenous leukemia.LeukRes,2006,30:407-13.
[98]Jang,B.C.,Lim,K.J.,Paik,J.H.,Cho,J.W.,Baek,W.K.,Suh,M.H.,Park,J.B.,Kwon,T.K.,Park,J.W.,Kim,S.P.,Shin,D.H.,Song,D.K.,Bae,J.H.,Mun,K.C.and Suh,S.I.Tetrandrine-induced apoptosis is mediated by activation of caspases and PKC-delta in U937 cells.Biochem Pharmacol,2004,67:1819-29.
[99]Chen,B.A.,Su,A.L.,Cheng,J.,Zhao,H.H.,Li,G.H.and Wang,X.M.[Effects of tetrandrine on nuclear factor-kappaB expression in leukemia multidrugresistant cell line K562/A02].ZhongXi Yi Jie He Xue Bao,2008,6:956-9.
[100]Ng,L.T.,Chiang,L.C.,Lin,Y.T.and Lin,C.C.Antiproliferative and apoptotic effects of tetrandrine on different human hepatoma cell lines.Am J Chin Med,2006,34:125-35.
[101]Liu,B.,Wang,T.,Qian,X.,Liu,G.,Yu,L.and Ding,Y.Anticancer effect of tetrandrine on primary cancer cells isolated from ascites and pleural fluids.Cancer Lett,2008,268:166-75.
[102]Chen， Y．， Chen， J．C．and Tseng， S．H．Tetrandrine suppresses tumor growth and angiogenesis of gliomas in rats．Int J Cancer．2008．
[103]Zhu， X．， Sui， M．and Fan， W．In vitro and in vivo characterizations of tetrandrine on the reversal of P-glycoprotein-mediated drug resistance to paclitaxel．Anticancer Res， 2005， 25： 1953-62．
[104]McCubrey， J．A．， Basecke， J．， Cervello， M．， Martelli， A．M．and Franklin， R．A．GSK-3beta is a critical mediator of tetrandrine induced cell cycle arrest and cytotoxicity．Cancer Biol Ther， 2008，7： 1079．
[105]Cho， H．S．， Chang， S．H．， Chung， Y．S．， Shin， J．Y．， Park， S．J．， Lee， E．S．， Hwang， S．K．， Kwon， J．T．， Tehrani， A．M．， Woo， M．， Noh， M．S．， Hanifah， H．， Jin， H．， Xu， C．X．and Cho， M．H．Synergistic effect of ERK inhibition on tetrandrine-induced apoptosis in A549 human lung carcinoma cells．J Vet Sci， 2009，10： 23-8．
[106]韩冬云，聂宏光，陈磊，宋志国，李金鸣．N-甲基小檗胺对大鼠肠系膜动脉平滑肌细胞钙激活钾电流的作用．中国药理学通报， 2006，22： 488-491．
[107]Zheng， G．H．， Chen， Z．L．and Jiang， J．H．[Effects of O-(4-ethoxyl-butyl)-berbamine on isolated frog hearts]．Zhongguo Yao Li Xue Bao， 1998，19： 497-500．
[108]Liu， J．， Qi， S．， Zhu， H．， Zhang， J．， Li， Z．and Wang， T．The effect of calmodulin antagonist berbaminederivative-EBB on hepatoma in vitro and in vivo．Chin Med J (Engl)， 2002，115： 759-62．
[109]张金红，许乃寒，徐畅，陈家童，刘惠，段江燕．小檗胺衍生物(EBB)体外抑制肺癌细胞增殖机制的初探．细胞生物学杂志， 2001， 23： 218-222．
[110]Fang， B．J．， Yu， M．L．， Yang， S．G．， Liao， L．M．， Liu， J．W．and Zhao， R．C．Effect of O-4-ethoxyl-butyl-berbamine in combination with pegylated liposomal doxorubicin on advanced hepatoma in mice．World J Gastroenterol， 2004，10：950-3．
[111]Cheng， Y．H．， Qi， J．， Xiong， D．S．， Liu， J．W．， Qi， S．L．， Pan， B．， Yang， C．Z．and Zhu， H．F．[Reversal of multidrug resistance in drug-resistant human breast cancer cell line MCF-7／ADR by calmodulin antagonist O-(4-ethoxyl-butyl)-berbamine]．Zhongguo Yi Xue Ke Xue Yuan Xue Bao， 2006，28： 164-8．
[112]Pan， B．， Zhou， Y．， Qi， J．， Xiong， D．S．， Liu， J．W．， Qi， S．L．， Cheng， Y．H．， Yang， C．Z．and Zhu， H．F．[Effect of calmodulin antagonist EBB on invasion of human fibrosarcoma cell HT1080]．Zhongguo Yi Xue Ke Xue Yuan Xue Bao， 2005，27：311-4．
[113]贡岳松，胡卓逸，沈子龙．四种小檗胺衍生物对钙调素诱导的肌球蛋白轻链激酶的抑制作用．中国药科大学学报， 1991，22：93-96．