中药雷公藤内酯醇、尿多酸肽对地塞米松耐药及敏感的多发性骨髓瘤细胞株的作用及机制的实验研究
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摘要
第一部分雷公藤内酯醇通过影响细胞生存信号通路及激素受体表达诱导对地塞米松耐药及敏感的多发性骨髓瘤细胞凋亡
目的:
探讨雷公藤内酯醇对地塞米松耐药及敏感的多发性骨髓瘤细胞株MM.1R、MM.1S细胞的作用机制并探讨其作用机制。
方法:
采用MTT比色法研究雷公藤内酯醇(TPL)对MM.1R、MM.1S细胞株以及对常规化疗方案VAD方案治疗无效的MM患者的骨髓单个核细胞的体外生长抑制作用;采用Wright-Giemsa染色、DNA凝胶电泳检测DNA Ladder的条带、流式细胞仪PI染色检测亚G1峰和Annexin V/PI双染色等方法检测细胞凋亡;采用流式细胞仪JC-1染色检测细胞线粒体膜电位的改变;Western-blot检测TPL作用后MM.1R、MM.1S细胞Caspase家族蛋白,凋亡相关蛋白Bcl-2家族,凋亡抑制蛋白IAPs家族蛋白的表达,并进一步加用Caspase-3抑制剂Z-DEVD-FMK来观察实验结果;提取核浆蛋白以观察TPL作用对NF-κB信号通路的影响,并应用免疫荧光方法来检测TPL对NF-κB核转位过程的影响;Western-blot检测TPL对MM.1R、MM.1S细胞PI3K/Akt和NF-κB信号通路相关生存蛋白的影响,并进一步加用PI3K抑制剂LY294002和NF-κB抑制剂PDTC来明确两条信号通路的相关性及机制;用RT-PCR方法检测TPL对编码PI3Kp110α、胰岛素样生长因子-1受体(Insulin-like growth factor-1 receptor,IGF-1R)蛋白的PIK3CA、IGF1R基因的影响,以明确TPL治疗的上游靶点。加用白介素-6(Interleukin-6,IL-6)来明确TPL抑制MM.1细胞生长的效应是否能被减弱,并探讨TPL增加MM.1R、MM.1S细胞株对Dex的敏感性与激素受体(Glucocorticoid Receptor,GR)之间的关系。从microRNA水平、mRNA、蛋白水平检测MM.1R、MM.1S细胞株的GR在TPL作用前后的改变。
结果:
①TPL可以抑制人MM细胞株MM.1S和对Dex耐药的MM.1R细胞株的生长;TPL还可抑制难治的MM患者原代细胞的生长,均呈浓度和时间依赖性。②TPL可以诱导MM.1R、MM.1S细胞凋亡,并呈浓度依赖性;TPL作用后可产生典型的DNALadder条带。③不同浓度TPL作用可以激活Caspase-9,-3,呈浓度依赖性,并伴有明显的线粒体膜电位下降;当TPL浓度为10ng/mL时激活Caspase-8,亦呈浓度依赖性;Caspase-3抑制剂可以抑制TPL诱导的MM.1R、MM.1S细胞凋亡。④不同浓度TPL作用可以下调Bcl-2家族抗凋亡蛋白Bcl-x1、Mcl-1、p-Bad和上调促凋亡蛋白Bax的表达,呈浓度依赖性;还可上调Smac的表达,呈浓度依赖性。⑤TPL可以抑制MM.1R、MM.1S细胞株IAPs家族中的cIAP1,xIAP和Survivin的表达,呈浓度依赖性。⑥TPL抑制MM.1R、MM.1S细胞株的IκBα磷酸化和NF-κB核转位过程。⑦TPL可降低IGF-1R基因及蛋白水平的表达,下调PIK3CA mRNA表达,抑制PI3K/Akt信号途径;PI3K抑制剂LY294002与TPL联用可以明显抑制p-Akt表达,并进一步影响其下游的信号途径,包括NF-κB信号通路的蛋白而诱导MM.1R、MM.1S细胞凋亡。⑧TPL抑制MM.1R、MM.1S细胞生长的效应不受IL-6的影响,还可以增加MM.1R、MM.1S细胞对地塞米松及蛋白酶体抑制剂PS341的敏感性。⑨不同浓度TPL处理后可降低GR相关的microRNA的表达,增加MM.1R、MM.1S细胞株的GR基因、p-GR(磷酸化GR)蛋白及GR直接靶基因:激素诱导锌指蛋白(glucocorticoidinducedleucine zipper——GILZ)的表达。
小结:
①TPL可以抑制MM.1S和对Dex耐药的MM.1R细胞株及MM原代细胞生长,呈时间和剂量依赖性,并且该抑制作用不能被IL-6减弱。②TPL通过启动内源性和外源性细胞凋亡途径诱导Caspase-3依赖型细胞凋亡,并以内源性(线粒体)途径为主,伴有明显的线粒体膜电位下降。③TPL通过下调Bcl-2家族蛋白Bcl-x1、Mcl-1、p-Bad,上调Bax蛋白的表达以及抑制IAPs家族中cIAP1、xIAP和Survivin的表达导致线粒体损伤和Caspase-9,-3激活,诱导细胞凋亡。④TPL通过抑制IκBα磷酸化,从而抑制NF-κB核转位;TPL对MM.1R、MM.1S细胞株NF-κB信号通路的影响主要通过对PI3K/Akt信号通路的调控来实现。⑤TPL降低IGF-1R基因及蛋白水平的表达;减少PIK3CA基因的表达而抑制PI3K/Akt信号通路,并进一步通过影响其下游的信号途径,如NF-κB信号通路等而诱导MM.1R、MM.1S细胞凋亡。⑥TPL下调MM.1R、MM.1S细胞内GR相关的成熟体microRNA(miR-181a、miR-142-5p)的表达,增加p-GR及GILZ的表达,并且该效应不能被IL-6拮抗,从而增加MM.1R、MM.1S细胞对Dex的敏感性。TPL还可增加MM.1R、MM.1S细胞对蛋白酶体抑制剂PS341的敏感性。
第二部分尿多酸肽(CDAⅡ)抗多发性骨髓瘤作用初步机制研究
目的:
探讨CDA-Ⅱ对多发性骨髓瘤的作用及机理,为CDA-Ⅱ应用于MM治疗提供理论依据。
方法:
采用MTT比色法研究CDA-Ⅱ对RPMI8226、U266、MM.1R、MM.1S细胞株以及对MM患者的骨髓单个核细胞的体外生长抑制作用;采用Wright-Giemsa染色、DNA凝胶电泳检测DNA Ladder条带、流式细胞仪Annexin V/PI双染色检测细胞凋亡;JC-1染色检测细胞线粒体膜电位的改变;用Western-blot方法检测CDA-Ⅱ作用后RPMI8226、U266、MM.1R、MM.1S细胞Caspase家族蛋白,凋亡相关蛋白Bcl-2家族,凋亡抑制蛋白IAPs家族蛋白的表达,并进一步加用Caspase-3抑制剂Z-DEVD-FMK来观察实验结果;应用免疫荧光方法来检测CDA-Ⅱ对NF-κB核转位过程的影响。
结果:
①CDA-Ⅱ可以明显抑制RPMI8226、U266、MM.1R、MM.1S细胞株生长,呈时间和剂量依赖性,而且对于患者原代细胞也具有抑制生长的作用,并呈时间和剂量依赖性,但不影响正常人骨髓单个核细胞的生长,对正常骨髓几乎无抑制作用。②CDA-Ⅱ可以诱导MM细胞株RPMI8226细胞凋亡,可产生典型的DNALadder条带。③CDA-Ⅱ通过启动内源性细胞凋亡途径诱导Caspase-3依赖型细胞凋亡,并伴有明显的线粒体膜电位下降。④CDA-Ⅱ通过下调Bcl-2家族蛋白Bcl-2,Mcl-1表达,上调Bax蛋白的表达以及抑制IAPs家族中xIAP和Survivin的表达导致线粒体损伤和Caspase-9,-3激活诱导细胞凋亡。⑤CDA-Ⅱ可以抑制NF-κB的核转位过程,从而使得MM细胞产生凋亡。
小结:
①CDA-Ⅱ可以明显抑制MM细胞株及患者原代细胞生长,呈时间和剂量依赖性。②CDA-Ⅱ可以诱导MM细胞凋亡。③CDA-Ⅱ引起明显的线粒体膜电位下降,启动内源性细胞凋亡途径诱导Caspase-3依赖型细胞凋亡。④CDA-Ⅱ可下调Bcl-2家族蛋白Bcl-2,Mcl-1表达,上调Bax蛋白的表达以及抑制IAPs家族中xIAP和Survivin的表达诱导细胞凋亡。⑤CDA-Ⅱ可以抑制NF-κB的核转位而抑制MM细胞生长。
结论:
TPL能抑制对Dex耐药及敏感的MM细胞生长,并且该抑制生长的作用不能被IL-6拮抗。其机理主要为抑制PI3K/Akt/NF-κB生存信号通路,诱导细胞凋亡;下调GR相关microRNA表达水平,增加GR基因及其p-GR蛋白表达,增加GILZ的表达,从而增加MM.1R、MM.1S细胞对Dex的敏感性。
CDA-Ⅱ可以通过抑制NF-κB的核转位过程,诱导Caspase-3依赖型细胞凋亡,为CDA-Ⅱ用于临床治疗MM提供实验和理论依据。
本研究显示雷公藤内醇酯、CDA-Ⅱ在体外均能抑制MM尤其是耐药MM的生长,提示该两种药物具有用于治疗MM及耐药MM的潜力。
Section 1 Triptolide(TPL) induces MM.1R and MM.1S cells into apoptosis via cell survival and glucocorticoid receptor signaling pathway
Objective:
The aim of this section was to investigate the antitumor activity of TPL against MM.1R and MM.1S cells and to determine if TPL-induced apoptosis of drug-resistant MM cells was associated with the PI3K/Akt and NF-κB survival signaling pathways, and if the activity of TPL-increasing the sensitivity of dexamethasone in MM.1R and MM.1S cells was associated with the glucocortioid receptor.
Methods:
We used MTT assay to investigate the effects of TPL on the MM.1R and MM.1S cells.We examine the effects of TPL on the MM.1R and MM.1S cells of induction of growth arrest and apoptosis.Apoptosis was measured by DNA Ladder and flow cytometry using the Annexin V-FITC apoptosis detection kit,according to the manufacturer's instructions.Alterations in the mitochondrial membrane potential were analyzed by flow cytometry using the specific dye JC-1.The cell-cycle analysis was done according to the PI staining by flow cytometry.Apoptotic proteins including Caspase-9,-8,-3,members of Bcl-2 family and IAPs family were studied by western-blot.Phosphoinositide 3-kinase(PI3K)/Akt and NF-κB survival signaling pathways were also examined using western-blot analysis.The caspase-3 inhibitor Z-DEVD-FMK was used to determine the involvement of caspase-3 and PARP.PI3K inhibitor LY294002 and NF-κB inhibitor PDTC were used to examine the involvement of PI3K/Akt and NF-κB signaling pathways.Immunofluorescent staining was used for observing the distribution of NF-κB in cells.The IGF-1R and PIK3CA genes,which expressed the IGF-1R and PI3Kp110αprotein,were examined by using RT-PCR to identify the target of TPL-induced apoptosis.We evaluate whether TPL-inhibited cell growth can be prevented by exogenous IL-6.We also evaluate the expression of miR181a,miR142-5p and the regulation of glucocorticoid receptor induced by TPL on MM.1R and MM.1S cells.
Results:
①TPL could inhibit the growth of MM.1R and MM.1S cell lines and the tumor cells isolated from three relapsed multiple myeloma patients.The cell growth was inhibited in a time- and dose-dependent manner,corresponding to the reduced cell viability.②TPL induced apoptosis of MM.1R and MM.1S cell lines in a dose-dependent manner.It showed that MM.1R and MM.1S cells after treated with 5ng/ml,10ng/ml TPL for 24h,a typical DNA Ladder was observed.③Treatment with TPL induced marked activation of caspase-9,-3 in a dose-dependent manner,however, caspase-8 was activated only at TPL concentrations greater than 10ng/mL.Pretreatment with Z-DEVD-FMK significantly decreased the apoptotic cells following TPL treatment. TPL also induced a loss in mitochondria transmembrane potential as evidence of mitochondria-mediated apoptosis.④Bcl-x1,Mcl-1 and p-Bad proteins were decreased, however,Bax and Smac proteins were increased in a dose-dependent manner after different concentrations of TPL treatment for 24h.⑤The xIAP,cIAP1 and Survivin proteins were down- regulated in a dose-dependent manner after TPL treatment.⑥TPL inhibited IκBαphosphorylation and prevented NF-κB(p65) nuclear translocation.⑦TPL significantly reduced expression of PI3K/Akt signaling pathway in a dose-dependent manner;the results were further confirmed by PIK3CA gene at mRNA level in a time- and dose-dependent manner,and TPL combined with LY294002 significantly inhibited the expression of p-IκBα;TPL reduced the expression of IGF-1R expression in a time-dependent and dose-dependent manner.⑧The effect of TPL-inhibited cell growth could not be affected by exogenous IL-6,and TPL could enhance the growth inhibitory effect of Dex and PS-341.⑨TPL reduced the expression of miR-181a,miR142-5p.And low expression of miR-181a and miR-142-5p resulted in an increased GR protein expression.TPL also enhanced the expression of glucocorticoid-induced leucine zipper(GILZ),a key component of GC-signaling.
Section 2:The mechanisms and the effects of Uroacitides(CDA-Ⅱ) on the multiple myeloma
Objective:
The aim of this section was to investigate the antitumor activity of CDA-Ⅱagainst MM cells and to determine if CDA-Ⅱ-induced apoptosis of MM cells was associated with NF-κB nuclear translocation.
Methods:
We used MTT assay to investigate the effects of CDA-Ⅱon the MM cells.We examine the effects of CDA-Ⅱon the MM cells of induction of apoptosis.Apoptosis was measured by DNA Ladder and flow cytometry using the Annexin V-FITC apoptosis detection kit,according to the manufacturer's instructions.Alterations in the mitochondrial membrane potential were analyzed by flow cytometry using the specific dye JC-1.Apoptotic proteins including Caspase-9,-3,Bcl-2 family members and IAPs family members were studied by western-blot.The caspase-3 inhibitor Z-DEVD-FMK was used to determine the involvement of caspase-3 and PARP.We exanmine the inhibition effect of CDA-Ⅱon the translocation of NF-κB using immunofluorescence analysis.
Results:
①CDA-Ⅱcould inhibit the growth of MM cells in a time- and dose-dependent manner,corresponding to the reduced cell viability.In contrasts,CDA-Ⅱdid not induce cytotoxicity in BMMCs from normal volunteers.②CDA-Ⅱinduced apoptosis of MM cells in a dose-dependent manner.It showed that RPMI8226 cells after treated with CDA-Ⅱfor 24h,a typical DNA Ladder was observed.③Treatment with CDA-Ⅱinduced marked activation of caspase-9,-3 in a dose-dependent manner. Pretreatment with Z-DEVD-FMK significantly decreased the apoptotic cells following CDA-Ⅱtreatment.CDA-Ⅱalso induced a loss in mitochondria transmembrane potentia as evidence of mitochondria-mediated apoptosis.④Bcl-2,Mcl-1 proteins were decreased,however,Bax protein was increased in a dose-dependent manner after CDA-Ⅱtreatment for 24h.The xIAP and Survivin proteins were down-regulated in a dose-dependent manner after CDA-Ⅱtreatment.⑤CDA-Ⅱprevented NF-κB nuclear translocation.
Conclusions:
CDA-Ⅱcould induce growth arrest and apoptosis of MM cells.The mechanism was related to the prevention NF-κB nuclear translocation,downregulation of IAPs family protein as well as involvement of Bcl-2 family members and triggered the mitochondrial pathway mediated caspase-3-dependent apoptosis. Summary:
TPL can induce growth arrest and apoptosis in MM.1R and MM.1S cell lines.TPL is an effective chinese medical herb with multiple targets,including involvement of PI3K/Akt/NF-κB survival signaling pathway directly,downregulation of the miRNA181a and miRNA142-5p,upregulation of the phosphorylated glucocorticoid receptor protein.TPL can enhance the expression of glucocorticoid-induced leucine zipper(GILZ),a key component of GC-signaling.These probably are the mechanisms of dexamethasone-induced apoptosis in dexamethasone-resistant multiple myeloma.
CDA-Ⅱcan induce growth arrest and apoptosis in MM cells,but not in normal BMMCs.CDA-Ⅱcan prevent NF-κB nuclear translocation and downregulate the expression of Bcl-2,Mcl-1 and IAPs family proteins,upregulation the expression of Bax. Different mechanisms of action acted concurrently to contribute to the TPL and CDA-Ⅱ-induced MM cells,especially Dex-resistant MM cells apoptosis.This research work may provide new insights for the treatment of drug-resistant MM.
目的:
探讨雷公藤内酯醇对地塞米松耐药及敏感的多发性骨髓瘤细胞株MM.1R、MM.1S细胞的作用机制并探讨其作用机制。
方法:
采用MTT比色法研究雷公藤内酯醇(TPL)对MM.1R、MM.1S细胞株以及对常规化疗方案VAD方案治疗无效的MM患者的骨髓单个核细胞的体外生长抑制作用;采用Wright-Giemsa染色、DNA凝胶电泳检测DNA Ladder的条带、流式细胞仪PI染色检测亚G1峰和Annexin V/PI双染色等方法检测细胞凋亡;采用流式细胞仪JC-1染色检测细胞线粒体膜电位的改变;Western-blot检测TPL作用后MM.1R、MM.1S细胞Caspase家族蛋白,凋亡相关蛋白Bcl-2家族,凋亡抑制蛋白IAPs家族蛋白的表达,并进一步加用Caspase-3抑制剂Z-DEVD-FMK来观察实验结果;提取核浆蛋白以观察TPL作用对NF-κB信号通路的影响,并应用免疫荧光方法来检测TPL对NF-κB核转位过程的影响;Western-blot检测TPL对MM.1R、MM.1S细胞PI3K/Akt和NF-κB信号通路相关生存蛋白的影响,并进一步加用PI3K抑制剂LY294002和NF-κB抑制剂PDTC来明确两条信号通路的相关性及机制;用RT-PCR方法检测TPL对编码PI3Kp110α、胰岛素样生长因子-1受体(Insulin-like growth factor-1 receptor,IGF-1R)蛋白的PIK3CA、IGF1R基因的影响,以明确TPL治疗的上游靶点。加用白介素-6(Interleukin-6,IL-6)来明确TPL抑制MM.1细胞生长的效应是否能被减弱,并探讨TPL增加MM.1R、MM.1S细胞株对Dex的敏感性与激素受体(Glucocorticoid Receptor,GR)之间的关系。从microRNA水平、mRNA、蛋白水平检测MM.1R、MM.1S细胞株的GR在TPL作用前后的改变。
结果:
①TPL可以抑制人MM细胞株MM.1S和对Dex耐药的MM.1R细胞株的生长;TPL还可抑制难治的MM患者原代细胞的生长,均呈浓度和时间依赖性。②TPL可以诱导MM.1R、MM.1S细胞凋亡,并呈浓度依赖性;TPL作用后可产生典型的DNALadder条带。③不同浓度TPL作用可以激活Caspase-9,-3,呈浓度依赖性,并伴有明显的线粒体膜电位下降;当TPL浓度为10ng/mL时激活Caspase-8,亦呈浓度依赖性;Caspase-3抑制剂可以抑制TPL诱导的MM.1R、MM.1S细胞凋亡。④不同浓度TPL作用可以下调Bcl-2家族抗凋亡蛋白Bcl-x1、Mcl-1、p-Bad和上调促凋亡蛋白Bax的表达,呈浓度依赖性;还可上调Smac的表达,呈浓度依赖性。⑤TPL可以抑制MM.1R、MM.1S细胞株IAPs家族中的cIAP1,xIAP和Survivin的表达,呈浓度依赖性。⑥TPL抑制MM.1R、MM.1S细胞株的IκBα磷酸化和NF-κB核转位过程。⑦TPL可降低IGF-1R基因及蛋白水平的表达,下调PIK3CA mRNA表达,抑制PI3K/Akt信号途径;PI3K抑制剂LY294002与TPL联用可以明显抑制p-Akt表达,并进一步影响其下游的信号途径,包括NF-κB信号通路的蛋白而诱导MM.1R、MM.1S细胞凋亡。⑧TPL抑制MM.1R、MM.1S细胞生长的效应不受IL-6的影响,还可以增加MM.1R、MM.1S细胞对地塞米松及蛋白酶体抑制剂PS341的敏感性。⑨不同浓度TPL处理后可降低GR相关的microRNA的表达,增加MM.1R、MM.1S细胞株的GR基因、p-GR(磷酸化GR)蛋白及GR直接靶基因:激素诱导锌指蛋白(glucocorticoidinducedleucine zipper——GILZ)的表达。
小结:
①TPL可以抑制MM.1S和对Dex耐药的MM.1R细胞株及MM原代细胞生长,呈时间和剂量依赖性,并且该抑制作用不能被IL-6减弱。②TPL通过启动内源性和外源性细胞凋亡途径诱导Caspase-3依赖型细胞凋亡,并以内源性(线粒体)途径为主,伴有明显的线粒体膜电位下降。③TPL通过下调Bcl-2家族蛋白Bcl-x1、Mcl-1、p-Bad,上调Bax蛋白的表达以及抑制IAPs家族中cIAP1、xIAP和Survivin的表达导致线粒体损伤和Caspase-9,-3激活,诱导细胞凋亡。④TPL通过抑制IκBα磷酸化,从而抑制NF-κB核转位;TPL对MM.1R、MM.1S细胞株NF-κB信号通路的影响主要通过对PI3K/Akt信号通路的调控来实现。⑤TPL降低IGF-1R基因及蛋白水平的表达;减少PIK3CA基因的表达而抑制PI3K/Akt信号通路,并进一步通过影响其下游的信号途径,如NF-κB信号通路等而诱导MM.1R、MM.1S细胞凋亡。⑥TPL下调MM.1R、MM.1S细胞内GR相关的成熟体microRNA(miR-181a、miR-142-5p)的表达,增加p-GR及GILZ的表达,并且该效应不能被IL-6拮抗,从而增加MM.1R、MM.1S细胞对Dex的敏感性。TPL还可增加MM.1R、MM.1S细胞对蛋白酶体抑制剂PS341的敏感性。
第二部分尿多酸肽(CDAⅡ)抗多发性骨髓瘤作用初步机制研究
目的:
探讨CDA-Ⅱ对多发性骨髓瘤的作用及机理,为CDA-Ⅱ应用于MM治疗提供理论依据。
方法:
采用MTT比色法研究CDA-Ⅱ对RPMI8226、U266、MM.1R、MM.1S细胞株以及对MM患者的骨髓单个核细胞的体外生长抑制作用;采用Wright-Giemsa染色、DNA凝胶电泳检测DNA Ladder条带、流式细胞仪Annexin V/PI双染色检测细胞凋亡;JC-1染色检测细胞线粒体膜电位的改变;用Western-blot方法检测CDA-Ⅱ作用后RPMI8226、U266、MM.1R、MM.1S细胞Caspase家族蛋白,凋亡相关蛋白Bcl-2家族,凋亡抑制蛋白IAPs家族蛋白的表达,并进一步加用Caspase-3抑制剂Z-DEVD-FMK来观察实验结果;应用免疫荧光方法来检测CDA-Ⅱ对NF-κB核转位过程的影响。
结果:
①CDA-Ⅱ可以明显抑制RPMI8226、U266、MM.1R、MM.1S细胞株生长,呈时间和剂量依赖性,而且对于患者原代细胞也具有抑制生长的作用,并呈时间和剂量依赖性,但不影响正常人骨髓单个核细胞的生长,对正常骨髓几乎无抑制作用。②CDA-Ⅱ可以诱导MM细胞株RPMI8226细胞凋亡,可产生典型的DNALadder条带。③CDA-Ⅱ通过启动内源性细胞凋亡途径诱导Caspase-3依赖型细胞凋亡,并伴有明显的线粒体膜电位下降。④CDA-Ⅱ通过下调Bcl-2家族蛋白Bcl-2,Mcl-1表达,上调Bax蛋白的表达以及抑制IAPs家族中xIAP和Survivin的表达导致线粒体损伤和Caspase-9,-3激活诱导细胞凋亡。⑤CDA-Ⅱ可以抑制NF-κB的核转位过程,从而使得MM细胞产生凋亡。
小结:
①CDA-Ⅱ可以明显抑制MM细胞株及患者原代细胞生长,呈时间和剂量依赖性。②CDA-Ⅱ可以诱导MM细胞凋亡。③CDA-Ⅱ引起明显的线粒体膜电位下降,启动内源性细胞凋亡途径诱导Caspase-3依赖型细胞凋亡。④CDA-Ⅱ可下调Bcl-2家族蛋白Bcl-2,Mcl-1表达,上调Bax蛋白的表达以及抑制IAPs家族中xIAP和Survivin的表达诱导细胞凋亡。⑤CDA-Ⅱ可以抑制NF-κB的核转位而抑制MM细胞生长。
结论:
TPL能抑制对Dex耐药及敏感的MM细胞生长,并且该抑制生长的作用不能被IL-6拮抗。其机理主要为抑制PI3K/Akt/NF-κB生存信号通路,诱导细胞凋亡;下调GR相关microRNA表达水平,增加GR基因及其p-GR蛋白表达,增加GILZ的表达,从而增加MM.1R、MM.1S细胞对Dex的敏感性。
CDA-Ⅱ可以通过抑制NF-κB的核转位过程,诱导Caspase-3依赖型细胞凋亡,为CDA-Ⅱ用于临床治疗MM提供实验和理论依据。
本研究显示雷公藤内醇酯、CDA-Ⅱ在体外均能抑制MM尤其是耐药MM的生长,提示该两种药物具有用于治疗MM及耐药MM的潜力。
Section 1 Triptolide(TPL) induces MM.1R and MM.1S cells into apoptosis via cell survival and glucocorticoid receptor signaling pathway
Objective:
The aim of this section was to investigate the antitumor activity of TPL against MM.1R and MM.1S cells and to determine if TPL-induced apoptosis of drug-resistant MM cells was associated with the PI3K/Akt and NF-κB survival signaling pathways, and if the activity of TPL-increasing the sensitivity of dexamethasone in MM.1R and MM.1S cells was associated with the glucocortioid receptor.
Methods:
We used MTT assay to investigate the effects of TPL on the MM.1R and MM.1S cells.We examine the effects of TPL on the MM.1R and MM.1S cells of induction of growth arrest and apoptosis.Apoptosis was measured by DNA Ladder and flow cytometry using the Annexin V-FITC apoptosis detection kit,according to the manufacturer's instructions.Alterations in the mitochondrial membrane potential were analyzed by flow cytometry using the specific dye JC-1.The cell-cycle analysis was done according to the PI staining by flow cytometry.Apoptotic proteins including Caspase-9,-8,-3,members of Bcl-2 family and IAPs family were studied by western-blot.Phosphoinositide 3-kinase(PI3K)/Akt and NF-κB survival signaling pathways were also examined using western-blot analysis.The caspase-3 inhibitor Z-DEVD-FMK was used to determine the involvement of caspase-3 and PARP.PI3K inhibitor LY294002 and NF-κB inhibitor PDTC were used to examine the involvement of PI3K/Akt and NF-κB signaling pathways.Immunofluorescent staining was used for observing the distribution of NF-κB in cells.The IGF-1R and PIK3CA genes,which expressed the IGF-1R and PI3Kp110αprotein,were examined by using RT-PCR to identify the target of TPL-induced apoptosis.We evaluate whether TPL-inhibited cell growth can be prevented by exogenous IL-6.We also evaluate the expression of miR181a,miR142-5p and the regulation of glucocorticoid receptor induced by TPL on MM.1R and MM.1S cells.
Results:
①TPL could inhibit the growth of MM.1R and MM.1S cell lines and the tumor cells isolated from three relapsed multiple myeloma patients.The cell growth was inhibited in a time- and dose-dependent manner,corresponding to the reduced cell viability.②TPL induced apoptosis of MM.1R and MM.1S cell lines in a dose-dependent manner.It showed that MM.1R and MM.1S cells after treated with 5ng/ml,10ng/ml TPL for 24h,a typical DNA Ladder was observed.③Treatment with TPL induced marked activation of caspase-9,-3 in a dose-dependent manner,however, caspase-8 was activated only at TPL concentrations greater than 10ng/mL.Pretreatment with Z-DEVD-FMK significantly decreased the apoptotic cells following TPL treatment. TPL also induced a loss in mitochondria transmembrane potential as evidence of mitochondria-mediated apoptosis.④Bcl-x1,Mcl-1 and p-Bad proteins were decreased, however,Bax and Smac proteins were increased in a dose-dependent manner after different concentrations of TPL treatment for 24h.⑤The xIAP,cIAP1 and Survivin proteins were down- regulated in a dose-dependent manner after TPL treatment.⑥TPL inhibited IκBαphosphorylation and prevented NF-κB(p65) nuclear translocation.⑦TPL significantly reduced expression of PI3K/Akt signaling pathway in a dose-dependent manner;the results were further confirmed by PIK3CA gene at mRNA level in a time- and dose-dependent manner,and TPL combined with LY294002 significantly inhibited the expression of p-IκBα;TPL reduced the expression of IGF-1R expression in a time-dependent and dose-dependent manner.⑧The effect of TPL-inhibited cell growth could not be affected by exogenous IL-6,and TPL could enhance the growth inhibitory effect of Dex and PS-341.⑨TPL reduced the expression of miR-181a,miR142-5p.And low expression of miR-181a and miR-142-5p resulted in an increased GR protein expression.TPL also enhanced the expression of glucocorticoid-induced leucine zipper(GILZ),a key component of GC-signaling.
Section 2:The mechanisms and the effects of Uroacitides(CDA-Ⅱ) on the multiple myeloma
Objective:
The aim of this section was to investigate the antitumor activity of CDA-Ⅱagainst MM cells and to determine if CDA-Ⅱ-induced apoptosis of MM cells was associated with NF-κB nuclear translocation.
Methods:
We used MTT assay to investigate the effects of CDA-Ⅱon the MM cells.We examine the effects of CDA-Ⅱon the MM cells of induction of apoptosis.Apoptosis was measured by DNA Ladder and flow cytometry using the Annexin V-FITC apoptosis detection kit,according to the manufacturer's instructions.Alterations in the mitochondrial membrane potential were analyzed by flow cytometry using the specific dye JC-1.Apoptotic proteins including Caspase-9,-3,Bcl-2 family members and IAPs family members were studied by western-blot.The caspase-3 inhibitor Z-DEVD-FMK was used to determine the involvement of caspase-3 and PARP.We exanmine the inhibition effect of CDA-Ⅱon the translocation of NF-κB using immunofluorescence analysis.
Results:
①CDA-Ⅱcould inhibit the growth of MM cells in a time- and dose-dependent manner,corresponding to the reduced cell viability.In contrasts,CDA-Ⅱdid not induce cytotoxicity in BMMCs from normal volunteers.②CDA-Ⅱinduced apoptosis of MM cells in a dose-dependent manner.It showed that RPMI8226 cells after treated with CDA-Ⅱfor 24h,a typical DNA Ladder was observed.③Treatment with CDA-Ⅱinduced marked activation of caspase-9,-3 in a dose-dependent manner. Pretreatment with Z-DEVD-FMK significantly decreased the apoptotic cells following CDA-Ⅱtreatment.CDA-Ⅱalso induced a loss in mitochondria transmembrane potentia as evidence of mitochondria-mediated apoptosis.④Bcl-2,Mcl-1 proteins were decreased,however,Bax protein was increased in a dose-dependent manner after CDA-Ⅱtreatment for 24h.The xIAP and Survivin proteins were down-regulated in a dose-dependent manner after CDA-Ⅱtreatment.⑤CDA-Ⅱprevented NF-κB nuclear translocation.
Conclusions:
CDA-Ⅱcould induce growth arrest and apoptosis of MM cells.The mechanism was related to the prevention NF-κB nuclear translocation,downregulation of IAPs family protein as well as involvement of Bcl-2 family members and triggered the mitochondrial pathway mediated caspase-3-dependent apoptosis. Summary:
TPL can induce growth arrest and apoptosis in MM.1R and MM.1S cell lines.TPL is an effective chinese medical herb with multiple targets,including involvement of PI3K/Akt/NF-κB survival signaling pathway directly,downregulation of the miRNA181a and miRNA142-5p,upregulation of the phosphorylated glucocorticoid receptor protein.TPL can enhance the expression of glucocorticoid-induced leucine zipper(GILZ),a key component of GC-signaling.These probably are the mechanisms of dexamethasone-induced apoptosis in dexamethasone-resistant multiple myeloma.
CDA-Ⅱcan induce growth arrest and apoptosis in MM cells,but not in normal BMMCs.CDA-Ⅱcan prevent NF-κB nuclear translocation and downregulate the expression of Bcl-2,Mcl-1 and IAPs family proteins,upregulation the expression of Bax. Different mechanisms of action acted concurrently to contribute to the TPL and CDA-Ⅱ-induced MM cells,especially Dex-resistant MM cells apoptosis.This research work may provide new insights for the treatment of drug-resistant MM.
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