新型PDK1抑制剂对多发性骨髓瘤的杀伤作用及分子机制研究

发布时间：2018-03-14 23:02

本文选题：多发性骨髓瘤　切入点：PDK1　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：第一部分PDK1抑制剂GSK2334470单药及联合mTOR抑制剂PP242的抗骨髓瘤作用研究目的:研究PDK1抑制剂GSK-470的抗骨髓瘤作用及其分子机制;通过体内外实验,探讨GSK2334470协同mTOR抑制剂PP242抑制骨髓瘤细胞生长和诱导细胞凋亡的作用和机制。方法:采用MTT法检测GSK2334470和/或PP242对多发性骨髓瘤(multiply myeloma,MM)细胞株和患者原代细胞的生长抑制作用;流式细胞仪AnnexinV/PI双染法检测细胞凋亡;Western blot 检测 Caspase-3/8/9、PARP、PI3K/PDK1/Akt/mTOR通路相关蛋白的表达情况。构建RPMI8226细胞荷瘤小鼠模型,检测GSK2334470和/或PP242对小鼠瘤体生长的影响。结果:1.GSK2334470可以抑制骨髓瘤细胞株及原代MM患者细胞生长,呈浓度依赖性;对正常细胞株无明显毒性作用。2.外源性IL-6和IGF-1不能逆转GSK2334470对骨髓瘤细胞株的杀伤作用。3.GSK2334470可以诱导MM细胞株和原代细胞发生凋亡,Caspase-3、Caspase-8、Caspase-9 和 PARP 被激活。4.GSK2334470通过抑制PDK1的磷酸化来调节PI3K/Akt/mTOR通路及其下游蛋白。5.GSK2334470可以协同PP242杀伤骨髓瘤细胞株和原代细胞,诱导细胞凋亡。6.PP242通过抑制mTORC2、下调Akt第473位点上丝氨酸的磷酸化来增强GSK2334470的抗骨髓瘤作用,两者联用完全抑制了 Akt和mTORC1/C2的活性。7.GSK2334470、PP242均可抑制MM荷瘤小鼠瘤体的生长,但联合组的抑制作用更明显,差异有统计学意义;联合组瘤体的mTORC1和mTORC2的活性都有明显下降。第二部分PTEN在PDK1抑制剂GSK2334470抗骨髓瘤作用中的重要性研究目的:研究和比较GSK2334470对四种MM细胞株杀伤作用的差异及其与PTEN基因的关系;通过腺病毒或药物改变骨髓瘤细胞中PTEN蛋白的水平来进一步证实PTEN在GSK2334470抗骨髓瘤作用中重要性;研究GSK2334470与蛋白酶体抑制剂MG132的协同抗骨髓瘤作用及其机制。方法:采用MTT法检测生长抑制作用;流式细胞仪Annexin V/PI双染法检测细胞凋亡;PCR和Western blot检测MM细胞株PTEN的本底表达;腺病毒转染使MM细胞株过表达或低表达PTEN;Western blot检测胞浆PTEN、胞核PTEN及PI3K/PDK1/Akt/mTOR通路相关蛋白的表达情况。激光共聚焦检测PTEN蛋白在细胞内的分布。结果:1.GSK2334470对四种骨髓瘤细胞杀伤作用有明显的差异,对ARP-1和MM.1R较敏感;ARP-1、MM.1R凋亡细胞比例也明显高于RPMI8226和OPM-2;与细胞株的PTEN表达呈正相关。2.通过腺病毒转染改变MM细胞株PTEN的表达,可改变GSK2334470的敏感性。3.MG132通过增加细胞中PTEN的表达,调节PI3K/Akt/mTOR通路及其下游蛋白,协同GSK2334470杀伤骨髓瘤细胞,诱导凋亡。4.GSK2334470可以使MM细胞株胞浆中高表达的PTEN向细胞核转移,明确了 GSK2334470抗骨髓瘤作用的其他机制,与MG132协同抗骨髓瘤的分子机制。结论:GSK2334470通过抑制PDK1的磷酸化来调节PI3K/Akt/mTOR通路,诱导骨髓瘤细胞凋亡;mTOR抑制剂PP242通过抑制mTORC2和p-Akt S473的磷酸化协同GSK2334470发挥抗骨髓瘤作用。GSK2334470的敏感性与MM细胞内PTEN的表达水平有关;可通过蛋白酶体抑制剂MG132或腺病毒转染增加细胞内PTEN的表达提高GSK2334470的杀伤作用;同时GSK2334470还可以使高表达的PTEN发生核转移,与MG132发挥协同抗MM作用。提示靶向抑制PDK1及联合mTOR抑制剂或蛋白酶体抑制剂可能是多发性骨髓瘤治疗的新的措施。
[Abstract]:Objective to study the antitumor action of bone marrow Part 1 PDK1 inhibitor GSK2334470 monotherapy and in combination with the mTOR inhibitor PP242: anti myeloma effect of PDK1 inhibitor GSK-470 and its molecular mechanism; in vivo. To explore the effect and mechanism of GSK2334470 cooperative mTOR inhibitor PP242 inhibits myeloma cell growth and induce cell apoptosis. Methods: MTT detection of GSK2334470 and / or PP242 on multiple myeloma (multiply myeloma, MM) inhibited the growth of primary cells and leukemic cells; flow cytometry AnnexinV/PI method to detect cell apoptosis; Western blot detection of Caspase-3/8/9, PARP, expression of PI3K/PDK1/Akt/mTOR pathway related proteins. Construction of RPMI8226 cell tumor bearing mice model effect, detection of GSK2334470 and / or PP242 on the growth of mouse tumors. Results: 1.GSK2334470 could inhibit the growth of myeloma cell lines and primary MM patients Cell growth in a concentration dependent manner; no obvious toxic effect of the killing effect of.3.GSK2334470.2. of exogenous IL-6 and IGF-1 could not reverse the GSK2334470 of myeloma cells can induce the apoptosis of MM cell lines and primary cells of normal cell lines Caspase-3, Caspase-8, Caspase-9 and PARP are activated by.4.GSK2334470 inhibited PDK1 phosphorylation to regulate the PI3K/Akt/mTOR pathway and its downstream protein.5.GSK2334470 can cooperate with PP242 anti myeloma cell lines and primary cells, induced apoptosis of.6.PP242 cells through inhibition of mTORC2, downregulation of Akt 473rd sites of serine phosphorylation to enhance the anti myeloma effect of GSK2334470 completely inhibited the activity of.7.GSK2334470, Akt and mTORC1/C2 combined with PP242 can inhibit the tumor bearing. The growth of MM in mice, but the inhibitory effect of combined group was more obvious, the difference was statistically significant; the joint group of tumor mTO RC1 and mTORC2 activity was decreased. The second part is the research purpose of the importance of PTEN in the PDK1 inhibitor GSK2334470 anti myeloma effect: To study and compare the differences of four kinds of GSK2334470 MM cell killing effect and its relationship with PTEN gene by adenovirus or drugs; change of PTEN protein level in myeloma cells further confirmed the importance of PTEN in the GSK2334470 anti myeloma effects; research of GSK2334470 and proteasome inhibitor MG132 synergistic anti myeloma effect and its mechanism. Methods: the growth inhibition was detected by MTT assay; flow cytometry Annexin V/PI method to detect cell apoptosis; PCR and Western blot in MM cells was detected PTEN basal expression; adenovirus transfected MM cell line that over expression or low expression of PTEN; Western blot detection of intracellular PTEN, the expression of nuclear PTEN and PI3K/PDK1/Akt/mTOR pathway related proteins induced. The distribution of PTEN protein was detected by confocal light in the cells. Results: the lethal effect of 1.GSK2334470 on four kinds of myeloma cells was significantly different, more sensitive to ARP-1 and MM.1R; ARP-1, the percentage of apoptotic cells was significantly higher than that of RPMI8226 and MM.1R OPM-2; Cheng Zhengxiang.2. expression by adenovirus transfection MM cell line PTEN cell strain PTEN expression and sensitivity of.3.MG132 GSK2334470 can be altered by increasing the expression of PTEN in the cells, regulating PI3K/Akt/mTOR pathway and its downstream proteins, Co GSK2334470 killing myeloma cells, apoptosis induced by.4.GSK2334470 can make the high expression of MM cells in the cytoplasm of PTEN translocates to the nucleus, the other mechanism of GSK2334470 anti myeloma effects, molecular the mechanism of synergistic anti myeloma with MG132. Conclusion: GSK2334470 can inhibit the phosphorylation of PDK1 to regulate PI3K/Akt/mTOR pathway, induce myeloma cell apoptosis; mTO R inhibitor PP242 inhibit the phosphorylation of S473 mTORC2 and p-Akt GSK2334470 expression level of sensitivity and play synergistic anti myeloma effects of MM cells in.GSK2334470 PTEN; by proteasome inhibitor MG132 or adenovirus transfection increased the expression of PTEN in endothelial cells enhance the killing effect of GSK2334470; while GSK2334470 can also make the high expression of PTEN nuclear transfer, play the role of anti MM in coordination with the MG132. Suggesting that targeted inhibition of PDK1 and combined with mTOR inhibitors or proteasome inhibitors may be the treatment of multiple myeloma with new measures.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R733.3

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