低剂量力达霉素诱导小鼠胚胎癌细胞分化的研究
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摘要
肿瘤干细胞的假说认为不是所有肿瘤细胞均具有增殖和维持肿瘤生长的能力，而仅有一小部分肿瘤细胞亚群，具有自我更新潜能和形成肿瘤维持肿瘤生长的能力，称之为肿瘤干细胞[1]。对于治疗反应敏感的肿瘤来说，传统的化疗及放疗所能杀伤的大部分肿瘤细胞，均属于已分化的终末细胞，残存的极少数肿瘤干细胞，成为肿瘤复发和转移的根源，直接影响肿瘤患者的生存率[2]。因此，肿瘤干细胞成为有效治疗的肿瘤新靶点[3]。
力达霉素(LDM，原名C-1027)，是一种由一个酸性蛋白和一个发色团组成的抗肿瘤抗生素。其不稳定发色团可直接断裂DNA双链，酸性蛋白作为载体靶向肿瘤组织。已经证实在体内、体外通过诱导细胞周期停滞和细胞凋亡抑制大多数肿瘤细胞的生长[4]。
P19胚胎癌（embryonic carcinoma, EC）细胞是从C3H/He鼠胚胎来源的畸胎瘤中分离得到的，具有与多能性的胚胎干细胞（embryonic stem cell,ES cell）极为相似的生物学特征，高表达囊胚内细胞团的标记物，如转录因子Oct4，是体外研究肿瘤干细胞增殖及分化的理想模型[5]。Oct4是维持胚胎干细胞自我更新和多能性的一种关键转录因子，其在全能性或多能性干细胞及一些实体肿瘤干细胞中高表达，但在正常体细胞和正常成体干细胞分化的子细胞中不表达。因此，有学者提出Oct4可以作为肿瘤干细胞的标志分子之一。
前期实验结果证实低剂量（0.01nmol）力达霉素处理P19EC细胞诱导G0/G1期阻滞而不引起凋亡,与其下调了胚胎干细胞样基因Oct4有关，提示力达霉素可作为研究胚胎干细胞分化的诱导剂[6]。
本研究发现:首先，小鼠P19EC细胞在诱导分化培养后，形态学符合神经细胞样改变，在mRNA、蛋白水平上检测到神经细胞的标志分子；其次，低剂量力达霉素处理小鼠P19EC细胞后，下调转录因子Oct4及上调p21的表达；其三，通过染色质免疫沉淀分析证实转录因子Oct4结合到p21的启动子的量逐渐减少，并且低剂量力达霉素通过下调转录因子Oct4，解除对其下游靶基因p21的抑制作用。
因此，我们得出结论：低剂量力达霉素可以诱导小鼠P19EC细胞向神经样细胞分化；低剂量力达霉素诱导小鼠P19EC细胞向神经样细胞分化与其下调转录因子Oct4的表达有关；转录因子Oct4的下调导致其靶基因p21的上调，促进肿瘤干细胞的分化；低剂量力达霉素通过诱导分化达到抗肿瘤作用，为其应用于临床提供理论基础和实验依据。
The cancer stem cell hypothesis suggests that not all the cells in thetumour have the ability to proliferate and maintain growth of the tumour, butonly a small subpopulation of cells in the tumour with a self-renewal potentialand the capacity to form a tumour and maintain its growth, called cancer stemcells. Conventional chemotherapy and radiotherapy are effective against thebulk cancer cells, but the presence of rare resistant CSCs may lead to tumorrecurrence and poor survival outcomes. Therefore, CSCs are considered ascrucial targets for curing the cancer.
Lidamycin (LDM, also known as C-1027) is an anticancer chromoproteinincluding an apoprotein and a chromophore. Its labile chromophore can directlybreak the DNA double-strand, and its noncovalently bound apoprotein acts as adelivery carrier targeting tumor tissue. LDM has been confirmed to inhibit thegrowth of most of cancers in vitro and in vivo by cell cycle arrest andapoptosis.
P19embryonal carcinoma (EC) cells derived from an embryo-derivedteratocarcinoma in C3H/He mice are multipotent. High expression of markersof the inner cell mass of blastocysts, such as transcription factors Oct4, is theideal model in vitroto linkage between the tumors and cell differentiation. Oct4,a crucial transcriptional factor for maintaining self-renewal and pluripotency, ishigh expressed in the totipotent or multipotent stem cells and solid tumor cells,but not expressed in normal somatic tissues and normal differentiated daughtersof the adult stem cells. Therefore, some scholars have proposed that Oct4canact as one of cancer stem cell markers.
Preliminary experimental results confirm that low dose of LDM induced G0/G1arrest of P19EC cells without apoptosis. Which relating todown-regulation of embryonic stem cell-like gene Oct4. This indicated thatLDM may be served as an inducer to study ES cell pluripotency anddifferentiation.
In this study, we found the following results. Firstly, After low-doselidamycin induced differentiation of P19EC cells, the morphology of P19cells undergoing neural differentiation, on the level of mRNA and proteindetected the markers of neural cells; Secondly, Lidamycin suppressedexpression of transcriptional factor Oct4and activated p21gene in mouse P19EC cells；Thirdly, by chromatin immunoprecipitation analysis confirmed thatOct4binding to p21gene promoter was decreased. These results suggested thatsuppression of Oct4by lidamycin led to activation of the p21gene.
We conclude that low-dose lidamycin induced neural differentiation inP19EC cell; low-dose lidamycin induced neuronal differentiation related todown regulation of Oct4;up regulation of p21played an important role incancer stem cell differentiation;low-dose lidamycin induced differentiation ofcells in chemotherapy,which provides a theoretical basis and experimentalevidence.
力达霉素(LDM，原名C-1027)，是一种由一个酸性蛋白和一个发色团组成的抗肿瘤抗生素。其不稳定发色团可直接断裂DNA双链，酸性蛋白作为载体靶向肿瘤组织。已经证实在体内、体外通过诱导细胞周期停滞和细胞凋亡抑制大多数肿瘤细胞的生长[4]。
P19胚胎癌（embryonic carcinoma, EC）细胞是从C3H/He鼠胚胎来源的畸胎瘤中分离得到的，具有与多能性的胚胎干细胞（embryonic stem cell,ES cell）极为相似的生物学特征，高表达囊胚内细胞团的标记物，如转录因子Oct4，是体外研究肿瘤干细胞增殖及分化的理想模型[5]。Oct4是维持胚胎干细胞自我更新和多能性的一种关键转录因子，其在全能性或多能性干细胞及一些实体肿瘤干细胞中高表达，但在正常体细胞和正常成体干细胞分化的子细胞中不表达。因此，有学者提出Oct4可以作为肿瘤干细胞的标志分子之一。
前期实验结果证实低剂量（0.01nmol）力达霉素处理P19EC细胞诱导G0/G1期阻滞而不引起凋亡,与其下调了胚胎干细胞样基因Oct4有关，提示力达霉素可作为研究胚胎干细胞分化的诱导剂[6]。
本研究发现:首先，小鼠P19EC细胞在诱导分化培养后，形态学符合神经细胞样改变，在mRNA、蛋白水平上检测到神经细胞的标志分子；其次，低剂量力达霉素处理小鼠P19EC细胞后，下调转录因子Oct4及上调p21的表达；其三，通过染色质免疫沉淀分析证实转录因子Oct4结合到p21的启动子的量逐渐减少，并且低剂量力达霉素通过下调转录因子Oct4，解除对其下游靶基因p21的抑制作用。
因此，我们得出结论：低剂量力达霉素可以诱导小鼠P19EC细胞向神经样细胞分化；低剂量力达霉素诱导小鼠P19EC细胞向神经样细胞分化与其下调转录因子Oct4的表达有关；转录因子Oct4的下调导致其靶基因p21的上调，促进肿瘤干细胞的分化；低剂量力达霉素通过诱导分化达到抗肿瘤作用，为其应用于临床提供理论基础和实验依据。
The cancer stem cell hypothesis suggests that not all the cells in thetumour have the ability to proliferate and maintain growth of the tumour, butonly a small subpopulation of cells in the tumour with a self-renewal potentialand the capacity to form a tumour and maintain its growth, called cancer stemcells. Conventional chemotherapy and radiotherapy are effective against thebulk cancer cells, but the presence of rare resistant CSCs may lead to tumorrecurrence and poor survival outcomes. Therefore, CSCs are considered ascrucial targets for curing the cancer.
Lidamycin (LDM, also known as C-1027) is an anticancer chromoproteinincluding an apoprotein and a chromophore. Its labile chromophore can directlybreak the DNA double-strand, and its noncovalently bound apoprotein acts as adelivery carrier targeting tumor tissue. LDM has been confirmed to inhibit thegrowth of most of cancers in vitro and in vivo by cell cycle arrest andapoptosis.
P19embryonal carcinoma (EC) cells derived from an embryo-derivedteratocarcinoma in C3H/He mice are multipotent. High expression of markersof the inner cell mass of blastocysts, such as transcription factors Oct4, is theideal model in vitroto linkage between the tumors and cell differentiation. Oct4,a crucial transcriptional factor for maintaining self-renewal and pluripotency, ishigh expressed in the totipotent or multipotent stem cells and solid tumor cells,but not expressed in normal somatic tissues and normal differentiated daughtersof the adult stem cells. Therefore, some scholars have proposed that Oct4canact as one of cancer stem cell markers.
Preliminary experimental results confirm that low dose of LDM induced G0/G1arrest of P19EC cells without apoptosis. Which relating todown-regulation of embryonic stem cell-like gene Oct4. This indicated thatLDM may be served as an inducer to study ES cell pluripotency anddifferentiation.
In this study, we found the following results. Firstly, After low-doselidamycin induced differentiation of P19EC cells, the morphology of P19cells undergoing neural differentiation, on the level of mRNA and proteindetected the markers of neural cells; Secondly, Lidamycin suppressedexpression of transcriptional factor Oct4and activated p21gene in mouse P19EC cells；Thirdly, by chromatin immunoprecipitation analysis confirmed thatOct4binding to p21gene promoter was decreased. These results suggested thatsuppression of Oct4by lidamycin led to activation of the p21gene.
We conclude that low-dose lidamycin induced neural differentiation inP19EC cell; low-dose lidamycin induced neuronal differentiation related todown regulation of Oct4;up regulation of p21played an important role incancer stem cell differentiation;low-dose lidamycin induced differentiation ofcells in chemotherapy,which provides a theoretical basis and experimentalevidence.
引文
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[3] Tai MH, Chang CC, Chang CC, et al. Oct4expression in adult humanstem cells: evidence in support of the stem cell theory of carcinogenesis[J].Carcinogenesis,2005,26(2):495–502
[4] Zhen YZ, Lin YJ, Shang BY, et al. Enediyne lidamycin induces apoptosisin human multiple myeloma cells through activation of p38mitogen-activated protein kinase and c-Jun NH2-terminal kinase[J]. Int J Hematol,2009,90(1):44–51
[5] Blelloch RH, Hochedlinger K, Yamada Y, et al. Nuclear cloning ofembryonic carcinoma cells[J]. Proc Natl Acad Sci USA,2004,101(39):13985–13990
[6] Zhen HY, He QH, Zhen YZ, et al. Inhibition of mouse embryoniccarcinoma cell growth by lidamycin through down-regulation ofembryonic stem cell-like genes Oct4,Sox2and Myc[J]. Invest New Drugs,2011,29(6):1188-1197
[7] Reya T, Morrison SJ, Clarke MF, et al. Stem cells, cancer, and cancer stemcells[J]. Nature,2001,414(6859):105-11
[8] Furth J, Kahn MC. The transmission of leukaemia of mice with a singlecell[J]. Am J Cancer,1937,31:276–282
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