诱导分化降低胶质瘤干细胞样细胞表型及其致瘤性
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摘要
目的通过诱导胶质瘤干细胞样细胞的分化,对分化细胞及原肿瘤细胞干细胞特性及其生物学特性的差异进行对比,比较其干细胞表型特性及致瘤性,评价分化治疗恶性胶质瘤的可行性。
方法采用干细胞条件下神经球方法培养胶质瘤细胞C6,胶质瘤原代细胞。利用添加全反式维甲酸(ATRA)诱导细胞分化,研究其不同的生长特点,包括细胞形态、自我更新能力等。RT-PCR、Western Blotting和流式细胞技术检测干细胞和分化细胞表面标志物的表达差异;Hoechst33342荧光染色流式细胞术检测其侧群细胞的含量;裸鼠颅内原位成瘤实验分析肿瘤干细胞及分化细胞肿瘤形成能力差异。采用统计学软件SPSS 17.0进行数据统计学分析。
结果在干细胞条件培养的C6及原代胶质瘤干细胞通过添加全反式维甲酸引导细胞分化,RT-PCR和Western Blot提示分化后CD133、Nestin表达降低,GFAP表达升高。流式细胞技术检测提示CD133、SP比例降低。裸鼠颅内原位成瘤实验提示胶质瘤干细胞样细胞种植的裸鼠生存时间明显比分化后细胞种植的裸鼠生存时间降低。
结论1.全反式维甲酸可以诱导胶质瘤干细胞样细胞的分化。
2.分化后的胶质瘤细胞在自我更新能力及表面标记物的表达上较干细胞样细胞降低,其致瘤性也明显降低。
3.分化治疗可能成为胶质瘤治疗的可行方法。
Objective Use ATRA to induce stem-like glioma cells to differentiate and compare stem-like cells properties and biologic characteristics between stem-like glioma cells, including C6 cell lines, primary glioma cells and their differentiated cells.
Methods Glioma cells were cultured with neurosphere-formation assay in serum-free supplemented medium. Stem-like glioma cells (SLGC) were differentiated with all-trans retinoic acid(ATRA)-containing medium. Growth characteristics, including morphological differences, capacity of self-renewal, were detected contrastively through neurosphere formation assay. Expressions of stem cell and differentiated cell markers were determined by reverse transcription polymerase chain reaction (RT-PCR), Western Blotting and Flow Cytometry(FCM). SLGC and differentiated cells were implanted stereotactically and intracranially into the Balb/c nude mice to contrast the capability of tumor initiation and the tumor models were detected by hematoxylin and eosin (HE) staining. All data analyses were performed with the SPSS software, version 17.0.
Results Our data suggest that in vitro differentiation of SLGCs with ATRA induces the growth characteristics changes of glioma cells ,down-regulation of CD133 and Nestin expression and up-regulation of GFAP. In vivo mouse model, differentiated cells inoculated mice have better survival outcome.
Conclusions
1.ATRA can induce the differentiation of SLGCs.
2.The tumorigenicity of stem-like glioma cells is reduced after ATRA differentiation.
3.Differentiation treatment (ATRA) targeted the stem-like cell population is a candidate for adjuvant therapy against malignant gliomas.
方法采用干细胞条件下神经球方法培养胶质瘤细胞C6,胶质瘤原代细胞。利用添加全反式维甲酸(ATRA)诱导细胞分化,研究其不同的生长特点,包括细胞形态、自我更新能力等。RT-PCR、Western Blotting和流式细胞技术检测干细胞和分化细胞表面标志物的表达差异;Hoechst33342荧光染色流式细胞术检测其侧群细胞的含量;裸鼠颅内原位成瘤实验分析肿瘤干细胞及分化细胞肿瘤形成能力差异。采用统计学软件SPSS 17.0进行数据统计学分析。
结果在干细胞条件培养的C6及原代胶质瘤干细胞通过添加全反式维甲酸引导细胞分化,RT-PCR和Western Blot提示分化后CD133、Nestin表达降低,GFAP表达升高。流式细胞技术检测提示CD133、SP比例降低。裸鼠颅内原位成瘤实验提示胶质瘤干细胞样细胞种植的裸鼠生存时间明显比分化后细胞种植的裸鼠生存时间降低。
结论1.全反式维甲酸可以诱导胶质瘤干细胞样细胞的分化。
2.分化后的胶质瘤细胞在自我更新能力及表面标记物的表达上较干细胞样细胞降低,其致瘤性也明显降低。
3.分化治疗可能成为胶质瘤治疗的可行方法。
Objective Use ATRA to induce stem-like glioma cells to differentiate and compare stem-like cells properties and biologic characteristics between stem-like glioma cells, including C6 cell lines, primary glioma cells and their differentiated cells.
Methods Glioma cells were cultured with neurosphere-formation assay in serum-free supplemented medium. Stem-like glioma cells (SLGC) were differentiated with all-trans retinoic acid(ATRA)-containing medium. Growth characteristics, including morphological differences, capacity of self-renewal, were detected contrastively through neurosphere formation assay. Expressions of stem cell and differentiated cell markers were determined by reverse transcription polymerase chain reaction (RT-PCR), Western Blotting and Flow Cytometry(FCM). SLGC and differentiated cells were implanted stereotactically and intracranially into the Balb/c nude mice to contrast the capability of tumor initiation and the tumor models were detected by hematoxylin and eosin (HE) staining. All data analyses were performed with the SPSS software, version 17.0.
Results Our data suggest that in vitro differentiation of SLGCs with ATRA induces the growth characteristics changes of glioma cells ,down-regulation of CD133 and Nestin expression and up-regulation of GFAP. In vivo mouse model, differentiated cells inoculated mice have better survival outcome.
Conclusions
1.ATRA can induce the differentiation of SLGCs.
2.The tumorigenicity of stem-like glioma cells is reduced after ATRA differentiation.
3.Differentiation treatment (ATRA) targeted the stem-like cell population is a candidate for adjuvant therapy against malignant gliomas.
引文
1. Ohgaki, H. and P. Kleihues. Epidemiology and etiology of gliomas. Acta Neuropathologica 2005:109(1): 93-108.
2. Lotolf, U. M., P. Kleihues, et al. Population-based study on incidence, survival rates, and genetic alterations of low-grade diffuse astrocytomas and oligodendrogliomas. Acta Neuropathologica 2004:108(1): 49-56.
3. Kopper L, Hajdu M. Tumor Stem Cells. Pathology Oncology Research 2004:10(2): 69-73.
4. Bjerkvig R, Tysnes B B, Aboody KS.The origin of the cancer stem cell: current controversies and new in sights. Nature Reviews (cancer)2005: 5 (11): 899 -904.
5. Dean, M, T. Fojo, et al. Tumor stem cells and drug resistance.Nature Reviews Cancer 2005:5(4): 275-284.
6. Reya T,Morrison SJ, ClarkeMF and WeissmanIL. Stem cells,Cancer,and Cancer stem cells. Nature 2001:414:105-111.
7. Hemmati, H. D. et al.Cancerous stem cells can arise from pediatric brain tumors.Proceedings of the National Academy of Sciences 2003:100(25): 15178-15183.
8. Singh S K, Clarke I D, Terasaki M, et al. Identification of a cancer stem cell in human brain tumors.Cancer Res 2003:63(18): 5821-5828.
9. Uchida N, Bcuk D W, He D, et al. Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A 2003:97(26): 14720-14725.
10. Reynolds BA, Weiss S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 1996:175:1-13.
11. Yuan, X., J. Curtin, et al. Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene 2004:23(58): 9392-9400.
12. Liu, Q., D. H. Nguyen, et al. Molecular properties of CD133+ glioblastoma stem cells derived from treatment-refractory recurrent brain tumors. Journal of Neuro-Oncology 2009:94(1): 1-19.
13. Michalczyk K, Ziman M. Nestin structure and predicted function in cellular cytoskeletal organization.Histol.Histopathol.2005:20 (2): 665–71.
14. Bidlingmaier, S., X. Zhu, et al. The utility and limitations of glycosylated human CD133 epitopes in defining cancer stem cells.Journal of Molecular Medicine 2008:86(9): 1025-1032.
15. Beier, D., P. Hau, et al. CD133+ and CD133- Glioblastoma-Derived Cancer Stem Cells Show Differential Growth Characteristics and Molecular Profiles. Cancer Research 2007:67(9): 4010-4015.
16. Singh SK, Hawkins C, Clarke ID et al. Identification of human brain tumour initiating cells. Nature 2004:432:396–401.
17. Zeppernick, F., R. Ahmadi, et al. Stem Cell Marker CD133 Affects Clinical Outcome in Glioma Patients. Clinical Cancer Research 2008:14(1): 123-129.
18. Challen, G. A. and M. H. Little. A Side Order of Stem Cells: The SP Phenotype. Stem Cells 2006:24(1): 3-12.
19. Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG. Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2– cancer cells are similarly tumorigenic. Cancer Res 2005:65:6207–6219.
20. Bleau, A.-M., D. Hambardzumyan, et al. PTEN/PI3K/Akt Pathway Regulates the Side Population Phenotype and ABCG2 Activity in Glioma Tumor Stem-like Cells. Cell Stem Cell 2009:4(3): 226-235.
21. Chiba, T., K. Kita, et al. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology 2006:44(1): 240-251.
22. Hirschmann-Jax, C.et al. A distinct "side population" of cells with high drug efflux capacity in human tumor cells. Proceedings of the National Academy of Sciences 2004:101(39): 14228-14233.
23. Harris, M. A., H. Yang, et al. Cancer Stem Cells Are Enriched in the Side Population Cells in a Mouse Model of Glioma. Cancer Research 2008:68(24): 10051-10059.
24. Wan F, Zhang S, Xie R, et al. The utility and limitations of neurosphere assay, CD133 immunophenotyping and side population assay in glioma stem cell research. BrainPathol 2010:20: 877–889.
25.唐珂,曹利等。全反式维甲酸对C6胶质瘤细胞增殖分化的影响。中南大学学报(医学版)2008:33(10):892-897
26. Karsy M, Albert L et al. All-trans retinoic acid modulates cancer stem cells of glioblastoma multiforme in an MAPK-dependent manner. Anticancer Research 2010:30:4915-4920.
27. Campos B, Wan F, Herold-Mende C.et al. Differentiation therapy exerts antitumor effects on stem-like glioma cells. Clin Cancer Res 2010:16(10): 2715–2728.
28. Gudas LJ, Wagner JA. Retinoids regulate stem cell differentiation. J Cell Physiol 2011:226:322-330.
29.綦斌,罗毅男,田宇等。全反式维甲酸对脑胶质瘤细胞Cyclin E,Caspase3表达的影响。中华实验外科杂志,2006:23(6):721-723.
30. Das, A., N. L. Banik, et al. Retinoids induced astrocytic differentiation with down regulation of telomerase activity and enhanced sensitivity to taxol for apoptosis in human glioblastoma T98G and U87MG cells. Journal of Neuro-Oncology 2007:87(1): 9-22.
31. Kaba SE, Kyritsis AP, Conrad C, et al. The treatment of recurrent cerebral gliomas with all-trans-retinoic acid tretinoin. J Neurooncol 997:34:145–51.
32. Phuphanich S, Scott C, Fischbach AJ, Langer C, Yung WK. Alltrans-retinoic acid: a phase II Radiation Therapy Oncology Group study RTOG 91-13 in patients with recurrent malignant astrocytoma. J Neurooncol 1997:34:193–200.
33.章翔,程光,费舟等。苦参碱对脑胶质瘤C6细胞系的诱导分化。[J]中华神经外科疾病研究杂志,2003.3(2):124-126
34. Karmakar, S., N. L. Banik, et al.Combination of all-trans retinoic acid and paclitaxel-induced differentiation and apoptosis in human glioblastoma U87MG xenografts in nude mice.Cancer 2008:112(3): 596-607.
1. Stieber VW, Mehta MP. Advances in radiation therapy for brain tumors. Neurol Clin 2007;25:1005–1033.
2. Westphal M, Hilt DC, Bortey E, et al. A phase 3 trial of local chemotherapy with biodegradable carmustine (BCNU) wafers (Gliadel wafers) in patients with primary malignant glioma. Neuro Oncol 2003;5:79–88.
3. Norden AD, Young GS, Setayesh K, et al. Bevacizumab for recurrent malignant gliomas: efficacy toxicity, and patterns of recurrence. Neurology 2008;70:779–787.
4. The Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 2008;455:1061–1068.
5. Verhaak RGW, Hoadley KA, Purdom E, et al. An integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR and NF1. Cancer Cell 2010;17:98–110.
6. Pelloski CE, et al. Epidermal growth factor receptor variant status defines clinically distinct subtypes of glioblastoma.J.Clin.Oncol.2007;25:2288-94
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2. Lotolf, U. M., P. Kleihues, et al. Population-based study on incidence, survival rates, and genetic alterations of low-grade diffuse astrocytomas and oligodendrogliomas. Acta Neuropathologica 2004:108(1): 49-56.
3. Kopper L, Hajdu M. Tumor Stem Cells. Pathology Oncology Research 2004:10(2): 69-73.
4. Bjerkvig R, Tysnes B B, Aboody KS.The origin of the cancer stem cell: current controversies and new in sights. Nature Reviews (cancer)2005: 5 (11): 899 -904.
5. Dean, M, T. Fojo, et al. Tumor stem cells and drug resistance.Nature Reviews Cancer 2005:5(4): 275-284.
6. Reya T,Morrison SJ, ClarkeMF and WeissmanIL. Stem cells,Cancer,and Cancer stem cells. Nature 2001:414:105-111.
7. Hemmati, H. D. et al.Cancerous stem cells can arise from pediatric brain tumors.Proceedings of the National Academy of Sciences 2003:100(25): 15178-15183.
8. Singh S K, Clarke I D, Terasaki M, et al. Identification of a cancer stem cell in human brain tumors.Cancer Res 2003:63(18): 5821-5828.
9. Uchida N, Bcuk D W, He D, et al. Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci U S A 2003:97(26): 14720-14725.
10. Reynolds BA, Weiss S. Clonal and population analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 1996:175:1-13.
11. Yuan, X., J. Curtin, et al. Isolation of cancer stem cells from adult glioblastoma multiforme. Oncogene 2004:23(58): 9392-9400.
12. Liu, Q., D. H. Nguyen, et al. Molecular properties of CD133+ glioblastoma stem cells derived from treatment-refractory recurrent brain tumors. Journal of Neuro-Oncology 2009:94(1): 1-19.
13. Michalczyk K, Ziman M. Nestin structure and predicted function in cellular cytoskeletal organization.Histol.Histopathol.2005:20 (2): 665–71.
14. Bidlingmaier, S., X. Zhu, et al. The utility and limitations of glycosylated human CD133 epitopes in defining cancer stem cells.Journal of Molecular Medicine 2008:86(9): 1025-1032.
15. Beier, D., P. Hau, et al. CD133+ and CD133- Glioblastoma-Derived Cancer Stem Cells Show Differential Growth Characteristics and Molecular Profiles. Cancer Research 2007:67(9): 4010-4015.
16. Singh SK, Hawkins C, Clarke ID et al. Identification of human brain tumour initiating cells. Nature 2004:432:396–401.
17. Zeppernick, F., R. Ahmadi, et al. Stem Cell Marker CD133 Affects Clinical Outcome in Glioma Patients. Clinical Cancer Research 2008:14(1): 123-129.
18. Challen, G. A. and M. H. Little. A Side Order of Stem Cells: The SP Phenotype. Stem Cells 2006:24(1): 3-12.
19. Patrawala L, Calhoun T, Schneider-Broussard R, Zhou J, Claypool K, Tang DG. Side population is enriched in tumorigenic, stem-like cancer cells, whereas ABCG2+ and ABCG2– cancer cells are similarly tumorigenic. Cancer Res 2005:65:6207–6219.
20. Bleau, A.-M., D. Hambardzumyan, et al. PTEN/PI3K/Akt Pathway Regulates the Side Population Phenotype and ABCG2 Activity in Glioma Tumor Stem-like Cells. Cell Stem Cell 2009:4(3): 226-235.
21. Chiba, T., K. Kita, et al. Side population purified from hepatocellular carcinoma cells harbors cancer stem cell-like properties. Hepatology 2006:44(1): 240-251.
22. Hirschmann-Jax, C.et al. A distinct "side population" of cells with high drug efflux capacity in human tumor cells. Proceedings of the National Academy of Sciences 2004:101(39): 14228-14233.
23. Harris, M. A., H. Yang, et al. Cancer Stem Cells Are Enriched in the Side Population Cells in a Mouse Model of Glioma. Cancer Research 2008:68(24): 10051-10059.
24. Wan F, Zhang S, Xie R, et al. The utility and limitations of neurosphere assay, CD133 immunophenotyping and side population assay in glioma stem cell research. BrainPathol 2010:20: 877–889.
25.唐珂,曹利等。全反式维甲酸对C6胶质瘤细胞增殖分化的影响。中南大学学报(医学版)2008:33(10):892-897
26. Karsy M, Albert L et al. All-trans retinoic acid modulates cancer stem cells of glioblastoma multiforme in an MAPK-dependent manner. Anticancer Research 2010:30:4915-4920.
27. Campos B, Wan F, Herold-Mende C.et al. Differentiation therapy exerts antitumor effects on stem-like glioma cells. Clin Cancer Res 2010:16(10): 2715–2728.
28. Gudas LJ, Wagner JA. Retinoids regulate stem cell differentiation. J Cell Physiol 2011:226:322-330.
29.綦斌,罗毅男,田宇等。全反式维甲酸对脑胶质瘤细胞Cyclin E,Caspase3表达的影响。中华实验外科杂志,2006:23(6):721-723.
30. Das, A., N. L. Banik, et al. Retinoids induced astrocytic differentiation with down regulation of telomerase activity and enhanced sensitivity to taxol for apoptosis in human glioblastoma T98G and U87MG cells. Journal of Neuro-Oncology 2007:87(1): 9-22.
31. Kaba SE, Kyritsis AP, Conrad C, et al. The treatment of recurrent cerebral gliomas with all-trans-retinoic acid tretinoin. J Neurooncol 997:34:145–51.
32. Phuphanich S, Scott C, Fischbach AJ, Langer C, Yung WK. Alltrans-retinoic acid: a phase II Radiation Therapy Oncology Group study RTOG 91-13 in patients with recurrent malignant astrocytoma. J Neurooncol 1997:34:193–200.
33.章翔,程光,费舟等。苦参碱对脑胶质瘤C6细胞系的诱导分化。[J]中华神经外科疾病研究杂志,2003.3(2):124-126
34. Karmakar, S., N. L. Banik, et al.Combination of all-trans retinoic acid and paclitaxel-induced differentiation and apoptosis in human glioblastoma U87MG xenografts in nude mice.Cancer 2008:112(3): 596-607.
1. Stieber VW, Mehta MP. Advances in radiation therapy for brain tumors. Neurol Clin 2007;25:1005–1033.
2. Westphal M, Hilt DC, Bortey E, et al. A phase 3 trial of local chemotherapy with biodegradable carmustine (BCNU) wafers (Gliadel wafers) in patients with primary malignant glioma. Neuro Oncol 2003;5:79–88.
3. Norden AD, Young GS, Setayesh K, et al. Bevacizumab for recurrent malignant gliomas: efficacy toxicity, and patterns of recurrence. Neurology 2008;70:779–787.
4. The Cancer Genome Atlas Research Network. Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 2008;455:1061–1068.
5. Verhaak RGW, Hoadley KA, Purdom E, et al. An integrated genomic analysis identifies clinically relevant subtypes of glioblastoma characterized by abnormalities in PDGFRA, IDH1, EGFR and NF1. Cancer Cell 2010;17:98–110.
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