Survivin反义寡核苷酸诱导白血病细胞K562凋亡及对化疗的增敏作用
摘要
白血病在当前是危害人们健康的十大肿瘤之一，在青少年恶性肿瘤中发病第一。化疗仍然是治疗的主要手段，但对正常细胞的毒性和肿瘤细胞耐药影响了治疗效果。近年来，迅速发展的细胞生物学使人们认识到细胞凋亡在肿瘤发生发展中起重要作用，降低肿瘤细胞凋亡阈，增加肿瘤细胞的凋亡敏感性是提高临床化疗的一个重要方法。研究发现，体内存在凋亡抑制基因，肿瘤细胞高表达这些抑制基因或蛋白是肿瘤细胞逃避药物作用的原因之一。在人类凋亡抑制蛋白(IAP)家族中生存素(Survivin)仅选择性地表达在肿瘤中，在正常成熟组织无表达，Survivin的这种特性使之可作为肿瘤治疗的靶点。本课题通过Survivin反义寡核苷酸(ASODN)封闭其基因表达，从而抑制肿瘤细胞增殖，增加肿瘤细胞凋亡，增加化疗的敏感性。
一、Survivin ASODN对白血病K562细胞的增殖和对化疗药物敏感性的影响
目的：探讨Survivin ASODN对白血病K562细胞的增殖和对化疗药物敏感性的影响。方法：人工合成Survivin硫代ASODN，通过脂质体(liposomes，Lip)转染K562细胞后，用MTT法检测对照组、Lip组、Lip+正义寡核苷酸(SODN)组、Lip+ASODN组，在不同浓度(200、400、
Leukemia is one of 10 cancers threathening people's health, which is the first one in ten-age people. Chemotherapy is still major method, but toxicity to normal cells and resistance to multidrugs influence therapeutic effect. Recently,cytobiology progress made people realize that apoptosis plays an important role in oncogenesis and advance.Decrease in apoptosis threshold and increase in cell apoptosis are important methods for improving clinic effect. Higher expression of inhibitors of apoptosis protein(IAP) ,which has been identified in human body, is one of the reasons for escaping anticancer drugs. In IAP family, survivin selectly expresses in tumor tissue, lacks of expression in normal adult tissue, which made survivin turn into a therapeutic target. In this study the expression of survivin gene was blocked by its antisense oligodeoxynucleotide(ASODN),which inhibited tumor cells' proliferation, increases tumor cells apoptosis aiming at enhancing chemotherapy sensitivity.
Part I. Effect of Survivin antisense oligodeoxynucleotide on inhibition rate and chemotherapy sensitivity of leukemic cell K562
Objective: To explore the effect of ASODN targeting survivin on inhibition rate and chemotherapy sensitivity of leukemic cell K562. Methods: Survivin ASODN was synthesized and transfected into K562 cells by liposome (Lip). MTT assay was used to detect cytotoxicity of
一、Survivin ASODN对白血病K562细胞的增殖和对化疗药物敏感性的影响
目的：探讨Survivin ASODN对白血病K562细胞的增殖和对化疗药物敏感性的影响。方法：人工合成Survivin硫代ASODN，通过脂质体(liposomes，Lip)转染K562细胞后，用MTT法检测对照组、Lip组、Lip+正义寡核苷酸(SODN)组、Lip+ASODN组，在不同浓度(200、400、
Leukemia is one of 10 cancers threathening people's health, which is the first one in ten-age people. Chemotherapy is still major method, but toxicity to normal cells and resistance to multidrugs influence therapeutic effect. Recently,cytobiology progress made people realize that apoptosis plays an important role in oncogenesis and advance.Decrease in apoptosis threshold and increase in cell apoptosis are important methods for improving clinic effect. Higher expression of inhibitors of apoptosis protein(IAP) ,which has been identified in human body, is one of the reasons for escaping anticancer drugs. In IAP family, survivin selectly expresses in tumor tissue, lacks of expression in normal adult tissue, which made survivin turn into a therapeutic target. In this study the expression of survivin gene was blocked by its antisense oligodeoxynucleotide(ASODN),which inhibited tumor cells' proliferation, increases tumor cells apoptosis aiming at enhancing chemotherapy sensitivity.
Part I. Effect of Survivin antisense oligodeoxynucleotide on inhibition rate and chemotherapy sensitivity of leukemic cell K562
Objective: To explore the effect of ASODN targeting survivin on inhibition rate and chemotherapy sensitivity of leukemic cell K562. Methods: Survivin ASODN was synthesized and transfected into K562 cells by liposome (Lip). MTT assay was used to detect cytotoxicity of
引文
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[03] Li F, Altier DC. The cancer antiapoptosis mouse survivin gene:characterization of locus and transcriptional requirements of basal and cell cyc-dependent expression. Cancer Res, 1999, 59(13):3143-3151.
[04] Li F, Ambrosini G, Chu EY, et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature, 1998,396(6711): 580-584.
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[09] Ikeguchi M, Ueda T, Sakatani T, et al. Expression of survivin messenger RNA correlates with poor prognosis in patients with hepatocellular carcinoma. Diagn Mol Pathol, 2002,11(1): 33-40.
[10] Monozo M, Rosell R, Felip E, et al. A novel anti-apoptosis gene: Re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers. J Clin Oncol, 1999,17(7): 2100-2104.
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[12] Altieri DC. The molecular basis and potential role of survivin in cancer diagnosis??and therapy. Trends Mol Med, 2001 ,7(12): 542-547.
[13] Trieb K, Lehner R, Stulnig T , et al. Survivin expression in human osteosarcoma is a marker for survival. Eur J Surg Oncol, 2003 ,29(4) :379-382.
[14] Kerr JF, Winterford CM, Harmon BV.Apoptosis, its significance in cancer and cancer therapy. Cancer, 1994,73(8): 2013-2026.
[15] Zamecnik PC, Stephenson ML. Inhibition of Rous sarcoma virus rep lication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci USA, 1978,75(1): 280 -284.
[16] Izant J, Weintraub HH. Inhibition of thymidine kinase gene expression by antisense RNA:A molecular approach to genetic analysis. Cell, 1984,36 (4): 1007 -1015.
[17] Yuen AR , Branimir LS. Clinical studies of antisense therapy in cancer. Front Biosci, 2000,5 (4) :D588-593.
[18] Altieri DC Marchisio PC Survivin apoptosis:an interloper between cell death and cell proliferation in cancer.Lab Invest, 1999,79(11): 1327-1333.
[19] Olie RA, Simoes-Wust AP, Baumann B, et al. A Novel Antisense Oligonucleotide Targeting Survivin Expression Induces Apoptosis and Sensitizes Lung Cancer Cells to Chemotherapy. Cancer Res, 2000,60(1): 2805-2809.
[20] Marks DC, Belov L, Davey MW, et al. The MTT cell viability assay for cytotoxicity testing in multidrug resistant human leukemia cells. Leuk Res, 1992, 10(12)1165-1173.
[21] Gutierrez Puente Y, Zapata Benavides P, Tari AM, et al. Bcl - 2 - related antisense therapy. Semin Oncol, 2002,29(3Suppl 11): 71 - 76.
[22] Mori A,Wada H, Nishimura Y, et al. Expression of the antiapoptosis gene survivin in human leukemia. Int J Haematol, 2002,75 (2) : 161 — 165.
[23] Kamihira S, Yamada Y, Hirakata Y, et al.Aberrant expression of caspase cascade regulatory genes in adult T-cell leukemia: survivin is an important determinant for prognosis. Br J Haematol, 2001,114 (1) : 63—69.
[24] Adida C, Recher C, Raffoux E, et al. Expression and prognostic significance of survivin in denovo acute myeloid leukemia. Br J Haematol, 2000,111 (1) :196— 203.
[25] Adida C, Berrebi D, Peuchmaur M, et al. Anti-apoptosis gene, survivin, andprognosis of neuroblastoma. Lancet, 1998,351(9106):882—883.
[26] Schimmer AD, Hedley DW, Penn LZ, et al. Receptor- and mitochondrial-mediated apoptosis in acute leukemia: a translational view. Blood, 2001, 98(13): 3541-3553.
[27] Fadeel B, Orrenius S, Zhivotovsky B. The most unkindest cut of all: on the multiple roles of mammalian caspases. Leukemia, 2000,14(8): 1514-1525.
[28] Rokhlin OW, Glover RA, Cohen MB. Fas-mediated apoptosis in human prostatic carcinoma cell lines occurs via activation of caspase-8 and caspase-7. Cancer Res, 1998,58(24): 5870-5875.
[29] Hu Y, Ding L, Spencer DM, et al. WD-40 repeat region regulates Apaf-1 self-association and procaspase-9 activation, J Biol Chem, 1998, 273(50): 33489-33494.
[30] Kang JJ, Schaber MD, Srinivasula SM, et al. Cascades of mammalian caspase activation in the yeast Saccharomyces cerevisiae. J Biol Chem, 1999, 274(5): 3189-3198.
[31] StennickeHR, Jurgensmeier JM, Shin H, et al. Pro-caspase-3 is a major physiologic target of caspase-8. J Biol Chem, 1998,273(42): 27084-27090.
[32] Raff M. Cell suicide for beginners . Nature, 1998,396 (6707) :119-122.
[33] Cohen GM. Caspase: the executioners of apoptosis. Biochem J , 1997,326(pt 1):1-16.
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