反义HIF-1α基因转染提高口腔癌放疗敏感性的相关基础研究
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摘要
氧依赖性转录活化细胞因子低氧诱导因子-1α(Hypoxia inducible factor-1α,HIF-1α)在恶性肿瘤普遍存在的低氧微环境中处于重要的中枢调节地位,其调控的下游靶基因近百种,除涉及肿瘤生长、发展、侵袭、肿瘤细胞凋亡、血管生成外,还导致肿瘤细胞对放化疗的抵抗,其表达水平对恶性肿瘤的放射治疗敏感性与预后具有重要的影响。
本研究拟通过免疫组化染色了解HIF-1α在人口腔癌中的表达及其与口腔癌微血管密度之间的关系,探讨口腔癌HIF-1α表达水平对放化疗敏感性和临床预后的影响;并通过口腔癌细胞~(192)Irγ射线近距离放射治疗体外研究,了解口腔癌~(192)Irγ射线近距离放射治疗的放射生物学效应及其敏感性,为口腔癌临床治疗提供放射生物学实验基础,并为针对口腔癌低氧状态下的处于中枢调节地位的HIF-1α治疗研究提供实验基础。在此基础上,应用反义基因治疗手段反义阻断HIF-1α表达,研究主动下调HIF-1α表达水平对口腔癌放射敏感性
Hypoxia inducible factor-1α, as an oxygen-dependent regulation transcription cell factor and regulates less than 100 putative target genes, is a master regulator of the transcription of many different factors involved in the biological progress of hypoxia malignant tumors. HIF-la is a vital important biomarker of malignant tumor prognosis and helps reduce tumor radiosensitivity. The purpose of this study was to investigate the expression of HIF-1α in oral cancer and its clinical and prognosis signification, to evaluate the radiobiological response in vitro of human oral cancer cell line TCa8113 and BCaCD885. Furthermore, we try to investigate the effect of in vitro transfection of antisense HIF-la oligonucleotides under normoxic condition on the radiosensitivity of the two tumor cell lines. 1. LsAB immunohistochemistry staining was applied in this study to evaluate the expression levels of HIF-1α and CD34 in oral cancers. The relationship within HIF-1α expression, clinical pathological
factors and tumor prognosis were investigated. 2. MTT assay was employed to detect the survival fraction of the two oral cancer cell lines after different dose of 192Ir high dose rate γ ray brachytherapy. The survival curves were synthesized according to the MTT results via Sigma Plot 8.0, and some of the main radiobiological parameters were determined by statistic program. The morphological and ultrastructure changes of the two cell lines after brachytherapy were also observed through both optical microscope and transmission electron microscope. 3. Antisense HIF-1α oligonucleotides were transfected into TCa8113 and BCaCD885 cells. Changes of the radiobiological reaction of the two cell lines via flow cytometry and MTT assay.The results show that 1. HIF-1α high expression was observed in both tongue and buccal carcinoma specimens. There was no significant relationship between HIF-1α expression and the pathological differentiation, while high expression of HIF-1α was correlated with higher microvessels density, the clinical stages and tumor prognosis. 2. Both TCa8113 and BCaCD885 cells showed low radiosensitivity to 192Ir γ ray, SF2 values of which were 0. 878 and 0. 862 respectively. 3. Antisense HIF-1α oligonucleotides transfection under normoxic condition increased the apoptosis rate to the two cell lines significantly,
the SER SF2 values of TCa8113 and BCaCD885 cells were 1.52 and 1.48 respectively.Based on these findigs, there exists over expression of HIF-1α in oral cancers, which is a vital important biomarker of oral cancer prognosis and helps reduce tumor radiosensitivity. In vitro antisense blocking of the HIF-1α mRNA expression under normoxic condition can significantly improve the radiosensitivity of oral cancer cells and increase the apoptosis rate of oral cancer cells after 192Ir y ray brachytherapy.
本研究拟通过免疫组化染色了解HIF-1α在人口腔癌中的表达及其与口腔癌微血管密度之间的关系,探讨口腔癌HIF-1α表达水平对放化疗敏感性和临床预后的影响;并通过口腔癌细胞~(192)Irγ射线近距离放射治疗体外研究,了解口腔癌~(192)Irγ射线近距离放射治疗的放射生物学效应及其敏感性,为口腔癌临床治疗提供放射生物学实验基础,并为针对口腔癌低氧状态下的处于中枢调节地位的HIF-1α治疗研究提供实验基础。在此基础上,应用反义基因治疗手段反义阻断HIF-1α表达,研究主动下调HIF-1α表达水平对口腔癌放射敏感性
Hypoxia inducible factor-1α, as an oxygen-dependent regulation transcription cell factor and regulates less than 100 putative target genes, is a master regulator of the transcription of many different factors involved in the biological progress of hypoxia malignant tumors. HIF-la is a vital important biomarker of malignant tumor prognosis and helps reduce tumor radiosensitivity. The purpose of this study was to investigate the expression of HIF-1α in oral cancer and its clinical and prognosis signification, to evaluate the radiobiological response in vitro of human oral cancer cell line TCa8113 and BCaCD885. Furthermore, we try to investigate the effect of in vitro transfection of antisense HIF-la oligonucleotides under normoxic condition on the radiosensitivity of the two tumor cell lines. 1. LsAB immunohistochemistry staining was applied in this study to evaluate the expression levels of HIF-1α and CD34 in oral cancers. The relationship within HIF-1α expression, clinical pathological
factors and tumor prognosis were investigated. 2. MTT assay was employed to detect the survival fraction of the two oral cancer cell lines after different dose of 192Ir high dose rate γ ray brachytherapy. The survival curves were synthesized according to the MTT results via Sigma Plot 8.0, and some of the main radiobiological parameters were determined by statistic program. The morphological and ultrastructure changes of the two cell lines after brachytherapy were also observed through both optical microscope and transmission electron microscope. 3. Antisense HIF-1α oligonucleotides were transfected into TCa8113 and BCaCD885 cells. Changes of the radiobiological reaction of the two cell lines via flow cytometry and MTT assay.The results show that 1. HIF-1α high expression was observed in both tongue and buccal carcinoma specimens. There was no significant relationship between HIF-1α expression and the pathological differentiation, while high expression of HIF-1α was correlated with higher microvessels density, the clinical stages and tumor prognosis. 2. Both TCa8113 and BCaCD885 cells showed low radiosensitivity to 192Ir γ ray, SF2 values of which were 0. 878 and 0. 862 respectively. 3. Antisense HIF-1α oligonucleotides transfection under normoxic condition increased the apoptosis rate to the two cell lines significantly,
the SER SF2 values of TCa8113 and BCaCD885 cells were 1.52 and 1.48 respectively.Based on these findigs, there exists over expression of HIF-1α in oral cancers, which is a vital important biomarker of oral cancer prognosis and helps reduce tumor radiosensitivity. In vitro antisense blocking of the HIF-1α mRNA expression under normoxic condition can significantly improve the radiosensitivity of oral cancer cells and increase the apoptosis rate of oral cancer cells after 192Ir y ray brachytherapy.
引文
1. Semenza GL, Wang GL. A nuclear factor induced by hypoxia via de novo protein synthesis binds to the human erythropoietin gene enhancer at a site required for transcriptional activation. Mol Cell Biol.1992 Dec; 12(12):5447-54.
2.邱尉六主编。口腔颌面外科学。第三版,北京:人民卫生出版社,1997,271
3.吴奇光主编。口腔组织病理学。第三版,北京:人民卫生出版社,1994,209
4.朱立平,陈学清主编。免疫学常用实验方法,第一版,北京:人民军医出版社,2000,382
5. Price P, McMillan TJ. The use of non-clonogenic assays in measuring the response of cells in vitro to raising radiation. Enr J Cancer. Review. Currt Radiat Oncol, 1994; 30A(6): 838-841
6.刘泰福.从肿瘤生物学研究放射治疗.中华放射肿瘤杂志,1993;2:209-211
7. Hua Zhong, De Marzo AM, Laughner E, et al. Overexpression of hypoxia-inducible factor1α in common human cancers and their metastases. Cancer Res. 1999, 59(22): 5830-5835
8. Talks KL, Turley H, Garter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-1a and HIF-2a in normal human tissues, cancers, and tumor-associated macrophages. Am J Patholo.2000; 157(2): 411-421.
9. Dai S, Huang ML, Hsu CY, et al. Inhibition of hypoxia inducible factor lalpha causes oxygen-independent cytotoxicity and induces p53 independent apoptosis in glioblastoma cells. Int J Radiat Oncol Biol Phys.2003; 55 (4): 1027-36
10. Beasley NJ, Leek R, Alam M, et al. Hypoxia-inducible factors HIF-1alpha and HIF-2alpha in head and neck cancer: relationship to tumor biology and treatment outcome in surgically resected patients. Cancer Res. 2002; 62 (9): 2493-7
11. Koukourakis MI, Giatromanolaki A, Fountzilas G, et al. Angiogenesis, thymidine phosphorylase and resistance of squamous cell head and neck cancer to cytotoxic and radiationtherapy. Clin Cancer Res 2000; 6 (2):381-389
12. Zhong H, Chiles K, Feldser D, et al. Modulation of HIF-1α expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res. 2000; 60(6): 1541-1545
13. Buchler P, Reber HA, Buchler M, et al. Hypoxia-inducible factor 1 regulates vascular endothelial growth factor expression in human pancreatic cancer. Pancreas. 2003;26(1):56-64
14. Stoeltzing O, McCarty MF, Wey JS, et al. Role of hypoxia-inducible factor lalpha in gastric cancer cell growth, angiogenesis, and vessel maturation. J Natl Cancer Inst. 2004;96(12):946-56
15. Sondergaard KL, Hilton DA, Penney M, et al. Expression of hypoxia-inducible factor 1 alpha in tumours of patients with glioblastoma. Neuropathol Appl Neurobiol. 2002;28(3):210-7.
16. Bos R, van der Groep P, Greijer AE, et al. Levels of hypoxia-inducible factor-1 alpha independently predict prognosis in patients with lymph node negative breast carcinoma. Cancer. 2003;97(6):1573-81.
17. Gunther Gruber, Richard H Greiner, Ruslan Hlushchuk, et al. Hypoxia-inducible factor 1 alpha in high-risk breast cancer: an independent prognostic parameter? Breast Cancer Research. 2000;6(3):191-198
18. Zhong H, De Marzo AM, Laughner E, et al. Overexpression of hypoxia-inducible factor 1α in common human cancers and their
matastasis. Cancer Res. 1999; 59(15): 5830-5835
19. Banasiak D, Barnetson AR, Odell RA, et al. Comparison between the clonogenic, MTT, and SRB assays for determining radiosensitivity in a panel of human bladder cancer cell lines and a ureteral cell line. Radiat Oncol Investig. 1999; 7(2): 77-85.
20.江浩,夏云飞,钱剑扬等.三种方法检测鼻咽癌细胞株SF2的结果分析.蚌埠医学院学报.2003;28(3):203-205.
21.汪俊,沈瑜,糜富顺。细胞存活曲线三种拟合方法的比较和改进.中华放射学与防护杂志.1988;8(2):117-119.
22.Manch PM, Loeffier JS, et al. Radiation Oncology: technology and biology. Philadelphia: WB Saunders Co. 1995; 532～545
23.刘建华,路梅,陈华.金黄地鼠颊囊鳞癌的192铱放射损伤超微结构研究.浙江大学学报(医学版).2000;29(1):16～18.
24.刘泰福主编.现代放射肿瘤学.上海:复旦大学出版社,2001,7-8
25. Pekkola-Heino K, Servomaa K, Kiuru A, et al. Increaed radiosensitivity is associated with P53 mutation in cell lines derived from oral cavity carcinoma. Acta Otolaryngol Stockh. 1996; 116(2):341
26. Fertil B, Malaise EP. Intrinsic radiosenstivty of human cell lines is correlated with radioresponsiveness of human tumors: analysis of 101 published survival curves. Int J Radiat Oncol Biol Phys. 1995; 11 (9): 1699
27. Gaussen A, Legal JD, Beron Gaillard N, et al. Radiosensitivity of human normal and tumor thyroid cells using fluorescence in situ hybridization and clonogenic survival. Int J Radiat Oncol Biol Phys.1999;44(3):683
28. Whitaker SJ, Ung YC, McMillan TJ. DNA bouble strand break induction and rejoining as determinants of human tumor cell radiosensitivity. A pulsed-field gel electrophoresis study. Int J Radiat Oncol Biol Phys. 1995;67(1):7
29. Kiltie AE, Orton CJ, Ryan AJ, et al. A correlation between residual DNA double strand break and clonogenic measurements of radiosensitivity in fibroblastos from preradiotherapy cervix cancer patients. Int J Radiat Oncol Biol Phys. 1997; 39(5): 1137
30.刘斌,司徒镇强,吴军正等.人粘液表皮样癌MEC-1细胞放射敏感性的研究.实用口腔医学杂志.1997;13(2):126-128
31. Hockel M, Schlenger K, Mitze M, et al. Hypoxia and radiation response in human tumors. Semin in Rdiat Oncol. 1996;6:3-9
32. Ciaccia AJ. Hypoxia stress proteins: survival of the fittest. Semin in Rdiat Oncol. 1996; 6: 46-58
33. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer. 2003;3 (10): 721-732
34. Moeller BJ, Cao Y, Li CY, et al. Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules. Cancer Cell. 2004; 5(5): 429-41.
35. Semenza GL. Intratumoral hypoxia, radiation resistance, and HIF-1. Cancer Cell. 2004; 5(5): 405-6.
36. Quintero M, Mackenzie N, Brennan PA. Hypoxia-inducible factor 1 (HIF-1) in caneer. Eur J Surg Oncol. 2004; 30(5): 465-8.
37. Weinmann M, Marini P, Jendrossek V, et al. Influence of hypoxia on TRAIL-induced apoptosis in tumor cells.Int J Radiat Oncol Biol Phys. 2004;58(2):386-96.
38. Sang N, Stiehl DP, Bohensky J, et al. MAPK signaling up-regulates the activity of hypoxia-inducible factors by its effects on p300. J. Biol.Chem. 2003; 278 (16): 14013-14019
39. Tanimoto K, Makino Y, Pereira T, et al. Mechanism of regulation of the hypoxia-inducible factor-1α by the von Hippel-Lindau tumor protein. EMBO J. 2000; 19 (16), 4298-4309
40. Caniggia I, Mostachfi H, Winter J, et al. Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGF β 3. J Clin Invest. 2000; 105(5): 577-587
41. Bardos JI, Ashcroft M. Hypoxia-inducible factor-1 and oncogenic signalling.Bioessays. 2004;26(3):262-9.
42. Sun X, Kanwar JR, Leung E, et al. Gene transfer of antisense hypoxia inducible factor-1 alpha enhances the therapeutic efficacy of cancer immunotherapy. Gene Ther. 2001 ;8(8): 638-45
43. Feldser D, Agani F, Iyer NV, et al. Reciprocal positive regulation of hypoxiainducible factor 1α and insulin-like growth factor 2. Cancer Res. 1999;59(16):3915-3918
2.邱尉六主编。口腔颌面外科学。第三版,北京:人民卫生出版社,1997,271
3.吴奇光主编。口腔组织病理学。第三版,北京:人民卫生出版社,1994,209
4.朱立平,陈学清主编。免疫学常用实验方法,第一版,北京:人民军医出版社,2000,382
5. Price P, McMillan TJ. The use of non-clonogenic assays in measuring the response of cells in vitro to raising radiation. Enr J Cancer. Review. Currt Radiat Oncol, 1994; 30A(6): 838-841
6.刘泰福.从肿瘤生物学研究放射治疗.中华放射肿瘤杂志,1993;2:209-211
7. Hua Zhong, De Marzo AM, Laughner E, et al. Overexpression of hypoxia-inducible factor1α in common human cancers and their metastases. Cancer Res. 1999, 59(22): 5830-5835
8. Talks KL, Turley H, Garter KC, et al. The expression and distribution of the hypoxia-inducible factors HIF-1a and HIF-2a in normal human tissues, cancers, and tumor-associated macrophages. Am J Patholo.2000; 157(2): 411-421.
9. Dai S, Huang ML, Hsu CY, et al. Inhibition of hypoxia inducible factor lalpha causes oxygen-independent cytotoxicity and induces p53 independent apoptosis in glioblastoma cells. Int J Radiat Oncol Biol Phys.2003; 55 (4): 1027-36
10. Beasley NJ, Leek R, Alam M, et al. Hypoxia-inducible factors HIF-1alpha and HIF-2alpha in head and neck cancer: relationship to tumor biology and treatment outcome in surgically resected patients. Cancer Res. 2002; 62 (9): 2493-7
11. Koukourakis MI, Giatromanolaki A, Fountzilas G, et al. Angiogenesis, thymidine phosphorylase and resistance of squamous cell head and neck cancer to cytotoxic and radiationtherapy. Clin Cancer Res 2000; 6 (2):381-389
12. Zhong H, Chiles K, Feldser D, et al. Modulation of HIF-1α expression by the epidermal growth factor/phosphatidylinositol 3-kinase/PTEN/AKT/FRAP pathway in human prostate cancer cells: implications for tumor angiogenesis and therapeutics. Cancer Res. 2000; 60(6): 1541-1545
13. Buchler P, Reber HA, Buchler M, et al. Hypoxia-inducible factor 1 regulates vascular endothelial growth factor expression in human pancreatic cancer. Pancreas. 2003;26(1):56-64
14. Stoeltzing O, McCarty MF, Wey JS, et al. Role of hypoxia-inducible factor lalpha in gastric cancer cell growth, angiogenesis, and vessel maturation. J Natl Cancer Inst. 2004;96(12):946-56
15. Sondergaard KL, Hilton DA, Penney M, et al. Expression of hypoxia-inducible factor 1 alpha in tumours of patients with glioblastoma. Neuropathol Appl Neurobiol. 2002;28(3):210-7.
16. Bos R, van der Groep P, Greijer AE, et al. Levels of hypoxia-inducible factor-1 alpha independently predict prognosis in patients with lymph node negative breast carcinoma. Cancer. 2003;97(6):1573-81.
17. Gunther Gruber, Richard H Greiner, Ruslan Hlushchuk, et al. Hypoxia-inducible factor 1 alpha in high-risk breast cancer: an independent prognostic parameter? Breast Cancer Research. 2000;6(3):191-198
18. Zhong H, De Marzo AM, Laughner E, et al. Overexpression of hypoxia-inducible factor 1α in common human cancers and their
matastasis. Cancer Res. 1999; 59(15): 5830-5835
19. Banasiak D, Barnetson AR, Odell RA, et al. Comparison between the clonogenic, MTT, and SRB assays for determining radiosensitivity in a panel of human bladder cancer cell lines and a ureteral cell line. Radiat Oncol Investig. 1999; 7(2): 77-85.
20.江浩,夏云飞,钱剑扬等.三种方法检测鼻咽癌细胞株SF2的结果分析.蚌埠医学院学报.2003;28(3):203-205.
21.汪俊,沈瑜,糜富顺。细胞存活曲线三种拟合方法的比较和改进.中华放射学与防护杂志.1988;8(2):117-119.
22.Manch PM, Loeffier JS, et al. Radiation Oncology: technology and biology. Philadelphia: WB Saunders Co. 1995; 532～545
23.刘建华,路梅,陈华.金黄地鼠颊囊鳞癌的192铱放射损伤超微结构研究.浙江大学学报(医学版).2000;29(1):16～18.
24.刘泰福主编.现代放射肿瘤学.上海:复旦大学出版社,2001,7-8
25. Pekkola-Heino K, Servomaa K, Kiuru A, et al. Increaed radiosensitivity is associated with P53 mutation in cell lines derived from oral cavity carcinoma. Acta Otolaryngol Stockh. 1996; 116(2):341
26. Fertil B, Malaise EP. Intrinsic radiosenstivty of human cell lines is correlated with radioresponsiveness of human tumors: analysis of 101 published survival curves. Int J Radiat Oncol Biol Phys. 1995; 11 (9): 1699
27. Gaussen A, Legal JD, Beron Gaillard N, et al. Radiosensitivity of human normal and tumor thyroid cells using fluorescence in situ hybridization and clonogenic survival. Int J Radiat Oncol Biol Phys.1999;44(3):683
28. Whitaker SJ, Ung YC, McMillan TJ. DNA bouble strand break induction and rejoining as determinants of human tumor cell radiosensitivity. A pulsed-field gel electrophoresis study. Int J Radiat Oncol Biol Phys. 1995;67(1):7
29. Kiltie AE, Orton CJ, Ryan AJ, et al. A correlation between residual DNA double strand break and clonogenic measurements of radiosensitivity in fibroblastos from preradiotherapy cervix cancer patients. Int J Radiat Oncol Biol Phys. 1997; 39(5): 1137
30.刘斌,司徒镇强,吴军正等.人粘液表皮样癌MEC-1细胞放射敏感性的研究.实用口腔医学杂志.1997;13(2):126-128
31. Hockel M, Schlenger K, Mitze M, et al. Hypoxia and radiation response in human tumors. Semin in Rdiat Oncol. 1996;6:3-9
32. Ciaccia AJ. Hypoxia stress proteins: survival of the fittest. Semin in Rdiat Oncol. 1996; 6: 46-58
33. Semenza GL. Targeting HIF-1 for cancer therapy. Nat Rev Cancer. 2003;3 (10): 721-732
34. Moeller BJ, Cao Y, Li CY, et al. Radiation activates HIF-1 to regulate vascular radiosensitivity in tumors: role of reoxygenation, free radicals, and stress granules. Cancer Cell. 2004; 5(5): 429-41.
35. Semenza GL. Intratumoral hypoxia, radiation resistance, and HIF-1. Cancer Cell. 2004; 5(5): 405-6.
36. Quintero M, Mackenzie N, Brennan PA. Hypoxia-inducible factor 1 (HIF-1) in caneer. Eur J Surg Oncol. 2004; 30(5): 465-8.
37. Weinmann M, Marini P, Jendrossek V, et al. Influence of hypoxia on TRAIL-induced apoptosis in tumor cells.Int J Radiat Oncol Biol Phys. 2004;58(2):386-96.
38. Sang N, Stiehl DP, Bohensky J, et al. MAPK signaling up-regulates the activity of hypoxia-inducible factors by its effects on p300. J. Biol.Chem. 2003; 278 (16): 14013-14019
39. Tanimoto K, Makino Y, Pereira T, et al. Mechanism of regulation of the hypoxia-inducible factor-1α by the von Hippel-Lindau tumor protein. EMBO J. 2000; 19 (16), 4298-4309
40. Caniggia I, Mostachfi H, Winter J, et al. Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGF β 3. J Clin Invest. 2000; 105(5): 577-587
41. Bardos JI, Ashcroft M. Hypoxia-inducible factor-1 and oncogenic signalling.Bioessays. 2004;26(3):262-9.
42. Sun X, Kanwar JR, Leung E, et al. Gene transfer of antisense hypoxia inducible factor-1 alpha enhances the therapeutic efficacy of cancer immunotherapy. Gene Ther. 2001 ;8(8): 638-45
43. Feldser D, Agani F, Iyer NV, et al. Reciprocal positive regulation of hypoxiainducible factor 1α and insulin-like growth factor 2. Cancer Res. 1999;59(16):3915-3918