氧化损伤对肿瘤细胞生长调控的研究
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摘要
以往研究显示：一氧化氮(NO)具有广泛的生物调节活性，可通过调节多种基因的表达，抑制肿瘤细胞的生长繁殖；自由基所致生物大分子的损伤可介导多种基因表达的改变。为进一步探讨自由基对肿瘤细胞生长繁殖的调控机制，通过研究光照核黄素、合成光核酸酶和L-精氨酸(L-arg)不同浓度对培养肿瘤细胞的生物学作用，探索了其对恶性肿瘤的抑制作用及机制。
一、L-精氨酸、光照核黄素及合成的光核酸酶浓度依赖性抑制肿瘤细胞生长繁殖 当培养肿瘤细胞系(Hela细胞)中加入中低浓度L-arg(0～30mmol／L)、核黄素(0.016 to32μmol／L)与光核酸酶(Br-Ph-ⅡP β Dp 0～205.2μmol／L，Br-Ph-PP β Dp 0～406.5μ mol／L)均能抑制肿瘤细胞的生长，其抑制率与药物浓度呈正相关；L-arg在较高浓度(30～100mmol／L各组)时对细胞的生长有促进的趋势，但其细胞形态发生明显变化，丧失贴壁能力；
二、光照核黄素可促进肿瘤细胞凋亡 经流式细胞仪检测核黄素给药组(32 μ mol／L，3.2 μ mol／L)与对照组(0μmol／L)凋亡指数(AI)依次为4.02，1.99，1.08，同MTT实验共同证明光照核黄素可诱发细胞凋亡；
三、光照核黄素可氧化DNA，产生8-羟基鸟嘌呤 采用8-羟基鸟嘌呤抗体免疫组化染色显示核黄素给药组(32μmol／L，3.2 μmol／L)细胞内有大量鸟嘌呤被氧化为8-羟基鸟嘌呤，且高剂
硕士学位论文
量时出现膜、核阳性，低剂量时显示核阳性;
四、光照时间与端粒的缩短呈正比在肿瘤细胞培养体系中，加入
核黄素，经不同光照时间后，采用经典方法Southem blot检测
端粒长度;发现端拉长度随给药后光照时间的延长而缩短，O，
0.5，l，2，4h光照后端粒长度分别约为4.61，4.36，4.10，3.92，
3 .79kb;
五、光照核黄素可导致与DNA损伤相关多种基因的表达发生变化
由高通量DNA损伤芯片筛选受光照核黄素处理的肿瘤细胞
mRNA后，发现GADD45，53BPI，BRCAZ，BTGZ，P55ede，
cEN甲一E，McAK，UNGZ等基因均有表达的改变，其中提高表
达的一些是与细胞周期及DNA损伤修复机制相关的基因的激
活，其中GADD45的提高表达可能提示GADD45在介导
Foxo3a修复DNA损伤前可能先激活p53或其它基因调节途
径，最终导致细胞的凋亡，抑制肿瘤细胞的生长.
总之，多种实验体系研究显示，自由基可抑制培养肿瘤细
胞的生长繁殖，其机制可能与自由基损伤DNA导致多种基因
表达异常及氧化切割缩短端粒有关。
The previous studies showed that nitric oxide possesses a wide range of biological characters, which involving in regulations on a lot kind of gene expressions, inhibition tumor cells proliferations, and so on; free radicals- inducing dysfunctions of biological macromolecules can mediate multi-changes of gene expressions. For the further research of regulations and mechanisms on tumor cells growth by free radicals, our experiments were designed to display ways of some photosensitive substance, such as riboflavin, composed photonuclease and L-arginine; those which act as donor of free radicals play different biological characters in different concentrations. In this research, the inhibition effects of oxidation on tumor cells and its mechanisms were studied.
1 The inhibition rate of tumor cells is dose-dependent on L-arginine\ riboflavin\ composed photonuclease MTT assay was used to estimate the inhibition rate of different drugs. Adding L-arginine in concentrations of 0 ~ 30mmol/L could depresss cells proliferation. The inhibition rate was positive correlation with the dose. Same with L-arginine, groups of riboflavin under 0.016 to 32 mol/L showed positive correlation with the dose. Groups of high L-arginine
dose(30 ~ 100mmol/L) have a trend of promoting cells proliferation.
2 Studies on photosensitive riboflavin induced cell apoptosis Riboflavin-given groups 32, 3.2 ju mol/L showed 4.02 and 1.99 in apoptosis index while the blank group is 1.08, which proved riboflavin could induce cell apoptosis.
3 Photosensitive riboflavin oxidizing guanine into 8-hydroxy-guanine Immnohistochemical studies by using Anti-8-hydroxyguanine antibody showed that guanine could be oxidized into 8-hydroxyguanine in both 32 and 3.2umol/L riboflavin- given groups. The high dose group appeared membranes and nucleolus positive while the low dose only showed nucleolus positive.
4 Telomere length is positive correlation with illumination time In cultured cell system, after given 32 u mol/L riboflavin and then illuminate different times, the telomere length was measured by southern blot and the results indicated that there was a continuous telomere shortens with time; in the time of 0, 0.5, 1, 2, 4h dealt with photosensitiv riboflavin, the telomere length are 4.61, 4.36, 4.10, 3.92, 3.79kb, respectively;
5 The DNA damage related gene expression changes had been found by Microarray after dealt with riboflavin The Hela cells dealt with 32 u mol/L riboflavin, and then isolated mRNA was analysised by GEArray. The results indicated that gene expressions of GADD45, 53Bpl, BRCA 2, BTG 2, P55cdc, CENP-E, MCAK, UNG 2 changed
obviously. Some of these expression changes were related to DNA damage and repair in response to stress stimuli. The data in our experiment showed that FOXO3a gene has no significant expression changes under our experimental condition. GADD45 expression may first not activate the FOXO3a-mediate DNA repair response but a p53 or other genes regulated pathway.
In conclusion, those experiment systems showed that free radicals could control the proliferations of tumor cells. The mechanism may relate with free radicals initiated damage of DNA, resulting in multi-gene expressions variation and oxidized incising and shorten telomere.
一、L-精氨酸、光照核黄素及合成的光核酸酶浓度依赖性抑制肿瘤细胞生长繁殖 当培养肿瘤细胞系(Hela细胞)中加入中低浓度L-arg(0～30mmol／L)、核黄素(0.016 to32μmol／L)与光核酸酶(Br-Ph-ⅡP β Dp 0～205.2μmol／L，Br-Ph-PP β Dp 0～406.5μ mol／L)均能抑制肿瘤细胞的生长，其抑制率与药物浓度呈正相关；L-arg在较高浓度(30～100mmol／L各组)时对细胞的生长有促进的趋势，但其细胞形态发生明显变化，丧失贴壁能力；
二、光照核黄素可促进肿瘤细胞凋亡 经流式细胞仪检测核黄素给药组(32 μ mol／L，3.2 μ mol／L)与对照组(0μmol／L)凋亡指数(AI)依次为4.02，1.99，1.08，同MTT实验共同证明光照核黄素可诱发细胞凋亡；
三、光照核黄素可氧化DNA，产生8-羟基鸟嘌呤 采用8-羟基鸟嘌呤抗体免疫组化染色显示核黄素给药组(32μmol／L，3.2 μmol／L)细胞内有大量鸟嘌呤被氧化为8-羟基鸟嘌呤，且高剂
硕士学位论文
量时出现膜、核阳性，低剂量时显示核阳性;
四、光照时间与端粒的缩短呈正比在肿瘤细胞培养体系中，加入
核黄素，经不同光照时间后，采用经典方法Southem blot检测
端粒长度;发现端拉长度随给药后光照时间的延长而缩短，O，
0.5，l，2，4h光照后端粒长度分别约为4.61，4.36，4.10，3.92，
3 .79kb;
五、光照核黄素可导致与DNA损伤相关多种基因的表达发生变化
由高通量DNA损伤芯片筛选受光照核黄素处理的肿瘤细胞
mRNA后，发现GADD45，53BPI，BRCAZ，BTGZ，P55ede，
cEN甲一E，McAK，UNGZ等基因均有表达的改变，其中提高表
达的一些是与细胞周期及DNA损伤修复机制相关的基因的激
活，其中GADD45的提高表达可能提示GADD45在介导
Foxo3a修复DNA损伤前可能先激活p53或其它基因调节途
径，最终导致细胞的凋亡，抑制肿瘤细胞的生长.
总之，多种实验体系研究显示，自由基可抑制培养肿瘤细
胞的生长繁殖，其机制可能与自由基损伤DNA导致多种基因
表达异常及氧化切割缩短端粒有关。
The previous studies showed that nitric oxide possesses a wide range of biological characters, which involving in regulations on a lot kind of gene expressions, inhibition tumor cells proliferations, and so on; free radicals- inducing dysfunctions of biological macromolecules can mediate multi-changes of gene expressions. For the further research of regulations and mechanisms on tumor cells growth by free radicals, our experiments were designed to display ways of some photosensitive substance, such as riboflavin, composed photonuclease and L-arginine; those which act as donor of free radicals play different biological characters in different concentrations. In this research, the inhibition effects of oxidation on tumor cells and its mechanisms were studied.
1 The inhibition rate of tumor cells is dose-dependent on L-arginine\ riboflavin\ composed photonuclease MTT assay was used to estimate the inhibition rate of different drugs. Adding L-arginine in concentrations of 0 ~ 30mmol/L could depresss cells proliferation. The inhibition rate was positive correlation with the dose. Same with L-arginine, groups of riboflavin under 0.016 to 32 mol/L showed positive correlation with the dose. Groups of high L-arginine
dose(30 ~ 100mmol/L) have a trend of promoting cells proliferation.
2 Studies on photosensitive riboflavin induced cell apoptosis Riboflavin-given groups 32, 3.2 ju mol/L showed 4.02 and 1.99 in apoptosis index while the blank group is 1.08, which proved riboflavin could induce cell apoptosis.
3 Photosensitive riboflavin oxidizing guanine into 8-hydroxy-guanine Immnohistochemical studies by using Anti-8-hydroxyguanine antibody showed that guanine could be oxidized into 8-hydroxyguanine in both 32 and 3.2umol/L riboflavin- given groups. The high dose group appeared membranes and nucleolus positive while the low dose only showed nucleolus positive.
4 Telomere length is positive correlation with illumination time In cultured cell system, after given 32 u mol/L riboflavin and then illuminate different times, the telomere length was measured by southern blot and the results indicated that there was a continuous telomere shortens with time; in the time of 0, 0.5, 1, 2, 4h dealt with photosensitiv riboflavin, the telomere length are 4.61, 4.36, 4.10, 3.92, 3.79kb, respectively;
5 The DNA damage related gene expression changes had been found by Microarray after dealt with riboflavin The Hela cells dealt with 32 u mol/L riboflavin, and then isolated mRNA was analysised by GEArray. The results indicated that gene expressions of GADD45, 53Bpl, BRCA 2, BTG 2, P55cdc, CENP-E, MCAK, UNG 2 changed
obviously. Some of these expression changes were related to DNA damage and repair in response to stress stimuli. The data in our experiment showed that FOXO3a gene has no significant expression changes under our experimental condition. GADD45 expression may first not activate the FOXO3a-mediate DNA repair response but a p53 or other genes regulated pathway.
In conclusion, those experiment systems showed that free radicals could control the proliferations of tumor cells. The mechanism may relate with free radicals initiated damage of DNA, resulting in multi-gene expressions variation and oxidized incising and shorten telomere.
引文
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[2] N.R. Webster J.F. Nunn Molecular structure of free radicals and their importance in biological reactions. Br. J. Anaesth. 1988, 60: 98-108
[3] J.M. Gutteridge, Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin. Chem. 1995, 41: 1819-1828
[4] Kinnula VL, Torkkeli T, Kristo P, et al. Ultrastructural and chromosomal studies of manganese superoxide dismutase in malignant mesothelioma Am J Respir Cell Mol Biol. 2004 Mar 23 [Epub ahead of print]
[5] Nam HY, Choi BH, Lee JY, et al. The role of nitric oxide in the particulate matter (PM2.5)-induced NFkappaB activation in lung epithelial cells. Toxicol Lett. 2004, 14; 148(1-2): 95-102
[6] Schafer A, Wiesmann F, Neubauer S, et al. Rapid regulation of platelet activation in vivo by nitric oxide. Circulation. 2004, 109(15): 1819-1822
[7] Wang YY, Tang ZY, Dong M, et al. Inhibition of platelet aggregation by polyaspartoyl L-arginine and its mechanism. Acta Pharmacol Sin. 2004,25(4): 469-473
[8] Dias KL, Da Silva Dias C, Barbosa-Filho JM, et al. Cardiovascular effects induced by reticuline in normotensive rats. Planta Med. 2004, 70(4): 328-333
[9] Chien YH, Bau DT, Jan KY. Nitric oxide inhibits DNA-adduct excision in nucleotide excision repair. Free Radic Biol Med. 2004, 36(8): 1011-1017
[10] Vega A, Chacon P, Monteseirin J,et al. A new role for monoamine oxidases in the modulation of macrophage-inducible nitric oxide synthase gene expression. J Leukoc Biol. 2004 Apr 1 [Epub ahead of print]
[11] Chen CJ, Raung SL, Liao SL, et al. Inhibition of inducible nitric oxide synthase expression by baicalein in endotoxin/cytokinestimulated microglia. Biochem Pharmacol. 2004, 67(5): 957-65
[12] Kim JY, Choi CY, Lee KJ, et al. Induction of inducible nitric oxide synthase and proinflammatory cytokines expression by o, p'-DDT in macrophages. Toxicol Lett. 2004, 147(3): 261-269
[13] Miller BH, Fratti RA, Poschet JF, et al. Mycobacteria Inhibit Nitric Oxide Synthase Recruitment to Phagosomes during Macrophage Infection. Infect Immun. 2004, 72(5): 2872-2878
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upregulated in acute immune complex induced inflammation. Biochem Biophys Res Commun, 1998, 9247(1): 84-87
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[20] Bernard DG, Gabilly ST, Dujardin G, et al. Overlapping specificities of the mitochondrial cytochrome c and c1 heme lyases. J Biol Chem. 2003, 278(50): 49732-49742
[21] Jin C, Barrientos A, Tzagoloff A. Yeast dihydroxybutanone phosphate synthase, an enzyme of the riboflavin biosynthetic pathway, has a second unrelated function in expression of mitochondrial respiration. J Biol Chem. 2003, 278(17): 14698-14703
[22] Shumyantseva VV, Bulko TV, Schmid RD, et al. Photochemical properties of a riboflavins/cytochrome P450 2B4 complex. Biosens
Bioelectron. 2002,17(3): 233-238
[23] Ito K, Inoue S, Yamamoto K et al. 8-Hydroxydeoxyguanosine formation at the 5' site of 5'-GG-3' sequences in double-stranded DNA by UV radiation with riboflavin. J Biol Chem. 1993, 268(18): 13221-13227
[24] LU Changyuan, HAN Zhenhui, LIU Guanshu, et al. Photophysical and photochemical processes of riboflavin (Vitamin B 2) by means of the transient absorption spectra in aqueous solution Science in China (Biochemistry) 2000(5): 428-436
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[2] N.R. Webster J.F. Nunn Molecular structure of free radicals and their importance in biological reactions. Br. J. Anaesth. 1988, 60: 98-108
[3] J.M. Gutteridge, Lipid peroxidation and antioxidants as biomarkers of tissue damage. Clin. Chem. 1995, 41: 1819-1828
[4] Kinnula VL, Torkkeli T, Kristo P, et al. Ultrastructural and chromosomal studies of manganese superoxide dismutase in malignant mesothelioma Am J Respir Cell Mol Biol. 2004 Mar 23 [Epub ahead of print]
[5] Nam HY, Choi BH, Lee JY, et al. The role of nitric oxide in the particulate matter (PM2.5)-induced NFkappaB activation in lung epithelial cells. Toxicol Lett. 2004, 14; 148(1-2): 95-102
[6] Schafer A, Wiesmann F, Neubauer S, et al. Rapid regulation of platelet activation in vivo by nitric oxide. Circulation. 2004, 109(15): 1819-1822
[7] Wang YY, Tang ZY, Dong M, et al. Inhibition of platelet aggregation by polyaspartoyl L-arginine and its mechanism. Acta Pharmacol Sin. 2004,25(4): 469-473
[8] Dias KL, Da Silva Dias C, Barbosa-Filho JM, et al. Cardiovascular effects induced by reticuline in normotensive rats. Planta Med. 2004, 70(4): 328-333
[9] Chien YH, Bau DT, Jan KY. Nitric oxide inhibits DNA-adduct excision in nucleotide excision repair. Free Radic Biol Med. 2004, 36(8): 1011-1017
[10] Vega A, Chacon P, Monteseirin J,et al. A new role for monoamine oxidases in the modulation of macrophage-inducible nitric oxide synthase gene expression. J Leukoc Biol. 2004 Apr 1 [Epub ahead of print]
[11] Chen CJ, Raung SL, Liao SL, et al. Inhibition of inducible nitric oxide synthase expression by baicalein in endotoxin/cytokinestimulated microglia. Biochem Pharmacol. 2004, 67(5): 957-65
[12] Kim JY, Choi CY, Lee KJ, et al. Induction of inducible nitric oxide synthase and proinflammatory cytokines expression by o, p'-DDT in macrophages. Toxicol Lett. 2004, 147(3): 261-269
[13] Miller BH, Fratti RA, Poschet JF, et al. Mycobacteria Inhibit Nitric Oxide Synthase Recruitment to Phagosomes during Macrophage Infection. Infect Immun. 2004, 72(5): 2872-2878
[14] Kettler M, Ikegagya N, Brees DK et al. L-arginine metabolism in immune mediated glomerulonephritis in the rat. Am J Kidney Dis, 1996, 28(6): 878-887
[15] Waddington SN, Mosley K, Cook HT et al. Arginase AI is
upregulated in acute immune complex induced inflammation. Biochem Biophys Res Commun, 1998, 9247(1): 84-87
[16] Johansson RK, Poljakovic M, Andersson KE et al. Expression of nitric oxide synthase in bladder smooth muscle cells: regulation by cytokines and L-arginine. J Urol 2002, 168(5): 2280-2285
[17] deRojas-Walker T, Tamir S, Ji H, Wishnok JS, et al. Nitric oxide induces oxidative damage in addition to deamination in macrophage DNA. Chem Res Toxicol. 1995, 8(3): 473-477
[18] Wink DA, Kasprzak KS, Maragos CM, et al. DNA deaminating ability and genotoxicity of nitric oxide and its progenitors. Science 1991, 254:1001-1003
[19] Oztezcan S, Kirgiz B, Unlucerci Y, et al. Heme oxygenase induction protects liver against oxidative stress in x-irradiated aged rats. Biogerontology. 2004, 5(2): 99-105
[20] Bernard DG, Gabilly ST, Dujardin G, et al. Overlapping specificities of the mitochondrial cytochrome c and c1 heme lyases. J Biol Chem. 2003, 278(50): 49732-49742
[21] Jin C, Barrientos A, Tzagoloff A. Yeast dihydroxybutanone phosphate synthase, an enzyme of the riboflavin biosynthetic pathway, has a second unrelated function in expression of mitochondrial respiration. J Biol Chem. 2003, 278(17): 14698-14703
[22] Shumyantseva VV, Bulko TV, Schmid RD, et al. Photochemical properties of a riboflavins/cytochrome P450 2B4 complex. Biosens
Bioelectron. 2002,17(3): 233-238
[23] Ito K, Inoue S, Yamamoto K et al. 8-Hydroxydeoxyguanosine formation at the 5' site of 5'-GG-3' sequences in double-stranded DNA by UV radiation with riboflavin. J Biol Chem. 1993, 268(18): 13221-13227
[24] LU Changyuan, HAN Zhenhui, LIU Guanshu, et al. Photophysical and photochemical processes of riboflavin (Vitamin B 2) by means of the transient absorption spectra in aqueous solution Science in China (Biochemistry) 2000(5): 428-436
[25] Dohno C, Nakatani K, Saito I. Guanine of the third strand of C.G~*G triplex serves as an effective hole trap. J Am Chem. Soc. 2002, 124(49): 14580-14585.
[26] Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology, and pharmacology Pharmacol Rev. 1991, 43: 109-142
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