IL-24对裸鼠人宫颈癌Hela细胞移植瘤抑制作用的探讨
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摘要
目的：探讨基因重组质粒pDC316-hIL-24对裸鼠人宫颈癌模型的肿瘤生长及凋亡的作用。
方法：于5周龄裸鼠腋窝外侧皮下注射1×107/mlHela细胞悬液100μl(约1×106),将构建成功的人宫颈癌Hela细胞裸鼠模型,随机分为3组,分别于瘤体内注射基因重组质粒pDC3.0-hIL-24,空质粒pDC3.0以及磷酸盐缓冲液(PBS),比较干预后移植瘤的体积及抑瘤率,应用PCR法测定移植瘤中hIL-24的表达,HE染色观察各组肿瘤组织形态学变化,免疫组化方法研究肿瘤细胞凋亡情况。
结果：经转染IL-24基因已成功转染入肿瘤细胞并在肿瘤细胞中已成功表达,至实验结束时,对照组和空质粒组移植瘤体积明显大于IL-24基因重组质粒组(F=63.395,P＜0.05)；IL-24重组质粒组的平均抑瘤率明显高于空质粒组(t=130.920,P＜0.01)；病理学观察见IL-24重组质粒组宫颈癌细胞有局灶性坏死,细胞变大,胞质疏松,核深染,可见核固缩、核溶解、核碎裂,染色质成团状；免疫组化结果显示,与其余两组相比,基因重组质粒组原癌基因Bcl-2表达降低(F=6.164,P＜0.05),而其同源蛋白Bax表达明显高于其他两组(F=58.426,P＜0.05)。
结论：基因重组质粒pDC316-hIL-24对宫颈癌裸鼠模型肿瘤生长具有明显的抑制及促进凋亡作用,IL-24可能通过激活细胞凋亡/抗凋亡通路而诱导肿瘤细胞核固缩碎裂、细胞凋亡,而且IL-24抗肿瘤作用对动物生活能力及质量无明显不良影响。
Method
Cervical cancer Hela cells were injected in a 106concentration into the central lateral subcutaneous of the left armpit of nude mice of 5 weeks age. Construction of nude mice model of human cervical cancer. The nude mice were randomly divided into 3 groups, and recombinant plasmids or empty plasmids as well as PBS were injected into transplanted tumor to interfere with the growth of transplanted tumor. Comparison of tumor volume and inhibitory rate after the intervention, determination of hIL-24 expression in transplanted tumor by PCR, observation of morphological changes of tumor tissues in each group by HE staining and as well as immunohistochemistry was employed for investigating tumor cell apoptosis in each group.
Result
After transfection IL-24 gene was successfully transfected into tumor cells and has been successfully expressed in tumor cells. To the end of the experiment, the control group and blank plasmid group tumor volume was significantly greater than IL-24 gene plasmid group (F=63.395, P<0.05); The average inhibition rate of IL-24 recombinant plasmid group was significantly higher than empty plasmid group (t=130.920, P<0.01); Pathological observation shows in plasmid IL-24 group cervical cancer cells have focal necrosis and cells become large, and loose cytoplasm, nucleus deeply stained, pyknosis, nuclear dissolution, nuclear fragmentation, chromatin lumps also can be found; Immunohistochemistry showed that, compared with the other two groups, recombinant plasmid group proto-oncogene Bcl-2 expression was decreased (F=6.164, P<0.05), but its homologous protein Bax expression was significantly increased (F=58.426, P<0.05).
Conclusion
Recombinant plasmid pDC316-hIL-24 has significant inhibition function on human cervical cancer nude mice model tumor growth. IL-24 may activate apoptosis/ anti-apoptosis pathway to induce tumor cell nuclear condensation, fragmentation and apoptosis, and anti-tumor function of IL-24 had no obvious adverse effects on the quality of animals'life.
方法：于5周龄裸鼠腋窝外侧皮下注射1×107/mlHela细胞悬液100μl(约1×106),将构建成功的人宫颈癌Hela细胞裸鼠模型,随机分为3组,分别于瘤体内注射基因重组质粒pDC3.0-hIL-24,空质粒pDC3.0以及磷酸盐缓冲液(PBS),比较干预后移植瘤的体积及抑瘤率,应用PCR法测定移植瘤中hIL-24的表达,HE染色观察各组肿瘤组织形态学变化,免疫组化方法研究肿瘤细胞凋亡情况。
结果：经转染IL-24基因已成功转染入肿瘤细胞并在肿瘤细胞中已成功表达,至实验结束时,对照组和空质粒组移植瘤体积明显大于IL-24基因重组质粒组(F=63.395,P＜0.05)；IL-24重组质粒组的平均抑瘤率明显高于空质粒组(t=130.920,P＜0.01)；病理学观察见IL-24重组质粒组宫颈癌细胞有局灶性坏死,细胞变大,胞质疏松,核深染,可见核固缩、核溶解、核碎裂,染色质成团状；免疫组化结果显示,与其余两组相比,基因重组质粒组原癌基因Bcl-2表达降低(F=6.164,P＜0.05),而其同源蛋白Bax表达明显高于其他两组(F=58.426,P＜0.05)。
结论：基因重组质粒pDC316-hIL-24对宫颈癌裸鼠模型肿瘤生长具有明显的抑制及促进凋亡作用,IL-24可能通过激活细胞凋亡/抗凋亡通路而诱导肿瘤细胞核固缩碎裂、细胞凋亡,而且IL-24抗肿瘤作用对动物生活能力及质量无明显不良影响。
Method
Cervical cancer Hela cells were injected in a 106concentration into the central lateral subcutaneous of the left armpit of nude mice of 5 weeks age. Construction of nude mice model of human cervical cancer. The nude mice were randomly divided into 3 groups, and recombinant plasmids or empty plasmids as well as PBS were injected into transplanted tumor to interfere with the growth of transplanted tumor. Comparison of tumor volume and inhibitory rate after the intervention, determination of hIL-24 expression in transplanted tumor by PCR, observation of morphological changes of tumor tissues in each group by HE staining and as well as immunohistochemistry was employed for investigating tumor cell apoptosis in each group.
Result
After transfection IL-24 gene was successfully transfected into tumor cells and has been successfully expressed in tumor cells. To the end of the experiment, the control group and blank plasmid group tumor volume was significantly greater than IL-24 gene plasmid group (F=63.395, P<0.05); The average inhibition rate of IL-24 recombinant plasmid group was significantly higher than empty plasmid group (t=130.920, P<0.01); Pathological observation shows in plasmid IL-24 group cervical cancer cells have focal necrosis and cells become large, and loose cytoplasm, nucleus deeply stained, pyknosis, nuclear dissolution, nuclear fragmentation, chromatin lumps also can be found; Immunohistochemistry showed that, compared with the other two groups, recombinant plasmid group proto-oncogene Bcl-2 expression was decreased (F=6.164, P<0.05), but its homologous protein Bax expression was significantly increased (F=58.426, P<0.05).
Conclusion
Recombinant plasmid pDC316-hIL-24 has significant inhibition function on human cervical cancer nude mice model tumor growth. IL-24 may activate apoptosis/ anti-apoptosis pathway to induce tumor cell nuclear condensation, fragmentation and apoptosis, and anti-tumor function of IL-24 had no obvious adverse effects on the quality of animals'life.
引文
[1]Bresalier RS,Ho SB,Schoepner HL,et al.Enhanced sialylation of mucin-associated carbohydrate structures in human colon cancer metastasis[J].Gastroenteology,1996, 110(5):1354-1367.
[2]JiangH, Lin JJ, Su ZZ, et al. Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression. Oncogene,1995, 11(12):2477-2486
[3]Caudell EG, Mumm JB, Poindexter N, et al. The protein product of the tumor suppressor gene, melanoma differentiation-associated gene 7, exhibits immunostimulatory activity and is designated IL-24. Immonol,2002, 168(12):6041-6046.
[4]Madireddi MT, Dent P, Fisher PB. AP-1 and C/EBP transcription factors contribute to mda-7 gene promoter activity during human melanoma differentiation. J Cell Physiol,2000,185(1):36-46
[5]Irina VL et al. Oncogene,2002;21(5):708
[6]Su ZZH et al. Proc Natl Acad Sci USA; 2001; 98(18):10332
[7]Gupta P,Su ZZ,Lebedeva IV,et al.mda-7/IL-24:Multifunctional cancer-specific apoptosis-inducing cytokine[J].Pharmacol Ther.2006,111(3):596-628
[8]Su Z,Emdad L,Sauane M,et al.Unique aspects of mda-7/IL-24 antitumorbystander activity:establishing a role for secretion of MDA-7/IL-24 protein by normal cells[J].Oncogene.2005,24(51):7552-7566
[9]Sauane M,Lebedeva IV,Su ZZ,et al.Melanoma differentiation associated gene-7/interleukin-24 promotes tumor cell-specific apoptosis through both secretory and nonsecretory pathways[J].Cancer Res.2004,64(9):2988-2993
[10]韩文玲,马大龙.新细胞因子IL-24研究进展[J].中华免疫学杂志.2003,19：211-212
[11]Mhashilkar AM, Schrock RD, Hindi M, et al. Melanoma differentiation associated gene-7(mda-7):a novel anti-tumor gene for cancer gene therapy. Mol Med,2001,7 (4):271-782.
[12]Wang M,Tan Z, Zhang R, et al Iterleuk in 24 (MDA-7/MOB-5) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2 J Biol Chem,2002,277 (9):7341-7347.
[13]曹涛,李莉,王赞宏等.IL-24对宫颈癌Hela细胞株增殖和凋亡的影响.中国妇产科临床杂志2009,(10)4：286-289.
1、 JiangH, Lin JJ, Su ZZ, et al. Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression. Oncogene,1995, 11(12):2477-2486
2、 Irina VL et al. Oncogene,2002;21(5):708
3、 Huang EY et al. Oncogene,2001;20(48):7051
4、 Zhang R et al. J Biol Chem,275(32):24436
5、 JiangH, Lin JJ, Su ZZ, et al. The melanoma differentiation associated gene mda-7 suppresses cancer cell growth [J]. Proc Natl Acad Sci USA,1996, 93(17):9160-9165
6、 Caudell EG, Mumm JB, Poindexter N, et al. The protein product of the tumor suppressor gene, melanoma differentiation-associated gene-7, exhibits immunostimulatory activity and is designated IL-24. J Immunol,2002,168 (12):6041-6046
7、 Sauane M, Gopalkrishnan RV, Sarkar D, et al. MDA-7/IL-24:Nov-el cancer growth suppressing and apoptosis inducing cytokine. Cytokine Growth Factor Rev,2003,14(1):35-51
8、 Su ZZH et al. Proc Natl Acad Sci USA; 2001; 98(18):10332
9、 Saeki T, Mhashilker A, Swanson X, et al. Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo. Oncogene, 2002,21(29):4558-4566
10、 RAMESH R, ITO I, GOPALAN B, et al. Ectopic production of MDA-7/IL-24 inhibits invasion and migration of human lung cancer cells [J]. Mol Ther,2004, 9(4):510-518.
11、 Ekmekcioglu S et al. Int J Cancer;2001;94(1):54
12、 Jiang H et al. Proc Natl Acad Sci USA,1996;93(17):9160
13、 SAUANE M, LEBEDEVA I V, SU Z Z, et al. Melanoma differentiation associated gene-7/interleukin-24 promotes tumor cell-specific apoptosis through both secretory and nonsecretory pathways [J]. Cancer Res,2004, 64(9):2988-2993.
14、 Ellerhorst JA, Prieto VG, Ekmekcioglu S, et al. Loss of MDA-7 expression with progression of melanoma. J Clin Oncol,2002,20(4):1069-1074.
15、 Mhashilkar AM, Stewart AL, Sieger K, et al.MDA-7 negatively regulates the beta-catenin and PI3K signaling pathways in breast and lung tumor cells. Mol Ther,2003,8(2):207-219.
16、 Saeki Tet al. Gene Ther,2000;7(23):2051
17、 SU Z, EMDAD L, SAUANE M, et al. Unique aspects of mda-7/IL-24 anti-tumor bystander activity:establishing a role for secretion of MDA-7/IL-24 by normal cells[J]. Oncogene,2005,24(51):7552-7566.
18、 Ekmekcioglu S et al. Int J Cancer; 2001;94(1):
19、 Sieger KA, Mhashilkar AM, Stewart A, et al. The tumor suppressor activity of MDA-7/IL-24 is mediated by intracellular protein expression in NSCLC cells. Mol Ther,2004,9(3):355-367.
20、 Gupta P, Walter MR, Su ZZ, et al.BiP/GRP78 is an intracellular target for MDA-7/IL-24 induction of cancer-specific apoptosis. Cancer Res,2006, 66(16):8182-8191.
21、 Fort MM et al. Immunity,2001; 15(6):985
22、 Yacoub A, Mitchell C, Lebedva IV, et al.mda-7 (IL-24) inhibits growth and enhances radiosensitivity of glioma cells in vitro via JNK signaling. Cancer Biol Ther,2003,2(4):347-353.
23、 程玉峰,乔乃安,业民等.剂量顺铂增加放疗诱导HeLa细胞 凋亡的研究[J].山东医药,2000,40(20)：20-21
24、 Cunningham CC, Chada S, Merritt JA, et al. Clinical and local biological effects of an intratumoral injection of mda-7(IL24;INGN241)in patients with advanced carcinoma:a phase Ⅰ study. Mol Ther,2005,11(1):149-159.
25、 Fisher PB, Gopalkrishnan RV, Chada S, et al.mda-7/IL-24,a novel cancer selective apoptosis inducing cytokine gene:from the laboratory into the clinic. Cancer Biol Ther,2003,2(4 Suppl 1):S23-37.
[2]JiangH, Lin JJ, Su ZZ, et al. Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression. Oncogene,1995, 11(12):2477-2486
[3]Caudell EG, Mumm JB, Poindexter N, et al. The protein product of the tumor suppressor gene, melanoma differentiation-associated gene 7, exhibits immunostimulatory activity and is designated IL-24. Immonol,2002, 168(12):6041-6046.
[4]Madireddi MT, Dent P, Fisher PB. AP-1 and C/EBP transcription factors contribute to mda-7 gene promoter activity during human melanoma differentiation. J Cell Physiol,2000,185(1):36-46
[5]Irina VL et al. Oncogene,2002;21(5):708
[6]Su ZZH et al. Proc Natl Acad Sci USA; 2001; 98(18):10332
[7]Gupta P,Su ZZ,Lebedeva IV,et al.mda-7/IL-24:Multifunctional cancer-specific apoptosis-inducing cytokine[J].Pharmacol Ther.2006,111(3):596-628
[8]Su Z,Emdad L,Sauane M,et al.Unique aspects of mda-7/IL-24 antitumorbystander activity:establishing a role for secretion of MDA-7/IL-24 protein by normal cells[J].Oncogene.2005,24(51):7552-7566
[9]Sauane M,Lebedeva IV,Su ZZ,et al.Melanoma differentiation associated gene-7/interleukin-24 promotes tumor cell-specific apoptosis through both secretory and nonsecretory pathways[J].Cancer Res.2004,64(9):2988-2993
[10]韩文玲,马大龙.新细胞因子IL-24研究进展[J].中华免疫学杂志.2003,19：211-212
[11]Mhashilkar AM, Schrock RD, Hindi M, et al. Melanoma differentiation associated gene-7(mda-7):a novel anti-tumor gene for cancer gene therapy. Mol Med,2001,7 (4):271-782.
[12]Wang M,Tan Z, Zhang R, et al Iterleuk in 24 (MDA-7/MOB-5) signals through two heterodimeric receptors, IL-22R1/IL-20R2 and IL-20R1/IL-20R2 J Biol Chem,2002,277 (9):7341-7347.
[13]曹涛,李莉,王赞宏等.IL-24对宫颈癌Hela细胞株增殖和凋亡的影响.中国妇产科临床杂志2009,(10)4：286-289.
1、 JiangH, Lin JJ, Su ZZ, et al. Subtraction hybridization identifies a novel melanoma differentiation associated gene, mda-7, modulated during human melanoma differentiation, growth and progression. Oncogene,1995, 11(12):2477-2486
2、 Irina VL et al. Oncogene,2002;21(5):708
3、 Huang EY et al. Oncogene,2001;20(48):7051
4、 Zhang R et al. J Biol Chem,275(32):24436
5、 JiangH, Lin JJ, Su ZZ, et al. The melanoma differentiation associated gene mda-7 suppresses cancer cell growth [J]. Proc Natl Acad Sci USA,1996, 93(17):9160-9165
6、 Caudell EG, Mumm JB, Poindexter N, et al. The protein product of the tumor suppressor gene, melanoma differentiation-associated gene-7, exhibits immunostimulatory activity and is designated IL-24. J Immunol,2002,168 (12):6041-6046
7、 Sauane M, Gopalkrishnan RV, Sarkar D, et al. MDA-7/IL-24:Nov-el cancer growth suppressing and apoptosis inducing cytokine. Cytokine Growth Factor Rev,2003,14(1):35-51
8、 Su ZZH et al. Proc Natl Acad Sci USA; 2001; 98(18):10332
9、 Saeki T, Mhashilker A, Swanson X, et al. Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo. Oncogene, 2002,21(29):4558-4566
10、 RAMESH R, ITO I, GOPALAN B, et al. Ectopic production of MDA-7/IL-24 inhibits invasion and migration of human lung cancer cells [J]. Mol Ther,2004, 9(4):510-518.
11、 Ekmekcioglu S et al. Int J Cancer;2001;94(1):54
12、 Jiang H et al. Proc Natl Acad Sci USA,1996;93(17):9160
13、 SAUANE M, LEBEDEVA I V, SU Z Z, et al. Melanoma differentiation associated gene-7/interleukin-24 promotes tumor cell-specific apoptosis through both secretory and nonsecretory pathways [J]. Cancer Res,2004, 64(9):2988-2993.
14、 Ellerhorst JA, Prieto VG, Ekmekcioglu S, et al. Loss of MDA-7 expression with progression of melanoma. J Clin Oncol,2002,20(4):1069-1074.
15、 Mhashilkar AM, Stewart AL, Sieger K, et al.MDA-7 negatively regulates the beta-catenin and PI3K signaling pathways in breast and lung tumor cells. Mol Ther,2003,8(2):207-219.
16、 Saeki Tet al. Gene Ther,2000;7(23):2051
17、 SU Z, EMDAD L, SAUANE M, et al. Unique aspects of mda-7/IL-24 anti-tumor bystander activity:establishing a role for secretion of MDA-7/IL-24 by normal cells[J]. Oncogene,2005,24(51):7552-7566.
18、 Ekmekcioglu S et al. Int J Cancer; 2001;94(1):
19、 Sieger KA, Mhashilkar AM, Stewart A, et al. The tumor suppressor activity of MDA-7/IL-24 is mediated by intracellular protein expression in NSCLC cells. Mol Ther,2004,9(3):355-367.
20、 Gupta P, Walter MR, Su ZZ, et al.BiP/GRP78 is an intracellular target for MDA-7/IL-24 induction of cancer-specific apoptosis. Cancer Res,2006, 66(16):8182-8191.
21、 Fort MM et al. Immunity,2001; 15(6):985
22、 Yacoub A, Mitchell C, Lebedva IV, et al.mda-7 (IL-24) inhibits growth and enhances radiosensitivity of glioma cells in vitro via JNK signaling. Cancer Biol Ther,2003,2(4):347-353.
23、 程玉峰,乔乃安,业民等.剂量顺铂增加放疗诱导HeLa细胞 凋亡的研究[J].山东医药,2000,40(20)：20-21
24、 Cunningham CC, Chada S, Merritt JA, et al. Clinical and local biological effects of an intratumoral injection of mda-7(IL24;INGN241)in patients with advanced carcinoma:a phase Ⅰ study. Mol Ther,2005,11(1):149-159.
25、 Fisher PB, Gopalkrishnan RV, Chada S, et al.mda-7/IL-24,a novel cancer selective apoptosis inducing cytokine gene:from the laboratory into the clinic. Cancer Biol Ther,2003,2(4 Suppl 1):S23-37.