Therapeutic Effect of Recombinant Adenovirus Containing Ad-EAFP-PALB/r-Caspase-3 on Primary Hepatocellular Carcinoma_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

WANG Wei , SHEN Haoyuan , LIU Meng.

Department of Hepatobiliary Surgery, The First Affiliated Hospital, Guangzhou Medical College, Guangzhou 510120, Guangdong Province, China;

Corresponding author：

Keywords：

Hepatocellular carcinoma; Caspase-3; Apoptosis; Implanted tumor; Nude mice

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ObjectiveTo evaluate therapeutic effect of the targeting recombinant adenovirus Ad-EAFP-PALB/r-Caspase-3 on primary hepatocellular carcinoma cells (HepG2) and subcutaneous implanted tumors of nude mice. MethodsHepG 2 cells, breast cancer MDA-MB-231 cells， and normal hepatic L-02 cells were infected with the previously constructed targeting recombinant adenovirus Ad-EAFP-PALB/r-Caspase-3, then morphological change was observed and apoptosis index was detected by cell morphologic assay. Tumor growth and pathological changes were observed after the implanted tumors model of nude mice were injected with the recombinant adenovirus. Results Both apoptosis and obvious apoptotic peak of cells were observed by flow cytometry (FCM) in HepG 2 cell group. The apoptosis index of MDA-MB-231 and L-02 cells was 0 and 7.3%, respectively, which were significantly lower than that of HepG2 cells (48.2%), P lt;0.01. The volume of subcutaneous tumor of nude mice was (0.26±0.31) cm 3 in HepG2 cell group, (0.25±0.15) cm 3 in MDA-MB-231 cell group， and (0.26±0.28) cm 3 in control group on two weeks after implantation of cancer cells, and no statistical difference was found among them (P gt;0.05). The volume of subcutaneous tumor of nude mice was (0.53±0.12) cm 3 in HepG 2 cell group, (0.49±0.22) cm 3 in MDA-MB-231 cell group， and (0.54±0.13) cm 3 in control group on four weeks after implantation of cancer cells, and the difference was not significant among them (P gt;0.05). But on eight weeks after implantation of cancer cells, the volume of tumor was (0.65±0.13) cm 3 in HepG 2 cell group, (1.27±0.32) cm 3 in MDA-MB-231 cell group， and (1.43±1.09) cm 3 in control group, which suggested that the growth of tumor in HepG 2 cell group slowed down significantly when compared with MDA-MB-231 cell group and control group (P lt;0.01), although the difference in the latter two groups was not significant (P gt;0.05). The necrosis regions were found with lymphocytic infiltration and apoptosis cells in HepG2 cell group after injection of recombinant adenovirus Ad-EAFP-PALB/r-Caspase-3, but no pathological change of cells was found in control group after injection. It was found that the cells in MDA-MB-231 cell group were solidly arranged with small glandular lumenlike structure, marked cellular atypia, multinucleated tumor cells and megakaryocytic tumor cells, which was not different from that before injection. ConclusionThe targeting recombinant adenovirus Ad-EAFP-PALB/r-Caspase-3 induces the apoptosis of primary hepatocellular carcinoma in vivo and in vitro, which may provide new ways of targeted gene therapy.

Citation： WANG Wei,SHEN Haoyuan,LIU Meng.. Therapeutic Effect of Recombinant Adenovirus Containing Ad-EAFP-PALB/r-Caspase-3 on Primary Hepatocellular Carcinoma. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2011, 18(7): 722-727. doi: Copy

1.	王炜, 程卫, 丘昶儒. 肝细胞肝癌靶向性rCaspase3重组腺病毒的构建与表达 [J]. 中国普外基础与临床杂志, 2008, 15(8): 589594.
2.	王炜, 沈浩元, 丘昶儒, 等. 靶向性反向半胱氨酸天冬氨酸蛋白酶3重组腺病毒诱导肝癌细胞凋亡的实验研究 [J]. 中华生物医学工程杂志, 2010, 16(4): 298303.
3.	陈汝福, 孙宏武, 邹倩. 反义硫代寡脱氧核苷酸抑制裸鼠人胆管癌生长的研究 [J]. 中华实验外科杂志, 2000, 17(1): 2223.
4.	毕允力, 高解春, 许德华. 5氟胞嘧啶对表达胞嘧啶脱氨酶的人肾母细胞瘤裸鼠异种移植瘤的疗效 [J]. 中华肿瘤杂志, 2000, 22(2): 113115.
5.	陈孝平, 张万广. 原发性肝癌面临的难点与对策 [J]. 中国普外基础与临床杂志, 2008, 25(4): 231233.
6.	Araida T, Higuchi R, Hamano M, et al. Hepatic resection in 485 R0 pT2 and pT3 cases of advanced carcinoma of the gallbladder: results of a Japanese Society of Biliary Surgery survey—a multicenter study [J]. Hepatobiliary Pancreat Surg, 2009, 16(2): 204215.
7.	Cescon M, Cucchetti A, Grazi GL, et al. Indication of the extent of hepatectomy for hepatocellular carcinoma on cirrhosis by a simple algorithm based on preoperative variables [J]. Arch Surg, 2009, 144(1): 5763.
8.	Ishizawa T, Hasegawa K, Kokudo N, et al. Risk factors and management of ascites after liver resection to treat hepatocellular carcinoma [J]. Arch Surg, 2009, 144(1): 4651.
9.	Cho JY, Han HS, Yoon YS, et al. Outcomes of laparoscopic liver resection for lesions located in the right side of the liver [J]. Arch Surg, 2009, 144(1): 2529.
10.	Miyazawa M, Aikawa M, Okada K, et al. Extended left hepatic lobectomy for hepatic hilar bile duct cancer: a novel surgical procedure in which the right hepatic duct is transected before the hepatoduodenal ligament is skeletonized [J]. Dig Surg, 2009, 26(1): 1519.
11.	Smerdou C, Menne S, HernandezAlcoceba R, et al. Gene therapy for HCV/HBVinduced hepatocellular carcinoma [J]. Curr Opin Investig Drugs, 2010, 11(12): 13681377.
12.	Sharapova OA, Pozdnykova NV, Laurinavichyute DK, et al. Highefficient expression, refolding and purification of functional recombinant Cterminal fragment of human alphafetoprotein [J]. Protein Expr Purif, 2010, 73(1): 3135.
13.	Lam PY, Sia KC, Khong JH, et al. An efficient and safe herpes simplex virus type 1 amplicon vector for transcriptionally targeted therapy of human hepatocellular carcinomas [J]. Mol Ther, 2007, 15(6): 11291136.
14.	Nussbaum T, Samarin J, Ehemann V, et al. Autocrine insulinlike growth factorII stimulation of tumor cell migration is a progression step in human hepatocarcinogenesis [J]. Hepatology, 2008, 48(1): 146156.
15.	Sadeghi H, Hitt MM. Transcriptionally targeted adenovirus vectors [J]. Curr Gene Ther, 2005, 5(4): 411427.
16.	Kanai F. Transcriptional targeted gene therapy for hepatocellular carcinoma by adenovirus vector [J]. Mol Biotechnol, 2001, 18(3): 243250.
17.	Srinivasula SM, Ahmad M, MacFarlane M, et al. Generation of constitutively active recombinant caspases3 and 6 by rearrangement of their subunits [J]. J Biol Chem, 1998, 273(17): 1010710111.
18.	王炜, 秦兆寅, 王剑利, 等. 重组的rCaspase3促凋亡基因体内外治疗胰腺癌的试验研究 [J]. 中华肝胆外科杂志, 2003, 9(5): 303305.
19.	王炜, 秦兆寅, 刘志国. 重组型Caspase3基因的构建及其在胰腺癌细胞中凋亡活性的观察 [J]. 中国普外基础与临床杂志, 2002, 9(4): 249251.
20.	Park HJ, Kim MJ, Ha E, et al. Apoptotic effect of hesperidin through caspase3 activation in human colon cancer cells, SNUC4 [J]. Phytomedicine, 2008, 15(12): 147151.
21.	Mouratidis PX, Colston KW, Dalgleish AG. Doxycycline induces caspasedependent apoptosis in human pancreatic cancer cells [J]. Int J Cancer, 2007, 120(4): 743752.
22.	Végran F, Boidot R, Oudin C, et al. Overexpression of caspase3s splice variant in locally advanced breast carcinoma is associated with poor response to neoadjuvant chemotherapy [J]. Clin Cancer Res, 2006, 12(19): 57945800.
23.	Aquino Esperanza JA, Aguirre MV, Aispuru GR, et al. In vivo 5fluorouracil[corrected] induced apoptosis on murine thymocytes: involvement of FAS, Bax and Caspase3 [J]. Cell Biol Toxicol, 2008, 24(5): 411422.
24.	易石坚, 李兰兰, 钟德许, 等. 裸鼠皮下人肝癌种植瘤模型的建立 [J]. 中国医药导报, 2008, 21(5): 2728.
25.	李立, 吴沛宏, 黄嘉凌, 等. 重组人内皮抑素腺病毒抑制肝癌裸鼠移植瘤生长 [J]. 中华肝脏病杂志, 2008, 16(9): 542545.
26.	Kurio N, Shimo T, Fukazawa T, et al. Antitumor effect in human breast cancer by TAE226, a dual inhibitor for FAK and IGFIR in vitro and in vivo [J]. Exp Cell Res, 2011, 317(8): 11341146.

1. 王炜, 程卫, 丘昶儒. 肝细胞肝癌靶向性rCaspase3重组腺病毒的构建与表达 [J]. 中国普外基础与临床杂志, 2008, 15(8): 589594.
2. 王炜, 沈浩元, 丘昶儒, 等. 靶向性反向半胱氨酸天冬氨酸蛋白酶3重组腺病毒诱导肝癌细胞凋亡的实验研究 [J]. 中华生物医学工程杂志, 2010, 16(4): 298303.
3. 陈汝福, 孙宏武, 邹倩. 反义硫代寡脱氧核苷酸抑制裸鼠人胆管癌生长的研究 [J]. 中华实验外科杂志, 2000, 17(1): 2223.
4. 毕允力, 高解春, 许德华. 5氟胞嘧啶对表达胞嘧啶脱氨酶的人肾母细胞瘤裸鼠异种移植瘤的疗效 [J]. 中华肿瘤杂志, 2000, 22(2): 113115.
5. 陈孝平, 张万广. 原发性肝癌面临的难点与对策 [J]. 中国普外基础与临床杂志, 2008, 25(4): 231233.
6. Araida T, Higuchi R, Hamano M, et al. Hepatic resection in 485 R0 pT2 and pT3 cases of advanced carcinoma of the gallbladder: results of a Japanese Society of Biliary Surgery survey—a multicenter study [J]. Hepatobiliary Pancreat Surg, 2009, 16(2): 204215.
7. Cescon M, Cucchetti A, Grazi GL, et al. Indication of the extent of hepatectomy for hepatocellular carcinoma on cirrhosis by a simple algorithm based on preoperative variables [J]. Arch Surg, 2009, 144(1): 5763.
8. Ishizawa T, Hasegawa K, Kokudo N, et al. Risk factors and management of ascites after liver resection to treat hepatocellular carcinoma [J]. Arch Surg, 2009, 144(1): 4651.
9. Cho JY, Han HS, Yoon YS, et al. Outcomes of laparoscopic liver resection for lesions located in the right side of the liver [J]. Arch Surg, 2009, 144(1): 2529.
10. Miyazawa M, Aikawa M, Okada K, et al. Extended left hepatic lobectomy for hepatic hilar bile duct cancer: a novel surgical procedure in which the right hepatic duct is transected before the hepatoduodenal ligament is skeletonized [J]. Dig Surg, 2009, 26(1): 1519.
11. Smerdou C, Menne S, HernandezAlcoceba R, et al. Gene therapy for HCV/HBVinduced hepatocellular carcinoma [J]. Curr Opin Investig Drugs, 2010, 11(12): 13681377.
12. Sharapova OA, Pozdnykova NV, Laurinavichyute DK, et al. Highefficient expression, refolding and purification of functional recombinant Cterminal fragment of human alphafetoprotein [J]. Protein Expr Purif, 2010, 73(1): 3135.
13. Lam PY, Sia KC, Khong JH, et al. An efficient and safe herpes simplex virus type 1 amplicon vector for transcriptionally targeted therapy of human hepatocellular carcinomas [J]. Mol Ther, 2007, 15(6): 11291136.
14. Nussbaum T, Samarin J, Ehemann V, et al. Autocrine insulinlike growth factorII stimulation of tumor cell migration is a progression step in human hepatocarcinogenesis [J]. Hepatology, 2008, 48(1): 146156.
15. Sadeghi H, Hitt MM. Transcriptionally targeted adenovirus vectors [J]. Curr Gene Ther, 2005, 5(4): 411427.
16. Kanai F. Transcriptional targeted gene therapy for hepatocellular carcinoma by adenovirus vector [J]. Mol Biotechnol, 2001, 18(3): 243250.
17. Srinivasula SM, Ahmad M, MacFarlane M, et al. Generation of constitutively active recombinant caspases3 and 6 by rearrangement of their subunits [J]. J Biol Chem, 1998, 273(17): 1010710111.
18. 王炜, 秦兆寅, 王剑利, 等. 重组的rCaspase3促凋亡基因体内外治疗胰腺癌的试验研究 [J]. 中华肝胆外科杂志, 2003, 9(5): 303305.
19. 王炜, 秦兆寅, 刘志国. 重组型Caspase3基因的构建及其在胰腺癌细胞中凋亡活性的观察 [J]. 中国普外基础与临床杂志, 2002, 9(4): 249251.
20. Park HJ, Kim MJ, Ha E, et al. Apoptotic effect of hesperidin through caspase3 activation in human colon cancer cells, SNUC4 [J]. Phytomedicine, 2008, 15(12): 147151.
21. Mouratidis PX, Colston KW, Dalgleish AG. Doxycycline induces caspasedependent apoptosis in human pancreatic cancer cells [J]. Int J Cancer, 2007, 120(4): 743752.
22. Végran F, Boidot R, Oudin C, et al. Overexpression of caspase3s splice variant in locally advanced breast carcinoma is associated with poor response to neoadjuvant chemotherapy [J]. Clin Cancer Res, 2006, 12(19): 57945800.
23. Aquino Esperanza JA, Aguirre MV, Aispuru GR, et al. In vivo 5fluorouracil[corrected] induced apoptosis on murine thymocytes: involvement of FAS, Bax and Caspase3 [J]. Cell Biol Toxicol, 2008, 24(5): 411422.
24. 易石坚, 李兰兰, 钟德许, 等. 裸鼠皮下人肝癌种植瘤模型的建立 [J]. 中国医药导报, 2008, 21(5): 2728.
25. 李立, 吴沛宏, 黄嘉凌, 等. 重组人内皮抑素腺病毒抑制肝癌裸鼠移植瘤生长 [J]. 中华肝脏病杂志, 2008, 16(9): 542545.
26. Kurio N, Shimo T, Fukazawa T, et al. Antitumor effect in human breast cancer by TAE226, a dual inhibitor for FAK and IGFIR in vitro and in vivo [J]. Exp Cell Res, 2011, 317(8): 11341146.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Therapeutic Effect of Recombinant Adenovirus Containing Ad-EAFP-PALB/r-Caspase-3 on Primary Hepatocellular Carcinoma

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