[Gene transfection of endostatin synergizes with doxorubicin to suppress human hepatocellular carcinomas: experiment with mice].
OBJECTIVE
To investigate whether endostatin, a potent antiangiogenic agent, synergizes with doxorubicin to suppress human hepatocellular carcinoma (HCC).


METHODS
An endostatin expression plasmid, Endo-cDNA3.1, was constructed and transfected into COS-1 cells. Human HCC cells of the line HepG2 and human umbilical vein endothelial cells of the line HUVEC were cultured and stimulated by the supernatant of the CoS-1 cells transfected with Endo-pcDNA3.1 and doxorubicin of different concentrations. MTT method was used to detect the proliferation of the cells. (How many) BALB/c mice were inoculated with HepG2 cells to establish HCC models, and then divided into 4 groups to undergo intratumoral injection of pcDNA3.1, End-pcDNA3.1, doxorubicin, or doxorubicin + Endo-pcDNA3.1. Other mice were used as untreated control group. Two weeks later 5 mice from each group were killed with the tumors taken out. Immunostaining was used to calculate the microvessel density and Western blotting was sued to detect the expression of endostatin, hypoxia-inducible factor (HIF) and vascular endothelial growth factor (VEGF).


RESULTS
The proliferation of the HUVEC cells, but not that of the HepG2 cells, transfected with Endo-pcDNA3.1 + doxorubicin was inhibited. Doxorubicin dose-dependently inhibited the proliferation of both HUVEC and HepG2 cells. Endostatin was strongly expressed in the cells treated with Endo-pcDNA3.1 the tumor size of the Endo-pcDNA3.1 and doxorubicin groups were (1545 +/- 180) mm(3) and (953 +/- 250) mm(3) respectively, both significantly lower than that of the untreated and pcDNA3.1 groups [(2360 +/- 330) mm(3) and (2235 +/- 268) mm(3), respectively, all P < 0.01], and the tumor size of the Endo-pcDNA3.1 + doxorubicin group was (426 +/- 87) mm(3), significantly lower than any other groups (all P < 0.01). The number of microvessels of the Endo-pcDNA, doxorubicin, and doxorubicin + Endo-pcDNA3.1 groups were all significantly less than those of the pcDNA3.1 and untreated groups (P < 0.05 or P < 0.01). The expression of HIF-1alphawas downregulated in the Endo-pcDNA3.1 and Endo-pcDNA3.1 + doxorubicin groups, but not in the doxorubicin group. The VEGF expression was down-regulated in the Endo-pcDNA3.1, doxorubicin, and Endo-pcDNA3.1 + doxorubicin groups, especially in the latter.


CONCLUSIONS
Endostatin gene therapy synergizes with doxorubicin to suppress HCC.
Liu
Feng-Jun
FJ
Department of General Surgery, Qilu Hospital of Shandong University, Jinan, China. lfj6671@163.com
Tan
Gang
G
Li
Jie
J
Sun
Xue-Ying
XY
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology / Q000627:therapeutic use; D015153:Blotting, Western; D019556:COS Cells; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D002460:Cell Line; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002522:Chlorocebus aethiops; D003131:Combined Modality Therapy; D004317:Doxorubicin / Q000494:pharmacology / Q000627:therapeutic use*; D043169:Endostatins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism; D007150:Immunohistochemistry; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008114:Liver Neoplasms, Experimental / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D023041:Xenograft Model Antitumor Assays
D000903:Antibiotics, Antineoplastic; D043169:Endostatins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D042461:Vascular Endothelial Growth Factor A; D004317:Doxorubicin