Effects of the C-terminal of endostatin on the tumorigenic potential of H22 cells

Abstract

Endostatin is an endogenous angiogenesis inhibitor whose specific functional site has not yet been determined. In the present experiment, 13 amino acids (LCIENSFMTSFSK) were selectively deleted from the C-terminal of endostatin and the resulting mutant endostatin was named EM13. To determine the effect of the C-terminal deletion on the biological activity of endostatin, EM13, wild-type endostatin and empty plasmid were transfected into H22 cells. After 48 h, the three types of transfected cells were harvested and injected into nude mice. The results demonstrated that there was no significant difference in tumor size, as determined by hematoxylin and eosin staining, between the EM13-transfected group and the endostatin and empty plasmid groups, although the nude mice that were injected with EM13-transfected H22 cells exhibited smaller tumors and lower density of blood vessels compared to those injected with endostatin- and empty plasmid-transfected H22 cells. The results suggested that the 13 amino acids of the C-terminal of endostatin do not play an important role in the tumorigenic potential of H22 cells. This experiment was unsuccessful in reproducing the results of several investigators. Therefore, the mechanism underlying the tumorigenesis of H22 cells remains to be elucidated.

Keywords: H22 cells; endostatin; tumorigenesis.

Figure 1

Extraction of total RNA and amplification of EM13. PCR amplification of EM13 gene: Lane 1, DL2000 DNA marker; lane 2, EM13; lane 3, endostatin.

Figure 2

(A) Construction of expressing plasmid. Screening for endostatin 13-T-positive clones by polymerase chain reaction (PCR): Lane 1, DL2000 DNA marker; Lanes 2–6, PCR products no. 1–5. (B) Screening for endostatin 13-T-positive clones by plasmid extracting: Lane 1, DL2000 DNA marker; lane 2, λ-HindIII DNA marker; and lane 3, plasmid of positive clone pMD18-T-EM13. (C) Digestion of endostatin 13-T, endostatin, pEGFP-N2 by SacII/XhoI: Lane 1, vector pEGFP-N2 digested by SacII/XhoI; lane 2, endostatin digested by SacII/XhoI; lane 3, pMD18-T-EM13 digested by SacII/XhoI; lane 4, DL2000 DNA marker; lane 5, λ-HindIII DNA marker; lane 6, purification of pMD18-T-EM13 digestion product; lane 7, purification of endostatin digestion product; and lane 8, purification of pEGFP-N2 digestion product. (D) Screening for positive clones by PCR: Lane 1, DL2000 DNA marker; lane 2–5, PCR products no. 11–14; lanes 6 and 11, negative control of respective PCR; lanes 8–10, PCR products no. 21–23. (E) Plasmid pEGFP-N2-EM13 and pEGFP-N2-endostatin. Lane 1, DL2000 DNA marker; lane 2, pEGFP-N2-EM13; lane 3, pEGFP-N2-endostatin; and lane 4, λ-HindIII DNA marker. (F) Plasmid pEGFP-N2-EM13 and pEGFP-N2-endostatin were digested with XhoI and SacII. Lane 1, DL2000 DNA marker; lane 2, pEGFP-N2-EM13 was digested with XhoI and SacII; lane 3, pEGFP-N2-endostatin was digested with XhoI and SacII; and lane 4, λ-HindIII DNA marker.

Figure 3

Endostatin inhibits tumor growth and blood vessel formation. After mice were sacrificed, the tumor density of blood vessels was observed under a light microscope following hematoxylin and eosin staining. (A) Untransfected, (B) transfected with pEGFP-N2-vector, (C) transfected with pEGFP-N2-endostatin and (D) transfected with pEGFP-N2-EM13.