J Korean Med Sci.  2005 Dec;20(6):1046-1052. 10.3346/jkms.2005.20.6.1046.

Antitumor Activity of TRAIL Recombinant Adenovirus in Human Malignant Glioma Cells

• Kim KU
• Seo SY
• Heo KY
• Yoo YH
• Kim HJ
• Lee HS
• Choi SS
• Hwang TH
• Lee HJ

• KMID: 2157760
• DOI: http://doi.org/10.3346/jkms.2005.20.6.1046

Abstract

Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) has been reported to specifically kill malignant cells but to be relatively nontoxic to normal cells. One of disadvantages to previous in vivo protocols was the need for large quantities of TRAIL recombinant protein to suppress tumor growth. To evaluate the antitumor activity and therapeutic value of the TRAIL gene, we constructed adenoviral vectors expressing the human TRAIL gene (Ad.hTRAIL) and transferred them into malignant glioma cells in vitro and tumors in vivo, as an alternative to recombinant soluble TRAIL protein. The results show that TRAIL-sensitive glioma cells infected Ad.hTRAIL undergo apoptosis through the production and expression of TRAIL protein. The in vitro transfer elicited apoptosis, as demonstrated by the quantification of viable or apoptotic cells and by the analysis of cleavage of poly (ADP-ribose) polymerase. Furthermore, in vivo administration of Ad.hTRAIL at the site of tumor implantation suppressed the outgrowth of human glioma xenografts in SCID mice. These results further define Ad.hTRAIL as an anti-tumor therapeutic and demonstrate its potential use as an alternative approach to treatment for malignant glioma.

Keyword

Glioma; TRAIL receptor 3; Adenoviral Vector; Gene Therapy; Mice, SCID

MeSH Terms

Adenoviridae/*genetics
Animals
Apoptosis
Apoptosis Regulatory Proteins/*genetics
Cell Line, Tumor
Gene Expression
Gene Therapy/*methods
Glioma/pathology/*therapy
Humans
Membrane Glycoproteins/*genetics
Mice
Mice, SCID
Neoplasm Transplantation
Research Support, Non-U.S. Gov't
Transplantation, Heterologous
Tumor Necrosis Factor-alpha/*genetics

Figure

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  Fig. 1 Production of adenovirus-encoding human TRAIL. (A) Ad.hTRAIL-infected U87MG cells express TRAIL protein according to MOI and period for incubation. Cell lysates from uninfected or Ad.hTRAIL-infected U87MG cells were prepared 24 hr after infection with different MOI (upper panel) and different incubation period with 300 MOI (lower panel). (B) Ad.hTRAIL-infected U87MG cells secret TRAIL into the media and secreted TRAIL was quantified by ELISA.

• 

  Fig. 2 Death of glioma cells after Ad.hTRAIL infection results from increased production of TRAIL protein. (A) U87MG cells. (B) U373MG cells. 96 well plates were seeded with 5×104 cells/well and allowed to adhere for at least 12 hr before infection with Ad.hTRAIL or Ad.lacZ at the indicated number of MOI. Cell viability was determined after 24 hr by MTT. Results shown are the mean±SEM from at least three independent experiments. *p<0.05, †p<0.01.

• 

  Fig. 3 Ad.hTRAIL-infected glioma cells undergo apoptotic cell death. (A) Nuclear fragmentation was examined by Hoechst nuclear staining in U87MG and U373MG cells treated with Ad.hTRAIL or Ad.lacZ. (B) Cleavage of PARP from 118 to 85 kDa occurs during apoptotic cell death. Cell lysates were prepared of various times after infection with 300 MOI and cellular proteins were determined by Western blot analysis.

• 

  Fig. 4 In vivo assessment of cell death by intratumoral delivery of Ad.hTRAIL. Ten days after tumor implantation, Ad.hTRAIL was administered by intratumoral injection. After 3 days, the tumor regions was excised and analyzed. (A & B) Tumor tissues were stained with H&E (×100) and Ad.lacZ-infected tissue (A) show well preserved and proliferated tumor cells. Ad.hTRAIL-infected tissue (B) showes markedly reduced tumor cells in the center and only small numbers of the cells are present at periphery. (C & D) TUNEL assay demonstrates that Ad.hTRAIL-infected cells (D) show positive stain for apoptotic cells at periphery and that Ad.lacZ-infected cells (C) are not stained for apoptotic cells (×100). (E & F) Immunohistochemistry using anti-TRAIL antibody shows that Ad.hTRAIL expressed TRAIL protein in vivo in Ad.hTRAIL-infected tumor cells (F). Ad.lacZ-infected tumor cells (E) are not detected by anti-TRAIL antibody.

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  Fig. 5 Ad.hTRAIL therapy significantly suppresses the growth of established tumors. Male SCID mice were injected subcutaneously with 1×106 U87MG cells. Animals were then given an injection of Ad.lacZ or Ad.hTRAIL at the site of tumor implantation on day 7. Tumor volume (µL) was monitored every other day for 30 days. Animals treated with Ad.hTRAIL have significantly smaller tumors than Ad.lacZ-treated animals (*p<0.05).

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