Bcl-XL antisense oligonucleotides coupled with antennapedia enhances radiation-induced apoptosis in pancreatic cancer.
BACKGROUND
Pancreatic cancer is highly resistant to radiation and chemotherapy, and its resistance reflects the enhancement of apoptosis inhibitory genes, including Bcl-2 family. Antennapedia (pAnt) is capable of almost 100% internalization into cells through the lipid bilayer without any cytotoxic effect. The aim of this study was to examine the effects of the Bcl-XL antisense oligonucleotide for radiosensitivity of in vitro and in vivo pancreatic cancer using oligonucleotide conjugated with antennapedia.


METHODS
In in vitro experiments, expression of Bcl-XL protein was examined in 5 pancreatic cancer cell lines. In AsPC-1 cells, internalization of the oligonucleotide was confirmed, and the effects of antennapedia-antisense (pAnt-AS) or antennapedia-scramble (pAnt-Scr) on Bcl-XL protein expression were examined. Cells were treated with pAnt-AS, pAnt-Scr or phosphorothioate antisense (S-AS) for 3 days, then the effects of irradiation on the cell survival, caspase-3 activity, and apoptotic index were evaluated. In AsPC-1 xenograft mice, pAnt-AS, pAnt-Scr, or S-AS was injected, and 5 or 10 Gy irradiation was added. Bcl-Xl protein expression was measured before irradiation. Apoptosis was evaluated at 48 hours after irradiation. On the 14th day after 10-Gy irradiation, tumor wet weight was measured, and tumor growth was estimated over 5 weeks.


RESULTS
In in vitro experiments, all pancreatic cancer cell lines expressed Bcl-XL protein. pAnt-AS was internalized into AsPC-1 cells within 2 hours. pAnt-AS at 10 mumol/L reduced more than 90% of the Bcl-XL protein in AsPC-1 cells, whereas pAnt-Scr or S-AS treatment at the same concentration reduced as much as 10% of the Bcl-XL protein. Treatment with pAnt-AS followed by irradiation significantly reduced cell viability when compared with that of pAnt-Scr or S-AS. Caspase-3 activity was significantly upregulated in the pAnt-AS-treated group (P = .033). The rate of nuclear fragmentation was significantly higher in the pAnt-AS group (P = .013). In in vivo experiments, Bcl-XL protein was reduced about 40% in the pAnt-AS-treated mice. Tumor doubling time of the pAnt-AS-treated mice was elongated by 10-Gy irradiation. The tumor wet weight of mice treated with pAnt-AS and 10-Gy irradiation was significantly reduced when compared with mice treated with pAnt-Scr and 10-Gy irradiation (P = .046). The apoptosis index at 48 hours after irradiation was significantly increased in pAnt-AS-treated mice (P < .01).


CONCLUSIONS
The results suggest that, when coupled with antennapedia, the antisense oligonucleotide against Bcl-XL could be a good therapeutic tool for radiosensitization of pancreatic cancer.
Masui
Toshihiko
T
Department of Surgery and Surgical Basic Science, Kyoto University, Kyoto, Japan.
Hosotani
Ryo
R
Ito
Daisuke
D
Kami
Kazuhiro
K
Koizumi
Masayuki
M
Mori
Tomohiko
T
Toyoda
Eiji
E
Nakajima
Sanae
S
Miyamoto
Yoshiharu
Y
Fujimoto
Koji
K
Doi
Ryuichiro
R
D000818:Animals; D051943:Antennapedia Homeodomain Protein / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000528:radiation effects; D018929:Cell Culture Techniques; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009368:Neoplasm Transplantation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D010190:Pancreatic Neoplasms / Q000378:metabolism* / Q000473:pathology / Q000532:radiotherapy; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism*
D051943:Antennapedia Homeodomain Protein; C494812:Bcl2l1 protein, mouse; D016376:Oligonucleotides, Antisense; D051020:bcl-X Protein