Enhanced therapeutic effects for human pancreatic cancer by application K-ras and IGF-IR antisense oligodeoxynucleotides.
OBJECTIVE
To investigate the combined effects of K-ras antisense oligodeoxynucleotide (K-ras ASODN) specific to GTT point mutation at codon 12 and type I insulin-like growth factor receptor (IGF-IR) antisense oligodeoxynucleotide (IGF-IR ASODN) on proliferation and apoptosis of human pancreatic cancer Patu8988 cells in vitro and in vivo.


METHODS
K-ras gene point mutation and its style at codon 12 of human pancreatic cancer cell line Patu8988 were detected by using polymerase chain reaction with special sequence primers (PCR-SSP) and sequence analysis. According to the mutation style, K-ras mutation ASODN specific to K-ras point mutation at codon 12 was designed and composed. After K-ras ASODN and IGF-IR ASODN treated on Patu8988 cells respectively or cooperatively, the proliferation and morphological change of Patu8988 cells were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony forming assay and transmission electron microscopy; the expression of K-ras and IGF-IR mRNA and protein in the treated cells was measured by reverse-transcript polymerase chain reaction (RT-PCR) and flow cytometry respectively; apoptosis was determined by flow cytometry. The combined antitumor activity of K-ras ASODN and IGF-IR ASODN was evaluated in BALB/c nude mice bearing human pancreatic cancer inoculated with Patu8988 cells.


RESULTS
The results of PCR-SSP and sequence analysis showed that the human pancreatic cancer cell line Patu8988 had point mutation at codon 12, and the mutation style was GGT-->GTT. 2-32 microg/mL K-ras ASODN and 2-32 microg/mL IGF-IR ASODN could inhibit Patu8988 cells' growth, induce apoptosis and decrease the expression of K-ras and IGF-IR mRNA and protein alone. However, there was much more effective inhibition of growth and induction of apoptosis by their combination than by each one alone. In tumor bearing mice, the combination of K-ras ASODN and IGF-IR ASODN showed a significant inhibitory effect on the growth of transplanted pancreatic cancer, resulting in a statistically significant difference compared with each alone.


CONCLUSIONS
It has been found that K-ras ASODN combined with IGF-IR ASODN could cooperatively inhibit the growth of Patu8988 cells, and induce their apoptosis via reinforcing specific down regulation of K-ras and IGF-IR mRNA and protein expression.
Shen
Yong-Mei
YM
Radioimmunoassay Center & Clinical Laboratory, Department of MRI, the Second Affiliated Hospital of Soochow University, Suzhou 215004, Jiangsu Province, China.
Yang
Xiao-Chun
XC
Yang
Chen
C
Shen
Jun-Kang
JK
D000230:Adenocarcinoma / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D015536:Down-Regulation / Q000187:drug effects; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology / Q000627:therapeutic use*; D010190:Pancreatic Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D017354:Point Mutation / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000235:genetics* / Q000378:metabolism
D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D017526:Receptor, IGF Type 1; D016283:Proto-Oncogene Proteins p21(ras)
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