[Combinational effects of K-ras and IGF-IR antisense oligodeoxynucleotide on proliferation and apoptosis of human pancreatic cancer Patu8988 cells].
OBJECTIVE
Point mutation of K-ras gene and overexpression of insulin-like growth factor receptor type 1 (IGF-IR) may contribute to the progression and aggressiveness of pancreatic cancer. Antisense oligodeoxynucleotide (ASODN) against K-ras mRNA and IGF-IR mRNA may inhibit the proliferation of pancreatic cancer cells. This study was to investigate the combinational effects of K-ras ASODN and IGF-IR ASODN on proliferation and apoptosis of human pancreatic cancer Patu8988 cells in vitro and in vivo.


METHODS
K-ras gene point mutation in Patu8988 cells was detected by polymerase chain reaction using special sequence primers (PCR-SSP) and sequence analysis. According to the mutation style, K-ras ASODN was designed and composed. K-ras ASODN and IGF-IR ASODN were transfected into Patu8988 cells alone or in combination. Cell proliferation was analyzed by MTT and colony forming assay. The morphologic changes of Patu8988 cells were assessed under transmission electron microscope. The expression of K-ras and IGF-IR mRNA and protein in Patu8988 cells was measured by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FCM). Cell apoptosis was determined by FCM. The combinational 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 point mutation of K-ras gene at codon 12 was detected in Patu8988 cells, and the mutation style was GGT-->GTT. Either 2-32 microg/mL K-ras ASODN or IGF-IR ASODN inhibited proliferation and induced apoptosis of Patu8988 cells. This effect was more obvious when K-ras ASODN and IGF-IR ASODN were used in combination than used alone (P<0.01). In tumor-bearing mice, the inhibitory effect on the growth of transplanted pancreatic cancer was more obvious when K-ras ASODN and IGF-IR ASODN were used in combination than used alone (P<0.01).


CONCLUSIONS
K-ras ASODN combined with IGF-IR ASODN could cooperatively inhibit the proliferation of Patu8988 cells and induce their apoptosis via down-regulating K-ras and IGF-IR expression.
Shen
Yong-Mei
YM
Department of Radioimmunoassay Center, The Second Affiliated Hospital, Soochow University, Suzhou, Jiangsu, 215004, People's Republic of China.
Yang
Xiao-Chun
XC
Zhang
Mei-Hua
MH
Shen
Jun-Kang
JK
Sun
Yi-Hui
YH
D000818:Animals; D017209:Apoptosis; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D011905:Genes, ras; D006801:Humans; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics*; D010190:Pancreatic Neoplasms / Q000378:metabolism / Q000473:pathology*; D017354:Point Mutation; D012333:RNA, Messenger / Q000378:metabolism; D017526:Receptor, IGF Type 1 / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D047368:Tumor Burden; D018631:ras Proteins / Q000235:genetics* / Q000378:metabolism
D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; D017526:Receptor, IGF Type 1; D018631:ras Proteins