Antitumor effect of bcl-2 antisense phosphorothioate oligodeoxynucleotides on human renal-cell carcinoma cells in vitro and in mice.
OBJECTIVE
Programmed cell death is a genetically regulated pathway that is altered in many cancers. This process is, in part, regulated by the bcl-2 oncogene. Antisense oligodeoxynucleotides (ODNs) targeted to specific oncogenes have been used with some therapeutic success in animal models of leukemia and melanoma cells and human Hodgkin's lymphoma. We evaluated the effects of antisense ODNs targeted to the bcl-2 oncogene on the proliferation of human renal-cell carcinoma (RCC) cells in vitro and on the growth of human RCC xenografts in BALBc nude (nu/nu) mice.


METHODS
Expression bcl-2 mRNA in five RCC cell lines (ACHN, Caki-1, RCZ, RCW, and OS-RC-2) was analyzed by reverse transcriptase-polymerase chain reaction. The effects of phosphorothioated ODNs containing human bcl-2 sense and bcl-2 antisense sequences that were transfected with Lipofectin on the proliferation and viability of cultures of established human RCC cell lines were determined by MTS assay. The expression of Bcl-2 protein in ACHN tumor cells following antisense bcl-2 (AS2) ODN treatment was evaluated by Western blot analysis, and the extent of apoptosis in these cells was determined by fluorescence-activated cell sorter (FACS) analysis. The antitumor activity in ACHN xenografts in nu/nu mice was monitored by measuring differences in tumor weight in treated and control mice.


RESULTS
Expression of bcl-2 mRNA was detected in all five RCC lines. Treatment with antisense bcl-2 ODNs inhibited the growth of all tested RCC cells and decreased Bcl-2 protein expression in ACHN cells. The AS2 antisense ODN complementary to the coding region of bcl-2 mRNA showed a superior antiproliferative effect compared with AS1 ODN complementary to the translation initiation region. Inhibition by antisense bcl-2 ODNs of ACHN cells was dose dependent. The FACS analysis revealed that growth inhibition was associated with the induction of programmed cell death. In vivo, AS2 ODN antitumor activity was noted in locally injected groups.


CONCLUSIONS
Treatment of human RCC with antisense ODNs targeted to bcl-2 inhibits growth and is associated with the induction of programmed cell death. These results suggest therapeutic use of antisense bcl2 in the treatment of RCC.
Uchida
T
T
Department of Urology, Kitasato University School of Medicine, Sagamihara, Kanagawa, Japan. tuchida@med.kitasato-u.ac.jp
Gao
J P
JP
Wang
C
C
Satoh
T
T
Itoh
I
I
Muramoto
M
M
Hyodo
T
T
Irie
A
A
Akahoshi
T
T
Jiang
S X
SX
Kameya
T
T
Baba
S
S
D000818:Animals; D017209:Apoptosis / Q000502:physiology; D002292:Carcinoma, Renal Cell / Q000473:pathology / Q000628:therapy*; D002455:Cell Division / Q000502:physiology; D002469:Cell Separation; D017690:Cell Transplantation; D005434:Flow Cytometry; D019254:Genes, bcl-2; D015316:Genetic Therapy; D006801:Humans; D007680:Kidney Neoplasms / Q000473:pathology / Q000628:therapy*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics / Q000627:therapeutic use*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D013873:Thionucleotides / Q000235:genetics / Q000627:therapeutic use*; D014162:Transfection; D014183:Transplantation, Heterologous; D014407:Tumor Cells, Cultured
D020319:Oligodeoxyribonucleotides, Antisense; D019253:Proto-Oncogene Proteins c-bcl-2; D013873:Thionucleotides