Inhibition of Ki-67 in a renal cell carcinoma severe combined immunodeficiency disease mouse model is associated with induction of apoptosis and tumour growth inhibition.
OBJECTIVE
To evaluate the effects of suppressing the expression of Ki-67 (expressed in proliferating cells) by antisense oligonucleotides (asON) directed against Ki-67 (which specifically inhibit the proliferation of tumour cells and tumour growth in cell culture and in subcutaneous murine tumour models) on the growth, cell viability and angiogenic activity of a preclinical renal cell carcinoma (RCC) severe combined immunodeficiency disease (SCID) mouse model.


METHODS
Human RCC cells (SK-RC-35) were incubated with asON and control ON in the presence of a cationic lipid in monolayer cell culture. To test Ki-67 as a target for antitumour therapy in more complex models, asON were administered to three-dimensional RCC (SK-RC-35) spheroid cultures and to SCID mice bearing subcutaneous SK-RC-35 xenografts. For animal studies, 1 x 10(6) SK-RC-35 cells were implanted subcutaneously. Subsequently, asON or ON were injected intraperitoneally daily for 14 days at 10 mg/kg/day. Tumour size, weight and status of metastasis were documented daily and after death, respectively. The number of apoptotic cells, Ki-67-positive cells and the microvessel density in tumour sections was determined immunohistochemically. Quantitative reverse transcription-polymerase chain reaction of Ki-67 mRNA was also assessed for the tumours.


RESULTS
Treatment of RCC cells with asON resulted in a specific inhibition of cell growth in monolayer and spheroid cell culture. Systemic administration of Ki-67-directed asON significantly decreased tumour growth (P = 0.009) in SCID mice. Immunohistochemical staining of tumour specimens showed stronger inhibition of Ki-67-positive cells in asON-treated tumours (mean 27.8%) than in controls (mean 42.5-57%). Furthermore, there were about twice as many apoptotic cells after asON treatment. There was no significant difference among treatment groups for microvessel density.


CONCLUSIONS
These results indicate that Ki-67 represents a suitable antiproliferative target, and that asON are a potent agent inhibiting tumour growth and apoptosis, but not tumour vascularization, in human RCC.
Kausch
Ingo
I
Department of Urology, University of Lubeck, Medical School, Ratzeburger Allee 160, 23538 Lubeck, Germany. IKausch@aol.com
Jiang
Hai
H
Ewerdwalbesloh
Nina
N
Doehn
Christian
C
Krüger
Stefan
S
Sczakiel
Georg
G
Jocham
Dieter
D
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002292:Carcinoma, Renal Cell / Q000098:blood supply / Q000378:metabolism / Q000473:pathology*; D002455:Cell Division / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D007150:Immunohistochemistry; D019394:Ki-67 Antigen / Q000378:metabolism*; D007680:Kidney Neoplasms / Q000098:blood supply / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D016513:Mice, SCID; D008954:Models, Biological; D009368:Neoplasm Transplantation; D016376:Oligonucleotides, Antisense / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D014407:Tumor Cells, Cultured
D019394:Ki-67 Antigen; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger