Chemosensitization of human renal cell cancer using antisense oligonucleotides targeting the antiapoptotic gene clusterin.
BACKGROUND
Renal cell cancer (RCC) is a chemoresistant disease with no active chemotherapeutic agent achieving objective response rates higher than 15%. Clusterin is a cell survival gene that increases in human renal tubular epithelial cells after various states of injury and disease. Downregulation of clusterin, using antisense oligonucleotides (ASO), has recently been shown to increase chemosensitivity in several prostate cancer models. The objectives in this study were to evaluate clusterin expression levels in human RCC and normal kidney tissue, and to test whether clusterin ASO could also enhance chemosensitivity in human RCC Caki-2 cells both in vitro and in vivo.


METHODS
Immunohistochemical staining was used to characterize clusterin expression in 67 RCC and normal kidney tissues obtained from radical nephrectomy specimens. Northern blot analysis was used to assess changes in clusterin mRNA expression after ASO and paclitaxel treatment. The effects of combined clusterin ASO and paclitaxel treatment on Caki-2 cell growth was examined using an MTT assay. Athymic mice bearing Caki-2 tumors were treated with clusterin ASO alone, clusterin ASO plus paclitaxel, and mismatch control oligonucleotides plus paclitaxel, over a period of 28 days with measurement of tumor volumes once weekly over 8 weeks.


RESULTS
Immunohistochemistry of normal and malignant kidney tissue sections of 67 patients demonstrated positive clusterin staining for almost all RCC (98%) and an overexpression, compared to normal tissue, in a majority of RCC (69%). Clusterin ASO, but not mismatch control oligonucleotides, decreased clusterin mRNA expression in Caki-2 cells in a dose-dependent and sequence-specific manner. Pretreatment of Caki-2 cells with clusterin ASO significantly enhanced chemosensitivity to paclitaxel in vitro. Characteristic apoptotic DNA laddering was observed after combined treatment with ASO plus paclitaxel, but not with either agent alone. In vivo administration of clusterin ASO plus paclitaxel acted synergistically to increase apoptosis and significantly delay Caki-2 tumor growth, compared to mismatch control oligonucleotide plus paclitaxel. In addition, TUNEL staining revealed increased apoptotic cells in tumors treated with clusterin ASO plus paclitaxel compared to treatment with either clusterin ASO or paclitaxel alone.


CONCLUSIONS
These findings confirm that the use of clusterin ASO may be a feasible strategy to enhance chemosensitivity for patients with advanced RCC.
Zellweger
T
T
The Prostate Centre, Vancouver General Hospital, Vancouver, British Columbia, Canada V6H 3Z6.
Miyake
H
H
July
L V
LV
Akbari
M
M
Kiyama
S
S
Gleave
M E
ME
D018262:Adenocarcinoma, Clear Cell / Q000188:drug therapy / Q000378:metabolism; D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000627:therapeutic use*; D017209:Apoptosis; D015152:Blotting, Northern; D002292:Carcinoma, Renal Cell / Q000188:drug therapy / Q000378:metabolism; D051152:Clusterin; D017931:DNA Primers / Q000737:chemistry; D015536:Down-Regulation; D006023:Glycoproteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D020287:In Situ Nick-End Labeling; D007668:Kidney / Q000187:drug effects / Q000378:metabolism; D007680:Kidney Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use*; D017239:Paclitaxel / Q000627:therapeutic use*; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D000972:Antineoplastic Agents, Phytogenic; C495242:CLU protein, human; C495243:Clu protein, mouse; D051152:Clusterin; D017931:DNA Primers; D006023:Glycoproteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D017239:Paclitaxel