Inhibition of bcl-2 enhances the efficacy of chemotherapy in renal cell carcinoma.
OBJECTIVE
Renal cell cancer (RCC) is highly resistant to chemotherapy. Increased expression of the antiapoptotic gene bcl-2 in tumors is known to be associated with poor responses to systemic treatment of cancer. Down-regulation of bcl-2 expression using antisense oligonucleotides (asON) has been shown to increase chemosensitivity in clinical phase I-III studies with various cancers. However, no studies on the efficacy of this approach in RCC have been reported so far. This study aimed to evaluate whether bcl-2 asON could enhance efficacy of chemotherapy in human RCC.


METHODS
Expression of bcl-2 mRNA and protein was analyzed in different RCC cell lines by RT-PCR and Western blot. Cells with high or low bcl-2 mRNA and protein expression were treated with different concentrations of bcl-2 asON in combination with cisplatin. AsON-induced down-regulation of bcl-2 mRNA and protein was documented by RT-PCR and Western blot. Treatment effects on cell viability were analyzed by colorimetric tetrazolium (MTT) assay. Immunohistochemical staining of M30-positive cells was performed for quantification of apoptotic cells.


RESULTS
Transfection of high bcl-2 expressing cells with bcl-2 asON alone induced no reduction of cell viability at a concentration range from 100-1000 nM. In combination therapy, pretreatment with asON significantly enhanced MTT reduction after cisplatin treatment. IC50 concentrations of cisplatin were 1 microg/ml with and 2.7 microg/ml without prior incubation. The marked reduction of cell viability correlated with an 8-fold increase of apoptotic cells after combination treatment. Only a minor increase of cisplatin effectivity was noted after asON preincubation of cells with lower bcl-2 expression.


CONCLUSIONS
The combination of cisplatin and bcl-2 antisense ON exerts significantly greater effects on cell viability and apoptosis than either agent used alone on human RCC cells. These data indicate that inhibition of bcl-2 expression may be an attractive therapeutic strategy in RCC tumors with high bcl-2 expression.
Kausch
I
I
Department of Urology, University of Lubeck, Medical School, Ratzeburger Allee 160, 23538 Lubeck, Germany. ikausch@aol.com
Jiang
H
H
Thode
B
B
Doehn
C
C
Krüger
S
S
Jocham
D
D
D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis / Q000235:genetics; D014408:Biomarkers, Tumor / Q000378:metabolism; D015153:Blotting, Western; D002292:Carcinoma, Renal Cell / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002945:Cisplatin / Q000627:therapeutic use; D003124:Colorimetry; D015536:Down-Regulation / Q000502:physiology; D004359:Drug Therapy, Combination; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects / Q000235:genetics*; D019254:Genes, bcl-2 / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D066298:In Vitro Techniques; D007680:Kidney Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D016376:Oligonucleotides, Antisense / Q000627:therapeutic use; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016896:Treatment Outcome
D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D002945:Cisplatin