Enhancement of death receptor 4 mediated apoptosis and cytotoxicity in renal cell carcinoma cells by subtoxic concentrations of doxorubicin.
OBJECTIVE
TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) triggers apoptosis in various tumor cells by engaging death receptors 4 and 5. We investigated the effect of chemotherapeutic agents on death receptor 4 mediated apoptosis in human renal cell carcinoma cells using HGS-ETR1, which is a human monoclonal agonistic antibody specific for death receptor 4.


METHODS
Cytotoxicity was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Synergy was assessed by isobolographic analysis.


RESULTS
Treatment of the ACHN human renal cell carcinoma cell line with HGS-ETR1 combined with 5-fluorouracil, vinblastine or gemcitabine did not overcome resistance to these agents. However, treatment with HGS-ETR1 combined with doxorubicin had a synergistic cytotoxic effect. Synergy was also achieved in another human renal cell carcinoma cell line, Caki-1, and in 5 freshly derived renal cell carcinoma cell cultures. A synergistic effect was also observed with HGS-ETR1 combined with the doxorubicin derivatives epirubicin, pirarubicin or amrubicin. The synergy achieved in cytotoxicity with HGS-ETR1 and doxorubicin was also achieved in apoptosis. Sequential treatment with doxorubicin followed by HGS-ETR1 induced significantly more cytotoxicity than reverse treatment or simultaneous treatment (p<0.05). Doxorubicin remarkably increased the cell surface expression of death receptor 4 in renal cell carcinoma cells. The combination of doxorubicin and HGS-ETR1 significantly activated the caspase cascade, including caspase-8, 9, 6 and 3, which are the downstream molecules of death receptors.


CONCLUSIONS
These findings indicate that doxorubicin sensitizes renal cell carcinoma cells to death receptor 4 mediated apoptosis through the induction of death receptor 4 and the activation of caspases, suggesting that combination therapy of doxorubicin and HGS-ETR1 might be effective as renal cell carcinoma therapy.
Jin
Xinghua
X
Department of Urology, Faculty of Medicine, Kagawa University, Kagawa, Japan.
Wu
Xiu-Xian
XX
Abdel-Muneem Nouh
Mohammed Ahmed
MA
Kakehi
Yoshiyuki
Y
D000903:Antibiotics, Antineoplastic / Q000008:administration & dosage*; D017209:Apoptosis / Q000187:drug effects*; D002292:Carcinoma, Renal Cell / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D045744:Cell Line, Tumor; D003601:Cytotoxicity Tests, Immunologic; D004305:Dose-Response Relationship, Drug; D004317:Doxorubicin / Q000008:administration & dosage*; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007680:Kidney Neoplasms / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D008856:Microscopy, Fluorescence; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand / Q000187:drug effects / Q000378:metabolism*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000187:drug effects / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; D053220:Receptors, TNF-Related Apoptosis-Inducing Ligand; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D004317:Doxorubicin