Agents that generate reactive oxygen species (ROS) are recognized to enhance MDA-7/IL-24 lethality. The present studies focused on clarifying how such agents enhanced MDA-7/IL-24 toxicity in renal cell carcinoma cells (RCCs). Infection of RCCs with a tropism-modified serotype 5/3 adenovirus expressing MDA-7/IL-24 (Ad.5/3-mda-7) caused plasma membrane clustering of CD95 and CD95 association with pro-caspase 8, effects that were enhanced by combined exposure to 17-N-allylamino-17-demethoxygeldanamycin (17AAG), As2O3, or fenretinide and that correlated with enhanced cell killing. Knockdown of CD95 or expression of cellular FADD (Fas-associated protein with death domain)-like interleukin-1β–converting enzyme inhibitory protein, short form (c-FLIP-s) blocked enhanced killing. Inhibition of ROS generation, elevated cytosolic Ca2+, or de novo ceramide synthesis blocked Ad.5/3-mda-7 ± agent-induced CD95 activation and the enhancement of apoptosis. Ad.5/3-mda-7 increased ceramide levels in a PERK-dependent fashion that were responsible for elevated cytosolic Ca2+ levels that promoted ROS generation; 17AAG did not further enhance cytokine-induced ceramide generation. In vivo, infection of RCC tumors with Ad.5/3-mda-7 suppressed the growth of infected tumors that was enhanced by exposure to 17AAG. Our data indicate that in RCCs, Ad.5/3-mda-7-induced ceramide generation plays a central role in tumor cell killing and inhibition of multiple signaling pathways may have utility in promoting MDA-7/IL-24 lethality in renal cancer.
This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant R01-DK52825] (to P.D.); the National Institutes of Health National Cancer Institute [Grants R01-CA108325, R01-CA141703, R01-CA150214] (to P.D.), [Grants R01-CA63753, R01-CA77141] (to S.G.), [Grants R01-CA097318, R01-CA127641, R01-CA134721] (to P.B.F.), and [Grant P01-CA104177] (to P.D., P.B.F., D.C., I.P.D.); The Jim Valvano “V” foundation (to P.D.); Department of Defense [Award DAMD17-03-1-0262] (to P.D.); the Leukemia Society of America [Grant 6405-97] (to S.G.); the Samuel Waxman Cancer Research Foundation (to P.B.F.); and the National Foundation for Cancer Research (to P.B.F.). P.D. is The Universal Inc. Professor in Signal Transduction Research. P.B.F. holds the Thelma Newmeyer Corman Chair in Cancer Research in the VCU Massey Cancer Center.
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
- renal cell carcinoma
- endoplasmic reticulum
- protein kinase R-like endoplasmic reticulum kinase
- glutathione transferase
- reactive oxygen species
- mitogen activated protein kinase
- coxsackie and adenovirus receptor
- N-(4-hydroxyphenyl) retinamide (fenretinide)
- dimethyl sulfoxide
- polyacrylamide gel electrophoresis
- multiplicity of infection
- small interfering
- terminal deoxynucleotidyl transferase dUTP nick-end labeling
- fetal bovine serum
- phosphate-buffered saline
- death-inducing signaling complex
- Von Hippel Lindau
- longevity assurance gene
- cytomegalovirus (empty vector)
- melanoma differentiation associated gene-7
- serotype 5/serotype 3 adenovirus
- Received October 18, 2010.
- Accepted November 30, 2010.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics