Abstract
Past studies have identified a number of distinct mechanisms that contribute to the resistance of melanoma cells against apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL). In this report we show that cystatin B is another endogenous inhibitor of TRAIL-induced apoptosis. Cystatin B-deficient melanoma cell lines established by shRNA knockdown displayed increased apoptosis that was associated with enhanced activation of caspase-8 induced by TRAIL. This was not related to the inhibitory effect of cystatin B on the lysosomal cysteine proteases, cathepsin B and L, as they did not have a role in TRAIL-induced apoptosis in most melanoma cell lines even when cystatin B was inhibited. Instead, sensitization of melanoma cells to TRAIL-induced apoptosis by inhibition of cystatin B appeared associated with decreased stability of FLIPL as the levels of FLIPL were reduced because of shortened half-life time in melanoma cells deficient in cystatin B. In contrast, over-expression of cystatin B increased the levels of FLIPL, decreased the amount of the E3 ligase Itch associated with FLIPL, and reduced FLIPL ubiquitination. Inhibition of Itch by siRNA restored the levels of FLIPL and blocked sensitization to TRAIL-induced apoptosis associated with deficiency in cystatin B. Taken together, these results indicate that cystatin B regulates Itch-mediated degradation of FLIPL and thereby TRAIL-induced apoptosis in melanoma cells.
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Abbreviations
- CA074Me:
-
L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl]-L-isoleucyl-L-proline methyl ester
- DISC:
-
death-inducing signaling complex
- FADD:
-
Fas-associated death domain
- FLIP:
-
FLICE-like inhibitory protein
- MAb:
-
monoclonal antibody
- MG132:
-
carbobenzoxy-L-leucyl-L-leucyl-L-leucinal
- ΔΨm:
-
mitochondrial membrane potential
- PI:
-
propidium iodide
- shRNA:
-
Short hairpin RNA
- siRNA:
-
Small interference RNA
- TRAIL:
-
TNF-related apoptosis-inducing ligand
- TRAIL-R:
-
TRAIL receptor
- z-IETD-fmk:
-
z-lle-Glu(Ome)-Thr-Asp(Ome)-CH2F
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Acknowledgements
This work was supported by the NSW State Cancer Council, the Melanoma and Skin Cancer Research Institute Sydney, the Hunter Melanoma Foundation, NSW, and the National Health and Medical Research Council (NHMRC), Australia. XD Zhang is a Cancer Institute NSW Fellow. The authors thank Dr. D-G Kim (Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea) for the pcDNA3.1 vector carrying cystatin B cDNA, and Dr H Nakano (Juntendo University School of Medicine, Tokyo, Japan) for the pCR vector carrying FLIPL cDNA and FLIPS cDNA.
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Yang, F., Tay, K., Dong, L. et al. Cystatin B inhibition of TRAIL-induced apoptosis is associated with the protection of FLIPL from degradation by the E3 ligase itch in human melanoma cells. Cell Death Differ 17, 1354–1367 (2010). https://doi.org/10.1038/cdd.2010.29
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DOI: https://doi.org/10.1038/cdd.2010.29
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