Downregulation of Bcl-2, FLIP or IAPs (XIAP and survivin) by siRNAs sensitizes resistant melanoma cells to Apo2L/TRAIL-induced apoptosis

Abstract

Melanoma cells are relatively resistant to Apo2L/TRAIL (TNFrelated apoptosis-inducing ligand). We postulated that resistance might result from higher expression of inhibitors of apoptosis including Bcl-2, FLIP (FLICE-like inhibitory protein) or IAPs such as XIAP (X-linked inhibitor of apoptosis) or survivin. Compared to scrambled or mismatch controls, targeting individual inhibitors with siRNA (si-Bcl-2, si-XIAP, si-FLIP or si-Surv), followed by Apo2L/TRAIL resulted in marked increase in apoptosis in melanoma cells. Compared to Bcl-2 or FLIP, siRNAs against XIAP and survivin were most potent in sensitizing melanoma cells. A similar substantial increase in apoptosis was seen in renal carcinoma cells (SKRC-45, Caki-2), following the inhibition of either XIAP or survivin by siRNAs. Apo2L/TRAIL treatment in IAP-targeted cells resulted in cleavage of Bid, activation of caspase-9 and cleavage of PARP (poly ADP-ribose polymerase). Thus, Apo2L/TRAIL resistance can be overcome by interfering with expression of inhibitors of apoptosis regulating both extrinsic (death receptor) or intrinsic (mitochondrial) pathways of apoptosis in melanoma cells.

Gene expression in Sinclair swine with malignant melanoma

“…The TYR gene associated with melanoma pathogenesis and significantly upregulated in this study is expressed at various levels in human melanoma cells and is considered an important biochemical marker of melanocytes Parkhurst et al, 1998;Overwijk et al, 1999), while the RAB28 gene, also overexpressed in this study, is expressed in melanocytes and is involved in melanosomal transport and docking (Jager et al, 2000) as well as in the proper sorting of tyrosinase-related protein 1 (TYRP1). The increased transcription of anti-apoptotic genes in this study, including BCL-2 that is elevated in a range of human cancers including melanomas, prostate carcinoma and gliomas (LaCasse et al, 1998;Altieri, 2003;Chawla-Sarkar et al, 2004), demonstrates further the similarity in the modulation of gene expression in Sinclair swine and human cancers.…”

Reversal of methylation silencing of Apo2L/TRAIL receptor 1 (DR4) expression overcomes resistance of SK-MEL-3 and SK-MEL-28 melanoma cells to interferons (IFNs) or Apo2L/TRAIL

“…Real-time RT-PCR (Table 1) and immunoblotting (data not shown) also identified cellspecific induction of XAF1 by IFNs, markedly in SK-MEL-28 but less in SK-MEL-3. XAF1 interacts with an inhibitor of apoptosis (XIAP) that may function as a competitive inhibitor of caspases to play a critical role in TRAIL-induced apoptosis (Deveraux et al, 1997;Liston et al, 2001;Leaman et al, 2002;Chawla-Sarkar et al, 2004). XAF1 has been silenced by DNA methylation in gastric cancer and is frequently reduced in expression in melanoma (Byun et al, 2003;Ng et al, 2004;Reu et al, 2006a).…”

Differential susceptibility to TRAIL of normal versus malignant human urothelial cells

“…26,27 In the case of FLIP, FLIP L and FLIP S isoforms, as well as a 43 kDa cleavage fragment (FLIP 43 ), bind to the DISC and block caspase-8 cleavage and hence activation of the apoptotic cascade. 28 Since CHX sensitized NHU and 253J cells to TRAILinduced apoptosis, we investigated the effects of CHX on FLIP, IAP-2 and XIAP expression.…”

“…Therefore, to determine whether FLIP specifically protects against TRAIL-induced apoptosis in resistant urothelial cells, we used siRNA technology to knockdown FLIP L expression. We used two small interfering RNAs (siRNAs) previously shown to knockdown FLIP L expression, 26,34 as detailed in Materials and Methods. FLIP L siRNA F1 did not reduce FLIP L protein expression (Figure 7a) and had no effect on TRAIL-mediated apoptosis (Figure 7b).…”