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

Center for Drug Discovery and Development, Taussig Cancer Center, Cleveland Clinic Foundation, Cleveland, OH 44195, USA.
Cell Death and Differentiation (Impact Factor: 8.18). 09/2004; 11(8):915-23. DOI: 10.1038/sj.cdd.4401416
Source: PubMed


Melanoma cells are relatively resistant to Apo2L/TRAIL (TNF-related 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.

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Available from: Mamta Chawla-Sarkar, Jan 06, 2015
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    • "In a classical approach by using antisense oligonucleotides (55), siRNA (56, 57) or morpholino-antisense (58), the decrease of the mRNA and protein levels of XIAP was shown to sensitize drug-resistant cancer cells to therapy-induced apoptosis or to induce even spontaneous cell death specifically in cancer cells. This is especially true for cancer cells with defective mitochondrial death signaling: repression of XIAP mRNA by RNAi or the specific XIAP-transcription-inhibitory compound Mithramycin A (59) overcomes TRAIL-resistance in carcinoma cells that show deregulation of the intrinsic apoptosis signaling pathway (60). "
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    ABSTRACT: Defects in apoptosis regulation are one main cause of cancer development and may result from overexpression of anti-apoptotic proteins such as inhibitor of apoptosis proteins (IAPs). IAPs are cell death regulators that, among other functions, bind caspases, and interfere with apoptotic signaling via death receptors or intrinsic cell death pathways. All IAPs share one to three common structures, the so called baculovirus-IAP-repeat (BIR)-domains that allow them to bind caspases and other proteins. X-linked inhibitor of apoptosis protein (XIAP) is the most potent and best-defined anti-apoptotic IAP family member that directly neutralizes caspase-9 via its BIR3 domain and the effector caspases-3 and -7 via its BIR2 domain. A natural inhibitor of XIAP is SMAC/Diablo, which is released from mitochondria in apoptotic cells and displaces bound caspases from the BIR2/BIR3 domains of XIAP thereby reactivating cell death execution. The central apoptosis-inhibitory function of XIAP and its overexpression in many different types of advanced cancers have led to significant efforts to identify therapeutics that neutralize its anti-apoptotic effect. Most of these drugs are chemical derivatives of the N-terminal part of SMAC/Diablo. These "SMAC-mimetics" either specifically induce apoptosis in cancer cells or act as drug-sensitizers. Several "SMAC-mimetics" are currently tested by the pharmaceutical industry in Phase I and Phase II trials. In this review, we will discuss recent advances in understanding the function of IAPs in normal and malignant cells and focus on approaches to specifically neutralize XIAP in cancer cells.
    Full-text · Article · Jul 2014 · Frontiers in Oncology
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    • "Permanent as well as inducible TRAIL-resistance seen in some melanomas may limit its applicability. The downregulation or malfunction of TRAIL receptors and initiator caspase 8 [5] [6], and/or upregulation of antiapoptotic molecules such as Flice-inhibitory protein (c-FLIP) [7] were listed as major causes of TRAIL treatment failure. Therefore, efforts have been undertaken to induce or increase cellular sensitivity for TRAIL-induced apoptosis by modulating death pathway at several levels. "
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    ABSTRACT: Tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL) has selective killing effect toward malignant cells; however some human melanomas are intrinsically resistant. In this study, we have shown that class I-specific histone deacetylase inhibitor (HDACi) MS-275 can synergize with TRAIL to induce apoptosis in TRAIL-resistant cell lines and to enhance susceptibility of sensitive cells. Conversely, class II-selective HDACi MC1575 has shown no effect on the resistance of melanoma cells and was able exclusively to increase TRAIL-induced cell death in responsive cells. Both the HDACis variably increased DR4, DR5, and procaspase 8 expression, regardless whether cells were TRAIL-sensitive or TRAIL-resistant. However, only MS-275 markedly decreased the expression levels of both the long and short c-FLIP isoforms. RNAi-mediated c-FLIP silencing resulted in caspase 8-dependent apoptosis in survivor cells which was comparable to that observed following MS-275 treatment. Accordingly, enforced expression of ectopic c-FLIP has abolished the cooperative induction of apoptosis by the combination of MS-275 and TRAIL. These data indicate that c-FLIP is a critical regulator of death ligand sensitivity in melanoma. Inhibition of class I HDAC isoenzymes 1, 2 and 3 has resulted to be functionally important for c-FLIP downregulation by MS-275. In contrast, knockdown of class II HDACs has had no effect on c-FLIP expression, thus explaining the dual incapacity of MC1575 to inhibit c-FLIP expression and sensitize cells resistant to TRAIL. The data reported here suggest that MS-275 represents a promising therapeutic approach in combination with TRAIL for treatment of cutaneous and uveal melanoma due to its ability to reduce c-FLIP expression.
    Full-text · Article · Jun 2014 · International Immunopharmacology
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    • "Therefore, IAPs have been proven to be closely related to therapy resistance, and strategies targeting IAPs may be effective for overcoming apoptosis resistance [35]. For example, overexpression of XIAP can increase resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), whereas down-regulation of XIAP restored the cell response to TRAIL [36]. Meanwhile, chemotherapeutic drugs such as DOX and docetaxel can induce the activation of the Ras/MEK/ERK pathway, and the activated cascade may regulate downstream factors that are involved in DNA repair and apoptosis, thereby contributing to drug resistance [11]. "
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    ABSTRACT: Drug resistance is one of the main hurdles for the successful treatment of breast cancer. The synchronous targeting of apoptosis resistance and survival signal transduction pathways may be a promising approach to overcome drug resistance. In this study, we determined that evodiamine (EVO), a major constituent of the Chinese herbal medicine Evodiae Fructus, could induce apoptosis of doxorubicin (DOX)-sensitive MCF-7 and DOX-resistant MCF-7/ADR cells in a caspase-dependent manner, as confirmed by significant increases of cleaved poly(ADP-ribose) polymerase (PARP), caspase-7/9, and caspase activities. Notably, the reversed phenomenon of apoptosis resistance by EVO might be attributed to its ability to inhibit the Ras/MEK/ERK pathway and the expression of inhibitors of apoptosis (IAPs). Furthermore, our results indicated that EVO enhanced the apoptotic action of DOX by inhibiting the Ras/MEK/ERK cascade and the expression of IAPs without inhibiting the expression and activity of P-glycoprotein (P-gp). Taken together, our data indicate that EVO, a natural product, may be useful applied alone or in combination with DOX for the treatment of resistant breast cancer.
    Full-text · Article · May 2014 · PLoS ONE
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