Article

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: 8.39). 09/2004; 11(8):915-23. DOI: 10.1038/sj.cdd.4401416
Source: PubMed

ABSTRACT 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.

Download full-text

Full-text

Available from: Mamta Chawla-Sarkar, Jan 06, 2015
0 Followers
 · 
97 Views
  • Source
    [Show abstract] [Hide abstract]
    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.
    International Immunopharmacology 06/2014; 21(2):439-446. DOI:10.1016/j.intimp.2014.05.024 · 2.71 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Induction of apoptosis plays a crucial role in the response of tumors to treatment. Thus, we investigated the pharmacodynamics and tumor saturation kinetics of a death receptor 5 antibody (anti-DR5) when combined with chemotherapeutics. For our investigations, we applied an imaging method that allows monitoring of apoptosis noninvasively in living mice. A stably transfected apoptosis reporter based on split luciferase technology facilitates to screen various chemotherapeutics and anti-DR5 on their ability to induce apoptosis in glioblastoma cells in vitro as well as in vivo. We found that doxorubicin (DOX) treatment in vitro led to significant apoptosis induction within 48 hours and to a 2.3-fold increased anti-DR5 binding to the cell surface. In contrast, cisplatin and 5-fluorouracil (5-FU) treatment altered anti-DR5 binding only marginally. Induction of apoptosis by treatment with anti-DR5 was dose- and time-dependent (both in vitro and in vivo). Simultaneous visualization of fluorescence-labeled anti-DR5 in tumor tissue and apoptosis revealed maximal apoptosis induction immediately after the compound had reached tumor site. Regarding combination therapy of anti-DR5 and DOX, we found that the sequential application of DOX before anti-DR5 resulted in synergistically enhanced apoptosis reporter activity. In striking contrast, anti-DR5 given before DOX did not lead to increased apoptosis induction. We suggest that DOX-induced recruitment of DR5 to the cell surface impacts the enhanced apoptotic effect that can be longitudinally monitored by apoptosis imaging. This study demonstrates that the combination of apoptosis and fluorescence imaging is an excellent method for optimizing dosing and treatment schedules in preclinical cancer models.
    Neoplasia (New York, N.Y.) 08/2013; 15(8):863-74. DOI:10.1593/neo.13932 · 5.40 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Sinclair swine develop an aggressive form of melanoma, which, in many cases, spontaneously regresses after a complete metastatic phase. We used Affymetrix GeneChip® Porcine Genome Arrays consisting of 24 123 probe sets to compare gene expression in white blood cells (WBCs) and various tissues including the liver, lungs, inguinal lymph nodes and spleen harvested from a Sinclair piglet afflicted by melanoma at birth and exhibiting metastatic lesions at weaning (6 weeks) with those from a full-sibling piglet that showed no incidence of melanoma at birth and weaning. The highest number (3489; ∼14%) of significantly upregulated transcripts (fold change in gene expression ≥2.0 and t-test P-value ≤0.05) was observed in the liver, while the spleen exhibited the lowest number of upregulated transcripts (528; ∼2%). Among significantly downregulated genes, the highest numbers were observed in the inguinal lymph nodes (3651; ∼15%) and the least in WBCs (730; ∼3%). Differentially expressed transcripts included genes involved in melanoma pathogenesis including SILV, TYR and RAB28. SILV was over-expressed 784-, 430- and 164-fold, while TYR was over-expressed 138-, 81- and 28-fold in the liver, lungs and inguinal lymph nodes, respectively. Quantitative real-time RT-PCR (qRT-PCR) confirmed the microarray data of 12 selected differentially expressed sequences. These results suggest that significant changes in gene expression occur during metastasis of malignant melanoma in the Sinclair swine model. In addition, qRT-PCR analysis of the above 12 differentially expressed sequences was carried out on liver samples collected from 22 pigs (12 of which had melanoma during the first 6 weeks of life), and an ANOVA test contrasting absolute RNA expression between pigs with regressing, progressing and without tumors was significant for TYR, TACSTD1, MATP, GPNMB and CYP4A22, with P-values of 0.034, 0.015, 0.007, 0.050 and 0.022, respectively.
    animal 02/2012; 6(2):179-92. DOI:10.1017/S1751731111001637 · 1.78 Impact Factor