Mithramycin A sensitizes cancer cells to TRAIL-mediated apoptosis by down-regulation of XIAP gene promoter through Sp1 sites

Abstract

Mithramycin A is a DNA-binding antitumor agent, which has been clinically used in the therapies of several types of cancer and Paget's disease. In this study, we investigated the combined effect of mithramycin A and tumor necrosis factor-A -related apoptosis-inducing ligand (TRAIL) on apoptosis of cancer cells. In Caki renal cancer cells, which are resistant to TRAIL, cotreatment with subtoxic doses of mithramycin A and TRAIL resulted in a marked increase in apoptosis. This combined treatment was also cytotoxic to Caki cells overexpressing Bcl-2 but not to normal mesengial cells. Moreover, apoptosis by the combined treatment with mithramycin A and TRAIL was dramatically induced in various cancer cell types, thus offering an attractive strategy for safely treating malignant tumors. Mithramycin A -stimulated TRAIL-induced apoptosis was blocked by pretreatment with the broad caspase inhibitor zVAD-fmk or Crm-A overexpression, showing its dependence on caspases. We found that mithramycin A selectively down-regulated XIAP protein levels in various cancer cells. Luciferase reporter assay and the chromatin immunoprecipitation assay using the XIAP promoter constructs show that mithramycin A down-regulates the transcription of XIAP gene through inhibition of Sp1 binding to its promoter. Although XIAP overexpression significantly attenuated apoptosis induced by mithramycin A plus TRAIL, suppression of XIAP expression by transfection with its small interfering RNA prominently enhanced TRAIL-induced apoptosis. We present here for the first time that mithramycin A -induced suppression of XIAP transcription plays a critical role in the recovery of TRAIL sensitivity in various cancer cells.

The Chinese Herb Isolate Isorhapontigenin Induces Apoptosis in Human Cancer Cells by Down-regulating Overexpression of Antiapoptotic Protein XIAP

“…Our results are con- sistent with a previous report showing that two putative SP1 binding sites (Ϫ144 and Ϫ25 bp) with the 5Ј-untranslated region were involved in the SP1-mediated regulation of XIAP expression (11). Based on our results from the ChIP assay, SP1 appears to be a major participant in transcription factor binding to the GC-box site of the XIAP promoter and is critically involved in down-regulating XIAP transcription upon ISO treatment.…”

“…5F). Considering that there are two SP1 binding sites between Ϫ214 and ϩ60 (Ϫ144 and Ϫ25) and that the previous report (11) showed that XIAP promoter activity is significantly decreased by double mutation of two SP1 sites at Ϫ144 and Ϫ25, we suggest that ISO down-regulation of XIAP transcription is mediated by its targeting and inhibiting of SP1 expression, transactivation, and specific binding to SP1 binding sites, particularly the two SP1 binding sites at Ϫ144 and Ϫ25 in the XIAP promoter region as shown in Fig. 5G.…”

Withaferin A sensitizes TRAIL-induced apoptosis through reactive oxygen species-mediated up-regulation of death receptor 5 and down-regulation of c-FLIP

“…RT-PCR analysis was done as previously described [13]. The cDNAs for DR5 and actin were amplified by PCR with specific primers.…”

“…Chromatin immunoprecipitation assays were done as previously described [13]. Purified DNA was subjected to PCR with primers specific for a region (+1 to +196) relative to the proposed transcriptional start site in the c-FLIP promoter spanning a putative NF-κB binding site.…”

Regulation of RON Tyrosine Kinase-mediated Invasion of Breast Cancer Cells

“…Mithramycin A was previously used to demonstrate the specificity of Sp1 binding to target promoters (22,23). To further confirm the dependence of RON gene expression on Sp1, additional experiments were performed by treating MDA MB 231 cells with mithramycin A, an inhibitor of Sp1 binding.…”