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

doi:10.1016/j.freeradbiomed.2009.03.022

Free Radical Biology and Medicine

Volume 46, Issue 12, 15 June 2009, Pages 1639-1649

https://doi.org/10.1016/j.freeradbiomed.2009.03.022 Get rights and content

Abstract

Withaferin A (Wit A) has reportedly shown cytotoxicity in a variety of tumor cell lines. Here, we show that cotreatment with subtoxic doses of Wit A and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in human renal cancer cells, Caki cells, but not in human normal mesangial cells. Moreover, the combined treatment with Wit A and TRAIL dramatically induces apoptosis in various cancer cell types, suggesting that this combined treatment might offer an attractive strategy for safely treating human cancers. Treatment of Caki cells with Wit A up-regulated death receptor 5 (DR5) in a C/EBP homologous protein (CHOP)-dependent manner. Interestingly, a Wit A-induced increase in ROS levels preceded the up-regulation of CHOP and DR5. The involvement of ROS in CHOP-mediated DR5 up-regulation was confirmed by the result that pretreatment with an antioxidant, NAC or catalase, inhibited Wit A-induced up-regulation of both CHOP and DR5. We also found that Wit A treatment down-regulated c-FLIP via NF-κB-mediated transcriptional control as well as ROS signaling pathways. Taken together, our results show that DR5 up-regulation and c-FLIP down-regulation contribute to the sensitizing effect of Wit A on TRAIL-mediated apoptosis in cancer cells.

Section snippets

Cells and materials

Caki, Huh7, SK-Hep1, and Hep3B cells were obtained from the American Type Culture Collection (Rockville, MD, USA). Primary culture of human renal glomerular mesangial cells (Cryo NHMC) and their corresponding growth medium (CC-3146 MsGM) were purchased from Clonetics (San Diego, CA, USA). The culture medium used throughout these experiments was Dulbecco's modified Eagle's medium, containing 10% FBS, 20 mM Hepes buffer, and 100 μg/ml gentamicin. Wit A (Sigma) was directly added to cell cultures

Wit A sensitizes cancer cells to TRAIL-mediated apoptosis

In an attempt to search for novel strategies to overcome TRAIL resistance in cancer cells, we investigated the effect of a combined treatment with Wit A and TRAIL in a human renal carcinoma cell line, Caki cells. Caki cells were treated with 1.2 μM Wit A alone, 100 ng/ml TRAIL alone, or the combination of Wit A and TRAIL for 24 h. Cotreatment of Caki cells with Wit A and TRAIL resulted in a markedly increased accumulation of sub-G1-phase cells and a typical ladder pattern of internucleosomal

Discussion

TRAIL is considered a highly promising candidate as an anti-cancer drug, because it has been shown to induce apoptosis specifically in malignant or transformed cells without any cytotoxicity toward a variety of normal cells [26], [27], [28]. However, considerable numbers of cancer cells are resistant to treatment with TRAIL, and this resistance may be caused by deregulated expression of antiapoptotic molecules [29], [30], [31], [32]. Therefore, novel therapeutic strategies are necessary to

Acknowledgments

This work was supported by the Korea Science and Engineering Foundation through the MRC at Keimyung University (R13-2002-028-03001-0) and BAERI-M20708630003-07B0863-00310 and Korea Research Foundation Grant KRF-2005-070-C00100.

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Sorafenib (Sora), a multi-target tyrosine kinase inhibitor, is widely recognized as a standard chemotherapy treatment for advanced hepatocellular carcinoma (HCC). However, drug resistance mechanisms hinder its anticancer efficacy. Derived from Withania somnifera, Withaferin A (WA) exhibits remarkable anti-tumor properties as a natural bioactive compound. This study aimed to examine the mechanisms that underlie the impacts of Sora and WA co-treatment on HCC.
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¹: These authors contributed equally to this work.

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

Abstract

Section snippets

Cells and materials

Wit A sensitizes cancer cells to TRAIL-mediated apoptosis

Discussion

Acknowledgments

Bioorg. Med. Chem. Lett.

J. Biol. Chem.

J. Biol. Chem.

Mol. Cell. Biol. Res. Commun.

Free Radic. Biol. Med.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Renal-cell carcinoma

N. Engl. J. Med.

An antagonist decoy receptor and a death domain-containing receptor for TRAIL

Science

Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors

Science

Assessment of differential gene expression patterns in human colon cancers

Ann. Surg.

Proteasome inhibition sensitizes hepatocellular carcinoma cells, but not human hepatocytes, to TRAIL

Hepatology

Withaferin A is a potent inhibitor of angiogenesis

Angiogenesis

Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic cell death of human myeloid leukemia HL-60 cells by a dietary compound withaferin A with concomitant protection by N-acetyl cysteine

Apoptosis

Withanolides potentiate apoptosis, inhibit invasion, and abolish osteoclastogenesis through suppression of nuclear factor-kappaB (NF-kappaB) activation and NF-kappaB-regulated gene expression

Mol. Cancer Ther.

Sodium butyrate sensitizes TRAIL-mediated apoptosis by induction of transcription from the DR5 gene promoter through Sp1 sites in colon cancer cells

Carcinogenesis

Sanguinarine-induced apoptosis: generation of ROS, down-regulation of Bcl-2, c-FLIP, and synergy with TRAIL

J. Cell. Biochem.

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

Mol. Cancer Ther.

Proteasome inhibitor MG132 induces death receptor 5 through CCAAT/enhancer-binding protein homologous protein

Cancer Res.

Tunicamycin enhances tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human prostate cancer cells

Cancer Res.

Curcumin sensitizes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through reactive oxygen species-mediated upregulation of death receptor 5 (DR5)

Carcinogenesis

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