Elsevier

Free Radical Biology and Medicine

Volume 53, Issue 10, 15 November 2012, Pages 1977-1987
Free Radical Biology and Medicine

Original Contribution
Capsazepine, a TRPV1 antagonist, sensitizes colorectal cancer cells to apoptosis by TRAIL through ROS–JNK–CHOP-mediated upregulation of death receptors

https://doi.org/10.1016/j.freeradbiomed.2012.08.012Get rights and content

Abstract

A major problem in clinical trials of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) as cancer therapy is the development of resistance to TRAIL. Therefore, agents that can overcome TRAIL resistance have great therapeutic potential. In this study, we evaluated capsazepine, a TRPV1 antagonist, for its ability to sensitize human colon cancer cells to TRAIL-induced apoptosis. Capsazepine potentiated the effect of TRAIL, as shown by its effect on intracellular esterase activity; activation of caspase-8,–9, and -3; and colony-formation assay. Capsazepine induced death receptors (DRs) DR5 and DR4, but not decoy receptors, at the transcriptional level and in a non-cell-type-specific manner. DR induction was dependent on CCAAT/enhancer-binding protein homologous protein (CHOP), as shown by (a) the induction of CHOP by capsazepine and (b) the abolition of DR- and potentiation of TRAIL-induced apoptosis by CHOP gene silencing. CHOP induction was also reactive oxygen species (ROS)-dependent, as shown by capsazepine's ability to induce ROS and by the quenching of ROS by N-acetylcysteine or glutathione, which prevented induction of CHOP and DR5 and consequent sensitization to TRAIL. Capsazepine's effects appeared to be mediated via JNK, as shown by capsazepine's ability to induce JNK and by the suppression of both CHOP and DR5 activation by inhibition of JNK. Furthermore, ROS sequestration abrogated the activation of JNK. Finally, capsazepine downregulated the expression of various antiapoptotic proteins (e.g., cFLIP and survivin) and increased the expression of proapoptotic proteins (e.g., Bax and p53). Together, our results indicate that capsazepine potentiates the apoptotic effects of TRAIL through downregulation of cell survival proteins and upregulation of death receptors via the ROS–JNK–CHOP-mediated pathway.

Highlights

► Capsazepine, a TRPV1 antagonist, sensitizes tumor cells to TRAIL. ► The sensitization effect of capsazepine is mediated by death receptor induction. ► Capsazepine can induce reactive oxygen species generation. ► Capsazepine can activate c-Jun N-terminal kinase. ► Capsazepine can induce the CHOP cell signaling pathway.

Introduction

Among the 18 members of the tumor necrosis factor (TNF)1 superfamily, TNF-related apoptosis-inducing ligand (TRAIL) is the only one being explored for its anticancer potential in the clinic. This is due in part to its lack of proinflammatory activity [1]. TRAIL−/− mice show an increased susceptibility to tumor initiation and metastasis [2], [3], which emphasizes the critical role of TRAIL in the defense against tumors. TRAIL mediates its anticancer effects through two transmembrane agonistic receptors and three antagonistic receptors. The two transmembrane agonistic receptors are TRAIL receptor 1 (DR4) [4] and TRAIL receptor 2 (DR5) [5]. The three antagonistic receptors include the transmembrane decoy receptor (DcR) 1 and DcR2 [6], [7] and the soluble receptor osteoprotegerin [8]. Engagement of DR4 or DR5 by TRAIL or agonistic antibodies can lead to activation of caspase-8 and caspase-3 and from there to apoptosis.

Unfortunately, one of the major problems with TRAIL in clinical trials is the development of resistance [9]. How tumor cells become resistant to TRAIL is open to interpretation. A wide variety of mechanisms have been proposed, including overexpression of antiapoptotic proteins such Bcl-xL [10], Bcl-2 [11], XIAP [12], survivin [13], and Mcl-1 [14]; activation of NF-κB [7], which controls the expression of several of these proteins; downregulation of DR4 [15], [16] and DR5 [17]; and upregulation of decoy receptors [18], [19], [20]. Thus, it has been proposed that agents that can modulate these various mechanisms of TRAIL resistance may potentially sensitize tumors to the cytokine.

One such agent is capsazepine (Fig. 1A), a capsaicin antagonist [21] that is now widely used as a selective vanilloid type 1 receptor (TRPV1) antagonist. Capsazepine can abolish osteosarcoma-induced hyperalgesia when administered subcutaneously at doses ranging from 3 to 10 mg/kg [22], block calcium channels [23], inhibit ovariectomy-induced bone loss in vivo [24], and suppress lipopolysaccharide-induced inducible nitric oxide synthase expression in macrophages through inactivation of NF-κB [25]. However, the mechanisms underlying the anticancer effects of capsazepine are not fully understood. Whether capsazepine can sensitize tumor cells to TRAIL-induced cell death is not known. Therefore, the objective of the study reported here was to determine whether capsazepine potentiates TRAIL-induced cancer apoptosis and, if so, how. We found that capsazepine effectively enhanced TRAIL-induced apoptosis by upregulating the activation of TRAIL receptors DR4 and DR5 via the reactive oxygen species (ROS)–JNK–CHOP pathway, by downregulating the expression of cell survival proteins, and by upregulating the expression of proapoptotic proteins.

Section snippets

Materials

Capsazepine (Sigma–Aldrich) was dissolved in dimethyl sulfoxide (50 mM) and stored at −20 °C until needed. Soluble recombinant human TRAIL/Apo2L was purchased from PeproTech. Penicillin, streptomycin, McCoy's 5A, Dulbecco's modified Eagle's medium (DMEM), and RPMI 1640 were purchased from Mediatech. Fetal bovine serum (FBS) was obtained from Atlanta Biologicals. Evodiamine was obtained from LKT Laboratories. Capsaicin, resiniferotoxin, Tris, glycine, NaCl, sodium dodecyl sulfate (SDS), bovine

Results

The objective of this study was to determine whether capsazepine can sensitize human tumor cells to TRAIL-induced apoptosis and, if so, the underlying molecular mechanism responsible for this effect. Most of the experiments reported here were carried out in the human colorectal cancer cell line HCT116 but were also conducted in other cell lines to confirm applicability to other types of cancer cells.

Discussion

Agents that can sensitize tumor cells to TRAIL have tremendous therapeutic potential. Our present results indicate that capsazepine is one such agent and that its ability to sensitize tumor cells to TRAIL involves multiple mechanisms. First, we found that selective upregulation of agonistic TRAIL receptors DR4 and DR5 had no effect on the antagonistic decoy receptors. Second, we found that the quenching of capsazepine-induced ROS generation abolished the sensitization of cancer cells to TRAIL.

Acknowledgments

We thank Jan Baker for carefully editing the manuscript. Dr. Aggarwal is the Ransom Horne, Jr., Professor of Cancer Research. This work was supported by a core grant from the National Institutes of Health (CA-106672), and a grant from the Center for Targeted Therapy of M.D. Anderson Cancer Center.

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