Elsevier

Biochemical Pharmacology

Volume 80, Issue 7, 1 October 2010, Pages 982-989
Biochemical Pharmacology

LW6, a novel HIF-1 inhibitor, promotes proteasomal degradation of HIF-1α via upregulation of VHL in a colon cancer cell line

https://doi.org/10.1016/j.bcp.2010.06.018Get rights and content

Abstract

Hypoxia-inducible factor HIF-1 is responsible for radiation resistance and poor prognosis in cancer therapy. As part of our drug discovery program, a novel HIF inhibitor, LW6, was identified as a small compound that inhibits the accumulation of HIF-1α. We found that LW6 decreased HIF-1α protein expression without affecting HIF-1β expression. MG132, a proteasome inhibitor, protected HIF-1α from LW6-induced proteasomal degradation, indicating that LW6 affects the stability of the HIF-1α protein. We found that LW6 promoted the degradation of wild type HIF-1α, but not of a DM-HIF-1α with modifications of P402A and P564A, at hydroxylation sites in the oxygen-dependent degradation domain (ODDD). LW6 did not affect the activity of prolyl hydroxylase (PHD), but induced the expression of von Hippel-Lindau (VHL), which interacts with prolyl-hydroxylated HIF-1α for proteasomal degradation. In the presence of LW6, knockdown of VHL did not abolish HIF-1α protein accumulation, indicating that LW6 degraded HIF-1α via regulation of VHL expression. In mice carrying xenografts of human colon cancer HCT116 cells, LW6 demonstrated strong anti-tumor efficacy in vivo and caused a decrease in HIF-1α expression in frozen-tissue immunohistochemical staining. These data suggest that LW6 may be valuable in the development of a HIF-1α inhibitor for cancer treatment.

Introduction

Hypoxia-inducible factor-1 (HIF-1) accumulates in many human tumors and is associated with increased vascular density and tumor grade severity, treatment failure, and a poor prognostic outcome with conventional therapies [1], [2]. In animal models, HIF-1 overexpression is associated with increased tumor growth, vascularization, and metastasis, whereas HIF-1 loss-of-function has the opposite effect [3]. HIF-1α has thus become an attractive therapeutic target [1], [4]. Under hypoxic conditions, HIF-1α translocates to the nucleus, where it interacts with HIF-1β, p300, and other transcription factors, and binds to hypoxic response element (HRE)-driven promoters [5], [6]. HIF-1α up-regulates a number of target genes involved in angiogenesis, survival, metastasis, and cell cycle regulation, to promote survival in low-oxygen conditions [7], [8].

HIF inhibitors have been extensively studied [9], [10], [11], [12]. A number of novel anticancer agents inhibit HIF-1α through a variety of molecular mechanisms, including transcriptional regulation, protein folding, stabilization, nuclear translocation, degradation, and trans-activation [3], [13]. Several mechanisms have been suggested to explain the degradation pathway of the HIF-1α protein. The first mechanism induces oxygen (O2)-dependent degradation of the HIF-1α subunit during normoxia, mediated by both prolyl hydroxylase (PHD) and the von Hippel-Lindau tumor suppressor (VHL)/Elongin-C/Elongin-B (VBC) E3 ubiquitin ligase complex. HIF-1α is hydroxylated at proline residues in the oxygen-dependent degradation domain (ODDD) by three prolyl hydroxylases, PHD1–3. These modifications allow HIF-1α to bind to VHL [14], [15], [16], which associates with VCB-Cul2 E3 ligase for proteasomal degradation [17], [18]. The second proposed mechanism involves p53-mediated accelerated degradation of HIF-1α[19]. Finally, the inhibition of heat-shock protein 90 (HSP90) leads to O2/PHD/VHL-independent degradation of HIF-1α, and the receptor of activated protein kinase C (RACK1) is shown to promote PHD/VHL-independent proteasomal degradation of HIF-1α. The HSP90 inhibitor, 17-allylaminogeldanamycin (17-AAG), degrades HIF-1α by binding directly to RACK, which binds to elongin C and promotes HIF-1α ubiquitination [20].

Previously, we reported the synthesis of a novel compound, an (aryloxyacetylamino)benzoic acid derivative, LW6, which inhibits the accumulation of HIF-1α[21]. Here, we present evidence that LW6 promotes HIF-1α degradation via upregulation of VHL. LW6 exhibited 53.6% tumor growth inhibition in a in vivo xenograft assay using the HCT116 cell line. These results suggest that LW6 may serve as a candidate in the development of a HIF-1α inhibitor for cancer therapy.

Section snippets

Materials

An (aryloxyacetylamino)benzoic acid derivative LW6 (Fig. 1A) was synthesized as described (compound 23 [21]). Chemicals were purchased from Life Technologies Inc. (Gaithersburg, MD, USA), and media was obtained from Sigma (St. Louis, MO, USA). Cell culture media and fetal bovine serum (FBS) were purchased from Gibco Laboratories (Grand Island, NY, USA). A Phototope-Horseradish Peroxidase Western Blot Detection Kit was obtained from Millipore (Billerica, MA, USA). The growth medium and the

LW6 inhibits HIF-1α protein accumulation and suppresses the expression of hypoxia-induced genes

HIF-1α protein expression increases under hypoxic conditions, while remaining at a baseline detectable level in normoxia. LW6, an (aryloxyacetylamino)benzoic acid derivative (Fig. 1A), inhibited accumulation of HIF-1α protein in human cancer cell lines, Caki-1, PC-3, SK-HEP1 and HCT-116 (Fig. 1B). Human colorectal carcinoma cells HCT116 was chosen for further study because HCT116 showed dramatic accumulation of HIF-1α protein during hypoxia and distinct reduction of HIF-1α protein in the

Discussion

Many attempts have been made to identify small molecules that inhibit the accumulation of HIF-1α, a key player in tumorigenesis under hypoxia. Herein, we present LW6, a HIF-1α inhibitor that abolished the accumulation of HIF-1α protein and led to suppression of its target genes under hypoxia. Prolyl hydroxylation at P564 and P402 in the ODDD is a major process for O2-dependent ubiquitination and degradation of HIF-1α. The increase in HRE-Luc activity, even during hypoxia, with increasing

Acknowledgments

We are grateful to Soo-Hyun Park and Won-Ki Yoon of the KRIBB for their technical assistance. This work was supported in part by a grant from KRIBB Initiative from the Korea Research Council of Fundamental Science and Technology and by the National Research Foundation of Korea (NRF) grant (2008-05676 and 2010-0000222 to K.L.).

References (44)

  • K.S. Chung et al.

    Rapid screen of human genes for relevance to cancer using fission yeast

    J Biomol Screen

    (2007)
  • J.O. Nam et al.

    Identification of the alphavbeta3 integrin-interacting motif of betaig-h3 and its anti-angiogenic effect

    J Biol Chem

    (2003)
  • M.S. Won et al.

    A novel benzimidazole analogue inhibits the hypoxia-inducible factor (HIF)-1 pathway

    Biochem Biophys Res Commun

    (2009)
  • H. Cho et al.

    A fluorescence polarization-based interaction assay for hypoxia-inducible factor prolyl hydroxylases

    Biochem Biophys Res Commun

    (2005)
  • U. Berchner-Pfannschmidt et al.

    Nuclear oxygen sensing: induction of endogenous prolyl-hydroxylase 2 activity by hypoxia and nitric oxide

    J Biol Chem

    (2008)
  • U. Berchner-Pfannschmidt et al.

    Nitric oxide modulates oxygen sensing by hypoxia-inducible factor 1-dependent induction of prolyl hydroxylase 2

    J Biol Chem

    (2007)
  • J. Zhou et al.

    PI3K/Akt is required for heat shock proteins to protect hypoxia-inducible factor 1alpha from pVHL-independent degradation

    J Biol Chem

    (2004)
  • G.L. Semenza

    Targeting HIF-1 for cancer therapy

    Nat Rev Cancer

    (2003)
  • H. Zhong et al.

    Overexpression of hypoxia-inducible factor 1alpha in common human cancers and their metastases

    Cancer Res

    (1999)
  • R. Pili et al.

    Is HIF-1 alpha a valid therapeutic target?

    J Natl Cancer Inst

    (2003)
  • R.S. Pollenz et al.

    The aryl hydrocarbon receptor and aryl hydrocarbon receptor nuclear translocator protein show distinct subcellular localizations in Hepa 1c1c7 cells by immunofluorescence microscopy

    Mol Pharmacol

    (1994)
  • A. Lal et al.

    Transcriptional response to hypoxia in human tumors

    J Natl Cancer Inst

    (2001)
  • Cited by (0)

    1

    These authors equally contributed to this work.

    View full text