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Normoxically Overexpressed Hypoxia Inducible Factor 1-Alpha is Involved in Arsenic Trioxide Resistance Acquisition in Hepatocellular Carcinoma

  • Translational Research and Biomarkers
  • Published:
Annals of Surgical Oncology Aims and scope Submit manuscript

Abstract

Background

The aim of this study was to examine the underlying signaling mechanisms of arsenic trioxide (ATO)-mediated anticancer effects and the responsible biomarker(s) for the acquired resistance in human heptatocellular carcinoma (HCC).

Materials and Methods

The therapeutic effects of ATO were examined using 2 characteristically distinct HCC cell lines, Hep-J5 (overexpressing HIF-1α/GRP78) and SK-Hep-1 (the matched control). ATO-mediated proliferation inhibition, oxidative stress, and apoptosis were analyzed using flowcytometric analysis and western blotting. The role of HIF-1α and GRP78 in HCC resistance to ATO treatment was determined using RNA silencing and inhibitor approaches.

Results

SK-Hep-1 cells, lacking both HIF-1α and GRP78 expressions were responsive to ATO-induced apoptosis via an oxidative-nitrosative mechanism. Intracellular glutathione depletion and lipid peroxidation have been identified as the early cascade of events preceding apoptosis via cytochrome c release and the severe drop of mitochondrial membrane potential (MMP). Conversely, Hep-J5 cells, with normoxic coexpression of HIF-1α and GRP78, were resistant to ATO-induced apoptosis. GRP78-silenced Hep-J5 cells remained resistant to ATO treatment. In contrast, ATO resistance in Hep-J5 cells was overcome by the addition of YC-1, a HIF-1α inhibitor.

Conclusions

HIF-1α was identified as the major positive modifier for ATO resistance acquisition in HCC, and it represents a prime molecular target for overcoming ATO resistance.

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Acknowledgment

This work is supported by National Science Council (NSC 99-2628-B-038-010-MY3) and TMU young investigator grant (TMU97-AE-1-B08) to ATHW and the Translational Research Laboratory, Cancer Center, Taipei Medical University Hospital. The other funding agency is Center of Excellence for Cancer Research, Taipei Medical University (TMU-CECR, DOH99-TD-C-111-008).

Author information

Authors and Affiliations

  1. Institute of Medicine, Chung Shan Medical University, Taichung, Taiwan

    Jia-Nien Tung MD & Ya-Wen Cheng PhD

  2. Department of Surgery, Tungs’ Taichung MetroHarbor Hospital, Taichung, Taiwan

    Jia-Nien Tung MD

  3. Department of Endocrinology and Metabolism, School of Medicine, Taipei Medical University, Taipei, Taiwan

    Chung-Huei Hsu MD, PhD

  4. Translational Research Laboratory, Cancer Center, Taipei Medical University Hospital, Taipei, Taiwan

    Tsan-Zon Liu PhD, Pei-Ying Hsieh MSc, Jeng-Fong Chiou MD, PhD & Alexander T. H. Wu PhD

  5. Department of Radiation, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

    Lai-Lei Ting MD, Hui-Ling Ko MD, Jeng-Fong Chiou MD, PhD & Alexander T. H. Wu PhD

  6. Department of Clinical Medicine, Taipei Medical University, Taipei, Taiwan

    Yu-Jia Chang PhD

  7. Center of Excellence for Cancer Research in Taipei Medical University (TMU-CECR), Taipei Medical University, Taipei, Taiwan

    Jeng-Fong Chiou MD, PhD & Alexander T. H. Wu PhD

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  1. Jia-Nien Tung MD
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Correspondence to Jeng-Fong Chiou MD, PhD or Alexander T. H. Wu PhD.

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Supplementary Fig. 1

GRP78 is not the major contributor to ATO resistance in J5 cells. Both control (vc-J5) and GRP78-silenced (si-J5) J5 cells were subjected to 30 μM ATO treatment for 48 and 72 h. Both groups remained resistant to ATO treatment. Experiments were performed in triplicate (EPS 1247 kb)

Supplementary Fig. 2

HIF-1α and GRP78 expression profile in 5 selected HCC cell lines. Western blotting analysis was performed on 5 HCC cell lines namely, HepG2 (G2), Hep3B (3B), HepJ5 (J5), Mahlavu (Ma), and Sk-Hep1. J5 cells uniquely demonstrated a significantly elevated expression in both HIF-1α and GRP78 under normoxic condition when compared with the other cell lines (EPS 1153 kb)

Supplementary Fig. 3

Control TUNEL assay. a SK cells were treated with HIF-1α inhibitor (YC-1) as a control. YC-1 treatment had minimal cytotoxic effect (approximately 5.7% cells underwent apoptosis) on SK cells without the addition of ATO treatment. b J5 cells which received YC-1 lone treatment did not show significant apoptotic signal (approximately 4.9% apoptotic population) as demonstrated by TUNEL assay (EPS 879 kb)

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Tung, JN., Cheng, YW., Hsu, CH. et al. Normoxically Overexpressed Hypoxia Inducible Factor 1-Alpha is Involved in Arsenic Trioxide Resistance Acquisition in Hepatocellular Carcinoma. Ann Surg Oncol 18, 1492–1500 (2011). https://doi.org/10.1245/s10434-010-1444-y

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  • DOI: https://doi.org/10.1245/s10434-010-1444-y

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