(The) Effect of a SIRT2 inhibitor on HIF-1alpha stability

다국어 초록 (Multilingual Abstract)

Sirtuin family proteins are involved in the regulation of hypoxic responses which are primarily dependent on a hypoxia-inducible factor (HIF). However, few studies have examined the use of sirtuin inhibitors to regulate HIF. The present study examined the effect of a SIRT2-specific inhibitor, AK-1, on hypoxic responses. Under hypoxic conditions, AK-1 increased the ubiquitination of HIF-1α in a VHL-dependent manner, leading to the degradation of HIF-1α via a proteasomal pathway. Downregulation of HIF-1α expression reduced its transcriptional activity and, eventually, reduced the expression of BNIP3, one of HIF-1 target genes, in AK-1-treated cells. The downregulation of BNIP3 was correlated with autophagy inhibition which was determined by p62 expression. These data demonstrate that SIRT2 inhibition attenuates hypoxic responses, and that SIRT2 inhibitors may have potential as treatments for hypoxia-associated pathological conditions.

국문 초록 (Abstract)

HIF-1 (Hypoxia-inducible factor-1)은 암의 저산소 상황을 조절하는 인자이다. Sirtuin은 NAD+의존적인 탈아세틸화 효소로써 HIF-1α 의존적 세포 내 과정을 조절한다. 따라서 Sirtuin 억제제가 암의 저산소 상황을 조절할 수 있다고 사료된다. 본 학위논문 연구에서는 특이적 SIRT2 억제제인 AK-1이 HIF-1α에 미치는 영향을 알아보고자 하였다. 그 결과, 다양한 암세포에서 AK-1 처리시 VHL (von Hippel-Lindau) 의존적으로 유비퀴틴화에 의해 프로테아좀에서 HIF-1α가 분해되었다. 이러한 HIF-1α의 감소는 그 전사활성을 낮춤으로서, 세포자살을 조절하는 유전자인 BNIP3 (BCL2/adenovirus E1B 19kDa protein-interacting protein 3)의 발현을 감소시켰다. 종합적으로, 본 연구는 SIRT2 억제제인 AK-1이 저산소 상태인 암에 존재하는 HIF-1α를 불안정화시킴으로써 AK-1이 암의 치료제로서 적용 가능함을 시사한다.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Materials and Methods 3
• Cell Culture, hypoxic condition and treatment 3
• Plasmid transfection 3
• Reverse transcription-polymerase chain reaction (RT-PCR) 3
• Immunofluorescence staining 4
• Preparation of crude cell extract and Western blotting 4
• In vivo ubiquitination assay 5
• Luciferase reporter assay 5
• Statistical analysis 5
• 3. Results 6
• SIRT2 inhibition induces the downregulation of HIF-1α expression 6
• HIF-1α protein undergoes proteasomal degradation in the presence of AK-1 9
• HIF-1α degradation is dependent of VHL in hypoxia following AK-1 treatment 13
• HIF-1 transcriptional activity and autophagy are decreased by AK-1 17
• 4. Discussion 20
• 5. References 23
• 6. 국문초록 28