Inhibitory mechanism of β-Lapachone

다국어 초록 (Multilingual Abstract)

β-Lapachone has been generally known for antitumor agents and the effects on anti-inflammatory, anti-bacterial, anti-fungal, anti-obesity and anti-diabetic has been reparted recently. This study was designed to figure out the inhibitory mechanism of β-Lapachone on melanogenesis. β-Lapachone inhibited melanin synthesis and tyrosinase activity at 0.8μM in melan-a melanocytes. But β-Lapachone did not inhibit mushroom tyrosinase activity and cell tyrosinase activity from Melan-a melanocyte directly. β-Lapachone also reduced the pigmentation of Melanoderm tissue at 40 μM. β-Lapachone inhibited α-glucosidase activity dose dependently (IC50 18.1 μM) but did not affect forming of immature tyrosinase. The expression of tyrosinase and tyrosinase-related protein-1 (TRP-1) were reduced by β-Lapachone at transcriptional and translational level. MITF protein level was also downregulated by β-Lapachone. The decreased expression of tyrosinase and TRP-1 might be resulted from the reduced MITF (Microphthalmia-associated trascription factor) which regulates major melanogenic proteins. Becuase the mRNA level of MITF was not changed by β-Lapachone treatment, we checked the effect of β-Lapachone on ERK phosphorylate which has been reported to regulated MITF degradation. The reduced level of MITF was associated with delayed ERK activation by β-Lapachone. These results show that β-Lapachone may be useful as a potential depigmenting agent for various hyperpigmentary disorders.

국문 초록 (Abstract)

β-Lapachone은 일반적으로 항암성물질로 잘 알려져 있으며 최근에는 항염증, 항균성, 항진균성, 항비만 그리고 항당뇨 효과들에 대해 보고되고 있다. 본 연구는 멜라닌 합성과정에서 β-Lapachone의 억제효과와 작용기전을 찾고자 하였다. β-Lapachone을 Melan-a melanocyte에 0.8 μM 처리하였을 때 멜라닌 합성과 Tyrosinase 활성을 억제하였다. 그러나 Mushroom tyrosinase나 Melan-a melanocyte로부터 얻은 세포 tyrosinase 활성을 직접적으로 억제하지는 않았다. β-Lapachone을 Melanoderm tissue에 40 μM 처리하였을 때 색소침착이 감소하였다. β-Lapachone은 α-glucosidase 활성을 농도 비례하게 억제하였지만 이에 따른 불안정한 tyrosinase 형성에는 영향을 주지 않았다. β-Lapachone을 Melan-a melanocyte에 처리하였을 때 tyrosinase와 tyrosinase-related protein-1 (TRP-1)의 단백질 및 유전자발현을 감소시켰으며 또한 MITF 단백질 수준을 감소시켰다. 따라서 이러한 Tyrosinase와 TRP-1의 발현이 감소된 이유는 주된 멜라닌 합성 단백질을 조절하는 MITF이 β-Lapachone에 의해 감소된 때문이라 생각된다. 한편 β-Lapachone 은 MITF의 유전자발현에 영향을 주지 않았기 때문에, MITF단백질 분해를 조절하는 것으로 알려진 ERK 활성화에 미치는 효과를 측정하였다. 그 결과 β-Lapachone 에 의해 ERK 활성화가 2시간까지 유지됨을 확인하였다. 따라서 앞선 실험들의 결과를 통해 β-Lapachone은 향후 다양한 과색소침착성 질병을 위한 색소침착억제제로서 매우 유용한 물질이 될 수 있을 것으로 생각된다.

목차 (Table of Contents)

• LIST OF FIGURES ⅲ
• ABSTRACTS 1
• KEY WORDS 2
• INTRODUCTION 3
• MATERIALS AND METHODS 8
• Compound and reagents 8
• Cell culture 8
• Cell proliferation assay 9
• Melanin assay 10
• α-Glucosidase activity assay 10
• Cellular tyrosinase activity assay 11
• Tyrosinase activity assay (extracted from melan-a melanocyte) 11
• Mushroom Tyrosinase Activity assay 12
• Melanoderm Culture 13
• Western Blot analysis 15
• Endoglycosidase H (Endo H) assay 16
• RT (reverse transcription)-PCR 17
• cAMP Enzyme Immunoassay 17
• RESULTS 19
• Effect of β-Lapachone on α-glucosidase activity 19
• Effect of β-Lapachone on melanin synthesis, tyrosinase activity in melan-a melanocytes 19
• Effect of β-Lapachone on Human epidermal equivalent
• (Melanoderm) 20
• Effect of β-Lapachone on tyrosinase activity 20
• Effect of β-Lapachone on forming an unstable tyrosinase
• in HM3KO melanoma cells 21
• Effect of β-Lapachone on the expression of tyrosinase,TRP-1, TRP-2 and MITF protein 21
• Effect of β-Lapachone on the expression of tyr, trp-1
• trp-2 and MITF mRNA level 22
• Effect of β-Lapachone on intracellular cAMP level 22
• Effect of β-Lapachone on the activation of ERK(1/2) 22
• DISCUSSION 24
• REFERENCE 30
• FIGURES AND LEGENDS 40
• FIGURE AND TABLE 45
• 국문초록 56