Inhibitory effect of anthraquinones isolated from marine fungus, Microsporum sp. on migration and invasion of cancer cells via inhibition of MMPs and cathepsins
- Alternative Title
- 해양진균 Microsporum sp.로부터 분리•정제한 anthraquinones의 MMP 및 cathepsin 활성저해를 통한 암세포의 이동과 침윤저해에 미치는 영향
- Abstract
- Cancer is a class of diseases with uncontrolled growth, invasion and metastasis characters, causes about 13% of all human deaths worldwide every year. Since cancer metastasis is the major cause of cancer mortality, a great deal of attention has been led to discover potential pharmaceutical compounds as anti-metastasis candidates. The marine environment is full of natural compounds with amazing biological and pharmacological activities. About 80% of the planet's animal and plant reside in the marine environment. Up to present, more than 20,000 new compounds have been isolated from marine organisms. The marine-derived fungi, one important member of marine organisms, have been regarded as a fresh source of secondary metabolites with significant therapeutic potential.
In the present study, chrysophanol (12.0 mg), physcion (10.0 mg), emodin (7.0 mg), gliotoxin (15.0 mg) were isolated from marine fungus, Microsporum sp. in the Guryongpo, Nam-gu, PoHang, Korea in 2009. These compounds did not show any significant cytotoxic effect in human fibrosarcoma cells (HT1080) and human breast cancer cells (MCF-7) below 100 μM. However, compounds influenced on migration and invasion of HT1080 cells in a concentration-dependent manner, and the number and length of stellate structures of HT1080 cells were significantly reduced in 3D culture condition by the treatment of compounds. Moreover, the expressions of MMP-2,-9 and cathepsin B, L were inhibited in a dose-dependent manner in HT1080 cells. Whereas MMP-2, -9 and cathepsin B, D enzymes activities were not influenced by compounds directly. AP-1 and NF-κB are major transcription factors that regulate MMP-2 and -9 expressions. Chrysophanol, physcion and emodin significantly inhibited both IκBα phosphorylation, p65 nuclear translocation, ERK and JNK phosphorylation and c-jun transactivation. Meanwhile, compounds also down-regulated TIMP-1 and -2 and up-regulated the RECK expression in a concentration-dependent manner. Since over expression of RECK decreases the amount of active MMP-2 and MMP-9, the up-regulation of RECK expression by those compounds is also involved in reducing the migration and invasion in HT1080 cells.
Chrysophanol, physcion and emodin h inhibitory effect on migration and invasion of cancer cells in present study, it is suggest that these compounds have potential to be utilized in nutraceutical and therapeutic agent for anti-metastasis therapy.
- Issued Date
- 2011
- Awarded Date
- 2011. 8
- Type
- Dissertation
- Publisher
- 부경대학교
- Department
- 대학원 화학과
- Advisor
- Se-Kwon Kim
- Table Of Contents
- Table of contents
Abstract i
Table of contents iv
List of figures vii
List of Abbreviations xi
Chapter 1 Introduction 1
Chapter 2 Isolation and characterization of anti-metastatic compounds from marine fungus, Microsporum sp. 8
1. INTRODUCTION 9
2. MATERIALS AND METHODS 11
2.1. Instruments and chemical reagents 11
2.2. Isolation, identification, and culture of the marine-derived fungus Microsporum sp. 12
2.3. Isolation, and purification of secondary metabolites from the marine-derived fungus Microsporum sp. 15
2.4. Cell culture 15
2.5. Cell viability assay 15
2.6. In vitro evaluation of the effect of fractions on cell migration by wound healing assay 16
2.7. Statistical analyses 17
3. RESULT 18
3.1 Extraction of anti-metastatic compounds from marine-derived fungus, Microsporum sp. 18
3.2. Isolation, and purification of anti-metastatic compounds from the marine-derived fungus, Microsporum sp. 20
3.3. Evaluation of cytotoxicity of the fractions (F6-F13) by MTT assay 22
3.4 In vitro effects of fractions (F6-F13) on cell migration by wound healing assay in HT1080 cells 24
3.5. Spectroscopic properties of compounds 1-4 isolated from extraction of the marine-derived fungus Microsporum sp. 26
4. SUMMARIES 44
Chapter 3 Anti-metastatic activity and mechanism of chrysophanol, physcion and emodin via the inhibition of MMP-2 and -9 in HT1080 cells 47
2. MATERIALS AND METHODS 53
2.1. Cell culture 53
2.2. Cell viability (MTT) assay 53
2.3. In vitro wound healing assay in HT1080 cells 54
2.4. In vitro invasion assay in HT1080 cells 54
2.5. Cellular morphology of HT1080 cells in three-dimensional (3D) culture 55
2.6. MMP-2 and 9 enzyme inhibitory assay 56
2.7. Measurement of total protein concentration 57
2.8. Gelatin Zymography 57
2.9. RNA extraction 58
2.10. Reverse-transcriptase polymerase chain reaction (RT-PCR) 59
2.11. Extraction of nuclear and plasma proteins and western blot analysis 60
2.12. Statistical analysis 61
3. RESULTS 62
3.1. Cell viability assay 62
3.2. Wound healing assay 62
3.3. In vitro invasion assay 70
3.4. Effects of compounds on HT1080 cells cellular morphology in 3D culture condition 70
3.5. MMP-2 and 9 enzyme inhibitory assay 78
3.6. Effects of chrysophanol, physcion and emodin on MMP-2 and -9 transcription and expression levels in HT1080 cell line 82
3.7. Effects of chrysophanol physcion and emodin influenced NF-κB, IκB, MAPK, AP-1, TIMPs and RECK expressions in HT1080 cells 92
4. DISCUSSION 103
Chapter 4 Effects of chrysophanol, physcion and emodin on activities and expressions of cathepsins in MCF-7 cells 109
1. INTRODUCTION 110
2. MATERIALS AND METHODS 113
2.1. Cell culture 113
2.2. Cell viability assay 113
2.3. In vitro wound healing assay in MCF-7 cells 114
2.4. Enzyme activity assay of cathepsin B and D 114
2.5. Reverse transcription-PCR 115
2.6. Extraction of proteins and western blot analysis 116
2.7. Statistical analysis 117
3. RESULTS 118
3.1. Cell viability assay 118
3.2. Effect of chrysophanol, physcion and emodin on MCF-7 cells migration 118
3.3. Enzyme activity assay of cathepsin B and D 121
3.4. Effects of chrysophanol, physcion and emodin on cathepsin B, D and L transcription and protein expression levels in MCF-7 cells 125
4. SUMMARIES AND DISCUSSION 132
Conclusions 136
References 140
- Degree
- Doctor
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