Tumor Suppressive Effect of Auriculasin Isolated from Flemingia Philippinensis in Human Prostate Cancer : 천근발로부터 분리된 auriculasin의 전립선암 억제효과

다국어 초록 (Multilingual Abstract)

Prostate tumors, the second most common type of tumor in men worldwide, characteristically spreads to secondary places, including bone, lymph node, and the central nervous system. To inhibit development of prostate tumors, chemotherapy drugs are used in various stages of prostate tumors. However, utilization of tumor therapeutics is known to cause significant toxicity to normal tissue and side effects. Therefore, effective regimens with non-toxic natural ingredients to prevent prostate tumors can be used as new molecular targets of therapy for prostate tumors.

Chapter 1. Comparison of proximate composition, functional components, and anti-tumor activity between F. philippinensis extracts
Flemingia philippinensis is a perennial herb that belongs to the legume family Fabacceae and is known as an herbal folk remedy and food ingredient in China. In this study, we conducted analysis of the proximate composition of Flemingia philippinensis root as well as free sugar, free amino acid, fatty acid, mineral, and total polyphenol contents of various extracts from Flemingia philippinensis. In addition, we compared the anti-tumor effects of F. philippinensis extracts on human prostate tumor cells. Although F. philippinensis extracts contained minor free sugars, free amino acids, fatty acids, and minerals, total polyphenol contents in F. philippinensis extracts were greater than 45.81 mg GAE/g with the highest content observed in 186.31 mg GAE/g in MeOH extract. EtOH, 70% EtOH, MeOH, and 70% MeOH extracts of F. philippinensis caused selective prostate tumor cell death without significant cytotoxicity in RWPE-1 human prostate epithelial cells. However, water extracts showed a weak inhibitory effect on prostate tumor cell proliferation. Furthermore, F. philippinensis extract-induced anti-tumor activities were related with cell cycle arrest and apoptosis. The overall results suggest that methanolic extract of F. philippinensis containing various bioactive compounds plays an effective role in prostate tumor suppression.

Chapter 2. Effect of AC on LNCaP prostate tumor cell death via induction of apoptosis
Recent studies have demonstrated that natural agents targeting the accumulation of reactive oxygen species (ROS) that selectively kill, leaving normal cells undamaged, can suppress prostate tumor. Here, we show that AC, derived from F. philippinensis, induces significant cell death and apoptosis via ROS generation in prostate tumor cells. AC treatment resulted in selective apoptotic cell death in LNCaP prostate tumor cells, characterized by DNA fragmentation, accumulation of sub-G1 cell population, cleavage of poly (ADP-ribose) polymerase (PARP), regulation of Bax/Bcl-2 ratio, increase of cytosolic apoptosis-inducing factor (AIF) and endonuclease G (EndoG), in addition to inhibiting tumor growth in a xenograft mouse model. Interestingly, AC-induced apoptosis did not result in caspase-3, -8, and -9 activations. We found that AC treatment decreased phosphorylation of AKT/mTOR/p70s6k in a dose- and time-dependent manner. Further, cellular ROS levels increased in LNCaP cells treated with AC and blocking ROS accumulation with ROS scavengers resulted in inhibition of AC-induced PARP cleavage, AIF increase, up-regulation of Bax/Bcl-2 ratio, and decrease in AKT/mTOR phosphorylation. Taken together, these data suggest that AC targets ROS-mediated caspase-independent pathways and suppresses PI3K/AKT/mTOR signaling, which leads to apoptosis and decreased tumor growth.

Chapter 3. Effect of AC on suppression of VEGF-induced angiogenesis in human umbilical vein endothelial cells
Angiogenesis plays an important role in various pathological conditions such as tumor via excessive delivery of oxygen and nutrients. Recent studies have demonstrated that understanding the molecular basis of natural agents in angiogenesis is critical for the development of promising tumor therapeutics. In this study, AC, an active component from F. philippinensis, was found to exert strong anti-angiogenesis activity. Treatment with AC suppressed proliferation of human umbilical vein endothelial cells (HUVECs) by modulating expression of Bcl-2, Bcl-XL, and vascular endothelial growth factor (VEGF). Further, AC inhibited VEGF-induced chemotactic migration, invasion, and capillary-like structure formation of endothelial cells. In addition, AC abrogated VEGF-induced vascular network formation around rat aortic rings as well as blocked accumulation of hemoglobin in the Matrigel plug of C57BL/6 mice. The inhibitory effect of AC on angiogenesis was well correlated with inhibition of VEGF receptor 2 (VEGFR2) activation as well as phosphorylation of intracellular downstream protein kinases of VEGFR2 containing Akt, mammalian target of rapamycin, phosphoinositide 3-kinase, p-38, extracellular signal-related kinase, and Src. Taken together, this study reports that AC potently inhibits angiogenesis by modulating VEGFR2-related signaling pathways, which further validates its great potential in clinical applications.

Chapter 4. Effect of AC on sensitization of TRAIL-induced apoptosis in RC-58T/h/SA#4 primary human prostate tumor cells
Increasing evidence suggests that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent due to its ability to induce apoptosis selectively in tumor cells. However, some prostate tumor cell lines are relatively resistant to TRAIL-induced apoptosis. In this study, AC acted as a synergistic TRAIL sensitizer by mediating expression of death receptor 5 (DR5) in RC-58T/h/SA#4 primary prostate tumor cells. Co-treatment with AC and TRAIL induced apoptotic death in RC-58T/h/SA#4 cells, as indicated by an increase in the apoptotic cell population, DNA fragmentation, and condensation of chromatin or apoptotic bodies. Combined treatment with AC and TRAIL potentially enhanced apoptosis in caspase-dependent and -independent manners. Further analysis revealed that combined treatment also activated death receptor 5, p53, and CHOP, and pre-treatment with a chimeric blocking antibody for DR5 reduced AC-TRAIL-induced apoptotic cell death. AC-suppressed the PI3K/Akt/mTOR signaling pathway also related with inhibition of proliferation in primary prostate tumor cells. In conclusion, our results suggest that AC sensitizes TRAIL-resistant primary prostate tumor cells to TRAIL-mediated apoptosis via up-regulation of DR5 and downstream signaling pathways.

국문 초록 (Abstract)

전 세계적으로 남성 발병률이 두번째로 높은 전립선 암은 특징적으로 뼈, 림프절, 중추신경계 등과 같은 2차 위치로 전이된다. 전립선 암의 발병과 발전을 억제하기 위해 다양한 화학약물요법이 이용되고 있으나 이러한 합성 항암제의 사용은 체내 정상 조직과 기관에 독성을 유발하는 것으로 알려져 있다. 따라서 인체 독성이 적은 전립선 암 예방 천연물 및 식품에 대한 연구가 활발히 진행되고 있으며 이들의 분자적 암 억제 기전은 전립선 암 치료를 위한 새로운 발전방향을 제시할 수 있다.

천근발 추출물의 일반성분, 기능성 성분 및 암세포 사멸 효과 비교
천근발은 다년생 콩과식물 중 하나로 예로부터 중국에서 식품 및 전통약재로 이용되어 왔다. 본 연구에서는 다양한 용매로 추출한 천근발 추출물의 유리당, 유리 아미노산 및 폴리페놀 함량을 분석하였으며, 전립선암 성장억제효과를 비교하였다. 천근발 추출물의 유리당, 유리 아미노산, 유리 지방산 및 무기물 함량은 소량이었으나 총 폴리페놀 함량은 45.81-186.31 mg GAE/g으로 나타났으며 메탄올 추출물에서 가장 높은 폴리페놀 함량을 나타내었다. 에탄올, 70% 에탄올, 메탄올 및 70% 메탄올 추출물에서 RWPE-1 전립선 정상세포의 유의적인 독성없이 LNCaP, RC-58T, PC-3 전립선 암세포의 선택적 사멸을 유도하였다. 하지만 물 추출물은 전립선 암세포 및 정상세포에서 모두 세포사멸효과를 확인할 수 없었다. 또한, 천근발 추출물의 전립선암 세포증식 억제효과는 세포주기 정지 및 apoptosis 유도와 관련이 있는 것으로 나타났다. 따라서 폴리페놀 함량이 풍부한 천근발 메탄올 추출물은 정상세포의 독성없이 효과적으로 전립선 암을 억제할 수 있었으며 이를 통해 기능성 식품 및 천연물 의약소재로서 이용할 수 있을 것으로 판단된다.

Auriculasin의 LNCaP 인체 전립선 암세포 사멸 효과
최근 천연물에 의해 유도된 암세포 내 활성산소종 (Reactive oxygen species, ROS)이 정상세포에는 독성없이 선택적으로 암의 발전을 억제할 수 있다는 연구가 지속적으로 보고되고 있다. 본 연구에서 천근발 (Flemingia philippinensis)로부터 분리된 프레닐화 이소플라본인 auriculasin은 전립선 암세포 내 ROS를 증가시키고 이를 통해 세포사멸 및 apoptosis를 유도하였다. Auriculasin은 LNCaP 전이성 전립선 암세포에서 DNA 분절, sub-G1 함량의 증가, poly (ADP-ribose) polymerase (PARP), Bax/Bcl-2 조절, apoptosis-inducing factor (AIF) 및 endonuclease G (Endo G)의 활성 증가를 통해 암세포 선택적 apoptosis를 유도하였다. 이는 전립선 암 이종이식 마우스 모델에서도 종양 성장 및 발전을 억제하는 것으로 나타났다. 흥미롭게도 auriculasin에 의한 LNCaP 전립선 암의 apoptosis는 caspase-3, -8 및 -9이 활성화 되지 않았으며 AKT/mTOR/p70s6k 경로 활성이 농도 및 시간의존적으로 억제되는 것을 확인하였다. auriculasin에 의해 증가된 세포 내 ROS는 항산화제에 의해 억제되었으며 이는 전립선 암세포 내 PARP 분절, AIF, Bax/Bcl-2 발현 비율을 억제하였고 AKT/mTOR 인산화 또한 감소시켰다. 따라서 본 연구의 결과는 auriculasin이 세포 내 ROS의 증가를 통해 caspase 비의존형 apoptosis를 유도하고 PI3K/AKT/mTOR 경로를 억제하여 전립선 암의 증식 및 종양성장을 억제하는 것을 나타낸다.

혈관내피세포 성장 인자에 의해 유도된 혈관내피세포에서 auriculasin의 혈관신생 억제 효과
혈관신생 (angiogenesis)은 암과 같은 병리학적 상태에서 과도한 산소와 영양분을 공급하는 역할을 수행한다. 최근 천연물에 의한 분자수준의 혈관신생 억제효과가 다양한 암의 치료에 뛰어난 효능을 보이는 것으로 보고되었다. 본 연구에서 auriculasin의 처리는 혈관내피세포 성장 인자 (vascular endothelial growth factor, VEGF)에 의해 촉진된 혈관내피세포 (human umbilical vein endothelial cells, HUVECs)의 혈관신생을 억제하는 것으로 확인되었다. AC은 혈관내피세포에서 Bcl-2, Bcl-XL 및 VEGF의 발현을 억제하였으며 VEGF에 의해 유도된 HUVECs의 이동, 침윤 및 모세혈관 유사 생성능을 억제하였다. 또한 쥐 동맥 주위의 모세혈관 생성과 C57BL/6 마우스 내 마트리겔 플러그 속의 헤모글로빈 함량도 감소시켰다. Auriculasin에 의한 혈관신생 억제효과는 VEGF 수용체 2 (VEGF receptor 2)의 인산화 억제, Akt, mammalian target of rapamycin (mTOR), phosphoinositide 3-kinase (PI3K), p-38, extracellular signal-related kinase (ERK) 및 Src의 발현을 억제와 관련이 있었다. 따라서 본 연구의 결과는 auriculasin이 VEGFR2 관련 신호전달 경로를 조절하여 혈관신생을 억제하는 것으로 확인되며 혈관신생 억제 효능을 갖는 천연물 암 억제제에 대한 기초자료를 제공한다.

RC-58T/h/SA#4 인체 전립선 원발암 세포에서 AC의 TRAIL 유도 apoptosis 민감성 향상 효과
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)은 정상세포에는 독성을 나타내지 않으며 암세포에서만 선택적으로 apoptosis를 유도하는 특성을 갖는 항암제이다. 하지만 전립선 암을 포함한 몇몇 암 종에서는 TRAIL에 의해 유도되는 apoptosis에 상대적으로 저항성을 갖는 것으로 보고되었다. 본 연구에서는 auriculasin의 처리가 TRAIL 저항성 전립선 원발암 세포에서 death receptor 5 (DR5)의 발현을 조절하여 TRAIL 민감성을 향상시키는 것으로 확인되었다. 전립선 원발암 세포에서 auriculasin과 TRAIL의 병용처리는 apoptosis 단계의 세포 증가, DNA 분절 및 염색체 및 apoptotic body 응축을 유도하였다. Auriculasin과 TRAIL의 병용처리는 전립선 원발암 세포에서 caspase 의존형 및 비의존형 apoptosis를 모두 유도하였으며 DR5, p53 및 CCAAT-enhancer-binding protein homologous protein (CHOP) 발현을 증가시키는 것으로 나타났다. 또한, auriculasin에 의한 TRAIL 유도 apoptosis의 증가는 DR5의 경쟁 결합단백질인 DR5 FC chimera protein의 처리에 의해 유의적으로 억제되었다. PI3K/AKT/mTOR 신호전달 경로의 활성이 auriculasin에 의해 농도의존적으로 감소하는 것으로 확인되었다. 결론적으로 본 연구의 결과는 auriculasin이 전립선 원발암 세포에서 DR5와 하위 신호전달 경로의 상향조절을 통해 TRAIL 유도 apoptosis를 증가시키는 것을 나타내며 이는 난치성 전립선 암세포의 사멸에 천연물의 이용가능성을 제시한다.

목차 (Table of Contents)

• Contents
• I. Introduction 1
• II. Materials and Methods 9
• 1. Chemicals 9
• 2. Methods 9
• 2.1. Preparation of F. philippinensis extract and solvent fractionation 10
• 2.2. Determination of proximate composition in F. philippinensis root 10
• 2.2.1. Determination of moisture content 10
• 2.2.2. Determination of crude protein 10
• 2.2.3. Determination of crude lipid 11
• 2.2.4. Determination of crude ash 11
• 2.2.5. Determination of carbohydrate 11
• 2.3. Isolation of AC 12
• 2.4. Free sugar contents determination 13
• 2.5. Free amino acid contents determination 14
• 2.6. Fatty acid contents determination 14
• 2.7. Mineral contents determination 15
• 2.8. Total phenolic contents determination 15
• 2.9. Cell culture and cell proliferation 16
• 2.10. Sulforhodamine B (SRB) assay 16
• 2.11. Sub-G1 population and cell cycle analysis 17
• 2.12. Annexin V staining assay 18
• 2.13. Detection of morphological apoptosis 19
• 2.14. Analysis of DNA fragmentation 19
• 2.15. Measurements of ROS 20
• 2.16. Mitochondria isolation 20
• 2.17. Wound-healing migration assay 20
• 2.18. Trans-well migration assay 21
• 2.19. Capillary-like tube formation assay 21
• 2.20. Rat aortic ring assay 22
• 2.21. Measurement of phosphorylated VEGFR2 concentration 22
• 2.22. Western blot analysis 23
• 2.23. Human prostate tumor xenograft mouse model 24
• 2.24. Angiogenesis animal study 25
• 2.25. Matrigel plug assay 25
• 2.26. Statistical analysis 25
• III. Results 27
• Chapter 1. Comparison of proximate composition, functional components, and anti-tumor activity between F. philippinensis extracts 27
• 1.1. Analysis of proximate composition in F. philippinensis root 27
• 1.2. Chemical characterization of F. philippinensis extracts in terms of free sugar, free amino acid, fatty acid, mineral and total phenolic contents 29
• 1.3. Effects of F. philippinensis extracts on proliferation of human prostate tumor cells, and human prostate endothelial 35
• 1.4. Effects of F. philippinensis extracts on induction of cell cycle arrest in human prostate tumor cells 37
• 1.5. Effects of F. philippinensis extracts on induction of apoptosis in human prostate tumor cells 39
• Chapter 2. Effect of AC on LNCaP prostate tumor cell death via induction of apoptosis 41
• 2.1. Prenylated isoflavones from methanolic extract of F. philippinensis inhibits prostate tumor cell growth 41
• 2.2. Effect of antitumor drugs on growth of prostate tumor and epithelial cells 43
• 2.3. Human prostate tumor cells are more sensitive to AC treatment compared with human prostate epithelial cells 45
• 2.4. AC induces apoptosis in LNCaP prostate tumor cells 48
• 2.5. AC-induced apoptosis in LNCaP cells is caspase-independent 52
• 2.6. AC inhibits growth of prostate tumor cells via suppression of the PI3K/AKT/mTOR signaling pathway 55
• 2.7. AC-induced ROS generation leads to apoptotic cell death in LNCaP prostate tumor cells 57
• 2.8. AC-induced ROS generation triggers activation of a caspase-independent pathway and inhibition of the PI3K/AKT/mTOR pathway 61
• 2.9. AC inhibits tumor growth in a xenograft athymic mouse model 63
• Chapter 3. Effect of AC on suppression of VEGF-induced angiogenesis in human umbilical vein endothelial cells 67
• 3.1. AC inhibits proliferation of HUVECs 67
• 3.2. AC suppresses VEGF-induced endothelial cell migration, invasion and differentiation into capillary-like structure in vitro 71
• 3.3. AC inhibits VEGF-induced microvessel sprouting ex vivo 75
• 3.4. AC inhibits activation of VEGFR2 77
• 3.5. AC inhibits growth of HUVECs via suppression of intracellular proangiogenic kinases 79
• 3.6. AC suppresses VEGF-induced angiogenesis in vivo 81
• Chapter 4. Effect of AC on sensitization of TRAIL-induced apoptosis in RC-58T/h/SA#4 primary human prostate tumor cells 84
• 4.1. AC sensitizes TRAIL-resistant RC-58T/h/SA#4 cells to TRAIL-induced cell death 84
• 4.2. AC sensitizes TRAIL-mediated apoptosis in RC-58T/h/SA#4 cells 86
• 4.3. Sensitization to TRAIL-mediated apoptosis by AC is dependent on caspases 89
• 4.4. Co-treatment of AC triggers caspase-independent cell death 92
• 4.5. AC sensitizes TRAIL-mediated apoptosis through up-regulation of DR5 95
• 4.6. AC sensitizes TRAIL-mediated cell death through suppression of PI3K/ AKT/mTOR pathway 99
• IV. Discussion 101
• Summary 115
• References 119
• Abstract in Korean 136