갈색거저리 유충 추출물의 간암세포에 대한 세포독성 효능

국문 초록 (Abstract)

본 연구에서 우리는 갈색거저리 유충 추출물의 암세포 선택적인 세포독성 활성을 암세포주를 대상으로 하는 in vitro 및 in vivo 실험으로 증명하였다. 먼저 갈색거저리 유충의 에탄올 추출물은 정상세포라 할 수 있는 primary hepatocyte에 대한 독성은 미미하였으나 암세포주들에 대한 세포독성과 함께 다른 정상세포인 primary cardiomyocyte에 대한 독성도 가지고 있었다. 에탄올 추출물을 hexane, butanol, ethyl acetate, 물을 이용하여 liquid-liquid partition으로 추가로 분획, 구성물질들을 분리하였고 이들 분획물 중에서 hexane 분획물은 다양한 암세포들(PC3, 22Rv1, HeLa, PLC/PRF5, HepG2, Hep3B, SK-HEP-1, HCT116, NCI-H460, MDA-MB231, SKOV3)에 대한 독성을 유지하면서 cardiomyocyte에 대한 독성이 상당히 줄어들었다. 0.4mg/mL 에탄올 추출물이 cardiomyocyte를 대부분 죽이는 독성을 보였으나 동일조건에서 hexane 분획물은 약 20%정도의 세포독성만을 보여주어 독성이 상당히 감소된 것을 확인하였다. 이렇게 비특이적인 세포독성이 물질분리 및 분획을 함으로써 줄어들 수 있다는 것을 확인하였다. 두 번째로 hexane과 ethyl acetate 분획물들이 아포토시스, 세포괴사, 오토파지와 같은 대표적인 세포죽음 기전들을 활성화시킬 수 있는 것으로 확인하였다. 더불어 hexane 분획물의 세포죽음 유도활성은 현재 임상에서 널리 처방되고 있는 항암물질들과 함께 간암세포주에 처리되었을 때 항암활성을 증대시킬 수 있는 것으로 확인하였다. 이와 같은 실험 결과를 바탕으로 갈색거저리 유충 추출물들이 단독으로 혹은 다른 세포독성 약물들과 함께 항암활성을 가질 수 있음을 확인하였다. 마지막으로 hexane 분획물의 항암활성을 in vivo xenograft 실험쥐 모델에서 확인하였는데, 간암세포주인 SK-HEP-1을 이식한 실험쥐에서 hexane 분획물을 15일간 복강주사 하였을 때 종양의 성장을 뚜렷하게 억제하는 것을 확인하였고, 앞선 정상세포에 대한 제한적인 영향과 일치하게 몸무게의 감소 등 부작용이라 할 수 있는 증상은 확인되지 않았다. 이상의 결과들을 종합하면 갈색거저리 유충 추출물의 항암활성을 in vitro와 in vivo에서 확인할 수 있었으며, 새로운 항암활성을 가지는 물질 발굴을 위해 추가적인 분획과 물질 분석이 필요하다고 사료된다.

다국어 초록 (Multilingual Abstract)

Various natural products or their derivatives, mostly originating from plants, fungi, and bacteria, have been exploited as therapeutic drugs to treat various human diseases. In addition to previously explored organisms, research on natural compounds has now expanded into unexamined living organisms in order to identify novel bioactive substances. Here, we determined whether or not the larval form of the mealworm beetle Tenebrio molitor, a species of darkling beetle, contains cytotoxic substances that exclusively affect cancer cell viability. Ethanol extract and its solvent partitioned fractions, hexane and ethyl acetate fractions, showed anticancer effects against various human cancer cells derived from the prostate (PC3 and 22Rv1), cervix (HeLa), liver (PLC/PRF5, HepG2, Hep3B, and SK-HEP-1), colon (HCT116), lung (NCI-H460), breast (MDA-MB231), and ovary (SKOV3). Cell death induced by the fractions was a mix of apoptosis, necrosis, and autophagy. The hexane fraction was administered intraperitoneally to nude mice bearing a hepatocellular carcinoma SK-HEP-1 and showed inhibition of tumor growth in vivo. Therefore, we concluded that worm extracts contain cytotoxic substances, which can be enriched by proper fractionation protocols, and further separation and purification could lead to the identification of novel molecules to treat human cancers.

목차 (Table of Contents)

• ABSTRACT
• 서론
• 재료 및 방법
• 결과 및 고찰
• 요약
• REFERENCES

참고문헌 (Reference)

1 Kamb A, "Why is cancer drug discovery so difficult?" 6 : 115-120, 2007

2 Kummar S, "Utilizing targeted cancer therapeutic agents in combination : novel approaches and urgent requirements" 9 : 843-856, 2010

3 Menendez JA, "The Warburg effect version 2. 0 : metabolic reprogramming of cancer stem cells" 12 : 1166-1179, 2013

4 Vistica DT, "Tetrazolium-based assays for cellular viability : a critical examination of selected parameters affecting formazan production" 51 : 2515-2520, 1991

5 Lee YJ, "Structure and expression of the tenecin 3 gene in Tenebrio molitor" 218 : 6-11, 1996

6 Mukhopadhyay P, "Simultaneous detection of apoptosis and mitochondrial superoxide production in live cells by flow cytometry and confocal microscopy" 2 : 2295-2301, 2007

7 Ravikumar B, "Regulation of mammalian autophagy in physiology and pathophysiology" 90 : 1383-1435, 2010

8 Moon HJ, "Purification and molecular cloning of cDNA for an inducible antibacterial protein from larvae of the coleopteran, Tenebrio molitor" 116 : 53-58, 1994

9 윤금주, "Oleic Acid and Linoleic Acid from Tenebrio molitor Larvae Inhibit BACE1 Activity in vitro: Molecular Docking Studies" 한국식품영양과학회 17 (17): 284-289, 2014

10 Patel MN, "Objective assessment of cancer genes for drug discovery" 12 : 35-50, 2013

11 Newman DJ, "Natural products as sources of new drugs over the 30 years from 1981 to 2010" 75 : 311-335, 2012

12 Galluzzi L, "Molecular definitions of cell death subroutines : recommendations of the Nomenclature Committee on Cell Death 2012" 19 : 107-120, 2012

13 Orr GA, "Mechanisms of Taxol resistance related to microtubules" 22 : 7280-7295, 2003

14 Bruix J, "Management of hepatocellular carcinoma : an update" 53 : 1020-1022, 2011

15 Clardy J, "Lessons from natural molecules" 432 : 829-837, 2004

16 Shen L, "Isolation and primary culture of rat hepatic cells" 64 : 3917-, 2012

17 Li WC, "Isolation and culture of adult mouse hepatocytes" 633 : 185-196, 2010

18 Jain MV, "Interconnections between apoptotic, autophagic and necrotic pathways : implications for cancer therapy development" 17 : 12-29, 2013

19 Dossey AT, "Insects and their chemical weaponry : new potential for drug discovery" 27 : 1737-1757, 2010

20 Pemberton RW, "Insects and other arthropods used as drugs in Korean traditional medicine" 65 : 207-216, 1999

21 Ratcliffe NA, "Insect natural products and processes : new treatments for human disease" 41 : 747-769, 2011

22 Youn K, "In vitro BACE1 inhibitory activity of geraniin and corilagin from Geranium thunbergii" 79 : 1038-1042, 2013

23 Mizushima N, "How to interpret LC3immunoblotting" 3 : 542-545, 2007

24 Hanahan D, "Hallmarks of cancer : the next generation" 144 : 646-674, 2011

25 한소리, "Evaluation of Genotoxicity and 28-day Oral Dose Toxicity on Freeze-dried Powder of Tenebrio molitor Larvae (Yellow Mealworm)" 한국독성학회 30 (30): 121-130, 2014

26 Simon E, "Elemental concentration in mealworm beetle(Tenebrio molitor L. )during metamorphosis" 154 : 81-87, 2013

27 Berenbaum MR, "Ecology. Bugs' bugs" 322 : 52-53, 2008

28 Wilhelm S, "Discovery and development of sorafenib : a multikinase inhibitor for treating cancer" 5 : 835-844, 2006

29 Denecker G, "Death receptor-induced apoptotic and necrotic cell death : differential role of caspases and mitochondria" 8 : 829-840, 2001

30 Edinger AL, "Death by design : apoptosis, necrosis and autophagy" 16 : 663-669, 2004

31 Siddik ZH, "Cisplatin : mode of cytotoxic action and molecular basis of resistance" 22 : 7265-7279, 2003

32 Lachance H, "Charting, navigating, and populating natural product chemical space for drug discovery" 55 : 5989-6001, 2012

33 Cherniack EP, "Bugs as drugs, Part 1: Insects: the "new"alternative medicine for the 21st century?" 15 : 124-135, 2010

34 Lord CJ, "Biology-driven cancer drug development : back to the future" 8 : 38-, 2010

35 Suh HJ, "Antioxidant activity of aqueous methanol extracts of Protaetia brevitarsis Lewis(Coleoptera : Scarabaedia)at different growth stages" 26 : 510-517, 2012

36 Minotti G, "Anthracyclines : molecular advances and pharmacologic developments in antitumor activity and cardiotoxicity" 56 : 185-229, 2004

37 Koopman G, "Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis" 84 : 1415-1420, 1994

38 Sreejit P, "An improved protocol for primary culture of cardiomyocyte from neonatal mice" 44 : 45-50, 2008