Korean J. Plant Res. 31(2):95-101(2018) https://doi.org/10.7732/kjpr.2018.31.2.095 Print ISSN 1226-3591 Online ISSN 2287-8203 Original Research Article 노종현, 이기호, 정호경, 이무진, 장지훈, 심미옥, 정자균, 정다은, 조현우 * 한약진흥재단 Role of Iridin Isolated from Iris koreana Nakai on Doxorubicin-induced Necrosis in HK-2 Cells, and Effect on Cancer Cells Jong Hyun Nho, Ki Ho Lee, Ho Kyung Jung, Mu Jin Lee, Ji Hun Jang, Mi Ok Sim, Ja Kyun Jung, Da Eun Jung and Hyun Woo Cho* National Development Institute of Korean Medicine, Jangheung-gun 59338, Korea Abstract - Doxorubicin is a anti-cancer drugs that interferes with the growth and spread of cancer cells in human body. Doxorubicin is used to treat different types of cancers that affect the ovary, thyoid and lungs, but induced side effect such as nephrotoxicity and cardiotoxicity. Thus, we investigated that the effect of iridin on doxorubicin-induced necrosis in HK-2 cells, a human proximal tubule cell. To confirm effect of iridin on doxorubicin-induced necrosis, HK-2 cells are treated with 10 μm doxorubicin and 80 μm iridin. 80 μm iridin reduced 10 μm doxorubicin-induced necrosis, the mitochondrial over activation and caspase-3 activation. However, iridin reduces anti-cancer effect of doxorubicin such as PARP1 and caspase-3 activation, checkpoint proteins (CDK4 and CDK6) in NCI-H1129 cells (Human non-small cell lung cancer cell). In HCT-116 cells (Human colorectan cancer cell), iridin do not increased protein expression of CDK4 and CDK6 decreased by doxorubicin. Results indicate that treatment of iridin was diminished doxorubicin-induced necrosis in HK-2 cells. However, iridin was decreased anti-cancer effect of doxorubicin on NCI-H1229, but not HCT-116. Thus, further experiment are required to iridin treatment on various cancer cells and animal models because effect of iridin different cell type. Key words - Doxorubicin, HK-2, Iridin, Iris koreana Nakai, Necrosis, Nephrotoxicity 서언 Doxorubicin 은 adriamycin 으로도잘알려져있으며, 암세포를억제하는항암제로사용되고있다. 이는암세포에서 DNA 사이에삽입 (intercalation) 되어 DNA 합성을방해할뿐만아니라 DNA 수리를담당하는 topoisomerase-2 를억제하며, 산화스트레스를유도함에따라암세포의사멸을유도한다 (Thorn et al., 2011). Doxorubicin 은이러한효과외에도암환자에게투여했을때심근독성과신장독성을일으킨다고알려져있다 (Yoon et al., 2011; Kim and Cho, 2016). 국내암환자수는매년증가되 * 교신저자 : thej01234@gmail.com Tel. +82-61-860-2873 Both authors contributed equally to this work. 는추세이고, 항암치료에사용되는항암제들은 doxorubicin 과마찬가지로대부분장기에독성을일으키는부작용이함께나타난다 (Tacar et al., 2013). 이러한부작용때문에현재항암제의부작용을줄이면서항암효능을증진시킬수있는천연물또는천연분리물질의탐색과연구가필요하다. Caspase-3 단백질은 caspase family 에속하는단백질로평소세포내부에서불활성화상태로존재하다가자극을받게되면활성화상태로바뀌게된다 (Yuan et al., 2016). 뿐만아니라 caspase-3 단백질은세포내부에서 apoptosis 또는 necrosis 가일어나는동안에 DNA 수리에관여하는 PARP1 단백질을절단해 DNA 수리를억제함에따라세포수복을억제하며다양한세포에서세포죽음에관련된중요한인자로이용되고있다 (Yoon et al., 2015). c 본학회지의저작권은 ( 사 ) 한국자원식물학회지에있으며, 이의무단전재나복제를금합니다. This is an Open-Access article distributed under the terms of the Creative Commons -95- Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
韓資植誌 Korean J. Plant Res. 31(2) : 95~101(2018) 진핵세포의세포주기는크게 G1, S, G2, M기로나누어져있으며, 각각단계마다다음단계로진행에관여하는체크포인트가존재한다. 이에관여하는여러단백질들의상호작용을통해세포주기가촉진되거나억제되며 (Park et al., 2013), CDK4 와 CDK6 단백질은 G1-S 진행에관여하는단백질이다 (Marcos, 2014). Doxorubicin 은암세포에서 CDK4 와 CDK6 의발현을억제하는것을통해암세포의세포주기를억제한다고알려져있다 (Tarasewicz et al., 2014; Tadesse et al., 2015). 최근천연소재개발연구가여러방면에서이루어지고있고, 예로부터사용된자원식물의항염증, 항산화등여러이로운효능이알려지면서자원식물에대한중요성이대두되고있다 (Park et al., 2017; Guo and Wang, 2017; Choi et al., 2017). 노랑붓꽃 (Iris koreana Nakai) 은우리나라중부이남에주로분포하는식물로변산반도와내장산인근에많이분포하고있는것으로알려져있으며 (Pi et al., 2016A), 자생지의식생현황과식생구조를분석을통해생태학적인기초자료를구축하는연구가활발히진행되고있지만 (Pi et al., 2016B) 노랑붓꽃에대한계통분류학적또는생장및개화에관한연구를제외하고본초학적효능및기전연구가전무한실정이다 (Lee et al., 2007; Lee and Park, 2013; Eom et al., 2013). 따라서본연구는 doxorubicin 에의해유발된신장독성세포모델에노랑붓꽃전초에서분리한 iridin을처리하여신장독성억제효과또는신장세포에대한 iridin의독성이나타나는지조사해보고자하였다. 재료및방법실험재료실험에사용한 iridin은노랑붓꽃전초에서분리된천연물질로, 천연물물질은행 (NPBANK, Gungsan, Korea) 에서파우더형태로분양받아사용하였다. Iridin은 0.9% NaCl 에녹여사용하였으며, doxorubicin (Sigma, St. Louis, Missouri, USA) 은 1% DMSO (dimethyl sulfoxide) 에완전히녹인후차광상태로냉동보관하고실험에사용하였다. 세포배양 HK-2 세포 (Human kidney proximal tubule cell), NCI-H1229 (Human non-small cell lung cancer cell) 과 HCT-116 (Human colorectal cancer cell) 은한국세포주은행 (KCLB, Seoul, Korea) 으로부터분양받아사용하였다. HK-2 세포와 NCI-H1229 세포는 PRMI 1640 배지 (Thermo, Waltham, Massachusetts, USA) 를, HCT-116 세포는 DMEM (Thermo, Waltham, Massachusetts, USA) 을사용하였고, 사용된배지에 10% FBS (fetal bovine serum), 1% penicilin/streptomycin 을첨가하여사용하였으며, 37, 5% CO 2 조건에서배양하고유지하였다. 세포생존율분석세포생존율은 CellTiter 96 AQueous One Solution Cell proliferation assay kit (Promega, Fitchburg, Wisconsin, USA) 를사용하였으며, 제조사의프로토콜에따라서측정하였다. HK-2 세포를 96 well plate 에 1 10 5 cells/ ml농도가되도록분주한뒤 37, 5% 배양기에서 24시간배양한후 iridin을각각 10, 20, 40 그리고 80 μm 농도로 24시간동안처리하여천연분리물질에대한독성을조사하였고, 이와같은농도로 1시간동안전처리한뒤 10 μm doxorubicin 을처리하고 24시간동안배양하였다. 24 시간뒤 MTS 시약 20 μl를넣고 2시간동안배양한후 microplate reader Infinite 200 PRO (TECAN, Mannedorf, Switzerland) 를이용하여 490 nm에서흡광도를측정하였다. 세포생존율은정상대조군에대한생존율로표시하였고, 이에따라보호효과가있는농도를확인하였다. 단백질분석 Doxorubicin 과 iridin 처리후배양이끝난 HK-2 세포는 PBS로세척한뒤 Pierce Protease and Phosphatase Inhibitor Mini Tablets (Thermo, Waltham, Massachusetts, USA) 가함유된 RIPA cell lysis buffer 2 (Enzo, Farmingdale, USA) 로 1시간동안용해했다. 세포용해액은원심분리기로 4, 13,000 g 조건에서 10분동안원심분리하였으며, Bradford 단백질정량법을이용해각각샘플에대한단백질의농도를측정했다. 20 μg의단백질샘플을 SDS-PAGE 로분리하였고, 분리된 SDS-PAGE 겔은 Thermo Scientific Pierce G2 Fast Blotter (Thermo, Massachusetts, USA) 를사용하여 PVDF membrane (GE healthcare life sciences, Little chalfont, Germany) 으로이동시켰다. Membrane 은 5% skim milk 가포함된 Tris buffered saline + 0.05% Tween-20으로 blocking 한뒤 1차항체를반응시키고, anti-rabbit 과 anti-mouse 2차항체를이용하여 D-Plus ECL Femto System (Donginbio, Souel, Korea) 를이용해단백질을확인하였다. Tunel 분석 Tunel 분석은 DeadEnd Fluorometric TUNEL System -96-
(Promega, Fitchburg, Wisconsin, USA) 을사용하였으며, 제조사의프로토콜에따라서수행하였다. HK-2 세포를 12 well plate 에서 cover slip 위에 2 10 5 cells/ ml농도가되도록분주한뒤, 37, 5% 배양기에서 24시간배양했다. 배양된세포는 80 μl iridin을 1시간동안전처리한뒤 10 μm doxorubicin 을처리하고 24시간동안배양하였다. 그후 10% neutralized buffered formalin (NBF) 로 4 에서 20분동안고정하였고, phosphate buffered saline (PBS) 로 2번씻어주었다. 다음 0.2% Triton X-100 in PBS 용액으로 5분간투과화한뒤 PBS 로 2번씻어주었다. 남은액체가없도록모두제거하고 Equilibration buffer 를 5분간처리하였으며, 100 μl rtdt incubation buffer 를처리하고 37 에서 1시간동안배양하였다. 그후 2 SSC buffer 를처리하고, PBS 로 2번씻은뒤 Prolong Gold Antifade Mountant with DAPI (Thermo, Waltham, Massachusetts, USA) 로봉입하여 Epi-fluorescence microscope (Carl Zeiss, Oberkochen, Germany) 를이용해형광정도를관찰하였다. 결과및고찰 Iridin 의신장세포보호효과 HK-2 세포에대한 iridin의세포독성과 doxorubicin 에대한세포보호효과를확인하기위해 MTS 분석을진행하였다. Iridin 을여러농도로 24시간동안처리하였을때, HK-2 세포에대한자체독성은나타나지않았다 (Fig. 1A). 다음으로 iridin을여러농도로전처리하고 10 μm doxorubicin 을함께처리하였을때, doxorubicin 단독처리군은정상대조군에비해세포생존율이 76.9 ± 3.0% 로감소하였다. 하지만 80 μm iridin 을전처리했을경우세포생존율이 94.6 ± 2.6% 까지회복되는결과를확인하였다 (Fig. 1B). 실험결과를종합해봤을때 iridin은 HK-2 세포에대해독성을나타내지않았으며, 10 μm doxorubicin 처리 Caspase-3 활성도측정 Caspase-3 의활성도는 Caspase-3 colorimetric detection kit (Enzo, Farmingdale, USA) 를이용해실험을진행했다. 단백질분석방법에서이용한방법으로세포를용해하였고, 20 μg의단백질을사용해제조사가제공한프로토콜에따라실험을진행했다. FACS 분석 Annxon V-FITC apoptosis detection kit (Enzo, Farmingdale, USA) 를사용하였다. HK-2 세포에약물처리후유도되는 apoptosis 의비율을구분하기위해제조사의프로토콜에따라 annexin V-FITC 와 propidium iodide (PI) 형광염료로염색시켰으며, mitochondrial membrane potential (MMP) 의비율을측정하기위해 JC-1 Dye (Thermo, Waltham, Massachusetts, USA) 를사용하여실험을진행하였다. 각각형광염료로염색된세포는유세포분석기 (FACS) 를이용하여분석하였다. 통계처리통계처리는평균 ± 표준편차 (mean ± SD) 로나타냈다. 유의성을검정하기위해 SPSS (Statistical Package for Social Science Inc., Chicago, IL, USA) 통계프로그램을사용하였다. 일원변량분석 (one way ANOVA) 을실시하였으며, 유의성이있는경우 p<0.05 수준에서, Duncan's Multiple Range Test (DMRT) 와 Tukey post-hoc test 를실시하였다. Fig. 1. Iridin reduces doxorubicin-induced cell death in HK-2 cells. (A) HK-2 cells were treated with iridin for various concentration. (B) After pretreatment with iridin for various concentration, HK-2 cells were treated with 10 μm doxorubicin for 24 h. Cell viability was measured by MTS assay. Means values ± SD from triplicate separated experiments are shown. *Means with difference letters are significantly different at p<0.05 by Duncan's Multiple Range Test. -97-
韓資植誌 Korean J. Plant Res. 31(2) : 95~101(2018) 에의한세포독성도 80 μm iridin 을전처리했을때세포생존율이회복되었다. Iridin이 doxorubicin 으로유도한 HK-2 세포독성에대한보호효과가있음을확인하였다. 그러나 MTS assay 는 apopotosis 와 necrosis 등다양한세포사멸경로를특이적으로분석할수없는단점이있기때문에 doxorubicin 에의한세포사멸경로와이에대한 iridin의억제효과가어떤단백질신호전달경로를통해일어나는지알아보기위해다음실험을진행하였다. Iridin 의 caspase-3 신호전달억제효과세포사멸에관여하는단백질인 poly (ADP-ribose] polymerase 1 (PARP1) 과 caspase-3 의단백질발현정도를확인하기위해 western blot을이용한단백질분석을진행하였다. 10 μm doxorubicin 을 24시간동안처리하였을때 caspase-3 와 PARP1 의 cleaved 형태가증가하였고 80 μm iridin 을전처리했을때억제되는결과를확인했다. Doxorubicin 은세포내부에서세포주기에관여하는 cyclin-dependent kinase 4 (CDK4) 와 cyclindependent kinase 6 (CDK6) 를억제한다고알려져있어 (Tarasewicz et al., 2014; Tadesse et al., 2015), 단백질발현량을확인해본결과 10 μm doxorubicin 처리했을때 CDK4 와 CDK6 의단백질발현량이조금감소되는경향이나타나고, 80 μm iridin 을전처리했을때약간회복되는것을확인할수있었지만이는유의적인차이가없었다 (Fig. 2A). 추가적으로 caspase-3 활성도를 확인해본결과앞선결과와마찬가지로 10 μm doxorubicin 을처리했을때정상대조군에비해 caspase-3 활성도가 143 ± 8.6% 까지증가되었으며, 이는 80 μm iridin 처리에의해 110 ± 4.4% 까지감소됨을확인하였다 (Fig. 2B). 결과를종합하자면 HK-2 세포에서 10 μm doxorubicin 처리는 caspase-3 단백질을활성과발현량을증가시켰지만이는 80 μm iridin 을전처리했을때모두차단되었다. 하지만 CDK4 와 CDK6 의단백질발현량뿐만아니라세포주기를확인하는추가적인실험이필요할것으로생각되며, 다음으로 iridin이어떤 apoptosis 또는 necrosis 와같은특정세포사멸기전에영향을미치는지알아보기위해다음실험을진행하였다. Iridin 의 HK-2 세포괴사, 미토콘드리아과활성억제효과 Tunel 분석은세포내부에서 apoptosis 또는 necrosis 에의해일어난 DNA 단편화 (DNA fragmentation) 를시각화하는방법으로, 10 μm doxorubicin 처리에의해증가된 tunel signal 이 80 μm iridin을전처리했을때감소되는결과를확인하였다 (Fig. 3A). 다음으로 iridin의세포보호효과가세포내부의 apoptosis 또는 necrosis 를억제하는지와그비율을알아보기위해 annexin-v/pi 염색을이용해실험을진행하였다. 그결과 10 μm doxorubicin 처리는세포내부의 necrosis 비율을 68.5% 까지증가시켰으나이는 80 μm iridin 처리에의해 34.5% 까지 Fig. 2. Iridin reduces doxorubicin-induced caspase-3/mst-1 signaling pathway in HK-2 cells. After pretreatment with 80 μm iridin for 1 h, HK-2 cells were treated with 10 μm doxorubicin for 24 h. Cell extracts were subjected to protein analysis using western blot and caspase-3 activity assay. (A-B) Data represent the mean± SEM of three independent experiments. **p<0.05 vs. control, *p<0.05 vs. 20 μm cisplatin. -98-
Fig. 3. Iridin ameliorates doxorubicin-induced necrosis and the mitochondrial over activation in HK-2 cells. After pretreatment with 80 μm iridin for 1 h, HK-2 cells were treated with 10 μm doxorubicin for 24 h. (A) HK-2 cell visualized by tunel assay. (B) Annexin V and propidium iodide staining and (C) JC-1 staining were analyzed by flow cytometry. Representative images were taken from at least three independent experiments. 감소되는결과를나타냈다 (Fig. 3B). 미토콘드리아의활성은 necrosis 로진행될때과활성화된다고알려져있으며 (Shin et al., 2015), 이에따라 JC-1 염색을통해미토콘드리아의활성을확인한결과앞선결과와마찬가지로, 10 μm doxorubicin 을 HK-2 세포에처리했을때증가된미토콘드리아활성이 80 μm iridin을처리했을때정상에가깝게억제되는것을확인하였다 (Fig. 3C). 결과를종합하자면 10 μm doxorubicin 처리에의해증가된 HK-2 세포의미토콘드리아의과활성과 necrosis, DNA 단편화는 80 μm iridin 전처리에의해감소되었다. 이는 iridin 이 doxorubicin 에의한 HK-2 세포의 necrosis 를억제하는것으로보이며, doxorubicin 에의해유도되는산화스트레스나 necrosis 초기에관여하는신호전달경로그리고세포주기의비율을추가적으로확인해야할뿐만아니라 doxorubicin 으로유도된신장독성동물모델에서도 iridin이보호효과를나타낼수있는지추가적인연구가필요하다고생각된다. 암세포에서 doxorubicin 에대한 iridin 의역할이상의결과를종합하면 iridin은 doxorubicin 에의해유도된신장세포의괴사를억제하는것으로사료된다. 이어같은조 건하에도 iridin이 doxorubicin 으로유도되는암세포의사멸에어떻게관여할것인지알아보기위해실험을진행하였다. 실험에사용된세포주는휴먼유래폐암세포주인 NCI-H1229 와대장암세포주인 HCT-116을이용하였다. HK-2 세포에서확인한단백질인자를추가로확인하였을때 NCI-H1229 세포에서 10 μm doxorubicin에의해 PARP1과 caspase-3 의 cleaved 형태가증가하였을뿐만아니라 CDK4 와 CDK6 의발현량이감소되었다. 하지만예상했던결과와다르게 80 μm iridin 처리에의하여정상대조군과가깝게회복되는결과를나타냈다 (Fig. 4A and 4C). 흥미롭게도 HCT-116 세포는 10 μm doxorubicin 처리에의해감소된 CDK4 와 CDK6 단백질이 NCI-H1229 세포와는다르게 80 μm iridin 처리에의해회복되지않았지만, caspase-3 의 cleaved 형태를조금회복시키는것으로나타났다 (Fig. 4B and 4C). 이는 iridin 처리가폐암세포에대해 doxorubicin 의효과를억제하는것으로나타났을뿐만아니라대장암세포에서 doxorubicin 처리에의한세포주기억제를제외한세포사멸효과에관여한다고생각된다. 이에따라폐암세포에서 doxorubicin 과 iridin의병용처리는어렵다고생각된다. 그러나대장암세포에서 caspase-3 의 cleaved 형태조금감소시키지만 CDK4 와 -99-
韓資植誌 Korean J. Plant Res. 31(2) : 95~101(2018) Fig. 4. Role of iridin on doxorubicin treatment in NCI-H1229 and HCT-116 cells. After pretreatment with 80 μm iridin for 1 h, HK-2 cells were treated with 10 μm doxorubicin for 24 h. (A) NCI-H1229 cells. (B) HCT-116 cells. (C) Relative optical density. Data represent the mean± SEM of three independent experiments. **p<0.05 vs. control, *p<0.05 vs. 10 μm doxorubicin. CDK6 를통한 doxorubicin 의세포성장억제를 iridin이방해하지않는것으로생각되지때문에, 신장독성동물모델과대장암관련동물실험을통해 iridin의역할을추가적으로확인해야된다고생각된다. 적요노랑붓꽃에서분리된 iridin의 doxorubicin 으로유도된신장세포괴사모델에대한보호효과및암세포에대한작용을알아 보기위해연구를수행하였다. Iridin 단일처리로는신장근위세뇨관세포주에대해독성을나타내지않았으며, 80 μm의농도에서 10 μm doxorubicin 처리에의한세포사멸을 94.6 ± 2.6% 까지회복시켰다. 또한 80 μm iridin 처리는 10 μm doxorubicin 처리에의해증가된 cleaved PARP1 과 cleaved caspase-3 를포함하는세포사멸신호전달을차단하였을뿐만아니라 DNA fragmentation, necrotic cell death 및 mitochondrial dysfunction 을개선시켰다. 마지막으로암세포에서 iridin의효과를확인해본결과, 폐암세포주인 NCI-H1229 세포에서 doxorubicin -100-
의항암효과를억제하는경향이나타났지만대장암세포주인 HCT-116 세포주에서는암세포에대한성장억제를방해하지않는것으로확인되었다. 따라서폐암세포에서 doxorubicin 과 iridin의병용처리는힘들다고판단되고, In vivo 수준에서신장독성및대장암관련실험을통해 iridin의역할을추가적으로확인해야한다고생각된다. 사사본연구는한의약의과학화, 한의약육성및산업진흥을통해국민의건강한삶과국가경제에기여할수있도록보건복지부한국토종자원의한약재기반구축사업에의해이루어진결과로이에감사드립니다. References Choi, K.M., J.H. Lee, G.O. Adam, S.J. Kim, H.S. Kang, Y.S. Yang and G.B. Kim. 2017. Effects of the Jinan red ginseng extract treatment on poloxamer 407-induced hyperlipidemia in rabbits. Korean J. Plant Res. 30:601-611. Eom, T.H., J.H. Kim, S.I. Lee and J.G. Jeong. 2013. A herbalogical study on the plants of Iridaceae in Korea. Korean J Herbol. 28:85-93. Guo, H.F. and M.H. Wang. 2017. Anti-inflammatory and anticancer effect of Stachys affinis tubers. Korean J. Plant Res. 30:679-685. Kim, Y.S. and J.H. Cho. 2016. Optimal treatment of advanced pancreatic neuroendocrine tumor. Korean J Pancreas Biliary Tract. 21:128-137. Lee, J.H., C.H. Lee, G.W. Park and C.Y. Song. 2007. Effect of forcing date and temperature on growth and flowering of Iris koreana and Iris minutoaurea. J Korean For Soc. 96:699-704. Lee, H.J. and S.J. Park. 2013. A phylogenetic study of Korean Iris L. based on pastid DNA (psba-trnh, trnl-f) sequences. Korean J PI Taxon. 43:227-235. Marcos, M. 2014. Cyclin-dependent kinases. Genome Biol. 15:122. Park, H.J., S.J. Jin, Y.N. Oh, S.G. Yun, J.Y. Lee, H.J. Kwon and B.W. Kim. 2013. Induction of G1 arrest by methanol extract of Lycopus lucidus in human lung adenocarcinoma A549 cells. J Life Sci. 23:1109-1117. Pi, J.H., J.G. Park, J.Y. Jung, J.S. Park, G.U. Suh and S.W. Son. 2016A. Habitats environmental and population characteristics of Iris koreana Nakia, a rare and endemic species in Korea. J Agric & Life Sci. 49:102-109. Pi, J.H., J.G. Park, J.Y. Jung, J.S., Park, H.H., Yang, G.U., Suh, C.H. Lee and S.W. Son. 2016B. Vegetation structure and flora of Iris koreana Nakia, endemic species in Korea. J Agric & Life Sci. 50:55-67. Park, G.H., H.M Song, S.B. Park, J.H. Park, M.S. Shin, H.J. Son, Y. Um and J.B. Jeong. 2017. Anti-proliferative activity of ethanol extracts from Taxilli ramulus (Taxillus chinensis (DC.) Danser) through cyclin D1 proteasomal degradation in human colorectal cancer cells. Korean J. Plant Res. 30:640-646. Shin, H.J., H.K. Kwon, J.H. Lee, X. Gui, A. Achek, J.H. Kim and S. Choi. 2015. Doxorubicin-induced necrosis is mediated by poly (ADP-ribose)polymerase 1 (PARP1) but is independent of p53. Sci Rep. 5:15798. Thorn, C.F., C. Oshiro, S. Marsh, T. Hernandez-Boussard, H. Mcleod, T.E. Klein and R.B. Altman. 2011. Doxorubicin pathways: pharmacodynamics and adverse effects. Phamacogenet Genomics 21:440-446. Tacar, O., P. Sriamornsak and C.R. Dass. 2013. Doxorubicin: an update on anticancer molecular action, toxicity and novel drug delivery systems. J Pharm Pharmacol. 65:157-170. Tarasewicz, E., R. Hamdan, J. Straehla, A. Hardy, O. Nunez, S. Zelivianski, D. Dokic and J.S. Jeruss. 2014. CDK4 inhibition and doxorubicin mediate breast cancer cell apoptosis through Smad3 and survivin. Cancer Biol Ther. 15:1301-1311. Tadesse, S., M. Yu, M. Kumarasiri, B.T. Le and S. Wang. 2015. Targeting CDK6 in cancer: State of the art and new insights. Cell Cycle 14:3220-3230. Yoon, G.A., S.Y. Chae and K.H. Kim. 2012. Synergistic anticancer activity on mouse sarcoma by mixture of doxorubicin and water extract of Albizzia julibrissin. Cancer Prev Res. 17:239-243. Yoon, J.H., S.H. Sohn, E.Y. Lee, G.S. Kim, S.E. Lee, D.Y. Lee, K.H. Seo, S.W. Lee and H.D. Kim. 2015. Protective effect of saururus chinensis ethanol extract against styrene in mouse spermatocyte cell line. Korean J Med Crop Sci. 25:45-51. Yuan, J., A. Najafov and B.F. Py. 2016. Roles of caspases in necrotic cell death. Cell 15:1693-1704. (Received 7 August 2017 ; Revised 17 November 2017 ; Accepted 12 December 2017) -101-