쪽 현탁배양(懸濁培養)에서 Indole이 세포 생장과 Indirubin 생성에 미치는 영향

정은숙,채영암 韓國藥用作物學會 1995 한국약용작물학회지 Vol.3 No.2

본 실험은 쪽 세포의 현탁배양에서 indirubin 생산을 위한 indole의 처리 시기와 처리 기간 및 첨가량을 찾기 위하여 수행하였으며 얻어진 결과는 다음과 같다. 1. Indole 첨가시 세포 생장량은 억제되었다. 2. Tryptophan 첨가시 세포 생장량의 증가는 크지 않았다. 3. Indole과 L-tryptophan을 고체레지에 첨가한 경우 indole을 첨가한 배지에서 만 indirubin이 검출되었다. 4. Indirubin 생산에 적절한 indole 첨가 농도는 고체배지에서 200mg으로 나타났다. 5. 현탁배양 중 indole 첨가시기를 달리한 결과 배양 20일 후 indole을 첨가한 배지에서 세포와 배지내 indirubin 농도가 높았다. 6. 고체배양보다 현탁배양에서 세포내에 축적된indirubin이 많았으며 배지 내 로 상당량의 indirubin이 유출되었다. This experiment was carried out to analyze the effect of indole on the synthesis of indirubin in suspension culture of Polygonum tinctorium. Adding indole and L-tryptophan into culture media was re­vealed that indirubin was synthesized in callus grown on solid medium containing indole and proper concentration of indole for indirubin production was decided as 200mg/1. Indirubin content in suspension culture was higher than in solid medium with considerable amount of indirubin secresed into media in suspension culture and highest quantity of indirubin was obtained when indole was added into medium after 20 days suspension culture.

Metabolic engineering of <i>Escherichia coli</i> for the production of indirubin from glucose

Du, Jikun,Yang, Dongsoo,Luo, Zi Wei,Lee, Sang Yup Elsevier 2018 Journal of biotechnology Vol.267 No.-

<P><B>Abstract</B></P> <P>Indirubin is an indole alkaloid that can be used to treat various diseases including granulocytic leukemia, cancer, and Alzheimer’s disease. Microbial production of indirubin has so far been achieved by supplementation of rather expensive substrates such as indole or tryptophan. Here, we report the development of metabolically engineered <I>Escherichia coli</I> strain capable of producing indirubin directly from glucose. First, the <I>Methylophaga aminisulfidivorans</I> flavin-containing monooxygenase (FMO) and <I>E. coli</I> tryptophanase (TnaA) were introduced into <I>E. coli</I> in order to complete the biosynthetic pathway from tryptophan to indirubin. Further engineering was performed through rational strategies including disruption of the regulatory repressor gene <I>trpR</I> and removal of feedback inhibitions on AroG and TrpE. Then, combinatorial approach was employed by systematically screening eight genes involved in the common aromatic amino acid pathway. Moreover, availability of the aromatic precursor substrates, phospho<I>enol</I>pyruvate and erythrose-4-phosphate, was enhanced by inactivating the <I>pykF</I> (pyruvate kinase I) and <I>pykA</I> (pyruvate kinase II) genes, and by overexpressing the <I>tktA</I> gene (encoding transketolase), respectively. Fed-batch fermentation of the final engineered strain led to production of 0.056 g/L of indirubin directly from glucose. The metabolic engineering and synthetic biology strategies reported here thus allows microbial fermentative production of indirubin from glucose.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Indirubin is a natural compound with a wide range of clinical applications. </LI> <LI> A <I>de novo</I> indirubin biosynthetic pathway was constructed. </LI> <LI> <I>E. coli</I> was engineered to produce indirubin directly from glucose. </LI> <LI> Fermentative production of indirubin directly from glucose was demonstrated. </LI> </UL> </P>

Improved Optimization of Indirubin Production from Bioreactor Culture of Polygonum tinctorium

Chung, Choong Sik,Kim, Kyung Il,Bae, Geun Won,Lee, Youn Hyung,Lee, Hyong Joo,Chae, Young Am,Chung, In Sik 한국응용생명화학회 2000 Journal of Applied Biological Chemistry (J. Appl. Vol.43 No.2

Effect of the two-stage operation and cell concentration on indirubin production was investigated using bioreactor culture of Polygonum tinctorium. Two-stage culture was operated successfully for 110 days without any adverse effects on continuous indirubin production. Maximum indirubin concentration was found to be at 80 mg/bioreactor. Initial cell concentration significantly affected indirubin production. The indirubin production at 29.2% PCV was improved by 845%, compared to that at 5% PCV. For high-density bioreactor culture of P. tinctorium, a maximum production rate of 10.2 mg indirubin/L day was obtained. Indirubin recovery for bioreactor operation was also examined using XAD-2, XAD-4, XAD-7, and solid silicon. XAD-4 was 1.6-fold more effective than that for solid silicon in indirubin recovery.

Effects of indirubin derivatives on the FLT3 activity and growth of acute myeloid leukemia cell lines

Han, Sun-Young,Ahn, Jin Hee,Shin, Chan Young,Choi, Sang-Un Wiley Subscription Services, Inc., A Wiley Company 2010 Drug development research Vol.71 No.4

<P>Indirubin is an active constituent of traditional Chinese preparations used for the treatment of chronic myelocytic leukemia (CML). In the present study, the inhibitory activity of indirubin and its derivatives toward Fms-like tyrosine kinase 3 (FLT3) was examined. Indirubin-3'-oxime (10) and 6-bromoindirubin-3'-oxime (BIO) had potent inhibitory activity against FLT3 (IC(50) = 79 nM and 254 nM, respectively). We also tested the cytotoxicity of these compounds against acute myeloid leukemia cell lines: MV4;11 cells harboring a constitutively activated form of FLT3, and RS4;11 cells with wild-type FLT3. IO and BIO potently inhibited the growth of MV4;11 cells with IC(50) values of 30 nM and 61 nM, respectively. Conversely, RS4;11 cells were far less sensitive to these compounds. IO arrested the cell cycle of MV4;11 cells at the G(1) phase, and increased the dead cell population at the sub-G(1) phase and annexin V-positive cells. From these results, derivatives of IO may have potential to be developed as antileukemic agents. Drug Dev Res 71:221-227, 2010. (C) 2010 Wiley-Liss, Inc.</P>

신경모세포종 세포배양에서 글루탐산염에 의해유발된 흥분성세포독성에 대한Indirubin-3'-monoxime의 보호 효과

한승연,정보현,이희수,유기연 대한구강해부학회 2014 대한구강해부학회지 Vol.35 No.1

신경연접틈새로 분비되어 글루탐산염 수용체에 결합하는 글루탐산염(glumate)은포유동물의 주요 흥분성 신경전달물질로알려져 있다1). 하지만, 글루탐산염에 의해전달되는 신경전달의 과도한 흥분은 신경독성(neurotoxic)을 나타내거나 세포사멸(cell death)을 유발할 수 있다2). 흥분성아미노산들(excitatory amino acids)은허혈(ischemia), 머리외상(head trauma), 뇌졸중(stroke), 알츠하이머병(Alzeimer’s disease) 및 파킨슨씨병(Parkinson’s disease)과 같은 급성 뇌손상이나 만성신경퇴행성 질환들의 병태생리에 관련이 있는 것으로 알려져 있다1). 이전의 글루탐산염에 관한 연구들은 calcium-dependent enzyme들의활성화, nitric oxide synthase 및 미토콘드리아 활성산소종 생성과 같이신경세포사멸을 일으키는 글루탐산염의 신경독성 유발 기전을 제시하였다3-5). 여러 종류의 배양 신경세포들이 글루탐산염에 의해 발생하는 분자 수준의 현상을규명하기 위해 사용되고 있는데, 그 중 신경모세포종(SH-SY5Y) 세포는 그들의 형태학적, 생화학적 그리고 기능적 특징들이신경모세포(neuroblast)와 유사하여neuronlike cell로써 세포자멸사 및 신경세포보호연구를위한유용한도구로써이용되고있다6). 또한, 신경모세포종 세포주는 ionotropic 및 metabotropic glutamate receptor를모두 발현하기 때문에 N-methyl-D-aspartate glutamate receptor (NMDA receptor)의 활성화를 통해 세포사멸을유발하는 연구에 자주 사용된다7). 본 연구에서도 IMX의 신경세포 보호효과를 확인하기 위하여 신경모세포종 세포주를 이용하였다. 사멸 세포에서 글루탐산염은 NMDA receptor의 흥분과 caspase-3의 활성화를통해 농도에 의존적으로 세포자멸사(apoptosis) 및 세포괴사(necrosis)를 유발하는 경향이 있다8,9). 이때 글루탐산염 수용체의 과흥분은 주로 NMDA receptor를통해 지속적으로 세포 내로 Ca2+를 유입시켜, Ca2+과 관련된 하위 기전에 과부하를유발해 신경세포를 사멸에 이르게 한다10). Polygonum tinctorium, Isatis indigotica 및 Isatis tinctoria와 와 같은식물들로부터 추출되는 남색 염료의 자주색성분인 인디루빈(Indiru- bin)은 항염증및 항백혈병 활성을 가지는 중국 한의약 처방의 활성 성분으로 알려져 있다11). 다양한연구에서 인디루빈은 cycling-dependent protein kinase (CDKs)11), glycogen synthetase kinase 3 (GSK3)11,12), c-Src kinase 및 c-Jun NH2-terminal kinase13,14)와 같은 중요한 protein kinase들의 억제 능력을 보고한 바 있다. 인디루빈에 대한 연구는 인디루빈의 유도체들에 대한 연구까지 확장되어 있는데 여러연구들에서 5-iodoi-ndirubin-3'-monoxime, 5-5'-dibromo-indirubin, iodoindirubin- 3'-mono-xime-5-sulfonate 및indirubin-3'-monoxime (IMX) 등의 인디루빈 유도체들을 합성하였으며, 이들유도체 또한 Cdk5, GSK3β, 및 tau 인산화 등을 억제하는 것으로 알려져 있다11). 여러 인디루빈 유도체 중에서 IMX는폐암 및 후두암에서 항암효과가 보고된 바있으며, 대식세포에서 LPS에 의해 유도된염증반응을 억제하는 것으로 알려져 있다. 뿐만 아니라 마우스를 이용한 알츠하이머병 모델에서 IMX의 처리는 Aβ 침착, tau 과인산화, Aβ plaque주변 미세아교세포와 별아교세포의 활성화 누적 등의 현저한 감소를 보인다는 연구결과도 제시된바 있다15). 하지만 아직까지 인디루빈 유도체들에 대한 연구 중 중추신경계 질환과관련된 연구가 많지 않으며, 더군다나 대표적인 인디루빈 유도체인 IMX의 경우가장 설득력있는 신경세포 사멸 기전 중글루탐산염에 의한 흥분성신경독성에 의한신경세포...

Synthesis and Anti-osteoporosis Potential of Two New Indirubin-3`-oxime Derivatives

( Nguyen Manh Cuong ),( Bui Huu Tai ),( Dang Hoang Hoan ),( Pham Quoc Long ),( Eun Mi Choi ),( Young Ho Kim ) 한국응용생명화학회 2010 Applied Biological Chemistry (Appl Biol Chem) Vol.53 No.1

Two new indirubin-3`-oxime derivatives, indirubin-3`-[O-(3-bromoprop-1-yl)-oxime] (2) and indirubin-3`-[O-(methoxycarbonylmethyl)-oxime] (3) were synthesized. Their structures were confirmed by ESI-MS and NMR spectroscopic method. Both of them (5 μg/mL) significantly caused a elevation of cell growth, alkaline phosphate activity, and mineralization in osteoblastic MC3T3-E1 cells (p<0.05).

5-Nitro-5'-hydroxy-indirubin-3'-oxime (AGM130), an indirubin-3'-oxime derivative, inhibits tumor growth by inducing apoptosis against non-small cell lung cancer in vitro and in vivo

Ahn, M.Y.,Kim, T.H.,Kwon, S.M.,Yoon, H.E.,Kim, H.S.,Kim, J.I.,Kim, Y.C.,Kang, K.W.,Ahn, S.G.,Yoon, J.H. Elsevier 2015 European journal of pharmaceutical sciences Vol.79 No.-

This study examined the anti-tumor effects of AGM130, a novel indirubin-3'-oxime derivative in A549 human non-small cell lung cancer cells. AGM130 significantly inhibited the proliferation and arrested the cell cycle of G2/M phase. Induction of apoptosis was detected in AGM130-treated A549 cells. The protein levels of Cytochrome c release, Bax, cleaved caspases and PARP were increased in AGM130 treated cells, whereas Bcl-2 levels were decreased. AGM130 inhibited Insulin-like growth factor 1 receptor (IGF1R), AKT/mTOR signaling and inactivated mitogen-activated protein kinases (MAPK). AGM130 also induced slight autophagy as pro-survival function and autophagy inhibition by chloroquine (CQ) induced necrosis. In vivo tumor xenograft model, AGM130 dose-dependently suppressed transplanted A549 cell tumor growth and induced the expression of proliferative cell nuclear antigen (PCNA). AGM130 also increased TUNEL positive apoptotic cell populations and the induction of glandular differentiation with mucin pool compared with vehicle-treated control in tumor tissue. These results suggest that AGM130 is an effective novel indirubin-3'-oxime derivative of anti-cancer drug and may be an attractive candidate for non-small cell lung cancer therapy.

Indirubin-3-monoxime Prevents Tumorigenesis in Breast Cancer through Inhibition of JNK1 Activity

Mi-Yeon Kim,Eun-Hye Jo,Yong-Chul Kim,Hee-Sae Park 대한의생명과학회 2021 Biomedical Science Letters Vol.27 No.3

c-Jun N-terminal kinases (JNKs) have a Janus face, regulating both cell apoptosis and survival. The present study focused on understanding the function of JNK in tumor development and the chemoresistance underlying JNK-mediated cancer cell survival. We identified an inhibitor of JNK1, an important regulator of cancer cell survival. Kinase assay data showed that JNK1-dependent c-Jun phosphorylation was inhibited by indirubin derivatives. In particular, indirubin-3-monoxime (I3M) directly inhibited the phosphorylation of c-Jun in vitro, with a half inhibition dose (IC50) of 10 nM. I3M had a significant inhibitory effect on JNK1 activity. Furthermore, we carried out assays to determine the viability, migration, and proliferation of breast cancer cells. Our results demonstrated that cell growth, scratched wound healing, and colony forming abilities were inhibited by the JNK inhibitor SP600125 and I3M. The combination of SP600125 and I3M significantly decreased cancer cell proliferation, compared with either SP600125 or I3M alone. Our studies may provide further support for JNK1-targeting cancer therapy using the indirubin derivative I3M in breast cancer.

Anti-inflammatory Effect of Indirubin-3'-Monoxime-5-Sulphonic Acid on Lipopolysaccharide-stimulated Murine Macrophage

Gang Baek Park,Hyun Jin Kim,Hye Seon Heo,Geun-Mook Park,Kyung Woo Park,Jin-Kyung Kim 대한의생명과학회 2011 Biomedical Science Letters Vol.17 No.3

Indirubin is the active ingredient of Danggui Longhui Wan, a mixture of plants that is used in traditional Chinese medicine to treat chronic diseases. In this study we investigated the anti-inflammatory effects of an indirubin derivative, indirubin-3'-monoxime-5-sulphonic acid (I3M-5S, C₁₆H₁₁N₃O₅S). We found that I3M-5S inhibits the production of various inflammatory mediators such as nitric oxide (NO) and prostaglandin E₂ (PGE₂) as well as inflammatory cytokines, tumor necrosis factor-α and interleukin-6 in lipopolysaccharide (LPS) stimulated murine macrophage, RAW264.7 cells. In addition, the expression of inducible nitric oxide synthase and cyclooxygenase-2, which are essential enzymes to produce NO and PGE₂, respectively, was blocked by I3M-5S treatment in LPS-stimulated RAW264.7 cells. Present data suggest that I3M-5S exhibits potent anti-inflammatory activity in cultured macrophages and merit further study as potential therapeutic agents for inflammatory disorders.

Synthesis and Anti-osteoporosis Potential of Two New Indirubin-3'-oxime Derivatives

Cuong, Nguyen Manh,Tai, Bui Huu,Hoan, Dang Hoang,Long, Pham Quoc,Choi, Eun-Mi,Kim, Young-Ho The Korean Society for Applied Biological Chemistr 2010 Applied Biological Chemistry (Appl Biol Chem) Vol.53 No.1

Two new indirubin-3'-oxime derivatives, indirubin-3'-[O-(3-bromoprop-1-yl)-oxime] (2) and indirubin-3'-[O-(methoxycarbonylmethyl)-oxime] (3) were synthesized. Their structures were confirmed by ESI-MS and NMR spectroscopic method. Both of them (5 ${\mu}g/mL$) significantly caused a elevation of cell growth, alkaline phosphate activity, and mineralization in osteoblastic MC3T3-E1 cells(p<0.05).