CC BY
12
16
оригинальные статьи
КЙ1аши1Й111Яид11пЙ11и11иКВи11Н1
doi: 10.24060/2076-3093-2017-7-4-16-20
molecular design of sugar-free migracin analog migracinal that inhibits ovarian cancer cell migration and invasion
Tamami Ukaji1, Naoki Koide1, Yinzhi Lin2, Kouji Banno3, Shamil Cantsev4, Kazuo Umezawa2
1 Department of Microbiology and Immunology, Aichi Medical University School of Medicine, Nagakute 480-1195, Japan
2 Department of Molecular Target Medicine, Aichi Medical University School of Medicine, Nagakute 480-1195, Japan
3 Department of Obstetrics and Gynecology, Keio University School of Medicine, Tokyo, 160-8582, Japan
4 Scientific Research Institute of Oncology, Bashkortostan State Medical University, Lenin Street 3, 450000 Ufa, Russian Federation
Tamami Ukaji - Department of Microbiology and Immunology, Aichi
Medical University School of Medicine, Nagakute
Naoki Koide - Department of Microbiology and Immunology, Aichi
Medical University School of Medicine, Nagakute
Yinzhi Lin - Department of Molecular Target Medicine, Aichi Medical
University School of Medicine, Nagakute
Kouji Banno - Department of Obstetrics and Gynecology, Keio
University School of Medicine, Tokyo,
orcid.org/0000-0003-2610-2833
Gantsev Shamil - Doctor of Medical Sciences, Professor, Chair of the Oncology Department with course of Oncology and Pathological Anatomy of Additional Professional Education Institution of Federal State Budgetary Educational Institution "Bashkir State Medical University" the Ministry of Health, the Russian Federation, Ufa, Russian Federation, orcid.org/0000-0003-2047-963X, e-mail: kafonco@mail.ru, tel.: +7 (347) 248-32-07
Kazuo Umezawa - Department of Molecular Target Medicine, Aichi
Medical University School of Medicine, Nagakute
Contacts: Kazuo Umezawa, E-mail: umezawa@aichi-med-u.ac.jp
Introduction. Cancer metastasis consists of several steps including detachment from the primary tumor, migration, invasion, transport in the blood or lymphatic vessels, attachment at the secondary site, and growth of secondary tumor. Migration and invasion areinvolved in the mechanism of all types of cancer metastasis. We previously isolated novel cellular migration inhibitor migracin A and B from a culture filtrate of Streptomyces sp. Migracin A was shown to inhibit IGF-1-mediated cellular migration and invasion in ovarian carcinoma cells. However, it is difficult to prepare large amount of migracin A. Migracin A consists of substituted benzene and an alkylated sugar moiety. In the present research, we have designed and synthesized a simplified dialdehydederivative of migracin called migracinal having no sugar moiety.
Material and methods. Migracinal was purchased from Techno Chem Co., Ltd., Tokyo, Japan. Migracinal was prepared from 2,4-dihydroxybenzaldehyde (2,4-DHBA). The structure was confirmed by proton and carbon NMR spectra and ESI mass spectroscopy. The antitumor activity of the new derivative was studied by standard tests under conditions in vitro.
Results. Migracinal inhibited cellular migration and invasion in ovarian clear cell carcinoma ES-2 cells. It also inhibited IGF-1 expression as migracin A. Moreover, it induced anoikis rather than apoptosis in ES-2 cells.
Conclusions. Migracinal is easier to prepare than migracins, and it may be useful for the mechanistic study and suppression of metastasis.
Keywords: Migracinal, Migracin, Migration, Invasion, Anoikis, Ovarian clear cell carcinoma
For citation: Ukaji T., Koide N., Lin Y, Banno K., Gantsev Sh, Umezawa K. Molecular design of sugar-free migracin analog migracinal that inhibits ovarian cancer cell migration and invasion. Creative surgery and oncology. 2017;7(4):16-20. DOI:10.24060/2076-3093-2017-7-4-16-20.
cr»eiii№eisiir.№iKBiimaliffiCiliii
оригинальные статьи
17
молекулярный дизайн не содержащего углеводного компонента аналога миграцина, препятствующего миграции и инвазии клеток рака яичников
Тамами Укаджи1, Иаоки Коиде1, йинжи Лин2, коуджи Банно3, Шамиль Ганцев4, кадзуо Умезава2
1 Кафедра микробиологии и иммунологии, Медицинский университет Айти, Нагакуте 480-1195, Япония
2 Кафедра целевой молекулярной медицины, Медицинский университет Айти, Нагакуте 480-1195, Япония
3 Отделение акушерства и гинекологии, Медицинский университет Кеио, Токио, 160-8582, Япония
4 Научно-исследовательский институт онкологии, Башкирский государственный медицинский университет, Россия, 450008, Уфа, ул. Ленина 3
Тамами Укаджи - кафедра микробиологии и иммунологии,
Медицинский университет Айти, Япония
Наоки Коиде - кафедра микробиологии и иммунологии,
Медицинский университет Айти, Япония
Йинжи Лин - кафедра целевой молекулярной медицины,
Медицинский университет Айти, Япония
Коуджи Банно - отделение акушерства и гинекологии,
Медицинский университет Кеио, Токио, Япония,
orcid.org/0000-0003-2610-2833
Шамиль Ганцев - д.м.н, профессор, заведующий
кафедрой онкологии с курсами онкологии и патологической
анатомии ИДПО ФГБОУ ВО БГМУ Минздрава России,
Уфа, Россия, orcid.org/0000-0003-2047-963X, e-mail: kafonco@mail.ru,
тел.: +7 (347) 248-32-07
Кадзуо Умезава - кафедра целевой молекулярной медицины, Медицинский университет Айти, Япония, e-mail: umezawa@aichi-med-u.ac.jp
Контакты: Кадзуо Умезава, e-mail: umezawa@aichi-med-u.ac.jp
Введение. Процесс метастазирования рака состоит из нескольких этапов: отсоединение клеток от первичной опухоли, миграцию, инвазию, перемещение в крови или лимфатических, сосудах, присоединение и рост вторичной опухоли. Механизмы миграции и инвазии универсальны для всех видов рака. Ранее из культуры Streptomyces SP мы выделили Migracin А и В - новые ингибиторы клеточной миграции. Было продемонстрировано как Migracin А ингибирует IGF-1-опосредованную миграцию и инвазию клеток рака яичников. Однако большое количество Migracin А, состоящего из замещенного бензола и алкилированного углеводного фрагмента, синтезировать трудоемко. В настоящем исследовании мы разработали и синтезировали упрощенное производное диальдегида Migracin, не имеющего углеводного компонента, названное Migracinal.
Материалы и методы. Migracin приобретался у компании «ТехноХим Co., Лтд» (Токио, Япония). Производное Migracinal получали взаимодействием Migracin с 2,4-дигидроксибензалдегидом. Структура была подтверждена спектрами ЯМР и масс-спектроскопией. Противоопухолевая активность нового производного изучалась стандартными тестами в условиях, in vitro.
Результаты. Установлено, что Migracinal ингибирует клеточную миграцию и инвазию клеток ES-2 рака яичника и аналогично Migracin A ингибирует IGF-1 экспрессию. Кроме того, он индуцировал аноикис, а не апоптоз в клетках. ES-2.
Заключение. Синтез Migracinal легче в сравнении с Migracin, а спектр противоопухолевой активности идентичен, что может быть использовано для подавления процессов метастазирования.
Ключевые слова: Migracinal, Migracin, миграции, инвазия, аноикис, рак яичника
Для цитирования: Укаджи Т., Коиде Н., Лин Й., Банно К., Ганцев Ш., Умезава К. Молекулярный дизайн не содержащего углеводного компонента аналога миграцина, препятствующего миграции и инвазии клеток рака яичников. Креативная хирургия и онкология. 2017;7(4):16-20. D0I:10.24060/2076-3093-2017-7-4-16-20.
18
оригинальные статьи
КЙ1аши1Й111Яид11пЙ11и11иКВи11Н1
introduction
Cancer metastasis consists of several steps including detachment from the primary tumor, migration, invasion, transport in the blood or lymphatic vessels, attachment at the secondary site, and growth of secondary tumor. Migration and invasion areinvolved in the mechanism of all types of cancer metastasis. Therefore, we looked for cellular migration inhibitors of low molecular weight from microbial culture filtrates. As a result, we discovered novel compounds, migracin A and B, from the culture filtrate of Streptomyces sp [1]. Migracin A and B are closely related in structure (Fig. lA), and show similar inhibitory activities. Migracin A (Fig. 1) and B inhibited migration of breast carcinoma MDA-MB-231, non-small cell lung carcinoma A549, and fibrosarcoma HT1080 cells without any toxicity. Migracin A and B showed similar activity.
CHO
I"'H I - iO
Migracin A Migracinal
Figure 1 - Molecular design of migracinal based on the structure of migracin A.
In one hand, ovarian cancer is the most common cause of gynecologic disease-related death. Ovarian carcinomas often metastasize to neighboring organs such as lung, liver and the peritoneal cavity through direct extension, migration, invasion, and lymphatic vessel transport [2,3]. Moreover, clear cell ovarian tumors are part of the epithelial tumor group of ovarian cancers, and these tumors may have a worse prognosis than other epithelial type-ovarian tumors [4]. Therefore, effective metastasis inhibitors with low toxicity should be useful for therapy against ovarian clear cell cancer.Later we have reported that migracin A inhibits migration and invasion of ovarian clear cell carcinoma ES-2 cells via inhibition of IGF-1 expression [5]. It inhibited the cellular Akt activity that is downstream signaling of IGF-1/IGF-1 receptor. However, migracin A did not inhibit colony formation of ES-2 cells. Although migracin A may become a candidate of anti-metastasis agent, yield by the producing organism is limited. Also chemical synthesis of migracin A appears not easy, since the structure is complicated. Therefore, we looked for a simpler derivative of migracin A. In the present research, we designed a sugar-free analog of migracin A. We named the synthesized compoundmigracinal (Fig. 1), since a new aldehyde moiety was added in the structure. We have also evaluated the cellular anti-metastasis and anoikis-inducing activities of this compound.
materials and methods
Migracinal. Migracinal was purchased from Techno Chem Co., Ltd., Tokyo, Japan. Migracinal was prepared
from 2,4-dihydroxybenzaldehyde (2,4-DHBA). The synthetic procedure of migracinal will be reported later. It was obtained as a brown oily residue. The structure was confirmed by proton and carbon NMR spectra and ESI mass spectroscopy.
Cell culture:
Human ovarian clear cell carcinoma ES-2 cells (ATCC, Manassas, VA) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and penicillin/ streptomycin at 37°C in a humidified incubator with 5% CO2.
Wound healing assay:
Cells in 24-well plates were allowed to reach confluence before the surface was uniformly scratched across the center of each well with a pipette tip. The wells were then rinsed twice with serum-free media to remove floating cells and growth media, after which the cells were cultured in serum-free media for 24 h. The initial wounded area and movement of the cells into the scratched area were recorded. Experiments were performed in triplicate in three independent experiments.
Cell invasion assay:
ES-2 cells were suspended in 500 l of serumfree medium containing migracin A or the DMSO and seeded into the upper chambers coated with BD Matrigel Basement Membrane Matrix (Corning Inc., Corning, NY). The lower chambers were filled with 750 pl of medium containing 10% FBS and incubated for 24 h at 37°C in a humidified CO2 incubator. Then, after fixation of theinvading cells, non-invading cells remaining on the upper surface were removed by wiping with a cotton swab. Invading cells attached to the underside were stained with Diff-Quick solution (Sysmex, Kobe, Japan), and counted.
Soft agar colony formation:
A soft agar colony formation assay was employed according to the manufacturer's instructions using CytoSelect 96-well Cell Transformation Assay kit (Cell Biolabs, San Diego, CA). Each well contained 50 pl of 0.6% agar in a complete medium as the bottom and feeder layer, and 75 l of 0.4% agar in a complete medium with 1 104 cells as the top layer. After 6 days colonies were lysed with lysis buffer and detected with CyQuant GR dye for the quantification of anchorage-independent growth andthe fluorescence was measured with a fluorometer using the 485/520 filter set SpectraMax M5 (Molecular Devices, Sunnyvale, CA).
Anoikis:
Cells death by anoikis were identified and quantified using the CytoSelect 24-well Anoikis Assay Kit (Cell Biolabs) according to the manufacturer's instructions. For the determination of anoikis, ovarian clear cell carcinoma ES-2 cells were cultured in serum-starved medium in normal or poly (2-hydroxyethyl methacrylate) (poly-HEMA)-coated 24-well plates. Anoikis-induced cell death was measured by ethidium homodimer (EthD-1) [6].
oiou
оригинальные статьи
results
Molecular design of migracinal: Migracin A has a similar structure of luminacin C. In case of luminacin C, a sugar-free dialdehydederivative was synthesized, and it showed the inhibitory activity on HUVEC tube formation [7]. Therefore, we designed the structure of migracinalas the luminacin analog having ethoxy moiety instead of methoxymoiety. Thus, migracinal(2,4-dihydroxy-5-(1-ethoxy-2-
methylpropyl)benzene-1,2-dicarbaldehyde) contains the ethoxy moiety instead of the methoxy moiety in theluminacin C derivative [7].
Inhibition of cellular migration and invasion: Migracinal did not decrease the viability of ES-2 cells below 10 g/ml (Fig. 2A). In one hand, it inhibited the cellular migration monitored by wound healing assay at 10 g/ml (Fig. 2B). Next, it inhibited the cellular invasion at 1-10 g/ml more effectively than migration in the Matrigel chamber assay (Fig. 2C). Previously, migracin A was found to lower the IGF-1 expression by the PCR array analysis to inhibit migration and invasion. Migracinal also inhibited the expression of IGF-1, as shown in Fig. 2D. It also inhibited the downstream signaling Akt phosphorylation.
10
10
Migracinal (цд/ml) IGF-1 p-actin
Migracinal (цд/ml) p-Akt Akt
Figure 2 - Inhibition of cellular migration and invasion by migracinal in ES-2 cells. (A)
Effect on viability. The cells were incubated for 24 h. (B) Inhibition of cellular migration. The cells were
incubated for 24 h, and the cellular migration was measured by wound healing assay. (C) Inhibition of cellular invasion. The cells were incubated for 24 h, and the cellular invasion was measured by Matrigel chamber assay. (D) Inhibition of IGF-1 expression and Akt phosphorylation. The cells were incubated for 24 h, and the expression was measured by PCR (IGF-1) or Western blotting (phosphorylated Akt). Statistical evaluation, *P<0.05, ** P<0.01.
Inhibition of Anoikis:
Inhibition of anoikis is important to inhibit cancer metastasis. Migracinal induced anoikis at 3-10 g/ml, as shown in Fig. 3. It did not induce apoptosis at the same concentrations.
□ Normal ■ poly-HEMA
□
о
Migracinal (цд/ml)
Figure 3 - Induction of anoikis. The cells were treated with migracinal for 24 h without or with poly-HEMA coating, and the cell death was measured as described in the Materials and methods. * P<0.05, ** P<0.01.
Inhibition of colony formation in soft agar: In one hand, migracin A did not inhibit colony formation of ES-2 cells in soft agar even at 100 g/ ml [5]. Interestingly, migracinal inhibited the colony formation effectively at 30-100 g/ml, as shown in Fig. 4. Then, migracinal may have cytostatic activity in addition to the anti-metastatic activity.
Figure 4 - Effect on growth in soft agar. The cells were incubated in soft agar for 6 days. Doxorubicin is a positive control. * P<0.01, ** P<0.001.
19
20
оригинальные статьи
КЙ1аши1Й111Яид11пЙ11и11иКВи11Н1
discussion
The structure of migracin A is closely related to that of luminacin C. Luminacin C was isolated from Streptomyces sp. as an inhibitor of capillary tube formation in human umbilical vein endothelial cells (HUVEC) [8, 9]. Since a sugar-free derivative of luminacin C did not lose the biological activity [7], we also prepared the similar structure based on migracin A.
Anoikis is the subset of apoptosis triggered by inadequate cell-matrix contacts [10]. Anoikis is essential for the regulation of tissue homeostasis in tissue remodeling, development, and especially tumor metastasis, so that the acquisition of anoikisresistance is considered to be important for achieving the successful metastasis for cancer cells. Because cancer cells should survive without binding to matrix during the metastasis. Therefore, anoikis-inducing agents may become new anti-metastasis agents. Migracinal inhibited the IGF-1 expression, as shown in Fig. 2 D. It has been reported that IGF-1 and the downstream Akt activity are essential to prevent anoikis in breast cancer cells [11].
In the present study, we employed the mixture of migracinaldiastereomers. We are planning to prepare stereo-specific migracinal for the further studies. Migracinal has simpler structure compared with migracin A, and can be prepared more easily. It may become a seed of anti-metastasis agents.
acknowledgments
The authors wish to thank Mr. Sadao Takehara and Mr. Junpei Takahashi, TechnoChem Co., Ltd., Tokyo, for the synthesis of migracinal. This work was supported in part by JSPS KAKENHI Grant Number 26350975 and 17K01967 from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. This work was also supported in part by MEXT-Supported Program for the Strategic Research Foundation at Private Universities, which is for Aichi Medical University 2011-2015 (S1101027).
Conflict of interest. The authors declare no conflict of interest.
references
1. Arai Y., Iinuma H., Ikeda Y., Igarashi M., Hatano M., Kinoshita N., et al. Migracins A and B new inhibitors of cancer cell migration, produced by Streptomyces so. J Antibiot (Tokyo). 2013;66(4):225-30. DOI: 10.1038/ ja.2012.112.
2. Naora H., Montell D.J. Ovarian cancer metastasis: integrating insights from disparate model organisms. Nat Rev Cancer. 2005;5(5):355-366. DOI: 10.1038/ nrc1611.
3. Kube ek O., Laco J., pa ek J., Petera J., Kopeck J., Kube kov A., et al. The pathogenesis, diagnosis, and management of metastatic tumors to the ovary: a comprehensive review. Clin Exp Metastasis. 2017, Jul 20. DOI: 10.1007/s10585-017-9856-8.
4. Chan J.K., Teoh D., Hu J.M., Shin J.Y., Osann K., Kapp D.S. Do clear cell ovarian carcinomas have poorer prognosis compared to other epithelial cell types? A study of 1411 clear cell ovarian cancers. Gynecol Oncol. 2008;109(3): 370-376. DOI: 10.1016/j. ygyno.2008.02.006.
5. Ukaji T., Lin Y., Banno K., Okada S., Umezawa K. Inhibition of IGF-1-mediated cellular migration and invasion by Migracin A in ovarian clear cell carcinoma cells. PLOS ONE. 2015;10(9):e0137663. DOI: 10.1371/ journal.pone.0137663.
6. Lee H.W., Joo K.M., Lim J.E., Cho H.J., Cho H.J., Park M.C., et al. Tpl2 kinase impacts tumor growth and metastasis of clear cell renal cell carcinoma. Mol Cancer Res. 2013;11(11):1375-86. DOI: 10.1158/1541-7786. MCR-13-0101-T.
7.Oneyama C., Agatsuma T., Kanda Y., Nakano H., Sharma S.V., Nakano S., et al. Synthetic inhibitors of proline-rich ligand-mediated protein-protein interaction: potent analogs of UCS15A. Chem Biol. 2003;10(5):443-51. PMID: 12770826.
8. Naruse N., Kageyama-Kawase R., Funahashi Y., Wakabayashi T. Luminacins: a family of capillary tube formation inhibitors from Streptomyces sp. I. Taxomony, fermentation, isolation, physico-chemical properties and structure elucidation. J Antibiot. 2000;53(6):579-590. PMID: 10966073.
9. Wakabayashi T., Kageyama-Kawase R., Naruse N., Funahashi Y., Yoshimatsu K. Luminacins: a family of capillary tube formation inhibitors from Streptomyces sp. II. Biological activities. J Antibiot. 2000;53(6):591-596. PMID: 10966074.
10.Grossmann J. Molecular mechanisms of «detachment-induced apoptosis--Anoikis». Apoptosis. 2002;7(3):247-60. PMID: 11997669.
11. Luey B.C., May F.E. Insulin-like growth factors are essential to prevent anoikis in oestrogen-responsive breast cancer cells: importance of the type I IGF receptor and PI3-kinase/Akt pathway. Mol Cancer. 2016;15:8. DOI:10.1186/s12943-015-0482-2.
