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THE NEW HETEROCYCLIC SCHIFF BASES AND THEIR COPPER COMPLEXES INDUCE MODIFICATIONS IN THE ERYTHROCYTE GLUTATHIONE SYSTEM

Research article
Sardari V.
Gulea A.
Tsapkov V.
Andronache L.
Graur V.
Shvets I.
Andronic L.
Issue: № 12 (114), 2021
Published:
2021/12/17
PDF

НОВЫЕ ГЕТЕРОЦИКЛИЧЕСКИЕ ШИФФОВЫЕ ОСНОВАНИЯ И ИХ МЕДНЫЕ КОМПЛЕКСЫ ВЫЗЫВАЮТ ИЗМЕНЕНИЯ В ГЛЮТАТИОНОВОЙ СИСТЕМЕ ЭРИТРОЦИТОВ

Научная статья

Пантя В.1, *, Сардарь В.2, Гуля А.3, Цапков В.4, Андронаке Л.5, Граур В.6, Швец И.7, Aндроник Л.8, Гудумак В.9

1 ORCID: 0000-0002-8835-6612;

2 ORCID: 0000-0002-1047-9145;

3 ORCID: 0000-0003-2010-7959;

4 ORCID 0000-0003-1732-3116;

5 ORCID: 0000-0002-8781-8037;

6 ORCID: 0000-0001-8153-2153;

7 ORCID: 0000-0002-7662-0973;

8 ORCID: 0000-0001-9494-4093;

9 ORCID: 0000-0001-9773-1878;

1, 2, 5, 7, 8 Государственный университет медицины и фармации им. Н. Тестемицану, Кишинёв, Молдова;

3, 4, 6 Молдавский Государственный Университет, Кишинёв, Молдова.

* Корреспондирующий автор (pantea.valeriana[at]usmf.md)

Аннотация

Изучены эффекты новых координационных соединений, производных тиосемикарбазида (кодовые соединения с химическим названием: (CMD-4) - 4-этил-2-[фенил(пиридин-2-ил)метилиден]гидразин-1-карботиоамид; (CMD-8)- Хлоро-{4-этил-2-[фенил (пиридин-2-ил) метилиден]гидразин-1-карботио амидо}медь; (CMJ-23) - 4-(3-метоксифенил)-2-[1-(пиридин-2-ил)этилиден]-гидразин-1-карботиоамид; и (CMJ-33) - Хлоро-{4-(3-метоксифенил)-2-[1-(пиридин-2-ил)этилиден]гидразин-1-карботиоамидо}медь) на основные показатели метаболизма глутатиона в эритроцитах - общий глутатион (tGSH), глутатионпероксидазу (GPO), глутатионредуктазу (GR) и ключевого фермента пентозофосфатного шунта - глюкозо-6-фосфатдегидрогеназу (G-6-PDH) при их инкубации с периферической кровью здоровых доноров.

Зарегистрированы выраженные нарушения в цикле глутатиона, которые проявлялись снижением уровня tGSH в эритроцитах под влиянием CMJ-33 и CMJ-23, снижением уровня GPO под влиянием CMD-4 и CMJ-23, снижением функционального уровня G-6-PDH под влиянием всех изученных соединений (CMD-4, CMD-8, CMJ-33 и CMJ-23), а также тенденцией к повышению активности GR. Обсуждается патогенетическое значение выявленных изменений и их возможное значение для разработки новых стратегий лечения заболеваний, связанных с нарушениями глутатионного цикла в эритроцитах.

Ключевые слова: координационные соединения меди, производные тиосемикарбазида, глутатионовая система эритроцитов.

THE NEW HETEROCYCLIC SCHIFF BASES AND THEIR COPPER COMPLEXES INDUCE MODIFICATIONS IN THE ERYTHROCYTE GLUTATHIONE SYSTEM

Research article

Pantea V.1, *, Sardari V.2, Gulea A.3, Tsapkov V.4, Andronache L.5, Graur V.6, Shvets I.7, Andronic L.8, Gudumac V.9

1 ORCID: 0000-0002-8835-6612;

2 ORCID: 0000-0002-1047-9145;

3 ORCID: 0000-0003-2010-7959;

4 ORCID 0000-0003-1732-3116;

5 ORCID: 0000-0002-8781-8037;

6 ORCID: 0000-0001-8153-2153;

7 ORCID: 0000-0002-7662-0973;

8 ORCID: 0000-0001-9494-4093;

9 ORCID: 0000-0001-9773-1878;

1, 2, 5, 7, 8 State University of Medicine and Pharmacy “Nicolae Testemitanu”, Chisinau, Republic of Moldova;

3, 4, 6 Moldavian State University, Chisinau, Republic of Moldova

* Corresponding author (valeriana.pantea[at]usmf.md)

Abstract

The effects of new coordination compounds, thiosemicarbazide derivatives (coded compounds with a chemical name: (CMD-4) - 4-ethyl-2- [phenyl (pyridin-2-yl) methylidene] hydrazine-1-carbothioamide]; (CMD-8) - Chloro- {4-ethyl-2- [phenyl (pyridin-2-yl) methylidene] hydrazine-1-carbothioamido} copper]; (CMJ-23) - 4- (3-methoxyphenyl) -2- [1- (pyridin-2-yl) ethylidene] hydrazine-1-carbothioamide] and (CMJ-33) - Chloro- {4- (3-methoxyphenyl) -2- [1- (pyridin-2-yl) ethylidene] hydrazine-1-carbothioamido} copper], on the main indices of erythrocytes glutathione metabolism - total glutathione (tGSH), glutathione peroxidase (GPO), glutathione reductase (GR) and the key enzyme of the pentose phosphate pathway - glucose-6-phosphate dehydrogenase (G-6-PDH) after incubation with the peripheral blood of healthy donors were studied. The significant disturbances in the erythrocyte glutathione cycle expressed by decreasing of the tGSH level by CMJ-33 and CMJ-23, diminushing of GPO by CMD-4 and CMJ-23, reduction of the functional level of G-6-PDH by the all studied compounds (CMD-4, CMD-8, CMJ-33 and CMJ-23), associated with increased tendency of GR activity were registered. The pathogenetic significance of the revealed changes and their possible importance for the development of new treatment strategies for diseases associated with disorders of the glutathione cycle in erythrocytes are discussed.

Keywords: copper coordination compounds, thiosemicarbazide derivatives, erythrocyte glutathione cycle.

Introduction

Glutathione and glutathione cycle enzymes have various and very important functions in the body: reduce and isomerize disulfide bonds, effectively protect the body from oxidative stress, maintain the functionality of membranes, fortify the resistance of cells to harmful actions, stimulate the activity of enzymes and other proteins, tissue proliferation and biosynthesis of nucleic acids, participate in the metabolism of eucosanoids and xenobiotics, influence gene expression, cell proliferation and apoptosis, signal transduction, immune response and protein glutathionylation [1], [2], [6], [7].

This explains the constant interest of scientists in the exploring of various aspects of thiol-disulfide metabolism [8], [9], [10], [13].

In the degradation of hydroperoxides, which are formed in the process of lipid peroxidation, the main role is played by the enzyme system - glutathione peroxidase (GPO) - glutathione reductase (GR). In the presence of glutathione, cytoplasmic GPO catalyzes the transformation of hydroperoxides into hydroxy acids: R-OOH + 2 G-SH ® R-OH + G-S-S-G, which contribute to the suppression of their toxic action on the cell membranes and prevent the initiation of lipid oxidation side reactions. Oxidized glutathione, formed in the reaction process, catalyzed by GPO, is reduced in the glutathione reductase reaction: 2 NADPH + G-S-S-G ® 2 NADP + 2 G-SH [14], [15], [16].

 The pentose phosphate pathway is distinguished by its important contribution in providing cells with NADPH, necessary for reductive biosynthetic reactions, including in the regeneration of reduced glutathione (GSH) [16]. The cardinal enzyme of the pentose phosphate pathway - glucose-6-phosphate dehydrogenase (G-6-PDH) catalyzes the transformation of glucose-6-phosphate into 6-phosphogluconolactone. The crucial role of this enzyme in the metabolism of various tissues and organs (erythrocytes, adrenal glands, adipose tissue, liver, etc.) is well known.

 A special attention is attributed to the bioactive new copper coordination compounds (CCC), thiosemicarbazide derivatives, obtained by the directed modification of some natural compounds in the Laboratory of advanced materials in biopharmaceutics and techniques, State University of Moldova [17,18,19,20], which show strong antitumor properties, but so far there is no in-depth and detailed research of their action on the erythrocyte glutathione system when testing under physiological conditions.

The aim of the study was to investigate the action mechanisms of copper coordination compounds, thiosemicarbazide derivatives (coded compounds - CMD-4, CMD-8, CMJ-23 and CMJ-33) on glutathione metabolism in the erythrocytes of practically healthy volunteer donors. 

Material and methods

In order to evaluate the particularities of the new CCC action, a series of in vitro experiments were performed on the peripheral blood samples collected from 8 practically healthy donors.

The blood was collected in the morning, by puncturing the ulnar vein, in an amount of 5 ml. Under sterile conditions, blood was placed in a vial containing 20 ml Dulbecco's modified Eagle nutrient medium (DMEM), heparin (2.5 un/ml), gentamicin (100 μg/ ml) and L-glutamine (0.6 mg/ml). To study the influence of new CCC on the erythrocyte glutathione system, 0.9 ml of this mixture was pipetted into the wells of the 24-well plate. In the first two parallel wells, to establish the initial level of the studied indices, 0.1 ml of 0.9% NaCl solution was poured. In the other wells were added the tested compounds (coded CMD-4; CMD-8; CMJ-23; CMJ-33) diluted in 0,1 ml of 0,9% NaCl, so that the final concentration was 0.05 µg/ml (0,05 mg/l): All dilutions were tested in duplicate.

Two new heterocyclic Schiff bases (CMD-4, and CMJ-23), and their copper (Cu2+) complexes (CMD-8, and CMJ-33) were used in the experiments. The names of the coded chemicals are prezented below in the table 1:

 

Table 1 – The names of the coded chemicals

Code Chemical name of the substance
CMD-4 4-ethyl-2- [phenyl (pyridin-2-yl) methylidene] hydrazine-1-carbothioamide
CMD-8 Chloro- {4-ethyl-2- [phenyl (pyridin-2-yl) methylidene] hydrazine-1-carbothioamido} copper
CMJ-23 4- (3-methoxyphenyl) -2- [1- (pyridin-2-yl) ethylidene] hydrazine-1-carbothioamide
CMJ-33 Chloro- {4- (3-methoxyphenyl) -2- [1- (pyridin-2-yl) ethylidene] hydrazine-1-carbothioamido} copper
 

After incubation in the CO2 incubator at 3.5% CO2 and 37°C for 24 h, the contents of the plates were carefully transferred into Eppendorf tubes with a volume of 2 ml; the samples were centrifuged for 5 min at 3000 rpm. Blood cells (erythrocytes) were washed 3 times with 0.9% NaCl, then hemolyzed with 1.0 ml of distilled H2O. In the obtained erythrocyte lysates, the following indices were evaluated by spectrophotometric micromethods adapted for Synergy H1 Hybrid Rider (BioTek Instruments, USA): total glutathione content (tGSH), activity of glutathione reductase (GR), glutathione peroxidase (GPO), as well as of glucose-6-phosphate dehydrogenase (G-6-PDH) [21], [22].

Data were subjected to statistical analysis in StatsDirect Statistical Software (version 1.9.5., 2001). The statistical analysis included calculation of mean and arithmetic mean error (M±m), U Mann-Whitney test (comparing iniţial level and the influence of tested compounds). The value equal or less than 0.05 was considered statistically significant.

The protocol of this study was approved by the Research Ethics Committee of the “Nicolae Testemitanu” State University of Medicine and Pharmacy (nr.81 of 19.09.2020). Eight conditionally healthy volunteer donors between the ages of 28 and 32 participated in the study. All participants gave their informed consent, both orally and in writing, in accordance with the principles of the Helsinki Treaty adopted in June 1964 with subsequent revisions and additions.

Results

The evaluation results of the glutathione system indices: tGSH, GR, GPO and G-6-PDH in the erythrocytes of practically healthy donors after treatment with new CCC are presented in the statistics of table 2.

 

Table 2 – Influence of CMD-4, CMD-8, CMJ-33 and CMJ-23 compounds on erythrocyte glutathione system in practically healthy donors

Nr. Study groups tGSH GPO GR G-6-PDH
μmol/g Hb % nM/s.g Hb % nM/s.g Hb % nM/s.g Hb %
1. Iniţial level 0,380±0,013 100 69,39±1,95 100 58,46±4,02 100 20,15±0,84 100
2. CMD-4 0,378±0,013 99 60,65±2,01* 87 62,90±4,0 108 14,87±1,49* 74
3. CMD-8 0,359±0,016 94 65,56±3,70 94 62,90±4,0 108 15,67±1,24* 78
4. CMJ-33 0,292±0,011*** 77 66,62±5,26 96 64,75±14,54 111 15,58±0,76** 77
5. CMJ-23 0,302±0,005*** 79 55,67±3,79** 80 51,96±6,68 89 15,97±1,03* 79

Note: Initial level - until the treatment of peripheral blood with the studied compounds; statistically significant difference toward the initial level: * - p <0,05; ** - p <0,01; *** - p <0,001

 

The chemical name of coded compounds: (CMD-4) - 4-ethyl-2- [phenyl (pyridin-2-yl) methylidene] hydrazine-1-carbothioamide]; (CMD-8) - Chloro- {4-ethyl-2- [phenyl (pyridin-2-yl) methylidene] hydrazine-1-carbothioamido} copper]; (CMJ-23) - 4- (3-methoxyphenyl) -2- [1- (pyridin-2-yl) ethylidene] hydrazine-1-carbothioamide] and (CMJ-33) - Chloro- {4- (3-methoxyphenyl) -2- [1- (pyridin-2-yl) ethylidene] hydrazine-1-carbothioamido} copper]

Research shows that the all tested compounds have a different influence on the tGSH content in the erythrocytes of practically healthy donors, the most pronounced changes being recorded in the case of treatment with CMJ-33 and CMJ-23 compounds, where the tGSH level statistically veracious decreases by 23% (p <0.001) and 21% (p<0.001), respectively, compared to the initial level. Under the influence of CMD-4 and CMD-8 compounds, the tGSH content is practically maintained at the initial level until the treatment of peripheral blood with the studied compounds.

The functional level of GPO is practically maintained at the initial level after treatment with CMD-8 and CMJ-33 compounds, and the use of CMD-4 and CMJ-23 compounds produces a statistically suggestive decrease of enzyme activity by 13% (p<0.05) and 20% (p<0.01), respectively.

At the same time, the changes of GR activity by compounds proved to be inconclusive. However, it should be noted that the use of compounds CMD-4, CMD-8, CMJ-33 produces a slight tendency of increasing by 8%-11% (p>0.5), and the compound CMJ-23 produces an insignificant decrease of GR enzyme activity by 11% compared to the initial values.

The evaluation results of the influence of the studied compounds on the activity of G-6-PDH show a statistically veritable decrease by 21-26% of this enzyme compared to the initial level (table 1).

Discussions

The heterocyclic Schiff base and their copper complexes represent key classes of medicinal compounds, having an enormous potential for biological activities [23], [24].

These compounds are thought to exert their effect by interaction with intracellular biomolecules, inhibition of enzymes, increasing lipophilicity and altering cell membrane functions, arresting the cell cycle, etc. [25]. A special role in the mechanisms of action of copper belongs to the concept of "redoxome", a proteomic network coupled to the redox reaction, which includes many states and species of sulfur oxidation and reactions of copper with sulfur-containing peptides, proteins and enzymes, and interaction with GSH producing glutathione disulfide (GSSG) (2GSH → GSSG) [26], [27].

The GSH / GSSG ratio has been found to be the most sensitive indicator of copper toxicity (and subsequent oxidative stress) [28]. The toxicity of copper excess in mammalian cells is explained by the obstruction of the control of the copper-sulfur "interactome" containing "redoxomes" [29]. According to some researchers, the complexation of thiosemicarbazone derivatives with Cu (II) ions improves their antitumor activity, which is explained by DNA damage and the G2 / M phase of cell cycle arrest, as well as with disorders of antioxidant enzyme expression. It should also be noted that the use of both a ligand and a copper salt is as effective as the use of a coordination compound [30].

Thus, the development and study of the biological properties of Schiff bases and their copper coordinating compounds is a promising and relevant field of medical chemistry, which will allow them to become widely used in medical practice.

It is known that GSH is the main component of cell buffer redox, which constantly maintains the reduced environment characteristic for cells and which participates in the control of thiol-redox activity of enzymes.

The veracious reduction of the total glutathione (tGSH) amount under the influence of CMJ-33 and CMJ-23 compounds could be caused by exacerbation of oxidative stress (OS). Erythrocyte glutathione is known to play a cardinal role in attenuating the harmful effects of circulating reactive oxygen species (ROS) and of products resulted from the permanent oxidation of erythrocyte hemoglobin. Reduced glutathione (GSH) can react with superoxide radicals and breakdown hydrogen peroxide and lipid peroxides through available enzyme systems (glutathione peroxidase, glutathione S-transferase) with the formation of water-soluble conjugated products which are then eliminated from the erythrocyte and excreted from the body [31].

Therefore, it is important in the future to study the content of reduced glutathione (GSH) and oxidized glutathione (GSSG), as well as the GSH / GSSG ratio for the evaluation of the intimate mechanisms of CCC action.

GPO is a family of antioxidant enzymes, present in many tissues under several isoforms. Inhibition of GPO by CMD-4 and CMJ-23 compounds, found in our research, decreases the antioxidant protection capacity of erythrocytes faced with excessive activation of free radical reactions at their initiation stages, thus showing a prooxidant effect. In erythrocytes the GPO activity is supported by the reduction of GSSG by GR, NADPH-dependent enzyme and in erythrocytes the main source of NADPH is the pentose phosphate pathway, in which the dehydrogenation of G-6-phosphate and of 6-phosphogluconate take place.

Studies in recent years suggest the idea that GPO is an unambiguous enzyme that can cause unambiguous responses [32]. GPO has a complex effect on the development and progression of cancer, due to its role as a modulator of intracellular ROS levels [33].

Therefore, the GPO level can be considered as a useful marker for evaluating the molecular mechanisms of action of new remedies, and their pathogenic significance.

The activity level of GR - an enzyme that reduces oxidized glutathione and thus plays an important role in recycling G-SH changes insignificantly, which illustrates that the conversion of oxidized glutathione to its reduced form does not change under the influence of the investigated compounds.

Decreased G-6-PDH activity, under the influence of the all the investigated compounds, can negatively influence the low NADPH background necessary to maintain the high cell reducing potential, as well as the cellular homeostasis [34].

To be noted that NADPH, regenerated by glucose-6-phosphate dehydrogenase is used for the GPO functioning. And for efficient functioning, GPO requires two secondary enzymes (GR and G-6-PDH) and cofactors (GSH, NADPH and glucose-6-phosphate). For these reasons, GR and G-6-PDH are considered secondary antioxidant enzymes because they do not act directly on ROS, but allow GPO to function efficiently [35].

The obvious decrease of G-6-PDH enzyme activity, found in our research, could lead to the inhibition or blocking of one of the important metabolic pathways of glucose use - the pentose phosphate pathway, diminishing the metabolic resources needed to maintain the cell's anabolic processes, and which could explain the antiproliferative effect of the tested compounds. It is not excluded that the tested compounds could influence the expression of transketolase genes - a key enzyme that controls the non-oxidative part of the pentose phosphate pathway, which plays crucial roles in tumorigenesis, metastasis and the evolution of several types of cancer [36], [37], so further studies are needed to strengthen these assumptions.

The results show that the new investigated CCC act by the modification of the main indices of erythrocytes glutathione cycles, and that their mechanisms of action are different, and are not only due to the presence of the thiol groups. Further studies are needed to consolidate these observations and to obtain through their exploitation an effective therapeutic approach strategy based on the obtained data.       

Conclusions

  1. The results of the study show the prooxidant effect of the tested CCC manifested by the reduction of GSH by CMJ-33 and CMJ-23, of GPO by CMD-4 and CMJ-23 and of G-6-PDH by - CMD-4, CMD -8, CMJ-33 and CMJ-23, as well as the antiproliferative effect of the researched compounds, manifested by the decrease of the functional level of G-6-PDH.
  2. The elucidation of the subtle mechanisms underlying the CCC action broadens the theoretical knowledge about the biological properties of a series of chemical compounds and also offers new possibilities to explore perspective objects in order to obtain new effective drug preparations.
Финансирование Исследование проведено при поддержке Государственной программы (2020-2023) Республики Молдова (исследовательский грант № 20.80009.5007.10). Funding This study was supported by the State Program (2020-2023) of the Republic of Moldova (research grant No. 20.80009.5007.10).
Конфликт интересов Не указан. Conflict of Interest None declared.

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  30. Pitucha, M. Influence of Complexation of Thiosemicarbazone Derivatives with Cu (II) Ions on Their Antitumor Activity against Melanoma Cells / M. Pitucha, A. Korga-Plewko, A. Czylkowska, et al. // Int. J. Mol. Sci. 2021, 22, 3104.
  31. Raftos J.E. Glutathione Synthesis and Turnover in the Human Erythrocyte. Alignment of a Model Based on Detailed Enzyme Kinetics With Experimental Data / Julia E. Raftos, Stephney Whillier, Philip W. Kuchel // J. Biol Chem. 2010, Vol. 285, No. 31, pp. 23557–23567.
  32. Lubos E. Glutathione peroxidase-1 in health and disease: from molecular mechanisms to therapeutic opportunities / Lubos E, Loscalzo J, Handy D.E. // Antioxidant Redox Signal. 2011; 15 (7): 1957-1997.
  33. Brigelius-Flohe R. Glutathione peroxidases in different stages of carcinogenesis / R. Brigelius-Flohe, A. Kipp //  Biochim Biophys Acta. 2009;1790:1555–1568.
  34. Townsend D.M. The importance of glutathione in human disease / D.M. Townsend, K.D. Tew, H. Tapiero // Biomed Pharmacother. 2003;57(3-4):145-155.
  35. Wu G. Glutathione Metabolism and Its Implications for Health / Guoyao Wu, Yun-Zhong Fang, Sheng Yang, et al. // The Journal of Nutrition, Volume 134, Issue 3, March 2004, Pages 489–492.
  36. Ahopelto K. Transketolase-like protein 1 expression predicts poor prognosis in colorectal cancer / Ahopelto K, Böckelman C, Hagström J, et al. // Cancer Biol Ther. 2016;17(2):163-8.
  37. Iris Ming-Jing Xu. TKT drives cancer development / Iris Ming-Jing Xu, Robin Kit-Ho Lai, Shu-Hai Lin, et al.// Proceedings of the National Academy of Sciences. 2016, 113 (6) E725-E734; DOI: 10.1073/pnas.1508779113.

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  29. Kardos, J. Copper signaling: causes and consequences / Kardos, J., Héja, L., Simon, Á. et al. // Cell Common Signal 16, 71 (2018).
  30. Pitucha, M. Influence of Complexation of Thiosemicarbazone Derivatives with Cu (II) Ions on Their Antitumor Activity against Melanoma Cells / M. Pitucha, A. Korga-Plewko, A. Czylkowska, et al. // Int. J. Mol. Sci. 2021, 22, 3104.
  31. Raftos J.E. Glutathione Synthesis and Turnover in the Human Erythrocyte. Alignment of a Model Based on Detailed Enzyme Kinetics With Experimental Data / Julia E. Raftos, Stephney Whillier, Philip W. Kuchel // J. Biol Chem. 2010, Vol. 285, No. 31, pp. 23557–23567.
  32. Lubos E. Glutathione peroxidase-1 in health and disease: from molecular mechanisms to therapeutic opportunities / Lubos E, Loscalzo J, Handy D.E. // Antioxidant Redox Signal. 2011; 15 (7): 1957-1997.
  33. Brigelius-Flohe R. Glutathione peroxidases in different stages of carcinogenesis / R. Brigelius-Flohe, A. Kipp //  Biochim Biophys Acta. 2009;1790:1555–1568.
  34. Townsend D.M. The importance of glutathione in human disease / D.M. Townsend, K.D. Tew, H. Tapiero // Biomed Pharmacother. 2003;57(3-4):145-155.
  35. Wu G. Glutathione Metabolism and Its Implications for Health / Guoyao Wu, Yun-Zhong Fang, Sheng Yang, et al. // The Journal of Nutrition, Volume 134, Issue 3, March 2004, Pages 489–492.
  36. Ahopelto K. Transketolase-like protein 1 expression predicts poor prognosis in colorectal cancer / Ahopelto K, Böckelman C, Hagström J, et al. // Cancer Biol Ther. 2016;17(2):163-8.
  37. Iris Ming-Jing Xu. TKT drives cancer development / Iris Ming-Jing Xu, Robin Kit-Ho Lai, Shu-Hai Lin, et al.// Proceedings of the National Academy of Sciences. 2016, 113 (6) E725-E734; DOI: 10.1073/pnas.1508779113.