Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2.
Aim.To determine cytotoxic effects towards a tumor cells culture of the heterocyclic derivative conjugated with lectins of different carbohydrate specificity. Methods. Conjugation of lectins (Pea seeds (PSA), peanuts (PNA) and erythroagglyutinin from bean seeds (PHA-E) with thiopyrano[2,3-d]thiazole...
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irk-123456789-1543832019-07-07T12:58:28Z Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2. Antonyuk, V.O. Skorohyd, N.R. Lozynskyi, A.V. Antonyuk, R.V. Lesyk, R.B. Stoika, R.S. Bioorganic Chemistry Aim.To determine cytotoxic effects towards a tumor cells culture of the heterocyclic derivative conjugated with lectins of different carbohydrate specificity. Methods. Conjugation of lectins (Pea seeds (PSA), peanuts (PNA) and erythroagglyutinin from bean seeds (PHA-E) with thiopyrano[2,3-d]thiazole Les-1895, biotesting these conjugates in the cell culture. Results. The conjugates of Les-1895 with specific lectins posses increased cytotoxic effects towards Jurkat cells — by 23 % for PSA-Les-1895, 34 % for PNA-Les-1895, and by 12 % for PHA-E-Les-1895. IC50 of Les-1895 conjugates was ≈ 10 μg/ml, whereas at using free Les-1895 – it was ≈ 30 μg/ml. The PNA and PHA-E conjugates with Les-1895 suppressed the viability of HCT 116 cells more considerably than the PSA conjugate. A cytotoxic action of PNA (IC50 = 4 μg/ml) and PHA-E (IC50 = 3 μg/ml) conjugates was more pronounced than the effect of free Les-1895 (IC50 = 10 μg/ml) or intact lectins. The conjugation of Les-1895 with human serum albumin (HSA) increased the water solubility, however, such conjugation decreased the cytotoxic effect towards Jurkat cells by 40 %. The pseudonormal cells line HEK 293 was less sensitive to the action of native lectins — PSA, PNA and PHA-E as well as to the action of conjugates of these lectins. Conclusions. Conjugation of Les-1895 with specific lectins enhanced a cytotoxic effect. The obtained results suggest a possibility of the addresed delivery of biologically active compounds to specific cells of tissues and organs of the human body. Мета. Визначення на культурі клітин біологічної активності кон’югатів сполуки з протипухлинною активністю з лектинами різної вуглеводної специфічності, де кон’югати використовували як векторні молекули для адресної доставки цих речовин. Методи. Кон’югування лектинів (насіння гороху (PSA), арахісу (PNA) та еритроаглютиніну з насіння квасолі звичайної (PHA-E) з похідним тіопірано[2,3-d]тіазолу Les-1895). Біотестування цих кон’югатів на культурі клітин різного походження Результати. Кон’югування сполуки Les-1895 з лектинами призводить до збільшення її цитотоксичних ефектів щодо клітин Jurkat на 23 % для PSA-Les-1895, на 34 % — для PNA-Les-1895 і на 12 % — для PHA-E-Les-1895. IC50 для кон’юґатів Les-1895 було ≈ 10 мкг/мл, тоді як для нативного Les-1895 ≈ 30 мкг/мл. Кон’югати Les-1895 з PNA та PHA-E пригнічують життєздатність клітин лінії HCT 116 значно сильніше, ніж аналогічний кон’югат з PSA. Цитотоксична дія кон’югатів з PNA (IC50 = 4 мкг/мл) і PHA-E(IC50 = 3 мкг/мл) на ці клітини була сильнішою, ніж дія вільного Les-1895 (IC50 = 10 мкг/мл) та інтактних лектинів. Приєднання Les-1895 до альбуміну сироватки крові людини (HSA) підвищує розчинність Les-1895 у воді, однак, на відміну від кон’югатів Les-1895 з лектинами, знижує на 40 % цитотоксичний ефект утвореного кон’югату, зокрема, для клітин лінії Jurkat. Псевдонормальні клітини лінії HEK 293 виявилися мало чутливими до дії нативних лектинів PSA, PNA і PHA-E та до кон’юґатів цих лектинів. Висновки. Кон’югування сполуки Les-1895 із специфічними лектинами посилює цитотоксичний ефект. Одержані результати є важливими для моделювання адресної доставки біологічно активних речовин в специфічні клітини тканин організму. Цель. Определение на культуре клеток биологической активности конъюгатов соединения с противоопухолевой активностью с лектинами различной углеводной специфичности, где конъюгаты использовали в качестве векторных молекул для адресной доставки этих веществ. Методы. Конъюгирование лектинов (семян гороха (PSA), арахиса (PNA) и эритроагглютинина из семян фасоли обыкновенной (PHA-E) из производным тиопирано[2,3-d]тиазола Les-1895). Биотестирование этих конъюгатов на культуре клеток различного происхождения. Результаты. Конъюгирование соединения Les-1895 с лектинами приводит к увеличению цитотоксических эффектов конъюгата для клеток Jurkat на 23 % для PSA-Les-1895, на 34 % — для PNA-Les-1895 и на 12 % — для PHA-E-Les -1895. IC50 для конъюгатов Les-1895 было ≈ 10 мкг/мл, тогда как для нативного Les-1895 ≈ 30 мкг/мл. Конъюгаты Les-1895 с PNA и PHA-E подавляют жизнеспособность клеток линии HCT 116 значительно сильнее, чем аналогичный конъюгат с PSA. Цитотоксическое действие конъюгатов c PNA (IC50 = 4 мкг/мл) и PHA-E (IC50 = 3 мкг/мл) на эти клетки было сильнее, чем действие свободного Les-1895 (IC50 = 10 мкг/мл) и интактных лектинов. Присоединение Les-1895 к альбумину сыворотки крови человека (HSA) повышает растворимость Les-1895 в воде, однако, в отличие от конъюгатов Les-1895 с лектинами, цитотоксический эффект образованного конъюгата снижается на 40 %, в частности, для клеток линии Jurkat. Псевдонормальные клетки линии HEK 293 оказались мало чувствительными к действию нативных лектинов PSA, PNA и PHA-E и к действию конъюгатов этих лектинов. Выводы. Конъюгирование Les-1895 со специфическими лектинами усиливает цитотоксический эффект. Полученные результаты показывают возможность адресной доставки биологически активных веществ в специфические клетки, ткани и органы тела человека. 2019 Article Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2. / V.O. Antonyuk, N.R. Skorohyd, A.V. Lozynskyi, R.V. Antonyuk, R.B. Lesyk, R.S. Stoika // Вiopolymers and Cell. — 2019. — Т. 35, № 1. — С. 54-63. — Бібліогр.: 12 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000994 http://dspace.nbuv.gov.ua/handle/123456789/154383 576.35+547.818+615.15 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Bioorganic Chemistry Bioorganic Chemistry |
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Bioorganic Chemistry Bioorganic Chemistry Antonyuk, V.O. Skorohyd, N.R. Lozynskyi, A.V. Antonyuk, R.V. Lesyk, R.B. Stoika, R.S. Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2. Вiopolymers and Cell |
| description |
Aim.To determine cytotoxic effects towards a tumor cells culture of the heterocyclic derivative conjugated with lectins of different carbohydrate specificity. Methods. Conjugation of lectins (Pea seeds (PSA), peanuts (PNA) and erythroagglyutinin from bean seeds (PHA-E) with thiopyrano[2,3-d]thiazole Les-1895, biotesting these conjugates in the cell culture. Results. The conjugates of Les-1895 with specific lectins posses increased cytotoxic effects towards Jurkat cells — by 23 % for PSA-Les-1895, 34 % for PNA-Les-1895, and by 12 % for PHA-E-Les-1895. IC50 of Les-1895 conjugates was ≈ 10 μg/ml, whereas at using free Les-1895 – it was ≈ 30 μg/ml. The PNA and PHA-E conjugates with Les-1895 suppressed the viability of HCT 116 cells more considerably than the PSA conjugate. A cytotoxic action of PNA (IC50 = 4 μg/ml) and PHA-E (IC50 = 3 μg/ml) conjugates was more pronounced than the effect of free Les-1895 (IC50 = 10 μg/ml) or intact lectins. The conjugation of Les-1895 with human serum albumin (HSA) increased the water solubility, however, such conjugation decreased the cytotoxic effect towards Jurkat cells by 40 %. The pseudonormal cells line HEK 293 was less sensitive to the action of native lectins — PSA, PNA and PHA-E as well as to the action of conjugates of these lectins. Conclusions. Conjugation of Les-1895 with specific lectins enhanced a cytotoxic effect. The obtained results suggest a possibility of the addresed delivery of biologically active compounds to specific cells of tissues and organs of the human body. |
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Article |
| author |
Antonyuk, V.O. Skorohyd, N.R. Lozynskyi, A.V. Antonyuk, R.V. Lesyk, R.B. Stoika, R.S. |
| author_facet |
Antonyuk, V.O. Skorohyd, N.R. Lozynskyi, A.V. Antonyuk, R.V. Lesyk, R.B. Stoika, R.S. |
| author_sort |
Antonyuk, V.O. |
| title |
Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2. |
| title_short |
Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2. |
| title_full |
Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2. |
| title_fullStr |
Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2. |
| title_full_unstemmed |
Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2. |
| title_sort |
use of lectins as vector molecules for delivery of drugs to cells and tissues. report 2. |
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Інститут молекулярної біології і генетики НАН України |
| publishDate |
2019 |
| topic_facet |
Bioorganic Chemistry |
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http://dspace.nbuv.gov.ua/handle/123456789/154383 |
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Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2. / V.O. Antonyuk, N.R. Skorohyd, A.V. Lozynskyi, R.V. Antonyuk, R.B. Lesyk, R.S. Stoika // Вiopolymers and Cell. — 2019. — Т. 35, № 1. — С. 54-63. — Бібліогр.: 12 назв. — англ. |
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Вiopolymers and Cell |
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54
V. O. Antonyuk, N. R. Skorohyd, A. V. Lozynskyi
© 2019 V. O. Antonyuk et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Bio-
polymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium,
provided the original work is properly cited
Bioorganic Chemistry ISSN 0233-7657
Biopolymers and Cell. 2019. Vol. 35. N 1. P 54–63
doi: http://dx.doi.org/10.7124/bc.000994
UDC: 576.35+547.818+615.15
Use of lectins as vector molecules for delivery of drugs to cells and
tissues. Report 2
V. O. Antonyuk1,2, N. R. Skorohyd1, A. V. Lozynskyi2, R. V. Antonyuk3,
R. B. Lesyk2,4, R. S. Stoika1
1 Institute of Cell Biology, NAS of Ukraine
14/16, Drahomanov Str., Lviv, Ukraine, 79005
2 Danylo Halytsky Lviv National Medical University
69, Pekarska Str., Lviv, Ukraine, 79010
3 Bureau of Forensic Medical Expertise of the Lviv Regional Council
61, Pekarska Str., Lviv, Ukraine, 79010
4 University of Information Technology and Management in Rzeszow
2, Sucharskiego Str., Rzeszow, Poland, 35-225
antonyukvo@gmail.com
Aim. To determine cytotoxic effects towards a tumor cells culture of the heterocyclic deriva-
tive conjugated with lectins of different carbohydrate specificity. Methods. Conjugation of
lectins (Pea seeds (PSA), peanuts (PNA) and erythroagglyutinin from bean seeds (PHA-E)
with thiopyrano[2,3-d]thiazole Les-1895, biotesting these conjugates in the cell culture.
Results. The conjugates of Les-1895 with specific lectins posses increased cytotoxic effects
towards Jurkat cells — by 23 % for PSA-Les-1895, 34 % for PNA-Les-1895, and by 12 %
for PHA-E-Les-1895. IC50 of Les-1895 conjugates was ≈ 10 μg/ml, whereas at using free
Les-1895 — it was ≈ 30 μg/ml. The PNA and PHA-E conjugates with Les-1895 suppressed
the viability of HCT 116 cells more considerably than the PSA conjugate. A cytotoxic action
of PNA (IC50 = 4 μg/ml) and PHA-E (IC50 = 3 μg/ml) conjugates was more pronounced than
the effect of free Les-1895 (IC50 = 10 μg/ml) or intact lectins. The conjugation of Les-1895
with human serum albumin (HSA) increased the water solubility, however, such conjugation
decreased the cytotoxic effect towards Jurkat cells by 40 %. The pseudonormal cells line HEK
293 was less sensitive to the action of native lectins — PSA, PNA and PHA-E as well as to
the action of conjugates of these lectins. Conclusions. Conjugation of Les-1895 with specific
lectins enhanced a cytotoxic effect. The obtained results suggest a possibility of the addresed
delivery of biologically active compounds to specific cells of tissues and organs of the human
body.
K e y w o r d s: pea lectin, peanut lectin, FGA-R, conjugates, thiopyrano[2,3-d]thiazoles, anti-
tumor activity.
55
Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2
Introduction
Various thiopyrano[2,3-d]thiazole derivatives
with the anticancer, antiviral, anti-inflamma-
tory, antimicrobial and antitrypanosomal ac-
tivities were synthezized at the Department of
the Pharmaceutical, Organic and Bioorganic
Chemistry of Danylo Halytsky Lviv National
Medical University [1]. A pharmaceutical po-
tential of these derivatives can be significant-
ly increased through the development of water
soluble forms that also creates the conditions
for their selective binding to the biological
targets. In previous study [2], we have conju-
gated pea seeds lectin in the alkaline medium
(pH 9.0) with the thiopyrano[2,3-d]thiazole
derivative through the interaction of the alde-
hyde group of this compound with the amino
groups of amino acids of the lectins. The bio-
logical activity of obtained conjugates towards
the mouse leukemia cells of L1210 line was
evaluated. It was shown that the obtained con-
jugates had a 2.5-fold increased antineoplastic
effect (calculated as the amount of substance
conjugated to the lectin) compared to the non-
conjugated compound. It was assumed that
such enhancement is caused by a selective
interaction of lectin with certain types of cells
in tissues. Thus, a targeted delivery of the
substance to potential sites of its action is pos-
sible. The application of lectin conjugates with
drugs might also have some disadvantages.
The main one is a protein nature of the lectin
carrier which can cause allergic reactions,
especially after its direct administration into
the bloodstream. At the same time, such con-
jugates could be useful at external application
into the body cavities (oral or nasal) or as
rectal suppositories. The advantage of lectins
as drug carriers compared to the synthetic
polymers is lectins biodegradability. There are
numerous literature data describing commer-
cially available lectins selectively binding to
normal or pathological tissues. It has been
established that peanut lectin (PNA) selec-
tively binds the T-antigen present in 71 % of
cancer cells and not detected by the monoclo-
nal antibodies in healthy donors [3, 4]. Our
investigation on binding capacity of the PNA-E
receptors with colon cancer cells revealed that
PNA-E interacted with the adenoma cells and
with adenocarcinomas of high degree of dif-
ferentiation. This lectin possesses a higher
selectivity to the adenoma cells compared to
the tumor cells with a low degree of differen-
tiation. Thus, the lectin binding can be used as
a selective histochemical marker of these pa-
thologies [5], as well as for the targeted deli-
very of drugs to the specific colon cells.
Therefore, the next step of our work was to
investigate the action of such conjugates on
the cells of other lines, in particular, the
T-lymphoblastic leukemia Jurkat cells, HCT
116 line of human colorectal carcinoma cells,
and HEK 293 pseudonormal cells of human
embryonic kidney. In this study, thiopyrano[2,3-
d]thiazole derivative (Les-1895) [6] was used
as an adduct attached to the lectins.
Materials and Methods
In Fig. 1, one can see a schematic reaction of
the thiopyrano[2,3-d]thiazole derivative (Les-
1895) conjugation with lectins isolated from
the pea seeds (PSA, Pisum sativum agglutinin),
peanut seeds (PNA, peanut agglutinin), and
erythroagglutinin of the common bean
(PHA-E). In control, Les-1895 was conju-
gated to human serum albumin (HSA). These
56
V. O. Antonyuk, N. R. Skorohyd, A. V. Lozynskyi et al.
conjugations were accomplished through the
aldehyde group of Les-1895 interacting with
the amino groups of amino acids of lectins
under the alkaline conditions (pH 9.0).
Conjugation. 10 mg of lectin or HSA were
dissolved in 1.0 ml of 1 % aqueous solution
of sodium bicarbonate with addition of 0.025-
0.2 ml of 1 % solution of Les-1895 in DMSO.
The obtained solution was stirred and kept for
3 h at room temperature. After stirring the
turbid solution was gradually highlighted, 5
mg of sodium borohydride were added to the
solution and the obtained mixture was kept for
12 h. To separate the unreacted Les-1895 from
its conjugate, the mixture was dialyzed against
50 ml of 20 % aqueous solution of DMSO,
and then against 50 ml of phosphate buffered
saline (PBS).
Investigation of the obtained conjugate. The
obtained conjugate was characterized for its
lectin activity measured as an ability to bind
specific carbohydrates and glycoproteins of the
plasma membrane. The content of protein and
Les-1895 attached to the lectin was also cal-
culated. The lectin’s activity before and after
conjugation was measured by determining the
agglutination titer of 2 % rabbit erythrocyte
suspension in PBS. The concentration of Les-
1895 in the conjugate was determined by mea-
suring the absorbance at 361 nm wavelength,
where this compound has a characteristic
maximum of absorption (Fig. 2). The content
of total protein was measured using Lowry
method with an absorbance at 740 nm.
Synthesis of Les-1895. Synthesis of
7-[4-(benzyloxy)phenyl]-2-oxo-3,5,6,7-tetra-
hydro-2H-thiopyrano[2,3-d][1,3]thiazole-
6-carbaldehyde (Les-1895) was accomplished
in the [4+2]-cyclocondensation reaction of
5 - ( 4 - b e n z y l o x y b e n z y l i d e n e ) - 4 -
thioxothiazolidinone-2 as a heterodiene and
acrolein as a dienophile under the refluxing for
1 h with a catalytic amount of the hydroqui-
none in a glacial acetic acid medium [6].
Cell culture. The human colorectal carci-
noma HCT 116 cells, T-lymphoblastic leukemia
Jurkat cells, and pseudonormal human embry-
onic kidney HEK 293 cells were obtained from
Cell culture collection at R.E. Kavetsky Institute
of Experimental Pathology, Oncology and
Lectin-NH2
Lectin-N = C
+
S
NH
S
O
O
H
O
S
NH
S
O
O
H
pH = 9
-H2O
Fig. 1. Conjugation of Les-1895 with lectins.
57
Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2
Radiobiology, National Academy of Sciences
of Ukraine (Kyiv). The HEK 293 cells were
cultured in the Dulbecco’s -modified Eagle’s
medium (DMEM, Sigma, USA) in the presence
of 10 % of the decomposed blood serum of
cattle embryos, using phenol red as an indicator
of pH, and 50 μg/ml of gentamicin antibiotic
(Sigma, USA). The cells of Jurkat and HCT 116
lines were cultured in the RPMI-1640 (Thermo
scientific, USA) medium in the presence of
10 % decomposed blood serum of cattle em-
bryos, phenol red, and 50 μg/ml of gentamicin
(Sigma, USA). To evaluate cytotoxicity of the
lectins (PSA, PNA, PHA-E), Les-1895, and the
conjugates with these lectins, the appropriate
samples were added in different concentrations
to the medium in which the cells are cultured.
After 24 h, the number of cells was counted in
the hemocytometric chamber using the Trypan
blue dye (DV-T10282, “Invitrogen”, USA) that
penetrates through the plasma membrane of the
damaged (dead) cells. The cells colored in blue
were considered as the necrotic ones. The num-
ber of cells in suspension was calculated ac-
cording to the formula: с = 12500n, where:
c — the number of cells in 1 ml of suspension,
n — the average number of cells in 5 large
squares of the hemocytometer chamber. The
evaluation of cytotoxicity and antiproliferative
activity of the tested compound was carried out
after 72 h of incubation using the MTT assay.
The cell viability was evaluated by measuring
Fig. 2. Absorption spectrum of Les-1895 (0.03 % solution in DMSO).
58
V. O. Antonyuk, N. R. Skorohyd, A. V. Lozynskyi et al.
a reduction of the MTT dye (3-(4,5-dimethyl-
thiazol-2-yl)-2,5-diphenyltetrazolium bromide)
(Sigma, USA) to water-insoluble formazan
which has a purple color.
Statistical analysis. Each experiment was
performed in triplicate and average values
were recorded. The data were evaluated statis-
tically using Student’s t-test, and a value of
p ≤ 0.05 was considered to be statistically
reliable.
Results and Discussion
Les-1895 is soluble only in organic solvents
such as dimethyl sulfoxide or dimethylfor-
mamide. Here we demonstrated that the con-
jugation of this compound with the lectin (see
Materials and Methods section) led to creation
of the water soluble product.
Comparing to the previously reported
thiopyrano[2,3-d]thiazole derivative, Les-1351
[2], an increase of the amount of Les-1895 in
the reaction mixture up to 1 mg per 10 mg of
pea lectin (10 %) did not lead to the lectin
inactivation. Noteworthy, Les-1895 present in
the reaction mixture was not completely im-
mobilized on lectin. According to our calcula-
tions, 10 mg of lectin bound 0.9 mg of Les-
1895 (9 % of total weight of the conjugate).
Similar results were obtained for PNA and
PHA-E, where 10 mg of PNA bound maximum
A
B
C
D
Fig. 3. The results of measuring the cytotoxic effect (Trypan blue exclusion test) of Les-1895, specific lectins, and
their conjugates towards human T-lymphoblastic leukemia Jurkat cells. The cytotoxic activity of the conjugates (Les-
1895 + PSA, PNA, PHA-E or HSA) was calculated taking into account the amount of the attached Les-1895.
* p < 0.05; ** p<0.01; *** p<0.001
59
Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2
0.5 mg of Les-1895 (5 % of total mass of the
conjugate), and 10 mg of PHA-E bound 8 %
of Les-1895 of total mass of the conjugate. At
the same time, 10 mg of HSA could bind up
to 1.6 mg Les-1895 (16 % of the total conju-
gate mass). The obtained conjugates of lectins
with Les-1895 were soluble in water, phos-
phate buffered saline (PBS), and 1 % NaHCO3
solution, whereas the initial Les-1895 com-
pound was practically insoluble in these media.
Unlike the murine leukemia L1210 cells [2]
that were insensitive to the pea lectin (PSA),
the T-lymphoblastic leukemia Jurkat cells were
subjected to a cytotoxic action of the PHA-E
and PNA lectins. IC50 for PNA and PSA was 80
and 37 μg/ml, respectively, and for PHA-E —
20 μg/ml. IC50 for Les-1895 was 32 μg/ml.
According to these data, PHA-E was more ef-
fective in targeting Jurkat cells than Les-1895.
The conjugates of the lectins with this com-
pound displayed a cytotoxic effect towards the
T-lymphoblastic leukemia Jurkat cells — 23 %
inhibition for PSA-Les-1895, 34 % — for PNA-
Les-1895, and 12 % — for PHA-E-Les-1895.
IC50 of the conjugates of lectins with Les-1895
was 10 μg/ml, whereas IC50 of free Les-1895
compound was 30 μg/ml. Though the conjuga-
tion of Les-1895 to HSA increased a water
solubility of Les 1895, it at the same time re-
duced by 40 % a cytotoxic effect of the obtained
conjugate towards the T-lymphoblastic leuke-
mia Jurkat cells (Fig. 3).
A
B
C
D
Fig. 4. The results of measuring cytotoxicity (Trypan blue exclusion test) of Les-1895, specific lectins, and their con-
jugates towards the human colorectal carcinoma cells of HCT 116 line.
* p < 0.05; ** p<0.01; *** p<0.001
60
V. O. Antonyuk, N. R. Skorohyd, A. V. Lozynskyi et al.
Noteworthy, PNA and PHA-E significantly
suppressed the viability of human colorectal
carcinoma HCT116 cells comparing to the ef-
fects of intact HSA and pea lectin. The cyto-
toxic action of the Les-1895 conjugates with
the PNA and PHA-E lectins was more potent
than the action of free Les-1895 and non-
conjugated lectins. IC50 of the PSA-Les-1895
conjugate was 28 μg/ml, PNA-Les-1895 —
4 μg/ml, and PHA-E-Les-1895 — 3 μg/ml.
IC50 of Les-1895 was approximately 10 μg/ml,
whereas the cytotoxic effect of the HSA-E-
Les-1895 and the PSA-Les-1895 conjugates
was weaker than that of free Les-1895 (Fig. 4).
Pseudonormal human embryonic kidney
HEK 293 cells were insensitive to the action
of PSA, PNA and PHA-E lectins, and at 30
mg/ml dose of these lectins there were ≈20 %
of dead cells. The conjugation of Les-1895
with these lectins did not significantly enhance
their cytotoxic effect as compared to the action
of the Les-1895 free form (Fig. 5).
Taking into account the obtained results, one
can suggest a positive correlation between the
level of lectin binding with cells and a cyto-
toxic action of the Les-1895 conjugates with
these lectins. This suggestion is in agreement
with our preliminary results on the binding of
specific lectins with histological specimens of
the human adenocarcinoma colon cells com-
pared to the cells of normal colon tissue [5].
Thus, the conjugation of Les-1895 with the
pea and peanut lectins increased the antineo-
plastic activity of the resulting conjugates in
the T-lymphoblastic leukemia Jurkat cells and
human adenocarcinoma HCT 116 cells. At the
same time, the pseudonormal HEK 293 line
cells were insensitive to the action of tested
lectins and their conjugates with Les-1895.
The human adenocarcinoma HCT 116 cells
were 2-fold more sensitive to the action of
studied conjugates of lectins and tested com-
pound. According to the literary data [7-9], an
increased toxicity of conjugates towards tumor
cells could be caused by a selective binding of
specific lectins to the carbohydrate-containing
receptors of the plasma membrane of cells.
The HSA-Les-1895 conjugate was not toxic
for the T-lymphoblastic leukemia Jurkat cells
and only slightly toxic for the human adeno-
carcinoma HCT 116 cells, comparing to a high
toxicity of the conjugates of Les-1895 with the
lectins. A cytotoxic action of conjugates Les-
1895 with lectins towards the pseudonormal
cells HEK 293 line was manifested after 72 h
only at high doses of the conjugates. Thus, the
action of the conjugates was more pronounced
for the adenocarcinoma HCT 116 cells than
for the pseudonormal cells HEK 293 line
(Fig. 6).
We had no the pseudonormal colon cells in
our collection, thus, we used the human em-
bryonic kidney cells that possess a similar
structure of the glycoconjugates on their sur-
face [10, 11]. Comparing to tumor cells, these
cells have a significantly less amount of ter-
minal sialic acids in the plasma membrane [12].
Lectins could interact specifically with the
individual cell surface glycoconjugates creat-
ing an increased local concentration of bound
substances. As noted in the Introduction sec-
tion, using lectins as medicines has certain
limitations. They can be used only externally
at treatment of patients with diseases of skin,
digestive tract, nasopharynx, lungs, or in the
rinses and sprays, suppositoria and ointment
bases. Lectin-containing medicines cannot be
recommended for administration into the
61
Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2
bloodstream because of possible allergic reac-
tions. However, their inclusion in the ointment
or utilizing in suppositories may be helpful for
the treatment of colorectal cancer.
Conclusions
1. Covalent attachment of the thiopyrano[2,3-
d]thiazole derivatives to proteins (lectins) is
accompanied by an increased solubility of
A
B
Fig. 6. The results of measuring viability (MTT assay) of Les-1895, specific lectins, and their conjugates towards the
pseudonormal human embryonic kidney HEK 293 cells and the human colorectal carcinoma HCT 116 cells.
* p < 0.05; ** p<0.01; *** p<0.001
A
B
C
D
Fig. 5. The results of measuring cytotoxicity (Trypan blue exclusion test) of Les-1895, specific lectins, and their con-
jugates towards the pseudonormal human embryonic kidney HEK 293 cells.
* p < 0.05; ** p<0.01; *** p<0.001
62
V. O. Antonyuk, N. R. Skorohyd, A. V. Lozynskyi et al.
formed conjugates in water, however, such
conjugation does not guarantee an enhacement
of the biological activity of the attached li-
gands.
2. Native lectins exhibit more pronounced
cytotoxic action on specific tumor cells, than
on the pseudonormal cells, and a degree of
such action depends on the carbohydrate spec-
ificity. Conjugation of lectins with a low mo-
lecular weight ligand posessing antitumor ac-
tivity leads to an increase in the cytotoxicity
of the resulting conjugates.
3. The cytotoxic effect of Les-1895 conju-
gates with lectin, which selectively binds to
the HCT 116 human intestinal cancers, was
dose-dependent. At the same time the effect of
the developed conjugates of lectins towards
the pseudonormal human embryonic kidney
HEK 293 cells was more pronounced after
24 h, although it was manifested only at high
doses of the conjugates.
REFERENCES
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Використання лектинів як векторних молекул
для доставки лікарських засобів до клітин
і тканин. Повідомлення 2
В. О. Антонюк, Н. Р. Скорохід, А. В. Лозинський,
Р. В. Антонюк, Р. Б. Лесик, Р. С. Стойка
Мета. Визначення на культурі клітин біологічної актив-
ності кон’югатів сполуки з протипухлинною активністю
з лектинами різної вуглеводної специфічності, де
63
Use of lectins as vector molecules for delivery of drugs to cells and tissues. Report 2
кон’югати використовували як векторні молекули для
адресної доставки цих речовин. Методи. Кон’югування
лектинів (насіння гороху (PSA), арахісу (PNA) та ери-
троаглютиніну з насіння квасолі звичайної (PHA-E) з
похідним тіопірано[2,3-d]тіазолу Les-1895).
Біотестування цих кон’югатів на культурі клітин різно-
го походження Результати. Кон’югування сполуки
Les-1895 з лектинами призводить до збільшення її ци-
тотоксичних ефектів щодо клітин Jurkat на 23 % для
PSA-Les-1895, на 34 % — для PNA-Les-1895 і на
12 % — для PHA-E-Les-1895. IC50 для кон’юґатів Les-
1895 було ≈ 10 мкг/мл, тоді як для нативного Les-1895
≈ 30 мкг/мл. Кон’югати Les-1895 з PNA та PHA-E при-
гнічують життєздатність клітин лінії HCT 116 значно
сильніше, ніж аналогічний кон’югат з PSA.
Цитотоксична дія кон’югатів з PNA (IC50 = 4 мкг/мл) і
PHA-E(IC50 = 3 мкг/мл) на ці клітини була сильнішою,
ніж дія вільного Les-1895 (IC50 = 10 мкг/мл) та інтактних
лектинів. Приєднання Les-1895 до альбуміну сироватки
крові людини (HSA) підвищує розчинність Les-1895 у
воді, однак, на відміну від кон’югатів Les-1895 з лекти-
нами, знижує на 40 % цитотоксичний ефект утвореного
кон’югату, зокрема, для клітин лінії Jurkat. Псевдо нор-
маль ні клітини лінії HEK 293 виявилися мало чутливи-
ми до дії нативних лектинів PSA, PNA і PHA-E та до
кон’юґатів цих лектинів. Висновки. Кон’югування
сполуки Les-1895 із специфічними лектинами посилює
цитотоксичний ефект. Одержані результати є важливи-
ми для моделювання адресної доставки біологічно ак-
тивних речовин в специфічні клітини тканин організму.
К л юч ов і с л ов а: лектин насіння гороху, лектин
арахісу, ФГА-Р, кон’югати, тіопірано[2,3-d]тіазоли,
протипухлинна активність
Использование лектинов как векторной
молекулы для доставки лекарственных средств
в клетки и ткани. Сообщение 2
В. О. Антонюк, Н. Р. Скорохид, А. В. Лозинский,
Р. В. Антонюк, Р. Б. Лесык, Р. С. Стойка
Цель. Определение на культуре клеток биологической
активности конъюгатов соединения с противоопухо-
левой активностью с лектинами различной углеводной
специфичности, где конъюгаты использовали в каче-
стве векторных молекул для адресной доставки этих
веществ. Методы. Конъюгирование лектинов (семян
гороха (PSA), арахиса (PNA) и эритроагглютинина из
семян фасоли обыкновенной (PHA-E) из производным
тиопирано[2,3-d]тиазола Les-1895). Биотестирование
этих конъюгатов на культуре клеток различного про-
исхождения. Результаты. Конъюгирование соедине-
ния Les-1895 с лектинами приводит к увеличению
цитотоксических эффектов конъюгата для клеток
Jurkat на 23 % для PSA-Les-1895, на 34 % — для PNA-
Les-1895 и на 12 % — для PHA-E-Les -1895. IC50 для
конъюгатов Les-1895 было ≈ 10 мкг/мл, тогда как для
нативного Les-1895 ≈ 30 мкг/мл. Конъюгаты Les-1895
с PNA и PHA-E подавляют жизнеспособность клеток
линии HCT 116 значительно сильнее, чем аналогичный
конъюгат с PSA. Цитотоксическое действие
конъюгатов c PNA (IC50 = 4 мкг/мл) и PHA-E (IC50 =
3 мкг/мл) на эти клетки было сильнее, чем действие
свободного Les-1895 (IC50 = 10 мкг/мл) и интактных
лектинов. Присоединение Les-1895 к альбумину
сыворотки крови человека (HSA) повышает раство-
римость Les-1895 в воде, однако, в отличие от
конъюгатов Les-1895 с лектинами, цитотоксический
эффект образованного конъюгата снижается на 40 %,
в частности, для клеток линии Jurkat. Псевдо нор маль-
ные клетки линии HEK 293 оказались мало чувст-
вительными к действию нативных лектинов PSA, PNA
и PHA-E и к действию конъюгатов этих лектинов.
Выводы. Конъюгирование Les-1895 со специфичес-
кими лектинами усиливает цитотоксический эффект.
Полученные результаты показывают возможность
адресной доставки биологически активных веществ
в специфические клетки, ткани и органы тела чело-
века.
К л юч е в ы е с л ов а: лектин семян гороха, лектин
арахиса, ФГА-Р, коньюґаты, тиопирано[2,3-d]тиазолы,
противоопухолевая активность
Received 08.09.2018
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