Enhancing effect of new biological response modifier sulfoethylated (1→3)-β-D-glucan on antitumor activity of cyclophosphamide in the treatment
Aim: One of the advanced methodologies of the tumor therapy is the application of the so-called biological response modifiers used for activation of the endogenous antitumor mechanisms and combined with classical cytotoxic agents. The aim of this work was the investigation of the effect of sulfoethy...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2006
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| Цитувати: | Enhancing effect of new biological response modifier sulfoethylated (1→3)-β-D-glucan on antitumor activity of cyclophosphamide in the treatment / T.A. Khalikova, T.A. Korolenko, S.Ya. Zhanaeva, V.I. Kaledin, G. Kogan // Experimental Oncology. — 2006. — Т. 28, № 4. — С. 308-313. — Бібліогр.: 34 назв. — англ. |
Репозитарії
Digital Library of Periodicals of National Academy of Sciences of Ukraine| _version_ | 1860058277778292736 |
|---|---|
| author | Khalikova, T.A. Korolenko, T.A. Zhanaeva, S.Ya. Kaledin, V.I. Kogan, G. |
| author_facet | Khalikova, T.A. Korolenko, T.A. Zhanaeva, S.Ya. Kaledin, V.I. Kogan, G. |
| citation_txt | Enhancing effect of new biological response modifier sulfoethylated (1→3)-β-D-glucan on antitumor activity of cyclophosphamide in the treatment / T.A. Khalikova, T.A. Korolenko, S.Ya. Zhanaeva, V.I. Kaledin, G. Kogan // Experimental Oncology. — 2006. — Т. 28, № 4. — С. 308-313. — Бібліогр.: 34 назв. — англ. |
| collection | DSpace DC |
| description | Aim: One of the advanced methodologies of the tumor therapy is the application of the so-called biological response modifiers used for activation of the endogenous antitumor mechanisms and combined with classical cytotoxic agents. The aim of this work was the investigation of the effect of sulfoethylated (1→3)-β-D-glucan (SEG) in the treatment of experimental murine leukoses in combination with cyclophosphamide (CPA) and its ability to modulate the activity of lysosomal enzymes in tumor tissues. Materials and Methods: The solid forms of inoculated murine leukoses P388 and L1210/1 were transplantated to male DBA/2 mice. The therapy was performed by treating animals with CPA (Biokhimik, Saransk, Russia) alone or in combination with SEG (Institute of Chemistry, Slovak Academy of Sciences, Slovakia). CPA was administered in saline as a single intraperitoneal (ip) injection on the 10th day after tumor transplantation; SEG was administered to mice ip 3 days after tumor transplantation with the intervals in 3 days. The therapy effect was estimated by measuring of solid tumor volume. Activity of the cysteine proteases — cathepsins B and L — was measured fluorometrically using fluorescent substrates Z-Arg-Arg-MCA and Z-Phe-Arg-MCA (Sigma, USA), respectively. The apoptosis was estimated evaluating the number of cells with fragmented nuclei using optical microscope. Results: It has been demonstrated that application SEG leads to inhibition of tumor growth and potentiates therapeutic action of CPA, especially at repeated administrations during the whole treatment/observation At addition of SEG, therapeutic effect of a one-half reduced dose of CPA is equal or higher than that of the full dose. Therapeutic action of CPA and SEG on the studied tumors is realized predominantly through induction of apoptosis and is accompanied by a substantial increase of the activity of cysteine proteases cathepsins B and L in tumor tissues. The highest cathepsin B and cathepsin L activity in tumor tissue accompanied with the strongest inhibition of tumor growth. It is suggested that this phenomenon is due to the infiltration of the macrophages rich in the named enzymes into the tumor, where they phagocytize the apoptotic cells and tissue debris. Conclusion: Utilization of this polysaccharide BRM, sulfoethylated (1→3)-β-D-glucan, might potentially enhance efficiency of antitumor therapy with standard cytostatics without a need of substantial increase of their dosage and hence avoiding their toxic side-effects.
Цель: одним из перспективных методов противоопухолевой терапии является использование так называемых модификаторов
биологического ответа, применяемых для активации эндогенных противоопухолевых механизмов и комбинируемых с
классическими цитотоксическими препаратами. Цель данной работы — исследование эффекта сульфоэтилированного (1→
3)-β-D-гликана (SEG) при лечении экспериментальных лейкозов мышей в комбинации с циклофосфаном (СРА) и его способность
модулировать активность лизосомных ферментов в опухолевой ткани. Материалы и методы: солидные формы перевиваемых
лейкозов мышей Р388 и L1210/1 трансплантировали мышам-самцам DBA/2. Для лечения использовали СРА (Биохимик,
Саранск, Россия) и его комбинацию с SEG (Институт химии Словацкой Академии Наук, Братислава, Словакия). СРА вводили
внутрибрюшинно на 10 сут после перевивки опухолей; SEG вводили внутрибрюшинно начиная с 3 сут после трансплантации
лейкозов с интервалом в 3 дня. Терапевтический эффект оценивали путем измерения объема солидной опухоли. Активность
цистеиновых протеаз — катепсинов В и L — определяли флюориметрическим методом, используя флюоресцентные субстраты
Z-Arg-Arg-MCA и Z-Phe-Arg-MCA (Sigma, США). Апоптоз оценивали по результатам подсчета клеток с фрагментированными
ядрами в световом микроскопе. Результаты: в работе показано, что использование SEG приводит к торможению опухолевого
роста и потенцирует терапевтический эффект СРА, особенно при повторном введении в течение всего лечения. В сочетании с SEG
терапевтический эффект половинной дозы СРА равнозначен или превышает действие полной дозы цитостатика. Воздействие
СРА и SEG на использованные в исследовании опухоли реализуется в основном через индукцию апоптоза и сопровождается
существенным повышением активности цистеиновых протеаз катепсинов В и L в опухолевой ткани. Наиболее высокая
активность катепсинов В и L сопровождается максимальным подавлением опухолевого роста. Предположительно это обусловлено
инфильтрацией опухолевой ткани макрофагами с высоким содержанием вышеназванных ферментов, где они фагоцитируют
клетки в апоптозе. Выводы: использование сульфоэтилированного (1→3)-β-D-гликана, нового модификатора биологического
ответа дает возможность существенно повысить эффективность противоопухолевой терапии стандартными цитостатиками без
повышения их дозы, что позволяет избежать побочных эффектов данных препаратов.
|
| first_indexed | 2025-12-07T17:02:45Z |
| format | Article |
| fulltext |
308 Experimental Oncology 28, 308–313, 2006 (December)
Increased incidence of oncological diseases has led
to the search of the new efficient therapies for malignant
neoplasia. One of the advanced methodologies of the
tumor therapy is development of such approaches that
involve not only a direct cytotoxic effect on the tumor
cells, but also consider activation of the endogenous
antitumor mechanisms [1]. One of the most efficient
applications was the use of the so-called biological
response modifiers (BRMs), such as the culture Co-
rinebacterium parvum, anti-tuberculosis vaccine BCG
[2], as well as the biologically аctive compounds mainly
of the polysaccharidic nature derived from bacteria and
fungi, such as prodigiosan, mannan, glucan, muramyldi-
peptide, etc [3]. In the last decade, much attention has
been given to the investigation of the antitumor activity
of the natural non-specific immunomodulators — fun-
gal (1→3)-β-D-glucans [4–7]. Glucans are structurally
distinct polysaccharides consisting of D-glucose units
linked by (1→3)-β and (1→6)-β glycosidic linkages. Many
experimental data corroborate the ability of (1→3)-β-
D-glucans to non-specifically stimulate cellular and
humoral components of the immune system [8]. One of
the (1→3,1→6)-β-D-glucans, lentinan, isolated from the
oriental edible mushroom shiitake or Lentinus edodes,
was admitted in Japan for application in clinical antitumor
therapy [9–11]. Another fungal glucan, (1→3,1→6)-β-D-
glucan isolated from the cell wall of baker’s yeast Sac-
charomyces cerevisiae has attracted attention of many
researchers due to its ability to enhance the functional
status of macrophages and neutrophils [12], modify
immunosuppression [13], increase resistance to infec-
tions by Gram-negative bacteria [14], as well as exert
antitumor activity [15, 16]. Since (1→3)-β-D-glucans
consist solely of glucose, they are not toxic to animals
and humans, however insolubility in water hinders their
application in animal models and humans. In order to
circumvent this obstacle, we have synthesized several
water-soluble derivatives of yeast β-D-glucan including
carboxymethylated and sulfoethylated ones [17, 18]. In a
Enhancing EffEct of nEw biological rEsponsE modifiEr
sulfoEthylatEd (1→3)-β-d-glucan on antitumor activity
of cyclophosphamidE in thE trEatmEnt of ExpErimEntal
murinE lEukosEs
T.A. Khalikova1, T.A. Korolenko1, *, S.Ya. Zhanaeva1, V.I. Kaledin2, G. Kogan3, *
1Institute of Physiology, Siberian Branch of Russian Academy of Medical Sciences,
630117 Novosibirsk, Russia
2Institute of Cytology and Genetics, Siberian Branch of Russian Academy of Sciences,
630090 Novosibirsk, Russia
3 Institute of Chemistry, Center of Excellence CEDEBIPO, Slovak Academy of Sciences,
845 38 Bratislava, Slovakia
Aim: One of the advanced methodologies of the tumor therapy is the application of the so-called biological response modifiers used for
activation of the endogenous antitumor mechanisms and combined with classical cytotoxic agents. The aim of this work was the inves-
tigation of the effect of sulfoethylated (1→3)-β-D-glucan (SEG) in the treatment of experimental murine leukoses in combination with
cyclophosphamide (CPA) and its ability to modulate the activity of lysosomal enzymes in tumor tissues. Materials and Methods: The solid
forms of inoculated murine leukoses P388 and L1210/1 were transplantated to male DBA/2 mice. The therapy was performed by treating
animals with CPA (Biokhimik, Saransk, Russia) alone or in combination with SEG (Institute of Chemistry, Slovak Academy of Sciences,
Slovakia). CPA was administered in saline as a single intraperitoneal (ip) injection on the 10th day after tumor transplantation; SEG was
administered to mice ip 3 days after tumor transplantation with the intervals in 3 days. The therapy effect was estimated by measuring of
solid tumor volume. Activity of the cysteine proteases — cathepsins B and L — was measured fluorometrically using fluorescent substrates
Z-Arg-Arg-MCA and Z-Phe-Arg-MCA (Sigma, USA), respectively. The apoptosis was estimated evaluating the number of cells with
fragmented nuclei using optical microscope. Results: It has been demonstrated that application SEG leads to inhibition of tumor growth
and potentiates therapeutic action of CPA, especially at repeated administrations during the whole treatment/observation At addition of
SEG, therapeutic effect of a one-half reduced dose of CPA is equal or higher than that of the full dose. Therapeutic action of CPA and SEG
on the studied tumors is realized predominantly through induction of apoptosis and is accompanied by a substantial increase of the activity
of cysteine proteases cathepsins B and L in tumor tissues. The highest cathepsin B and cathepsin L activity in tumor tissue accompanied
with the strongest inhibition of tumor growth. It is suggested that this phenomenon is due to the infiltration of the macrophages rich in the
named enzymes into the tumor, where they phagocytize the apoptotic cells and tissue debris. Conclusion: Utilization of this polysaccharide
BRM, sulfoethylated (1→3)-β-D-glucan, might potentially enhance efficiency of antitumor therapy with standard cytostatics without a
need of substantial increase of their dosage and hence avoiding their toxic side-effects.
Key Words: murine leukosis, cathepsins B, L and D, yeast glucan, cyclophosphamide.
Received: November 17, 200.
*Correspondence: Fax: +7 383 3324254
��mai�: �.�.�oro�en�o�ip�.ma.nsc.r���mai�: �.�.�oro�en�o�ip�.ma.nsc.r�
Abbreviations used: CP� — cyc�op�osp�amide; S�G — s��foet�y�
�ated (1→3)�β�d�g��can; BRMs — bio�ogica� response modifiers;
Z��rg��rg�MC� — Z�arginine�arginine�met�y�c�mari�amide;— Z�arginine�arginine�met�y�c�mari�amide; Z�arginine�arginine�met�y�c�mari�amide;
Z�P�e��rg�MC� — Z�p�eny��arginine�met�y�c�mari�amide.— Z�p�eny��arginine�met�y�c�mari�amide. Z�p�eny��arginine�met�y�c�mari�amide.
Exp Oncol 2006
28, 4, 308–313
Experimental Oncology 28, 308–313, 2006 (December) 30928, 308–313, 2006 (December) 309December) 309) 309 309
previous paper, we have demonstrated the possibility of
enhancement of antitumor activity of cyclophosphamide
by means of addition of the carboxymethylated (1→3)-β-
D-glucan in the Lewis lung carcinoma model [1]. In this
work, we have investigated the effect of another soluble
(1→3)-β-D-glucan derivative, sulfoethylated (1→3)-β-D-
glucan (SEG), in the treatment of experimental murine
leukoses, its synergistic action with cyclophosphamide,
as well as its ability to modulate the activity of lysosomal
enzymes in tumor tissues in the process of therapy of
leukoses.
matErials and mEthods
Preparation of SEG. The water-insoluble (1→3)-
β-D-glucan was isolated from the commercial baker’s
yeast biomass purchased from Slovlik (Trenčín,
Slovakia). Yeast cells were treated with 6% NaOH at
60 °C followed by 4% phosphoric acid extraction at
room temperature as previously described [19]. After
the removal of all soluble material, β-D-glucan was
left as the insoluble residue. Sulfoethylation of the
insoluble β-D-glucan was performed according to
Chorvatovičová et al. [20].
Animals and tumors. Male DBA/2 mice, 3–4 months
of age used in the experiments were obtained from
Research Institute of Pharmacology Siberian Branch of
RAMS (Tomsk, Russia). The animals were kept in plastic
cages in groups of 8–10 at natural illumination and had free
access to a standard pellet diet (Laboratorsnab, Moscow,
Russia) and water. Two experimental tumor models were
used: transplantable murine leukoses P-388 and L1210/1.
The tumors were received from the experimental animal
laboratory of the Institute of Cytology and Genetics Si-
berian Branch of RAS (Novosibirsk, Russia). Leukosis
L1210/1 is a version of the leukosis L1210 obtained after
a series of in vitro passages of initial tumor and charac-
terized by rather benign behavior and absence of visible
macroscopic signs of generalization [21]. The work with
animals was approved by Ethic committee.
Tumor transplantation and animal treatment.
The cryoconserved suspensions of tumor cells were
thawed and implanted into abdominal cavity of DBA/2
mice and the developed ascites were used for trans-
plantation to the experimental animals. The ascites
were diluted with 20 volumes of physiological saline
and 1 ml of a suspension (1.5–1.7 × 106 tumor cells)
was inoculated intramuscularly (im) into animal’s right
thigh. In each experiment, shortly after tumor trans-
plantation the animals were divided into four groups,
one of which (group 1) was a control group and three
other groups (2 to 4) were subjected to therapy. The
therapy was performed by treating animals with cyc-
lophosphamide (CPA, Biokhimik, Saransk, Russia)
alone (groups 2 and 3) or in combination with SEG
(group 4). CPA was administered in saline as a single
intraperitoneal (ip) injection on the 10th day after tumor
transplantation at the doses of 20 and 40 mg/kg in one
experiment and 25 and 50 mg/kg in other experiments.
SEG was dissolved in saline and administered to mice
ip 3 days after tumor transplantation three times in one
experiment and 5–7 times in other experiments. Each
single dose of SEG was 25 mg/kg and the intervals
between the individual injections were 3 days.
Estimation of tumor growth. Three perpendicular
diameters of the tumor were measured with a caliper
and tumor volume was calculated by multiplication
of their values. When the mice were sacrificed, the
excised tumor nodules were homogenized in buffered
0.1% Triton X-100 solution for subsequent determina-
tion of proteases activity.
Cysteine protease assay. Activity of the cysteine
proteases — cathepsins B and L — was measured
fluorometrically as previously described by Svechnikova
et al. [22]. Shortly activity of cathepsins B and L were as-activity of cathepsins B and L were as-
sessed in the tumor tissue using fluorescent substrates
Z-Arg-Arg-MCA and Z-Phe-Arg-MCA (Sigma, USA),
respectively. At assay of cathepsin L a selective inhibitor
of cathepsin B — CA-074 (kind gift of Prof. N. Katunuma,
Japan) was added into incubation mixture. Fluores-
cence was measured at 355 nm (excitation) and 460 nm
(emission) using a fluorescent spectrophotometer Per-
kin-Elmer 650–10S (Japan). The results were expressed
as nmol of methylcoumarylamide (MCA)/ min per mg
protein. The measurements were carried out in theThe measurements were carried out in the
treated animals on the 3rd day after CPA administration,
i. e. 13 days after tumor implantation.
Apoptosis assay. In order to evaluate apoptosis,
mice were euthanized 3 days after administration of
CPA, tumors were extracted and minced with scissors
in physiological solution. The resulting suspension was
filtered through nylon sieve. Upon 3 min centrifuga-
tion, sedimented cells were fixed in Carnois fluid, dyed
with acetocarmine in 40% acetic acid and the number
(percentage) of cells with fragmented nuclei (cells in
the preterminal apoptosis phase) was evaluated using
optical microscope. It should be however pointed out
that this method yields underestimated data since it
does not take into account cells totally disintegrated
to apoptotic corpuscles [23].
Statistical analysis. Statistical analysis was
performed using Wilcoxon — Mann — Whitney non-
parametrical criteria.
rEsults
Fig. 1 illustrates the results of the first experiment
with leukosis Р-388. As can be seen, on the 10th day
after tumor transplantation its size was smaller in mice
treated with SEG in comparison with those, which
did not receive the polysaccharide. During 3–4 days
upon administration of CPA at all modes of treatment
applied, the tumor size has decreased, however it
started to grow again, and this growth was especially
pronounced in mice of the second group, which were
administered CPA alone in a low dose (25 mg/kg). Mice
from the fourth group, which received the same dose
of CPA in combination with SEG revealed smaller size
of tumor up to the 14th day of the experiment than the
mice from the third group treated with higher dose of
CPA alone (50 mg/kg), while on the 14th day the size of
tumor was similar in mice of these two groups. How-
310 Experimental Oncology 28, 308–313, 2006 (December)
ever, afterwards tumors in mice of the fourth group
grew faster than in the animals of group 3 (see Fig. 1).
Thus, application of low dose of CPA after three admin-
istrations of SEG exerted better effect than higher dose
of CPA only during the early phase of the experiment,
while later this advantage disappeared.
fig. 1. Effect of cyclophosphamide and sulfoethylated (1→3)-β-
D glucan on tumor volume in mice with leukosis P-388
*P < 0.05 compared to the untreated mice. # P < 0,05 compared
to the mice treated with CPA 50 mg/kg. $ P < 0.05 compared to
the mice treated with CPA 25 mg/kg + SEG. 7–10 mice in each
group. CPA was administered to mice on the 10th day after tumor
transplantation. SEG was administered to mice on the 3rd, 6th, and
9th days after tumor transplantation (25 mg/kg). Tumor volume
was calculated as the product of multiplication of 3 perpendicular
diameters of tumor node in murine thigh.
Taking this into account, in the following experi-
ment SEG was applied both before (on the 3rd, 6th, and
9th days), as well as after (on the 12th and 15th days)
administration of CPA, which was similarly to the first
experiment injected in a dose of 25 mg/kg on the 10th
day after transplantation of leukosis Р-388. At this
scheme of treatment, therapeutic effect of the com-
bined application of low dose CPA and SEG was simi-
lar to that of the treatment using higher dose of CPA
throughout whole duration of the experiment. Tumor
volume at the end of the monitoring (on the 17th day)
was 0.42 ± 0.125 cm3 (combined CPA and SEG appli-
cation) and 0.48 ± 0.090 cm3 (application of 50 mg/kg
CPA alone), while in mice that received 25 mg/kg CPA
alone tumor volume reached 1.61 ± 0.223 cm3 at the
end of the experiment and all control (untreated) ani-
mals did not survive till the 17th day.
Similar results have been obtained in the experi-
ments involving leukosis L1210/1. Using this tumor
model, CPA in doses of 20 mg/kg and 40 mg/kg
was administered once on the 10th day after tumor
transplantation, while taking into account results of
the preceding experiments, SEG was applied to the
animals, which received 40 mg/kg CPA, seven times
during the whole monitoring — on the 3rd, 6th, 9th, 12th,
15th, 18th, and 21st days after tumor implantation.
As can be seen in Fig. 2, implants of L1210/1 tumor
continued to grow during the first three days after
injection of CPA, while within the following three days
tumor size decreased to one half (at 20 mg/kg dose)
or to one fourth of the initial size (at 40 mg/kg) and the
tumor began to grow again after 4 or 7 days, respec-
tively, with practically similar rate as can be judged
upon the slope of the curves. In contrast to that, in
mice, which were administered CPA and SEG together,
tumors started to diminish immediately upon applica-
tion of CPA and at the end of the experiment, when in
the group of mice treated with CPA alone tumor size
doubled in two days, at a combined administration of
CPA and SEG tumor grew only gradually and the size
increase was insignificant (see Fig. 2).
fig. 2. Effect of CPA and SEG on tumor volume in mice with
leukosis L1210/1
*P < 0,05 compared with the untreated mice.#P < 0,05 compared
with the mice treated with CPA 40 mg/kg + SEG. 7–10 mice in each
group. CPA was administered to mice on the 10th day after tumor
transplantation. SEG was administered to mice on the 3rd, 6th, 9th,
12th, 15th, 18th, and 21rd days after tumor transplantation (25 mg/kg).
Tumor volume was calculated as the product of multiplication of
3 perpendicular diameters of tumor node in murine thigh.
The results of the investigation of the activity of
the cysteine proteases — cathepsins B and L — in the
tumor tissue of mice with implanted leukoses Р-388
and L1210/1 on the 3rd day after CPA injection are pre-
sented in the Table. As can be seen, untreated animals
with either of the tumors revealed practically equal
activities of the individual cathepsins and after the on-
set of treatment activity of cathepsins increased in all
experimental groups, while the observed increase was
more pronounced with Р-388 tumor in comparison with
the L1210/1. The highest cathepsin B and cathepsin L
activity in tumor tissue accompanied with the strongest
inhibition of tumor growth was detected in the animals
treated with CPA and SEG (Table).
Table. ��mor weig�t and cysteine proteases activity in t�mor transp�ants
of �e��osis P388 and L1210/1 (M ± S.D.)
Gro�p of anima�s ��mor weig�t
(g)
Cat�epsin B
(nmo� MC�/min
per mg of protein)
Cat�epsin L (nmo�
MC�/min per mg
of protein)
Le��osis P388
1. Untreated anima�s 1.6 ± 0.11
(100%)
0.16 ± 0.040
(100%)
0.04 ± 0.004
(100%)
2. CP�, 50 mg/�g × 1 1.0 ± 0.13***
(62.5%)
0.51 ± 0.159
(319%)
0.11 ± 0.025*
(275%)
3. CP�, 25 mg/�g ×
1 + S�G, 25 mg/�g × 4
0.6 ± 0.14***
(37.5 %)
0.74 ± 0.060***
(463%)
0.13 ± 0.009***
(325%)
Le��osis 1210/1
1. Untreated anima�s 4.7 ± 0.31
(100%)
0.18 ± 0.017
(100%)
0.04 ± 0.004
(100%)
2. CP�, 40 mg/�g × 1 3.1 ± 0.25**
(66.0%)
0.35 ± 0.041**
(194%)
0.06 ± 0.004***
(150%)
3. CP�, 40 mg/�g ×
1 + S�G, 25 mg/�g × 4
1.8 ± 0.19***##
(38.3%)
0.46 ± 0.022***#
(256%)
0.07 ± 0.002***#
(175%)
Notes: CP was administered to mice on t�e 10t� day after t�mor trans�
p�antation; S�G was administered on t�e 3rd, 6t�, 9t�, and 12t� days after
t�mor transp�antation. In parent�eses: percentage re�ative�y to t�e �n�
treated gro�p. ��e n�mber of anima�s in eac� gro�p was 7–10. *P < 0.05,
**P < 0.01, ***P < 0.001 compared to t�e �ntreated gro�p, #P < 0.05,
##P < 0.01 compared to t�e CP��treated gro�p.
Experimental Oncology 28, 308–313, 2006 (December) 31128, 308–313, 2006 (December) 311December) 311) 311 311
Shape of the curves in Fig. 2 (increase of tumor
volume in the first 2–3 days after CPA injection and the
subsequent regression) may suggest that the therapeutic
effect in the used model is to a significant extent medi-
ated through induction of apoptosis of the tumor cells
[23]. Upon performing the analysis of cell suspensions
obtained from the implanted L1210/1 tumor on the 3rd day
after beginning of the treatment, it has been found that
indeed the predominant portion of the cells contains
picnotic and fragmented nuclei, i.e. are in apoptotic state.
Number of such cells in the tumors of the treated mice
was 3–4 times higher than in the control animals, and in
mice treated with CPA after three SEG administrations
this number was significantly higher than in those that
received CPA alone (Fig. 3, а). As can be seen in Fig. 3, b,
leukosis P388 is also susceptible to CPA-induced apop-
tosis (effect of a combined administration of CPA and
SEG was not studied in this case).
fig. 3. Apoptosis of the tumor cells in mice with L1210/1 and
Р-388 leukoses 3 days after CPA administration: a) L1210/1
leukosis, CPA 40 mg/kg; b) Р-388 leukosis, CPA 50 mg/kg
discussion
The results presented in the study indicate that the
prepared sulfoethylated derivative of yeast cell wall
(1→3)-β-D-glucan possesses an ability to significantly
augment the therapeutic action of CPA in the murine
lymphoid tumors. Multiple applications of SEG revealed
enhanced effect in comparison to its single admin-
istration simultaneously with CPA (data not shown)
or its three-time application prior to injection of CPA.
Since protective activity of the glucans is known to be
mediated via the stimulation of the immune system of
the host rather than through direct interaction of the
polysaccharide with the infective agent [4, 5, 8] it was
not anticipated that application of SEG alone could
affect tumor growth and therefore there was no ex-
perimental group of animals that received SEG alone.
Nevertheless, it was observed that besides potentiating
the therapeutic action of CPA, SEG itself was capable
of exerting inhibitory effect on tumor growth: upon its
three-time administration a certain retardation of the
implanted tumor growth was observed already prior to
the onset of cytostatics treatment (Fig. 1 and 2). The
biological effects of (1→3)-β-D-glucans are initiated
through their recognition and binding to the specific
cell surface receptors: CR3 [24] lactosylceramide [25]
scavenger receptors [26] and dectin-1 [27]. Besides
monocytes and macrophages, presence of β-D-glucan
receptors has been established also on the neutrophiles
[25, 28], NK-cells [29–31], fibroblasts [32] and some
tumor cells [33]. Previously we demonstrated that SEG
alone suppressed growth of susceptible to CPA murine
lymphosarcoma [18].
What mechanism could be involved in its inhibitory
activity on tumor growth? The antitumor action of SEG
could be ascribed to the ability of yeast β-D-glucan
to stimulate release of tumor necrosis factor TNF-α
from monocytes/macrophages, as we have previously
demonstrated [34]. Some indications of the additional
mechanism are provided by the results of the current
investigation of the mechanism of death of the tumor
cells and of the activities of cathepsins B and L in tumor
tissues of the treated animals. Increased content of
apoptotic cells in the tumors subjected to the treatment
by a combination of CPA and SEG in comparison with
those treated with CPA alone implies that SEG is able
to induce apoptosis in susceptible cells. Augmented
activity of the lysosomal proteases in tumor tissues is
justified if these enzymes take part in the induction or
realization of apoptosis. However as a more reason-
able explanation appears to be a suggestion that the
observed post-treatment enhancement of activity of
cathepsins B and L in tumor tissues results not from
their activation in tumor cells, but rather is due to the
increased number of macrophages infiltrating the
tumor cells to resorb dying apoptotic tumor cells. We
are currently attempting to verify this hypothesis.
In conclusion, combined application of CPA and
SEG resulted in the enhanced apoptosis of the leu-
kemic cells and was accompanied by a substantial
increase of the activity of cysteine proteases cathep-
sins B and L in tumor tissues.The results obtained in
the present work demonstrate that at addition of SEG,
therapeutic effect of a one-half reduced dose of CPA is
equal or higher than that of the full dose (Fig. 1 and 2).
Thus, utilization of this polysaccharide BRM might
potentially enhance efficiency of antitumor therapy
with standard cytostatics without a need of substan-
tial increase of their dosage and hence avoiding their
toxic side-effects. These results together with the
previously published data on the beneficial effect of
administration of the derivatives of yeast β-D-glucan
in the antitumor therapy of various types of cancer
indicate possible utilization of these preparations
especially at a combined application with classical
anticancer agents.
312 Experimental Oncology 28, 308–313, 2006 (December)
acknowlEdgmEnts
Financial support from the INTAS grant 2001-0592
and from the VEGA grant 2/4143/26 of the Slovak
Academy of Sciences and Ministry of Education of
Slovak Republic is gratefully acknowledged.
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Experimental Oncology 28, 308–313, 2006 (December) 31328, 308–313, 2006 (December) 313December) 313) 313 313
ПОТЕНЦИРУЮЩИЙ ЭФФЕКТ СУЛЬФОЭТИЛИРОВАННОГО
(1→3)-β-d-ГЛИКАНА НА ПРОТИВООПУХОЛЕВУЮ АКТИВНОСТЬ
ЦИКЛОФОСФАНА ПРИ ЭКСПЕРИМЕНТАЛЬНЫХ ЛЕЙКОЗАХ
МЫШЕЙ
Цель: одним из перспективных методов противоопухолевой терапии является использование так называемых модификаторов
биологического ответа, применяемых для активации эндогенных противоопухолевых механизмов и комбинируемых с
классическими цитотоксическими препаратами. Цель данной работы — исследование эффекта сульфоэтилированного (1→
3)-β-D-гликана (SEG) при лечении экспериментальных лейкозов мышей в комбинации с циклофосфаном (СРА) и его способность
модулировать активность лизосомных ферментов в опухолевой ткани. Материалы и методы: солидные формы перевиваемых
лейкозов мышей Р388 и L1210/1 трансплантировали мышам-самцам DBA/2. Для лечения использовали СРА (Биохимик,
Саранск, Россия) и его комбинацию с SEG (Институт химии Словацкой Академии Наук, Братислава, Словакия). СРА вводили
внутрибрюшинно на 10 сут после перевивки опухолей; SEG вводили внутрибрюшинно начиная с 3 сут после трансплантации
лейкозов с интервалом в 3 дня. Терапевтический эффект оценивали путем измерения объема солидной опухоли. Активность
цистеиновых протеаз — катепсинов В и L — определяли флюориметрическим методом, используя флюоресцентные субстраты
Z-Arg-Arg-MCA и Z-Phe-Arg-MCA (Sigma, США). Апоптоз оценивали по результатам подсчета клеток с фрагментированными
ядрами в световом микроскопе. Результаты: в работе показано, что использование SEG приводит к торможению опухолевого
роста и потенцирует терапевтический эффект СРА, особенно при повторном введении в течение всего лечения. В сочетании с SEG
терапевтический эффект половинной дозы СРА равнозначен или превышает действие полной дозы цитостатика. Воздействие
СРА и SEG на использованные в исследовании опухоли реализуется в основном через индукцию апоптоза и сопровождается
существенным повышением активности цистеиновых протеаз катепсинов В и L в опухолевой ткани. Наиболее высокая
активность катепсинов В и L сопровождается максимальным подавлением опухолевого роста. Предположительно это обусловлено
инфильтрацией опухолевой ткани макрофагами с высоким содержанием вышеназванных ферментов, где они фагоцитируют
клетки в апоптозе. Выводы: использование сульфоэтилированного (1→3)-β-D-гликана, нового модификатора биологического
ответа дает возможность существенно повысить эффективность противоопухолевой терапии стандартными цитостатиками без
повышения их дозы, что позволяет избежать побочных эффектов данных препаратов.
Ключевые слова: лейкозы мышей, катепсины, дрожжевые гликаны, циклофосфан.
Copyrig�t © �xperimenta� Onco�ogy, 2006
|
| id | nasplib_isofts_kiev_ua-123456789-137933 |
| institution | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| issn | 1812-9269 |
| language | English |
| last_indexed | 2025-12-07T17:02:45Z |
| publishDate | 2006 |
| publisher | Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| record_format | dspace |
| spelling | Khalikova, T.A. Korolenko, T.A. Zhanaeva, S.Ya. Kaledin, V.I. Kogan, G. 2018-06-17T19:16:38Z 2018-06-17T19:16:38Z 2006 Enhancing effect of new biological response modifier sulfoethylated (1→3)-β-D-glucan on antitumor activity of cyclophosphamide in the treatment / T.A. Khalikova, T.A. Korolenko, S.Ya. Zhanaeva, V.I. Kaledin, G. Kogan // Experimental Oncology. — 2006. — Т. 28, № 4. — С. 308-313. — Бібліогр.: 34 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/137933 Aim: One of the advanced methodologies of the tumor therapy is the application of the so-called biological response modifiers used for activation of the endogenous antitumor mechanisms and combined with classical cytotoxic agents. The aim of this work was the investigation of the effect of sulfoethylated (1→3)-β-D-glucan (SEG) in the treatment of experimental murine leukoses in combination with cyclophosphamide (CPA) and its ability to modulate the activity of lysosomal enzymes in tumor tissues. Materials and Methods: The solid forms of inoculated murine leukoses P388 and L1210/1 were transplantated to male DBA/2 mice. The therapy was performed by treating animals with CPA (Biokhimik, Saransk, Russia) alone or in combination with SEG (Institute of Chemistry, Slovak Academy of Sciences, Slovakia). CPA was administered in saline as a single intraperitoneal (ip) injection on the 10th day after tumor transplantation; SEG was administered to mice ip 3 days after tumor transplantation with the intervals in 3 days. The therapy effect was estimated by measuring of solid tumor volume. Activity of the cysteine proteases — cathepsins B and L — was measured fluorometrically using fluorescent substrates Z-Arg-Arg-MCA and Z-Phe-Arg-MCA (Sigma, USA), respectively. The apoptosis was estimated evaluating the number of cells with fragmented nuclei using optical microscope. Results: It has been demonstrated that application SEG leads to inhibition of tumor growth and potentiates therapeutic action of CPA, especially at repeated administrations during the whole treatment/observation At addition of SEG, therapeutic effect of a one-half reduced dose of CPA is equal or higher than that of the full dose. Therapeutic action of CPA and SEG on the studied tumors is realized predominantly through induction of apoptosis and is accompanied by a substantial increase of the activity of cysteine proteases cathepsins B and L in tumor tissues. The highest cathepsin B and cathepsin L activity in tumor tissue accompanied with the strongest inhibition of tumor growth. It is suggested that this phenomenon is due to the infiltration of the macrophages rich in the named enzymes into the tumor, where they phagocytize the apoptotic cells and tissue debris. Conclusion: Utilization of this polysaccharide BRM, sulfoethylated (1→3)-β-D-glucan, might potentially enhance efficiency of antitumor therapy with standard cytostatics without a need of substantial increase of their dosage and hence avoiding their toxic side-effects. Цель: одним из перспективных методов противоопухолевой терапии является использование так называемых модификаторов
 биологического ответа, применяемых для активации эндогенных противоопухолевых механизмов и комбинируемых с
 классическими цитотоксическими препаратами. Цель данной работы — исследование эффекта сульфоэтилированного (1→
 3)-β-D-гликана (SEG) при лечении экспериментальных лейкозов мышей в комбинации с циклофосфаном (СРА) и его способность
 модулировать активность лизосомных ферментов в опухолевой ткани. Материалы и методы: солидные формы перевиваемых
 лейкозов мышей Р388 и L1210/1 трансплантировали мышам-самцам DBA/2. Для лечения использовали СРА (Биохимик,
 Саранск, Россия) и его комбинацию с SEG (Институт химии Словацкой Академии Наук, Братислава, Словакия). СРА вводили
 внутрибрюшинно на 10 сут после перевивки опухолей; SEG вводили внутрибрюшинно начиная с 3 сут после трансплантации
 лейкозов с интервалом в 3 дня. Терапевтический эффект оценивали путем измерения объема солидной опухоли. Активность
 цистеиновых протеаз — катепсинов В и L — определяли флюориметрическим методом, используя флюоресцентные субстраты
 Z-Arg-Arg-MCA и Z-Phe-Arg-MCA (Sigma, США). Апоптоз оценивали по результатам подсчета клеток с фрагментированными
 ядрами в световом микроскопе. Результаты: в работе показано, что использование SEG приводит к торможению опухолевого
 роста и потенцирует терапевтический эффект СРА, особенно при повторном введении в течение всего лечения. В сочетании с SEG
 терапевтический эффект половинной дозы СРА равнозначен или превышает действие полной дозы цитостатика. Воздействие
 СРА и SEG на использованные в исследовании опухоли реализуется в основном через индукцию апоптоза и сопровождается
 существенным повышением активности цистеиновых протеаз катепсинов В и L в опухолевой ткани. Наиболее высокая
 активность катепсинов В и L сопровождается максимальным подавлением опухолевого роста. Предположительно это обусловлено
 инфильтрацией опухолевой ткани макрофагами с высоким содержанием вышеназванных ферментов, где они фагоцитируют
 клетки в апоптозе. Выводы: использование сульфоэтилированного (1→3)-β-D-гликана, нового модификатора биологического
 ответа дает возможность существенно повысить эффективность противоопухолевой терапии стандартными цитостатиками без
 повышения их дозы, что позволяет избежать побочных эффектов данных препаратов. Financial support from the INTAS grant 2001-0592
 and from the VEGA grant 2/4143/26 of the Slovak
 Academy of Sciences and Ministry of Education of
 Slovak Republic is gratefully acknowledged. en Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України Experimental Oncology Original contributions Enhancing effect of new biological response modifier sulfoethylated (1→3)-β-D-glucan on antitumor activity of cyclophosphamide in the treatment Потенцирующий эффект сульфоэтилированного (1→3)-β-d-гликана на противоопухолевую активность циклофосфана при экспериментальных лейкозах мышей Article published earlier |
| spellingShingle | Enhancing effect of new biological response modifier sulfoethylated (1→3)-β-D-glucan on antitumor activity of cyclophosphamide in the treatment Khalikova, T.A. Korolenko, T.A. Zhanaeva, S.Ya. Kaledin, V.I. Kogan, G. Original contributions |
| title | Enhancing effect of new biological response modifier sulfoethylated (1→3)-β-D-glucan on antitumor activity of cyclophosphamide in the treatment |
| title_alt | Потенцирующий эффект сульфоэтилированного (1→3)-β-d-гликана на противоопухолевую активность циклофосфана при экспериментальных лейкозах мышей |
| title_full | Enhancing effect of new biological response modifier sulfoethylated (1→3)-β-D-glucan on antitumor activity of cyclophosphamide in the treatment |
| title_fullStr | Enhancing effect of new biological response modifier sulfoethylated (1→3)-β-D-glucan on antitumor activity of cyclophosphamide in the treatment |
| title_full_unstemmed | Enhancing effect of new biological response modifier sulfoethylated (1→3)-β-D-glucan on antitumor activity of cyclophosphamide in the treatment |
| title_short | Enhancing effect of new biological response modifier sulfoethylated (1→3)-β-D-glucan on antitumor activity of cyclophosphamide in the treatment |
| title_sort | enhancing effect of new biological response modifier sulfoethylated (1→3)-β-d-glucan on antitumor activity of cyclophosphamide in the treatment |
| topic | Original contributions |
| topic_facet | Original contributions |
| url | https://nasplib.isofts.kiev.ua/handle/123456789/137933 |
| work_keys_str_mv | AT khalikovata enhancingeffectofnewbiologicalresponsemodifiersulfoethylated13βdglucanonantitumoractivityofcyclophosphamideinthetreatment AT korolenkota enhancingeffectofnewbiologicalresponsemodifiersulfoethylated13βdglucanonantitumoractivityofcyclophosphamideinthetreatment AT zhanaevasya enhancingeffectofnewbiologicalresponsemodifiersulfoethylated13βdglucanonantitumoractivityofcyclophosphamideinthetreatment AT kaledinvi enhancingeffectofnewbiologicalresponsemodifiersulfoethylated13βdglucanonantitumoractivityofcyclophosphamideinthetreatment AT kogang enhancingeffectofnewbiologicalresponsemodifiersulfoethylated13βdglucanonantitumoractivityofcyclophosphamideinthetreatment AT khalikovata potenciruûŝiiéffektsulʹfoétilirovannogo13βdglikananaprotivoopuholevuûaktivnostʹciklofosfanapriéksperimentalʹnyhleikozahmyšei AT korolenkota potenciruûŝiiéffektsulʹfoétilirovannogo13βdglikananaprotivoopuholevuûaktivnostʹciklofosfanapriéksperimentalʹnyhleikozahmyšei AT zhanaevasya potenciruûŝiiéffektsulʹfoétilirovannogo13βdglikananaprotivoopuholevuûaktivnostʹciklofosfanapriéksperimentalʹnyhleikozahmyšei AT kaledinvi potenciruûŝiiéffektsulʹfoétilirovannogo13βdglikananaprotivoopuholevuûaktivnostʹciklofosfanapriéksperimentalʹnyhleikozahmyšei AT kogang potenciruûŝiiéffektsulʹfoétilirovannogo13βdglikananaprotivoopuholevuûaktivnostʹciklofosfanapriéksperimentalʹnyhleikozahmyšei |