Effect of antitumor drugs in low concentrations on the biological, immunophenotypic and cytogenetic characteristics of human colon cancer cells in vitro
Objective: To estimate the impact of the low-dose anticancer drugs (ACD) with the different mechanisms of action and human interferon (IFN) alpha 2b on the biological properties, immunophenotypic and cytogenetic characteristics of colon cancer cells in vitro. Materials and Methods: The study was per...
Saved in:
| Date: | 2017 |
|---|---|
| Main Authors: | , , , , , , , |
| Format: | Article |
| Language: | English |
| Published: |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2017
|
| Series: | Experimental Oncology |
| Subjects: | |
| Online Access: | https://nasplib.isofts.kiev.ua/handle/123456789/137627 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Journal Title: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Cite this: | Effect of antitumor drugs in low concentrations on the biological, immunophenotypic and cytogenetic characteristics of human colon cancer cells in vitro / N. Bezdieniezhnykh, O. Kovalova, O. Lykhova, R. Kocherga, A. Vorontsova, V. Zhylchuk, G. Maksimyak, Yu. Kudryavets // Experimental Oncology. — 2017 — Т. 39, № 1. — С. 17-24. — Бібліогр.: 27 назв. — англ. |
Institution
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
nasplib_isofts_kiev_ua-123456789-137627 |
|---|---|
| record_format |
dspace |
| spelling |
nasplib_isofts_kiev_ua-123456789-1376272025-02-23T17:10:05Z Effect of antitumor drugs in low concentrations on the biological, immunophenotypic and cytogenetic characteristics of human colon cancer cells in vitro Bezdieniezhnykh, N. Kovalova, O. Lykhova, O. Kocherga, R. Vorontsova, A. Zhylchuk, V. Maksimyak, G. Kudryavets, Yu. Original contributions Objective: To estimate the impact of the low-dose anticancer drugs (ACD) with the different mechanisms of action and human interferon (IFN) alpha 2b on the biological properties, immunophenotypic and cytogenetic characteristics of colon cancer cells in vitro. Materials and Methods: The study was performed on human colon cancer cell lines COLO 205, HT-29 and 3C-P treated with ACD and IFN in subtoxic concentrations. Expression of CD44, N-cadherin, vimentin, β-catenin, ERCC1 and Slug was assessed by immunocytochemical method. Using cytogenetic analysis, the numbers of mitoses, cells with micronuclei, apoptotic cells and cells with nuclear protrusions were studied. Results: The prolonged exposure (up to 30 days) of colon cancer cells to low-dose ACD (0.2–0.5 µg/ml cisplatin and 0.1–0.2 µg/ml irinotecan) in combination with IFN (500–1000 IU/ml) led to 37-fold decreased colony-forming activity of these cell and 10-fold reduction of the number of cells expressing mesenchymal protein markers (N-cadherin, vimentin). Also, in COLO 205 cells treated with ACD and IFN the number of SLUG- and CD44-positive cells decreased by 92 and by 85%, respectively. Long-term cultivation of HT-29 cells in the presence of cisplatin and IFN resulted in 5-fold suppression of ERCC1 expression. The cytogenetic analysis has shown that the ACD, IFN and their combinations in subtoxic concentrations caused significant genotoxic effect, suppression of cell proliferation and accumulation of cells with micronuclei. The sensitivity of colon cancer cells to ACD in standard cytotoxic concentrations did not change after prolonged low-dose exposure. Conclusion: The data showed that the prolonged action of the low doses of ACD on human colon cancer cells resulted in the suppression of cell proliferation, colony-forming activity in soft agar, expression of epithelialmesenchymal transition-associated markers and significant cytogenetic changes. 2017 Article Effect of antitumor drugs in low concentrations on the biological, immunophenotypic and cytogenetic characteristics of human colon cancer cells in vitro / N. Bezdieniezhnykh, O. Kovalova, O. Lykhova, R. Kocherga, A. Vorontsova, V. Zhylchuk, G. Maksimyak, Yu. Kudryavets // Experimental Oncology. — 2017 — Т. 39, № 1. — С. 17-24. — Бібліогр.: 27 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/137627 en Experimental Oncology application/pdf Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| topic |
Original contributions Original contributions |
| spellingShingle |
Original contributions Original contributions Bezdieniezhnykh, N. Kovalova, O. Lykhova, O. Kocherga, R. Vorontsova, A. Zhylchuk, V. Maksimyak, G. Kudryavets, Yu. Effect of antitumor drugs in low concentrations on the biological, immunophenotypic and cytogenetic characteristics of human colon cancer cells in vitro Experimental Oncology |
| description |
Objective: To estimate the impact of the low-dose anticancer drugs (ACD) with the different mechanisms of action and human interferon (IFN) alpha 2b on the biological properties, immunophenotypic and cytogenetic characteristics of colon cancer cells in vitro. Materials and Methods: The study was performed on human colon cancer cell lines COLO 205, HT-29 and 3C-P treated with ACD and IFN in subtoxic concentrations. Expression of CD44, N-cadherin, vimentin, β-catenin, ERCC1 and Slug was assessed by immunocytochemical method. Using cytogenetic analysis, the numbers of mitoses, cells with micronuclei, apoptotic cells and cells with nuclear protrusions were studied. Results: The prolonged exposure (up to 30 days) of colon cancer cells to low-dose ACD (0.2–0.5 µg/ml cisplatin and 0.1–0.2 µg/ml irinotecan) in combination with IFN (500–1000 IU/ml) led to 37-fold decreased colony-forming activity of these cell and 10-fold reduction of the number of cells expressing mesenchymal protein markers (N-cadherin, vimentin). Also, in COLO 205 cells treated with ACD and IFN the number of SLUG- and CD44-positive cells decreased by 92 and by 85%, respectively. Long-term cultivation of HT-29 cells in the presence of cisplatin and IFN resulted in 5-fold suppression of ERCC1 expression. The cytogenetic analysis has shown that the ACD, IFN and their combinations in subtoxic concentrations caused significant genotoxic effect, suppression of cell proliferation and accumulation of cells with micronuclei. The sensitivity of colon cancer cells to ACD in standard cytotoxic concentrations did not change after prolonged low-dose exposure. Conclusion: The data showed that the prolonged action of the low doses of ACD on human colon cancer cells resulted in the suppression of cell proliferation, colony-forming activity in soft agar, expression of epithelialmesenchymal transition-associated markers and significant cytogenetic changes. |
| format |
Article |
| author |
Bezdieniezhnykh, N. Kovalova, O. Lykhova, O. Kocherga, R. Vorontsova, A. Zhylchuk, V. Maksimyak, G. Kudryavets, Yu. |
| author_facet |
Bezdieniezhnykh, N. Kovalova, O. Lykhova, O. Kocherga, R. Vorontsova, A. Zhylchuk, V. Maksimyak, G. Kudryavets, Yu. |
| author_sort |
Bezdieniezhnykh, N. |
| title |
Effect of antitumor drugs in low concentrations on the biological, immunophenotypic and cytogenetic characteristics of human colon cancer cells in vitro |
| title_short |
Effect of antitumor drugs in low concentrations on the biological, immunophenotypic and cytogenetic characteristics of human colon cancer cells in vitro |
| title_full |
Effect of antitumor drugs in low concentrations on the biological, immunophenotypic and cytogenetic characteristics of human colon cancer cells in vitro |
| title_fullStr |
Effect of antitumor drugs in low concentrations on the biological, immunophenotypic and cytogenetic characteristics of human colon cancer cells in vitro |
| title_full_unstemmed |
Effect of antitumor drugs in low concentrations on the biological, immunophenotypic and cytogenetic characteristics of human colon cancer cells in vitro |
| title_sort |
effect of antitumor drugs in low concentrations on the biological, immunophenotypic and cytogenetic characteristics of human colon cancer cells in vitro |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2017 |
| topic_facet |
Original contributions |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/137627 |
| citation_txt |
Effect of antitumor drugs in low concentrations on the biological, immunophenotypic and cytogenetic characteristics of human colon cancer cells in vitro / N. Bezdieniezhnykh, O. Kovalova, O. Lykhova, R. Kocherga, A. Vorontsova, V. Zhylchuk, G. Maksimyak, Yu. Kudryavets // Experimental Oncology. — 2017 — Т. 39, № 1. — С. 17-24. — Бібліогр.: 27 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
AT bezdieniezhnykhn effectofantitumordrugsinlowconcentrationsonthebiologicalimmunophenotypicandcytogeneticcharacteristicsofhumancoloncancercellsinvitro AT kovalovao effectofantitumordrugsinlowconcentrationsonthebiologicalimmunophenotypicandcytogeneticcharacteristicsofhumancoloncancercellsinvitro AT lykhovao effectofantitumordrugsinlowconcentrationsonthebiologicalimmunophenotypicandcytogeneticcharacteristicsofhumancoloncancercellsinvitro AT kochergar effectofantitumordrugsinlowconcentrationsonthebiologicalimmunophenotypicandcytogeneticcharacteristicsofhumancoloncancercellsinvitro AT vorontsovaa effectofantitumordrugsinlowconcentrationsonthebiologicalimmunophenotypicandcytogeneticcharacteristicsofhumancoloncancercellsinvitro AT zhylchukv effectofantitumordrugsinlowconcentrationsonthebiologicalimmunophenotypicandcytogeneticcharacteristicsofhumancoloncancercellsinvitro AT maksimyakg effectofantitumordrugsinlowconcentrationsonthebiologicalimmunophenotypicandcytogeneticcharacteristicsofhumancoloncancercellsinvitro AT kudryavetsyu effectofantitumordrugsinlowconcentrationsonthebiologicalimmunophenotypicandcytogeneticcharacteristicsofhumancoloncancercellsinvitro |
| first_indexed |
2025-11-24T02:34:30Z |
| last_indexed |
2025-11-24T02:34:30Z |
| _version_ |
1849637387065360384 |
| fulltext |
Experimental Oncology 39, 17–24, 2017 (March) 17
EFFECT OF ANTITUMOR DRUGS IN LOW CONCENTRATIONS
ON THE BIOLOGICAL, IMMUNOPHENOTYPIC AND CYTOGENETIC
CHARACTERISTICS OF HUMAN COLON CANCER CELLS in vitro
N. Bezdieniezhnykh1, *, O. Kovalova1, O. Lykhova1, R. Kocherga1, A. Vorontsova1, V. Zhylchuk2,
G. Maksimyak2, Yu. Kudryavets1
1R.E. Kavetsky Institute of Experimental Pathology, Oncology and Radiobiology, NAS of Ukraine, Kyiv 03022, Ukraine
2Rivne Regional Oncology Center, Rivne 33003, Ukraine
Objective: To estimate the impact of the low-dose anticancer drugs (ACD) with the different mechanisms of action and human interferon
(IFN) alpha 2b on the biological properties, immunophenotypic and cytogenetic characteristics of colon cancer cells in vitro. Materials
and Methods: The study was performed on human colon cancer cell lines COLO 205, HT-29 and 3C-P treated with ACD and IFN
in subtoxic concentrations. Expression of CD44, N-cadherin, vimentin, β-catenin, ERCC1 and Slug was assessed by immunocyto-
chemical method. Using cytogenetic analysis, the numbers of mitoses, cells with micronuclei, apoptotic cells and cells with nuclear protru-
sions were studied. Results: The prolonged exposure (up to 30 days) of colon cancer cells to low-dose ACD (0.2–0.5 µg/ml cisplatin and
0.1–0.2 µg/ml irinotecan) in combination with IFN (500–1000 IU/ml) led to 37-fold decreased colony-forming activity of these cell and
10-fold reduction of the number of cells expressing mesenchymal protein markers (N-cadherin, vimentin). Also, in COLO 205 cells
treated with ACD and IFN the number of SLUG- and CD44-positive cells decreased by 92 and by 85%, respectively. Long-term cultiva-
tion of HT-29 cells in the presence of cisplatin and IFN resulted in 5-fold suppression of ERCC1 expression. The cytogenetic analysis
has shown that the ACD, IFN and their combinations in subtoxic concentrations caused significant genotoxic effect, suppression of cell
proliferation and accumulation of cells with micronuclei. The sensitivity of colon cancer cells to ACD in standard cytotoxic concentrations
did not change after prolonged low-dose exposure. Conclusion: The data showed that the prolonged action of the low doses of ACD on hu-
man colon cancer cells resulted in the suppression of cell proliferation, colony-forming activity in soft agar, expression of epithelial-
mesenchymal transition-associated markers and significant cytogenetic changes.
Key Words: colon cancer, tumor cells, epithelial-mesenchymal transition, malignant phenotype, anticancer drugs, ERCC1, micronuclei.
In clinical practice the standard chemotherapy
of cancer patients is limited by its high toxicity and
the development of anticancer drug (ACD) resistance.
Therefore, it is considered important to implement
metronomic chemotherapy regimen, in which the ACD
are administered in low doses at short intervals [1–4].
The ACD doses vary in rather wide range and depend
on the ACD, tumor type and stage etc., but such doses
are always much lower (5–20 times) than the doses
used in standard chemotherapy. Metronomic chemo-
therapy regimen allows reducing the level of the overall
toxicity of chemotherapy and side effects. It is known
that low doses of ACD had no cytostatic effect toward
tumor cells, but retain high activity as inhibitors of neo-
angiogenesis [5]. It is believed that metronomic low-
dose regimen (MLDR) is targeted not against tumor
cells, which are genetically unstable and form resistant
to chemotherapy phenotypic variants, but against ge-
netically stable and highly sensitive to chemotherapy
normal endothelial cells required for tumor growth
[6, 7]. The authors consider following mechanisms
of MLDR action: its antiproliferative effect against en-
dothelial cells, blockage of VEGF, inhibiting of matrix
metalloprotei nases and tissue plasminogen activator,
and increased endogenous thrombospondin 1 result-
ing in apoptosis of CD36-positive endothelial cells
[8]. By our opinion, this list of MLDR mechanisms
could be continued, because even in the absence
of direct cytotoxic effect of ACD one could not exclude
its effect on tumor cells by modifying their biological
characteristics. It is logical to assume that the ACD
as a biologically active substance will affect not only
tumor cells through induction of cell death, but also
through modification of their characteristics, includ-
ing the reversal of their malignancy. In particular, our
previous studies have shown that type I interferon (IFN)
at the doses that do not affect the growth acti vity,
proliferative potential and viability of cells in gene ral,
exerts long-term effects on tumor cells caused by sup-
pression of their malignant potential through inhibition
of proteins and transcription factors associated with
epithelial-mesenchymal transition (EMT) [9, 10]. Bio-
logical characteristics and immunophenotype of tumor
cells to a large extent determine the rate of tumor
growth, its drug sensiti vity, and intensity of metasta-
sis. In turn, the EMT plays a key role in tumorigenicity,
metastatic cascade and drug resistance of tumors.
Accordingly, the assessment of MLDR impact into bio-
logical characteristics of tumor cells and the expres-
sion of proteins involved in EMT regulation is an actual
problem. In our previous study [3], a positive result
of MLDR in the treatment of patients with metastatic
colorectal cancer associated with its antiangiogenic
therapeutic effect was demonstrated. It was shown
that the mostly effective MLDR schemes were based
on the use of platinum drugs and camptothecin deriva-
Submitted: February 18, 2017.
*Correspondence: E-mail: beznalia@mail.ru
Abbreviations used: ACD — anticancer drugs; CFA — colony-form-
ing activity; CP — cisplatin; EMT — epithelial-mesenchymal transi-
tion; IFN — interferon; IT — irinotecan; FBS — fetal bovine serum;
MLDR — metronomic low-dose regimen; MN — micronuclei.
Exp Oncol 2017
39, 1, 17–24
ORIGINAL CONTRIBUTIONS
18 Experimental Oncology 39, 17–24, 2017 (March)
tives in combination with IFN, and resulted in disease
stabilization, a significant increase in survival period
of patients with metastatic colorectal cancer and pro-
vided a relatively high quality of their life.
These important clinical results of MLDR use and
new data about activity of IFN as an inhibitor of EMT
in tumor cells were the prerequisite for the in vitro study
of influence of IFN and ACD with different mechanisms
of action, including derivatives of camptothecin and
platinum drugs, on the biological characteristics and
immunophenotypic profile of colon cancer cells. In this
case, special attention was focused on the influence
of ACD at the doses that corresponded to these
in the blood of patients treated with MLDR — much
lower than the IC50, on EMT-associated proteins,
and consequently, on the biological and cytogenetic
characteristics of tumor cells.
MATERIALS AND METHODS
Cell lines. Cells of human colon cancer (COLO 205,
HT-29 cell lines) and new stable cell line from primary
culture of ascitic fluid of the patient with colorectal
adenocarcinoma generated by 20 passages in vitro
(3С-Р cell line) were obtained from the Bank of Cell
Lines of Human and Animal Tissues, R.E. Kavetsky In-
stitute of Experimental Pathology, Oncology and Radio-
biology, NAS of Ukraine (Kyiv, Ukraine). COLO 205 cells
were maintained in RPMI-1640 medium (Sigma, USA)
supplemented with 10% fetal bovine serum (FBS)
(PAA, Austria) and 40 μg/ml gentamicin (Sigma, USA).
HT-29 and 3C-P cell lines were maintained in plastic
flasks (TPP, Italy) in DMEM with 4 mmol/l L-glutamine
(PAA, Austria) and 10% of FBS (PAA, Austria) and
40 μg/ml gentamicin (Sigma, USA). The cells were
incubated at 37 °C in a humidified atmosphere with 5%
CO2. Cells were detached with EDTA solution (Sigma,
USA) and cultured by the standard method [11].
Modified cell sublines were obtained in our labora-
tory by treatment of COLO 205, HT-29 and 3С-Р cells
with subtoxic doses (10–20 times lower than ІС50 es-
tablished for each culture) of cisplatin (CP), irinotecan
(IT) and human recombinant IFN-α 2b for 30 days. The
final concentration of drugs were as following: platinum
drugs (cisplatin) — 0.2–0.5 μg/ml (ІС50 — 8 μg/ml),
camptothe cin derivative (irinotecan) — 0.1–0.2 μg/ml
(ІС50 — 2.5 μg/ml), and IFN (laferobion) — 500–
1000 IU/ml (ІС50 — 10,000 IU/ml).
Analysis of growth kinetic of tumor cells. Stu-
died cells (1•104 cells/ml) were seeded in 24-wells
plates (TPP, Italy) in 1.5 ml of complete DMEM
or RPMI-1640 and incubated for 8 days. Every 24 h the
cells were detached with EDTA solution (BioTestMed,
Ukraine) from three wells at each time point, stained
with trypan blue solution (HyClone, USA) and counted
in hemocytometer.
Semisolid agar colony assay. The assay was
conducted in parallel wells of 6-well plates. Base lay-
ers of 4 ml of 0.5% agar (Difco, USA) were prepared
on RPMI-1640 medium with 10% FBS. A 2 ml over-
layer of 0.33% agar containing studied cells (2•103)
in DMEM medium with 10% FBS and 40 μg/ml genta-
micin was then applied and the plates were incubated
at 37 °C in a humidified atmosphere containing 5% CO2.
After 14 days of the incubation, the plating colonies
were stained with 0.2% methylthiasoliletetrazolium
solution (Sigma, USA) at 37 °C for 2 h. After that, the
number of colonies was counted in each well using bi-
nocular magnifiers BM-51–2 (LOMO, Russia). Colony-
forming activity (CFA) of studied cells was determined
by the formula:
CFA = (A/B) • 100%,
wherein A — the number of colonies per well; B —
the number of cells planted in semisolid agar per well.
immunocytochemical assay. Phenotypic
changes of the studied cells after their prolonged
treatment with ACD and IFN were determined by count-
ing the cells expressing CD44, N-cadherin, vimentin,
β-catenin, ERCC1 and Slug. Control and modified cells
were placed on the glass slides (cytospin samples) and
the analysis was made by the standard method with the
use of monoclonal antibodies against CD44 (Diagnos-
tic BioSystems, USA), N-cadherin, vimentin, β-catenin
(ThermoScientific, USA), ERCC1 (Аbcam, USA), Slug
(GenTex, USA). Ultra Vision LPValue Detection system
(Thermo Scientific, USA) was used to visualize the
reaction of antigen/ antibody. DAB Quanto (3,3-di-
aminobenzydyn) (Thermo Scientific, USA) was used
as a chromogen. Stained preparations were analyzed
using binocular Axiostar Plus microscope and immer-
sion oil at the magnification × 100 and photographed
using camera Canon PowerShot G5. Expression
of mentioned proteins was evaluated semiquantitavely
using H-score system accounting the number and
intensity of the stained cells. The percentage of the
stained cells was multiplied by score number corre-
sponding to the staining intensity (0 = none, 1 = weak,
2 = moderate, 3 = strong). The resulting score ranged
from 0 (no stained cells) to 300 (diffuse intense stain-
ing of cells), and H-score was calculated as (percent-
age of cells stained at intensity 1×1) + (percentage
of cells stained at intensity 2×2) + (percentage of cells
stained at intensity 3×3) [12].
Cytogenetic analysis. Studied cells were de-
tached with EDTA solution and incubated for 40 min
in hypotonic solution of KCl (0.54%) (Reahim, Ukraine)
at 37 °C. After that the cells were fixed with methanol
(Reahim, Ukraine) and acetic acid (Chemlaborreactiv,
Ukraine) at a ratio of 3:1. The procedure of fixing was
performed in triplicate. Cell suspension in the fixing
solution was dripped on the cold wet glass slides and
dried. Samples were stained with Giemsa stain (Merck,
Germany). The cytogenetic preparations were ana-
lyzed using binocular Axiostar Plus microscope (Сarl
Zeiss, Germany) at the magnification × 100 and pho-
tographed using camera Canon PowerShot G5 (UK).
The following characteristics were studied: number
of mitoses, cells with micronuclei (MN), apoptotic
cells and cells with nuclear protrusions. These indica-
tors were counted per 1000 cells, and the results are
expressed in per mille (‰) [13].
Experimental Oncology 39, 17–24, 2017 (March) 19
Statistical analysis. Statistical analysis of the
data was performed by the Student t-test using Sta-
tistica 6.0 program. The data are presented as M ±
SD. Statistical significance was set at p ≤ 0.05.
RESULTS AND DISCUSSION
To investigate the impact of subtoxic doses
of cytostatics on colorectal cancer cells, as potential
modifiers of their malignant phenotype, we have used
the ACD usually applied in the clinic for the treatment
of patients with colorectal cancer — camptothecin
derivative and a platinum drug. These drugs were
used in mono- or combined regimes because they
have different mechanisms of action and could exert
a synergistic effect [14, 15].
For example, CP is a DNA crosslinker that changes
the DNA structure and functions, inhibits its synthesis,
thereby leading to cell death. IT is a semi-synthetic
derivative of camptothecin that is a specific inhibitor
of topoisomerase-1, and causes damage to the single-
stranded DNA and blocks its replication. Also, we have
investigated the impact of the low doses of IFN on colon
cancer cells. IFN — multifunctional cytokine, which exerts
antiproliferative and antiangiogenic effects and is capa-
ble to modify the phenotype of tumor cells, reduce their
tumorigenicity [9] and increase their sensitivity to ACD.
Firstly, we have studied the morphology, growth
and immunophenotypic patterns of COLO 205 cells
after their exposure to the abovementioned agents.
This model was selected because of its initial biological
chara cteristics, such as the dominance of mesenchymal
features. Our observations showed that after addition
of the investigated drugs or their combinations, the
number of COLO 205 living cells did not change statisti-
cally significant compared to control cells within the first
10 days of cultivation, but after 15 days of cultivation, the
rate of cell growth started to decline. It should be men-
tioned that the most significant antiproliferative effect
was observed in the cells exposed to low-dose IT (Fig. 1).
0
20
40
60
80
100
120
Control IFN IT
*
*
*
*
*
*
*
*
*
*
IT + IFN CP CP +
IFN
IT + CP IT +
CP + IFN
Nu
m
be
r o
f l
ivi
ng
c
el
ls
, %
10 days of cultivation (2 passage)
15 days of cultivation (3 passage)
Fig. 1. Percentage of COLO 205 living cells after their cultivation
with subtoxic doses of ACD compared to control. *p < 0.05 com-
pared with control cells
Significant changes in the morphology of these
cells and their adhesion to the substrate was observed
only after 2 weeks of COLO 205 cultivation in the pre-
sence of CP, IT, and IFN. The most significant impact
on the morphology of COLO 205 cells was observed
in the case of treatment with IFN and CP (Fig. 2).
b
a
c
Fig. 2. Morphology of live COLO 205 cells: control cells (a), cells
treated with IFN (b) or CP (c) within 14 days, × 320
The malignancy of these cells was studied in vitro
by semisolid agar colony assay. Our investigation has
shown that treatment of COLO 205 cells with MLDR and
IFN has resulted in a significant inhibition of their abi-
lity to form colonies in semisolid agar (Table 1). In this
case, the most significant effect was caused by IT and
complex action of these ACD and cytokine.
Thereby, the results of semisolid agar colony assay
in vitro showed the suppression of the features of tu-
mor cell malignancy upon their exposition to subtoxic
doses of ACD and IFN. Also, we have studied immu-
nophenotypic features of the cells, including adhesion
and cytoskeleton proteins associated with EMT such
as Е- and N-cadherins, CD44, β-catenin, transcription
20 Experimental Oncology 39, 17–24, 2017 (March)
factor Slug, which is directly related to the regulation
of cell adhesion, invasion and migration, and protein
that is responsible for drug resistance of tumor cells
to some ACD — ERCC1 (DNA excision repair protein)
(Table 2, Fig. 3, 4).
Table 1. CFA of COLO 205 cells after their prolonged exposure to low
doses of ACD and IFN
Treatment CFA, %
Control 9.25 ± 0.62
IFN 7.25 ± 0.48
IT 1.66 ± 0.33**
CP 3.25 ± 0.25*
IT + CP 1.25 ± 0.25**
IT + IFN 1.00 ± 0***
CP + IFN 2.50 ± 0.29*
IT + CP + IFN 0.25 ± 0.25***
Note: The difference is statistically significant compared with control
COLO 205 cells; *р < 0.005; **р < 0.002;***р < 0.001.
Table 2. Immunophenotypic profile of COLO 205 cells after prolonged ex-
posure to the subtoxic doses of ACD and IFN
Antigen
Drugs
Co
nt
ro
l
ІFN ІТ CP ІТ +
CP
ІТ +
ІFN
CP +
ІFN
ІТ +
CP +
ІFN
Points (Н-Score)
N-cadherin 108 ±
22
< 10 < 10 32 ±
6*
< 10 < 10 54 ± 9 < 10
CD44 118.6±
3.6
8.8 ±
2.2***
105.4±
4.8
25 ±
1.7***
72.2 ±
2.3 **
5.3 ±
2.3***
12.3 ±
2.2***
14.6 ±
1.7***
Slug 300 < 10 < 10 194 ±
3.2***
< 10 < 10 < 10 < 10
Note: The difference is statistically significant compared with control
COLO 205 cells; *р < 0.05; **р < 0.002, ***р < 0.001.
As it was recently accepted, these proteins de-
termine the EMT status of cells and participate in the
formation of their invasive-metastatic phenotype. For
exa mple, N-cadherin is a Ca-dependent transmem-
brane protein of classical cadherin family, regulating
cell adhesion, but in contrast to E-cadherin, it promotes
the separation of cells from the original tumor mass,
their migration, and initiates the metastatic cascade.
The other adhesion molecules, CD44 antigen also has
direct relevance to the invasive and migratory activity
of tumor cells and is used as a marker of progression
and metastasis in many cancer types, including breast
cancer cells with the phenotype of CD44+ CD24−
considered as “cancer stem cells” [16]. A potential
regulator of cell adhesion and migration, transcription
factor Slug plays an important role in the physiological
(embryogenesis) and pathological (malignancy) EMT
of cells and is a suppressor of E-cadherin promoter
through its inhibition [17]. It is known that an increased
expression of Slug provides tumor cells with the mesen-
chymal phenotype and high metastatic potential [18].
To study the effects of subtoxic doses of ACD on tumor
cell phenotype, expression of abovementioned proteins
was assessed in COLO 205, HT-29, and 3C-P cells.
The results of the study of immunophenotypical
characteristics of the cells are presented in Tables 2, 3.
As it is shown in Table 2, the prolonged exposure of the
COLO 205 cells to IT and IFN in monoregimen or in com-
binations resulted in significant reduction of the number
of N-cadherin-positive cells; the action of CP also de-
creased the expression of this antigen but at the lower
level. Regarding expression of CD44, stem cell marker,
the lowest effect was observed in the case of low-dose
IT: the expression of CD44 decreased by 11.1%, while
the most significant effect was registered in the case
of combined action of IFN and the cytostatics. The
most interesting observation was almost complete
inhibition the Slug expression in COLO 205 cells under
the influence of subtoxic doses of the studied drugs
used either in monoregimen or in combination; only
the influence of CP did not reduce significantly number
of Slug-positive cells (35.4%).
0
50
100
150
200
250
300
Control IFN ІТ ІТ + ІFN CP CP +
IFN
ІТ +
CP
ІТ +
CP + IFN
H-
Sc
or
e
sy
st
em
, p
oi
nt
s
* *
*
*
*
*
Fig. 3. Immunocytochemical analysis of ERCC1 expression
in COLO 205 cells after prolonged (30 days) exposure to the
subtoxic doses of ACD. *p < 0.05 compared with control cells
Another studied antigen was ERCC1, a protein that
takes part in DNA repair. According to the literature,
an increased expression of this protein in colon cancer
cells is associated with their high resistance to plati-
num-based chemotherapy [19, 20] and is the basis for
the indication of IT (camptothecin) to such patients.
As we have shown (see Fig. 3, 4), the use of IFN and
its combination with IT and CP resulted in the almost
complete disappearance of ERCC1-positive cells. The
action of CP and CP combined with IFN led to significant
decrease the number of ERCC1-positive cells, while
the prolonged action of IT and CP combination resulted
in an increased number of ERCC1-positive cells.
The changes of immunophenotypic features of HT-
29 cells with predominantly epithelial characteristics
are shown in Table 3. Immunocytochemical analysis
of ERCC1 expression in HT-29 cells revealed signifi-
cantly increased the number of ERCC1-positive cells
Table 3. Immunophenotypic profile of HT-29 cells after prolonged exposure to the subtoxic doses of ACD and IFN
Antigen
Drugs
Control ІFN ІТ CP ІТ + CP ІТ + ІFN CP + ІFN ІТ + CP + ІFN
Points (Н-Score)
CD44 120.0 ± 11.6 110.0 ± 4.0 255.0 ± 16.5** 205.0 ± 9.0* 225.0 ± 11.0** 230.0 ± 17.5* 210.0 ± 8.4** 198.0 ± 2.0**
Slug (localization
in cytoplasm)
34.0 ± 1.6 < 10 21.0 ± 7.0 134.0 ± 8.6 *** 174.0 ± 11.0*** < 10 < 10 62.0 ± 4.0**
Note: The difference is statistically significant compared with control HT-29 cells; *р < 0.02; **р < 0.01; ***р < 0.002.
Experimental Oncology 39, 17–24, 2017 (March) 21
ba
c d
fe
g h
Fig. 4. Immunocytochemical analysis of ERCC1 expression in COLO 205 cells after prolonged (30 days) exposure to the subtoxic
doses of ACD. a: control cells; b: + IFN; c: + IT; d: IT + IFN; e: + CP; f: CP + IFN; g: IT + CP; h: IT + CP + IFN (× 1000)
22 Experimental Oncology 39, 17–24, 2017 (March)
exposed to IT or IT + CP (Fig. 5). The same trend was ob-
served in COLO 205 cells, although in the case of com-
bined action of IT with IFN a significant reduction in the
number of ERCC1-positive cells was noted, whereas
in HT-29 cells such effect wasn’t observed. A significant
decrease of ERCC1-positive cells was observed only
if IFN was used in monoregimen or in combination with
CP. Expression of E-cadherin did not differ significantly
between the control cells and cells treated with ACD
(approximately 180–220 points by the H-Score system).
We suppose that this fact evidences on a higher stability
of cells with the dominance of epithelial characteristics
vs these with mesenchymal characteristics.
0
50
100
150
200
250
Control IFN ІТ ІТ + ІFN CP CP +
IFN
ІТ + CP ІТ +
CP + IFN
H-
Sc
or
e
sy
st
em
, p
oi
nt
s
*
*
*
*
*
*
*
Fig. 5. Immunocytochemical analysis of ERCC1 expression
in HT-29 cells after prolonged (30 days) exposure to the subtoxic
doses of ACD. *p < 0.05 compared with control cells
The study of CD44 expression in HT-29 cells has
shown significantly increased number of CD44-posi-
tive cells treated with CP and IT, but not IFN (Table 3)
compared to the cells of mesenchymal phenotype
(COLO 205) in which the number of CD44-positive
significantly decreased after exposure to CP and ACD
combined with IFN (Table 2).
The analysis revealed the location of transcription
factor Slug only in the cytoplasm of HT-29 cells and
in control cells — at a low level (since this factor
is chara cteristic of mesenchymal cells), while after
the action of CP the number of Slug-positive cells
significantly increased. The additional effect of IFN
significantly reduced the number of Slug express-
ing cells. A similar trend was observed in the study
of COLO 205 cells: the highest number of cells with
Slug expression was observed after their exposure
to CP (although lower than in control cells), which was
significantly reduced by the additional effect of IFN
(Table 2). These facts require further investigations
of crosstalk between IFN system and reparation and
adhesion proteins, as far as they indicate the capability
of IFN to exert antitumor action dependent on the EMT
status of tumor cells.
Also, we have investigated the influence of ACD
on a newly established cell line 3C-P derived from
ascitic fluid of the patient with colon cancer. Its growth
kinetics after more than 20 passages in vitro is shown
in Fig. 6. The cells of 3C-P line are characterized
by quick proliferation with a maximal growth density
on day 4 and then a significant reduction in the number
of living cells was observed.
3C-P cells were exposed to low-dose IFN, CP,
IT and then immunocytochemical analysis of the
markers associated with EMT has been conducted
(Table 4). This investigation was of special impor-
tance because these cells underwent a small number
of doublings in vitro, yet are heterogeneous and did
not accumulate a large number of mutations. Interest-
ingly, in the case of IFN combined with IT in subtoxic
concentrations total cell death was registered, but not
if these agents were used in monoregimen.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
1 2 3 4 5 6 7
Nu
m
be
r o
f l
ivi
ng
c
el
ls
, ×
10
6 /w
el
l
Day of cultivation
Fig. 6. Kinetics of growth (number of live cells) of the newly
established 3C-Р cell line
Similarly, to the cells with a mesenchymal pheno-
type, COLO 205, a trend for reduction of the number
of CD44-positive cells after exposure of 3C-P to IFN
was observed, while other ACD had no effect on the
CD44 expression. The number of β-catenin-positive
cells significantly increased after exposure of 3C-P cells
to IFN (and in the case of its combination with CP) and
IT, while CP did not influence the marker expression
(Table 4). Significantly decreased number of vimentin-
positive cells after the action of IFN was registered,
while the action of IT resulted in increased number
of cells expressing this antigen.
Таble 4. Immunophenotypic profile of 3C-P cells after prolonged exposure
to the subtoxic doses of ACD and IFN
Аntigen
Drugs
Control CP ІT ІFN CP + ІFN
Points (H-Score)
СD44 19 4 ± 6 210 ± 15 192 ± 8 124 ± 10** 215 ± 22
β-catenin 32 ± 11 24 ± 2 180 ± 15*** 140 ± 4*** 125 ± 15*
Vimentin 180 ± 11 132 ± 3* 262 ± 7** 80 ± 14* 112 ± 4*
Note: The difference vs control 3C-P cells is statistically significant; *р < 0.02;
**р < 0.01; ***р < 0.005.
Thus, immunocytochemical analysis showed that
in 3C-P cells IFN inhibited manifestation of the me-
senchymal phenotype, while IT and CP had a multidi-
rectional impact on the expression of EMT markers.
Next, we analyzed whether long exposure of the
cells to low-dose ACD in vitro affected their drug resis-
tance. For this purpose, we compared the IC50 values
of colon cancer cells to standard (high) doses ACD
before and after long-term low-dose treatment and
revealed that IC50 values did not differ significantly
in all studied cell lines (data not shown).
At last, the cytogenetic analysis of COLO 205 cells
(number of mitoses, a number of cells with MN, apop-
totic cells and cells with nuclear protrusions) has been
conducted (Fig. 7). As markers of genotoxic events
Experimental Oncology 39, 17–24, 2017 (March) 23
and chromosomal instability, there are used counting
of cells with MN and other nuclear abnormalities, such
as nucleoplasmic bridges, nuclear buds, nuclear “tails”,
dumbbell nucleus [21]. MN are formed mainly from acen-
tric chromosomal fragments or individual chromosomes
that are not included into the daughter nuclei during
telophase [22]. Since MN and various nuclear abnormali-
ties are the results of clastogenic and aneugenic events,
the counting these abnormalities in cells is a convenient
method for evaluation of cytogenetic instability in vitro
after exposure to different chemical agents [23–27].
ba
c d
Fig. 7. Cytogenetic characteristics of COLO 205 cell line: a) mi-
tosis, b) cell with MN, c) apoptosis, d) nuclear protrusion
In our study, it was found that control COLO 205 cells
have a relatively high cytogenetic stabi lity — the num-
ber of cells with MN was 1‰, cells with protrusions —
9‰, relatively high proliferative activity of cells with
mitosis frequency 14 ‰ (Table 5). After cultivations
with CP, IT, and IFN at subtoxic doses, the significant
increase of the COLO 205 cells with MN and inhibition
of mitosis (р < 0.05) as well as a trend for accumula-
tion of the cells with nuclear abnormalities were found
(Table 5).
Table 5. Cytogenetic characteristics of COLO 205 cells after their pro-
longed exposure to subtoxic doses of ACD and IFN
Cytogenetic
characteristics
Drugs
Control CP ІТ ІFN
Frequency of cells (‰)
Cells with MN 1.33 ± 1.5 7.00 ± 1.7* 7.00 ± 1.0* 7.00 ± 1.0*
Mitosis 13.67 ± 1.5 4.33 ± 1.5* 6.67 ± 1.1* 2.67 ± 0.6*
Apoptosis 8.67 ± 1.5 9.00 ± 1.0 7.67 ± 1.5 10.33 ± 0.6
Nuclear protrusions 9.33 ± 1.5 12.33 ± 1.1 9.67 ± 1.1 10.67 ± 1.5
Note: The difference is statistically significant compared with control
COLO 205 cells; *р < 0.05; **р < 0.01.
Thus, such exposure on the cells resulted in geno-
toxic events. The IT + IFN combination had the most
significant genotoxic effect in COLO 205 cells causing
the decrease of mitotic activity (р < 0,05) and accumula-
tion of cells with MN (p < 0.001) which is almost 40 times
higher than the value in the control cells (Table 6).
The combination of IT + CP caused lower cytogenetic
disorders possibly due to the induction of apoptosis,
which is 2 times higher than in control cells (p < 0.001),
however, led to significant genotoxic effect, in par-
ticular, decrease of proliferative activity (p < 0.05) and
high frequency of cells with MN (p < 0.05). The com-
bination of IFN + CP had the lowest genotoxicity of all
drug combinations (Table 6). So, prolonged treatment
of COLO 205 cells with ACD in subtoxic doses resulted
in genotoxic effects, i.e. reduction of cell proliferation
and accumulation of cells with MN.
Table 6. Cytogenetic characteristics in COLO 205 cell line after prolonged
exposure to subtoxic doses of ACD, their combinations and IFN
Drugs
Cytogenetic
characteris-
tics
Control CP + IFN ІТ + ІFN CP + ІТ CP + ІТ +
ІFN
Frequency of cells (‰)
Cells with MN 1.33 ± 1.5 6.00 ± 0.00* 38.67 ± 2.5** 14.67 ± 2.0* 12.00 ± 1.7*
Mitosis 13.67 ± 1.5 3.5 ± 0.41* 3.33 ± 0.6* 3.33 ± 0.6* 3.33 ± 1.1*
Apoptosis 8.67 ± 1.5 9.00 ± 0.82 8.00 ± 1.0 18.67 ± 1.5** 9.67 ± 0.67
Nuclear pro-
trusions 9.33 ± 1.5 10.50 ± 0.41 8.00 ± 1.1 11.00 ± 1.0 7.00 ± 1.0
Note: The difference is statistically significant compared with control
COLO 205 cells; *р < 0.05; **р < 0.01.
CONCLUSION
The data showed that the direct prolonged ac-
tion of the low doses of ACD on human colon cancer
cells resulted in the suppression of cell proliferation,
colony-forming activity in soft agar, expression of EMT-
associated markers and significant cytogenetic
changes. The sensitivity of colon cancer cells to ACD
in standard cytotoxic concentrations did not change
after prolonged low-dose exposure.
REFERENCES
1. Zhukov NV. The current state of anti-angiogenic
therapy. Targeted therapy without a target? Prakt Oncol 2007;
8: 164–73 (in Russian).
2. Chubenko VА. Perspective methods of treatment of ma-
lignant neoplasms. Prakt Oncol 2007; 8: 228–34 (in Russian).
3. Chekhun VF, Maksim’yak GI, Zhylchuk VE, et al. The
ways to improve drug therapy of rectal cancer patients with
liver metastases: advantages of metronomic regime of chemo-
therapy. Oncologiya 2011; 13: 229–33 (in Russian).
4. Loven D, Hasnis E, Bertolini F, Shaked Y. Low-dose met-
ronomic chemotherapy: from past experience to new paradigms
in the treatment of cancer. Drug Discov Today 2013; 18: 193–201.
5. Hanahan D, Bergers G, Bergsland E. Less is more,
regularly: metronomic dosing of cytotoxic drugs can target
tumor angiogenesis in mice. J Clin Invest 2000; 105: 1045–7.
6. Bocci G, Nicolaou KC, Kerbel RS. Protracted low-dose
effects on human endothelial cell proliferation and survival
in vitro reveal a selective antiangiogenic window for various
chemotherapeutic drugs. Cancer Res 2002; 62: 6938–43.
7. He K, Jin K, Wang H, Teng L. Anti-angiogenic therapy
for colorectal cancer: on the way to getting better! Hepatogas-
troenterology 2012; 59: 1113–7.
8. Bocci G, Francia G, Man S, et al. Thrombospondin 1,
a mediator of the antiangiogenic effects of low-dose metronomic
chemotherapy. Proc Natl Acad Sci USA 2003; 100: 12917–22.
9. Lykhova A, Bezdenezhnykh N, Semesiuk N, et al. Loss
of malignancy in mouse melanoma cells by long-term impact
of recombinant interferon-beta in vitro is associated with N-
and VE-cadherins suppression without inhibition expression
of epithelial-mesenchymal transition transcription factors
Twist and Slug. J Anal Oncol 2014; 3: 136–45.
24 Experimental Oncology 39, 17–24, 2017 (March)
10. Kudryavets YuI, Bezdenezhnykh NO, Lykhova OO,
et al. The role of interferon as a modifier of epithelial-mesen-
chymal transition in tumor cells. Exp Oncol 2011; 33: 178–81.
11. Animal Cell Culture. In: Freshney RI, eds. A practical
approach. Moscow: Binom, 2010. 714 p. (in Russian).
12. McClelland RA, Wilson D, Leake R, et al. A multicen-
tre study into the reliability of steroid receptor immunocyto-
chemical assay quantification. British Quality Control Group.
Eur J Cancer 1991; 27: 711–5.
13. Kovalova O, Bezdeneznykh N, Kudryavets Yu. Non-
chromosomal cytogenetic analysis of mammal somatic cells.
Biopolym Cell 2013; 29: 33–41.
14. Allegrini G, Di Paolo A, Cerri E, et al. Irinotecan
in combination with thalidomide in patients with advanced
solid tumors: a clinical study with pharmacodynamic and
pharmacokinetic evaluation. Cancer Chemother Pharmacol
2006; 58: 585–93.
15. Ogata Y, Mori S, Ishibashi N, et al. Metronomic
chemotherapy using weekly low-dosage CPT-11 and UFT
as postoperative adjuvant therapy in colorectal cancer at high
risk to recurrence. J Exp Clin Cancer Res 2007; 26: 475–82.
16. Liu S, Cong Y, Wang D, et al. Breast cancer stem cells
transition between epithelial and mesenchymal states reflective
of their normal counterparts. Stem Cell Reports 2013; 2: 78–91.
17. Wang Y, Shi J, Chai K, et al. The Role of Snail
in EMT and tumorigenesis. Curr Cancer Drug Targets 2013;
13: 963–72.
18. Kalluri R, Weinberg RA. The basics of epithelial-
mesenchymal transition. J Clin Invest 2009; 119: 1420–8.
19. Viguier J, Boige V, Miquel C, et al. ERCC1 codon
118 polymorphism is a predictive factor for oxaliplatin/5-
fluorouracil combination chemotherapy in patients with
advanced colorectal cancer. Clin Cancer Res 2005; 11: 6212–7.
20. Milovic-Kovacevic M, Srdic-Rajic T, Radulovic S,
et al. Expression of ERCC1 protein in biopsy specimen pre-
dicts survival in advanced ovarian cancer patients treated with
platinum-based chemotherapy. J BUON 2011; 16: 708–14.
21. Fenech М, Kirsch-Volders M, Natarajan AT, et al.
Molecular mechanisms of micronucleus, nucleoplasmic bridge
and nuclear bud formation in mammalian and human cells.
Mutagenesis 2011; 26: 125–32.
22. Fenech M. The lymphocyte cytokinesis-block micro-
nucleus cytome assay and its application in radiation biodo-
simetry. Health Phys 2010; 98: 234–43.
23. Gisselsson D. Classification of chromosome segrega-
tion errors in cancer. Chromosoma 2008; 117: 511–9.
24. Zyss D, Gergely F. Centrosome function in can-
cer: guilty or innocent? Trends Cell Biol 2009; 19: 334–46.
25. Surrallés J, Catalán J, Creus A, et al. Micronuclei
induced by alachlor, mitomycin-C and vinblastine in human
lymphocytes: presence of centromeres and kinetochores
and influence of staining technique. Mutagenesis 1995;
10: 417–23.
26. Wu J, Lyons GH, Graham RD, Fenech MF. The ef-
fect of selenium, as selenomethionine, on genome stability
and cytotoxicity in human lymphocytes measured using the
cytokinesis-block micronucleus cytome assay. Mutagenesis
2009; 24: 225–32.
27. Duan H, Leng S, Pan Z, et al. Biomarkers measured
by cytokinesis-block micronucleus cytome assay for evaluating
genetic damages induced by polycyclic aromatic hydrocarbons.
Mutat Res 2009; 677: 93–9.
Copyright © Experimental Oncology, 2017
|