Mitochondria do not play a major role in landomycin E-induced ROS burst and circumvention of multiple drug resistance in HL-60 leukemia cells
Aim. To study the molecular mechanisms of reactive oxygen species (ROS) involvement in circumventing the cancer drug resistance by novel angucycline antibiotic landomycin E in HL-60 human leukemia cells and its drug-resistant sublines HL-60/adr and HL-60/vinc. Methods. MTT assay, trypan blue exclusi...
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nasplib_isofts_kiev_ua-123456789-1528242025-02-09T16:17:48Z Mitochondria do not play a major role in landomycin E-induced ROS burst and circumvention of multiple drug resistance in HL-60 leukemia cells Мітохондрії не відіграють основної ролі в індукції оксидативного стресу при доланні множинної лікарської стійкості в лейкозних клітинах лінії HL-60 ландоміцином Е Митохондрии не играют основной роли в индукции оксидативного стресса при преодолении множественной лекарственной устойчивости в лейкозных клетках линии HL-60 ландомицином Е Panchuk, R.R. Lehka, L.V. Rohr, J. Berger, W. Stoika, R.S. Genomics, Transcriptomics and Proteomics Aim. To study the molecular mechanisms of reactive oxygen species (ROS) involvement in circumventing the cancer drug resistance by novel angucycline antibiotic landomycin E in HL-60 human leukemia cells and its drug-resistant sublines HL-60/adr and HL-60/vinc. Methods. MTT assay, trypan blue exclusion test, DCFDA and JC-1 staining of cells. Results. Landomycin E (LE) leads to a massive hydrogen peroxide production in HL-60 cell line already 1h after the drug addition to the cell culture, while depolarization of mitochondria is observed only at 6–12h, which indicates on the extra-mitochondrial ROS production by LE. The drug-resistant cells of HL-60/vinc (P-gp+) despite 100-fold resistance to doxorubicin (Dx) action, demonstrated no difference in the resistance to LE compared to the parental cell line, while HL-60/adr line (MRP-1+), which was found to be 200-fold resistant to Dx action, had shown a weak (2-fold) decrease in sensitivity to LE. Circumvention of drug resistance by LE in HL-60/adr cells was accompanied by a 2-fold higher level of H2O2 compared to the wild-type cells, but the mitochondrial respiratory chain inhibitors had no impact on this phenomenon. Conclusions. LE-induced cell death is accompanied by massive hydrogen peroxide production, consisting of two peaks – major one at 1h and secondary at 12h after drug treatment. The highest ROS production was observed in HL-60/adr cells, which have shown a 2-fold increase of resistance to LE compared to parental cells. However, mitochondria seem to play secondary role in this process, due to the fact that the use of specific inhibitors of mitochondrial respiratory chain did not affect the early ROS burst, induced by LE in tumor cells. Мета. Молекулярні механізми участі активних форм кисню (АФК) в доланні множинної лікарської стійкості злоякісних клітин новим ангуцикліновим антибіотиком ландоміцином E в лейкозних клітинах людини лінії HL-60 і її сублініях HL-60/adr і HL-60/vinc, резистетних до хіміотерапії. Методи. МТТ тест, тест на життєздатність клітин з трипановим синім, фарбування клітин флуоресцентними барвниками DCFDA і JC-1. Результати. Ландоміцин Е (ЛЕ) призводить до значного зростання продукції пероксиду водню в клітинах лінії HL-60 вже на 1 год після додавання препарату до культури клітин, в той час як деполяризация мітохондрій спостерігаються тільки на 6-12 год, що вказує на позамітохондріальне джерело АФК за дії цього антибіотика. Клітини лінії HL-60/vinc (P-глікопротеїн +), незважаючи на 100-кратне зростання стійкості до доксорубіцину (Dx), продемонстрували ідентичну чутливість до ЛЕ в порівнянні з вихідною клітинною лінією, в той час як клітини лінії HL-60/adr (MRP- 1+), які проявляли 200-кратне зростання стійкості до дії Dx, показали слабке (в 2 рази) зниження чутливості до ЛЕ. Долання стійкості до ліків ландоміцином Е у клітинах лінії HL-60/adr супроводжувалося 2-кратним зростанням рівня Н2О2 в порівнянні з клітинами дикого типу, але мітохондріальні інгібітори дихального ланцюга не проявляли ніякого впливу на це явище. Висновки. ЛЕ-індукована загибель пухлинних клітин супроводжується масовим виробництвом перекису водню, що складається з двох піків – основного (1 год) і вторинного (на 12 год дії препарату). Найвища продукція АФК спостерігалася в клітинах лінії HL-60/adr, які показали 2-кратне збільшення стійкості до ЛЕ в порівнянні з вихідною лінією HL-60. Мітохондрії, очевидно, відіграють вторинну роль в цьому процесі, оскільки використання специфічних інгібіторів дихального ланцюга мітохондрій ніяк не вплинуло на ранню індукцію АФК за дії ЛЕ в пухлинних клітинах. Цель. Молекулярные механизмы участия активных форм кислорода (АФК) в преодолении множественной лекарственной устойчивости злокачественных клеток новым ангуциклиновым антибиотиком ландомицином E в лейкозных клетках человека линии HL-60 и ее сублиниях HL-60/adr и HL-60/vinc, резистентных к химиотерапии. Методы. МТТ тест, тест на жизнеспособность клеток с трипановым синим, окраска клеток флуоресцентными красителями DCFDA и JC-1. Результаты. Ландомицин Е (ЛЕ) приводит к значительному росту продукции пероксида водорода в клетках линии HL-60 уже на 1 ч после добавления препарата к культуре клеток, в то время как деполяризация митохондрий наблюдаются только на 6–12 ч, что указывает на внемитохондриальный источник АФК происхождения при действии этого антибиотика. Клетки линии HL-60 vinc (P-гликопротеин+), несмотря на 100-кратный рост устойчивости к доксорубицину (Dx), продемонстрировали идентичную чувствительность к ЛЕ по сравнению с исходной клеточной линией, в то время как клетки линии HL-60/adr (MRP-1+), которые проявляли 200-кратный рост устойчивости к действию Dx, показали слабое (в 2 раза) снижение чувствительности к ЛЕ. Преодоление устойчивости к лекарствам ландомицином Е в клетках линии HL-60/adr сопровождалось 2-кратным ростом уровня Н2О2 по сравнению с клетками дикого типа, но митохондриальные ингибиторы дыхательной цепи не проявляли никакого влияния на это явление. Выводы. ЛЕ-индуцированная гибель опухолевых клеток сопровождается массовым производством перекиси водорода, состоящей из двух пиков – основного (1 час) и вторичного (на 12 ч действия препарата). Самая высокая продукция АФК наблюдалась в клетках линии HL-60/adr, которые показали 2-кратное увеличение устойчивости к ЛЕ по сравнению с исходной линией HL-60. Митохондрии, очевидно, играют вторичную роль в этом процессе, поскольку использование специфических ингибиторов митохондриальной дыхательной цепи никак не повлияло на раннюю индукцию АФК при действии ЛЕ на опухолевые клетки. This work was partially supported by bilateral UkraineAustria grants for 2011-2014 years; Grant of President of Ukraine Gp/F32/192-2011; CRDF Global Grant # FSCX-14-60609-0; Ministry of Science and Education of Ukraine grant #M/122-2015; grants of WestUkrainian Biomedical Research Center (WUBMRC) in 2008-2016, given to R. Panchuk and L. Lehka. 2016 Article Mitochondria do not play a major role in landomycin E-induced ROS burst and circumvention of multiple drug resistance in HL-60 leukemia cells / R.R. Panchuk, L.V. Lehka, J. Rohr, W. Berger, R.S. Stoika // Вiopolymers and Cell. — 2016. — Т. 32, № 3. — С. 190-202. — Бібліогр.: 25 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000920 https://nasplib.isofts.kiev.ua/handle/123456789/152824 577.22 + 577.181.5 en Вiopolymers and Cell application/pdf Інститут молекулярної біології і генетики НАН України |
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Genomics, Transcriptomics and Proteomics Genomics, Transcriptomics and Proteomics |
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Genomics, Transcriptomics and Proteomics Genomics, Transcriptomics and Proteomics Panchuk, R.R. Lehka, L.V. Rohr, J. Berger, W. Stoika, R.S. Mitochondria do not play a major role in landomycin E-induced ROS burst and circumvention of multiple drug resistance in HL-60 leukemia cells Вiopolymers and Cell |
| description |
Aim. To study the molecular mechanisms of reactive oxygen species (ROS) involvement in circumventing the cancer drug resistance by novel angucycline antibiotic landomycin E in HL-60 human leukemia cells and its drug-resistant sublines HL-60/adr and HL-60/vinc. Methods. MTT assay, trypan blue exclusion test, DCFDA and JC-1 staining of cells. Results. Landomycin E (LE) leads to a massive hydrogen peroxide production in HL-60 cell line already 1h after the drug addition to the cell culture, while depolarization of mitochondria is observed only at 6–12h, which indicates on the extra-mitochondrial ROS production by LE. The drug-resistant cells of HL-60/vinc (P-gp+) despite 100-fold resistance to doxorubicin (Dx) action, demonstrated no difference in the resistance to LE compared to the parental cell line, while HL-60/adr line (MRP-1+), which was found to be 200-fold resistant to Dx action, had shown a weak (2-fold) decrease in sensitivity to LE. Circumvention of drug resistance by LE in HL-60/adr cells was accompanied by a 2-fold higher level of H2O2 compared to the wild-type cells, but the mitochondrial respiratory chain inhibitors had no impact on this phenomenon. Conclusions. LE-induced cell death is accompanied by massive hydrogen peroxide production, consisting of two peaks – major one at 1h and secondary at 12h after drug treatment. The highest ROS production was observed in HL-60/adr cells, which have shown a 2-fold increase of resistance to LE compared to parental cells. However, mitochondria seem to play secondary role in this process, due to the fact that the use of specific inhibitors of mitochondrial respiratory chain did not affect the early ROS burst, induced by LE in tumor cells. |
| format |
Article |
| author |
Panchuk, R.R. Lehka, L.V. Rohr, J. Berger, W. Stoika, R.S. |
| author_facet |
Panchuk, R.R. Lehka, L.V. Rohr, J. Berger, W. Stoika, R.S. |
| author_sort |
Panchuk, R.R. |
| title |
Mitochondria do not play a major role in landomycin E-induced ROS burst and circumvention of multiple drug resistance in HL-60 leukemia cells |
| title_short |
Mitochondria do not play a major role in landomycin E-induced ROS burst and circumvention of multiple drug resistance in HL-60 leukemia cells |
| title_full |
Mitochondria do not play a major role in landomycin E-induced ROS burst and circumvention of multiple drug resistance in HL-60 leukemia cells |
| title_fullStr |
Mitochondria do not play a major role in landomycin E-induced ROS burst and circumvention of multiple drug resistance in HL-60 leukemia cells |
| title_full_unstemmed |
Mitochondria do not play a major role in landomycin E-induced ROS burst and circumvention of multiple drug resistance in HL-60 leukemia cells |
| title_sort |
mitochondria do not play a major role in landomycin e-induced ros burst and circumvention of multiple drug resistance in hl-60 leukemia cells |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2016 |
| topic_facet |
Genomics, Transcriptomics and Proteomics |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/152824 |
| citation_txt |
Mitochondria do not play a major role in landomycin E-induced ROS burst and circumvention of multiple drug resistance in HL-60 leukemia cells / R.R. Panchuk, L.V. Lehka, J. Rohr, W. Berger, R.S. Stoika // Вiopolymers and Cell. — 2016. — Т. 32, № 3. — С. 190-202. — Бібліогр.: 25 назв. — англ. |
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190
R. R. Panchuk, L. V. Lehka, J. Rohr
© 2016 R. R. Panchuk et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers 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
UDC 577.22 + 577.181.5
Mitochondria do not play a major role in landomycin E-induced
ROS burst and circumvention of multiple drug resistance
in HL-60 leukemia cells
R. R. Panchuk1, L. V. Lehka1, J. Rohr2, W. Berger3, R. S. Stoika1
1 Institute of Cell Biology NAS of Ukraine
14/16, Drahomanov St., Lviv, Ukraine, 79005
2 University of Kentucky, College of Pharmacy, Lexington, USA,
789 S. Limestone St. Lexington
3 Institute of Cancer Research and Comprehensive Cancer Center, Medical University Vienna,
8a, Borschkegasse, Vienna 1090, Austria
rpanchuk@ukr.net
Aim. To study the molecular mechanisms of reactive oxygen species (ROS) involvement in circumventing the
cancer drug resistance by novel angucycline antibiotic landomycin E in HL-60 human leukemia cells and its
drug-resistant sublines HL-60/adr and HL-60/vinc. Methods. MTT assay, trypan blue exclusion test, DCFDA
and JC-1 staining of cells. Results. Landomycin E (LE) leads to a massive hydrogen peroxide production in
HL-60 cell line already 1h after the drug addition to the cell culture, while depolarization of mitochondria is
observed only at 6-12h, which indicates on the extra-mitochondrial ROS production by LE. The drug-resistant
cells of HL-60/vinc (P-gp+) despite 100-fold resistance to doxorubicin (Dx) action, demonstrated no difference
in the resistance to LE compared to the parental cell line, while HL-60/adr line (MRP-1+), which was found to
be 200-fold resistant to Dx action, had shown a weak (2-fold) decrease in sensitivity to LE. Circumvention of
drug resistance by LE in HL-60/adr cells was accompanied by a 2-fold higher level of H2O2 compared to the
wild-type cells, but the mitochondrial respiratory chain inhibitors had no impact on this phenomenon.
Conclusions. LE-induced cell death is accompanied by massive hydrogen peroxide production, consisting of
two peaks – major one at 1h and secondary at 12h after drug treatment. The highest ROS production was ob-
served in HL-60/adr cells, which have shown a 2-fold increase of resistance to LE compared to parental cells.
However, mitochondria seem to play secondary role in this process, due to the fact that use of specific inhibitors
of mitochondrial respiratory chain did not affect the early ROS burst, induced by LE in tumor cells.
K e y w o r d s: landomycin E, ROS, mitochondria, cancer drug resistance, apoptosis
Introduction
Despite advancement of modern pharmacology in the
development of efficient anticancer drugs, the overall
ratio of successful results of the metastatic cancer che-
motherapy still remains rather low. Compared to the
golden chemotherapy standards (anthracyclines and
vinca alkaloids), developed in early 1960s, over the
last decade the specificity of modern targeted medi-
cines became markedly higher and their side effects
significantly diminished. However, the average sur-
vival time of patients with disseminated cancer did
not considerably increase] [1].
Such ineffectiveness of cancer chemotherapy is
explained by the rapid development of acquired
multi-drug resistance (MDR) of tumor cells to the
anticancer drugs, which is found in 30-50% of can-
cer patients already in a year after the treatment
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2016. Vol. 32. N 3. P 190–202
doi: http://dx.doi.org/10.7124/bc.000920
191
Role mitochondria in landomycin E-induced ROS burst and circumvention of MDR in HL-60
start [2]. The MDR phenotype is characterized by
the simultaneous resistance of cancer cells to the
pharmacologically and structurally diverse drugs [3].
It could be a cause of the poor prognosis and lower
survival rate in several types of cancer such as gas-
tric cancer, breast cancer, ovarian cancer, pancreatic
cancer, and leukemia [4, 5]
MDR is related to an increased production of the
ATP-Binding Cassette (ABC) transport proteins. the
membrane-bound proteins that extrude a variety of
anticancer drugs from the tumor cells using ATP hy-
drolysis. Until now, 48 human ABC genes have been
identified and classified into seven subfamilies based
on the sequence homology and domain structure [1].
In addition to their physiological expression in normal
tissues, these proteins were found to be over-ex-
pressed in the human tumors, which significantly low-
ers their response to the chemotherapy treatment.
Although all types of ABC transporters are involved
in drug resistance, most studies on the ABC transport-
er protein in cancer have focused on three proteins:
P-gp (p-glycoprotein), MRP-1 (multidrug-resistance
associated protein), and BCRP (breast cancer resis-
tance protein). Each of the above drug transporters is
specific to a certain type of substrates. In particular,
P-glycoprotein is responsible for resistance to colchi-
cine, dexamethasone, doxorubicin, vinblastine, and
etoposide, whereas the over-expression of MRP-1
protein results in resistance to vincristine and doxoru-
bicin[6, 7]. In turn, bcrp, besides resistance to metho-
trexate and mitoxantrone, is also responsible for the
cancer cell resistance to targeted chemotherapies,
such as imatinib and lapatinib, which makes it espe-
cially important in modern cancer chemotherapy [8].
Thus, the development of novel anticancer drugs,
capable of circumventing the cancer drug resistance
in different ways, which could not be inhibited by any
of known drug resistance mechanisms, remains an
important task of the current pharmacology and medi-
cine. Here, one of promising approaches is searching
for natural compounds, the anticancer activities of
which could not be affected by the ABC-transporter
proteins. Landomycins are a novel group of angucy-
cline antibiotics possessing a strong antineoplastic
potential. All natural landomycins identified to date,
share the same aglycon (landomycinone) group and
vary only in their oligosaccharide chain, a linear gly-
cosidic chain containing only di- and trideoxysugars
(β-D-olivose and α-L-rhodinose [9]. They show a
broad activity against many cancer cell lines, with the
general tendency that the compounds with longer sac-
charide chains possess a higher antitumor poten-
tial [10, 11].
Previously, we studied the cellular and molecular
mechanisms of antitumor activity of one landomycin
family member – landomycin E, possessing three
sugar residues in its saccharide chain. It was shown
[12, 13] that this drug could overcome the multidrug
resistance of cancer cells to chemotherapy treatment
due to the over-expression of ABC-transporters. It
wasalso shown by us, that the anticancer activity of
another member of this family – landomycin A – is
accompanied by massive ROS burst at early time-
points of its action, but the molecular mechanisms of
this phenomenon still remain unclear [14].
The main aim of the current study was to investi-
gate the ROS profile in HL-60 leukemia cells and the
drug-resistant HL-60/adr and HL-60/vinc sublines
under LE treatment in vitro, and to evaluate a possi-
ble role of mitochondria in circumvention of cancer
drug resistance by LE using specific mitochondrial
respiratory chain inhibitors. A well-known antican-
cer drug doxorubicin was used for the comparison .
Materials and Methods
Materials
LE-overproducing Streptomyces globisporus 1912
strain was obtained in the laboratory of Prof. B.
Matselyukh (D.K. Zabolotny Institute of Micro bio-
logy and Virology, National Academy of Sciences of
Ukraine, Kyiv). LE (99.5% purity, according to
HPLC data) was prepared in the laboratory of Prof.
J. Rohr (University of Kentucky, USA) and dis-
solved in absolute ethanol to obtain a 4 mg/ml stock
solution. Doxorubicin hydrochloride was obtained
from Pfizer (New York, NY). Diphenylene Iodonium
(DPI), antimycin A (AMA), oligomycin A (OM),
192
R. R. Panchuk, L. V. Lehka, J. Rohr et al.
N-acetylcysteine (NAC) and sodium pyruvate were
purchased from Sigma-Aldrich (St. Louis, MO).
Cell culture and treatments
Human leukemia cells of HL-60 line, its drug-resistant
sublines HL-60/adr (MRP-1 overexpression) and HL-
60/vinc (P-glycoprotein overexpression) were ob-
tained from the cell culture collections at Vienna
Medical University, Institute of Cancer Research, from
ATCC. Cells were cultured in RPMI medium, supple-
mented with 10% fetal calf serum (Sigma-Aldrich), 50
µg/ml streptomycin (Sigma-Aldrich), 50 units/ml pen-
icillin (Sigma-Aldrich) in 5% CO2-containing humidi-
fied atmosphere at 37 °C. For experiments[, the] cells
were seeded into 24-well tissue culture plates (Greiner
Bio-one, Germany). Short-term (24h) cytotoxic effect
of antitumor drugs was studied under the Evolution
300 Trino microscope (Delta Optical, Poland) after
cell staining with trypan blue dye (0.1%).
DPI (3 mM stock solution) was dissolved in
DMSO, while antimycin A and oligomycin A were
dissolved in absolute ethanol to obtain 2 mM stock
solutions. NAC (0.5 M) and D-mannitol (10 M)
were dissolved in 1x phosphate buffered saline
(PBS). Modulators were added to cell culture 30 min
before addition of anticancer drugs, and final con-
centration of NAC was 1 mM, sodium pyruvate
10 mM, DPI and Oligomycin A – 4 µM. Stock solu-
tions of aforementioned reagents were dissolved in
PBS before addition to cell culture.
For long-term (72h) cytotoxicity assays Jurkat
cells were plated (5x103) in 100 µl per well in 96-
well plates, and allowed to grow for 24 h. Drugs
were added in another 100 µl culture medium (final
concentration of DPI – 0.5 µM, oligomycin A –
1 µM, antimycin A – 2 µM, sodium pyruvate –
10 mM, NAC – 1 mM) and cells were exposed for
72 h. The proportion of viable cells was determined
by EZ4U assay according to the manufacturer’s rec-
ommendations (EZ4U, Biomedica, Vienna, Austria).
Cytotoxicity was expressed as IC50 values calculated
from full dose-response curves (drug concentration
including 50% reduction in cell survival comparing
to the control cultured in parallel without drug).
Flow cytometric assays
Analyses were performed using a FACScalibur flow
cytometer (BD Biosciences, San Jose, CA) and
Summit v3.1 software (Cytomation, Inc., Fort Collins,
CO). HL-60, HL-60/adr and HL-60/vinc leukemia
cells were treated as indicated in the figures.
Cellular ROS contents were measured by incubat-
ing the control or drug-treated cells with fluorescent
dye dihydrodichlorofluoresceindiacetate (H2DCFDA,
H2O2-specific) in concentration 10 μM of H2DCFDA
at 37 °C for 30 min. After incubation with the
fluorochrome, cells were washed with PBS and im-
mediately analyzed at FL1 channel of FACSCalibur
flow cytometer (BD Biosciences, San Jose, CA)
Breakdown of ΔΨm mitochondrial membrane po-
tential was determined by FACS analysis using JC-1
(5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimid-
azolylcarbocyanine iodide). For this purpose the
Mitochondrial Membrane Potential Detection Kit
(Stratagene, La Jolla, CA, USA) was used, as de-
scribed in the manufacturer’s instruction. 106 HL-60
or HL-60/adr cells were treated for 1, 3, 6, 12 and
24 h with the tested drugs. After PBS washing, cells
were incubated for 10 min in freshly prepared JC-1
solution (10 mg/ml in culture medium) at 37 °C.
Spare dye was removed by PBS washing and cell-
associated fluorescence was measured with FACS.
Statistical analysis
If not stated otherwise, data are expressed as mean
± SD. The results were analyzed using GraphPad
Prism software. Statistical analyses were performed
using t-test or two-way analysis of variance
(ANOVA). To examine differences between the drug
treatment responses, Bonferroni post-tests were con-
ducted. P values below 0.05 were considered as sta-
tistically significant and marked with stars: * p<0.05;
** p<0.01; *** p<0.001.
Results and Discussion
At the first stage of our study, the sensitivity of HL-
60 leukemia cells and their drug-resistant sublines to
the action of landomycin E (LE) and well-known
anticancer drug doxorubicin (Dx) was studied in de-
193
Role mitochondria in landomycin E-induced ROS burst and circumvention of MDR in HL-60
tail (Fig.1). It was revealed that overexpression of
P-glycoprotein in HL-60/vinc cells increased their
resistance profile to Dx 100-fold, while HL-60/adr
cell line, overexpressing MRP-1, was found to be
200-fold more resistant to the Dx action compared to
parental HL-60/wt line (Fig. 1). On [the] contrary,
no difference in sensitivity of HL-60/wt, HL-60/adr
and HL-60/vinc cells to [the] LE action was ob-
served at 72h drug incubation time, according to
MTT tests (Fig.1).
Short-time drug toxicity experiments (24h incu-
bation), analyzed by trypan blue exclusion test, have
revealed similar tendencies – 20-fold resistance in-
crease of HL-60/vinc cell line, and 40-fold resistance
increase of Hl-60/adr cell line towards Dx (Fig.1),
while for LE the only cells which showed a weak
(~2-fold) decrease in sensitivity to this drug, were of
HL-60/adr subline. Thus, the cytotoxic activity of
LE is almost unaffected by ABC transporters, which
makes it a promising compound for treatment of
drug-resistant leukemia.
Previously we demonstrated that one of the lando-
mycin family members – LA, possessing 6 sugar
residues instead of 3 residues in LE, and thus higher
Fig. 1. Comparison of anticancer activities of LE and Dx towards human leukemia cells of HL-60 line and its drug-resistant sublines
HL-60/adr (MRP-1+) and HL-60/vinc (P-gp+). Cell viability was analyzed by MTT assay after 72 h exposure to indicated
concentrations of LE and Dx, or by trypan blue exclusion assay after 24 h exposure to aforementioned drugs. Graphs represent the
mean +/- SD of three independent experiments done in triplicates
** p<0.01, *** p<0.001 relative to HL-60/wt cell line, 2-way ANOVA, Bonferroni post-test.
194
R. R. Panchuk, L. V. Lehka, J. Rohr et al.
cytotoxic activity, leads to specific two-phase ROS
burst in Jurkat T-leukemia cells (Lehka et al, 2015),
which consists of two peaks 1h and 12h after the tar-
get cell treatment with LA. Mitochondria, being the
main energetic stations in the eukaryotic cells, are
also considered to be the main source of ROS [15].
Thus, for the identification of nature and source of
LE-induced ROS, the joint treatment of HL-60/adr/
vinc cells with LE and various ROS scavengers
(N-acetylcysteine, sodium pyruvate) and mitochon-
drial respiratory chain inhibitors (antimycin A, oli-
gomycin A, DPI) was addressed.
For studying potential impact of mitochondria on
the cytotoxic activity of LE in vitro several specific
inhibitors of mitochondrial respiratory chain were
used, namely DPI (NADPH dehydrogenase inhibitor
[16]), antimycin A (complex III inhibitor [17]) and
oligomycin A (ATP-synthetase inhibitor [18]).
However, it was revealed that all these compounds,
except antimycin A (2 µM), are toxic to HL-60 leu-
kemia cells, even in very low concentrations (1 µM
for oligomycin A and 0.5 µM for DPI) (Fig. 2). The
same tendency was observed also for HL-60/adr
cells, while HL-60/vinc cell line demonstrated a
high internal resistance to the action of all tested mi-
tochondrial inhibitors, thus indicating an insignifi-
cant role of mitochondria in the regulation of their
proliferation and apoptosis. We did not observe any
statistically significant impact of aforementioned
compounds on inhibition or stimulation of LE cyto-
toxic action, mainly due to a high toxicity of oligo-
mycin A and DPI towards the tested cell lines at 72h
incubation. However, in HL-60/vinc cell line, where
no toxic activity of antimycin A, oligomycin A and
DPI was observed, they possessed a little, still statis-
tically insignificant impact on lowering antineoplas-
tic action of LE. Thus, mitochondria seem to play
rather secondary role in the LE-induced cell death.
For better understanding a role of specific ROS in
LE-involved cell death, specific hydrogen peroxide
scavenger sodium pyruvate [19] and wide-scale
ROS scavenger N-acetylcysteine [20] were ad-
dressed. It was revealed that sodium pyruvate pos-
sessed the moderate cytoprotective activity towards
the LE action (P<0.05 for HL-60 cells and P<0.01
for HL-60/adr cells), whereas NAC almost com-
pletely blocked its activity towards all tested cell
lines (P<0.001). Such unexpectedly high efficiency
of the NAC action may be explained not only by its
ROS scavenging activity, but also by its indirect im-
pact on the glutathione level in the cells [20].
Due to the fact that ROS are short-living mole-
cules, MTT assays for 72h drug incubation and try-
pan blue tests for 24h drug treatment may not show
all the dynamics of cellular processes, taking place
immediately after drug entry into the cells. For better
understanding a role of specific ROS and mitochon-
dria at early timepoints of LE action, a level of hy-
drogen peroxide in the cells was measured by flow
cytometry using DCFDA dye at 1,3,6,12,24h after
LE addition to studied cell lines. As seen in Fig. 3-5,
LE leads to the] appearance of typical two-peak
H2O2 burst in wild-type HL-60 cells, like previously
observed by us in LA in Jurkat T-cells (Lehka et al,
2015). At the beginning (1h) the ROS level increases
3-fold (for 2 µM dose of LE) or 5.5-fold (for 4 µM
LE), and this process is partially inhibited by
N-acetylcysteine (see Fig. 3)
Then at 3h the ROS level under LE treatment rap-
idly drops, almost reaching a control level at 6h, and
then slowly recovers at 12h, slightly decreasing
again at 24h (Fig. 3). Mitochondrial respiratory
chain inhibitors DPI and oligomycin A have no sta-
tistically significant impact on the LE-derived ROS
production, as seen in Fig. 3, thus confirming our
previous MTT data (see Fig.2).
However, in the HL-60/adr cell line, which was
found 2-fold more resistant to the LE action, the
drug leads to a massive ROS production, which is
approximately 2-fold higher compared to HL-60/wt
cells (Fig. 4), and NAC strongly reduces it almost to
a control level. Surprisingly, the ROS level under LE
treatment in HL-60/adr cells does not drop as rapidly
as observed in parental HL-60 line (Fig.4). Instead,
it slowly decreases from 1h till 24h, so no a “para-
dox” double peak is observed here. Despite major
H2O2 production, neither DPI, nor oligomycin
A have significant impact on the ROS production
195
Role mitochondria in landomycin E-induced ROS burst and circumvention of MDR in HL-60
Fig. 2. Study of modulating effect of mitochondrial respiratory chain inhibitors on cytotoxic activities of LE towards human leuke-
mia cells of HL-60 line and its drug-resistant sublines HL-60/adr (MRP-1+) and HL-60/vinc (P-gp+). Cell viability was analyzed by
MTT assay after 72 h exposure to indicated concentrations of LE. Graphs represent the mean +/- SD of three independent experiments
done in triplicates
* p<0.05, ** p<0.01, *** p<0.001 relative to LE, 2-way ANOVA, Bonferroni post-test.
DPI – diphenylene iodonium, NAC – N-acetylcyteine, Pyruvate – sodium pyruvate
196
R. R. Panchuk, L. V. Lehka, J. Rohr et al.
Fig. 3. LE leads to significant increase of hydrogen peroxide
level in human leukemia cells of HL-60 line already at 1h after
addition to cell culture, which is partially inhibited by N-acetyl-
systeine, but not by mitochondrial respiratory chain inhibitors
DPI and oligomycin A. The cells were pre-treated for 30 min
with NAC, DPI and OM then treated with either vehicle, LE
(2 µM and 4 µM) and then stained with DCFDA for analysis by
flow cytometry. Bar graphs show an increase (over vehicle con-
trol) in the percentage of cells with positive DCF fluorescence in
each sample. Baseline level of hydrogen peroxide in control
cells is taken as 100%.
DPI – diphenylene iodonium, NAC – N-acetylcyteine, OM –
oligomycin A
* p<0.05, ** p<0.01 relative to control, unpaired t-test.
p<0.05, relative to LE, unpaired t-test.
197
Role mitochondria in landomycin E-induced ROS burst and circumvention of MDR in HL-60
Fig. 4. LE leads to over 2-fold increase of hydrogen peroxide
level in human leukemia cells of HL-60/adr line, overexpressing
MRP-1, in comparison to HL-60 parental line already at 1h after
addition to cell culture, which is strongly inhibited by N-acetyl-
systeine, but not by mitochondrial respiratory chain inhibitors
DPI and oligomycin A. Cells were pre-treated for 30 min with
NAC, DPI and OM, then treated with either vehicle, LE (2 µM
and 4 µM) and then stained with DCFDA for analysis by flow
cytometry. Bar graphs show an increase (over a vehicle control)
in the percentage of cells with positive DCF fluorescence in each
sample. A baseline level of hydrogen peroxide in control cells is
taken as 100%.
DPI – diphenylene iodonium, NAC – N-acetylcyteine, OM –
oligomycin A
* p<0.05, ** p<0.01 relative to control, unpaired t-test.
p<0.05, relative to LE, unpaired t-test.
198
R. R. Panchuk, L. V. Lehka, J. Rohr et al.
Fig. 5. No major differences in ROS production under LE action
between P-gp overexpressing HL-60/vinc cell line and HL-60
parental cells are observed. NADPH oxidase inhibitor DPI par-
tially decreases LE-driven ROS at 24h cells incubation with the
drug. Cells were pre-treated for 30 min with NAC, DPI and OM,
then treated with either vehicle, LE (2 µM and 4 µM) and then
stained with DCFDA for analysis by flow cytometry. Bar graphs
show an increase (over vehicle control) in the percentage of cells
with positive DCF fluorescence in each sample. Baseline level
of hydrogen peroxide in control cells is taken as 100%.
DPI – diphenylene iodonium, NAC – N-acetylcyteine, OM –
oligomycin A
* p<0.05, ** p<0.01 relative to control, unpaired t-test.
p<0.05, relative to LE, unpaired t-test.
199
Role mitochondria in landomycin E-induced ROS burst and circumvention of MDR in HL-60
under the LE action, thus indicatin their weak in-
volvement in this process (Fig. 4).
The only slight ROS-inhibitive effect of DPI to-
wards the LE action was observed in the HL-60/vinc
cell line, characterized by P-gp overexpression
(Fig. 5). However, it was much lower compared to
NAC alone and statistically insignificant. Thus, all
studied mitochondrial respiratory chain inhibitors
have a slight effect on the LE-induced ROS produc-
tion, which may indicate the fact that LE leads to the
extra mitochondrial ROS production.
For checking this hypothesis, the status of mito-
chondrial membrane in HL-60/wt and HL-60/adr
cells was tested using JC-1 staining. Unfortunately,
JC-1, being a mitochondrial stain, was also found by
us to be a perfect substrate for P-glycoprotein, thus
the measurement of JC-1 fluorescence in HL-60/
vinc cells was technically impossible due to a mas-
Fig.6. LE leads to late depolarization of mitochondrial membrane in HL-60 and HL-60/adr cells, which takes place
mainly at 12h after incubation with the drug. DPI possesses strong synergistic effect with LE on depolarization of mi-
tochondria, but this effect is also observed much later (6h) than massive ROS burst at 1h under LE treatment. Cells were
pre-treated for 30 min with NAC, Oligomycin A or DPI, then treated with either vehicle, LE (2 µM and 4 µM) and then
stained with JC-1. Intracellular JC-1 fluorescence was measured by FACS and analyzed by Cytomation Summit
software.
A
C
B
D
200
R. R. Panchuk, L. V. Lehka, J. Rohr et al.
sive dye extrusion from the cells. As seen in Fig. 6,
LE leads to the late depolarization of mitochondria
in HL-60 and HL-60/adr cells, reaching its maxi-
mum at 12h timepoint – time, which coincides with
the second ROS burst observed under the LE action
(see Fig.3). However, no signs of mitochondria de-
polarization were observed at 1h timepoint in both
cell lines, where the major ROS peak was observed
by us (Fig. 3-5). This fact further indicates a second-
ary role of mitochondria in the LE-induced ROS
production, and corresponds to the second ROS
burst, but not the first one (1h). At a combination of
LE and mitochondrial respiratory chain inhibitors it
was revealed that ATP-synthase inhibitor oligomy-
cin A had no statistically significant impact on the
LE-induced mitochondrial membrane depolarization
at 1h, 3h and 6h timepoints, whereas DPI strongly
enhanced the LE-induced mitochondrial membrane
depolarization, increasing it 2.5-5-fold depending on
drug concentration and incubation time (Fig. 6). It is
known that DPI, blocking mitochondrial Complex I,
can lead to subsequent oxidative stress in the cells
[16]. The synergistic action of DPI and LE at 6h
timepoint on depolarization of mitochondria (Fig. 6)
clearly indicates that both compounds induce the
mitochondrial-mediated ROS production. It takes
place much later (12h) compared to a massive ROS
burst, caused by LE in both sensitive, and even high-
er, –in doxorubicin-resistant HL-60 cells at 1h. As
DPI was shown to be a broad NAPDH oxidase in-
hibitor, blocking activity not only of Complex I, but
also of other membrane-bound NADPH-oxidases
[16], the LE-induced an early ROS burst cannot be
explained by involvement of either mitochondria or
of the plasma membrane ROS-producing enzymes.
The only possible explanation of this early ROS
burst is the hydrogen peroxide production by drug
itself during its entry into the target cell. It has been
already shown that all compounds containing qui-
none motif in their structure, may be the source of
ROS itself as the catalyzers of the continuous redox
cycles [21]. These ROS may be inhibited by cellular
antioxidant systems, and glutathione system is con-
sidered to be one of the most efficient [22].
The GSH level in HL-60/adr cells was shown to be
2-fold lower in comparison to HL-60/wt cells [23]. This
may explain the nature of a 2-fold higher ROS burst,
induced by LE in HL-60/adr cells, as this antioxidant at
a lower level inside the cells is unable to quickly scav-
enge all hydrogen peroxide, produced by LE.
Several studies have already demonstrated a tight
connection between MRP-1 pump and intracellular
GSH pool, which plays an important role in the func-
tioning of this drug transporter [24]. In particular,
GSH depletion by buthionine sulphoximine (BSO)
increases the accumulation of DNR and Rhodamine
123 (Rh123) in several MRP1-positive cell lines [25],
thus partially reverting their resistance to these drugs.
The observed connection between the glutathione
antioxidant system and the MRP-1 expression in
HL-60/adr cells may explain their higher resistance
profile to LE. HL-60/vinc cells, which have the basal
GSH level, do not demonstrate any significant resis-
tance to the LE action, even despite P-gp overex-
pression. Thus, joint targeting of tumor cells by LE
together with GSH-depleting compounds (such as
BSO) may be a promising approach in the treatment
of drug-resistant tumors.
Conclusions
LE effectively circumvents drug resistance of HL-60
leukemia cells in vitro, caused by P-gp overexpres-
sion, whereas in HL-60/adr cells, characterized by
MRP-1 overexpression, the activity of this drug drops
2-fold compared to the parental cell line. The LE-
induced cell death is accompanied by a massive ROS
burst, consisting of two peaks of hydrogen peroxide
production – major one at 1h and secondary at 12h
after drug treatment. The highest ROS production was
observed in HL-60/adr cells, which have shown 2-fold
increase of resistance to LE compared to the parental
cells. However, mitochondria seem to be involved
only at tetherminal stages of LE-induced apoptosis
(12h), indicating the extra-mitochondrial ROS produc-
tion by LE at early timepoints. The mitochondrial re-
spiratory chain inhibitors failed to inhibit the LE-
induced ROS burst at both early and terminal stages of
the LE-induced cell death, and only one of them (DPI)
201
Role mitochondria in landomycin E-induced ROS burst and circumvention of MDR in HL-60
was shown to have synergistic effect with LE on the
mitochondrial membrane depolarization at 6h of cell
incubation with the drug. All these data indicate that
there was no involvement of mitochondria at early
stages of the LE action, which are accompanied by
ROS burst, possibly produced by the drug itself due to
its specific chemical structure. A low glutathione level
in HL-60/adr cells, which is a well-known antioxidant,
may explain a 2-fold higher ROS burst, observed un-
der the cytotoxic action of LE towards these cells.
Funding
This work was partially supported by bilateral Ukraine-
Austria grants for 2011-2014 years; Grant of President
of Ukraine Gp/F32/192-2011; CRDF Global Grant #
FSCX-14-60609-0; Ministry of Science and Education
of Ukraine grant #M/122-2015; grants of West-
Ukrainian Biomedical Research Center (WUBMRC)
in 2008-2016, given to R. Panchuk and L. Lehka
REFERENCES
1. Gottesman MM, Fojo T, Bates SE. Multidrug resistance in
cancer: role of ATP-dependent transporters. Nat Rev Can-
cer. 2002;2(1):48-58.
2. Young RC. Drug resistance: the clinical problem. Cancer
Treat Res. 1989;48:1–12.
3. Ford JM, Hait WN. Pharmacology of drugs that alter multi-
drug resistance in cancer. Pharmacol Rev. 1990;42(3):155–99.
4. Jamroziak K, Robak T. Pharmacogenomics of MDR1/
ABCB1 gene: the influence on risk and clinical outcome
of haematological malignancies. Hematology. 2004;9(2):
91–105.
5. Leighton JC Jr, Goldstein LJ. P-glycoprotein in adult solid
tumors. Expression and prognostic significance. Hematol
Oncol Clin North Am. 1995;9(2):251–73.
6. Eberl S, Renner B, Neubert A, Reisig M, Bachmakov I,
König J, Dörje F, Mürdter TE, Ackermann A, Dormann H,
Gassmann KG, Hahn EG, Zierhut S, Brune K, Fromm MF.
Role of p-glycoprotein inhibition for drug interactions: evi-
dence from in vitro and pharmacoepidemiological studies.
Clin Pharmacokinet. 2007;46(12):1039–49.
7. Munoz M, Henderson M, Haber M, Norris M. Role of the
MRP1/ABCC1 multidrug transporter protein in cancer.
IUBMB Life. 2007;59(12):752–7.
8. Kodaira H, Kusuhara H, Ushiki J, Fuse E, Sugiyama Y. Ki-
netic analysis of the cooperation of P-glycoprotein (P-gp/
Abcb1) and breast cancer resistance protein (Bcrp/Abcg2)
in limiting the brain and testis penetration of erlotinib, fla-
vopiridol, and mitoxantrone. J Pharmacol Exp Ther. 2010;
333(3):788–96.
9. Henkel T, Rohr J, Beale JM, Schwenen L. Landomycins,
new angucycline antibiotics from Streptomyces sp. I. Struc-
tural studies on landomycins A-D. J Antibiot (Tokyo). 1990;
43(5):492–503.
10. Luzhetskyy A, Zhu L, Gibson M, Fedoryshyn M, Dürr C,
Hofmann C, Hoffmeister D, Ostash B, Mattingly C,
Adam s V, Fedorenko V, Rohr J, Bechthold A. Generation of
novel landomycins M and O through targeted gene disrup-
tion. Chembiochem. 2005;6(4):675–8.
11. Shaaban KA, Srinivasan S, Kumar R, Damodaran C, Rohr J.
Landomycins P-W, cytotoxic angucyclines from Streptomy-
ces cyanogenus S-136. J Nat Prod. 2011;74(1):2–11.
12. Panchuk R, Korynevska A, Ostash B, Osyp Y, Fedorenko V,
Stoika R. Study of mechanisms of landomycin E action on
mammalian cells. Visn L’viv Univ Ser Biol. 2004; 35:54–9.
13. Korynevska A, Heffeter P, Matselyukh B, Elbling L, Mick-
sche M, Stoika R, Berger W. Mechanisms underlying the
anticancer activities of the angucycline landomycin E. Bio-
chem Pharmacol. 2007;74(12):1713–26.
14. Lehka LV, Panchuk RR, Berger W, Rohr J, Stoika RS. The
role of reactive oxygen species in tumor cells apoptosis in-
duced by landomycin A. Ukr Biochem J. 2015;87(5):72–82.
15. Dröse S, Brandt U. Molecular mechanisms of superoxide
production by the mitochondrial respiratory chain. Adv Exp
Med Biol. 2012;748:145–69.
16. Riganti C, Gazzano E, Polimeni M, Costamagna C, Bo-
sia A, Ghigo D. Diphenyleneiodonium inhibits the cell re-
dox metabolism and induces oxidative stress. J Biol Chem.
2004;279(46):47726–31.
17. Chen Q, Vazquez EJ, Moghaddas S, Hoppel CL, Lesnef-
sky EJ. Production of reactive oxygen species by mitochon-
dria: central role of complex III. J Biol Chem. 2003;
278(38):36027–31.
18. Shchepina LA, Pletjushkina OY, Avetisyan AV, Bakeeva LE,
Fetisova EK, Izyumov DS, Saprunova VB, Vyssokikh MY,
Chernyak BV, Skulachev VP. Oligomycin, inhibitor of the
F0 part of H+-ATP-synthase, suppresses the TNF-induced
apoptosis. Oncogene. 2002;21(53):8149–57.
19. Jagtap JC, Chandele A, Chopde BA, Shastry P. Sodium py-
ruvate protects against H(2)O(2) mediated apoptosis in hu-
man neuroblastoma cell line-SK-N-MC. J Chem Neuro-
anat. 2003;26(2):109–18.
20. Samuni Y, Goldstein S, Dean OM, Berk M. The chemistry
and biological activities of N-acetylcysteine. Biochim Bio-
phys Acta. 2013;1830(8):4117–29.
21. Minotti G, Menna P, Salvatorelli E, Cairo G, Gianni L. An-
thracyclines: molecular advances and pharmacologic devel-
opments in antitumor activity and cardiotoxicity. Pharma-
col Rev. 2004;56(2):185–229.
22. Liou GY, Storz P. Reactive oxygen species in cancer. Free
Radic Res. 2010;44(5):479–96.
202
R. R. Panchuk, L. V. Lehka, J. Rohr et al.
23. Lutzky J, Astor MB, Taub RN, Baker MA, Bhalla K, Gerva-
soni JE Jr, Rosado M, Stewart V, Krishna S, Hindenburg AA.
Role of glutathione and dependent enzymes in anthracycline-
resistant HL60/AR cells. Cancer Res. 1989;49(15):4120–5.
24. Legrand O, Simonin G, Beauchamp-Nicoud A, Zittoun R,
Marie JP. Simultaneous activity of MRP1 and Pgp is cor-
related with in vitro resistance to daunorubicin and with in
vivo resistance in adult acute myeloid leukemia. Blood.
1999;94(3):1046–56.
24. Legrand O, Zittoun R, Marie JP. Role of MRP1 in multi-
drug resistance in acute myeloid leukemia. Leukemia.
1999;13(4):578–84.
25. Versantvoort CH, Broxterman HJ, Bagrij T, Scheper RJ,
Twentyman PR. Regulation by glutathione of drug transport
in multidrug-resistant human lung tumour cell lines overex-
pressing multidrug resistance-associated protein. Br J Can-
cer. 1995;72(1):82–9.
Мітохондрії не відіграють основної ролі в індукції
оксидативного стресу при доланні множинної
лікарської стійкості в лейкозних клітинах лінії HL-60
ландоміцином Е
Р. Р. Панчук, Л. В. Легка, Ю. Рор, В. Бергер, Р. С. Стойка
Мета. Молекулярні механізми участі активних форм кисню
(АФК) в доланні множинної лікарської стійкості злоякісних
клітин новим ангуцикліновим антибіотиком ландоміцином E в
лейкозних клітинах людини лінії HL-60 і її сублініях HL-60/adr
і HL-60/vinc, резистетних до хіміотерапії. Методи. МТТ тест,
тест на життєздатність клітин з трипановим синім, фарбування
клітин флуоресцентними барвниками DCFDA і JC-1.
Результати. Ландоміцин Е (ЛЕ) призводить до значного зрос-
тання продукції пероксиду водню в клітинах лінії HL-60 вже
на 1 год після додавання препарату до культури клітин, в той
час як деполяризация мітохондрій спостерігаються тільки на
6-12 год, що вказує на позамітохондріальне джерело АФК за дії
цього антибіотика. Клітини лінії HL-60/vinc (P-глікопротеїн +),
незважаючи на 100-кратне зростання стійкості до доксорубі-
цину (Dx), продемонстрували ідентичну чутливість до ЛЕ в
порівнянні з вихідною клітинною лінією, в той час як клітини
лінії HL-60/adr (MRP- 1+), які проявляли 200-кратне зростання
стійкості до дії Dx, показали слабке (в 2 рази) зниження чутли-
вості до ЛЕ. Долання стійкості до ліків ландоміцином Е у клі-
тинах лінії HL-60/adr супроводжувалося 2-кратним зростан-
ням рівня Н2О2 в порівнянні з клітинами дикого типу, але міто-
хондріальні інгібітори дихального ланцюга не проявляли нія-
кого впливу на це явище. Висновки. ЛЕ-індукована загибель
пухлинних клітин супроводжується масовим виробництвом
перекису водню, що складається з двох піків – основного (1
год) і вторинного (на 12 год дії препарату). Найвища продукція
АФК спостерігалася в клітинах лінії HL-60/adr, які показали
2-кратне збільшення стійкості до ЛЕ в порівнянні з вихідною
лінією HL-60. Мітохондрії, очевидно, відіграють вторинну
роль в цьому процесі, оскільки використання специфічних ін-
гібіторів дихального ланцюга мітохондрій ніяк не вплинуло на
ранню індукцію АФК за дії ЛЕ в пухлинних клітинах.
К л юч ов і с л ов а: ландоміцин Е, ROS, мітохондрії, стій-
кість пухлин до ліків, апоптоз
Митохондрии не играют основной роли в индукции
оксидативного стресса при преодолении множественной
лекарственной устойчивости в лейкозных клетках
линии HL-60 ландомицином Е
Р. Р. Панчук, Л. В. Легкая, Ю. Рор, В. Бергер, Р. С. Стойка
Цель. Молекулярные механизмы участия активных форм кис-
лорода (АФК) в преодолении множественной лекарственной
устойчивости злокачественных клеток новым ангуциклино-
вым антибиотиком ландомицином E в лейкозных клетках че-
ловека линии HL-60 и ее сублиниях HL-60/adr и HL-60/vinc,
резистентных к химиотерапии. Методы. МТТ тест, тест на
жизнеспособность клеток с трипановым синим, окраска кле-
ток флуоресцентными красителями DCFDA и JC-1.
Результаты. Ландомицин Е (ЛЕ) приводит к значительному
росту продукции пероксида водорода в клетках линии HL-60
уже на 1 ч после добавления препарата к культуре клеток, в то
время как деполяризация митохондрий наблюдаются только на
6-12 ч, что указывает на внемитохондриальный источник АФК
происхождения при действии этого антибиотика. Клетки ли-
нии HL-60 vinc (P-гликопротеин+), несмотря на 100-кратный
рост устойчивости к доксорубицину (Dx), продемонстрирова-
ли идентичную чувствительность к ЛЕ по сравнению с исход-
ной клеточной линией, в то время как клетки линии HL-60/adr
(MRP-1+), которые проявляли 200-кратный рост устойчивости
к действию Dx, показали слабое (в 2 раза) снижение чувстви-
тельности к ЛЕ. Преодоление устойчивости к лекарствам лан-
домицином Е в клетках линии HL-60/adr сопровождалось
2-кратным ростом уровня Н2О2 по сравнению с клетками дико-
го типа, но митохондриальные ингибиторы дыхательной цепи
не проявляли никакого влияния на это явление. Выводы. ЛЕ-
индуцированная гибель опухолевых клеток сопровождается
массовым производством перекиси водорода, состоящей из
двух пиков – основного (1 час) и вторичного (на 12 ч действия
препарата). Самая высокая продукция АФК наблюдалась в
клетках линии HL-60/adr, которые показали 2-кратное увели-
чение устойчивости к ЛЕ по сравнению с исходной линией
HL-60. Митохондрии, очевидно, играют вторичную роль в
этом процессе, поскольку использование специфических ин-
гибиторов митохондриальной дыхательной цепи никак не по-
влияло на раннюю индукцию АФК при действии ЛЕ на опухо-
левые клетки.
К л юч е в ы е с л ов а: ландомицин Е, ROS, митохондрии,
устойчивость опухолей к лекарствам, апоптоз
Received 21.02.2016
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