β-lapachone induces growth inhibition and apoptosis in bladder cancer cells by modulation of BCL-2 family and activation of caspases
Aim: To study in vitro the molecular mechanism of apoptosis caused by b-lapachone, a quinone obtained from the bark of the lapacho tree (Tabebuia avellanedae). Materials and Methods: The study was carried out on human bladder carcinoma T24 cell line. Determination of cell viability was done using tr...
Gespeichert in:
| Datum: | 2006 |
|---|---|
| Hauptverfasser: | , , , , , , |
| Format: | Artikel |
| Sprache: | English |
| Veröffentlicht: |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
2006
|
| Schriftenreihe: | Experimental Oncology |
| Schlagworte: | |
| Online Zugang: | https://nasplib.isofts.kiev.ua/handle/123456789/134525 |
| Tags: |
Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
|
| Назва журналу: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Zitieren: | β-lapachone induces growth inhibition and apoptosis in bladder cancer cells by modulation of BCL-2 family and activation of caspases / J.I. Lee, D.Y. Choi, H.S. Chung, H.G. Seo, H.J. Woo, B.T. Choi, Y.H. Choi // Experimental Oncology. — 2006. — Т. 28, № 1. — С. 30-35. — Бібліогр.: 22 назв. — англ. |
Institution
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
nasplib_isofts_kiev_ua-123456789-134525 |
|---|---|
| record_format |
dspace |
| spelling |
nasplib_isofts_kiev_ua-123456789-1345252025-02-09T11:33:23Z β-lapachone induces growth inhibition and apoptosis in bladder cancer cells by modulation of BCL-2 family and activation of caspases β-лапакон индуцирует подавление роста и апоптоз опухолевых клеток мочевого пузыря путем модуляции семейства BCL-2 и активации каспаз Lee, J.I. Choi, D.Y. Chung, H.S. Seo, H.G. Woo, H.J. Choi, B.T. Choi, Y.H. Original contributions Aim: To study in vitro the molecular mechanism of apoptosis caused by b-lapachone, a quinone obtained from the bark of the lapacho tree (Tabebuia avellanedae). Materials and Methods: The study was carried out on human bladder carcinoma T24 cell line. Determination of cell viability was done using trypan blue exclusion method, apoptosis quantitative estimation — by DAPI staining and agarose gel electrophoresis for DNA fragmentation. Flow cytometry analysis, RT-PCR and Western blot analysis, colorimetric assay of caspase activity were applied as well. Results: It was found that in micromolar range of concentrations b-lapachone inhibited the viability of T24 cells by inducing apoptosis, which could be proved by formation of apoptotic bodies and DNA fragmentation. Treatment of T24 cells with b-lapachone resulted in a down-regulation of Bcl-2 expression and up-regulation of Bax expression. b-lapachone-induced apoptosis was also associated with activation of caspase-3 and caspase-9, inhibition of IAP expression, and degradation of poly (ADP-ribose) polymerase, phospholipase C-g1 and b-catenin proteins. At the same time Fas and FasL levels were inhibited upon treatment with b-lapachone in a concentration-dependent manner. Conclusion: b-lapachone-induced apoptosis in T24 cells is mediated, at least in part, by the mitochondrial-signaling pathway. Цель: изучить механизмы апоптоза клеток карциномы мочевого пузыря человека Т24 при действии β-лапакона, хинона из коры дерева Tabebuia avellanedae. Материалы и методы: для определения жизнеспособности клеток использовали окраску трипановым синим; окрашивание DAPI и электрофоретический анализ фрагментации ДНК в агарозном геле, метод проточной цитометрии (для количественной оценки апоптоза); полимеразную цепную реакцию в режиме реального времени (РВ-ПЦР) и Вестерн блот-анализ (для оценки уровня экспрессии генов и белков), а также колориметрический анализ активности каспаз. Результаты: выявлено, что в микромолярных концентрациях β-лапакон понижает жизне- способность клеток линии Т24 путем активации апоптоза, что подтверждается формированием апоптотических тел и фрагментацией ДНК. Результаты РВ-ПЦР и иммуноблоттинга указывают на то, что обработка клеток β-лапаконом приводит к снижению экспрессии Bcl-2 и к активации Bax. Апоптоз, индуцированный β-лапаконом, также сопровож- дается активацией каспаз -3 и -9, ингибированием экспрессии семейства IAP, а также деградацией поли-(ADP-рибозо) полимеразы, фосфатазы C-γ1 и β-катенина. Тем не менее, уровень экспрессии Fas и FasL снижался при увеличении концентрации β-лапакона. Выводы: апоптоз, индуцированный при действии β-лапакона в клетках Т24, может быть час- тично опосредован митохондриальным сигнальным каскадом. 2006 Article β-lapachone induces growth inhibition and apoptosis in bladder cancer cells by modulation of BCL-2 family and activation of caspases / J.I. Lee, D.Y. Choi, H.S. Chung, H.G. Seo, H.J. Woo, B.T. Choi, Y.H. Choi // Experimental Oncology. — 2006. — Т. 28, № 1. — С. 30-35. — Бібліогр.: 22 назв. — англ. 1812-9269 https://nasplib.isofts.kiev.ua/handle/123456789/134525 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 Lee, J.I. Choi, D.Y. Chung, H.S. Seo, H.G. Woo, H.J. Choi, B.T. Choi, Y.H. β-lapachone induces growth inhibition and apoptosis in bladder cancer cells by modulation of BCL-2 family and activation of caspases Experimental Oncology |
| description |
Aim: To study in vitro the molecular mechanism of apoptosis caused by b-lapachone, a quinone obtained from the bark of the lapacho tree (Tabebuia avellanedae). Materials and Methods: The study was carried out on human bladder carcinoma T24 cell line. Determination of cell viability was done using trypan blue exclusion method, apoptosis quantitative estimation — by DAPI staining and agarose gel electrophoresis for DNA fragmentation. Flow cytometry analysis, RT-PCR and Western blot analysis, colorimetric assay of caspase activity were applied as well. Results: It was found that in micromolar range of concentrations b-lapachone inhibited the viability of T24 cells by inducing apoptosis, which could be proved by formation of apoptotic bodies and DNA fragmentation. Treatment of T24 cells with b-lapachone resulted in a down-regulation of Bcl-2 expression and up-regulation of Bax expression. b-lapachone-induced apoptosis was also associated with activation of caspase-3 and caspase-9, inhibition of IAP expression, and degradation of poly (ADP-ribose) polymerase, phospholipase C-g1 and b-catenin proteins. At the same time Fas and FasL levels were inhibited upon treatment with b-lapachone in a concentration-dependent manner. Conclusion: b-lapachone-induced apoptosis in T24 cells is mediated, at least in part, by the mitochondrial-signaling pathway. |
| format |
Article |
| author |
Lee, J.I. Choi, D.Y. Chung, H.S. Seo, H.G. Woo, H.J. Choi, B.T. Choi, Y.H. |
| author_facet |
Lee, J.I. Choi, D.Y. Chung, H.S. Seo, H.G. Woo, H.J. Choi, B.T. Choi, Y.H. |
| author_sort |
Lee, J.I. |
| title |
β-lapachone induces growth inhibition and apoptosis in bladder cancer cells by modulation of BCL-2 family and activation of caspases |
| title_short |
β-lapachone induces growth inhibition and apoptosis in bladder cancer cells by modulation of BCL-2 family and activation of caspases |
| title_full |
β-lapachone induces growth inhibition and apoptosis in bladder cancer cells by modulation of BCL-2 family and activation of caspases |
| title_fullStr |
β-lapachone induces growth inhibition and apoptosis in bladder cancer cells by modulation of BCL-2 family and activation of caspases |
| title_full_unstemmed |
β-lapachone induces growth inhibition and apoptosis in bladder cancer cells by modulation of BCL-2 family and activation of caspases |
| title_sort |
β-lapachone induces growth inhibition and apoptosis in bladder cancer cells by modulation of bcl-2 family and activation of caspases |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2006 |
| topic_facet |
Original contributions |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/134525 |
| citation_txt |
β-lapachone induces growth inhibition and apoptosis in bladder cancer cells by modulation of BCL-2 family and activation of caspases / J.I. Lee, D.Y. Choi, H.S. Chung, H.G. Seo, H.J. Woo, B.T. Choi, Y.H. Choi // Experimental Oncology. — 2006. — Т. 28, № 1. — С. 30-35. — Бібліогр.: 22 назв. — англ. |
| series |
Experimental Oncology |
| work_keys_str_mv |
AT leeji blapachoneinducesgrowthinhibitionandapoptosisinbladdercancercellsbymodulationofbcl2familyandactivationofcaspases AT choidy blapachoneinducesgrowthinhibitionandapoptosisinbladdercancercellsbymodulationofbcl2familyandactivationofcaspases AT chunghs blapachoneinducesgrowthinhibitionandapoptosisinbladdercancercellsbymodulationofbcl2familyandactivationofcaspases AT seohg blapachoneinducesgrowthinhibitionandapoptosisinbladdercancercellsbymodulationofbcl2familyandactivationofcaspases AT woohj blapachoneinducesgrowthinhibitionandapoptosisinbladdercancercellsbymodulationofbcl2familyandactivationofcaspases AT choibt blapachoneinducesgrowthinhibitionandapoptosisinbladdercancercellsbymodulationofbcl2familyandactivationofcaspases AT choiyh blapachoneinducesgrowthinhibitionandapoptosisinbladdercancercellsbymodulationofbcl2familyandactivationofcaspases AT leeji blapakoninduciruetpodavlenierostaiapoptozopuholevyhkletokmočevogopuzyrâputemmodulâciisemejstvabcl2iaktivaciikaspaz AT choidy blapakoninduciruetpodavlenierostaiapoptozopuholevyhkletokmočevogopuzyrâputemmodulâciisemejstvabcl2iaktivaciikaspaz AT chunghs blapakoninduciruetpodavlenierostaiapoptozopuholevyhkletokmočevogopuzyrâputemmodulâciisemejstvabcl2iaktivaciikaspaz AT seohg blapakoninduciruetpodavlenierostaiapoptozopuholevyhkletokmočevogopuzyrâputemmodulâciisemejstvabcl2iaktivaciikaspaz AT woohj blapakoninduciruetpodavlenierostaiapoptozopuholevyhkletokmočevogopuzyrâputemmodulâciisemejstvabcl2iaktivaciikaspaz AT choibt blapakoninduciruetpodavlenierostaiapoptozopuholevyhkletokmočevogopuzyrâputemmodulâciisemejstvabcl2iaktivaciikaspaz AT choiyh blapakoninduciruetpodavlenierostaiapoptozopuholevyhkletokmočevogopuzyrâputemmodulâciisemejstvabcl2iaktivaciikaspaz |
| first_indexed |
2025-11-25T21:55:28Z |
| last_indexed |
2025-11-25T21:55:28Z |
| _version_ |
1849801032741158912 |
| fulltext |
30 Experimental Oncology 28, 30–35, 2006 (March)
Apoptosis, programmed cell death, plays important
role in controlling cells fate in normal physiological
state and upon carcinogenesis [4, 5, 12]. Several
genes’ products have been demonstrated to be critical
in the regulation of apoptosis. Caspases are essential
for the execution of cell death upon various apoptotic
stimuli [20].
Caspase activation is often regulated by various
cellular proteins, including members of the inhibitor
of apoptosis protein (IAP), the Bcl-2 family and/or the
Fas/FasL system [4, 5, 13, 18]. Emerging evidence
has demonstrated that anticancer activities of certain
chemotherapeutic agents are involved in the induction
of apoptosis, which is regarded as the preferred way
to manage cancer [4, 12].
β-lapachone (3,4-dihydro-2,2-dimenthyl-2H-nap-
thol [1,2-b]pyran-5,6-dione) is natural plant quinone
obtained from the bark of the lapacho tree (Tabebuia
avellanedae) which is native to South America [21].
This compound inhibits reverse transcriptase and DNA
polymerase-α, and blocks DNA repair thus sensitizing
tumor cells to DNA — damaging agents [2, 21]. Several
studies suggested that β-lapachone could directly target
DNA topoisomerases and inhibit their activity [8, 15, 16].
However, its inhibitory mode is distinct from that of other
typical topoisomerase inhibitors, such as camptothecin
and related compounds [15]. β-lapachone exhibits
anti-bacterial, anti-fungal and anti-trypanocidal action
[10, 11, 17], which are linked to the formation of reactive
oxygen species [7, 19]. In addition, it has been known
that β-lapachone induces apoptotic cell death in human
cancer cells, however, the molecular mechanisms of its
anti-proliferative action are not known completely.
In this study we determined the antiproliferative
activity of β-lapachone, and examined its effect on
apoptosis in the human bladder carcinoma cell line
T24. Furthermore, to establish the anticancer mecha-
nism of β-lapachone, we assayed the levels of several
important genes, which are strongly associated with
apoptosis and influence the sensitivity of tumor cells
to anticancer agents.
MATERIALS AND METHODS
Cell culturing. Human bladder carcinoma cell line
T24 was purchased from the American Type Culture
Collection (Rockville, USA), and cultured in RPMI
1640 medium supplemented with 10% heat-inacti-
vated fetal bovine serum (FBS), 2 mM L-glutamine,
100 µg/mL streptomycin, and 100 µg/mL penicillin
(Gibco BRL, USA) at 37 °C and 5% CO2. β-lapachone
was purchased from Biomol (Plymouth Meeting, PA,
USA) and dissolved in ice-cold absolute alcohol as
a stock solution at 10 mM concentration, and stored
in aliquots at –20 °C. Cells were cultured in the ab-
sence and presence of variable concentrations of
β-lapachone for 48 h, then trypsinized, washed with
phosphate-buffered saline (PBS), and the viable cells
were scored using hemocytometer and the exclusion
of trypan blue.
β-LAPACHONE INDUCES GROWTH INHIBITION AND APOPTOSIS IN
BLADDER CANCER CELLS BY MODULATION OF BCL-2 FAMILY AND
ACTIVATION OF CASPASES
J.I. Lee1, D.Y. Choi1, H.S. Chung1, H.G. Seo1, H.J. Woo2, B.T. Choi2, Y. H. Choi2, 3, *
1R&E Program, Korea Science Academy, Busan, South Korea
2Department of Oriental Medicine Dongeui University, Busan, South Korea
3Department of Biomaterial Control, Dongeui University, Busan, South Korea
Aim: To study in vitro the molecular mechanism of apoptosis caused by β-lapachone, a quinone obtained from the bark of the la-
pacho tree (Tabebuia avellanedae). Materials and Methods: The study was carried out on human bladder carcinoma T24 cell line.
Determination of cell viability was done using trypan blue exclusion method, apoptosis quantitative estimation — by DAPI staining
and agarose gel electrophoresis for DNA fragmentation. Flow cytometry analysis, RT-PCR and Western blot analysis, colorimetric
assay of caspase activity were applied as well. Results: It was found that in micromolar range of concentrations β-lapachone inhibited
the viability of T24 cells by inducing apoptosis, which could be proved by formation of apoptotic bodies and DNA fragmentation.
Treatment of T24 cells with β-lapachone resulted in a down-regulation of Bcl-2 expression and up-regulation of Bax expression.
β-lapachone-induced apoptosis was also associated with activation of caspase-3 and caspase-9, inhibition of IAP expression, and
degradation of poly (ADP-ribose) polymerase, phospholipase C-γ1 and β-catenin proteins. At the same time Fas and FasL levels
were inhibited upon treatment with β-lapachone in a concentration-dependent manner. Conclusion: β-lapachone-induced apoptosis
in T24 cells is mediated, at least in part, by the mitochondrial-signaling pathway.
Key Words: β-lapachone, T24 bladder cancer cells, apoptosis, Bcl-2 family, caspases.
Received: October 19, 2005.
*Correspondence: Fax: +82-51-853-4036;
E-mail: choiyh@deu.ac.kr
Abbreviations used: cIAP — cellular inhibitor of apoptotis
protein; DAPI — 4,6-diamidino-2-phenylindile; ECL — enhanced
chemiluminescence; EtBr — ethidium bromide; FBS — fetal bovine
serum; GAPDH — glyceraldehyde-3-phosphate dehydrogenase;
IAP — inhibitor of apoptosis protein; PARP — poly (ADP-ribose)
polymerase; PBS — phosphate-buffered saline; PCR — polymerase
chain reaction; PI — propidium iodide; PLC- γ1 — phospholipase
C-γ1; pNA — p-nitroaniline; XIAP — X-linked inhibitor to apoptotis.
Exp Oncol 2006
28, 1, 30–35
Experimental Oncology 28, 30–35, 2006 (March) 31
Nuclear staining with DAPI. Cells were washed with
cold PBS and fixed with 3.7% paraformaldehyde in
PBS for 10 min at room temperature. Fixed cells were
washed with PBS, and stained with 4, 6-diamidino-
2-phenylindile (DAPI, Sigma Chemical Co., USA)
solution for 10 min at room temperature. The cells
were washed two more times with PBS and analyzed
by fluorescent microscopy.
DNA fragmentation assay. After treatment with
β-lapachone, cells were lysed in a buffer containing
10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA
and 0.5% Triton X-100 for 30 min on ice. Lysates were
vortexed and cleared by centrifugation at 10.000 g
for 20 min. Fragmented DNA in the supernatant was
extracted with an equal volume of neutral phenol :
chloroform : isoamylalcohol (25 : 24 : 1, v/v/v) and
analyzed electrophoretically on 1% agarose gel con-
taining ethidium bromide (EtBr, Sigma).
Flow cytometry assay. After treatment with β-
lapachone, cells were collected, washed with cold
PBS, and resuspended in PBS. DNA contents of cells
were measured using a DNA staining kit (CycleTESTTM
PLUS Kit, Becton Dickinson, USA). Propidium iodide
(PI) — stained nuclear fractions were obtained by
following the kit protocol. Fluorescence intensity was
determined using a FACScan flow cytometer and ana-
lyzed by CellQuest software (Becton Dickinson).
RNA extraction and RT-PCR. Total RNA was
isolated according to the method [3]. Single-stranded
cDNA was synthesized from 2 µg of total RNA using
M-MLV reverse transcriptase (Gibco BRL). The mRNAs
were amplified by polymerase chain reaction (PCR)
with desired primers (see Table). Conditions for PCR
reaction were 1 х (94 °C, 3 min); 35 х (94 °C, 45 s;
58 °C, 45 s; and 72 °C, 1 min) and 1 х (72 °C, 10 min).
Amplification products obtained by PCR were electro-
phoretically separated on 1% agarose gel.
Table 1. Gene-specific primers for RT-PCR
Name Sequence of primers
Bax sence 5΄-ATG-GAC-GGG-TCC-GGG-GAG-3΄
antisence 5΄-TGG-AAG-AAG-ATG-GGC-TGA-3΄
Bcl-2 sence 5΄-CAG-CTG-CAC-CTG-ACG-3΄
antisence 5΄-GCT-GGG-TAG-GTG-CAT-3΄
Bcl-XL sence 5΄-CAG CTG CAC CTG ACG-3΄
antisence 5΄-GCT GGG TAG GTG CAT-3΄
XIAP sence 5΄-GAA-GAC-CCT-TGG-GAA-CAA-CA-3΄
antisence 5-CGC-CTT-AGC-TGC-TCT-CTT-CAG-T-3΄
cIAP-1 sence 5΄-TGA-GCA-TGC-AGA-CAC-ATG-C-3΄
antisence 5΄-TGA-CGG-ATG-AAC-TCC-TGT-CC-3΄
cIAP-2 sence 5΄-CAG-AAT-TGG-CAA-GAG-CTG-G-3΄
antisence 5΄-CAC-TTG-CAA-GCT-GCT-CAG-G-3΄
Fas sence 5΄-TCT-AAC-TTG-GGG-TGG-CTT-TGT-CTT-C-3΄
antisence 5΄-GTG-TCA-TAC-GCT-TTC-TTT-CCA-T-3΄
Fas sence 5΄-GGA-TTG-GGC-CTG-GGG-ATG-TTT-CA-3΄
antisence 5΄AGC-CCA-GTT-TCA-TTG-ATC-ACA-AGG-3΄
GAPDH sence 5΄-CGG-AGT-CAA-CGG-ATT-TGG-TCG-TAT-3΄
antisence 5΄-AGC-CTT-CTC-CAT-GGT-GGT-GAA-GAC-3΄
Gel electrophoresis and Western blot analysis.
The cells were harvested, lyzed, and protein concen-
trations were measured using the Bio Rad protein
assay (BioRad Lab., USA), according to protocol of
manufacturer. An equal amount of protein was subject-
ed to electrophoresis on SDS-polyacrylamide gels and
transferred to nitrocellulose membranes (Schleicher &
Schuell, USA) by electroblotting. Blots were incubated
with the desired antibodies for 1 h, incubated with di-
luted enzyme-linked secondary antibodies, and then
visualized by the enhanced chemiluminescence (ECL)
according to the recommended procedure (Amersham
Corp., USA). The primary antibodies were purchased
from Santa Cruz Biotechnology Inc. (USA). Peroxi-
dase-labeled donkey anti-rabbit immunoglobulin and
peroxidase-labeled sheep anti-mouse immunoglobu-
lin were purchased from Amersham.
In vitro caspase activity assay. Caspases activity
was determined by colorimetric assay using the cas-
pase-3 and caspase-9 activation kits from Clontech
Lab. (USA) and R&D Systems (USA), respectively,
following the manufacturer’s protocol. The kits used in
the present study utilize synthetic tetrapeptides, DEVD
for caspase-3 and LEHD for caspase-9, labeled with
p-nitroaniline (pNA). Briefly, β-lapachone-treated and
untreated cells were lyzed in the supplied lysis buffer.
The supernatants were collected and incubated with
the supplied reaction buffer containing dithiothreitol
and substrates at 37 °C. The reaction was measured by
changes in absorbance at 405 nm using the VERSAmax
tunable microplate reader (Molecular devices, USA).
RESULTS AND DISCUSSION
Inhibition of viability and induction of apop-
tosis by β-lapachone. Cells were cultured in 10%
FBS-containing medium, with or without β-lapachone
for 48 h, and cell viability was evaluated by hemocy-
tometer counts. As shown in Fig. 1, a, β-lapachone had
a marked dose — dependent inhibitory effect on T24
cell viability. Then we examined whether β-lapachone
may influence apoptosis of T24 cells. By morphological
analysis with DAPI staining, the untreated control cells
displayed intact nuclear structures, while in cells treated
with β-lapachone chromosomal condensation and the
formation of apoptotic bodies were observed, indicat-
ing that β-lapachone induced apoptotic cell death in a
concentration-dependent manner (Fig.1, b).
Another hallmark of apoptosis is degradation of
chromosomal DNA at internucleosomal linkages [22].
So, we analyzed whether DNA fragmentation was
induced by β-lapachone in T24 cells. After treatment
of T24 cells with β-lapachone, a typical ladder pat-
tern of internucleosomal fragmentation was observed
(Fig. 2, a). To evaluate an apoptosis rate, we analyzed
the amount of sub-G1 population by the flow cytometry
of fixed nuclei. As shown in Fig. 2, b, the addition of
β-lapachone to T24 cells resulted in a markedly in-
creased accumulation of cells on the sub-G1 phase in
a dose-dependent manner. These results suggest that
upon treatment with β-lapachone, T24 cells underwent
apoptosis, and that there is a good correspondence
between apoptosis rate and inhibition of cell viability.
32 Experimental Oncology 28, 30–35, 2006 (March)
Fig. 1. Viability of T24 cells treated with β-lapachone (1–5 µM)
for 48 h (a) and induction of apoptotic bodies (b). (a) Results
are expressed as the means ± S.E. of three independent
experiments. (b) Cells were incubated with β-lapachone
(1–5 µM) for 48 h. A number in the right corner responds to
concentration of β-lapachone. Cells were stained with DAPI,
stained nuclei were observed in fluorescent microscope using
a blue filter. Magnification, X 400
Modulation of Bcl-2 family and activation of
caspases by β-lapachone. Apoptotic cell death is
known to be regulated by pro-apoptotic and anti-ap-
optotic modulators, including these of Bcl-2 family [5,
20]. To analyze whether β-lapachone induces cell death
by modulating the expression of Bcl-2 family proteins,
after exposure to different concentrations of β-lapa-
chone, total RNAs and cellular proteins were isolated,
and RT-PCR and immunoblotting were performed.
The results indicated that treatment of cells with β-la-
pachone resulted in a down-regulation of expression
of anti-apoptotic Bcl-2 protein and up-regulation of
pro-apoptotic Bax (Fig. 3, a and b).
Recent studies have identified caspases as impor-
tant mediators of apoptosis induced by various apop-
totic stimuli [5, 20]. To determine the role of caspases
in β-lapachone-induced apoptosis, we determined the
levels of caspase-3 and caspase-9 expression in T24
cells treated with β-lapachone. As shown in Fig. 4, a,
β-lapachone-induced apoptosis was associated with a
decreased expression of the pro-caspase-3 and pro-
caspase-9 protein; however, we did not observed the
active subunits of both caspases. To further quantify
the proteolytic activities of caspase-3 and caspase-9,
lyzates equalized for protein from cells treated with
β-lapachone were assayed for in vitro caspase-3 and
caspase-9 activity using as substrates DEVD-pNA
and LEHD-pNA, respectively. The results showed
that exposure of cells β-lapachone increased both
caspase-3 and caspas-9 activity in a concentration-
dependent manner.
Activated caspases induce a limited proteolysis in
a number of cellular proteins, which are degraded and
have been used as a marker of chemotherapy-induced
apoptosis [1, 9, 14]. Here, we examined whether the
substrate proteins such as poly (ADP-ribose) polymer-
ase (PARP), phospholipase C-γ1 (PLC- γ1) and β-cate-
nin were cleaved in cells treated with β-lapachone.
A dose-dependent degradation of mentioned proteins
was observed (Fig. 4, b), being in correlation with
caspases activation upon treatment with β-lapachone.
Because the anti-apoptotic IAP family proteins bind
to caspases leading to their inactivation in eukaryotic
cells [5, 6], we further examined the involvement of
Fig. 2. Induction of DNA fragmentation (a) and flow cytometry
analysis of T24 cells treated by β-lapachone (1–5 µM). (b) Each
point represents the mean of two independent experiments
Experimental Oncology 28, 30–35, 2006 (March) 33
the IAP family in β-lapachone-dependent apoptosis.
As shown in Fig. 5, the levels of both X-linked inhibitor
to apoptosis (XIAP) and cellular inhibitor of apoptosis
protein (cIAP)-1 expression were decreased upon
treatment with β-lapachone and cIAP-2 protein was
cleaved in a concentration dependent fashion. These
results suggest that β-lapachone-induced apoptosis
in T24 cells is mediated, at least in part, by the mito-
chondrial-signaling pathway.
Effects of β-lapachone on the levels of Fas and
FasL expression. Fas/FasL system is a key signaling
transduction pathway of apoptosis in cells and tissues
[13, 18]. Ligation of Fas by an agonistic antibody or its
mature ligand induces receptor oligomerization and for-
mation of death-inducing signaling complex, followed by
activation of caspase-8, with further activation of caspase
cascade resulting in cell apoptotic death [5, 20]. How-
ever, the role of Fas/Fas ligand system in the control of
apoptosis in bladder cancer is controversial. We further
examined the involvement of the Fas/FasL system in T24
cells treated with β-lapachone. As shown in Fig. 6, Fas
and Fas-L expression decreased upon the treatment with
β-lapachone in a concentration dependent fashion.
In summary, we have shown here that β-lapachone
induces apoptosis in T24 cell line, which appears to
account for its anti-proliferating activity. These apop-
totic mechanisms were mediated by an activation of
caspases and an upregulating of the ratio Bax/Bcl-2,
and associated with a down-regulation of IAP family.
Our study has demonstrated that β-lapachone may
be further studied as a promising agent for treatment
of bladder cancer.
Fig. 3. Influence of β-lapachone (1–5 µM) on expression of Bcl-2 and Bax in T24 cells on mRNA (a) and protein (b) levels evaluated
by RT-PCR and Western-blotting, respectively
Fig. 4. Activation of caspase-3 and caspase-9 (a) and degradation of PARP, PLC-γ1 and β–catenin protein (b) upon treatment of
T24 cells β-lapachone (1–5 µM). Data represent the relative mean values of two independent experiments
34 Experimental Oncology 28, 30–35, 2006 (March)
REFERENCES
1. Bae SS, Perry DK, Oh YS, Choi JH, Galadari SH,
Ghayur T, Ryu SH, Hannun YA, Suh PG. Proteolytic cleav-
age of phospholipase C-γ1 during apoptosis in Molt-4 cells.
FASEB J 2000; 14: 1083–92.
2. Boothman DA, Trask DK, Pardee AB. Inhibition of
potentially lethal DNA damage repair in human tumor cells
by β-lapachone, an activator of topoisomerase I. Cancer Res
1989; 49: 605–12.
3. Chorna I, Fedorenko O, Datsyuk L, Stoika R. Expression
of mRNA coding for TGF-β and its receptors in irradiated hu-
man breast carcinoma MCF-7 cells differing in their sensitivity
to doxorubicin. Exp Oncol 2005; 27: 156–8.
4. D’Agostini F, Izzotti A, Balansky RM, Bennicelli C, De
Flora S. Modulation of apoptosis by cancer chemopreventive
agents. Mutat Res 2005; 591: 173–86.
5. Debatin KM. Apoptosis pathways in cancer and cancer
therapy. Cancer Immunol Immunother 2004; 53: 153–9.
6. Deveraux QL, Reed JC. IAP family proteins-suppressors
of apoptosis. Genes Dev 1999; 13: 239–52.
7. Fernandez VS, Stoppani AO, Dubin M. Redox cycling
of β-lapachone and structural analogues in microsomal
and cytosol liver preparations. Methods Enzymol 2004;
378: 67–87.
8. Frydman B, Marton LJ, Sun JS, Neder K, Witiak DT,
Liu AA, Wang HM, Mao Y, Wu HY, Sanders MM, Liu LF.
Induction of DNA topoisomerase II-mediated DNA cleavage
by β-lapachone and related naphthoquinones. Cancer Res
1997; 57: 620–27.
9. Fukuda K. Apoptosis-associated cleavage of β-catenin
in human colon cancer and rat hepatoma cells. Int J Biochem
Cell Biol 1999; 31: 519–29.
10. Goijman SG, Stoppani AO. Effects of β-lapachone, a
peroxide-generating quinone, on macromolecule synthesis
and degradation in Trypanosoma cruzi. Arch Biochem Biophys
1985; 240: 273–80.
11. Guiraud P, Steiman R, Campos-Takaki GM, Seigle-
Murandi F, Simeon de Buochberg M. Comparison of antibac-
terial and antifungal activities of lapachol and β-lapachone.
Planta Med 1994; 60: 373–4.
12. Hengartner MO. The biochemistry of apoptosis. Na-
ture 2000; 407: 770–6.
13. Jiang C, Yang YF, Cheng SH. Fas ligand gene ther-
apy for vascular intimal hyperplasia. Curr Gene Ther 2004;
4: 33–9.
14. Lazebnik YA, Kaufmann SH, Desnoyers S, Poirier GG,
Earnshaw WC. Cleavage of poly(ADP-ribose polymerase by a
proteinase with properties like ICE. Nature 1994; 371: 346–7.
15. Li CJ, Averboukh L, Pardee AB. β-lapachone, a novel
DNA topoisomerase I inhibitor with a mode of action different
from camptothecin. J Biol Chem 1993; 268: 22463–8.
16. Li CJ, Wang C, Pardee AB. Induction of apoptosis
by β-lapachone in human prostate cancer cells. Cancer Res
1995; 55: 3712–5.
17. Lopes JN, Cruz FS, Docampo R, Vasconcellos ME,
Sampaio MC, Pinto AV, Gilbert B. In vitro and in vivo evalua-
tion of the toxicity of 1,4-naphthoquinone and 1,2-naphtho-
quinone derivatives against Trypanosoma cruzi. Ann Trop Med
Parasitol 1978; 72: 523–31.
18. O’Brien DI, Nally K, Kelly RG, O’Connor TM, Shana-
han F, O’Connell J. Targeting the Fas/Fas ligand pathway in
cancer. Expert Opin Ther Targets 2005; 9: 1031–44.
19. Pardee AB, Li YZ, Li CJ. Cancer therapy with β-lapa-
chone. Curr Cancer Drug Targets 2002; 2: 227–42.
Fig. 5. Influence of β-lapachone (1–5 µM) on expression of XIAP, cIAP-1 and cIAP-2 in T24 cells on mRNA (a) and protein (b) levels
evaluated by RT-PCR and Western-blotting, respectively
Fig. 6. Influence of β-lapachone (1–5 µM) on expression of Fas and FasL in T24 cells on mRNA (a) and protein (b) levels evaluated
by RT-PCR and Western-blotting, respectively
Experimental Oncology 28, 30–35, 2006 (March) 35
20. Philchenkov A, Zavelevich M, Kroczak TJ, Los M.
Caspases and cancer: mechanisms of inactivation and new
treatment modalities. Exp Oncol 2004; 26: 82–97.
21. Schaffner-Sabba K, Schmidt-Ruppin KH, Wehrli W,
Schuerch AR, Wasley JW. β-lapachone: synthesis of de-
rivatives and activities in tumor models. J Med Chem 1984;
27: 990–4.
22. Walker PR, Sikorska M. New aspects of the mecha-
nism of DNA fragmentation in apoptosis. Biochem Cell Biol
1997; 75: 287–99.
Copyright © Experimental Oncology, 2006
β-ЛАПАКОН ИНДУЦИРУЕТ ПОДАВЛЕНИЕ РОСТА И АПОПТОЗ
ОПУХОЛЕВЫХ КЛЕТОК МОЧЕВОГО ПУЗЫРЯ ПУТЕМ
МОДУЛЯЦИИ СЕМЕйСТВА BCL-2 И АКТИВАЦИИ КАСПАЗ
Цель: изучить механизмы апоптоза клеток карциномы мочевого пузыря человека Т24 при действии β-лапакона, хинона
из коры дерева Tabebuia avellanedae. Материалы и методы: для определения жизнеспособности клеток использовали
окраску трипановым синим; окрашивание DAPI и электрофоретический анализ фрагментации ДНК в агарозном геле,
метод проточной цитометрии (для количественной оценки апоптоза); полимеразную цепную реакцию в режиме реального
времени (РВ-ПЦР) и Вестерн блот-анализ (для оценки уровня экспрессии генов и белков), а также колориметрический
анализ активности каспаз. Результаты: выявлено, что в микромолярных концентрациях β-лапакон понижает жизне-
способность клеток линии Т24 путем активации апоптоза, что подтверждается формированием апоптотических тел и
фрагментацией ДНК. Результаты РВ-ПЦР и иммуноблоттинга указывают на то, что обработка клеток β-лапаконом
приводит к снижению экспрессии Bcl-2 и к активации Bax. Апоптоз, индуцированный β-лапаконом, также сопровож-
дается активацией каспаз -3 и -9, ингибированием экспрессии семейства IAP, а также деградацией поли-(ADP-рибозо)
полимеразы, фосфатазы C-γ1 и β-катенина. Тем не менее, уровень экспрессии Fas и FasL снижался при увеличении
концентрации β-лапакона. Выводы: апоптоз, индуцированный при действии β-лапакона в клетках Т24, может быть час-
тично опосредован митохондриальным сигнальным каскадом.
Ключевые слова: β-лапакон, клетки рака мочевого пузыря линии T24, апоптоз, семейство Bcl-2, каспазы.
|