From the Department of Dermatology, Case Western Reserve University and Research Institute of University Hospitals of Cleveland, Cleveland, Ohio 44106
Received for publication, August 1, 2000, and in revised form, December 21, 2000
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ABSTRACT |
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Photodynamic therapy (PDT), a promising treatment
modality, is an oxidative stress that induces apoptosis in many cancer
cells in vitro and tumors in vivo.
Understanding the mechanism(s) involved in PDT-mediated apoptosis may
improve its therapeutic efficacy. Although studies suggest the
involvement of multiple pathways, the triggering event(s) responsible
for PDT-mediated apoptotic response is(are) not clear. To investigate
the role of Bcl-2 in PDT-mediated apoptosis, we employed
Bcl-2-antisense and -overexpression approaches in two cell types
differing in their responses toward PDT apoptosis. In the first
approach, we treated radiation-induced fibrosarcoma (RIF 1) cells,
which are resistant to silicon phthalocyanine (Pc 4)-PDT apoptosis,
with Bcl-2-antisense oligonucleotide. This treatment resulted in
sensitization of RIF 1 cells to PDT-mediated apoptosis as demonstrated
by i) cleavage of poly(ADP-ribose) polymerase, ii) DNA ladder
formation, iii) terminal deoxynucleotidyl transferase-mediated dUTP
nick end labeling (TUNEL)-positive cells, and iv) DEVDase activity. This treatment also resulted in oligonucleotide
concentration-dependent decrease in cell viability and
down-regulation of Bcl-2 protein with a concomitant increase in
apoptosis. However, the level of Bax, a pro-apoptotic member of
Bcl-2 family, remained unaltered. In the second approach, an
overexpression of Bcl-2 in PDT apoptosis-sensitive human epidermoid
carcinoma (A431) cells resulted in enhanced apoptosis and up-regulation
of Bax following PDT. In both the approaches, the increased Bax/Bcl-2
ratio was associated with an increased apoptotic response of PDT. Our
data also demonstrated that PDT results in modulation of other Bcl-2
family members in a way that the overall ratio of pro-apoptotic and
anti-apoptotic member proteins favors apoptosis.
Photodynamic therapy
(PDT)1 with the
phthalocyanine photosensitizer Pc 4 (HOSiPcOSi(CH3)2(CH2)3N(CH3)2)
is an oxidative stress that induces cell death, mainly through
apoptosis, in many tumor cells in vitro and during tumor
shrinkage in vivo (1-6). PDT, a United States FDA-approved
modality for the treatment of esophageal and lung cancer and actinic
keratosis, is currently undergoing clinical trials for the treatment of
many other solid cancers as well as many non-malignant conditions
(1-6). PDT relies on a bimodal protocol in which visible light of an
appropriate wavelength activates tumor cell-associated photosensitizer
to produce reactive oxygen species. The involvement of apoptosis has
been shown as an early response of PDT, both during in vitro
tumor cell killing and in vivo situations during tumor
ablation (7-11). Studies from this laboratory have shown the
involvement of i) an increased generation of nitric oxide (12), ii) a
WAF1/p21-mediated inhibition of the cyclin-cdk network (10), and iii) a
deregulation of pRb/E2F-DP machinery (13) during Pc 4-PDT-mediated
apoptosis. Studies have shown the involvement of phospholipases A2 and
C (8), intracellular Ca2+ (8, 14), ceramide (15, 16),
caspases (17, 18), c-Jun N-terminal kinase (JNK)/p38 MAPK (19, 20), and
cytochrome c release (17, 18) during PDT-mediated apoptotic
cell death. Thus, it has become clear that multiple pathways are
involved in PDT-mediated apoptosis. This offers exciting opportunities to take advantage of these pathways in improving PDT treatment protocol. The understanding of the mechanism(s) of triggering event(s)
of PDT-mediated apoptosis is far from complete. Mitochondrial damage
has been suggested as an early event in PDT-mediated apoptosis, which
could result in the release of apoptotic factors like cytochrome c, which in turn, activates the downstream targets in the
apoptotic pathway (21). Members of Bcl-2 family of proteins are
critical regulators of the apoptotic pathway. Bcl-2, an antiapoptotic
member of Bcl-2 family inhibits the release of cytochrome c
from mitochondria whereas Bax and Bid proteins, pro-apoptotic
members of Bcl-2 family has been shown to release cytochrome
c from the mitochondria thereby enhancing apoptotic response
(22-24). The role of Bcl-2 protein in PDT-mediated apoptosis is not
convincingly established (4, 25-28). In the present study, employing
two cell lines differing in their response to Pc 4-PDT-induced
apoptosis and antisense treatment and overexpression approaches we show
that Bcl-2 plays an important role in PDT-mediated apoptosis.
Furthermore, the ratio of Bax (pro-apoptotic) and Bcl-2 (antiapoptotic)
proteins appears to determine the response to PDT-induced apoptosis.
Cells--
Radiation-induced fibrosarcoma (RIF 1) and human
epidermoid carcinoma (A431) cells were maintained in Eagle's minimal
essential medium supplemented with 15% fetal bovine serum and 1%
penicillin/streptomycin and Dulbecco's modified Eagle's medium (10%
fetal bovine serum, 1% penicillin/streptomycin) and kept in an
atmosphere of 95% air/5% CO2 in a 37 °C humidified
incubator. In all experiments, 70-80% confluent cells were used.
Antisense Treatment--
Antisense oligonucleotides directed
against the coding region of Bcl-2 protein were custom-synthesized from
Operon Biotechnologies Inc. (Alameda, CA).
Phosphorothiote-modified 20-mer antisense (ATTCCTCCCCCAGTTCACCC)
and scrambled mismatched control oligo (CTCATTCCTACCGACACCCC) were used
in this study. Oligonucleotides were diluted with HEPES-buffered saline
to a final concentration of 5.0 µg/ml or unless otherwise mentioned.
Lipofectin reagent (Sigma Chemical Co., St. Louis, MO) was mixed with
diluted DNA (at a concentration of 10 µg/ml/µg of DNA) and
incubated at room temperature for 30 min. This mixture was further
diluted in 5 ml of Eagle's minimal essential medium and added to cells
in 100-mm plates followed by incubation at 37 °C for 12 h.
Transfection of A431 Cells--
The A431 cells were transfected
with a eukaryotic expression vector (pSFFV/neo) containing an
EcoRI fragment of human Bcl-2 cDNA. The cells were
seeded at 2 × 105 cells in 100-mm plates and were
allowed to attach overnight. On the following day, cells were
transfected with 1.0 µg of vector using Lipofectin transfection
reagents according to the manufacturer's protocol (Sigma Chemical
Co.). The transfectants were selected on neomycin-containing media and
were allowed to grow on the selection media for 48 h and then
subjected to PDT as described below.
PDT--
Cells were washed with Hanks' balanced salt solution
(HBSS, with Ca2+ and Mg2+) and were treated
with Pc 4 (0.5 µM in complete media) overnight in 100-mm
disposable culture plates. Next morning, the cells were washed with
HBSS, irradiated with 15 kJ/m2 light, as measured by a
digital photometer (Tektronix, Beaverton, OR), using a 300-watt halogen
lamp. The light was filtered through a Lee primary red filter (no. 106, Vincent Lighting, Cleveland, OH) to remove light with wavelengths of
<600 nm. Following irradiation, the cells were incubated in darkness
for selected time in a humidified incubator at 37 °C. Appropriate
controls, as specified at appropriate places, were also included. After
the specified times, the medium was aspirated, the cells were washed
with cold PBS (10 mM, pH 7.4), and processed as desired.
Preparation of Cell Lysate--
Following the treatments, cells
were washed with PBS (10 mM, pH 7.4) and incubated in lysis
buffer (150 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 20 mM NaF, 100 mM Na3VO4, 0.5% Nonidet P-40, 1%
Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml leupeptin) on ice for 30 min. The plates were scrapped, and cell lysates were collected in a centrifuge tube and
passed through a 21-gauge needle to break the cell aggregates. Cell
lysates were centrifuged at 14,000 × g at 4 °C, and
the supernatants (total cell lysate) were collected and stored at
Immunoblot Analysis--
50-100 µg of protein was resolved
over SDS/12% polyacrylamide gels and electrotransferred to
nitrocellulose membrane. The blot was blocked in blocking buffer (5%
nonfat dry milk, 1% Tween-20; in 20 mM Tris-buffered
saline, pH 7.5) for 1 h at room temperature, incubated with
appropriate monoclonal or polyclonal primary antibody (Bcl-2, Bax,
Bcl-xs, Bcl-xL and Bak from Oncogene Research
Products Cambridge, MA; PARP and DNA fragmentation factor from
Upstate Biotechnology, Lake Placid, NY; Bad from Calbiochem-Novabiochem Corp., San Diego, CA; Bid from Santa Cruz Biotechnology, Inc., Santa
Cruz, CA) in blocking buffer for 1 h to overnight at 4 °C. Blots were then incubated with anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibody and detected by
chemiluminescence and autoradiography using ECL Hyperfilm
(Amersham Pharmacia Biotech. Piscataway, NJ).
DNA Isolation, Electrophoresis, and Quantification of Apoptosis
by Flow Cytometry--
After treatment, the cells were washed with PBS
and suspended in cytoplasm extraction buffer (10 mM Tris,
150 mM NaCl/5 mM MgCl2/0.5%
Triton-X), left over ice for 30 min, and then centrifuged at
14,000 × g at 4 °C. The resultant pellet was
discarded, and the supernatant was incubated with RNase H (0.2 mg/ml)
overnight at 4 °C and then with Proteinase K (0.1 mg/ml) at 37 °C
for 2 h. DNA was extracted by treatment of the supernatant with
phenol:chloroform (1:1) and then precipitated with 95% ethanol for
2 h at DEVDase Assay--
Caspase activity was measured by DEVDase
assay. In brief, the cell lysates were collected at different time
points and protein was estimated. Then, 50 µg of protein was mixed
with the substrate solution and incubated at room temperature for
1 h. At the end of the incubation time, 940 µl water was added
to the tubes and placed on ice. Samples were analyzed for AFC
florescence spectrofluorometrically using 460-nm excitation and 505-nm
emission wavelengths.
Cell Viability Assay--
The cytotoxic effect of antisense
oligonucleotide was assessed by the
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT)
assay. The cells were seeded at a density of 1 × 105
cells/well in a 96-well microtiter plate and allowed to attach overnight. Cells were grown to 70% confluency and then treated with
different concentrations of antisense oligonucleotide (1-5 µg of
DNA) overnight. Scrambled oligonucleotide was used as positive control.
Next day, the media containing DNA was aspirated and washed with HBSS
and then subjected to Pc 4-PDT. Cells were incubated for 6 h in
complete media and then the incubation media was aspirated the cells
were incubated with 50 µl of MTT (1.0 mg/ml) reagent at 37 °C for
6 h. MTT reagent was aspirated and washed with PBS, 150 µl of
Me2SO was added, and the mixture was placed on a shaker for
10 min. Optical density was read at 540 nm in ELISA plate reader, and
the data was expressed as percent viable cells. Each assay was
performed in triplicate.
Estimation of Bcl-2 Level by ELISA--
The level of Bcl-2 was
estimated by a Bcl-2 ELISA kit obtained from Endogen Inc. (Woburn, MA)
as per the manufacturer's protocol.
Effect of Antisense Bcl-2 Oligonucleotide on RIF 1 Cell Survival
after PDT--
The radiation-induced fibrosarcoma cells (RIF 1) are
resistant to photodynamic therapy (PDT)-mediated apoptosis (12). To determine whether treatment of antisense oligonucleotide, directed against the coding region of Bcl-2 protein, induces cell death in RIF 1 cells following PDT, we treated the cells with different concentrations
of antisense oligonucleotide. As shown in Fig. 1A, treatment of cells with
antisense oligonucleotide resulted in a significant decrease in the
viability of cells in a concentration-dependent manner as
compared with cells treated with scrambled nucleotide. Cells treated
with scrambled control oligonucleotide did not show any significant
reduction in the cell survival.
Induction of Apoptosis with Antisense Bcl-2 in RIF 1 Cells--
Because our data showed that the antisense oligonucleotide
treatment resulted in oligonucleotide
concentration-dependent decrease in cell survival in RIF 1 cells, we investigated whether this decreased cell survival is due to
apoptosis. As shown by DNA ladder assay (Fig. 1B), treatment
of PDT apoptosis-resistant RIF 1 cells with antisense Bcl-2
oligonucleotide resulted in a significant induction of apoptosis,
6 h following PDT. Next, we quantified the extent of apoptosis by
flow cytometric analysis of the cells labeled with dUTP and
propidium iodide. The PDT of the RIF 1 cells treated with antisense
resulted in 39.2% TUNEL-positive cells at 6 h post-PDT compared
with only 3.0% TUNEL-positive cells for similarly treated cells
without antisense. Consistent with our previous observation, the
untreated control cells or cells treated with light alone or Pc 4 alone
did not result in any appreciable apoptosis (Fig. 1C). These
data clearly establish that the effects are photodynamic
treatment-mediated.
Caspase activation is an important event in the apoptotic pathway and
can be used as a marker of apoptosis. Therefore, to further
substantiate our findings, we measured the effect of antisense treatment on PDT-mediated apoptosis by caspase activity assay (DEVDase
assay). As shown by the data in Fig. 1D, the antisense treatment was found to result in enhanced caspase activity following PDT as compared with the cells treated with PDT alone. The increase in
caspase activity was maximum at 3 h post-PDT and was found to
declined at 6 h post-PDT.
Activation of apoptotic pathway leads to a cleavage of
poly(A)DP ribose polymerase (PARP) that is involved in DNA repair and is extensively used as a marker of apoptosis. Therefore, we
investigated the effect of antisense oligonucleotide on PDT-mediated
modulation in PARP. As shown in Fig. 1E, the antisense
treatment resulted in a significant PARP cleavage at 6 h
post-PDT.
Effect of Antisense Bcl-2 Oligonucleotide on the Protein Expression
of Bcl-2 in RIF 1 Cells--
To assess the effect of antisense Bcl-2
oligonucleotide on the protein expression of Bcl-2, we performed
immunoblot analysis. As shown by Fig.
2A, the immunoblot analysis
revealed a down-regulation of Bcl-2 protein in RIF 1 cells treated with
antisense oligonucleotide followed by PDT whereas the level of Bcl-2
remains unaltered in cells without antisense treatment. Treatment of
cells with scrambled control oligonucleotide showed no effect on the
Bcl-2 level in RIF 1 cells after PDT (data not shown). The
densitometric analysis showed almost 2-fold decrease in the protein
expression of Bcl-2 in cells treated with antisense followed by PDT
(Fig. 2B). This observation suggested that constitutively
high levels of Bcl-2 might be a reason for the known PDT
apoptosis-resistant property of RIF 1 cells.
To further establish the role of Bcl-2 in PDT-mediated apoptosis,
we assessed the level of Bcl-2 by ELISA and compared it with the extent
of apoptosis as assessed by TUNEL assay, in the cells treated with
antisense followed by PDT. The estimation of Bcl-2 by ELISA
demonstrated a concentration-dependent decrease in the
level of Bcl-2 with a concomitant increase in the extent of apoptosis,
as evident from flow cytometric determination of TUNEL-positive cells
(Fig. 3A). Therefore, our data
demonstrated that the decrease in Bcl-2 levels is correlated with the
increased the sensitivity of RIF 1 cells toward PDT apoptosis,
following antisense oligonucleotide treatment (Fig. 3A). We
also assessed the effect of antisense oligonucleotide treatment on
PDT-mediated modulation in the relative ratio of Bax/Bcl-2 proteins by
immunoblot analysis (Fig. 3B). The antisense oligonucleotide
treatment of RIF 1 cells followed by PDT resulted in a down-modulation
of Bcl-2 protein whereas the levels of Bax protein remained unaltered
(Fig. 3B). However, the densitometric analysis showed that
with the increase in the Bax/Bcl-2 ratio there is increase in the
apoptotic response (Fig. 3C).
Effect of PDT on the Protein Expression of Bcl-2 in A431
Cells--
Human epidermoid carcinoma cells are sensitive to
PDT-mediated apoptosis (10). Therefore, we were interested in
evaluating the effect of Bcl-2 during PDT-mediated apoptosis in these
cells. We assessed the levels of Bcl-2 protein following PDT in these cells. As shown in Fig. 4A,
PDT of these cells resulted in a significant time-dependent
down-regulation of Bcl-2 protein.
Effect of Bcl-2 Overexpression on Apoptotic Response of A431
Cells--
The data in RIF 1 cells suggested that the down-regulation
of Bcl-2 induces apoptosis in PDT apoptosis-resistant cells. In another
approach, to investigate the role of Bcl-2 in PDT-mediated apoptosis,
we overexpressed the Bcl-2 protein in the A431 cells. In these
Bcl-2-transfected A431 cells, as expected, the Bcl-2 protein was
overexpressed (Fig. 4B) and PDT resulted in a
down-regulation of Bcl-2 protein levels (Fig. 4B).
Interestingly, as shown by TUNEL assay (Fig.
5A), the Bcl-2 overexpression
resulted in an increase in apoptosis by PDT as compared with normal
wild type A431 cells. To evaluate the effect of overexpression of Bcl-2 on caspase activity, we performed DEVDase assay in both wild type and
Bcl-2-overexpressing cells. As shown by data in Fig. 5B, the overexpression of Bcl-2 was found to enhance the PDT-mediated caspase
activity indicative of increased apoptosis. The increase in caspase
activity was highest at 3 h post-PDT and declined thereafter.
Effect of Bcl-2 Overexpression on Bax Protein in A431
Cells--
Bax is a pro-apoptotic member of the Bcl-2 protein family.
The relative amounts or equilibrium between the pro- and anti-apoptotic proteins influences the susceptibility of cells to apoptosis. Therefore, in the next set of experiments, we assessed the effect of
PDT on the status of Bax protein in Bcl-2-overexpressing A431 cells.
The immunoblot analysis showed that PDT resulted in an increased level
of Bax protein in Bcl-2-overexpressing cells as compared with normal
A431 cells (Fig. 6A). The
densitometric analysis demonstrated that Bcl-2-overexpressing cells
have high Bax/Bcl-2 ratio as compared with their wild type counterparts
(Fig. 6B).
Modulations in Other Bcl-2 Family Members in RIF 1 and A431 Cells
Subjected to PDT--
The Bcl-2 family of proteins consists of
apoptosis regulators with both anti- and pro-apoptotic
effect. The anti-apoptotic group includes Bcl-2 and Bcl-xL,
whereas the pro-apoptotic group includes Bax, Bad, Bid, Bik, Bak,
and Bcl-xS. Therefore, we assessed the effect of PDT on
these family members in both the systems, viz. RIF 1 cells
(without and with antisense Bcl-2 oligonucleotide treatment) and A431
cells (wild type and transiently transfected with Bcl-2). As shown by
data in Fig. 7, PDT was found to result in an increase in Bcl-xs protein levels in RIF 1 cells
subjected to oligonucleotide antisense treatment at all the time points studied, as compared with untreated cells. An increasing trend in the
protein levels of Bid was also observed in the cells treated with
oligonucleotide. Another pro-apoptotic protein Bak was not affected at
early times (1 h and 3 h) following PDT, but its level was found
to be elevated at 6 h following PDT. On the other hand, the
protein expression of Bad was found to be up-regulated at all the time
points following PDT (Fig. 7). The interesting observation of this
experiment, however, was the observed increasing trend in the protein
expression of anti-apoptotic protein Bcl-xL in cells
treated with oligonucleotide, compared with the cells without oligonucleotide treatment (Fig. 7).
We also assessed the effect of PDT on modulations in other Bcl-2 family
members in A431 cells overexpressing Bcl-2. As shown by data in Fig. 7,
in Bcl-2-overexpressing A431 cells, PDT was found to result in a
significant increase in the levels of Bcl-xS at 1 h
post-PDT and was found to diminish at later time points. The level of
Bid was not found to be significantly affected following PDT. PDT was
also found to result in significant up-regulation of Bak (at 1 h)
and Bad (at 1 h, 3 h, and 6 h post-PDT) in
Bcl-2-overexpressing cells compared with their wild type counterparts
(Fig. 7).
PDT, a new treatment modality for many cancer types and for
certain non-malignant diseases, has been shown to induce apoptosis of
cancer cells both in vitro during tumor cell killing and
in vivo during tumor ablation. Understanding of the
mechanisms involved in PDT-mediated apoptosis is far from complete.
This study was designed to investigate the hypothesis that the Bcl-2
family of proteins plays a critical role in PDT-mediated apoptosis. The Bcl-2 gene, initially recognized as a proto-oncogene
in human follicular B-cell lymphoma, is the prototype of a novel class of oncogenes that contribute to neoplastic progression by enhancing tumor cell survival through inhibition of apoptotic cell death (22,
23). The product of Bcl-2 is known to play a role in promoting cell
survival and inhibiting apoptosis following variety of stimuli,
including In the present study, we evaluated the role of Bcl-2 protein in
PDT-mediated apoptosis of cancer cells. For this reason, we employed
two independent unique approaches. First, we used radiation-induced fibrosarcoma cells (RIF 1), which are resistant to PDT-mediated apoptosis under the conditions employed in this study. As assessed by
multiple methods, the treatment of RIF 1 cells with antisense oligonucleotides directed against the coding region of Bcl-2 protein was found to sensitize these PDT apoptosis-resistant cells to PDT
apoptosis. Because Pc 4 alone, light alone, or scrambled nucleotide alone had no effect on apoptotic response, the observed effects are
photodynamic treatment-mediated.
Bcl-2 is an upstream effector molecule in the apoptotic pathway and is
identified as a potent suppressor of apoptosis. Bcl-2 is found at
inappropriately high levels in more than half of all human tumors (23).
Several mechanisms are proposed for the antiapoptotic function of
Bcl-2. Bcl-2 might act as a regulator of Ca2+ homeostasis
(31) or as an antioxidants (32). Bcl-2 has been show to form a
heterodimer with the pro-apoptotic member Bax and might thereby
neutralize its pro-apoptotic effects (33, 34). In addition, Bcl-2 is
also known to prevent the release of potent mitochondrial activators of
the cytosolic death effector proteases, the caspase family, which
mediates the intracellular proteolysis characteristics of apoptosis
(35). The antisense treatment of RIF 1 cells followed by PDT resulted
in down-regulation of Bcl-2 in RIF 1 cells, which might initiate the
apoptotic pathway sensitizing the RIF 1 cells to PDT-mediated
apoptosis. Caspases are known to cleave many proteins, including the
inhibitor of nuclear DNase, the DNA fragmentation factor, the
ICAD, the DNA repair enzyme poly(A)DP-ribose polymerase (PARP), and
DNA-dependent protein kinase subunit and structural
proteins such as To further substantiate our findings, in the second approach, we
overexpressed Bcl-2 in A431, which readily undergoes apoptosis with
PDT (10). Immunoblot analysis revealed a decrease in Bcl-2 levels
following PDT in a time-dependent manner in A431 cells. Interestingly, the overexpression of Bcl-2 resulted in an increased apoptotic response in A431 cells compared with their normal wild type
cells as evident by TUNEL assay and caspase activity assay. Immunoblot
analysis revealed the high level of Bax (a pro-apoptotic member of
Bcl-2 family) in Bcl-2-overexpressing cells. Earlier reports have
suggested that the overexpression of Bcl-2 may stabilize Bax protein
(28), which, in turn, may promote apoptosis by triggering the
pore-forming activity in the mitochondrial membrane (38) and can
promote release of cytochrome c from mitochondria (35). It
was reported previously that caspases mediated conversion of Bcl-2 in a
Bax-like molecule can also increase the apoptotic response (39, 40).
High Bax level (stabilization effects of Bcl-2) shifting the ratio
between Bax/Bcl-2 toward Bax, and high caspase activity may be
responsible for the enhanced PDT-mediated apoptotic response in
Bcl-2-overexpressing cells.
Earlier reports have revealed a dual identity for Bcl-2 protein in
PDT-mediated cell death. The first report on PDT-mediated apoptosis (8)
suggested that mitochondrial damage could result in degradation of
Bcl-2 and related proteins that normally function as apoptotic
suppressors. He et al. (25) reported that Bcl-2 transfection
of Chinese hamster ovary cells leads to partial resistance to
PDT-mediated apoptosis. Consistent with this finding, Granville et al. (26) have reported that overexpression of Bcl-2
protein blocks the activation of caspases and downstream events
instigated by PDT. Meanwhile using antisense retroviral vector-mediated
reduction of Bcl-2, Zhang et al. (27) have shown increased
phototoxicity and sensitivity to apoptosis induced by 2-BA-2-DMHA PDT
in human gastric adenocarcinoma, further supporting the role of Bcl-2
in PDT-mediated apoptosis. However, in a recent report Kim et
al. (28) have reported contradictory findings. In this study, the overexpression of Bcl-2 in a human breast cell line resulted in enhanced apoptosis by PDT.
Based on our findings, the controversy regarding the role of Bcl-2
protein in PDT-mediated apoptosis can be put in proper context.
Down-regulation of Bcl-2 with antisense oligonucleotide resulted in
sensitization of PDT apoptosis-resistant cells to apoptosis; however,
the overexpression of Bcl-2 in a PDT apoptosis-sensitive cell line
further enhances the response. This study clearly suggested that the
shift in the ratio of pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) in favor of pro-apoptotic protein (Bax) is an important factor
in determining the response toward PDT.
Our data also demonstrated that PDT results in modulation of other
Bcl-2 family members in a way that the overall ratio of pro-apoptotic
and anti-apoptotic member proteins remains in favor of pro-apoptotic
proteins. We found that PDT of RIF 1 cells (treated with Bcl-2
antisense oligonucleotide) caused an up-regulation of
Bcl-xs, Bid, Bak, and Bad, the pro-apoptotic members of the Bcl-2 family. The up-regulation of these pro-apoptotic proteins by PDT
in RIF 1 cells (treated with Bcl-2 antisense oligonucleotide) was found
to be an early response for Bcl-xs and Bad (occurring as
early as 1 h post-PDT), whereas it was a late response for Bak
(occurring at 6 h post-PDT). An interesting observation of this
experiment was the observed increasing trend in the levels of
anti-apoptotic Bcl-xL as a result of PDT in RIF 1 cells
treated with Bcl-2 antisense oligonucleotide. It is likely that,
despite this increasing trend in the levels of anti-apoptotic
Bcl-xL protein, the high levels of pro-apoptotic members
were able to overcome its anti-apoptotic activity that ultimately
results in apoptotic death of RIF 1 cells following PDT.
Like RIF 1 cells treated with Bcl-2 antisense oligonucleotide, we also
found that PDT of Bcl-2-overexpressing A431 cells caused an
up-regulation of protein expression in pro-apoptotic members, viz. Bcl-xs, Bak, and Bad, but not of Bid. The
protein expression of anti-apoptotic Bcl-xL was found to
decrease as a result of PDT in these cells. Thus, it seems that in
these cells PDT results in a general up-regulation in the protein
expression of the pro-apoptotic members while down-modulating the
anti-apoptotic proteins, shifting the balance in favor of apoptosis.
Another interesting observation was the decreased protein expression of
some of the molecules studied as a result of antisense or
overexpression treatments. The reason for this decrease is not clear at
present, however, it is clear that alterations of Bcl-2 levels in our
experimental setup modulates many Bcl-2 family members. This
interesting observation could also be explained by the fact that the
members of Bcl-2 family proteins have the ability to form hetero- or
homodimers (41). In fact, when the apoptotic pathway is activated or
altered, these proteins either form homodimers or heterodimers or
undergo post-translational modifications. For example, upon activation of the apoptotic pathway, Bid protein undergoes cleavage and the cleaved fragment moves to the mitochondrial membrane and/or form heterodimers with either Bax or Bcl-2, resulting in apoptotic cell
death (42, 43). Similar behavior has been reported for Bax (44). Thus,
because the Bcl-2 antisense oligonucleotide treatment causes an
activation of apoptotic pathway in RIF 1 cells, the levels of these
proteins are decreased as compared with the control, which have high
levels of these proteins possibly because of the absence of apoptotic
signal. We plan to direct our future efforts to investigate these
queries in detail.
Extensive experimental evidences suggest that antisense Bcl-2
oligonucleotides induce apoptotic cell death in various types of
malignant cell lines in vitro, including leukemia, lymphoma, myeloma, small cell lung carcinoma, and cholangiocarcinoma.
Furthermore, combined use of antisense Bcl-2 oligonucleotide with
chemotherapeutic agents resulted in additive inhibition of small cell
lung cancer cells in vitro and melanoma cells in
vitro and in vivo (45). Our results demonstrate that
Bcl-2 plays an important role in PDT-mediated apoptosis and the shift
of balance between pro-apoptotic and anti-apoptotic members of the
Bcl-2 protein family in favor of pro-apoptotic proteins decides the
susceptibility of cells to PDT-mediated apoptosis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
80 °C. The protein of cell lysate was determined by DC Bio-Rad
assay using manufacturer's protocol (Bio-Rad Laboratories, Hercules, CA).
80 °C. The DNA precipitate was centrifuged at
14,000 × g at 4 °C for 15 min. The DNA pellet was
air-dried and resuspended in 40 µl of TE buffer (10 mM
Tris-HCl, pH 8.0/1 mM EDTA). Total DNA was resolved on
1.5% agarose gel, containing 0.3 µg of ethidium bromide in Tris
borate EDTA (1× TBE). Bands were visualized under a UV transilluminator followed by Polaroid photography. The quantification of apoptosis was performed using an APODIRECT flow cytometry kit (Phoenix Flow Systems, San Diego, CA) as per the manufacturer's protocol.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Antisense Bcl-2 oligonucleotide induces
apoptosis in RIF 1 cells. A, cell survival in RIF 1 cells after antisense treatment followed by PDT. Cells were treated
with Bcl-2 antisense- or scrambled-oligonucleotide followed by PDT as
described under "Materials and Methods." Cell survival was assessed
by MTT assay at 6 h post-PDT. The data shown are means ± S.D. of three independent experiments. B, DNA ladder
formation in RIF 1 cells. Cells were treated with Bcl-2 antisense
oligonucleotide followed by PDT as described under "Materials and
Methods," and DNA was isolated at 6 h post-PDT and subjected to
gel electrophoresis followed by visualization of bands.
M = molecular weight marker. The data shown here are
representative of three independent experiments with similar results.
C, quantification of apoptosis by flow cytometry. Cells were
treated with Bcl-2 antisense oligonucleotide (2.5 µg/ml) followed by
PDT. Cells were harvested after 6 h post-PDT and labeled with dUTP
using terminal deoxynucleotide transferase and propidium iodide and
analyzed by flow cytometry. The details are described under
"Materials and Methods." The data shown here are representative of
two independent experiments with similar results. D, effect
of Bcl-2 antisense oligonucleotide treatment followed by PDT on DEVDase
activity in RIF 1 cells. DEVase activity was measured using AFC-DEVD as
substrate and analyzed by a fluorescence spectrophotometer. Details are
described under "Materials and Methods." The data shown are
means ± S.D. of three independent experiments. Statistical
analysis was done by Student's t test and p < 0.001 was considered significant. E, immunoblot analysis
of PARP cleavage in RIF 1 cells treated with Bcl-2 antisense
oligonucleotide followed by PDT. The cells lysate were prepared at the
specified times following the treatments, and 50 µg of protein was
subjected to 12% Tris-glycine gel electrophoresis followed by
immunoblot analysis and chemiluminescence detection as described under
"Materials and Methods." NT = untreated control.
The data shown here are representative of three independent experiments
with similar results.
View larger version (27K):
[in a new window]
Fig. 2.
Down-regulation of Bcl-2 by Bcl-2 antisense
oligonucleotide following by PDT in RIF 1 cells. A,
immunoblot analysis. B, densitometric analysis of the bands.
The cells were treated with antisense oligonucleotide (2.5 µg/ml),
and the cell lysate was prepared at specified times following the
treatments. 50 µg of protein was subjected to 12% Tris-glycine gel
electrophoresis followed by immunoblot analysis and chemiluminescence
detection as described under "Materials and Methods." Equal loading
was confirmed by stripping the membrane and reprobing it for -actin.
The bands were quantified by densitometric analysis using Scion Image
software (Scion Corp., Frederick, MD) and normalized to
-actin. The
data shown here are representative of three independent experiments
with similar results. n = untreated control,
DO = drug only control, LO = light only
control.
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[in a new window]
Fig. 3.
Effect of antisense Bcl-2 oligonucleotide on
levels of Bcl-2 and Bax proteins, and extent of apoptosis in RIF 1 cells. A, down-regulation of Bcl-2 induces apoptosis.
Cells were treated with different concentrations of Bcl-2 antisense
oligonucleotide, subjected to PDT, harvested at 6 h post-PDT and
then labeled with dUTP using terminal deoxynucleotide transferase and
propidium iodide and TUNEL-positive (apoptotic) cells were analyzed by
flow cytometry. The level of Bcl-2 was estimated by Bcl-2 ELISA kit
obtained from Endogen Inc. (Woburn, MA) as per the manufacturer's
protocol. For a comparison, the TUNEL-positive cells are plotted with
the levels of Bcl-2 protein. The data shown here are representative of
two independent experiments with similar results. B, effect
of antisense oligonucleotides on Bax and Bcl-2 protein levels. Cells
were subjected to PDT alone or first treated with Bcl-2 oligonucleotide
followed by PDT, cell lysates were prepared at 6 h post-PDT and
proteins (50 µg) were resolved over 12% Tris-glycine gels followed
by immunoblot analysis and chemiluminescence detection as detailed
under "Materials and Methods." NT = untreated
control. The data shown here are representative of two independent
experiments with similar results. C, Bax/Bcl-2 ratio
determines the apoptotic response of PDT. This figure shows a
correlation between the apoptosis and Bax/Bcl-2 ratio. The extent of
apoptosis was estimated by flow cytometry as described earlier. The
ratio between Bax and Bcl-2 proteins was determined by densitometric
analysis of the immunoblots shown in B, using Scion Image
software (Scion Corp., Frederick, MD).
View larger version (46K):
[in a new window]
Fig. 4.
PDT down-regulates Bcl-2 protein expression
in wild type A431 and Bcl-2 overexpressing-A431 cells.
A, effect of PDT on Bcl-2 protein in A431 cells. The cells
were subjected to PDT and harvested at 1, 3, 6, 9, and 12 h
post-PDT. Total cell lysates were prepared, and 50 µg of protein was
subjected to 12% Tris-glycine gel electrophoresis followed by
immunoblot analysis and chemiluminescence detection. Equal loading was
confirmed by stripping the membrane and reprobing it for -actin. The
details are given under "Materials and Methods." The data shown
here are representative of three independent experiments with similar
results. NT = untreated control, DO = drug only control, LO = light only control.
B, effect of PDT on Bcl-2 protein levels in
Bcl-2-overexpressing A431 cells. As described under "Materials and
Methods," the cells were transiently transfected with human cDNA
carrying plasmid and then subjected to Pc 4-PDT. Cell lysates were
prepared and immunoblot analysis was done as described earlier. The
data shown here are representative of two independent experiments with
similar results. NT = untreated control,
WT = wild type A431 cells, T = A431
cells transiently transfected with Bcl-2.
View larger version (17K):
[in a new window]
Fig. 5.
Overexpression of Bcl-2 enhances PDT-mediated
apoptosis in A431 cells. A, quantification of apoptosis
by flow cytometry. Cells were transiently transfected with Bcl-2 as
described under "Materials and Methods" followed by PDT. Cells were
harvested at specified time points post-PDT and labeled with dUTP using
terminal deoxynucleotide transferase and propidium iodide and analyzed
by flow cytometry. The data shown here are representative of two
independent experiments with similar results. n = untreated control. B, DEVDase activity assay. The caspase
activity was measured in both wild type and Bcl-2-transfected cells at
different time points following PDT. Caspase activity was measured by
AFC-DEVDase assay as described under "Materials and Methods." Data
represents means ± S.D. of three independent experiments.
Statistical analysis was performed by Student's t test, and
p < 0.005 was considered significant.
NT = untreated control.
View larger version (33K):
[in a new window]
Fig. 6.
Overexpression of Bcl-2 stabilizes Bax
protein following Pc 4-PDT in A431 cells. A, effect of
PDT on Bax protein levels in wild type and Bcl-2-overexpressing A431
cells. A431 cells were transiently transfected with Bcl-2 followed by
PDT. Cell lysates were prepared at specified times following PDT and 50 µg of protein was subjected to 12% Tris-glycine gel electrophoresis
followed by immunoblot analysis and chemiluminescence detection as
detailed under "Materials and Methods." The data shown here are
representative of three independent experiments with similar results.
n = untreated control. B, effect of PDT on
Bax/Bcl-2 ratio in wild type and Bcl-2-transfected cells. The ratio of
Bax/Bcl-2 was determined by densitometric analysis of bands in
immunoblots for Bax (A) and Bcl-2 (Fig. 4A). The
data shown here are means ± S.E. from three independent
experiments. NT = untreated control.
View larger version (45K):
[in a new window]
Fig. 7.
Modulations in other Bcl-2 family members in
RIF 1 and A431 cells subjected to PDT. A, immunoblot
analysis; B, quantification of proteins by densitometry. The
treatment of cells with oligonucleotides and/or PDT is detailed under
"Materials and Methods." Following treatments, cell lysates were
prepared at specified times post-PDT, and 50 µg of protein was
subjected to Tris-glycine gel electrophoresis followed by immunoblot
analysis and chemiluminescence detection. Equal loading was confirmed
by stripping the immunoblot and reprobing it for -actin. The
quantification of protein was performed by densitometric analysis using
Scion Image software (Scion Corp.). The immunoblots shown here are
representative of three independent experiments with similar results.
The densitometry data represent means ± S.E. from three
immunoblots and are shown as relative density of protein bands
(relative to background) normalized to
-actin. NT = untreated control, WT = wild type A431 cells,
T = A431 cells transiently transfected with
Bcl-2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-radiation, glucocorticoid, hypothermia, growth factor
withdrawal, and chemotherapeutic agents (23, 29, 30).
-fodrin. These proteins are required by
cells for protection against apoptosis (36, 37). Our results
demonstrate high caspase activity and cleavage of PARP with antisense
treatment of RIF 1 cells followed by PDT. Our study also showed
unaltered levels of Bax (a pro-apoptotic member of the Bcl-2 family)
with the antisense treatment. Antisense treatment resulted in decreased
levels of Bcl-2 protein in RIF 1 cells, thus shifting the ratio in
favor of Bax protein that initiates the apoptotic pathway sensitizing
the RIF 1 cells to PDT-mediated apoptosis.
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FOOTNOTES |
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* This work was supported by United States Public Health Services Grants RO1 CA 51802, PO1 CA 48735, P30 CA 43703, and P30 AR 39750.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Career Development Award from Dermatology
Foundation, USA.
§ To whom correspondence should be addressed: Dept. of Dermatology, Case Western Reserve University, 11100 Euclid Ave., Cleveland, OH 44106. Tel.: 216-368-1127; Fax: 216-368-0212; E-mail: hxm4@po.cwru.edu.
Published, JBC Papers in Press, January 31, 2001, DOI 10.1074/jbc.M006920200
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ABBREVIATIONS |
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The abbreviations used are: PDT, photodynamic therapy; Pc 4, phthalocyanine photosensitizer (HOSiPcOSi(CH3)2(CH2)3N(CH3)2); JNK, c-Jun NH2-terminal kinase; RIF 1, radiation-induced fibrosarcoma cells; HBSS, Hanks' balanced salt solution; PARP, poly(ADP-ribose) polymerase; DEVD, N-acetyl-Asp-Glu-Val-Asp; AFC, 7-amino-4-trifluoromethyl coumarin; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; ELISA, enzyme-linked immunosorbent assay; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling.
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