Cell apoptosis induced by UV irradiation is a highly complex process in which different molecular signaling pathways
are involved. JNK has been proposed as an important regulator in UV irradiation-induced apoptosis. However, the
molecular mechanism through which JNK regulates apoptosis, especially how JNK activates Bax in response to UV
irradiation is still controversial. In this study, using real-time single-cell analysis, we studied the machinery of Bax
activation during UV-induced apoptosis. UV treatment resulted in a series of events: phosphorylation of JNK,
mitochondrial translocation of Bim, and subsequent activation of Bax. The activation of Bim and Bax could be inhibited
in the presence of SP600125 (a specific inhibitor of JNK), suggesting that UV irradiation activated the JNK/Bim/Bax
pathway.
Photodynamic therapy (PDT) employing photosensiter N-aspartyl chlorin e6 (NPe6) can induce
lysosome disruption and initiate apoptotic pathway. Apoptosis signal-regulating kinase (ASK1) is an
important regulator of apoptosis in response to various stresses, such as reactive oxygen species (ROS),
endoplasmic reticulum (ER) stress, lipopolysaccharide (LPS) and calcium influx. In this study, we
investigated the molecular mechanisms of apoptosis induced by
NPe6-PDT in ASTC-a-1 cells. The
results showed that the activities of ASK1 increased in response to NPe6-PDT. Over-expression of
wild-type or activated mutant of ASK1 could obviously decrease cell viability and increase cell death;
while inhibition of ASK1 significantly decreased cell apoptosis. These results suggested that ASK1
plays an important role in apoptosis induced by NPe6-PDT.
Cardiolipin is a unique and ubiquitous diphosphatidylglycerol phospholipid, located exclusively in inner
membrane of mitochondria and particularly intermembrane contact sites. Cardiolipin is essential for
mitochondrial to maintain its functions. Numerous mitochondrial proteins and processes require the presence of
cardiolipin. Recent researches gradually confirm that cardiolipin participates in several mitochondria-dependent
apoptotic steps: interactions between cardiolipin and cytochrome c, Bid and caspase-8 have now been
established. These functions are associated with the redistribution of cardiolipin in mitochondria. However, the
exact mechanism of the redistribution, which happens at the early stage of apoptosis, is still controversial. In this
study, we used 10-N-nonyl-3, 6-bis (dimethylamino) acridine (10-N-nonyl acridine orange), a specific probe for
cardiolipin to monitor changes of cardiolipin redistribution during apoptosis. We demonstrated that during
apoptosis cardiolipin moves to the outer leaflet of mitochondrial inner membrane from the inner leaflet, where it
used to be riched in. We also found that ROS (reactive oxygen species) may have association with the
redistribution of cardiolipin.
Lysosomal photosensitizers have been used in photodynamic therapy (PDT). Combination of such photosensitizers and
light causes lysosomal photodamage, inducing cell death. The lysosomal disruption can lead to apoptosis but its
signaling pathways remain to be elucidated. In this study, we selected N-aspartyl chlorin e6 (NPe6), an effective
photosensitizer which preferentially accumulates in lysosomes, to study the mechanism of apoptosis caused by
lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells treated by NPe6-PDT was studied using
real-time single-cell analysis. In this study, the fluorescence probes Cyto c-GFP and DsRed-Mit were used to detect the
spatial and temporal changes of cytochrome c in real-time in sub-cell level; the Rhodamine 123 dyes were used to
monitor the changes of mitochondrial membrane potential. The results showed that, after PDT treatment,the
mitochondrial membrane potential decreased, and cytochrome c released from mitochondria; The caspase-3 was
activated obviously. These results suggested that lysosomal photodamage activates mitochondrial apoptotic pathway to
induce cell death.
Lysosomal photosensitizers have been used in photodynamic therapy (PDT). Combination of such photosensitizers and
light causes lysosomal photodamage, inducing cell death. The lysosomal disruption can lead to apoptosis but its
signaling pathways remain to be elucidated. In this study, we selected N-aspartyl chlorin e6 (NPe6), an effective
photosensitizer which preferentially accumulates in lysosomes, to study the mechanism of apoptosis caused by
lysosomal photodamage. Apoptosis in living human lung adenocarcinoma cells treated by NPe6-PDT was studied using
real-time single-cell analysis. Confocal imaging of cells transfected with BimL-GFP demonstrated that BimL
translocated to mitochondria after NPe6-PDT treatment for about 150 min, and then sequestered into clusters associated
with the mitochondira within 30 min. The activation of BimL proved to be an important event in the apoptotic machinery,
as demonstrated by the significant protection of cell death in samples suppressed the expression level of endogenous
BimL. This study demonstrates that BimL activation was involved in the cell death induced by PDT with lysosomal
photosensitizer.
Cisplatin, an efficient anticancer agent, can trigger multiple apoptotic pathways in cancer cells. However, the signal
transduction pathways in response to cisplatin-based chemotherapy are complicated, and the mechanism is not fully
understood. In current study, we showed that, during
cisplatin-induced apoptosis of human lung adenocarcinoma cells,
both the caspase-dependent and -independent pathways were activated. Herein, we reported that after cisplatin treatment,
the activities of caspase-9/-3 were sharply increased; pre-treatment with Z-LEHD-fmk (inhibitor of caspase-9),
Z-DEVD-fmk (inhibitor of caspase-3), and Z-VAD-fmk (a pan-caspase inhibitor) increased cell viability and decreased
apoptosis, suggesting that caspase-mediated apoptotic pathway was activated following cisplatin treatment. Confocal
imaging of the cells transfected with AIF-GFP demonstrated that AIF release occurred about 9 h after cisplatin treatment.
The event proceeded progressively over time, coinciding with a nuclear translocation and lasting for more than 2 hours.
Down-regulation of AIF by siRNA also significantly increased cell viability and decreased apoptosis, these results
suggested that AIF-mediated caspase-independent apoptotic pathway was involved in cispatin-induced apoptosis. In
conclusion, the current study demonstrated that both
caspase-dependent and -independent apoptotic pathways were
involved in cisplatin-induced apoptosis in human lung adenocarcinoma cells.
Photodynamic therapy (PDT) employing photosensiter N-aspartyl chlorin e6 (NPe6) can induce lysosome disruption and
initiate the intrinsic apoptotic pathway. Bax, a member of the Bcl-2 family of proteins, is an essential regulator of
apoptosis. Bax is normally found in the cytosol of healthy cells, and translocates to mitochondria in response to many
apoptotic stimuli. In this study, using real-time single-cell analysis, we have investigated the kinetics of Bax
distribution during NPe6-induced apoptosis in ASTC-a-1 cells. In order to monitor Bax subcellular distribution, cells
were stained with GFP-Bax and Mito Tracker Red. The results show that Bax redistribution occurred at about 170 min
after treated with NPe6-PDT, and then sequestered into clusters associated with the mitochondira within 30 min. Our
data clearly showed the spatial and temporal changes in Bax distribution in living cells during NPe6-induced apoptosis.
Mitochondrial apoptosis inducing factor (AIF) on activation can translocate to the nucleus and induce cell death via
caspase-independent pathway in cisplatin-induced apoptosis. Yet the precise signal transduction pathway(s) which
regulates AIF-induced apoptotic pathway still remains poorly understood. In this study, we investigated the molecular
mechanism of AIF release and redistribution in cisplatin-induced apoptosis in living ASTC-a-1 cells, as assessed by
real-time anlysis. Herein, We report that during cisplatin-induced apoptosis, calpain activation, as measured in intact
cells by a fluorescent substrates, is an early event, taking place well before AIF release and caspase-3 activation.
Confocal imaging of the cells transfected with AIF-GFP demonstrated that AIF release occurred about 9 h after cisplatin
treatment. The event proceeded progressively over time, coinciding with a nuclear translocation and lasting for more
than 2 hours. AIF release and redistribution were effectively inhibited in samples co-treated with calpeptin and
PD150606, two selective calpain inhibitors. Therefore, our results clearly show the kinetics of AIF release and
redistribution in cisplatin-induced apoptosis in living ASTC-a-1 cells, and calpain played a crucial role in these events.
UV irradiation is a DNA-damage agent that triggers apoptosis through both the membrane death receptor and the
mitochondrial apoptotic signaling pathways. Bid and Bax are two important proapoptotic members of the Bcl-2 family,
localize largely in the cytoplasm and redistribute to mitochondria in response to most apoptotic stimuli. Cells deficient in
Bax are resistant to UV-induced apoptosis, cells deficient in Bid are less susceptible than normal cells in response to
DNA damage. Thus, studying characteristics of Bid and Bax translocation by UV irradiation is very important for us to
understand the cellular signaling mechanisms mediating UV-induced apoptosis. In this study, to investigate Bid and Bax
translocation in real time in a single cell by UV irradiation, we transfected Bid-CFP, YFP-Bax and DsRed-Mit into
human lung adenocarcinoma cells (ASTC-a-1), then observed temporal and spatial characteristics of Bid and Bax
translocation by laser confocal scanning microscope imaging technique. Our results showed that Bax translocation was
earlier than Bid translocation and the average duration of Bax translocation was about 20-30 min during UV-induced
apoptosis.
The current advances in fluorescence microscopy, coupled with the development of new fluorescent probes, make
fluorescence resonance energy transfer (FRET) a powerful technique for studying molecular interactions inside living
cells with improved spatial and temporal resolution, distance range, and sensitivity and a broader range of biological
applications. In recent years, a large number of studies have been conducted aiming to understand the process of
apoptosis during tumor therapy on a molecular basis. Here, we utilized a recombinant FRET Bid probe to determine
the kinetics of Bid cleavage during Cisplatin-induced apoptosis in ASTC-a-1 cells. Cells treated with Cisplatin (20 μM)
showed a cleavage of the Bid-FRET probe, occurring at about 4-5 h after onset of the Cisplatin exposure, and lasted
about 1.5 h. Our data clearly showed the kinetics of Bid cleavage during Cisplatin-induced apoptosis. We also used
FRET technique to measure the dynamics of caspase-3 activation during apoptosis induced by high fluence low-power
laser irradiation (LPLI). The data showed that we can detect caspase-3 activation sensitively and effectively, by using
the FRET probe SCAT3.
Cancer cell apoptosis can be induced by cisplatin, an efficient anticancer agent. However, its mechanism is not fully
understood. Bcl-2 homology domain (BH) 3-only proteins couple stress signals to mitochondrial apoptotic pathways.
Calpain-mediated cleavage of the BH3-only protein Bid into a 14 kD truncated protein (tBid) has been implicated in
cisplatin-induced apoptotic pathway. We utilized a recombinant fluorescence resonance energy transfer (FRET) Bid
probe to determine the kinetics of Bid cleavage during cisplatin-induced apoptosis in ASTC-a-1 cells. The cells were
also co-transfected with Bid-CFP and DsRed-Mit to dynamically detect tBid translocation. Cells showed a cleavage of
the Bid-FRET probe occurring at about 4-5 h after treated with 20 µM cisplatin. Cleavage of the Bid-FRET probe
coincided with a translocation of tBid from the cytosolic to the mitochondria, and the translocation lasted about 1.5 h.
Using real-time single-cell analysis, we first observed the kinetics of Bid cleavage and translocation to mitochondria in
living cells during cisplatin-induced apoptosis.
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.