Lysosomal enzymes promote mitochondrial oxidant production, cytochrome c release and apoptosis
Exposure of mammalian cells to oxidant stress causes early (iron catalysed) lysosomal rupture followed by apoptosis or necrosis. Enhanced intracellular production of reactive oxygen species (ROS), presumably of mitochondrial origin, is also observed when cells are exposed to nonoxidant pro-apoptotic agonists of cell death. We hypothesized that ROS generation in this latter case might promote the apoptotic cascade and could arise from effects of released lysosomal materials on mitochondria. Indeed, in intact cells (J774 macrophages, HeLa cells and AG1518 fibroblasts) the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH) causes lysosomal rupture, enhanced intracellular ROS production, and apoptosis. Furthermore, in mixtures of rat liver lysosomes and mitochondria, selective rupture of lysosomes by MSDH promotes mitochondrial ROS production and cytochrome c release, whereas MSDH has no direct effect on ROS generation by purifed mitochondria. Intracellular lysosomal rupture is associated with the release of (among other constituents) cathepsins and activation of phospholipase A2 (PLA2). We find that addition of purified cathepsins B or D, or of PLA2, causes substantial increases in ROS generation by purified mitochondria. Furthermore, PLA2 - but not cathepsins B or D - causes rupture of semipurified lysosomes, suggesting an amplification mechanism. Thus, initiation of the apoptotic cascade by nonoxidant agonists may involve early release of lysosomal constituents (such as cathepsins B and D) and activation of PLA2, leading to enhanced mitochondrial oxidant production, further lysosomal rupture and, finally, mitochondrial cytochrome c release. Nonoxidant agonists of apoptosis may, thus, act through oxidant mechanisms.
Lysosomal enzymes promote mitochondrial oxidant production,
release and apoptosis
, Fernando Antunes
, John W. Eaton
and Ulf T. Brunk
Division of Pathology II, Faculty of Health Sciences, Linko
ping University, Sweden;
Grupo de Bioquı
mica e Biologia Teo
Instituto Bento da Rocha Cabral and Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, Portugal;
James Graham Brown Cancer Center, University of Louisville, Louisville, KY, USA
Exposure of mammalian cells to oxidant stress causes early
(iron catalysed) lysosomal rupture followed by apoptosis
or necrosis. Enhanced intracellular production of reactive
oxygen species (ROS), presumably of mitochondrial origin,
is also observed when cells are exposed to nonoxidant pro-
apoptotic agonists of cell death. We hypothesized that ROS
generation in this latter case might promote the apoptotic
cascade and could arise from eﬀects of released lysosomal
materials on mitochondria. Indeed, in intact cells (J774
macrophages, HeLa cells and AG1518 ﬁbroblasts) the
lysosomotropic detergent O-methyl-serine dodecylamide
hydrochloride (MSDH) causes lysosomal rupture, enhanced
intracellular ROS production, and apoptosis. Furthermore,
in mixtures of rat liver lysosomes and mitochondria, selective
rupture of lysosomes by MSDH promotes mitochondrial
ROS production and cytochrome c release, whereas MSDH
has no direct eﬀect on ROS generation by purifed mito-
chondria. Intracellular lysosomal rupture is associated with
the release of (among other constituents) cathepsins and
activation of phospholipase A2 (PLA2). We ﬁnd that addi-
tion of puriﬁed cathepsins B or D, or of PLA2, causes
substantial increases in ROS generation by puriﬁed mito-
chondria. Furthermore, PLA2 ) but not cathepsins B or
D ) causes rupture of semipuriﬁed lysosomes, suggesting an
ampliﬁcation mechanism. Thus, initiation of the apoptotic
cascade by nonoxidant agonists may involve early release of
lysosomal constituents (such as cathepsins B and D) and
activation of PLA2, leading to enhanced mitochondrial
oxidant production, further lysosomal rupture and, ﬁnally,
mitochondrial cytochrome c release. Nonoxidant agonists
of apoptosis may, thus, act through oxidant mechanisms.
Keywords: apoptosis; cathepsins; lysosomes; lysosomotropic
detergents; oxidative stress.
In the last two decades, the phenomenon of apoptosis has
attracted great interest and many intricate molecular events
underlying the process have been elucidated [1–8]. Several
crucial steps are thought to involve mitochondrial release
of pro-apoptotic factors, although the exact mechanisms
involved in this release are less well understood.
In this regard, there is substantial evidence that, at least
in some circumstances, the discharge into the cytosol of
lysosomal constituents may be an early and, perhaps,
initiating event in apoptosis, and that mitochondrial release
of pro-apoptotic factors might be a consequence of earlier
lysosomal destabilization [9–18]. In further, albeit indirect,
support of this, it was recently found that activation of the
pro-apoptotic tumour supressor protein, p53, also results
in early lysosomal rupture, although through still unknown
In the case of simple oxidant-induced apoptosis, lyso-
somal rupture occurs in two sequential phases [19,20], where
the second one includes activation of phospholipase A2
(PLA2) with production of free arachidonic acid (AA)
[21,22]. Theoretically, released lysosomal enzymes, PLA2,
and AA all might be capable of destabilizing mitochondrial
membranes. Interestingly, over-expression of the anti-
apoptotic protein, Bcl-2, abrogates the secondary phase of
lysosomal rupture, the activation of PLA2, and the
mitochondrial release of cytochrome c [19,21,22]. However,
the precise mechanisms through which Bcl-2 mediates these
effects are presently unknown.
Remarkably, in apoptosis caused by a number of
nonoxidative agents, there appears to be increased intracel-
lular generation of reactive oxygen species (ROS), probably
of mitochondrial origin [23–30]. Although the mechanisms
responsible for enhanced mitochondrial ROS production
during the process of apoptosis remain unknown, this
phenomenon raises the possibility that internally generated
ROS, like exogenously added oxidants, may act through a
common pathway–lysosomal destabilization.
The present investigations were aimed at identifying
intracellular events that might connect exposure of cells to
nonoxidative agonists of apoptosis and intracellular ROS
production. As mentioned above, there is abundant evi-
dence that ) at least in some circumstances ) lysosomal
rupture might be an early, perhaps even initiating, event in
Correspondence to M. Zhao, Division of Pathology II,
Faculty of Health Sciences, Linko
SE-581 85 Linko
Fax: +46 13 22 15 29, Tel.: +46 13 22 15 15,
Abbreviations: AA, arachidonic acid; DHE, dihydroethidium; HRP,
horseradish peroxidase; LE, lysosomal enzymes; LEF, lysosome-
mitochondria enriched fraction; MSDH, O-methyl-serine
dodecylamide hydrochloride; PLA2, phospholipase A2;
ROS, reactive oxygen species.
(Received 28 April 2003, revised 11 July 2003,
accepted 24 July 2003)
Eur. J. Biochem. 270, 3778–3786 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03765.x
the apoptotic cascade. Therefore, in the present investiga-
tions we have used a synthetic lysosomotropic detergent,
O-methyl-serine dodecylamide hydrochloride (MSDH) to
speciﬁcally induce lysosomal rupture and ensuing apoptosis
[12,31,32]. This was done in order to determine whether
internal oxidative stress of mitochondrial origin might arise
as a consequence of lysosomal rupture and act as an
amplifying loop causing further lysosomal breach. Here, we
present evidence that released lysosomal enzymes ) both
directly and through activation of PLA2 ) may trigger
enhanced mitochondrial production of superoxide and
hydrogen peroxide, and cause the release of cytochrome c.
Materials and methods
Chemicals were from Sigma unless stated otherwise. RPMI
1640 medium, Hepes, foetal bovine serum, glutamine,
penicillin, and streptomycin were from Gibco. BODIPY
FL phallacidin and dihydroethidium (DHE) were from
Molecular Probes. Monoclonal anti-cytochrome c Igs were
from Pharmingen, and horseradish peroxidase (HRP)-
conjugated goat anti-mouse Igs were from DAKO. Percoll
was from Amersham Pharmacia Biotech.
Human foreskin ﬁbroblasts (AG-1518, passages 14–20;
Coriell Institute, Camden, NJ, USA), J774 cells (a murine
histiocytic lymphoma cell line), and human epithelial cells
(HeLa) were cultured at 37 C in humidiﬁed air with 5%
in RPMI 1640 medium supplemented with 2 m
glutamine, 50 IUÆmL
penicillin-G, 50 lgÆmL
mycin, and 10% foetal bovine serum. Cells were subcul-
tured once a week. Twenty-four hours before experiments,
cells were trypsinized and seeded into 35-mm Petri dishes or
96-well plates (Costar, Cambridge, MA, USA) at a density
of 10 000 cells per cm
DNA fragmentation was assessed using propidium iodide
staining of nuclear DNA, essentially as described by
Nicoletti et al. . Brieﬂy, cell pellets from individual
wells were gently resuspended in 1.5 mL of a hypotonic
and membrane-disrupting solution of propidium iodide
in 0.1% sodium citrate with 0.1% Triton
X-100) in 12 · 75 mm polypropylene tubes. The tubes were
kept overnight in the dark at 4 C before ﬂow-cytometric
analyses. The propidium iodide-induced red ﬂuorescence of
suspended individual nuclei was measured by ﬂow cyto-
ﬂuorometry, using the FL3 channel. Nuclei with partly
degraded DNA were counted, and their frequency was
expressed as a percentage of the total number of nuclei
analysed in at least 10 000 cells.
AG1518 ﬁbroblasts were seeded in 35-mm Petri dishes and
cultured for 24 h before being exposed to 30 l
ordinary medium for 3 h. Cellular actin was stained with
BODIPY FL phallacidin. Cells were ﬁxed for 10 min in 4%
formaldehyde in NaCl/P
, permeabilized for 10 min in
0.3% Triton X-100 in phosphate-buffered saline (NaCl/P
and stained for 30 min with BODIPY FL phallacidin
(ﬁnal concentration 0.6 lgÆmL
)at37C. After staining,
cells were washed twice in NaCl/P
, and visualized and
495 nm; k
520 nm) using a Nikon
microphot-SA ﬂuorescence microscope with a Hamamatsu
ORCA-100 color digital camera and Adobe
Evaluation of oxidative stress
AG1518 ﬁbroblasts, J774 and HeLa cells were seeded in
96-well plates and cultured for 24 h under standard
conditions before being exposed to 30 l
DHE (in complete medium). Fluorescence intensity,
indicating oxidation of DHE was assayed at various periods
of time after addition of MSDH and DHE on a VICTOR
1420 (Wallac Sverige AB, Upplands Va
ﬂuorescent plate-reader (k
485 nm; k
620 nm). In
some experiments, cells were observed and documented
under green light excitation (k
546 nm; k
using ﬂuorescence microscopy as described above.
Preparation of rat liver lysosome-mitochondria
Livers were removed from 160–200-g female Sprague–
Dawley rats (starved overnight), homogenized in 0.3
sucrose (1 : 9, w/v) and centrifuged at 500 g for 10 min.
The supernatants were again centrifuged at 3500 g for
10 min, the pellets discarded, and the lysosome/mitochon-
dria-containing supernatants centrifuged at 10 000 g for
10 min. The pellets were washed, suspended and re-centri-
fuged at 10 000 g for 10 min and ﬁnally resuspended in
the sucrose solution to a protein concentration of
. The resultant lysosome/mitochondria
enriched fraction (LEF) was found to be stable (no release
of lysosomal enzymes) for up to 4 h in the homogenization
solution at 4 C, while some release of lysosomal enzymes
Preparation of a puriﬁed mitochondria fraction
Mitochondria were puriﬁed from rat liver using a combi-
nation of differential and Percoll gradient centrifugation
[34,35]. All procedures were carried out at 4 C. Brieﬂy,
fresh liver was minced and homogenized in M-SHE buffer
sucrose, 10 m
Hepes pH 7.4,
EDTA, 1 m
EGTA, 0.15 m
spermine, 0.75 m
spermidine). Unbroken cells and nuclei were pelleted at
500 g for 10 min. The supernatant was centrifuged at
10 000 g to pellet mitochondria and lysosomes which were
resuspended and washed twice with M-SHE buffer. A 2-mL
suspension was then layered onto 37.5 mL of Percoll
solution (50% Percoll, 50% 2 · M-SHE) and centrifuged
for 1 h at 50 000 g in a Ti-60 rotor. The brown
mitochondrial band was collected, either by fractionating
the gradient or by direct syringe aspiration. The puriﬁed
mitochondria were pooled, diluted 10-fold with M-SHE
buffer, again pelleted by centrifugation and, ﬁnally,
FEBS 2003 Lysosomes, apoptosis and mitochondria-mediated oxidative stress (Eur. J. Biochem. 270) 3779
resuspended in M-SHE buffer to a protein concentration
of 1.5 mgÆmL
. The degree of lysosomal contamination
of the puriﬁed mitochondria fraction was estimated by
assaying b-galactosidase/protein and compared to that
Enzymatic detection of lysosomal integrity
and estimation of fraction purity
The integrity of lysosomes in the LEF preparation was
assessed by assaying released b-galactosidase. LEF
(200 lL) was incubated for 3 h at 37 C with either
PLA2 (0.2 UÆmL
), 30 l
MSDH, 2.5 lgÆmL
sin B, or 2.5 lgÆmL
cathepsin D and then centrifuged at
14 000 g for 10 min. Stock solutions of the cathepsins were
made up in NaCl/P
pH 6.0, whereas MSDH and PLA2
were in NaCl/P
pH 7.4. The supernatants were removed,
and 1 mL distilled water with Triton X-100 (ﬁnal concen-
tration 0.1%) was added to the pellets to cause complete
lysis of remaining intact lysosomes. Activities of b-galac-
tosidase were measured as described previously  on the
ruptured lysosomal pellet and on the supernatant. The
results were expressed as percentage released over total
Mitochondrial generation of H
Mitochondrial production of H
was assayed essentially
as described elsewhere . Brieﬂy, 1.33 UÆmL
q-hydroxyphenylacetate, 0.013 mgÆmL
superoxide dismutase, and 1 mg mitochondrial protein
were added to 2.4 mL respiratory buffer (0.07
mannitol, 30 m
Tris/HCl, 4 m
EDTA, 0.5% BSA, pH 7.4) in a spectro-
ﬂuorophotometer cuvette at 37 C. Succinate (ﬁnal concen-
tration 6.67 m
) and antimycin A (ﬁnal concentration
) were added, and H
320 nm; k
400 nm) during the ﬁrst 10 min
Western blotting for cytochrome
Two-hundred microlitres LEF, or puriﬁed mitochondria,
were incubated for 3 h at 37 C with either 30 l
MSDH, PLA2 (0.2 UÆmL
), 2.5 lgÆmL
cathepsin B, or
cathepsin D. Stock solutions of the cathepsins
were made up in NaCl/P
pH 6.0, while MSDH and PLA2
were in NaCl/P
pH 7.4. Following centrifugation at
14 000 g for 10 min, the supernatants were separated by
Fig. 1. MSDH induces apoptosis and stress
ﬁbre formation in ﬁbroblasts. (A) Cells were
seeded into 35-mm Petri dishes at a density of
10 000 cellsÆcm
was added to complete culture medium
(2 mL), and cells were incubated for another
10 h under standard culture conditions. The
Nicoletti technique for apoptotic nuclei was
applied. One representative experiment out of
three is shown. (B) Cells were seeded in 35-mm
Petri dishes and kept for 24 h before being
exposed to 30 l
MSDH for 3 h. Actin
staining was then performed as described in
Materials and methods.
3780 M. Zhao et al. (Eur. J. Biochem. 270) FEBS 2003
SDS/PAGE (12% acrylamide) and transferred onto Immo-
bilon membranes (2 h; 200 mA). Membranes then were
incubated at room temperature for 1 h in blocking buffer
[5% low-fat milk powder in Tris-buffered saline (TBS)] and
for another 2 h in dilution buffer (0.5% low-fat milk
powder in TBS) containing a 1 : 400 dilution of a mono-
clonal anti-cytochrome c Ig. After washing in TBS with
0.06% Tween 20, Immobilon membranes were incubated
for 1 h at room temperature in a buffer containing a
1 : 1500 dilution of peroxidase-conjugated secondary
antibodies. After washing, peroxidase-dependent chemilu-
minescence was detected by using enhanced chemilumines-
cence Western blotting reagents and hyperﬁlm according to
the manufacturer’s instructions (Amersham Pharmacia
All experiments were repeated at least three times. Values
are given as arithmetic mean ± SD. Signiﬁcance
Fig. 2. MSDH induces intracellular ROS production. Cells were seeded into 96-well plates at a density of 10 000 cellsÆcm
. After 24 h, cells were
exposed simultaneously to 30 l
MSDH and 10 l
DHE under otherwise standard culture conditions while control cells were exposed to DHE
only. (A) Fluorescence intensity arising from oxidized dihydroethidium in J774, HeLa and AG1518 cells was measured at indicated time points.
(B) J774 cells were visualized and photographed after 3 h exposure to MSDH (n ¼ 3). Very similar results were obtained with HeLa and AG1518
cells under the same conditions although detectable oxidant generation occurred earlier.
FEBS 2003 Lysosomes, apoptosis and mitochondria-mediated oxidative stress (Eur. J. Biochem. 270) 3781
of differences between groups was determined using
Student’s two-tailed t-test. *P £ 0.05; **P £ 0.01;
***P £ 0.001.
Cultured cells exposed to the synthetic lysosomotropic
detergent, MSDH, undergo lysosomal rupture and ensuing
apoptosis or necrosis depending upon the extent of
lysosomal destabilization . In the present experiments,
we induced apoptosis in ﬁbroblasts, J774 cells, and HeLa
cells by exposing them to 30 l
MSDH. After 8 h of
MSDH exposure, nuclear propidium iodide staining and
ﬂow cytometry (to detect DNA fragmentation) revealed
apoptotic nuclei appearing as a broad, hypodiploid DNA
smear in front of a narrow peak of diploid DNA (Fig. 1A
shows results in ﬁbroblasts). At an early stage in this
process, well before the appearance of frank apoptosis,
ﬁbroblasts showed signiﬁcantly increased numbers of stress
ﬁbres (Fig. 1B).
Fig. 3. MSDH induces mitochondrial ROS production by rupturing lysosomes. Puriﬁed mitochondria (1.0 mg proteinÆmL
) or a lysosome/mito-
chondria-enriched fraction (1.0 mg proteinÆmL
) were incubated with either of MSDH (30 l
), PLA2 (0.2 UÆmL
), or cathepsin B or D
production, (B) cytochrome c release, and (C) lysosomal stability were assayed as described in Materials
and methods (n ¼ 3).
3782 M. Zhao et al. (Eur. J. Biochem. 270) FEBS 2003
Because oxidative stress has been reported to induce
stress ﬁbre formation , we suspected that the MSDH
exposure might be causing increased intracellular generation
of ROS. This latter was monitored by following changes in
DHE-induced ﬂuorescence. When oxidized, this compound
intercalates into DNA and RNA, resulting in an increase in
quantum yield. Fluorescence intensity was measured kineti-
cally at indicated time points. Increased ROS production
occurred after 1 h of MSDH-exposure in ﬁbroblasts
(AG1518) and epithelial cells (HeLa), but was signiﬁcant
only after 3 h in macrophages (J774) (Fig. 2A). Note that in
ﬁbroblasts and HeLa cells, and also in J774 cells (results not
shown), the oxidation of DHE eventually reached a steady
state consistent with only a transient production of ROS.
Figure 2B shows DHE-exposed J774 cells after 3 h expo-
sure to MSDH, when there were still no morphological
signs of apoptosis.
Theoretically, the increased oxidant generation might
arise from effects of released lysosomal enzymes (directly or
by activation of PLA2) on mitochondrial ROS production
or, alternatively, from direct effects of MSDH on the
mitochondria. To discriminate between these possibilities,
we added MSDH to puriﬁed rat liver mitochondria (4.5-
fold puriﬁed from lysosomal contamination as compared to
the LEF preparation, results not shown). Under these
conditions, no changes in mitochondrial production of
(Fig. 3A) or release of cytochrome c (Fig. 3B) took
place. Because we previously observed that lysosomal
contents cause activation of PLA2 in J774 cells , we
also exposed mitochondria to that enzyme and found it to
enhance mitochondrial production of ROS (Fig. 3A) and to
release cytochrome c as well (Fig. 3B). These ﬁndings
strongly suggest that MSDH affects mitochondria by ﬁrst
destabilizing lysosomes and causing the release of hydrolytic
enzymes which, in turn, attack mitochondria or activate
PLA2. Activated PLA2 may further promote this cascade
of events, attacking both mitochondrial and lysosomal
membranes and causing further lysosomal rupture. This
supposed sequence of events was conﬁrmed by adding
MSDH to a lysosome/mitochondria-enriched rat liver
fraction, where it was found to induce enhanced mito-
chondrial production of H
(Fig. 3A), release of cyto-
chrome c (Fig. 3B), and lysosomal rupture (Fig. 3C).
To test further the idea that released lysosomal
hydrolases might enhance mitochondrial ROS production,
release of cytochrome c, and activation of PLA2 (all of
which may promote the apoptotic cascade), we tested the
effects of two lysosomal cathepsins (cathepsin B, a
cysteine protease, and cathepsin D, an aspartic protease)
on puriﬁed mitochondria. Both proteases caused substan-
tial increases in mitochondrial production of H
(Fig. 3A) and release of cytochrome c (Fig. 3B). However,
neither cathepsin B nor D caused detectable lysosomal
rupture in LEF preparations (Fig. 3C), although, as
expected, both MSDH and PLA2 did induce lysosomal
rupture (Fig. 3C).
Thus, cathepsins B and D do not directly cause rupture of
lysosomes in an LEF preparation. However, the possibility
remains that the intracellular release of other lysosomal
hydrolases may do so, or that lysosomal proteases might
secondarily destabilize lysosomes through, for example,
enhanced oxidative stress or activation of PLA2 following
Fig. 4. The lysosomal/mitochondrial axis theory of apoptosis. Both the internal and external pathways may involve lysosomal rupture. Released
lysosomal enzymes (LE) may: (a) attack mitochondria directly, inducing oxidative stress and release of cytochrome c (this study and [12,20–22,49–
52]); (b) activate lytic pro-enzymes, such as PLA2, which may attack both mitochondria or lysosomes (this study and ); (c) activate Bid ;
(d) directly activate caspases [15,16,54,55]. It is also possible that released lysosomal enzymes backﬁre on still intact lysosomes, causing further
rupture. Caspase 8 may somehow induce lysosomal rupture [56,57] or the activation of death receptors may cause production of sphingosine ,
which is a lysosomotropic detergent ; while p53 causes lysosomal labilization by unknown mechanisms . Other mechanisms may also be
involved in lysosomal labilization in relation to apoptosis.
FEBS 2003 Lysosomes, apoptosis and mitochondria-mediated oxidative stress (Eur. J. Biochem. 270) 3783
mitochondrial attack by cathepsins and PLA2. Indeed, low,
steady-state oxidative stress has been shown to destabilize
lysosomes  and relocation of lysosomal enzymes to the
cytosol was earlier shown to activate PLA2 .
We previously suggested that oxidative stress-induced
apoptosis might be initiated by iron-catalysed lysosomal
rupture [9,10]. It has since been found that early release to
the cytosol of lytic lysosomal enzymes may be characteristic
of apoptosis caused by a variety of stimuli [10,12–14,
19,21,22,38–40]. In these latter circumstances, it appears
that relocation of lysosomal enzymes to the cytosol may, as
in the case of oxidant-induced apoptosis, precede changes
of mitochondrial membrane potential, release of cyto-
chrome c, and all the morphological signs of apoptosis.
These considerations raised the question of whether there
might be some ROS-dependent mechanisms common to
apoptosis caused by oxidants and that caused by nonoxi-
In most cells, the predominant source of intracellular
ROS generation is the mitochondrial electron transport
chain which, even under normal conditions, may leak
1–2% of all electrons as ROS [41–43] (although there is
controversy regarding this estimate and the absolute
percentage may well be lower ). Not only will exogenous
oxidants, such as H
, directly induce apoptosis, but
enhanced intracellular production of ROS occurs when cells
are exposed to a number of pro-apoptotic agents including
tumour necrosis factor-a , ceramide , growth factor
withdrawal, HIV infection, and lipopolysaccharide [25–30].
In these cases it is unclear whether such oxidative stress is
the cause or an effect of apoptosis.
We hypothesized that released lysosomal enzymes or
PLA2 directly or indirectly activated by such enzymes 
might attack mitochondria and induce not only release
of cytochrome c, but also enhanced formation of ROS.
Released arachidonic acid may further exaggerate this
process . These ROS of mitochondrial origin could
promote further lysosomal rupture but could also have the
secondary effect of maintaining any cytochrome c released
by the mitochondria in the oxidized form (although we
should note that the cellular cytoplasm contains an abun-
dance of reducing agents which could counteract this).
Cytochrome c is involved in the activation of caspase-9
[7,46] and is considered a key component of the apoptotic
cascade. Ordinarily, any cytochrome c released from
mitochondria in oxidized form would rapidly be reduced
by the reductive cytosolic milieu. However, it has been
proposed that cytochrome c needs to remain oxidized
in order to promote apoptosis , and the oxidizing
equivalents generated by mitochondria may have precisely
MSDH is a lysosomotropic detergent that rapidly induces
speciﬁc lysosomal rupture and therefore is a very useful tool
for detailed kinetic studies of the consequences of lysosomal
rupture. The pKa of MSDH is 5.8–5.9 [31,32], allowing it to
accumulate in charged form intralysosomally (pH 4.5)
due to proton trapping , while its accumulation in the
cytosol (pH 7.2) is negligible. In protonated, charged
form MSDH acts as a much stronger detergent than when
uncharged, further targeting the action of this agent to the
lysosomal compartment .
We previously reported that released lysosomal enzymes
activate PLA2 causing further lysosomal fragmentation
. The new data presented here conﬁrm and extend those
ﬁndings and show that relocated lysosomal enzymes work
in concert with activated PLA2, causing the release of
cytochrome c, enhanced mitochondrial formation of ROS,
and promoting further lysosomal degradation. With regard
to the mechanisms involved in enhanced mitochondrial
ROS production, one particularly likely possibility is that of
generation of free fatty acids. At least in pancreatic beta cell
mitochondria, free fatty acids have been shown to increase
ROS generation, perhaps through electron leak involving
complex I of the respiratory chain . Whether the
progressive lysosomal destabilization is dependent exclu-
sively on upstream actions of cathepsins B and D, or
whether other lysosomal constituents might similarly desta-
bilize mitochondria and lysosomes is not yet clear.
Our present understanding concerning the involvement
of lysosomes in apoptosis is summarized in Fig. 4. As
shown, the initiation of apoptosis by exogenous oxidants,
and by at least some other agonists, may involve early
lysosomal rupture. The release of lysosomal enzymes (LE)
into the cell cytoplasm may set off a cascade of intracellular
degradative events. These LE may: (a) attack mitochondria
directly, inducing release of cytochrome c; (b) directly and/
or indirectly cause enhanced formation of mitochondrial
ROS (and further oxidant-induced lysosomal destabiliza-
tion); (c) activate lytic pro-enzymes, such as PLA2, which in
turn would attack both mitochondria and lysosomes;
(d) activate Bid and/or other pro-apoptotic proteins; and
(e) directly activate pro-caspases. Notably, this sequence of
early events (except for cytochrome c release) may be
relatively independent of the classical apoptotic cascade
involving caspase activation. In many circumstances, this
lysosomal-mitochondrial axis apoptotic pathway, invol-
ving combined effects of caspases, lysosomal hydrolases and
mitochondrial ROS generation, may be of central import-
ance in the ﬁnal execution of the apoptotic cascade wherein
a lysosomal/mitochondrial cross-talk may constitute an
We thank G. Dubowchik (Bristol-Myers Squibb; Pharmaceutical
Research Institute) for the kind gift of MSDH. This study was
supported by a grant from the Swedish Cancer Foundation (grant no.
4296). JWE was the recipient of a visiting professorship from the
ping University Hospital and is supported by The Common-
wealth of Kentucky Research Challenge Trust Fund.
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