Iranian Journal of
Medical Hypotheses and Ideas
Original article
Lysosome: as a proposed target for rose bengal in inducing cell death in
melanoma cells
Seyed Hadi Mousavi 1,2 and Seyed Hamed Moosavi2
Address:
1
Assistant Professor of Pharmacology, Department of Pharmacology and Pharmacological Research Centre of Medicinal Plants, School of
Medicine, Mashhad University of Medical Sciences, Mashhad, IRAN
2
Medical Toxicology Research Center, Mashhad University of Medical Sciences, Mashhad, IRAN
Corresponding author:
Dr. Seyed Hadi Mousavi
School of Medicine, Mashhad University of Medical Sciences Mashhad, IRAN
Email: [email protected], [email protected]
Tel: 0098 9155199598
Fax: 0511 8828567
Received: 29 Feb 2008
Accepted: 5 May 2008
Published: 11 May 2008
Iran J Med Hypotheses Ideas, 2008, 2:12
© 2008 Seyed Hadi Mousavi and Seyed Hamed Moosavi ; licensee Tehran Univ. Med. Sci.
Abstract
We have previously shown that rose bengal (RB) itself and not as a photosensitiser could
induces dual modes of cell death in melanoma cells and has clinical activity against
melanoma. But the mechanisms of RB-induced cell death are unclear. Recently lysosome
was reported to initiate different kinds of cell death including necrosis, caspase-dependent
and -independent apoptosis. The hypothesis of this study is to investigate the role of
lysosome in mediating RB-induced cell death in melanoma cells focusing on lysosomal
protease cathepsin B (CB). We present two lines of evidence indicating a central role for
the CB in mediating cell death. First, inhibition of CB would result in a strong protection
against drug-induced cell death and apoptosis in melanoma cells. Simultaneous inhibition
of caspases would determine that CB acts upstream or downstream the caspase cascade.
Second, we show RB triggers disruption of lysosomes leading to release and activation of
CB using an engineered yellow fluorescent protein-tagged CB.
It is hypothesized that RB-induced cell death in melanoma cells is mediated through
lysosomal CB in this novel cell death pathway. This idea points to a new tumorsuppressive role for lysosomes which may be less affected by chemotherapy-induced
resistance mechanisms.
Keywords
Lysosome, Cathepsin B, Melanoma, Rose Bengal
Introduction
Over the past decade, it has become known that
many therapeutic agents kill cancer cells by
inducing apoptosis or necrosis (1,2). Apoptosis is a
programmed cell death is characterized by distinct
morphological features including; chromatin
condensation, cell and nuclear shrinkage,
membrane blebbing and oligonucleosomal DNA
fragmentation. Three major apoptotic pathways
originating from separate subcellular compartments
have been identified the death receptor,
mitochondrial and the endoplasmic reticulum
pathway (3,4,5). Although each pathway is initially
Irn J Med Hypotheses Ideas 2008, 2:12
mediated by different mechanisms, they share a
common final phase of apoptosis, consisting of
activation of the executioner caspases and
dismantling of substrates critical for cell survival
(6,7).
However, apoptosis signaling mechanisms induced
by many agents are impaired in tumor cells, leading to
resistance against therapy. Lysosomes and the
endoplasmic reticulum (ER) hold promise as drug
targets and mediators of apoptosis signaling which
may be less affected by intrinsic or chemotherapyinduced resistance mechanisms (8,9). Recently
lysosome was reported to initiate different kinds of
cell death including necrosis, caspase-dependent
and -independent apoptosis. Lysosomes proteases
such as cathepsins may account for these
alternative types of PCD leading to a new tumorsuppressive role for lysosomes which may be less
affected by resistance mechanisms (10,11,12).
Hypotheses
Rose Bengal (RB) has been used as a systemic
diagnostic of hepatic function, ophthalmic
diagnostic and photosensitiser in photodynamic
treatment. We have previously found that RB itself
and not as photosensitiser could induces dual
modes of cell death (apoptotic and non-apoptotic
cell death) in melanoma cells and has clinical
activity against melanoma (13,14,15). RB-induced
apoptosis was both caspase-dependent and independent. This toxicity was also independent of
Reactive Oxygen Species (ROS) production.
Taking together the mechanisms of RB-induced
toxicity are unclear (14,15).
Lysosome could be considered as a one of the
proposed targets for the selective RB-induced
toxicity in melanoma cells. Rupture of lysosomes,
leading to the release of their cathepsins content,
has long been recognized as potentially harmful to
the cell (16). IT has been shown that lysosomal
cathepsins including B, D, and L translocate from
the lysosomal lumen to the cytosol in response to a
variety of signals such as TNF receptor ligation,
p53 activation, oxidative stress and also by
lysosomotropic agents such as cyprofloxacin and
hydroxychloroquine (11). In this study lysosome is
considered as a target for RB-induced toxicity in
melanoma cells focusing on CB. We present two
lines of evidence indicating a central role for the
CB in mediating cell death. First, inhibition of CB
would result in a strong protection against druginduced cell death and apoptosis in melanoma
cells. Simultaneous inhibition of caspases would
determine that CB acts upstream or downstream the
caspase cascade. Second, we show RB triggers
disruption of lysosomes leading to release and
activation of CB using an engineered yellow
fluorescent protein-tagged CB
.
Seyed Hadi Mousavi and Seyed Hamed Moosavi
Evaluation of the hypotheses
Human melanoma cell line, Sk-Mel-28, described
previously is cultured in DMEM containing 5% FCS.
(13,14). Cells are seeded overnight, and then
incubated with RB in the dark. SK 28 cells are treated
with pan-caspase inhibitor z-VAD-fmk, or CA-074
Me as a selective inhibitor of CB, 1 h before adding
RB (200 µM) for another 24 h.
Cell viability is measured by MTT cell
proliferation assay according to the literature.
(13,14) The extent of apoptosis is determined by
flow cytometry, using propidium iodide (Sigma)
staining of hypodiploid DNA-content, as
previously described (12,13,17).
Lysosomal integrity assays are performed with
the lysosomotropic probe Lysotracker Red
according to the literature (18). For studying of CB
release, a yellow fluorescent protein-tagged CB is
cloned into the expression vector pEYFPC1, to
allow easy detection by fluorescence microscopy.
First full length human CB cDNA is generated by
reverse transcription-PCR (5'-ATC TGT TCT CGA
GCT ATG TGG CAG CTC TGG GCC TCC-3' and
5'[-ATG GTA GGA TCC TTA GAT CTT TTC
CCA GTA CTG-3'). Then it is cloned into the
pEYFPC1 vector. Cells are seeded onto glass
coverslips in a 6-well tray and are transfected with
1.5 µg of plasmid DNA using the FuGene6
transfection
reagent
(Roche
Molecular
Biochemicals), according to the manufacturer’s
protocol (19).
CB activity is determined fluorimetrically using
the methyl-coumarylamide substrate z-Arg-ArgNHMec at pH 6.0, as described in literature (20).
Fluorescence is measured with an excitation
wavelength of 360 nm and emission wavelength of
460 nm. One unit of enzyme activity is defined as
the release of 1 µmol of product/min.
Experimental data
Effect of pan-caspase and CB inhibitor on RBinduced cell death
To elucidate pathways mediating cell death
induced by RB, the contribution of CB to the
cytotoxic potential of RB is evaluated. For this
purpose, RB-induced cell death and apoptosis are
studied in presence of pan-caspase inhibitor zVAD-fmk or CA-074 Me as a selective inhibitor
of CB. CA-074 Me is a highly specific inhibitor of
CB in vitro and easily penetrates into cells (21).
The percentage of apoptosis in the presence of zVAD-fmk, is considered as caspase- independent
apoptosis.
Effect of RB on lysosome integrity, CB release and
activity
To assess whether treatment with RB results in a
lysosomal membrane permeabilization, lysosomal
integrity assays is performed using the
Page 2 of 5 (page number not for citation purposes) Irn J Med Hypotheses Ideas 2008, 2:12
lysosomotropic fluorescence probe Lysotracker
Red. In cells treated with RB, increased number of
cells with a weak fluorescence (so-called “pale
cells”) is detected, a phenomenon reported to be
due to leakage of the probe into the cytoplasm,
indicating lysosomal rupture. Next we would show
whether the disruption of the lysosomal integrity is
consistent with release of CB using an expression
plasmid encoding yellow fluorescent proteintagged CB. IF there is a diffuse staining pattern of
the fusion protein throughout the cells after
treatment, indicates CB is released from the
lysosomes of melanoma cells on incubation with
RB. To examine the activation of CB after its
release into the cytoplasm, CB activity is
performed. Increased CB activity after treatment
with RB indicates role of CB in RB-induced cell
death.
Discussion
Cross-resistance of cells to cytotoxic effects of
natural and synthetic anticancer drugs is the wellknown phenomenon called multidrug resistance
(MDR). The sensitisation of MDR-resistant cells to
anticancer drugs by lysosomotropic compounds
recommends scrutinizing the potential of
lysosomotropic drugs in cancer therapy (22).
In regard to melanoma there has been little
progress in the medical treatment of metastatic
melanoma because of its resistance to current
chemotherapeutic agents (23). We have recently
reported that RB triggers pronounced cell death in
melanoma cells via apoptotic- and non-apoptotic
routs by unknown mechanism. In this study, we
proposed lysosome as a target for RB in melanoma
cells. If Inhibition of CB strongly protects against
cell death, indicates that CB is the mediator of RBinduced cell death in melanoma cells. If treatment
with RB, results in disruption of lysosomal
Seyed Hadi Mousavi and Seyed Hamed Moosavi
membrane but not activation of CB, role of other
cathpsins such as D, and L, should be explored. We
have shown that both caspase-dependent and
-independent pathways were induced by RB in
melanoma cells (14,15). If CB inhibitor completely
inhibits RB-induced apoptosis, it indicates CB
triggers both caspase-dependent and caspaseindependent pathways in melanoma cells and
caspase activation is downstream to CB activation
(Figure 1). CB has been reported to contribute to
apoptosis via induction of mitochondrial membrane
permeabilization, possibly via cleavage of Bid, in
some systems, thereby acting upstream of the
caspase cascade (24, 25, 26). If inhibition of CB,
only blocks the caspase-independent apoptosis, in
this respect it could be conclude that RB initiate
only caspase-independent apoptosis pathways
which has been shown by other studies (27,28).
Strong evidence is now accumulating for the
involvement of alternative proteases, such as CB,
in PCD (29), but the molecular identity of the
mediators and the necessity of activation of the
apoptotic pathways remain to be elucidated in most
cases and may vary on the type of cells and the
applied death stimulus (30).
Taken together, if this idea is verified, it
emphasizes using of lysosomotropic drugs in
cancer therapy. It is concluded that RB-induced
cell death in melanoma cells is mediated through
lysosomal CB in the novel cell death pathway. This
hypothesis points to a new tumor-suppressive role
for lysosomes which may be less affected by
chemotherapy-induced resistance mechanisms.
Acknowledgements
The authors would like to thank Dr Peter Heresy
(Professor of Immunology and Oncology, Newcastle,
Australia) for supervising the previous studies.
Page 3 of 5 (page number not for citation purposes) Irn J Med Hypotheses Ideas 2008, 2:12
Seyed Hadi Mousavi and Seyed Hamed Moosavi
Fig. 1. Proposed model for RB‐induced different kind of cell death in melanoma cells: Role of cathepsin B z‐VAD‐fmk: pan‐caspase inhibitor CA‐074 Me: selective cathepsin B inhibitor AIF: apoptosis inducing factors
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