Glucose-regulated protein 78 antagonizes cisplatin and adriamycin in human melanoma cells
Advance Access publication October
Glucose-regulated protein 78 antagonizes cisplatin and adriamycin in human melanoma cells
Chen Chen Jiang 0
Zhi Gang Mao 0
Kelly A.Avery-Kiejda 0
Margaret Wade 0
Peter Hersey 0
Xu Dong Zhang 0
0 Immunology and Oncology Unit, Room 443, Newcastle Misericordiae Hospital , David Maddison Clinical Sciences Building, Corner King and Watt Streets, Newcastle, New South Wales 2300 , Australia
Resistance of melanoma cells to chemotherapeutics remains a major obstacle to successful treatment of melanoma once it has spread beyond locoregional sites. We report in this study that activation of the unfolded protein response (UPR) is involved in resistance of melanoma cells to two chemotherapeutic drugs, cisplatin (CDDP) and adriamycin, and this is associated with glucose-regulated protein 78 (GRP78)-mediated inhibition of activation of caspase-4 and -7. The UPR was constitutively activated in cultured melanoma cell lines and fresh melanoma isolates as evidenced by elevated expression levels of the GRP78 protein and the active form of x-box-binding protein 1 messenger RNA. Treatment with CDDP or adriamycin further increased the levels, indicative of induction of endoplasmic reticulum stress and activation of the UPR by the drugs. Inhibition of GRP78 by small-interference RNA (siRNA)-sensitized melanoma cells to CDDP- and adriamycin-induced apoptosis. This was associated with enhanced caspase-4 and -7 activation as siRNA knockdown of the caspases blocked induction of apoptosis. In contrast, overexpression of GRP78 attenuated activation of caspase-4 and -7 and induction of apoptosis by the drugs. CDDP- and adriamycininduced activation of caspase-4 and -7 appeared to be mediated by calpain activity in that it was blocked by the calpain inhibitors calpeptin and PD150606 even when GRP78 was inhibited by siRNA. These results provide new insights into resistance mechanisms of melanoma cells to CDDP and adriamycin and identify GRP78 as a potential target for enhancing chemosensitivity in melanoma.
Melanoma continues to increase in incidence in many parts of the
world, but there is currently no curative treatment once the disease has
spread beyond the primary site because of the absence of effective
systemic therapies. This is believed to be largely due to resistance of
melanoma cells to induction of apoptosis by available
chemotherapeutic drugs and biological reagents (
). Inappropriate activation of
survival signaling pathways such as those mediated by extracellular
signal-regulated kinase (ERK) kinase (MEK)/ERK and
phosphoinositide 3-kinase (PI3K)/Akt, either as consequences of genetic
alterations or resulting from environmental stimulations, is believed to
play a central role in resistance of melanoma to apoptosis (
However, the potential effect of signaling pathways initiated by the
endoplasmic reticulum (ER) upon stress stimulations remains undefined.
Abbreviations: CDDP, cisplatin; ER, endoplasmic reticulum; ERK,
extracellular signal-regulated kinase; GRP78, glucose-regulated protein 78; MAb,
monoclonal antibody; MEK, ERK kinase; mRNA, messenger RNA; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; siRNA,
smallinterference RNA; TM, tunicamycin; UPR, unfolded protein response; XBP1,
x-box-binding protein 1.
The ER responds to stress conditions by activation of a range of
signaling pathways that couples the ER protein folding load with the
ER protein folding capacity and is termed the unfolded protein
response (UPR) (
). The UPR of mammalian cells is initiated by
three ER transmembrane proteins—activating transcription factor 6,
inositol-requiring enzyme 1 and double-stranded RNA-activated
protein kinase-like ER kinase that act as proximal sensors of ER stress
). Under unstressed conditions, the luminal domains of these
sensors are occupied by the ER chaperone glucose-regulated protein
78 (GRP78). Upon ER stress, sequestration of GRP78 by unfolded
proteins activates these sensors by inducing phosphorylation and
homodimerization of inositol-requiring enzyme 1 and protein
kinaselike ER kinase and relocalization of activating transcription factor 6
to the Golgi where it is cleaved by site 1 and site 2 proteases, leading
to its activation as a transcriptional factor (
The UPR is fundamentally a cytoprotective response, but excessive or
prolonged UPR can result in apoptosis. This involves many of the same
molecules that have important roles in other apoptotic cascades (
Among them, caspase-12 in rodents and its human homologue
caspase4 are thought to be key mediators, whereas activation of other caspases,
including caspase-2, -3, -7, -8 and -9, may also be involved (
Although several recent studies have questioned the role of caspase-12
and -4 (
), we have found that the pharmacological ER stress
inducers tunicamycin (TM) and thapsigargin can induce
caspase-4-mediated apoptosis in human melanoma cell lines when the MEK/ERK
pathway is inhibited (13). Caspase-4 is otherwise bound to and
inhibited by GRP78 (
). The latter may also protect cells from apoptosis
by a number of other mechanisms such as maintenance of ER calcium
homeostasis and prevention of caspase-7 activation (
There is increasing evidence that the UPR is activated in various
solid tumors, e.g. elevated expression of GRP78 has been reported in
a number of cancers such as breast cancer and prostate cancer (
It seems that some cancer cells may have adapted to ER stress by
activation of the UPR without resulting in cell death. Persistent
expression of proteins that facilitate cell survival such as GRP78 may be
a central feature of adaptation to ER stress (
). In support of this,
GRP78 expression is known, in some cases, to be associated with
tumor development and growth and correlated with resistance to
certain forms of chemotherapy (
). In particular, GRP78 is known to
protect against various DNA-damaging agents, including the
topoisomerase II inhibitors etoposide and adriamycin, the topoisomerase
I inhibitor camptothecin and the alkylating agent temozolomide
). On the other hand, induction of ER stress has been reported
to be involved in induction of apoptosis in enucleated melanoma cells
by the alkylating agent cisplatin (CDDP) (
We have studied the activation status of the UPR and its role in
responses to CDDP and adriamycin in human melanoma cells. We
show in this report that the GRP78 protein and the active form of
xbox-binding protein 1 (XBP1) messenger RNA (mRNA) are expressed
at elevated levels in cultured melanoma cell lines and fresh melanoma
isolates, and the expression levels can be further increased by CDDP
and adriamycin. We demonstrate that the UPR plays a role in
protection of melanoma cells against the drugs, and this is, at least
in part, due to GRP78-mediated inhibition of the activation of
caspase4 and -7. These results provide new insights into resistance of
melanoma to CDDP and adriamycin and identify GRP78 as a potential
target for sensitizing melanoma cells to chemotherapy.
Materials and methods
Human melanoma cell lines Mel-RM, MM200, IgR3, Mel-CV, Me4405,
Sk-Mel-28, Mel-FH and Me1007 have been described previously (
Among them, MM200, Me4405, Me1007 and IgR3 were from primary
melanoma. Mel-RM, Mel-CV, Sk-Mel-28 and Mel-FH were from metastatic
melanoma (21). They were cultured in Dulbecco’s modified Eagle’s medium
containing 5% fetal calf serum (Commonwealth Serum Laboratories,
Melbourne, Australia). The cultured human melanocyte line HEMn-MP was
purchased from Banksia Scientific (Bulimba, Queensland, Australia) and the
cells were cultured in medium supplied by Clonetics (Edward Kellar, Victoria,
Fresh melanoma isolates
Isolation of melanoma cells from fresh surgical specimens was carried out as
described previously (
). All fresh melanoma isolates were from
Antibodies, recombinant proteins and other reagents
CDDP and adriamycin were supplied by Pharmacia Upjohn (Sydney,
New South Wales, Australia). TM was purchased from Sigma Chemical Co.
(Castle Hill, Australia). The cell-permeable general caspase inhibitor
Z-ValAla-Asp(OMe)-CH2F (z-VAD-fmk) and the caspase-3-specific inhibitor
Z-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-CH2F (z-DEVD-fmk) were purchased
from Calbiochem (La Jolla, CA). The caspase-4-specific inhibitor
Z-LeuGlu-Val-Asp-fmk (z-LEVD-fmk) was from BioVision (Mountain View, CA).
The mouse monoclonal antibody (MAb) against caspase-4 was from Abcam
(Cambridge, UK). The rabbit MAb against GRP78 was purchased from Santa
Cruz Biotechnology (Santa Cruz, CA). Isotype control antibodies used were
the ID4.5 (mouse IgG2a) MAb against Salmonella typhi supplied by Dr L.Ashman
(Institute for Medical and Veterinary Science, Adelaide, Australia), and the
107.3 mouse IgG1 MAb was purchased from PharMingen (San Diego, CA)
and rabbit IgG from Sigma Chemical Co.
Cell viability assays
The cytotoxic effect of CDDP on melanoma cells was determined using
3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays as
described previously (
). Briefly, cells were seeded at 5000 per well onto
flatbottomed 96-well culture plates and allowed to grow for 24 h followed by the
desired treatment. Cells were then labeled with MTT from the Vybrant MTT
Cell Proliferation Assay Kit (Molecular Probes, Eugene, OR) according to the
Quantitation of apoptotic cells by measurement of sub-G1 DNA content using
the propidium iodide method or by Annexin-V staining was carried out as
described elsewhere (
Western blot analysis
Western blot analysis was carried out as described previously (
bands were detected by Immun-StarTM HRP Chemiluminescent Kit, and
images were captured and the intensity of the bands was quantitated with the
Bio-Rad VersaDocTM image system (Bio-Rad, Regents Park, New South
Detection of XBP1 mRNA splicing
The method used for detection of unspliced and spliced XBP1 mRNAs was as
described previously (
). Briefly, reverse transcription–polymerase chain reaction
products of XBP1 mRNA were obtained from total RNA extracted using primers
5#-cggtgcgcggtgcgtagtctgga-3# (sense) and 5#-tgaggggctgagaggtgcttcct-3#
(antisense). Because a 26 bp fragment containing an Apa-LI site is spliced upon
activation of XBP1 mRNA, the reverse transcription–polymerase chain reaction
products were digested with Apa-LI to distinguish the active spliced form from the
inactive unspliced form. Subsequent electrophoresis revealed the inactive form as
two cleaved fragments and the active form as a non-cleaved fragment.
Immunostaining on intact and permeabilized cells was carried out as described
previously. Analysis was carried out using a Becton Dickinson (Mountain
View, CA) FACScan flow cytometer (
Caspase activity assay
Measurement of caspase activities by fluorometric assays was performed as
described previously (
). The specific substrates z-DEVD-AFC,
Ac-LEVDAFC and z-LEHD-AFC were used to measure caspase-3, -4 and -9 activities,
respectively (Calbiochem). The generation of free AFC was determined using
Fluostar OPTIMA (LABTECH, Offenburg, Germany) set at an excitation
wavelength of 400 nm and an emission wavelength of 505 nm.
Calpain activity assay
Measurement of calpain activities by fluorometric assays was performed as
described previously (
). Briefly, cells were suspended in cold lysis buffer (10
mM N-2-hydroxyethylpiperazine-N#-2-ethanesulfonic acid, pH 7.4, 5 mM
MgCl2, 42 mM KCl and 0.32 M sucrose) and lysed by repeated passage
through a 27 gauge syringe. For each sample, 40 lg of protein was incubated
with the calpain substrate N-Suc-Leu-Tyr-AMC (Suc) (Sigma Chemical Co.)
in assay buffer (10 mM N-2-hydroxyethylpiperazine-N#-2-ethanesulfonic acid,
pH 7.4, 1% Triton X-100 and 100 lM CaCl2) at 37 C for 2 h. The generation of
free AMC was determined using Fluostar OPTIMA (LABTECH) set at an
excitation wavelength of 380 nm and an emission wavelength of 460 nm.
Melanoma cells were seeded at 4 104 cells per well in 24-well plates and
allowed to reach 50% confluence on the day of transfection. The
smallinterference RNA (siRNA) constructs used were obtained as the siGENOME
SMARTpool reagents (Dharmacon, Lafayette, CO), the siGENOME
SMARTpool GRP78 (M-008198-01), the siGENOME SMARTpool caspase-4
(M004404-00) and the siGENOME SMARTpool caspase-7 (M-004407-02).
The non-targeting siRNA control SiConTRolNon-targeting SiRNA pool
(D-001206-13-20) was also obtained from Dharmacon. Cells were transfected
with 50–100 nM siRNA in Opti-MEM Medium (Invitrogen, Carlsbad, CA)
with 5% fetal calf serum using Lipofectamine Reagent (Invitrogen) according
to the manufacturer’s transfection protocol. Twenty-four hours after
transfection, the cells were switched into medium containing 5% fetal calf serum and
were treated as designed before quantitation of apoptotic cells by measurement
of sub-G1 DNA content using the propidium iodide method in flow cytometry.
Efficiency of siRNA was measured by western blot analysis.
Expression of GRP78 in melanoma cell lines and fresh melanoma
To study if melanoma cells may express increased levels of GRP78,
we examined GRP78 expression in a panel of melanoma cell lines in
western blot analysis. These include four melanoma cell lines
established from primary (MM200, Me4405, Me1007 and IgR3) and
another four from metastatic melanomas (Mel-RM, Mel-CV, Sk-Mel-28
and Mel-FH). A cultured melanocyte line was included as a control.
The results showed that in comparison with the melanocyte line,
melanoma cell lines expressed varying but generally higher levels
of GRP78 (Figure 1A). Quantitation of western blot band densities
indicated that the relative levels of GRP78 expression differed by up
to four times among the melanoma cell lines (supplementary Figure 1
is available at Carcinogenesis Online), but there was no significant
difference in the levels of GRP78 between melanoma cell lines from
primary and those from metastatic melanomas (P . 0.05, two-tailed
student’s t-test). Similarly, there was no apparent association between
the levels of GRP78 expression and p53 or BRAF status in the
melanoma cell lines (data not shown).
We examined if melanoma cells in vivo also express elevated levels
of GRP78. Five metastatic melanoma specimens from patients
undergoing surgery before adjunctive treatment were processed, and the
resulting fresh melanoma isolates were tested for GRP78 expression.
Compared with melanocytes, all the fresh isolates expressed increased
levels of GRP78, which were even higher than those in most cultured
melanoma cell lines (Figure 1A and supplementary Figure 1 is
available at Carcinogenesis Online). These results indicate that GRP78 is
commonly expressed at increased levels in melanoma cells,
conceivably as a consequence of activation of the UPR due to chronic ER
stress. To confirm this, expression of another indicator of UPR
activation, the spliced XBP1 mRNA (
), was analyzed by polymerase
chain reaction. As shown in Figure 1B, the spliced XBP1 mRNA could
not be detected in the melanocyte line but was observed to varying
degrees in all the melanoma cell lines and fresh melanoma isolates.
CDDP and adriamycin upregulate GRP78 in melanoma cells
We studied whether CDDP and adriamycin impinge on ER stress and
GRP78 expression in melanoma cells. Mel-RM and MM200 cells were
treated with the drugs for varying periods ranging from 6 to 36 h. Figure
2A shows that, while the established ER stress inducer TM induced
marked increases in the levels of GRP78 in both cell lines (
CDDP and adriamycin also caused upregulation of GRP78 with similar
kinetics, albeit to a lesser extent. We also monitored the levels of the
spliced XBP1 mRNA after exposure to the drugs and found that
treatment with CDDP or adriamycin resulted in upregulation of the levels
(Figure 2B). Induction of GRP78 by CDDP and adriamycin was
confirmed in another four melanoma cell lines treated with the drugs for
24 h (Figure 2C). These results indicate that both CDDP and adriamycin
can induce ER stress and activation of the UPR in melanoma cells.
GRP78 protects melanoma cells against killing induced by CDDP and
GRP78 is known to be an important prosurvival factor in cells under
ER stress (
). We examined whether it also protects melanoma
cells against cytotoxicity mediated by CDDP and adriamycin. First,
the cytotoxic potentials of the drugs were tested in the panel of
melanoma cell lines. As shown in Figure 3A, exposure to CDDP or
adriamycin for 48 h reduced cell viability to varying degrees as
measured in MTT assays. This was associated with externalization of
phosphatidylserine, activation of caspase-3 and accumulation
of sub-G1 DNA content (supplementary Figure 2 is available at
Carcinogenesis Online) and could be inhibited by the general caspase
inhibitor z-VAD-fmk (Figure 3B), indicating that the cytotoxicity of
the drugs against melanoma cells was largely mediated by induction
of apoptosis. We analyzed the relationship between the GRP78 levels
and sensitivities of melanoma cell lines to CDDP and adriamycin but
did not find any significant correlation (regression analysis,
X2 5 0.2161 and 0.0176; P . 0.05, respectively) (supplementary
Figure 3 is available at Carcinogenesis Online).
To study the role of GRP78 in regulating sensitivity of melanoma
cells to the drugs, we transfected a siRNA pool for GRP78 into
MelRM and MM200, two melanoma cell lines with moderate levels of
GRP78 expression. Western blot analysis showed that the levels of
GRP78 were markedly reduced in cells transfected with the GRP78
siRNA in comparison with those with the control siRNA (Figure 3C).
Assessment of apoptosis induction indicated that inhibition of GRP78
by siRNA resulted in significant increases in sensitivity of both cell
lines to killing induced by CDDP or adriamycin (P , 0.01, two-tailed
student’s t-test) (Figure 3C).
To confirm the role of GRP78 in protection of melanoma cells
against CDDP and adriamycin, Mel-RM and MM200 cells were
transfected with complementary DNA encoding GRP78. Western blot
analysis verified that GRP78 expression was markedly increased in
cells transfected with GRP78 complementary DNA but not in those
transfected with the vector alone (Figure 3D). Overexpression of
GRP78 markedly inhibited CDDP- or adriamycin-induced killing in
both cell lines (P , 0.01, two-tailed student’s t-test) (Figure 3D).
Taken together, these results indicate that GRP78 antagonizes
cytotoxic effects of cysplatin and adriamycin in melanoma cells.
GRP78 inhibits activation of the caspase cascade induced by CDDP
We have shown previously that GRP78 can bind to caspase-4 and
inhibit its activation in melanoma cells submitted to ER stress (
In addition, GRP78 is also known to interact with caspase-7 and
suppress its activation (
). We therefore studied whether
GRP78-mediated protection of melanoma cells against CDDP and
adriamycin is associated with inhibition of activation of caspase-4
and -7. First, we examined if CDDP and adriamycin induce activation
of caspase-4 and caspase-7 in melanoma cells by western blot analysis
of whole-cell lysates from Mel-RM and MM200 cells treated with the
drugs. Figure 4A shows that both drugs induced activation of
caspase4 and -7, as evidenced by reduction in the proenzyme levels and
appearance of smaller cleaved forms of the caspases. Activation
of caspase-4 and -7 was also confirmed in fluorometric assays
using caspase-4-specific substrate Ac-LEVD-AFC and the caspase-7
substrate Ac-DEVD-AFC, respectively (Figure 4A).
To further study the role of caspase-4 and -7 in CDDP- and
adriamycin-mediated killing of melanoma cells, we silenced
caspase-4 and -7 by specific siRNA pools in Mel-RM and MM200
cells, respectively. Figure 4B shows that while the caspase-4 siRNA
significantly reduced the levels of caspase-4 but not caspase-7
expression, the caspase-7 siRNA similarly decreased the levels of caspase-7
but not caspase-4 expression. CDDP- and adriamycin-mediated
killing of Mel-RM and MM200 cells were significantly inhibited in cells
transfected with the caspase-4 and -7 siRNA, respectively, in
comparison with those transfected with the control siRNA (P , 0.01,
twotailed student’s t-test) (Figure 4B).
We next examined the role of GRP78 in regulating activation of
caspase-4 and -7 induced by CDDP or adriamycin. As shown in
Figure 4C, inhibition of GRP78 by siRNA knockdown (Figure 3C)
enhanced, whereas overexpression of GRP78 (Figure 3D) attenuated
caspase-4 and -7 activities induced by CDDP and adriamycin as
measured in fluorometric assays. Inhibiting activation of caspase-4 and -7
by overexpression of GRP78 was also confirmed in western blot
analysis (supplementary Figure 4 is available at Carcinogenesis
Online). Collectively, these results demonstrate that GRP78 protects
melanoma cells from CDDP- and adriamycin-induced killing
through, at least in part, inhibition of caspase-4 and -7.
Calpain activity contributes to activation of caspase-4 and -7 induced
by CDDP or adriamycin
Calpain activity has been reported to contribute to CDDP-induced
caspase-7 activation in melanoma cells (
). We examined if calpain
activity similarly plays a role in caspase-4 activation induced by
CDDP and if it is involved in activation of the caspases by adriamycin
in melanoma cells. As expected, treatment with CDDP resulted in
increased calpain activity (Figure 5A). Similarly, exposure to
adriamycin also led to increases in calpain activity in melanoma cells
(Figure 5A). Inhibition of calpain activity by the inhibitor
PD150606 or calpeptin (Figure 5A) partially blocked activation of
caspase-4 and -7 and apoptosis induced by CDDP and adriamycin
(Figure 5B and C). These results indicate that calpain activity is an
initiating factor for activation of ER-associated caspases in melanoma
cells induced by CDDP and adriamycin.
Resistance of melanoma cells to chemotherapeutics is a major
obstacle to successful treatment of melanoma once it has spread beyond
locoregional sites. In the present study, we show that activation of the
UPR contributes to resistance of melanoma cells against the
chemotherapeutic drugs CDDP and adriamycin, and this is, at least in part,
due to inhibition of activation of the ER-associated caspases,
caspase4 and -7, by GRP78. These results provide new insights into resistance
mechanisms of melanoma cells to chemotherapy and may have
important therapeutic applications in the treatment of melanoma.
Although elevated GRP78 expression levels have been reported in
a number of solid cancers (
), the present study appears to be the
first to show that GRP78 is expressed at relatively high levels in both
cultured melanoma cell lines and fresh melanoma isolates. The
elevated levels of the GRP78 protein, along with the increased levels of
the spliced XBP1 mRNA, suggest that the UPR in melanoma cells is
constitutively activated (
). In support of this, we found in a
separate study on tissue sections from a large panel of melanocytic
tumors demonstrated that GRP78 was expressed at relatively high levels
on most melanoma tissue sections (data not shown). Given the highly
malignant nature of melanoma, it is conceivable that the rapid growth
rate and perhaps inadequate vascularization would create a
microenvironment with hypoxia, glucose deprivation and acidosis, which in
turn results in chronic ER stress. In addition, increased glycolytic
activity in melanoma cells may also contribute to ER stress
). In support of this, increased lactate dehydrogenase levels,
indicative of increased glycolytic activity, are common in metastatic
). Whether there are other properties of melanoma
cells that predispose to ER stress remains unknown. It has recently
been reported that ER stress was induced at early stages of melanoma
initiation by transfection of melanocytes with oncogenic forms of
HRAS (HRASG12V) (33).
GRP78 appeared to protect melanoma cells against cytotoxic
effects of CDDP and adriamycin. This was largely due to inhibition of
induction of apoptosis, as killing of melanoma cells by the drugs was
associated with hallmarks of apoptosis such as externalization of
phosphatidylserine, accumulation of sub-G1 DNA content and
activation of caspase-3, which could be efficiently inhibited by a general
caspase inhibitor. Although DNA adducts are generally believed to be
shown are the mean ± SE of three individual experiments. (C) Inhibition of
calpain by PD150606 partially blocks apoptosis of melanoma cells induced
by CDDP and adriamycin. Mel-RM and MM200 cells were treated with
CDDP (10 lg/ml) or adriamycin (1 lM) for 48 h in the presence or absence
of PD150606 (40lM). Apoptosis was measured by the propidium iodide
method using flow cytometry. The data shown are the mean ± SE of three
the key toxic lesions induced by CDDP (
), a number of recent
studies have shown that CDDP can exert cytotoxicity independently
of its DNA-damaging activity (
). Particularly, CDDP induced
ER stress-mediated apoptotic signaling in enucleated cells of the
human melanoma cell line 224 (
). We observed in this study that
CDDP induced increases in the GRP78 protein and the spliced XBP1
mRNA levels, indicating that induction of ER stress and activation of
the UPR is a general effect of CDDP on melanoma cells. Similarly, we
found, for the first time, that the topoisomerase II inhibitor adriamycin
also caused ER stress and activation of the UPR in melanoma cells.
Together, these results suggest that CDDP and adriamycin, two
chemotherapeutic drugs that were conventionally regarded as
DNA-damaging agents, can exert their cytotoxicity in melanoma cells by
inducing ER stress. Their cytotoxicity is, however, attenuated by
GRP78 as a consequence of activation of the UPR. Intriguingly, there
was no correlation between the GRP78 expression levels and
sensitivities of melanoma cell lines to CDDP or adriamycin, suggesting
that, besides GRP78, other mechanisms may also contribute to
regulation of responses of melanoma cells to the drugs. For example,
expression of adenosine triphosphate-binding cassette transporters
and increased DNA repair are known to contribute to resistance of
cancer cells against CDDP and adriamycin (
). In addition,
activation survival signaling pathways such as the PI3K/Akt and MEK/
ERK pathways is a common cause for resistance of melanoma to
). It seems that resistance of melanoma cells to these
drugs is afforded by multiple mechanisms including that mediated by
GRP78. Whether GRP78 plays a similar role in regulating sensitivity
of melanoma cells to other DNA-damaging agents remains to be
studied. Inhibition of GRP78 is known to cause increased sensitivity
of glioma cells to the DNA-alkylating drug temozolomide (
We have shown previously that caspase-4 plays an important role in
induction of apoptosis of melanoma cells by TM and thapsigargin
when the MEK/ERK pathway is inhibited (
). Similarly, we found
in this study that caspase-4 was also involved in CDDP- or
adriamycin-mediated killing of melanoma cells. Importantly, inhibition of
GRP78 by siRNA enhanced, whereas overexpression of GRP78
inhibited, caspase-4 activation. This is consistent with our previous
finding that GRP78 can bind to and inhibit caspase-4 (
has been shown to be localized to the ER membrane and mitochondria
in human neuroblastoma cells (
). The physical association between
GRP78 and caspase-4 suggests that caspase-4 may also be present in
the ER in melanoma cells (
). This was supported by the punctate
staining pattern of caspase-4 in immunofluorescence studies, indicative
of organelle localization as shown previously (
Another caspase that appeared to involve in CDDP- and
adriamycininduced apoptosis was caspase-7. This was shown by activation of
caspase-7 by the drugs and inhibition of apoptosis by siRNA
knockdown of the caspase. Although caspase-7 is an executor caspase (
has been shown to be located to the ER membrane where it was bound
to and inhibited by GRP78 in human leukemia and bladder carcinoma
). Similarly, we found that inhibition of GRP78 by siRNA
increased, whereas overexpression of GRP78 inhibited, caspase-7
activation induced by CDDP and adriamycin, suggesting a possible
association of caspase-7 and GRP78 in melanoma cells.
CDDP has been reported to activate caspase-7 in human melanoma
cells via induction of calpain activity (
). The present study
demonstrated that calpain activity was also responsible for caspase-4
activation induced by CDDP and adriamycin in melanoma cells. In the
murine system, a number of mechanisms have been suggested to be
responsible for caspase-12 activation (
). For example,
caspase-12 could be activated by a direct association with the ER stress
transducer inositol-requiring enzyme 1a and the adapter protein
TRAF2 (40). In addition, caspase-12 could be directly activated
downstream of caspase-7 upon ER stress (
). Our observations
did not support a role of caspase-7 in activation of caspase-4 by CDDP
and adriamycin in melanoma cells, as caspase-7 and -4 were activated
with similar kinetics, and siRNA inhibition of caspase-7 did not block
caspase-4 activation (data not shown). It is possible that these
caspases may be simultaneously activated by calpain activity, at least in
the case of treatment with CDDP or adriamycin.
Besides inhibition of caspase-4 and -7, GRP78 may also protect
melanoma cells against CDDP and adriamycin by other mechanisms
). For example, as a protein chaperone, GRP78 can bind to
unfolded/misfolded protein and thereby protecting cells against ER
). GRP78 is also known to act as a calcium-binding
protein in the ER, thus preventing calcium efflux into the cytosol and
inhibiting apoptosis (
). Similarly, activation of the UPR may
provide other protective mechanisms against CDDP and adriamycin
apart from induction of GRP78 (
In summary, we demonstrate that activation of the UPR plays an
important part in protection of melanoma cells against cytotoxic
effects of CDDP and adriamycin. This is mediated, at least in part, by
GRP78-mediated inhibition of caspase-4 and -7. Therefore, agents
that target GRP78, such as the macrocyclic compound versipelostatin
and the green tea extract epigallocatechin, both of which are natural
products and in development for clinical use (
), could be
expected to have a significant role for sensitizing melanoma cells to
these chemotherapeutic drugs.
Supplementary Figures 1–4 can be found at http://carcin.oxfordjournals.
New South Wales State Cancer Council; Melanoma and Skin Cancer
Research Institute Sydney; Hunter Melanoma Foundation, New South
Wales; National Health and Medical Research Council, Australia.
X.D.Z. is a Cancer Institute New South Wales fellow.
Conflict of Interest Statement: None declared.
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Received June 27, 2008; revised August 13 , 2008 ; accepted September 12, 2008