ORIGINAL PAPER

TRAIL-induced apoptosis of human melanoma cells involvesactivation of caspase-4

Zhi Gang Mao • Chen Chen Jiang • Fan Yang •

Rick F. Thorne • Peter Hersey • Xu Dong Zhang

Published online: 1 June 2010

� Springer Science+Business Media, LLC 2010

Abstract Although it is conventionally regarded as an

inflammatory caspase, recent studies have shown that

caspase-4 plays a role in induction of apoptosis by endo-

plasmic reticulum (ER) stress. We report here that activa-

tion of caspase-4 is also involved in induction of apoptosis

by TNF-related apoptosis-inducing ligand (TRAIL) in

human melanoma cells. Treatment with TRAIL resulted in

activation of caspase-4. This appeared to be mediated by

caspase-3, in that caspase-4 was activated later than cas-

pase-8, -9, and -3, and that inhibition of caspase-3 blocked

TRAIL-induced caspase-4 activation. Notably, TRAIL

triggered ER stress in melanoma cells as shown by up-

regulation of the GRP78 protein and the spliced form of

XBP-1 mRNA. This seemed to be necessary for activation

of caspase-4, as activation of caspase-3 by agents that did

not trigger ER stress did not cause activation of caspase-4.

Importantly, inhibition of caspase-4 also partially blocked

caspase-3 activation, suggesting that activation of caspase-

4 may be positive feed-back mechanism to further enhance

caspase-3 activation. Collectively, these results show that

activation of caspase-4 contributes to TRAIL-induced

apoptosis and is associated with induction of ER stress by

TRAIL in melanoma cells, and may have important

implications for improving therapeutic efficacies of TRAIL

in melanoma.

Keywords TRAIL � Caspase-4 � Melanoma � Apoptosis

Introduction

Tumor necrosis factor (TNF)-related apoptosis-inducing

ligand (TRAIL) is a member of the TNF family that

induces apoptosis by binding to two death receptors (Rs),

TRAIL-R1 and -R2 [1, 2]. This in turn orchestrates the

assembly of adapter components such as Fas-associated

death domain (FADD) and pro-caspase-8, leading to the

formation of the death-inducing signaling complex (DISC)

in which pro-caspase-8 is activated by dimerization and

subsequent autocatalytic cleavage [3, 4]. The activated

caspase-8 can then activate downstream effect or caspases

such as caspase-3 either directly or indirectly by recruit-

ment of the mitochondrial apoptotic pathway through

activation of the BH3-only protein Bid, leading eventually

to apoptosis [3–5]. Another BH3-only protein that has been

shown to be involved in TRAIL-induced activation of the

mitochondrial apoptotic pathway is Bim [6, 7]. Moreover,

caspase-2 activation is known to contribute to TRAIL-

induced apoptosis in some cells by functioning up-stream

of cleavage of Bid [8, 9].

Caspase-4 is a member of the inflammatory caspase

group [10, 11]. It is closely related to caspase-1 with 53%

shared sequence homology and exhibits similar inhibitor

preference for CrmA and p35 [10, 11]. Although the main

substrates for caspase-1 are known to be prointerleukin

(proIL)-1b and proIL-18, two related cytokines that

play critical roles in inflammation, no specific substrates

for caspase-4 have been identified so far [10, 11].

Z. G. Mao � C. C. Jiang � F. Yang � R. F. Thorne �P. Hersey (&) � X. D. Zhang (&)

Immunology and Oncology Unit, Newcastle Misericordiae

Hospital, Room 443, David Maddison Clinical Sciences

Building, Cnr. King and Watt Streets, Newcastle,

NSW 2300, Australia

e-mail: Peter.Hersey@newcastle.edu.au

X. D. Zhang

e-mail: Xu.Zhang@newcastle.edu.au

123

Apoptosis (2010) 15:1211–1222

DOI 10.1007/s10495-010-0513-9

Nevertheless, caspase-4 has been shown to activate cas-

pase-3 in vitro [11, 12]. Caspase-4 also shares 48%

sequence homology with caspase-12 in rodents [10, 13].

The latter is not expressed in functional form in human

due to polymorphisms resulting from the interruption of

the gene from frame shifts, premature stop codon, and

amino acid substitution in the critical site for its activity

[14].

Both caspase-12 in rodents and caspase-4 in human have

been shown to be predominantly located to the outer

membrane of the endoplasmic reticulum (ER), and to play

important roles in ER stress-induced apoptosis [13, 15, 16].

Activation of other caspases, including caspase-2, -3, -7,

-8, and -9, may also be involved in apoptosis induced by

ER stress [15, 17]. Although several recent studies have

challenged the role of caspase-12 and -4 [18, 19], we have

demonstrated that the pharmacological ER stress inducers

tunicamycin (TM) and thapsigargin (TG) induce apoptosis

in human melanoma cell lines by activation of caspase-4

providing the inhibitory effect of the ER chaperon glucose-

regulated protein 78 (GRP78) is removed [16]. Otherwise,

caspase-4 is bound to and inhibited by GRP78 that is

substantially induced by TM and TG [16]. Moreover,

GRP78 is also known to exert its anti-apoptotic effect in

cells under ER stress by other mechanisms, such as binding

to the unfolded proteins and/or calcium, thus alleviating

ER stress conditions [20, 21].

Release of caspase-4 from GRP78 itself is known not to

be sufficient to cause its activation in melanoma cells [16],

but the mechanism(s) directly responsible for activation of

caspase-4 remains unknown. It is clear however that acti-

vation of caspase-4 by TM and TG in melanoma cells is

independent of caspase-8, and occurs up-stream of the

mitochondrial apoptotic events and activation of caspase-9

and -3 [16]. In the murine system, several mechanisms

have been suggested to be responsible for ER stress-

induced caspase-12 activation [15, 22, 23]. For instance,

the protease calpain, upon activation by calcium released

from ER, can activate caspase-12 [22]. In addition, cas-

pase-12 has also been reported to be activated by a direct

association with the ER stress transducer IRE1a and the

adaptor protein TRAF2 [23].

Our previous studies have shown that the mitochondrial

apoptotic pathway plays an important role in TRAIL-

induced apoptosis of melanoma cells [7, 24, 25]. In the

present study, we have studied the potential involvement of

the ER and caspase-4 in TRAIL-induced apoptosis in

melanoma cell lines. We demonstrate in this report that

caspase-4 is involved in TRAIL-induced apoptosis of

melanoma cells. Moreover, we show that TRAIL-induced

activation of caspase-4 takes place downstream of caspase-

3, which in turn plays a role in enhancing caspase-3

activation.

Materials and methods

Cell lines

Human melanoma cell lines Mel-RM, MM200, IgR3, Mel-

CV, Me4405, Sk-Mel-28, and Me1007 have been described

previously [16, 25]. They were cultured in DMEM con-

taining 5% FCS (Commonwealth Serum Laboratories,

Melbourne, Australia).

Antibodies, recombinant proteins, and other reagents

Recombinant human TRAIL was supplied by Immunex

(Seattle, WA, USA). The preparation was supplied as a

leucine zipper fusion protein, which required no further

cross-linking for maximal activity. TM was purchased

from Sigma Chemical Co. (Castle Hill, Australia). It was

dissolved in DMSO and made up in a stock solution

of 1 mM. The cell-permeable general caspase inhibitor

Z-Val-Ala-Asp(OMe)-CH2F (z-VAD-fmk), the caspase-3

specific inhibitor Z-Asp(OMe)-Glu(OMe)Val-Asp(OMe)-

CH2F (z-DEVD-fmk), the caspase-9 specific inhibitor

Z-Leu-Glu(OMe)His-Asp(OMe)CH2F (z-LEHD-fmk),

and the caspase-8 specific inhibitor Z-lle-Glu(OMe)-

Thr-Asp(OMe)-CH2F (z-IETD-fmk) were purchased from

Calbiochem (La Jolla, CA). The caspase-4 specific inhib-

itor Z-Leu-Glu-Val-Asp-FMK (z-LEVD-fmk) was from

Bio-Vision (Mountain View, CA). The rabbit polyclonal

Abs against caspase-3, -8, -2, and -9 were from Stressgen

(Victoria, BC, Canada). The mouse MAb against caspase-4

was from Abcam (Cambridge, UK). The rabbit MAb

against GRP78 was purchased from Santa Cruz Biotech-

nology (Santa Cruz, CA). Isotype control Abs used were

the ID4.5 (mouse IgG2a) MAb against Salmonella typhi

supplied by Dr. Ashman (Institute for Medical, Veterinary

Science, Adelaide, Australia), the 107.3 mouse IgG1 MAb

purchased from PharMingen (San Diego, CA), and rabbit

IgG from Sigma Chemical Co. (Castle Hill, Australia).

Apoptosis

Quantitation of apoptotic cells by measurement of sub-G1

DNA content using the propidium iodide (PI) method was

carried out as described elsewhere [16, 25].

Caspase activity assay

Measurement of caspase activities by fluorometric assays

was performed as described previously [16]. The specific

substrates z-DEVD-AFC, Ac-LEVD-AFC, z-IETD-AFC,

and z-LEHD-AFC were used to measure caspase-3, -4, -8,

and -9 activities, respectively (Calbiochem, La Jolla, CA).

The generation of free AFC was determined using Fluostar

1212 Apoptosis (2010) 15:1211–1222

123

OPTIMA (LABTECH, Offenburg, Germany) set at an

excitation wavelength of 400 nm and an emission wave-

length of 505 nm.

Western blot analysis

Western blot analysis was carried out as described previ-

ously [16, 26]. Labeled 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, NSW, Australia).

Detection of XBP-1 mRNA splicing

The method used for detection of unspliced and spliced

XBP-1 mRNAs was as described previously [16, 27].

Briefly, RT-PCR products of XBP-1 mRNA were obtained

from total RNA extracted using primers 50-cggtgcgcg

gtgcgtagtctgga-30 (sense) and 50-tgaggggctgagaggtgcttc

ct-30 (anti-sense). Because a 26 bp fragment containing an

Apa-LI site is spliced upon activation of XBP-1 mRNA,

the RT-PCR products were digested with Apa-LI to dis-

tinguish 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.

Plasmid vector and transfection

Stable Mel-CV and MM200 transfectants of Bcl-2 were

established by electroporation of the pEF-puro vector car-

rying human Bcl-2 provided by Dr. David Vaux (Walter,

Eliza Hall Institute, Melbourne, Victoria, Australia) and

described elsewhere [28]. GRP78 cDNA cloned into

pcDNA3-Flag was provided by Dr. Amy Lee (Keck School

of Medicine of the University of Southern California, Los

Angeles, USA) and described elsewhere [29].

Small RNA interference (siRNA)

Melanoma cells were seeded at 4 9 104 cells/well in

24-well plates and allowed to reach approximately 50%

confluence on the day of transfection. The siRNA constructs

used were obtained as the siGENOME SMARTpool

reagents (Dharmacon, Lafayette, CO), the siGENOME

SMARTpool caspase-8 (M-003466-04), the siGENOME

SMARTpool caspase-3 (M-004307-03), siGENOME SMART-

pool caspase-9 (M-003309-00), the siGENOME SMARTpool

caspase-4 (M-004404-00), the siGENOME SMARTpool Bim

(M-008198-01), and the siGENOME SMARTpool GRP78

(M-008198-01). The non-targeting siRNA control, SiCon-

TRolNon-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 Lipofect-

amine reagent (Invitrogen, Carlsbad, CA) according to the

manufacturer’s transfection protocol. 24 h after transfec-

tion, the cells were switched into medium containing 5%

FCS and were treated as designed before quantitation of

apoptotic cells by measurement of sub-G1 DNA content

using the PI method in flow cytometry. Efficiency of siRNA

was measured by Western blot analysis.

Results

Inhibition of caspase-4 partially blocks TRAIL-induced

apoptosis of melanoma cells

Although caspase-4 is conventionally regarded as a caspase

that is mainly involved in regulation of the inflammatory

response [10, 11], recent studies have shown that it plays a

role in induction of apoptosis under various circumstances

[13, 15, 16]. We examined if caspase-4 is involved in

TRAIL-induced apoptosis in a panel of melanoma cell

lines by pre-treating the lines with the caspase-4 inhibitor

z-LEVD-fmk for 1 h before the addition of TRAIL. The

general caspase inhibitor z-VAD-fmk and the caspase-8

inhibitor z-IELD-fmk were included as controls. As shown

in Fig. 1a, while z-VAD-fmk and z-IELD-fmk inhibited

TRAIL-induced apoptosis by *80%, respectively, z-LEVD-

fmk decreased TRAIL-induced apoptosis by at least 30% in

most of the lines. The melanoma cell line ME1007 was

known to resist to apoptosis induced by TRAIL due to lack

of caspase-8 expression [24, 25]. The specificity of the

caspase-4 inhibitor z-LEVD-fmk was evidenced by its

failure to block staurosporine-induced activation of cas-

pase-8, -9, and -3 and apoptosis known to be independent

of caspase-4 [13, 16, 30] (Fig. 1b).

To confirm the results from studies with the caspase

inhibitors, we silenced caspase-4 and -8 by transfecting

specific siRNA pools for caspase-4 and caspase-8 into

MM200 and Mel-CV, two melanoma cell lines relatively

sensitive to TRAIL-induced apoptosis. Figure 1c shows

that caspase-4 siRNA significantly reduced the levels of

caspase-4 but not caspase-8 expression, whereas the cas-

pase-8 siRNA decreased the levels of caspase-8 but not

caspase-4 expression, suggesting that the siRNA pools

were relatively specific for individual caspases. The levels

of apoptosis induced by TRAIL were reduced by *80 and

30% in cells transfected with the caspase-8 and -4 siRNA,

respectively, in comparison with those transfected with the

control siRNA (Fig. 1d). The specific effect of caspase-4

knockdown on TRAIL-induced apoptosis was confirmed

by using separate caspase-4 siRNAs as described by

Apoptosis (2010) 15:1211–1222 1213

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previously (data not shown) [13]. These results indicate

that caspase-4 is involved in apoptotic signaling transduc-

tion induced by TRAIL. Consistently, caspase-4 was found

to be activated similar to caspase-8, -9, and -3 in melanoma

cells after treatment with TRAIL as evidenced by both

Western blot analysis and fluorometric assays detecting its

activity with a caspase-4 specific substrate (Fig. 1e) [25].

TRAIL induces ER stress in melanoma cells

We have previously shown that ER stress induces caspase-

4-mediated apoptosis in melanoma cells when the inhib-

itory effect of GRP78 is removed [16]. This led us

to test whether involvement of caspase-4 in TRAIL-

induced apoptosis is associated with induction of ER stress.

1214 Apoptosis (2010) 15:1211–1222

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We monitored the levels of the GRP78 protein and the

spliced XBP-1 mRNA (two indicators of activation of the

ER stress response) in melanoma cells after exposure to

TRAIL [31, 32]. The classic ER stress inducer TM was

used as a control. As shown in Fig. 2a, while TM-induced

marked up-regulation of these indicators, treatment with

TRAIL also resulted in, albeit to a lesser extent, increases

in GRP78 and spliced XBP-1 mRNA, indicative of

induction of ER stress by TRAIL.

GRP78 in melanoma cells is known to bind to caspase-4

and to inhibit its activation after exposure to TM [16]. To

test if GRP78 similarly inhibit TRAIL-induced caspase-4

activation, we transfected cDNA encoding GRP78 into

MM200 and Mel-CV cells. As shown in Fig. 2b, over-

expression of GRP78 blocked TRAIL-induced activation

of caspase-4. Consistently, TRAIL-induced apoptosis was

also partially inhibited in cells over-expressing GRP78

(Fig. 2c). The role of GRP78 in governing TRAIL-induced

caspase-4 activation was tested in MM200 and Mel-CV

cells transfected with a GRP78 siRNA pool as shown in

Fig. 2d. Inhibition of GRP78 by siRNA sensitized the cells

to TRAIL-induced apoptosis (Fig. 2c). Similarly, siRNA

knockdown of GRP78 resulted in increases in caspase-4

activation in both MM200 and Mel-CV cells (Fig. 2d).

Taken together, these results suggest that TRAIL induces

ER stress that may be associated with TRAIL-induced

activation of caspase-4.

TRAIL-induced activation of caspase-4 occurs later

than activation of caspase-8, -9, and -3

We studied the kinetics of activation of caspase-4 in relation

to activation of caspase-8, -9, and -3 induced by TRAIL.

Whole cell lysates from MM200 and Mel-CV treated with

TRAIL for varying periods (0, 10, 30, 60, and 120 min)

were subjected to Western blot analysis. Figure 3a shows

that while activation of caspase-8, -9, and -3 was detected at

10 min, activation of caspase-4 could not be observed until

30 min after treatment with TRAIL (Fig. 3a).

We also monitored the kinetics of activation of caspase-4,

-8, -9, and -3 in fluorometric assays using specific substrates

in whole cell lysates from Mel-CV cells. Similar to the

kinetics shown by Western blot analysis, the increases in

activities of caspase-8, -9, and -3 were observed as early as

10 min after exposure to TRAIL, but an increase in caspase-

4 activity could only be detected by 30 min after treatment

(Fig. 3b). It is of note that the levels of the processed form of

caspase-4 at 2 h appeared to be lower than those at 30 and

60 min after exposure to TRAIL. This was presumably due

to further cleavage of the processed form into even smaller

forms that could not be detected by the antibody used. Col-

lectively, these results indicate that activation of caspase-8,

-9, and -3 occurs prior to activation of caspase-4 induced by

TRAIL in melanoma cells. Moreover, they suggest that the

mechanism(s) by which TRAIL induces activation of cas-

pase-4 differs from that by the classic ER stress inducers TM

and TG when GRP78 is inhibited. The latter is known to take

place up-stream of caspase-3 and -9 [16].

Inhibition of caspase-8 blocks activation of caspase-4

induced by TRAIL

To study the mechanism(s) of activation of caspase-4

induced by TRAIL, we examined the relationship between

activation of caspase-4 and -8 by testing the effects of the

caspase-4 inhibitor z-LEVD-fmk and the caspase-8 inhib-

itor z-IELD-fmk on activation of the caspases in MM200

and Mel-CV cells. As shown in Fig. 4a, the caspase-8

inhibitor blocked activation of both caspase-8 and -4, but

the caspase-4 inhibitor did not block activation of caspase-

8, although it inhibited activation of caspase-4 induced by

TRAIL in both cell lines. Blockage of activation of cas-

pase-4 by inhibition of caspase-8 was also demonstrated by

measurement of caspase-4 activity in fluorometric assays in

Mel-CV cells with caspase-8 knocked down by siRNA

Fig. 1 Inhibition of caspase-4 partially blocks TRAIL-induced

apoptosis of melanoma cells. a The caspase-4 inhibitor z-LEVD-

fmk partially blocks TRAIL-induced apoptosis of melanoma cells.

Melanoma cells were treated with the general caspase inhibitor

z-VAD-fmk (30 lM), the caspase-4 inhibitor z-LEVD-fmk (30 lM),

or the caspase-8 inhibitor z-IELD-fmk (20 lM) 1 h before the

addition of TRAIL (200 ng/ml) for a further 24 h. Apoptosis was

measured by the PI method using flow cytometry. The data shown are

the mean ± SE of three individual experiments. b The general

caspase inhibitor z-VAD-fmk but not the caspase-4 inhibitor

z-LEVD-fmk inhibits staurosporine-induced activation of caspase-8,

-9, and -3. IgR3 melanoma cells were treated with z-VAD-fmk

(30 lM) or z-LEVD-fmk (30 lM) 1 h before the addition of

staurosporine (1 lM) for a further 24 h. Whole cell lysates were then

subjected to Western blot analysis. The data shown are representative

of three individual experiments. c Inhibition of caspase-8 (left panel)and -4 (right panel) by siRNA. MM200 and Mel-CV cells were

transfected with either the control siRNA, the caspase-8 siRNA (leftpanel), or the caspase-4 siRNA (right panel). 24 h later, whole cell

lysates were subjected to Western blot analysis. The arrow-pointedbands in figures for caspase-4 are presumably either non-specific

bands or intermediately cleaved caspase-4. The data shown are

representative of three individual experiments. d siRNA knockdown

of caspase-4 partially inhibited TRAIL-induced apoptosis of mela-

noma cells. MM200 and Mel-CV cells were transfected with either the

control siRNA or the caspase-8 or caspase-4 siRNA. 24 h later, cells

were treated with TRAIL (200 ng/ml) for a further 24 h. Apoptosis

was measured by the PI method using flow cytometry. The data shown

are the mean ± SE of three individual experiments. e TRAIL induces

activation of caspase-4. Whole cell lysates from MM200 and Mel-CV

cells treated with TRAIL (200 ng/ml) for 3 h were subjected to either

Western blot analysis of caspase-4 (upper panel) or measurement of

caspase-4 activity with a specific substrate in fluorometric assays

(lower panel). The values of activity in the cells without treatment

were arbitrarily designated as 1. The data shown are representative of

three individual experiments (upper panel) or the mean ± SE of three

individual experiments (lower panel)

b

Apoptosis (2010) 15:1211–1222 1215

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(Fig. 4b). In contrast, inhibition of caspase-4 by siRNA did

not reduce caspase-8 activity induced by TRAIL (Fig. 4b).

These results suggest that activation of caspase-4 is,

directly or indirectly, dependent on activation of caspase-8.

Over-expression of Bcl-2 inhibits TRAIL-induced

activation of caspase-4 in melanoma cells

The mitochondrial apoptotic pathway is known to play a

critical role in TRAIL-induced apoptosis of melanoma cells.

To further study the mechanism by which TRAIL induces

activation of caspase-4 in melanoma cells, we examined the

effect of Bcl-2 on activation of caspase-4 induced by TRAIL

in MM200 and Mel-CV that had been stably transfected with

cDNA encoding Bcl-2. As shown in Fig. 5a and b, Over-

expression of Bcl-2 did not inhibit activation of caspase-8, but

markedly blocked activation of caspase-4 in both cell lines.

Over-expression of Bcl-2 is known to inhibit TRAIL-induced

activation of caspase-9 and -3 in melanoma cells [24, 25].

To confirm that caspase-4 is activated by TRAIL

downstream of mitochondria in melanoma cells, we tested

the effect of activation of caspase-9 on activation of cas-

pase-4 induced by TRAIL in Mel-CV cells. Figure 5c

shows that the caspase-9 inhibitor z-LEHD-fmk blocked

activation of caspase-4, but the caspase-4 inhibitor

z-LEVD-fmk had no notable effect on TRAIL-induced

activation of caspase-9. Similarly, transfection of a cas-

pase-9 siRNA pool efficiently reduced caspase-4 activity,

but the caspase-4 siRNA did not decrease caspase-9

activity induced by TRAIL (Fig. 5d). Together, these

results suggest that activation of caspase-4 is a post-mito-

chondrial event in TRAIL-induced apoptosis of melanoma

cells.

Inhibition of caspase-3 blocks activation of caspase-4,

whereas inhibition of caspase-4 attenuates activation

of caspase-3 induced by TRAIL

We examined the relationship between TRAIL-induced

activation of caspase-3 and -4 in MM200 and Mel-CV cells

with the caspase-4 inhibitor z-LEVD-fmk and the caspase-

3 inhibitor z-DEVD-fmk. Western blot analysis showed

that while the caspase-3 inhibitor blocked activation of

caspase-4, the caspase-4 inhibitor also blocked, albeit only

partially, activation of caspase-3 induced by TRAIL

(Fig. 6a). We also tested the effects of siRNA knockdown

of caspase-4 and -3 on activation of the caspases in

MM200 and Mel-CV cells, respectively in fluorometric

caspase activity assays. Figure 6b shows that inhibition of

caspase-3 by siRNA reduced TRAIL-induced caspase-4

activity by *85%, whereas siRNA inhibition of caspase-4

decreased caspase-3 activity by *30% in both cell lines.

Therefore, TRAIL-induced activation of caspase-4 is not

only dependent on activation of caspase-3, but also nec-

essary for optimal activation of caspase-3.

To further evaluate if caspase-3-mediated activation of

caspase-4 is specific to TRAIL-induced apoptosis, we tested

whether other apoptotic stimuli may also induce caspase-4

activation downstream of caspase-3. The agents tested were

the chemotherapeutic drug cisplatin, the microtubulin-tar-

geting drug vincristine, and the broad kinase inhibitor

staurosporine, all of which are known to activate caspase-3

[30, 33, 34]. Figure 6c shows that, similar to TRAIL, cis-

platin-induced activation of caspase-4. In contrast, little or

no caspase-4 activity could be detected in cells after treat-

ment with vincristine or staurosporine. As shown in Fig. 6d,

siRNA knockdown of caspase-3 partially inhibited activa-

tion of caspase-4 induced by cisplatin, whereas inhibition of

caspase-4 by siRNA also partially inhibited cisplatin-

induced activation of caspase-3. Of note, cisplatin, but not

vincristine or staurosporine, was found to induce ER stress,

as evidenced by up-regulation of the GRP78 protein and the

spliced XBP-1 mRNA to moderate levels as does by TRAIL

(data not shown). These results suggest that activation of

caspase-4 by caspase-3 may be a common mechanism in

apoptosis mediated by agents that can induce ER stress, but

do not elicit strong induction of GRP78.

Fig. 2 TRAIL induces ER stress in melanoma cells. a TRAIL up-

regulates GRP78 (upper panel) and activates XBP-1 (lower panel) in

melanoma cells. Upper panel whole cell lysates from MM200 and

Mel-CV cells treated with TRAIL (200 ng/ml) and TM (3 lM),

respectively, for indicated periods were subjected to Western blot

analysis. Lower panel RT-PCR products of XBP-1 mRNA from

MM200 and Mel-CV cells treated with TRAIL (200 ng/ml) and TM

(3 lM), respectively, for indicated periods were digested with Apa-LI

for 90 min followed by electrophoresis. The longer fragment derived

from the active form of XBP-1 mRNA and two shorter bands derived

from the inactive form are indicated. The data shown are represen-

tative of three individual experiments. b Over-expression of GRP78

partially blocks TRAIL-induced activation of caspase-4. Upper panelwhole cell lysates from MM200 and Mel-CV cells transfected with

cDNA encoding GRP78 or vector alone were subjected to Western

blot analysis. Lower panel MM200 and Mel-CV cells transfected with

cdna encoding GRP78 or vector alone were treated with TRAIL

(200 ng/ml) for 3 h. Whole cell lysates were subjected to Western

blot analysis. The data shown are representative of three individual

experiments. c Left panel MM200 and Mel-CV cells transfected with

either the cDNA encoding GRP78 or vector alone as shown in Fig. 2b

were treated with TRAIL (200 ng/ml) for 24 h before apoptosis was

measured by the PI method. Right panel MM200 and Mel-CV cells

transfected with either the GRP78 or the control siRNA as shown in

figure. d Inhibition of GRP78 by siRNA enhances TRAIL-induced

activation of caspase-4 in melanoma cells. Upper panel MM200 and

Mel-CV cells were transfected with either the control or GRP78

siRNA. 24 h later, cells were treated with TRAIL (200 ng/ml) for a

further 6 h. Whole cell lysates were subjected to Western blot

analysis. Lower panel MM200 and Mel-CV cells were transfected

with either the control or GRP78 siRNA. 24 h later, cells were treated

with TRAIL (200 ng/ml) for a further 3 h. Whole cell lysates were

then subjected to Western blot analysis. The data shown are

representative of three individual experiments

c

1216 Apoptosis (2010) 15:1211–1222

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Discussion

The above results demonstrate that activation of caspase-4,

a predominantly ER localized caspase [13, 16], is required

for optimal induction of apoptosis by TRAIL in human

melanoma cells. They also show that activation of caspase-

4 by TRAIL is associated with induction of moderate levels

of ER stress but the mechanism of activation of caspase-4

induced by TRAIL differs from that induced by the

classic ER stress inducers TM and TG. The latter activate

Apoptosis (2010) 15:1211–1222 1217

123

caspase-4 independently of other caspases including cas-

pase-8, -9, and -3 [16] but TRAIL-induced activation of

caspase-4 is mediated by signaling downstream of activa-

tion of caspase-3.

Caspase-4 belongs to the interleukin-1-converting

enzyme family of proteins that were believed to be mainly

involved in cytokine maturation and inflammation [10, 11].

However, recent studies have shown that it plays an

important role in induction of apoptosis by ER stress in

various cell types [13, 15, 16]. We have also demonstrated

that, providing the inhibitory effect of GRP78 is removed,

caspase-4 can potently induce apoptosis in melanoma cells

undergoing ER stress [16]. Moreover, caspase-4 activity

has been shown to participate in induction of apoptosis by

Fas in Hela cells [12], and by TRAIL in human rheumatoid

arthritis synovial fibroblasts [35]. In the present study, both

the caspase-4 inhibitor z-LEVD-fmk and knockdown of

caspase-4 produced partial but significant inhibition of

Fig. 4 Inhibition of caspase-8 blocks TRAIL-induced activation of

caspase-4. a The caspase-8 inhhibitor z-IETD-fmk blocks TRAIL-

induced activation of caspase-4. MM200 and Mel-CV cells were

treated with the caspase-8 inhibitor z-IETD-fmk (30 lM) and the

caspase-4 inhibitor z-LEVD-fmk (20 lM) for 1 h before the addition

of TRAIL (200 ng/ml) for a further 3 h. Whole cell lysates were then

subject to Western blot analysis. The data shown are representative of

three individual experiments. b siRNA knockdown of caspase-8

reduced caspase-4 activity induced by TRAIL. Mel-CV cells were

transfected with either the control siRNA, the caspase-8 siRNA, or

the caspase-4 siRNA as shown in Fig. 1b. 24 h later, cells were

treated with TRAIL for a further 3 h. Whole cell lysates were

harvested and caspase activities were measured with specific

substrates for caspase-8 and -4 in fluorometric assays. The values

of activity in the cells without treatment were arbitrarily designated as

1. The values of activity in cells treated with TRAIL were compared

with those in cells without treatment, and are expressed as the fold

increase. The data shown are the mean ± SE of three individual

experiments

Fig. 3 TRAIL-induced activation of caspase-4 occurs later than

activation of caspase-8, -9, and -3. a Whole cell lysates from MM200

and Mel-CV cells treated with TRAIL (200 ng/ml) for indicated

periods were subjected to Western blot analysis. The data shown are

representative of three individual experiments. b Mel-CV cells were

treated with TRAIL for indicated periods. Whole cell lysates were

harvested and caspase activities were measured with specific

substrates for caspase-8, -3, -4, and -9, respectively, in fluorometric

assays. The values of activity in the cells without treatment were

arbitrarily designated as 1. The values of activity in cells treated with

TRAIL were compared with those in cells without treatment, and are

expressed as the fold increase. The data shown are the mean ± SE of

three individual experiments

1218 Apoptosis (2010) 15:1211–1222

123

TRAIL-induced apoptosis of the melanoma cells, indicat-

ing that caspase-4 is involved in apoptotic signaling

transduction induced by TRAIL, and is necessary for

maximal induction of apoptosis by TRAIL in human

melanoma cells.

Activation of caspase-4 by TRAIL suggests that TRAIL

may impinge on ER stress, as caspase-4 is primarily

located to the exterior membrane of the ER [13]. Induction

of ER stress by TRAIL was shown by up-regulation of the

GRP78 protein and the spliced XBP-1 mRNA, both of

which are indicators of the ER stress response [30, 31].

In previous studies on ER stress-induced apoptosis it was

found necessary to inhibit GRP78 before caspase-4-

induced apoptosis could be detected [16]. This was not

necessary in TRAIL-induced apoptosis most likely due to

the markedly lower levels of GRP78 induced by TRAIL

compared to those induced by TM or TG. In support of

this, over-expression of GRP78 inhibited, whereas siRNA

knockdown of GRP78 enhanced, TRAIL-induced activa-

tion of caspase-4 in melanoma cells. These results are of

Fig. 5 Activation of caspase-4 occurs downstream of mitochondria.

a Over-expression of Bcl-2 in MM200 and Mel-CV cells. Whole cell

lysates from MM200 and Mel-CV cells transfected with cDNA

encoding Bcl-2 or vector alone were subjected to Western blot

analysis. The data shown are representative of three individual

experiments. b Over-expression of Bcl-2 inhibits TRAIL induces

activation of caspase-4. MM200 and Mel-CV cells transfected with

cdna encoding Bcl-2 or vector alone were treated with TRAIL

(200 ng/ml) for 3 h were subjected to Western blot analysis. The data

shown are representative of three individual experiments. c The

caspase-9 inhibitor z-LEHD-fmk blocked activation of caspase-4.

Mel-CV cells were treated with the caspase-9 inhibitor z-LEHD-fmk

(30 lM) or the caspase-4 inhibitior z-LEVD-fmk (20 lM) for 1 h

before the addition of TRAIL (200 ng/ml) for a further 3 h. Whole

cell lysates were then subject to Western blot analysis. The data

shown are representative of three individual experiments. d siRNA

knockdown of caspase-9 blocks TRAIL-induced activation of

caspase-4. Upper panel Mel-CV cells were transfected with either

the control siRNA or the caspase-9 siRNA. 24 h later, whole cell

lysates were subjected to Western blot analysis. Lower panel Mel-CV

cells were transfected with either the control siRNA or the caspase-9

siRNA. 24 h later, cells were treated with TRAIL (200 ng/ml) for a

further 3 h. Whole cell lysates were harvested and caspase activities

were measured with specific substrates for caspase-9 and -4 in

fluorometric assays. The values of activity in the cells without

treatment were arbitrarily designated as 1. The values of activity in

cells treated with TRAIL were compared with those in cells without

treatment, and are expressed as the fold increase. The data shown are

other the representative of three individual experiments (upper panel)or the mean ± SE of three individual experiments (lower panel)

Apoptosis (2010) 15:1211–1222 1219

123

particular interest in that they suggest that targeting

GRP78 may be useful in sensitizing melanoma cells to

TRAIL-induced apoptosis. Notably, it has been recently

reported that interaction of extracellular prostate apoptosis

response-4 (Par-4) and cell surface GRP78 is necessary for

TRAIL-induced apoptotic signaling in other cell types [36].

Although the role of Par-4 in TRAIL-induced apoptosis of

melanoma cells remains to be established, it is conceivable

that GRP78 may have dual effects on TRAIL-induced

apoptosis. When it is located to the ER membrane, it

antagonizes TRAIL death signaling, but when it is

expressed on the cell surface, it facilitates TRAIL death

signaling transduction.

Activation of caspase-4 by TM and TG is known to be

independent of activation of caspase-8 and is up-stream of

activation of caspase-9 and -3 [16]. However, activation of

caspase-4 by TRAIL took place downstream of activation

of the initiator caspases, caspase-8 and -9, and the effector

caspase, caspase-3. This was demonstrated by the kinetics

of activation of the caspases. In addition, inhibition of

caspase-8, -9, and -3 respectively by specific inhibitors and

siRNA blocked TRAIL-induced activation of caspase-4.

Moreover, over-expression of Bcl-2 inhibited activation of

caspase-4 along with caspase-9 and -3 by TRAIL. These

observations indicated that activation of caspase-4 was

downstream of caspase-3. Activated caspase-3 is known to

1220 Apoptosis (2010) 15:1211–1222

123

enhance activation of caspase-8 and -9 in a positive feed-

back fashion [37, 38]. Caspase-3 was also reported to

directly activate caspase-2 in TRAIL-induced apoptosis

[8]. It is therefore conceivable that activation of caspase-4

induced by TRAIL in melanoma cells may be a direct

consequence of activation of caspase-3.

Intriguingly, activation of caspase-3 did not appear to

result in activation of caspase-4 in all scenarios. We found

that although caspase-3 was activated by both vincristine

and staurosporine, activation of caspase-4 was not induced

by these compounds. In contrast, activation of caspase-3

induced by cisplatin resulted in caspase-4 activation in

melanoma cells. Similar to TRAIL, cisplatin also induced

moderate levels of ER stress, but the levels of GRP78 after

treatment with cisplatin were also substantially lower than

those induced by TM (data not shown). It seems that during

high levels of ER stress-induced by TM or TG, apoptosis is

inhibited by GRP78 binding of caspase-4 but when GRP78

is inhibited, caspase-4 is activated by an ER stress-induced

factor(s). When ER stress is at low levels as seen after

exposure to TRAIL or cisplatin, release of caspase-4 from

GRP78 is not sufficient by itself to cause activation of

caspase-4 but exposes caspase-4 to activation by activated

caspase-3 [16]. In the absence of ER stress, caspase-4 may

not be exposed to activated caspase-3 as in the case of

treatment with vincristine and staurosporine.

Despite the need for activated caspase-3 to activate

caspase-4, inhibition of caspase-4 by the specific inhibitor

z-LEVD-fmk and siRNA partially blocked caspase-3 acti-

vation induced by TRAIL. These results indicate that

caspase-4 is another caspase (in addition to caspase-8 and

-9) that contributes to activation of caspase-3 in TRAIL-

induced apoptosis of melanoma cells [24, 25]. In support of

this, caspase-4 has been shown to activate caspase-3

directly in in vitro assays [12]. To our knowledge, these

studies are the first to show that the ER is another intra-

cellular organelle (in addition to the mitochondrion, prob-

ably the lysosome) that is involved in TRAIL-induced

apoptosis in melanoma cells. In addition, the results may

have important implications in treatment of melanoma with

TRAIL in that co-treatment with agents that facilitate the

ER stress-induced apoptotic pathway could be expected to

increase cell death induced by TRAIL. We have shown

previously that high levels of ER stress-induced by TM or

TG up-regulates the expression of TRAIL receptor-2

(TRAIL-R2) which are down-regulated in most melanoma

[38, 39]. These dual effects could be expected to enhance

the effectiveness of TRAIL.

Acknowledgments This work was supported by the NSW State

Cancer Council, the Melanoma and Skin Cancer Research Institute

Sydney, the Hunter Melanoma Foundation, NSW, and the National

Health and Medical Research Council, Australia. X. D. Zhang is a

Cancer Institute NSW Fellow.

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