Increased level of exogenous zinc induces cytotoxicity and up-regulates the expression of the ZnT-1 zinc transporter gene in pancreatic cancer cells.

Abstract

A balance between zinc uptake by ZIP (SLC39) and efflux of zinc from the cytoplasm into subcellular organelles and out of the cell by ZnT (SLC30) transporters is crucial for zinc homeostasis. It is not clear whether normal and cancerous pancreatic cells respond differently to increased extracellular zinc concentrations. Use of flow cytometry-based methods revealed that treatment with as little as 0.01 mM zinc induced significant cytotoxicity in two human ductal adenocarcinoma cell lines. In contrast, normal human pancreatic islet cells tolerated as high as 0.5 mM zinc. Insulinoma cell lines of mouse and rat origin also succumbed to high concentrations of zinc. Exposure to elevated zinc concentrations enhanced the numbers of carcinoma but not primary islet cells staining with the cell-permeable zinc-specific fluorescent dye, FluoZin-3, indicating increased zinc influx in transformed cells. Mitochondrial membrane depolarization, superoxide generation, decreased antioxidant thiols, intracellular acidosis and activation of intracellular caspases characterized zinc-induced carcinoma cell death. Only the antioxidant glutathione but not inhibitors of enzymes implicated in apoptosis or necrosis prevented zinc-induced cytotoxicity in insulinoma cells. Immunoblotting revealed that zinc treatment increased the ubiquitination of proteins in cancer cells. Importantly, zinc treatment up-regulated the expression of ZnT-1 gene in a rat insulinoma cell line and in two human ductal adenocarcinoma cell lines. These results indicate that the exposure of pancreatic cancer cells to elevated extracellular zinc concentrations can lead to cytotoxic cell death characterized by increased protein ubiquitination and up-regulation of the zinc transporter ZnT-1 gene expression.

Full text

Page 1

Increased level of exogenous zinc induces cytotoxicity and up-regulates the
expression of the ZnT-1 zinc transporter gene in pancreatic cancer cells☆
Arathi K. Jayaraman, Sundararajan Jayaraman⁎
Department of Surgery, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612 USA
Received 22 August 2009; received in revised form 30 November 2009; accepted 3 December 2009
Abstract
A balance between zinc uptake by ZIP (SLC39) and efflux of zinc from the cytoplasm into subcellular organelles and out of the cell by ZnT (SLC30)
transporters is crucial for zinc homeostasis. It is not clear whether normal and cancerous pancreatic cells respond differently to increased extracellular zinc
concentrations. Use of flow cytometry-based methods revealed that treatment with as little as 0.01 mM zinc induced significant cytotoxicity in two human ductal
adenocarcinoma cell lines. In contrast, normal human pancreatic islet cells tolerated as high as 0.5 mM zinc. Insulinoma cell lines of mouse and rat origin also
succumbed to high concentrations of zinc. Exposure to elevated zinc concentrations enhanced the numbers of carcinoma but not primary islet cells staining with
the cell-permeable zinc-specific fluorescent dye, FluoZin-3, indicating increased zinc influx in transformed cells. Mitochondrial membrane depolarization,
superoxide generation, decreased antioxidant thiols, intracellular acidosis and activation of intracellular caspases characterized zinc-induced carcinoma cell
death. Only the antioxidant glutathione but not inhibitors of enzymes implicated in apoptosis or necrosis prevented zinc-induced cytotoxicity in insulinoma cells.
Immunoblotting revealed that zinc treatment increased the ubiquitination of proteins in cancer cells. Importantly, zinc treatment up-regulated the expression of
ZnT-1 gene in a rat insulinoma cell line and in two human ductal adenocarcinoma cell lines. These results indicate that the exposure of pancreatic cancer cells to
elevated extracellular zinc concentrations can lead to cytotoxic cell death characterized by increased protein ubiquitination and up-regulation of the zinc
transporter ZnT-1 gene expression.
© 2011 Elsevier Inc. All rights reserved.
Keywords: Cytotoxicity; Islets; Mitochondria; Pancreatic carcinoma; Zinc transporters
1. Introduction
Pancreatic canceris the fifth commonmost causeof cancer-related
deaths in western countries and accounts for about 3% of all cancers
[1]. In some cases, pancreatic cancer is also associated with pancreatic
insufficiency-diabetes [2]. Only 15% to 20% of pancreatic cancers can
be surgically removed when diagnosed at advanced or metastatic
stage, and chemotherapeutic agents gemcitabine and 5-fluorouracil
provide benefits only to some patients [3]. A number of clinical trials
including the use of signal transduction inhibitors and ablation of the
hormonal influence produced marginal effects when combined with
the standard cytostatic drugs [1,3]. However, some of these combina-
tions displayed severe and sometimes fatal toxicity. Thus, pancreatic
carcinomas still remain a great challenge for the oncologists, and the
continuoussearchforbettertreatmentmodalitiesishighlywarranted.
Zinc, normally found in cells, is important for the regulation of
gene expression, protein synthesis and intracellular protein traffick-
ing [4].In insulin-producingβ cells,large amountsofzincare required
for insulin synthesis and storage [5], and zinc is dissociated from
insulin after exocytosis [6]. High concentrations of zinc appear to
modulate the function of neighboring glucagon-producing α cells [7].
Although exposure to extracellular zinc has been shown to induce
necrosis in the mouse insulinoma cell line, Min6 [8,9], the underlying
mechanisms have not been fully elucidated. Zinc homeostasis is
achieved by two groups of proteins with opposing functions, solute-
linked carrier genes, SLC39 (ZIP- Zrt- and Irt-like proteins) and SLC30
(ZnT-Zinc transporters) [10–12]. ZIP family consists of 14 members
that allow the influx of extracellular zinc and transport of zinc from
the lumen of subcellular organelles into the cytoplasm. In insulinoma
cell lines, zinc uptake is predominantly mediated by the L-type Ca2+
channels [13,14]. The cation diffusion facilitators have been termed as
ZnTs and consist of 10 members implicated in reducing cytosolic zinc
concentrations, either through efflux across the plasma membrane or
through sequestration into intracellular compartments such as Golgi
and endoplasmic reticulum [10–12]. ZnT-1 is ubiquitously expressed
on the plasma membrane of a variety of cells including rat pancreatic
islets [10–12,15]. In the mouse, ZnT-1 was reported to be present in
pancreatic α but not β cells [16]. ZnT-2 has a vesicular localization in
pancreatic acinar cells but not in pancreatic β cells [16]. Although
ZnT-4 is abundantly expressed in the mammary gland and important
for zinc transport into milk, it is also expressed in the brain, small
intestine and pancreatic islets [10,15]. While ZnT-5 is present in a
Available online at www.sciencedirect.com
Journal of Nutritional Biochemistry 22 (2011) 79–88
☆This work was in part supported by the Department of Surgery, UIC.
⁎Corresponding author. Tel.: +1 312 355 5133; fax: +1 312 355 1497.
E-mail address: anue2468@uic.edu (S. Jayaraman).
0955-2863/$ – see front matter © 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jnutbio.2009.12.001

Page 2

number of tissues, it is abundantly expressed in human pancreatic β
cells but not in glucagon-producing α cells or the exocrine acinar cells
[17]. ZnT-8 was first described to be located in insulin secretory
granules of pancreatic β cells [18]. ZnT-8 was also suggested to be an
autoantigenin type 1 diabetes [19]. However,recent studies indicated
the presence of ZnT-8 in α cells and other secretory cell types [20], as
wellasin peripheralbloodlymphocytes [21].The expression ofZnT-3,
ZnT-6 and ZnT-7 has not been reported in the pancreas [10–12].
In contrast to the distribution of ZnTs in normal pancreatic tissues,
little is known about their expression in pancreatic cancers. Impor-
tantly,itisnotknownwhetherpancreaticcancercellsdisplayaberrant
zinc homeostasis unlike normal pancreatic cells, such as β cells. A
previous report indicated that clinical pancreatic adenocarcinoma
specimens overexpressed the zinc influx transporter, ZIP-4, suggest-
ing that this may lead to enhanced zinc uptake in cancer cells and
possibly contribute to carcinogenesis [22]. However, the conse-
quences of altered zinc homeostasis in pancreatic cancer cells with
referencetothemodulationofZnTsthatfacilitatetheeffluxofzincout
of the cell and sequestration into subcellular organelles have not been
reported. Our study reveals previously undocumented characteristics
of pancreatic cancer cells, up-regulation of selected ZnT genes and
induction of cell death by cytotoxic mechanisms in response to a
challenge with zinc. These results suggest that the ZnT transporter(s)
may serve as a molecular target for pancreatic cancer therapy.
2. Methods and materials
2.1. Cells
NIT-1 cells (ATCC) were grown in F-12K medium, while RINm5F (provided by
Louis Philipson, University of Chicago), CD18 and S2013 (obtained from David
Benstrem, Northwestern University) and L929 cells were cultured in RPMI medium
(Invitrogen) substituted with 10% fetal bovine serum [23]. Islets from cadaveric human
pancreata (n=8) were isolated at the University of Pennsylvania, Washington
University in St. Louis and the City of Hope, Duarte. Isolated islets were made available
for this study as a part of the ICR Basic Science Islet Distribution Program. Islets were
cultured at 37°C in CMRL 1066 medium (Mediatech) supplemented with antibiotics,
nicotinamide, insulin and 0.25% human serum albumin as described earlier [24].
2.2. Cell treatment
Adherent cell lines were dislodged from the culture flasks by treating with 0.05%
trypsin–EDTA for 5 min at 37°C [24], and 1×106cells/ml were resuspended in complete
medium and treated with zinc chloride (Sigma) that was dissolved in plain RPMI
medium to minimize sequestration of zinc by serum proteins. Islets were treated with
zinc chloride overnight and then dispersed by trypsin treatment and vice versa in some
experiments. L-Glutathione (GSH, Sigma) and L-glutathione monoethyl ester, GSH-
MEE (Calbiochem), were dissolved, respectively, in plain RPMI medium and dimethyl
sulfoxide (DMSO). The following inhibitors were dissolved at 10 mM concentrations in
DMSO: Q-VD-OPH (R&D Systems), necrostatin-1, SB203580, SP600125 and U0126 (all
from Biomol International). NIT-1 cells were incubated with 10 μM final concentrations
of inhibitors for 45 min prior to treatment with zinc chloride. N-Acetyl cysteine (NAC,
Sigma) was dissolved in plain RPMI medium and used at concentrations indicated in
the text. In some experiments, cells were incubated with GSH or GSH-MEE for 45 min
at 37°C, centrifuged and resuspended in complete media and incubated further with or
without zinc chloride overnight.
2.3. Flow cytometry
Viability was determined by incubating with 50 nM tetramethylrhodamine ethyl
ester (TMRE, Invitrogen) and 100 μM monochlorobimane (mBcl, Invitrogen) at 37°C for
30 min as described [23,24]. Islet cells were also incubated with 1 μM FluoZin-3-AM
(Invitrogen) and analyzed by flow cytometry [24]. Activation of caspases was
determined by staining with 10 μM FITC-VAD-FMK (Promega, Madison, WI) and
propidium iodide (PI) as described before [25]. Cells were also incubated with 10 μM
MitoSox Red (Invitrogen) for 30 min at 37°C or with 5 μM SNARF-1 (carboxy-
seminaphthorhodafluor-1, Invitrogen) for 20 min at 37°C [26]. Cells were analyzed on a
BD LSR (Becton-Dickinson) or a CyAn ADP (DakoCytomation) flow cytometer, and
10,000 events were collected and analyzed using FlowJo 6.3.4 software (Treestar).
2.4. Western blotting
Human pancreatic cancer cells were cultured in media or treated with 0.25 mM
zinc chloride for 4 h at 37°C. Total cell lysates were prepared and 10 μg of proteins were
separated on a 12% sodium dodecyl sulfate (SDS) polyacrylamide gel, transferred to a
polyvinylidene fluoride (PVDF) membrane and probed with rabbit antisera against Bcl-
XL, total ERK-1/2, caspase-9 poly ADP ribose polymerase (PARP) (all from Santa Cruz)
or phosphorylated ERK-1/2 (Cell Signaling). Monoclonal antibodies against C-Abl
(Oncogene Research Products), XIAP (MBL), Cbl-b and ubiquitin (Santa Cruz) as well as
goat antiserum against β actin (Santa Cruz) were used for blotting. Immunoblots were
developed using the ECL Plus system (GE Health Care).
2.5. Real-time real-time reverse transcription-polymerase chain reaction
(RT-PCR) analysis
Total RNA was isolated using TRIzol (Invitrogen) and treated with DNase either
using SV Total RNA Isolation System (Promega) or TURBO DNA-free kit (Applied
Biosystems). TotalRNA isolatedfrom rat pancreaswasobtainedfrom BioChain Institute
(Hayward, CA). DNase-treated RNA (5–10 μg) was reverse transcribed into cDNA using
High-Capacity cDNA Reverse Transcription kit (Applied Biosytems). Real-time quanti-
tative RT-PCR was performed on an ABA Prism 7500 Real-Time polymerase chain
reaction(PCR) system(Applied Biosystems)using 1μlof cDNAequivalentto250 to500
ng of RNA using 2× SYBR Premix Ex Taq (Perfect Real Time) reagent (Takara-Clontech).
AllratzntandhumanGAPDHprimersweredesignedusingPRIMEREXPRESSversion2.0
software (Applied Biosciences). The primers for human ZNT1, ZNT5 and ZNT7 were
synthesized as described earlier [27]. All primers were synthesized at Integrated DNA
Technologies (Coralville, IA) except that the rat znt4 primers were synthesized at
Invitrogen and used at 0.4-μM concentrations. Primer sequences are shown in Table 1.
Relative quantitation for all assays used GAPDH as the normalizer. Quantitation of gene
expression in comparison to GAPDH and fold change in gene expression due to zinc
treatment were calculated using the comparative threshold cycle method [28].
2.6. Statistical analysis
The statistical analysis was performed by unpaired, two-tailed Student's t
test between the groups using GraphPad Prism 4.0c software.
3. Results
3.1. Pancreatic cancer cells are vulnerable to extracellular zinc
Previous studies indicated that the mouse insulinoma cell line,
Min6, underwent necrosis when exposed to high concentrations of
zinc [8–9]. However, it is not clear whether this is unique to mouse
insulinoma or other pancreatic cancer cells from different phylogenic
origins can also undergo cell death in response to increases in
extracellular zinc. To address this possibility, pancreatic cancer cells
derived from mouse, rat and human were treated with 0.01 to 0.5 mM
zinc concentrations, considered to be physiological in mammalian
cells [11]. After overnight culture, viability was determined by
incubating cells with the mitochondrial membrane potential indica-
tor, TMRE [23] and mBcl, which binds to intracellular antioxidant
thiols [24]. Dead cells and debris were excluded based on forward
Table 1
Sequences of the primers used in quantitative real-time RT-PCR assays
Gene nameSequence
Rat znt1
Forward 5′-ATC CAG CCT GAA TTC GCT AGC-3′
Reverse 5′-CGT GTC CCA CAG CAC TGC T-3′
Forward 5′-GAC CCC ATC TGC ACC TTC CT-3′
Reverse 5′-CCT TTG GGA GTC CCT TCC AT-3′
Forward 5′-GAT CGG AGA GCT TGT AGG TGG ATA C-3′
Reverse 5′-AAC ATG GTG TCC CCT TTG ATC TC-3′
Forward 5′-TGG CTA AGA TGG CCG AAC AC-3′
Reverse 5′-CCA GGA AGG CGA TAG CTG TAT AAA-3′
Forward 5′-CTT GAG AGG ACT TAC CTT GTG-3′
Reverse 5′-GTG AAG GCA TAC ATC TTG GTG-3′
Forward 5′-TGA TGC TGG TGC TGA GTA TGT CGT-3′
Reverse 5′-TTG TCA TTG AGA GCA ATG CCA GCC-3′
Forward 5′-GAG ATG CCT TGG GTT CAG TGA TTG-3′
Reverse 5′-GGT CAG GGA AAC ATG GAT TCA CAC-3′
Forward 5′-GGA GGC ATG AAT GCT AAC ATG AGG-3′
Reverse 5′-GTG GAT ACG ATC ACA CCA ATG CTG-3′
Forward 5′-TTT CTT CCT GTG CCT GAA CCT CTC-3′
Reverse 5′-GAG TCG GAA ATC AAG CCT AAG CAG-3′
Forward 5′-TTG CCA TCA ATG ACC CCT TCA-3′
Reverse 5′-CGC CCC ACT TGA TTT TGG A-3′
Rat znt2
Rat znt4
Rat znt5
Rat znt8
Rat gapdh
Human ZNT1
Human ZNT5
Human ZNT7
Human GAPDH
80
A.K. Jayaraman, S. Jayaraman / Journal of Nutritional Biochemistry 22 (2011) 79–88

Page 3

angle and side scatter light properties, and intact cells were
electronically gated (Fig. 1A) and analyzed for the retention of both
TMRE and mBcl (Fig. 1B). When the commonly used mouse
insulinoma cell line NIT-1 was treated with 0.25 mM zinc chloride,
absolute numbers of viable cells were reduced from 59% in controls to
21.5% after zinc treatment (Fig. 1A). Substantial reduction in viable
cells retaining both TMRE and mBcl was also noted after zinc
treatment (from 96.7% to 22.7%, Fig. 1B, upper right quadrants).
Significant cytotoxicity was detectable after treatment of NIT-1 cells
with as little as 0.1 mM zinc, and enhanced cell death was observed
with increasing zinc concentrations (Fig. 1C). The rat insulinoma cell
line, RINm5F, also displayed similar levels of sensitivity to zinc
(Fig. 1C).In addition,the mouse insulinomacell line,Min6, andthe rat
insulinoma cell line, βTC3, exhibited comparable levels of sensitivity
to zinc (data not shown). Thus, rodent insulinomas succumbed to
concentrations of extracellular zinc considered to be physiological in
mammalian cells [11].
It was next examined whether human pancreatic ductal
adenocarcinomas, in comparison to normal human islet cells,
could also display enhanced sensitivity to increased concentrations
of extracellular zinc, like rodent insulinomas. To this end, freshly
isolated normal human islets were treated overnight with various
concentrations of zinc. Islets were mildly dispersed using trypsin,
and β cells were identified by staining with FluoZin-3-AM [24], a
cell-permeant dye with higher affinity to Zn2+(KD=15 nM) and
higher quantum yield than other zinc-sensitive dyes [29]. Data in
Fig. 1D show that normal human β (FluoZin-3+) cells were resistant
to treatment with 0.1- to 0.5 mM zinc concentrations, as indicated
by normal levels of mitochondrial membrane potential and
intracellular thiols among FluoZin-3+(β) cells. Resistance of β
cells to these zinc concentrations was consistently observed in eight
different islet preparations cultured either in CMRL 1066 medium
supplemented with human serum albumin (medium used for islet
transplantation) or in RPMI medium supplemented with fetal
bovine serum (data not shown). In some experiments, islets were
first dispersed by trypsin treatment and then treated overnight with
0.1 to 0.5 mM zinc. Islet cells were again treated with trypsin just
before staining to avoid clumping. Under these conditions also, no
increase in cytotoxicity was observed in β (FluoZin-3+) cells treated
with 0.5 mM zinc (% viability: control — 48% ± 8 vs. treated — 45%
± 9; n=3), indicating that trypsin treatment did not simply render
β cells more sensitive to high concentrations of zinc. In sharp
contrast to normal β cells, human pancreatic ductal adenocarcinoma
cell lines, CD18 and S2013 [30] underwent significant cell death
when treated with as little as 0.01 mM zinc (Fig. 1D). Although the
underlying mechanisms are not clear, the human pancreatic
carcinoma cells were more sensitive to extracellular zinc concentra-
tions than rodent insulinomas.
Kinetic experiments revealed that zinc treatment induced mito-
chondrial membrane depolarization in both CD18 and S2013 cell lines
within 1 h, as indicated by the reduction in TMRE retention (Fig. 1E).
Concurrent reduction in intracellular thiol levels as detected by
staining with mBcl also occurred within 1 h of zinc treatment in both
CD18 and S2013 cancer cells. A concomitant increase in the plasma
membrane permeability was seen, as indicated by the uptake of PI in
zinc-treated cells. Taken together, these results indicate that trans-
formed but not normal human pancreatic cells are highly susceptible
to physiological concentrations of zinc, 0.01 to 0.5 mM [11], leading to
cell death characterized by rapid induction of mitochondrial dysfunc-
tion, intracellular oxidation and plasma membrane permeability.
3.2. Events during zinc-induced cell death
We next examined whether differential influx of Zn2+could
explain the various outcomes of exposure to extracellular zinc in
normal and cancerous pancreatic cells. To test this possibility, we
stained cells treated with various concentrations of zinc with the cell-
permeable FluoZin-3-AM, which binds to labile Zn2+following the
cleavage of the acetomethyl ester (AM) moiety by nonspecific
esterases present in viable cells [24,29]. As can be seen in Fig. 2A,
incubation of CD18 and S2013 carcinoma cells with low concentra-
tions of zinc (0.01–0.1 mM) resulted in significantly increased
numbers of FluoZin-3+cells. In contrast, treatment of primary islet
cells with higher concentrations of zinc (0.1–0.5 mM) increased the
frequency of FluoZin-3+cells slightly but not significantly (Fig. 2B). In
data not shown, we observed that zinc treatment resulted in
comparable levels of zinc influx into primary β cells and pancreatic
cancer cells, as indicated by similar mean fluorescence intensities in
thesecells.Takentogether,theseresultsindicatethatexposuretohigh
concentrations ofzinc canleadtothe increasein free intracellularzinc
levels in more malignant cells than in normal pancreatic cells.
Zinc treatment increases the level of reactive oxygen species
(ROS) in neuronal cells [31,32] and Hep-2 tumor cells [33]. Therefore,
it was next examinedwhetherzinc-induced cytotoxicity in pancreatic
cancer cells was also associated with the generation of ROS. To this
end, cells were stained with MitoSox Red, a cell-permeant dye that
becomes highly fluorescent when oxidized by superoxide and
subsequent binding to nucleic acids [29]. Flow cytometric analysis
revealed that zinc treatment substantially increased the frequency of
cells stained with MitoSox Red (Fig. 3). Since MitoSox Red is oxidized
by superoxide but not by other reactive oxygen or nitrogen species
[29], the data indicate that zinc causes a significant increase in
mitochondrial superoxide production in insulinoma cells.
Both caspase-dependent and -independent cell death are associ-
ated with intracellular acidosis [34]. Therefore, intracellular pH was
analyzed by staining cells with SNARF-1, an emission-rationing dye
that shows a clear pH-dependent shift in its absorbance and emission
spectra [26]. Data shown in Fig. 3 also indicate that zinc treatment
significantly induced intracellular acidification as shown by the
decrease in the frequency of NIT-1 cells exhibiting pH-sensitive
SNARF-1 fluorescence. Collectively, the data indicate that zinc
treatment resulted in zinc influx, mitochondrial superoxide genera-
tion and intracellular acidification in mouse insulinoma cells.
3.3. Protection against zinc-induced insulinoma cell death
by antioxidants
Since extracellular [35,36] and intracellular [36] presence of
reduced GSH prevents zinc-induced cell death in astrocytes, it was
next examined whether they could similarly prevent zinc-induced
cytotoxicity in insulinomas. Data shown in Fig. 4A indicate that
continuous incubation of NIT-1 cells with 10 mM GSH or GSH-MEE, a
cell permeable form of GSH, could prevent zinc-induced cell death
significantly. However, removal of cell-impermeable GSH as well as
the cell-permeant GSH-MEE antioxidants after 45 min of incubation
abrogated protection against zinc-induced cytotoxicity. Similar
results were obtained using rat insulinomas, RINm5F and βTC3
(data not shown). Protection was efficient at 10 mM but not at 5 or 1
mM concentrations of either of these antioxidants (data shown),
indicating a requirement for certain stoichiometry between zinc and
the antioxidants for effective neutralization of toxicity [37].
Coincubation of NIT-1 cells with GSH and zinc also significantly
prevented mitochondrial production of ROS, as indicated by the
reduction in MitoSox Red fluorescence significantly (Fig. 4B). In
addition, zinc-induced activation of intracellular caspases was
substantially reduced by GSH treatment, as indicated by decreased
staining of cells with the FITC-conjugated polycaspase inhibitor, VAD-
FMK, that binds to all intracellular activated caspases [25]. Activation
of caspases by zinc in NIT-1 cells is consistent with previous studies
showingzinc-inducedactivationofcaspase-3and-9inHL-60[38]and
81
A.K. Jayaraman, S. Jayaraman / Journal of Nutritional Biochemistry 22 (2011) 79–88

Page 4

Ramos B and Jurkat cells [39]. However, zinc-induced death could not
be blocked by pretreating NIT-1 cells with the potent polycaspase
inhibitor, Q-VD-OPH (Fig. 4C) [40]. In addition, the inhibitor of
necrosis, necrostatin [41], failed to prevent zinc-induced cell death. In
data not shown, we observed that the necrostatin inhibited tumor
necrosis factor (TNF)-α-induced necrosis of L929 fibroblasts, as
reported [41]. In addition, the p38 mitogen-activated protein kinase
(MAPK)inhibitorSB203580andtheselectiveERK-1/2inhibitorU0126
[42] failed to block zinc-induced apoptosis in NIT-1 cells. SP600125,
inhibitor of JNK-1, -2 and -3 as well as 13 other protein kinases [43]
failed to prevent zinc-mediated cytotoxicity. Although the precursor
ofGSH,NAC[37],hasbeenshowntopreventzinc-inducedcelldeathin
82
A.K. Jayaraman, S. Jayaraman / Journal of Nutritional Biochemistry 22 (2011) 79–88

Page 5

retinal pigment epithelial cells [44], addition of NAC failed to protect
NIT-1cellsfromcelldeath,asreportedin epithelial typeIIcells[45].In
addition,incubationwithmelatonin,whichcandetoxifyoxidants[37],
also failed to protect NIT-1 cells from zinc-induced cell death.
Collectively, these data demonstrate that zinc treatment rapidly
induced irreversible damage to mitochondria and activated caspases,
and only the sequestration of extracellular zinc with the antioxidant
GSH can protect the insulinoma cells from cytotoxicity.
3.4. Zinc-induced carcinoma cell death involves increased
protein ubiquitination
To gain insights into the mechanisms of zinc-induced cell death,
whole cell lysates were prepared from human pancreatic carcino-
mas treated with zinc and immunoblotted with well-characterized
antibodies. Data shown in Fig. 5 indicate that zinc treatment did not
alter the levels of the survival factors, Bcl-XLexpressed in certain
pancreatic carcinomas [46], XIAP that directly prevents the
activation of caspases 3 and 7 [47] and c-Abl [48]. Interestingly,
the prototype survival factor, Bcl-2, expressed in some but not all
pancreatic carcinomas [49], could not be detected in CD18 and
S2013 cells but detected in the T cell leukemia, Jurkat (data not
shown). Signaling via the MAPK family member, ERK-1/2 has been
implicated in the survival of pancreatic carcinomas [50]. However,
zinc treatment neither prevented the signaling event, phosphory-
lation of ERK1/2, nor reduced the absolute amounts of ERK1/2 in
these carcinomas. Zinc treatment also failed to reduce the level of
pro-caspase-9, an apical caspase involved in mitochondria-depen-
dent intrinsic apoptotic pathway, and activates effector caspase -3
and -7, leading to the proteolytic cleavage of PARP [51]. Consis-
tently, zinc treatment did not result in the cleavage of PARP,
implicated in DNA damage and apoptosis [51]. The level of Cbl-b, an
E3 ubiquitin ligase, involved in the ubiquitination of proteins [52],
was also not altered by zinc treatment. Notably, enhanced
ubiquitination of proteins, critical for cell death [53], was evident
in both of the carcinoma cell lines treated with zinc. Collectively,
these data indicate that zinc treatment induced cytotoxicity and not
caspase-dependent apoptosis in pancreatic carcinomas. Although
cytotoxicity was not associated with proteolytic cleavage of the
prosurvival factors analyzed, substantial increase in protein ubiqui-
tination was evident.
3.5. Zinc-induced cytotoxicity is associated with the up-regulation
of ZnTs
Since the retention of zinc was considerably increased in
pancreatic cancer cells but not in primary islet cells treated with
Fig. 1. Pancreatic cancer cell lines were sensitive to zinc treatment than normal β cells. (A) The mouse insulinoma cell line, NIT-1 treated with 0.25 mM ZnCl2, was analyzed
by flow cytometry after overnight incubation. Note that the intact cells gated based on forward angle light scatter are reduced after zinc treatment. (B) Intact cells were
electronically gated and analyzed for viability after staining with TMRE and mBcl. The frequency of viable cells is shown in upper right quadrants. Representative data from
12 experiments are shown. (C) Dose-dependent toxicity of mouse insulinoma, NIT-1 (n=6) and rat insulinoma, RINm5F (n=6) cells are shown. The differences between
untreated vs. cells treated with 0.25 and 0.5 mM zinc were significant in both NIT-1 and RINm5F cell lines (Pb.05). (D) Freshly isolated human islets (n=8) and human
ductal adenocarcinoma cell lines CD18 and S2013 (n=5) were treated with indicated concentrations of ZnCl2and analyzed for viability. Insulin-producing β cells were gated
based on staining with FluoZin-3 and analyzed for the retention of TMRE and mBcl. The viability of cells treated with zinc was not significantly altered (control vs. 0.1 mM
Zn, P=.2, control vs. 0.25 mM Zn, P=.09, control vs. 0.5 mM Zn, P=.06). Viability of carcinoma cells was based on retention of TMRE and mBcl. The differences between
untreated and zinc-treated CD18 and S2013 cells were significant (N0.01 mM: Pb.05). (E) CD18 and S2013 carcinoma cell lines were treated with 0.25 mM ZnCl2 and
harvested at indicated time points and analyzed for viability and plasma membrane integrity, respectively, by staining with TMRE + mBcl and PI. Data are pooled from three
experiments. Zinc treatment significantly (Pb.05) reduced the viability and concomitantly increased the plasma membrane permeability within 1 h of treatment in both CD18
and S2013 cells.
Fig. 2. Increased zinc influx in pancreatic carcinoma cells. (A) Human ductal
adenocarcinoma cell lines CD18 and S2013 were treated overnight with indicated
concentrations of zinc. Cells were stained with the zinc-specific dye FluoZin-3 and
assessed by flow cytometry. The increase in FluoZin-3+cells after treatment with
0.025, 0.05 and 0.1 mM zinc was significant (Pb.01) in both of the cell lines, as
indicated by asterisks (n=3). (B) Normal human islets were treated overnight
with various concentrations of zinc and analyzed for intracellular free Zn2+as
detected by the binding of FluoZin-3. The numbers of FluoZin-3+cells increased
slightly but not significantly after zinc treatment. Data shown are pooled from
eight separate islet preparations.
Fig. 3. Zinc treatment induced ROS generation and intracellular acidosis in insulinoma
cells. NIT-1 cells were treated with 0.25 mM zinc chloride overnight and then stained
with MitoSox Red and analyzed for the generation of mitochondrial ROS by flow
cytometry (n=3). Cells were stained with SNARF-1 for the determination of
intracellular pH and analyzed by flow cytometry (n=3). P values are shown.
83
A.K. Jayaraman, S. Jayaraman / Journal of Nutritional Biochemistry 22 (2011) 79–88

Page 6

zinc, cytotoxicity in cancer cells could be due to perturbed zinc
homeostasis. Altered expression of ZnTs, implicated in the efflux of
zinc ions out of the cell and sequestration into intracellular vesicles
[10–12], may contribute to aberrant zinc homeostasis in cancer
cells. To test this possibility, total RNA was extracted from zinc-
treated rat insulinoma cell line, RINm5F and cDNA was synthesized
and quantitative real-time RT-PCR was performed using selected
primer sets and SYBR Green dye. As shown in Fig. 6A, both rat
whole pancreas and the RINm5F insulinoma expressed comparable
levels of znt1, znt2, znt4, znt5 and znt8 mRNA. Treatment of
RINm5F cells with 0.25 mM zinc chloride increased the expression
of znt1 and znt2 significantly when compared to the level of znt8
mRNA (Fig. 6B). However, the abundance of ubiquitously
expressed znt4 [10–12] and the β cell selective znt5 [17] was not
significantly altered when compared to znt8 in RINm5F insulinoma
cells following zinc treatment.
We next determined whether human pancreatic cancer cells
could also differentially regulate the expression of selected ZNT
genes in response to zinc treatment. A comparison was made with
similarly treated normal human islets (n=3) containing 60% to 80%
viable β cells, as determined by staining with a combination of
FluoZin-3, TMRE and mBcl [24]. The levels of the ubiquitously
expressed ZNT1 were not different among untreated islets and
pancreatic ductal carcinoma cells (Fig. 6C), while the expression of
the β cell-selective ZNT5 was lower in CD18 (Pb.05) but not in S2013
carcinoma in comparison to islets. The expression of ZNT7 mRNA
was several-fold lower in both of the carcinomas than in islets
(Pb.05). After overnight culture of human islets with 0.25 mM zinc,
the expression of ZNT5 and ZNT7 mRNA was significantly reduced,
but the level of ZNT1 remained unaltered. In sharp contrast, zinc
treatment increased the abundance of ZNT1 mRNA in both CD18 and
S2013 cell lines significantly. Interestingly, the level of ZNT7 mRNA
was increased by zinc treatment in S2013 but not in CD18
carcinoma, indicating variations in ZNT expression among different
pancreatic cancer cells. Collectively, these results indicate that zinc-
induced cytotoxicity is associated with consistent up-regulation of
ZNT1 in pancreatic cancer cells, and other ZNT genes may also be
up-regulated in a cell type-specific manner.
Fig. 4. Antioxidants protected insulinoma cells from zinc-induced cytotoxicity. (A) NIT-1 cells were incubated with 10 mM GSH or the cell-permeable GSH-MEE for 45 min and then
removed by centrifugation. Cells were then incubated with0.25 mM zinc. Aliquots of cells were also simultaneously incubated withGSH/GSH-MEE plus zinc overnight. Viability of cells
was assessed by flow cytometry (n=4). Addition of GSH or GSH-MEE significantly prevented zinc-induced cell death (Pb.05). However, removal of GSH and GSH-MEE after a brief
period of incubation did not restore viability. (B) Intracellular activated caspases were determined by incubating NIT-1 cells with FITC-VAD-FMK, and PI was added to assess plasma
membrane permeability. GSH was added simultaneously with zinc (n=3). The addition of GSH to zinc decreased ROS production and activation of caspases significantly (Pb.05). (C)
NIT-1 cells were incubated with 10 mM GSH, with 200 μM NAC or melatonin or with 10 μM concentrations of the cell-permeable inhibitors of caspases, Q-VD-OPH, necrostatin or
MAPK inhibitors for 45 min prior to the addition of 0.25 mM zinc. Controls included treatment of cells only with GSH or the inhibitors or DMSO. After overnight incubation, cells were
stained with TMRE and mBcl, and analyzed by flow cytometry (n=4).
84
A.K. Jayaraman, S. Jayaraman / Journal of Nutritional Biochemistry 22 (2011) 79–88

Page 7

Fig. 5. Zinc-induced protein ubiquitination in carcinoma cells. Human pancreatic ductal adenocarcinoma cells were treated with 0.025 mM zinc for 4 h, and cell lysates were
subjected to electrophoresis and the separated proteins were blotted using indicated antibodies. Apparent molecular weights of proteins are shown. Equal protein loading was
verified by immunoblotting with an antibody against actin. Note that zinc treatment increased the amounts of ubiquitinated proteins. Representative data from three to five
experiments are shown.
Fig. 6. Zinc treatment induced the up-regulation of selected ZnT genes in pancreatic cancer lines. (A) Quantitative real-time RT-PCR was performed with cDNA synthesized using RNA
extracted from rat whole pancreas and the rat insulinoma cell line, RINm5F. Relative expression of indicated znt genes was determined using gapdh as the normalizer. Determinations
were performed in triplicates in each experiment, and the data are the mean±SD of all three experiments. The levels of expression of indicated genes were not significantly different
between rat pancreas and rat insulinoma cells. (B) The expression levels of indicated znt genes were determined separately in untreated RINm5F cells and those treated overnight with
zinc (0.25 mM), using gapdh as the normalizer. Fold change in the expression of indicated genes due to zinc treatment was then calculated and expressed as described [28]. Assays
were done in triplicates, and the data were pooled from three to four experiments. The expression levels of znt1, znt2, znt4 and znt5 were compared with that of znt8. ⁎⁎P=.0001,
⁎P=.008 and ns, not significant. (C) Changes in relative mRNA expression levels of selected ZnT transporters after zinc treatment were determined using GAPDH as the normalizer in
normal human islet preparations and in human pancreatic cancer cell lines, CD18 and S2013. Data are expressed as the means ± SD of triplicate values of a representative of three
separate islet preparations. Data for CD18 and S2013 were pooled from four independent experiments. The P values between untreated and zinc-treated cells are as follows: 1,
P=.0038; 2, P=.0016; 3, Pb.0001; 4, P=.0007; and 5, P=.005.
85
A.K. Jayaraman, S. Jayaraman / Journal of Nutritional Biochemistry 22 (2011) 79–88

Page 8

4. Discussion
The data presented herein demonstrate that treatment with
physiological concentrations of zinc (0.01–0.5 mM) can induce
cytotoxicity in human ductal adenocarcinoma cell lines but not in
normal human insulin-producing β cells. Insulinoma cell lines
derived from rat and mouse also displayed enhanced sensitivity to
higher concentrations of extracellular zinc, indicating the conserva-
tion of this characteristic in transformed pancreatic cells through
phylogeny. Zinc-induced cytotoxicity in pancreatic cancer cells was
associated with rapid mitochondrial dysfunction, generation of ROS,
intracellular acidification, compromise in plasma membrane integ-
rity and increased protein ubiquitination, without apparent reduc-
tion in prosurvival factors. Notably, zinc treatment consistently
increased the abundance of ZnT-1 mRNA in a rat insulinoma and two
human ductal adenocarcinoma cell lines but not in normal human
islet cells.
Zinc treatment has been shown to induce cell death in a number of
transformed cell lines [21,27,54] including insulinomas [8,9]. Our data
demonstrate for the first time that ZnT-1 gene is up-regulated in both
rat insulinoma and human pancreatic ductal adenocarcinomas but
not in normal islet cells challenged with physiological concentrations
of zinc. Thus, a correlation between ZNT1 gene up-regulation and
induction of cytotoxicity is evident in zinc-treated pancreatic cancer
cells. Our data are consistent with the observation that ZnT-1,
ubiquitously expressed in a variety of transformed cells including
THP-1, Molt-4 and Raji [54], HeLa cells [27] and Epstein-Barr (EB)
virus transformed human lymphoblastoid cells [21], is the most up-
regulated zinc transporter gene in response to enhanced extracellular
zinc concentrations. Interestingly, rat mammary tumor [55] and
human breast cancer grown in nude mice [56] also responded to zinc
supplementation by increasing the level of ZnT-1 mRNA. However, it
is not known whether zinc supplementation can lead to cytotoxic
death of these tumor cells without affecting normal cells in vivo. Our
data indicate that unlike the primary islet cells, pancreatic ductal
adenocarcinomas succumbed to very low concentrations of exoge-
nous zinc accompanied by ZnT-1 mRNA up-regulation in vitro. This
finding may have important implications to the treatment of cancers
including pancreatic cancers with dietary zinc supplementation or via
pharmacological maneuvers to enhance ZnT-1 expression preferen-
tially in transformed cells.
Exposure to higher concentrations of zinc increased the frequen-
cy of transformed but not primary islet cells binding to the zinc-
specific dye, FluoZin-3. Impaired zinc homeostatic mechanisms in
transformed cells may facilitate the retention of excess intracellular
Zn2+, which may explain the increase in the frequency of FluoZin-3+
cells. A majority of islet cells (60–85%) are insulin-producing β cells
and bind FluoZin-3 with high affinity due to high levels of
intracellular zinc [24]. However, the remaining non-β cells present
in islets may not retain excess zinc as the transformed cells, which
may explain the lack of increase in FluoZin-3+cells when islet cells
were exposed to high doses of zinc. Further work is necessary to fully
elucidate the homeostatic mechanisms involved in the regulation of
intracellular levels of Zn2+in normal islet cells and their alteration
during transformation.
Our observation that the association of exaggerated expression of
ZNT1 and cell death in transformed pancreatic cells respondingto zinc
treatment is in apparent contradiction to the previous observation
that overexpression of ZNT1 in baby hamster kidney cells increased
the zinc efflux, reduced the intracellular steady-state zinc concentra-
tion and conferred resistance to zinc-mediated toxicity [57]. Although
ZnT-1 is located on the plasma membrane [10–12,54], it is also found
in intracellular compartments of rat enterocytes [10] and in the
insulinoma cell line, INS-1E [58]. It is possible that increased
intracellular expression of ZnT-1 may facilitate the accumulation of
toxic levels of zinc leading to mitochondrial damage and cytotoxicity
in transformed cells. Imaging studies are required to determine
whether exaggerated expression of ZnT-1 in intracellular organelles
could explain the aberrant zinc homeostasis in transformed pancre-
atic cells.
Our study has also revealed the expression of ZNT7 mRNA in
human islets and pancreatic ductal adenocarcinomas. Interestingly,
zinc treatment decreased the expression of ZNT7 in human islets but
increased it in S2013 carcinoma. The abundance of znt2 was
increased by zinc treatment in the rat insulinoma cell line, RINm5f.
Since ZnT-2 and ZnT-7 are, respectively, located in vesicles and
perinuclear vesicles such as Golgi apparatus [10,12], they could
transport zinc into intracellular vesicles. Increased concentrations of
zinc induce the dissipation of mitochondrial transmembrane
potential and elevation of ROS in neuronal cells [31,32] and Hep-2
cells [33]. Exposure of pancreatic cancer cells to high levels of
extracellular zinc may lead to overloading of zinc into mitochondria,
and subsequent mitochondrial membrane depolarization, genera-
tion of ROS, intracellular acidification and eventual cell death. Since
ZnT-1 is consistently up-regulated in rodent and human-trans-
formed pancreatic cells and other ZnT genes in a cell type-specific
manner, the relative contribution of these zinc transporters to
pancreatic cancer cell death may be unraveled by siRNA-mediated
ablation of ZNT1, ZNT2 and ZNT7 genes.
A comparison of the expression profiles of ZNT1 to ZNT9 in THP-1,
Molt-4 and Raji cell lines indicated lower expression of these genes
than in primary human T and B cells [54]. Similarly, a lower steady-
state level of ZNT1 mRNA expression was reported in chemically
induced mammary tumors compared to normal rat mammary gland
tissues [55]. However, we found that the expression profile of znt1,
znt2, znt4, znt5 and znt8 in the rat insulinoma cell line RINm5F was
similar to whole rat pancreas. While the expression levels of ZNT1 and
ZNT5weresimilar in purifiedhumanisletsandpancreatic carcinomas,
the steady-state level of ZNT7 mRNA was comparably lower in human
pancreatic carcinomas. Although it was reported that the steady-state
level of ZIP4 was higher in human pancreatic cancer tissues [22], our
data do not indicate up-regulation of the ZNT genes analyzed in rat
and human-transformed pancreatic cell lines compared to, respec-
tively, whole pancreas and purified islets. A thorough analysis of the
expressionprofiles ofthese ZNT genes in human pancreaticcarcinoma
biopsies may unravel additional useful biomarker(s) for the diagnosis
as well as prognosis of pancreatic cancers.
Our data show that zinc treatment induced cytotoxic but not
apoptotic death in pancreatic carcinomas and insulinomas as
indicated by the lack of proteolytic processing of pro-caspase-9
and PARP cleavage, characteristic features of apoptosis [51]. In
addition, the survival factors Bcl-XL, XIAP, C-Abl and Cbl-b were not
affected by zinc treatment. Although the MAPK family member ERK-
1/2 has been implicated in survival of pancreatic cancer cells [50],
zinc-induced cytotoxicity was not associated with either degrada-
tion of ERK-1/2 or its phosphorylation. In addition, only the
antioxidant GSH but not the inhibitors of enzymes implicated in
apoptosis could block zinc-induced carcinoma cell death even
though caspases were activated during zinc treatment as indicated
by the binding of FITC-VAD-FMK [25]. These data are consistent with
the notion that transformed pancreatic ductal adenocarcinoma cells
do not undergo caspase-dependent apoptosis, similar to the
observation in Hep-2 cells [59]. However, zinc has been reported
to induce caspase-dependent apoptosis in neuronal cells [31], HL-60
cells [38] and leukemia cells [39]. In addition, our data are also at
variance with a previous report showing the participation of MAPK
in zinc-induced cell death in HL-60 cells [39]. Taken together, these
data suggest that zinc can engage caspase-dependent and
-independent cell death pathways in a cell type-specific manner.
Importantly, our data indicate that zinc treatment led to increased
86
A.K. Jayaraman, S. Jayaraman / Journal of Nutritional Biochemistry 22 (2011) 79–88

Page 9

ubiquitination of proteins, a process crucial for cell death [53,60]. It
is possible that zinc influx may facilitate ubiquitination of survival
factors not investigated herein and accelerate their degradation via
the proteasomal pathway. Further work is necessary to determine
the identity of target proteins for ubiquitination during zinc-induced
cytotoxicity in pancreatic cancer cells.
Our data indicating that normal human β cells are resistant to
treatment with extracellular zinc are at variance with a previous
report that proposed that zinc is toxic to normal pancreatic cells [8].
Although the basis of this discrepancy is not clear, the striking
difference is that in the previous study, a small number (n=3) of
frozen islet preparations were studied [8] while we analyzed eight
different freshly isolated islet preparations. Freezing and thawing of
dispersed islet cells, as in the previous study [8], could render islet
cells more susceptible to cell death upon subsequent culture and zinc
treatment. We verified that the primary β cells, as stained by using
FluoZin-3 [24], are not susceptible to physiological zinc concentra-
tions, up to 0.5 mM. Therefore,our results do not support the previous
proposal that zinc is a paracrine effector in normal β cell death and
therefore mayplay a rolein diabetes[8]. Rather,our datademonstrate
that pancreatic carcinoma cells and not primary β cells are exquisitely
sensitive to increases in extracellular zinc concentrations. This raises
an interesting possibility of modulating pancreatic carcinomas by
using the transition metal zinc and without causing undue toxicity to
normal β cells.
Acknowledgments
Susan Camasta, Hinsdale South High, Darien, IL, is gratefully
acknowledged for her interest in this work. Kumar Sripathirathan,
Department of Pharmacology, University of Illinois at Chicago, is
acknowledged for the generous supply of some of the materials used
in this study. The generous supply of cell lines and islets by various
investigators mentioned in the text is gratefully acknowledged.
Annette Bruno, DNA Services at the University of Illinois at Chicago,
is acknowledgedfor helpwithdesigningsomeof theprimersetsused.
References
[1] Bachet JB, Mitry E, Lepère C, Declety G, Vaillant JN, Parlier H, et al. Chemotherapy
as initial treatment of locally advanced unresectable pancreatic cancer: a valid
option? Gastroenterol Clin Biol 2007;31:151–6.
[2] Chari ST, Leibson CL, Rabe KG, Timmons LJ, Ransom J, de Andrade M, et al.
Pancreatic cancer-associated diabetes mellitus: prevalence and temporal associ-
ation with diagnosis of cancer. Gastroenterology 2008;134:95–101.
[3] Zalatnai A. Novel therapeutic approaches in the treatment of advanced pancreatic
carcinoma. Cancer Treat Rev 2007;33:289–98.
[4] Maret W, Krezel A. Cellular zinc and redox buffering capacity of metallothionein/
thionein in health and disease. Mol Med 2007;13:371–5.
[5] Emdin SO, Dodson GG, Cutfield JM, Cutfield SM. Role of zinc in insulin
biosynthesis. Some possible zinc–insulin interactions in the pancreatic B-cell.
Diabetologia 1980;19:174–82.
[6] Grodsky GM, Schmid-Formby F. Kinetic and quantitative relationships between
insulin release and 65Zn efflux from perifused islets. Endocrinology 1985;117:
704–10.
[7] Ishihara H, Maechler, Gjiinovci A, Herrera PL, Wolheim CB. Islet beta-cell secretion
determines glucagon release from neighbouring alpha-cells. Nat Cell Biol 2003;5:
330–5.
[8] Kim BJ, Kim YH, Kim S, Kim JW, Koh JY, Oh SH, et al. Zinc as a paracrine effector in
pancreatic islet cell death. Diabetes 2000;49:367–72.
[9] Chang I, Cho N, Koh JY, Lee MS. Pyruvate inhibits zinc-mediated pancreatic islet
cell death and diabetes. Diabetologia 2003;46:1220–7.
[10] Liuzzi JP, Cousins RJ. Mammalian zinc transporters. Annu Rev Nutr 2004;24:
151–72.
[11] Eide DJ. Zinc transporters and the cellular trafficking of zinc. Biochim Biophys Acta
2006;1763:711–22.
[12] Sekler I, Sensi SL, Hershfinkel M, Silverman WF. Mechanism and regulation of
cellular zinc transport. Mol Med 2007;13:337–43.
[13] Priel T, Hershfinkel M. Zinc influx and physiological consequences in the beta-
insulinoma cell line, Min6. Biochem Biophys Res Commun 2006;346:205–12.
[14] Gyulkhandanyan AV, Lee SC, Bikopoulos G, Dai F, Wheeler MB. The Zn2+-
transporting pathways in pancreatic beta-cells: a role for the L-type voltage-gated
Ca2+ channel. J Biol Chem 2006;281:9361–72.
[15] Clifford KS, MacDonald MJ. Survey of mRNAs encoding zinc transporters and other
metal complexing proteins in pancreatic islets of rats from birth to adulthood:
similar patterns in the Sprague-Dawley and Wistar BB strains. Diabetes Res Clin
Pract 2000;49:77–85.
[16] Liuzzi P, Bobo JA, Lichten LA, Samuelson DA, Cousins RJ. Responsive transporter
genes within the murine intestinal–pancreatic axis form a basis of zinc
homeostasis. Proc Natl Acad Sci U S A 2004;101:14355–60.
[17] Kambe T, Narita H, Yamaguchi-Iwai Y, Hirose J, Amano T, Sugiura N, et al.
Cloning and characterization of a novel mammalian zinc transporter, Zinc
Transporter 5, abundantly expressed in pancreatic β cells. J Biol Chem 2002;
277:19049–55.
[18] Chimienti F, Devergnas S, Favier A, Seve M. Identification and cloning of a cell-
specific zinc transporter, ZnT-8, localized into insulin secretory granules. Diabetes
2004;53:2330–7.
[19] Wenzlau JM, Juhl K, Yu L, Moua O, Sarkar SA, Gottlieb P, et al. The cation efflux
transporter ZnT8 (Slc30A8) is a major autoantigen in human type 1 diabetes. Proc
Natl Acad Sci U S A 2007;104:17040–5.
[20] Murgia C, Devirgiliis C, Mancinin E, Donadel G, Zalewski P, Perozzi G. Diabetes-
linked zinc transporter ZnT8 is a homodimeric protein expressed by distinct
rodent endocrine cell types in the pancreas and other glands. Nutr Metab
Cardiovasc Dis 2009;19:431–9.
[21] Andree KB, Kim J, Kirschke CP, Gregg JP, Paik H, Joung H, et al. Investigation of
lymphocyte gene expression for use as biomarkers for zinc status in humans.
J Nutr 2004;134:1716–23.
[22] Li M, Zhang Y, Liu Z, Bharadwaj U, Wang H, Wang X, et al. Aberrant expression of
zinc transporter ZIP4 (SLC39A4) significantly contributes to human pancreatic
cancer pathogenesis and progression. Proc Natl Acad Sci U S A 2007;104:
18636–41.
[23] Jayaraman S. Flow cytometric determination of mitochondrial membrane
potential changes during apoptosis of T lymphocytic and pancreatic beta cell
lines: comparison of tetramethylrhodamineethylester (TMRE), chloromethyl-X-
rosamine (H2-CMX-Ros) and MitoTracker Red 580 (MTR580). J Immunol
Methods 2005;306:68–79.
[24] Jayaraman S. A novel method for the detection of viable human pancreatic beta
cells by flow cytometry using fluorophores selective for intracellular labile zinc,
mitochondrial membrane potential and protein thiols. Cytometry A 2008;73:
615–25.
[25] Jayaraman S. Intracellular determination of activated caspases (IDAC) by flow
cytometry using a pancaspase inhibitor labeled with FITC. Cytometry A 2003;56:
104–12.
[26] Bassnett S, Reinisch L, Beebe DC. Intracellular pH measurement using
single excitation-dual emission fluorescence ratios. Am J Physiol 1990;258:
C171–8.
[27] Devergnas S, Chimienti F, Naud N, Pennequin A, Coquerel Y, Chantegrel J, et al.
Differential regulation of zinc transporters ZnT-1, ZnT-5 and ZnT-7 gene
expression by zinc levels: a real-time RT-PCR study. Biochem Pharmacol 2004;
68:699–709.
[28] Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T)
method. Nat Protoc 2008;3:1101–8.
[29] Haugland RP. Handbook of fluorescent probes and research products. Eugene,
Oregon: Molecular Probes; 2001.
[30] Salabat MR, Melstrom LG, Strouch MJ, Ding XZ, Milam BM, Ujiki MB, et al. Geminin
is overexpressed in human pancreatic cancer and downregulated by the
bioflavanoid apigenin in pancreatic cancer cell lines. Mol Carcinog 2008;47:
835–44.
[31] Sensi SL, Ton-That D, Sullivan PG, Jonas EA, Gee KR, Kaczmarek LK, et al.
Modulation of mitochondrial function by endogenous Zn2+ pools. Proc Natl Acad
Sci U S A 2003;100:6157–62.
[32] Dineley KE, Richards LL, Votyakova TV, Reynolds IJ. Zinc causes loss of membrane
potential and elevates reactive oxygen species in rat brain mitochondria.
Mitochondrion 2005;5:55–65.
[33] Rudolf E, Rudolf K, Cervinka M. Zinc induced apoptosis in HEP-2 cancer cells: the
role of oxidative stress and mitochondria. BioFactors 2005;23:107–20.
[34] Matsuyama S, Reed J. Mitochondria-dependent apoptosis and cellular pH
regulation. Cell Death Differ 2000;7:1155–65.
[35] Cho IH, Im JY, Kim D, Kim KS, Lee JK, Han PL. Protective effects of extracellular
glutathione against Zn2+-induced cell death in vitro and in vivo. J Neurosci Res
2003;74:736–43.
[36] Kim D, Joe CO, Han PL. Extracellular and intracellular glutathione protects
astrocytes from Zn2+-induced cell death. Neuroport 2003;14:187–90.
[37] Moriarty-Craige SE, Jones DP. Extracellular thiols and thiol/disulfide redox in
metabolism. Annu Rev Nutr 2004;24:481–509.
[38] Kondoh M, Tasaki E, Araragi S, Takiguchi M, Higashimoto M, Watanabe Y, et al.
Requirement of caspase and p38MAPK activation in zinc-induced apoptosis in
human leukemia HL-60 cells. Eur J Biochem 2002;269:6204–11.
[39] Mann JJ, Fraker PJ. Zinc pyrithione induces apoptosis and increases expression of
Bim. Apoptosis 2005;10:369–79.
[40] Chauvier D, Ankri S, Charriaut-Marlangue C, Casimir R, Jacotot E. Broad-spectrum
caspase inhibitors: from myth to reality? Cell Death Differ 2007;14:387–91.
[41] Degterev A, Huang Z, Boyce M, Li Y, Jagtap P, Mizushima N, et al. Chemical
inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain
injury. Nature Chem Biol 2005;1:112–9.
[42] Roux PP, Blenis J. ERK and p38 MAPK-activated protein kinases: a family of
protein kinases with diverse biological functions. Microbiol Mol Biol Rev 2004;
68:320–44.
87
A.K. Jayaraman, S. Jayaraman / Journal of Nutritional Biochemistry 22 (2011) 79–88

Page 10

[43] Bain J, MCLauchlan H, Elliott M, Cohen P. The specificities of protein kinase
inhibitors: an update. Biochem J 2003;317:199–204.
[44] Song J, Lee SC, Kim SS, Koh HJ, Kwon OW, Kang JJ, et al. Zn2+-induced cell death is
mediated by the induction of intracellular ROS in ARPE-19 cells. Curr Eye Res
2004;28:195–201.
[45] Walther UI, Walther SC, Temruck O. Effect of enlarged glutathione on zinc-
mediated toxicity in lung-derived cell lines. Toxicol In Vitro 2007;21:380–6.
[46] Ghaneh P, Kawesha A, Evans JD, Neoptolemos JP. Molecular prognostic markers in
pancreatic cancer. J Hepatobiliary Pancreat Surg 2002;9:1–11.
[47] Dubrez-Daloz L, Dupoux A, Cartier J. IAPs: more than just inhibitors of apoptosis
proteins. Cell Cycle 2008;7:1036–46.
[48] Genua M, Pandini G, Cassarino MF, Messina RL, Frasca F. c-Abl and insulin receptor
signaling. Vitam Horm 2009;80:77–105.
[49] Campini D, Esposito I, Boggi U, Cecchetti D, Menicagli M, De Negri F, et al. Bcl-2
expression in pancreas development and pancreatic cancer progression. J Pathol
2001;194:444–50.
[50] Boucher MJ, Morisset J, Vachon PH, Reed JC, Lainé J, Rivard N. MEK/ERK
signaling pathway regulates the expression of Bcl-2, Bcl-XLand Mcl-1 and
promotes survival of human pancreatic cancer cells. J Cell Biochem 2000;79:
355–69.
[51] Cullen SP, Martin SJ. Caspase activation pathways: some recent progress. Cell
Death Differ 2009;16:935–8.
[52] Thien CB, Langdon WY. C-Cbl and Cbl-b ubiquitin ligases: substrate diversity
and the negative regulation of signaling responses. Biochem J 2005;391:
153–66.
[53] Hoeller D, Dikic I. Targeting the ubiquitin system in cancer therapy. Nature 2009;
458:438–44.
[54] Overbeck S, Uciechowski P, Ackland ML, Ford D, Rink L. Intracellular zinc
homeostasis in leukocyte subsets is regulated by different expression of zinc
exporters ZnT-1 to ZnT-9. J Leukoc Biol 2008;83:368–80.
[55] LeeR,WooW,WuB,KummerA,DuminyH,XuZ.ZincaccumulationinN-methyl-N-
nitrosourea-inducedratmammarytumorsisaccompaniedbyanalteredexpression
of ZnT-1 and metallothionein. Exp Biol Med (Maywood) 2003;228:689–96.
[56] SunD,Zhang L,Wang Y,WangX,HuX,CuiFA,etal.Regulation ofzinc transporters
by dietary zinc supplement in breast cancer. Mol Biol Rep 2007;34:241–7.
[57] Palmiter RD, Findley SD. Cloning and functional characterization of a mammalian
zinc transporter that confers resistance to zinc. EMBO J 1995;14:639–49.
[58] Sondergaard LG, Brock B, Stoltenberg M, Flyvbjerg A, Schmitz O, Smidt K, et al.
Zinc fluxes during acute andchronic exposure of INS-1E cells to increasing glucose
levels. Horm Metab Res 2005;37:133–9.
[59] Rudolf E, Cervinka M. Cytoskeletal changes in non-apoptotic cell death. Acta
Medica 2006;49:123–8.
[60] Bader M, Steller H. Regulation of cell death by the ubiquitin-proteasome system.
Curr Opin Cell Biol 2009;21:1–7.
88
A.K. Jayaraman, S. Jayaraman / Journal of Nutritional Biochemistry 22 (2011) 79–88