Rhein Induces Apoptosis through Induction of Endoplasmic Reticulum by mikeholy


									ANTICANCER RESEARCH 27: 3313-3322 (2007)

      Rhein Induces Apoptosis through Induction of Endoplasmic
      Reticulum Stress and Ca2+-dependent Mitochondrial Death
         Pathway in Human Nasopharyngeal Carcinoma Cells
                      YUNG-TSUAN HO5, CHIOU-YING YANG2 and JING-GUNG CHUNG6

    1School   of Medical Laboratory Science and Biotechnology, 5Graduate Institute of Chinese Medical Science, and
            6Departments  of Biological Science and Technology, China Medical University, Taichung, Taiwan;
                  2Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan;
                     3Medical Laboratory Science and Biotechnology and 4Institute of Life Sciences,

                    Central Taiwan University of Science and Technology, Taichung, Taiwan, R.O.C.

Abstract. Apoptosis is a physiological mechanism for                    together, these results suggest that ER stress and Ca2+-
eliminating malignant cells, including cancer cells, without            dependent mitochondrial death pathway may be involved in
eliciting damage to normal cells or surrounding tissues. Here,          rhein-induced apoptosis in NPC cells.
we report that rhein (4,5-dihydroxyanthraquinone-2-carboxylic
acid), a major constituent in the rhizome of rhubarb, induced           Cancer is a major public health problem worldwide because
apoptosis of human nasopharyngeal carcinoma (NPC) cells.                of its significantly high rates of morbidity and mortality.
Rhein induced apoptosis in NPC cells as demonstrated by                 Nasopharyngeal carcinoma (NPC) is a rare tumor arising
increased nuclear condensation and DNA fragmentation.                   from the epithelium of the nasopharynx (1). It occurs with
Moreover, for the first time in NPC cells it was demonstrated           high frequency in Asian populations, especially among
that the pathway involved in rhein-induced apoptosis is                 Chinese people (2, 3). The incidence of NPC among people
caspase-dependent, presumably through the endoplasmic                   in Hong Kong is about 15 to 25 per 100,000 (4). Of note,
reticulum (ER) stress pathway, as shown by an increase in the           the average age of NPC patients is usually ten years younger
levels of glucose-regulated protein 78 (GRP 78), PKR-like ER            than those of patients with other head and neck cancers.
kinase (PERK), activating transcription factor 6 (ATF6) and             Clinically, this cancer exhibits a high incidence of lymph
CCAAT/enhancer-binding protein homologous protein                       node spread as well as distant metastasis that contribute to
(CHOP) as well as the activation of caspase-3, -8, -9 and -12.          its poor prognosis (5).
This increased susceptibility to ER stress-induced apoptosis               Use of chemopreventative agents is now regarded as a
may be due to an increased accumulation of reactive oxygen              promising strategy against cancer development (6) and
species (ROS). Rapid accumulation of calcium (Ca2+) and a               many natural or dietary substances have been shown to
decrease in the mitochondrial membrane potential (MMP)                  inhibit carcinogenesis in vitro and in vivo (7). Apoptosis is a
were also observed. Cytochrome c and apoptosis-inducing                 physiological mechanism for eliminating malignant cells or
factor (AIF) were released upon treatment with rhein. Taken             cancer cells without eliciting damage to normal cells or
                                                                        surrounding tissues (8-10). Thus, induction of apoptosis in
                                                                        target cells is a key mechanism by which anticancer therapy
Correspondence to: Dr. J.-G. Chung, Departments of Biological Science   works (8). Rhein is isolated from the rhizome of rhubarb
and Technology, China Medical University, No 91, Hsueh-Shih Road,       (Rheum palmatum. or Rheum tanguticum maxim), which is
Taichung City 40402, Taiwan, R.O.C. Tel: +886 4 22053366 2161, Fax:     a Chinese medicinal plant used traditionally for treatment
+886 4 22053764, e-mail: jgchung@mail.cmu.edu.tw/Dr. C-Y. Yang,         of chronic liver disease in mainland China (11). In vivo
Institute of Molecular Biology, National Chung Hsing University, No     experimental studies show that rhein inhibits the growth of
250, Kuo Kuang Road, Taichung City 404, Taiwan, R.O.C. Tel: +886
                                                                        tumor cells in rat liver (12). In addition, it has also been
4 22840485241, e-mail: cyyang@dragon.nchu.edu.tw
                                                                        shown to induce apoptosis in several cancer cell lines such
Key Words: Rhein, reactive oxygen species, caspase-3,                   as human colonic adenocarcinoma cells, promyelocytic
mitochondrial membrane potential, apoptosis, nasopharyngeal             leukemia cell (HL-60), and cervical cancer Ca Ski cells (13-
carcinoma cells.                                                        15). These results suggest that rhein should have a great

0250-7005/2007 $2.00+.40                                                                                                          3313
                                       ANTICANCER RESEARCH 27: 3313-3322 (2007)

potential serving as a cancer chemopreventative agent.             Research and Development Institute (Hsinchu, Taiwan) and
However, the molecular mechanisms of its anti-cancer               cultured in minimum essential medium (MEM) supplemented with
activity are still poorly understood.                              10% FBS. Both cell lines were grown in 10 cm tissue culture dish at
                                                                   37ÆC in a humidified incubator containing 5% CO2.
   The endoplasmic reticulum (ER) is the principal organelle
for the synthesis of proteins, as well as being a regulator of     Chemicals and reagents. Rhein, propidium iodide (PI), Tris-HCl,
intracellular Ca2+ homeostasis. An imbalance between the           4'-6-diamidino-2-phenylindole (DAPI) and Triton X-100 were
rate of protein synthesis and the folding capacity of the ER       obtained from Sigma Chemical Co. (St. Louis, MO, USA).
induces ER stress. Cells initially adapt to the accumulation       Dimethyl sulfoxide (DMSO) and potassium phosphate were
of unfolded proteins by inducing the expression of ER-             purchased from Merck Co. (Darmstadt, Germany). DMEM, FBS,
resident molecular chaperones such as glucose-regulated            penicillin-streptomycin, trypsin-EDTA and glutamine were
                                                                   obtained from Gibco BRL (Grand Island, NY, USA). Caspase-3,
protein 78 (GRP 78) (16, 17). However, excessive and
                                                                   and -9 activity assay kits were purchased from OncoImmunin, Inc
prolonged stress leads cells to apoptosis associated with the      (Gaithersburg, MD, USA). Inhibitors of caspase-3 (Z-DQMD-
induction of the CCAAT/enhancer-binding protein                    FMK) was purchased from Calbiochem (San Diego, CA, USA) and
homologous protein (CHOP) (18). The current view is that           was dissolved in DMSO. Western blotting Luminol reagent was
three ER transmembrane proteins, activating transcription          purchased from Santa Cruz Co. (La Cruz, CA, USA).
factor 6 (ATF6), inositol-requiring 1 (IRE1) and PKR-like
ER kinase (PERK) are considered to be sensors of ER                Isolation of peripheral blood mononuclear cells (PBMC). Isolation of
stress (19). In the case of PERK, it is responsible for the        PBMC was performed by concentration on a Ficoll-Hypaque (Sigma,
                                                                   St Louis, MO, USA) gradient according to the protocol provided by
increased phosphorylation of the translation elongation
                                                                   the manufacturer. Briefly, heparinized venous blood (5 ml) was
initiation factor 2· (eIF2·) for the inhibition of synthesis of    layered over Ficoll-Hypaque and centrifuged at 375 xg for 30 min.
new proteins after exposure to ER stress (19-21). It has           Cells at the interface were removed and washed three times with PBS.
been shown that prolonged ER Ca2+ depletion triggers ER
stress and activates PERK (22). X box binding protein 1            Antibodies. ATF-6·, ATF-6‚, GRP 78, PERK, CHOP, and AIF
(XBP1), a key transcription factor of the unfolded protein         antibodies were purchased from Santa Cruz Co. (La Cruz, CA,
response (UPR), is activated by ATF6 and an active form            USA) and cytochrome c and caspase-12 antibodies were purchased
                                                                   from BD PharMingen (San Diego, CA, USA). Anti-mouse, -goat
of XBP1 is activated by IRE1-mediated splicing of its
                                                                   and -rabbit IgG peroxidase-conjugated secondary antibodies were
mRNA in response to ER stress (23). Previous reports show          purchased from Jackson ImmunoResearch Laboratory (West
that increases in the intracellular level of Ca2+ was              Grove, Pennsylvania, USA).
accompanied by the activation of the ER resident caspase-
12 (24). Moreover, Ca2+ released from the ER enters                Cell viability assay. Cells were seeded at a density of 1x105 cells per
mitochondria leading to depolarization of the inner                well into 12-well plate before drug treatment. Drugs were added to
membrane and cytochrome c release (25, 26). The results            medium at various times (12, 24, 36 and 48 h) and concentrations
also indicate that ER stress may lead to cell death via            (120, 140, 160, 180 and 200 ÌM). Cells were harvested and stained
                                                                   with 10 Ìg/ml of propidium iodide (PI) for the determination of
accumulation of ROS (27). ROS induction has also been
                                                                   cell viability. Samples were analyzed on a fluorescence-activated
shown to activate the down-stream apoptosis cascade or             cell sorter (FACS; BD Biosciences, Franklin Lakes, NJ, USA). Cell
induce the alteration of mitochondrial proteins, such as the       Quest software was applied to analyze the results (BD Biosciences,
voltage-dependent anion channel (VDAC) and/or the                  Franklin Lakes, NJ, USA).
adenine nucleotide translocase (ANT) that can induce the
pro-apoptotic mitochondrial membrane permabilization (28,          DAPI staining for the analysis of nuclear morphological changes. Cells
29). Furthermore, ER and mitochondria cross-talk                   were seeded at a density of 1x105 cells per well into 12-well plates.
apoptotic pathways induced by ER stress resulted in the            Cells were cultured with 180 mM rhein or 180 mM rhein and
                                                                   caspase-3 inhibitor at 37ÆC for 36 h. After treatment, cells were fixed
activation of caspase-9, -12 and/or cytochrome c release
                                                                   with 4% paraformaldehyde for 20 min, permeabilized with 0.1%
from mitochondria (30-32).                                         Triton X-100 and stained with 1 mg/ml of DAPI for 10 min at room
   In the present study, we investigated the possible              temperature (RT). Cells were then washed twice with PBS. The
involvement of the ER stress and mitochondrial death               morphological changes were observed using an Olympus
pathway in rhein-induced NPC apoptosis.                            fluorescence microscope (Olympus 1 X 71, Inc., Melville, NY, USA).

Materials and Methods                                              Comet assay. This assay was performed as described elsewhere (34).
                                                                   Briefly, treated cells or control cells were pelleted and resuspended
Human NPC cell lines. The nasopharyngeal carcinoma-derived cell    in 0.5% low melting point agarose at 37ÆC then layered on a
line NPC-039 was cultured routinely in Dulbecco's modified         frosted microscope slide previously coated with a thin layer of 0.5%
Eagle's medium (DMEM) supplemented with 10% fetal bovine           normal melting agarose and kept for 5 min at 4ÆC. After
serum (FBS) (33). WI-38 (human embryonic fibroblast, lung-         solidification, slides were immered in lysing solution (2.5 M NaCl,
derived cell line) cell line was obtained from the Food Industry   100 mM EDTA, 10 mM Tris-HCl (pH 10.5), 1% Tritin X-100 and

                          Lin et al: Rhein-induced Apoptosis in Human Nasopharyngeal Carcinoma Cells

10% dimethyl sulphoxide) for 1 h at 4ÆC. Slides were washed with        of 2,7-dichlorodihydrofluorescein diacetate (10 ÌM) and incubated
0.4 M Tris-HCl (pH 7.5) and stained with DAPI (1 mg/ml), then           at 37ÆC for 30 min. The level of ROS of the NPC cells was
observed under an Olympus fluorescence microscope.                      examined by flow FACS.

Western blot analysis. Rhein-treated cells were lysed in sodium         Detection of the intracellular Ca2+ level. Intracellular Ca2+ levels
dodecyl sulfate (SDS) sample buffer (62.5 mM Tris-HCl (pH 6.8),         were determined as described elsewhere (38). Briefly, rhein-treated
2% SDS, 10% glycerol, 50 mM dithiothreitol and 0.1%                     NPC cells were incubated with 3 Ìg/ml of Indo 1/AM (Calbiochem;
bromophenol blue) containing a cocktail of protease inhibitors.         La Jolla, CA, USA) and incubated at 37ÆC for 30 min. After
Protein concentration was determined by the Bradford method             washing twice with assay buffer, cells were suspended in assay
(BioRad, Laboratories, Richmond, CA, USA) as described in the           buffer. The level of Ca2+ of the NPC cells was determined by flow
manufacturer’s instructions. GRP 78, PERK, ATF6·, ATF6‚,                cytometry (FACS Calibur, BD Bioscience, Fanklin, NJ, USA).
CHOP and caspase-12 were analyzed by electrophoresis on a 10%
SDS-PAGE gel and then transferred to a PVDF membrane                    Measurement of mitochondrial membrane potential (MMP). MMP
(Millipore, Immobilon-P; Bedford, MA, USA). Immunoblot                  was determined by measuring the retention of the dye 3,3’-
analysis was performed by blocking overnight with a phosphate-          dihexyloxacarbocyanine (DiOC6) (39). Briefly, rhein-treated NPC
buffered saline (PBS) buffer containing 3% skimmed milk and then        cells were incubated with 4 mol/l of DiOC6 for 30 min at 37ÆC.
incubated with primary antibodies. Proteins were visualized by          Cells were then pelleted by centrifugation at 1,000 rpm for 10 min.
staining with horseradish peroxidase-conjugated goat anti-mouse,        The cell pellet was re-suspended and washed twice with PBS. The
goat anti-rabbit or donkey anti-goat antibodies (Jackson                MMP was determined by flow cytometry (FACS Calibur, BD
ImmunoResearch Laboratories, Inc., West Grove, Pennsylvania,            Bioscience, Fanklin, NJ, USA).
USA) and Western Blotting Luminol Reagent. Á-Tubulin was used
as internal control for sample loading.                                 Statistical analysis of data. Statistical calculations of the data were
                                                                        performed using an unpaired Student’s t-test and ANOVA analysis.
Detection of cytochrome c and AIF release. Cytosolic protein extracts   Statistical significance between control and experimental groups
were prepared by the method of Zuppinin et al. (35). Cells were         was set at p<0.05.
homogenized in ice-cold buffer containing 75 mM NaCl, 1 mM
NaH2PO4, 8 mM Na2HPO4, 250 mM sucrose and 190 Ìg/ml                     Results
digitonin. The cytosolic fractions were then centrifuged at 13,000 xg
for 5 min at 4ÆC. The assay for the release of AIF was performed as
                                                                        Apoptosis is induced in NPC cells by rhein. Since rhein was
described elsewhere (36). Cells were first lysed in isotonic
mitochondrial buffer (210 mM mannitol, 70 mM sucrose, 1 mM
                                                                        found to inhibit tumor growth and to induce apoptosis in
EDTA and 10 mM HEPES (pH 7.5)) and homogenized for 30 to                several cancer cell lines (12-14), the first study served to
40 strokes with a Dounce homogenizer (Dounce; Bellco Glass Co.,         determine whether rhein exhibited cytotoxicity on NPC cells.
Vineland, NJ, USA). Nuclei and unbroken cells were removed by           To quantitate the effect of rhein on cell growth, cell viability
centrifugation at 500 xg for 5 min in microcentrifuge at 4ÆC.           was determined by PI staining and FACS analysis. As shown
Supernatants were centrifuged at 10,000 xg for 30 min at 4ÆC, and       in Figure 1 (panel A), NPC cells exhibited a clear dose- and
the resulting supernatants were stored as the cytosolic fraction.
                                                                        time-dependent growth inhibition with rhein. In contrast,
Twenty Ìg of total proteins underwent electrophoresis on a 12%
SDS-polyacrylamide gel and transferred onto a PVDF membrane.
                                                                        human peripheral blood mononuclear cells (PBMC) and
The membrane was incubated with rabbit anti-cytochrome c                normal fibroblasts show no significant reduction in viability
antibody at 4ÆC overnight. After incubation with goat anti-rabbit       following treatment with rhein (Figure 1, panels B and C).
horseradish peroxidase antibody, the signal was visualized by           Figure 2A illustrate that apoptotic bodies increased with
Western Blotting Luminol reagent.                                       increasing concentrations of rhein up to 180 ÌM after 48 h
                                                                        of exposure to rhein. Furthermore, after 12, 24, 36 and 48 h
Assay for caspase-3, -8 and -9 activity. The activities of caspase-3,   of 180 ÌM rhein treatment apoptotic bodies were found but
-8 and -9 were measured using the PhiPhiLux G1D2, CaspaLux
                                                                        the control cells were not affected (Figure 2B). These data
L1D2 and CaspaLux M1D2 kits (OncoImmunin, Inc., College
Park, MD, USA), respectively, according to the protocol provided
                                                                        suggest that rhein-treated NPC cells undergo cell death and
by the manufacturer. Briefly, rhein-treated NPC cells were              exhibit morphological features suggestive of apoptosis.
incubated with PhiPhiLux fluorogenic caspase substrate at 37ÆC for         To further investigate whether the induction of cell death
1 h. After incubation, samples were washed three times with PBS.        by rhein could be linked to apoptosis in NPC cells, single
Subsequently, 500 Ìl of ice-cold flow cytometry dilution buffer was     cell gel electrophoresis (Comet assay) was performed and
added to this reaction mixture and cells were analyzed by FACS as       nuclear morphological changes examined. As expected,
described elsewhere (37).
                                                                        rhein-treated cells showed well-formed comets while the
                                                                        control cells did not demonstrate any comet-like appearance
Detection of reactive oxygen species (ROS). ROS production was
measured as described elsewhere (37). The NPC cells (1x105 cells)
                                                                        (Figure 3A). NPC cells also showed a remarkable change in
were treated with or without 180 ÌM rhein for 0.5, 1, 2, 3 and 6 h to   nuclear morphology after treatment with rhein (Figure 3B),
detect the changes of ROS. After treatment, cells were harvested        whereas the caspase-3 inhibitor (Z-DQMD-FMK) had
and washed twice with PBS. Cells were then re-suspended in 500 Ìl       significant effect on blocking nuclear morphological change

                                             ANTICANCER RESEARCH 27: 3313-3322 (2007)

Figure 1. Cytotoxicity of rhein on human NPC cells. The effect of rhein on cell viability. Each point is mean±S.D. of three experiments. *p<0.05.

Figure 2. Rhein induces morphological changes in NPC cells. Arrows indicate dead cells. Photographs were taken under x200 light microscopy.

(Figure 3B). Based on the above results, it was indicated                   ER stress markers are activated by rhein. To explore
that rhein could induce apoptosis in NPC cells.                             whether rhein induces apoptosis by activation of caspase,
                                                                            we used flow cytometry to analyze the activity of caspase-
Rhein-induced ER stress provokes CHOP expression. To                        3, a marker of caspase-dependent apoptosis (41).
investigate whether apoptosis is induced by ER stress, we                   Compared with control cells, rhein treatment caused the
evaluated the translation levels of ER stress genes in NPC                  activation of caspase-3 (Figure 5A). In order to confirm
cell lines exposed to rhein. First, we analyzed the expression              the induction of ER stress in NPC cells, we next examined
of GRP 78 protein which is an ER-resident molecular                         caspase-8 and -9 activities. Caspase-8 and -9 were
chaperone whose expression serves as a good marker of ER                    significantly activated at 6 h and continued to 36 h (Figure
stress (40). Immunoblot analysis showed that GRP 78                         5B and C). The changes in the caspase-8 and -9 activities
expression was augmented after rhein treatment (Figure                      at 12 h may have resulted from the activation of ER
4A). Consistently, ER stress sensors ATF6 and PERK were                     stress-induced CHOP (Figure 4C) and caspase-3 (Figure
also significantly induced after 6 h treatment with rhein                   5A). Western blot was then performed to examine the
(Figure 4B). It has been shown that induction of ER stress-                 cleavage of procaspase-12 during rhein-induced apoptosis
associated apoptosis factor CHOP is regulated by ATF6 and                   of NPC cells. Figure 5D shows that the cleaved form of
PERK. Here, a marked increase in CHOP protein levels                        caspase-12, p42/35, was noted at 12 h after rhein
was also observed after 12 and 24 h in rhein-treated NPC                    treatment and increased up to 36 h. These results indicate
cells (Figure 4C). These results suggest that rhein induces                 that the ER resident caspase-12 plays an important role
cell apoptosis through an ER stress pathway.                                in rhein-induced apoptosis.

                           Lin et al: Rhein-induced Apoptosis in Human Nasopharyngeal Carcinoma Cells

Figure 3. DNA fragmentation induced by rhein. (A) Rhein treated cells showed well-formed comet tails. (B) Nuclear morphological changes in rhein-
treated NPC cells. Photographs were taken under x200 light microscopy.

Cross-talk between ER stress- and mitochondrial-mediated cell
apoptosis. We investigated whether rhein treatment affects
the cytosolic Ca2+ level, MMP and the cytochrome c level.
The levels of cytosolic Ca2+, ROS and MMP were
determined by flow cytometric assay. As shown in Figures
6A and 6B, the levels of Ca2+ and ROS increased in NPC
cells after being treated with rhein, and the levels of Ca2+
were maintained in the following treatment periods up to 6
h. Rhein-induced apoptosis of NPC cells was also
accompanied by a significant decrease in MMP (Figure 6C).
   Western blot results showed that cytochrome c was
significantly increased in the cytosol in a time-dependent
manner (Figure 7A) and AIF was released at the same time
(Figure 7B), suggesting that rhein is sufficient to induce the
release of cytochrome c and AIF but not Endo G (data not
shown). Thus these results indicate that the mitochondrial
death pathway may also be involved in rhein-induced
apoptosis in NPC cells.

Discussion                                                                Figure 4. Induction of ER stress marker proteins by Rhein. The protein
                                                                          levels of GRP 78, ATF6, PERK and CHOP in cell lysates after incubation
Despite the possible involvement of rhein in the apoptosis                with 180 ÌM rhein were analyzed by using specific antibodies; Á-tubulin
                                                                          was used as internal controls for sample loading.
of human cancer cells, the pathway of rhein-induced
apoptosis has not been fully elucidated. In the present
paper, we demonstrated that rhein induces nuclear
morphological changes and reduces the percentage of viable                evidence that ER stress induced by rhein was correlated
NPC cells through apoptosis via ER stress and the Ca2+-                   with the augmented expression of GRP 78 and CHOP as
dependent mitochondrial death pathway. Here, we provide                   well as the cleavage of procaspase-12. Indeed, NPC cells

                                             ANTICANCER RESEARCH 27: 3313-3322 (2007)

Figure 5. The ER stress-specific caspase cascade is activated during rhein treatment. (A-C) Rhein induced the activation of caspase-3, -8 and -9 in NPC
cells treated with 180 ÌM of rhein. The caspase activities were measured as described in materials and methods. The results are shown as means±SD of
the three independent experiments. (D) Rhein induced the cleavage of caspase-12 in NPC cells treated with 180 ÌM of rhein. Cytosolic protein extracts
were subjected to Western blot with antibody to caspase-12.

exposed to rhein demonstrated a dramatic increase in                         activation. The activated caspase-8 then initiates the activity
mitochondrial dysfunction, including the loss of MMP and                     of downstream caspase-3 (42). The other is the intrinsic
the release of cytochrome c and AIF.                                         pathway, in which the release of cytochrome c from
  Two pathways of caspase-dependent apoptosis have been                      mitochondria to the cytosol during the early stages of
identified. The first is death receptor-mediated caspase-8                   apoptosis triggers the activation of procaspase-9 initiating

                             Lin et al: Rhein-induced Apoptosis in Human Nasopharyngeal Carcinoma Cells

Figure 6. Rhein-induced changes in the levels of Ca2+, ROS and MMP. (A) Changes in the cytosolic Ca2+ level induced by rhein. Cytosolic level of Ca2+
in NPC cells following treatment with 180 ÌM rhein was monitored by measuring increased fluorescence of Indo-1 by flow cytometry as described in Materials
and Methods. The zero concentration was defined as control. The time course of Indo-1 fluorescence change in the presence of 180 ÌM rhein is shown. (B)
Rhein-induced increases in the intracellular ROS level. Generation of ROS in NPC cells following treatment with 180 ÌM rhein was monitored by measuring
increased fluorescence of DCF by flow cytometry as described in Materials and Methods. The time course of DCF fluorescence change in the presence of 180
ÌM rhein is shown. (C) Reduction of MMP in rhein-treated (180 ÌM) NPC cells. After treatment, cells were harvested for measurement of MMP. The
decrease of fluorescence DiOC6 was measured by flow cytometry as described in Materials and Methods. The time course of DiOC6 fluorescence changes
in the presence of 180 ÌM rhein is shown. The zero concentration was defined as control. *Significantly different from control at p<0.05.

an apoptosome formation composed of Apaf-1, dATP,                              the release of cytochrome c into the cytosol and the
procaspase-9 and cytochrome c (43). Apoptosome                                 occurrence of downstream caspase activation (47). It was
formation leads to the activation of executioner caspase-3,                    shown that the release of Endo G and AIF from the
-6 and -7 (44, 45). Previous studies have demonstrated that                    mitochondria in response to proapoptotic stimuli occurs in
MMP is a key factor for apoptosis induction (46), triggering                   a caspase-dependent manner (36).

                                               ANTICANCER RESEARCH 27: 3313-3322 (2007)

                                                                                to cell apoptosis (51), whereas caspase-9 activation is
                                                                                associated with the mitochondrial apoptotic pathway. In
                                                                                several experiments, it was found that induction of
                                                                                apoptosis in cancer cells by anticancer drugs was mediated
                                                                                by up-regulation of Fas ligand and Fas (52, 53).
                                                                                Accordingly, we cannot exclude the possibility that rhein
                                                                                activates caspase-8 via a Fas death receptor pathway.
                                                                                Further studies are required to elucidate the mechanism.
                                                                                    Cellular Ca 2+ homeostasis is critical for maintaining
                                                                                normal cell function; depletion of ER Ca2+ stores causes
                                                                                growth arrest and apoptosis (54). In this regard, a
                                                                                previous report has shown that an increase in the level of
                                                                                intracellular Ca 2+ is thought to converge on
                                                                                mitochondrial dysfunction, which in turn results in the
                                                                                overproduction of ROS (55). Moreover, experimental
                                                                                data indicated that ER stress causes accumulation of ROS
Figure 7. Increase in the release of cytochrome c and AIF by rhein. Cytosolic   leading to cell death, the generation of ROS being derived
extracts (A) and supernatant fractions (B) were analyzed by immunoblotting      from the ER and mitochondria (27). The relationship
with specific antibodies against cytochrome c, AIF or Á-tubulin.
                                                                                between ROS or Ca 2+ and apoptosis has been broadly
                                                                                investigated in several cancer cell lines (37, 56). Little is
                                                                                known about the role of ROS and Ca2+ in the induction
                                                                                of apoptosis caused by rhein. Our results show that rhein
   In our present study, the release of cytochrome c was                        increases the production of ROS and intracellular Ca2+.
observed quickly after cells were exposed to rhein (Figure                      Despite the accumulation of high concentrations of ROS
7A). Indeed, a significant amount of AIF was released                           after cells were treated with rhein, the cells were able to
(Figure 7B). Unlike AIF, the release of Endo G was                              survive with low levels of intracellular Ca2+ (Figures 6A
unaffected (data not shown). The rapid loss in MMP of                           and B). Therefore, it is unclear whether or not rhein may
NPC cells upon treatment with rhein was significantly                           interact directly with a Ca2+ channel that is responsible
correlated with the release of cytochrome c (Figures 6C                         for the final demise of the cell which leads to prevent its
and 7A). Therefore, these findings indicate that rhein-                         opening. Moreover, it could be that they may prevent the
induced mitochondrial membrane permeabilization is                              signaling mechanism between high ROS levels and the
sufficient to induce the release of cytochrome c and AIF.                       opening of the Ca2+ channel.
The release of cytochrome c, which occurred at the same                            It was reported that GSH levels in cells are associated
time as the processing of caspase-3, -8 and -9, suggests that                   with ROS levels and loss in cellular GSH up to 85% of the
cytochrome c release is a preceding event for the                               control level causes only a 5- to 10-fold increase in levels of
activation of caspase cascades. Rhein-induced nuclear                           ROS (57). The interesting point is that a greater GSH loss
morphological change was completely inhibited by                                may stimulate mitochondria to produce a 100-fold increase
preincubation with a caspase-3 inhibitor (Figure 3B).                           in ROS which causes cell death (57). Whether or not rhein
Taken together, our results suggest that the rhein-induced                      can act as an antioxidant needs further investigation.
apoptosis process occurred in a caspase-dependent                                  The present chemotherapy agents for cancer are usually
manner. Interestingly, processing of caspase-8 by rhein was                     toxic to normal cells, often resulting in punishing side-
also observed in the present assay. A recent study also                         effects such as temporary hair loss, nausea and vomiting. In
demonstrated similar findings that rhein-induced caspase-                       this paper, we show that rhein significantly inhibited the
8 activation may be associated with apoptosis of human                          growth of and induced apoptosis of NPC cells, without
cervical cancer CaSki cells (15). Although no evidence                          toxicity to the human PBMC and normal fibroblasts. These
supports the direct action of rhein in the activation of                        data highlight its potential as a treatment for NPC.
caspase-8, it is speculated that rhein may activate caspase-                       In summary, this is the first report of rhein-induced
8 by elevating the expression of Fas ligand. Caspase-12, an                     apoptosis in human NPC cells. Rhein-induced apoptosis is
ER resident caspase, is activated by ER stress and can lead                     an ER stress and mitochondria-dependent pathway which
to the cleavage and activation of caspase-9 (48, 49).                           also involves Ca2+ levels in these cells. Understanding the
Activated caspase-9 consequently leads to the activation of                     molecular mechanism involved in rhein-induced apoptosis
the caspase cascade, such as caspase-3 (50). Caspase-8 is a                     may provide essential information for the development of
key component of the Fas death receptor pathway leading                         novel therapies to treat nasopharyngeal carcinoma.

                          Lin et al: Rhein-induced Apoptosis in Human Nasopharyngeal Carcinoma Cells

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