1,4-Diselenophene-1,4-diketone triggers caspase-dependent apoptosis in human melanoma A375 cells through induction of mitochondrial dysfunction

Article abstract of DOI:10.1248/cpb.59.1227

Epidemiological, preclinical and clinical studies have supported the role of selenocompounds as potential cancer chemopreventive and chemotherapeutic agents. In this study, a novel selenophene-based compound, 1,4-diselenophene-1, 4-diketone (DSeD), has been synthesized by Double Friedel - Crafts reaction and identified as a potent antiproliferative agent against a panel of six human caner cell lines. Despite this potency, DSeD was relatively nontoxic toward human normal cells, HS68 fibroblasts and HK-2 kidney cells. These results suggest that DSeD possesses great selectivity between cancer and normal cells. Induction of apoptosis in human melanoma A375 cells by DSeD was evidenced by accumulation of sub-G1 cell population, DNA fragmentation and nuclear condensation. Activation of caspase-9 and depletion of mitochondrial membrane potential indicated the initiation of the mitochondria-mediated apoptosis pathway. Pretreatment of cells with general caspase inhibitor z-VAD-fmk and caspase-9 inhibitor z-LEHD-fmk significantly suppressed the cell apoptosis, demonstrating the important roles of caspase and mitochondria in DSeD-induced apoptotic cell death. Furthermore, DSeD-induced apoptosis was found independent of reactive oxygen species generation. Taken together, our results suggest that DSeD induces caspase-dependent apoptosis in A375 cells through activation of mitochondria-mediated apoptosis pathway.

Full text of DOI:10.1248/cpb.59.1227

October 2011
Regular Article
Chem. Pharm. Bull. 59(10) 1227—1232 (2011)
1227
1,4-Diselenophene-1,4-diketone Triggers Caspase-Dependent Apoptosis in
Human Melanoma A375 Cells through Induction of Mitochondrial
Dysfunction
a
b
Yi LUO,a Xiaoling LI,a,b Xiaochun HUANG, Yum-Shing WONG, Tianfeng CHEN, ,a Yibo ZHANG,a and
*
,a
*
Wenjie ZHENG
a Department of Chemistry, Jinan University; Guangzhou, Guangdong Province 510632, China: and b School of Life
Sciences, The Chinese University of Hong Kong; Hong Kong, China.
Received May 23, 2011; accepted July 3, 2011; published online July 7, 2011
Epidemiological, preclinical and clinical studies have supported the role of selenocompounds as potential
cancer chemopreventive and chemotherapeutic agents. In this study, a novel selenophene-based compound,
1,4-diselenophene-1,4-diketone (DSeD), has been synthesized by Double Friedel–Crafts reaction and identified as
a potent antiproliferative agent against a panel of six human caner cell lines. Despite this potency, DSeD was rel-
atively nontoxic toward human normal cells, HS68 fibroblasts and HK-2 kidney cells. These results suggest that
DSeD possesses great selectivity between cancer and normal cells. Induction of apoptosis in human melanoma
A375 cells by DSeD was evidenced by accumulation of sub-G1 cell population, DNA fragmentation and nuclear
condensation. Activation of caspase-9 and depletion of mitochondrial membrane potential indicated the initia-
tion of the mitochondria-mediated apoptosis pathway. Pretreatment of cells with general caspase inhibitor z-
VAD-fmk and caspase-9 inhibitor z-LEHD-fmk significantly suppressed the cell apoptosis, demonstrating the
important roles of caspase and mitochondria in DSeD-induced apoptotic cell death. Furthermore, DSeD-induced
apoptosis was found independent of reactive oxygen species generation. Taken together, our results suggest that
DSeD induces caspase-dependent apoptosis in A375 cells through activation of mitochondria-mediated apoptosis
pathway.
Key words selenium; apoptosis; caspase activation; mitochondria; reactive oxygen species
Selenium (Se) is an essential trace element with funda- of periodic table of elements, it is feasible to substitute
mental importance to humans and animals.1) Epidemiologi- sulfur atom in thiophene-based compounds with Se. Recent
cal, preclinical and clinical studies have supported the role of studies have reported that substituting sulfur with Se in es-
selenocompounds as potential cancer chemopreventive and tablished chemopreventive agents may lead to more effective
chemotherapeutic agents.2,3) The chemical forms and meta- analogs.8—10) D-501036, a novel selenophene-based trihetero-
bolic activity are determinants of anticancer activities of se- cycle, has been identified as a promising anticancer com-
lenocompounds. Comparing with inorganic Se, organosele- pound with potential for therapy of human cancers.9) Das and
nium compounds show several advantages, such as higher co-workers10) synthesized a novel Se analog (Se-PBIT)
absorptivity, better anticancer activity and lower toxicity. of a chemopreventive agent S,Sꢀ-(1,4-phenylenebis[1,2-
Therefore, during the past decade, a number of potent ethanediyl])bisisothiourea (PBIT) and found that Se-PBIT
organoselenium compounds have been designed to achieve was superior to PBIT as remarkable inducer of apoptosis and
greater chemopreventive efficacy and minimal side effects by inhibitor of cancer cell growth. Moreover, Desai and co-
structural modifications, such as ebselen, selenocyanate, se- workers verified that substitution of sulfur with Se increased
lenobetaine and Se analogues of amino acids and other sulfur the compound’s potency by several folds in different cancer
compounds with known antitumor activity.3) For instance, cell lines by inhibition of inducible nitric oxide synthase
1,2,5-selenadiazolo-[3,4-d]pyrimidine-5,7-(4H,6H)-dione (iNOS)/Akt.11)
has been identified as a potent antiproliferative agent against
Malignant melanoma is a rapidly spreading skin tumor
human breast carcinoma MCF-7 cells, human hepatoma with a high invasive capacity and growing incidence.12) Al-
HepG2 cells and human melanoma A375 cells.4) Dhimant though great improvement in survival rate of disseminated
Desai et al. found that Se analogs of suberoylanilide hydrox- melanoma by using the contemporary therapeutic strategies,
amic was potent histone deacetylase inhibitors.5) 1-Benzyl-3- severe side effects, such as thrombocytopenia, neutropenia
(5-hydroxymethyl-2-furyl)selenolo[3,2-c]pyrazole deriva- and anemia are unavoidable. Therefore, searching for new
tives have also been synthesized and found to display favor- agents capable of selectively killing melanoma cells consti-
able anticancer activity.6) Nowadays, the application of syn- tutes an urgent priority in the field of cancer research. Apo-
thetic organoselenium compounds in chemoprevention and ptosis, an active mode of cell death, plays a vital role in the
chemotherapy is a fascinating field for cancer research.
development, homeostasis, and prevention of cancer. The
Thiophenes are sulphur-containing compounds widely dis- role of apoptosis in action of anticancer drugs is becoming
tributed in Asteraceae (Compositae), a plant with known me- more and more clear.13) Generally, apoptosis could occur via
dicinal use.7) Several naturally occurring and synthetic death receptor-dependent (extrinsic) or mitochondria-medi-
dithiophenes and terthiophenes have been proved to possess ated (intrinsic) pathway. The extrinsic pathway is triggered
insecticidal, bactericidal, antifungal, antiviral and anticancer through the formation of death inducing signaling complex,
activities.8) Since sulfur and Se are in the same group (VIA) which subsequently activates initiator caspase-8 and then
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed. e-mail: tchentf@jnu.edu.cn; tzhwj@jnu.edu.cn

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Vol. 59, No. 10
cleaves executioner caspases.14) Intrinsic apoptotic cell death
is initiated by the release of cytochrome c from mitochondria
into cytosol and subsequent activation of caspase-9.13) Mito-
chondria play a central role in cellular metabolism and con-
trol of cell apoptosis.15) Disruption of mitochondrial mem-
brane potential and release of apoptogenic factors are impor-
tant for activation of both caspase-dependent and caspase-in-
dependent apoptosis pathways.15) Apoptosis has been found
as one of the most critical mechanisms for anticancer action
of selenocompounds.3) We have previously showed that, se-
lenocystine, a nutritionally available selenoamino acid, ex-
hibited potent antiproliferative effects on human melanoma
cells through induction of apoptosis with the involvement of
reactive oxygen species (ROS) generation and p53-mediated
mitochondrial dysfunction.16) Dietary supplementation of se-
lenomethionine and high-Se soy protein was also found be
able to reduce the metastasis of melanoma cells in mice.17)
Moreover, apoptosis was identified as the major mode of cell
death induced in melanoma cells by synthetic selenadiazole
derivatives.18) However, so far very little information about
the apoptosis-inducing activities of selenophene-based com-
pounds and the underlying mechanisms are available.
Table 1. Growth Inhibitory Effects of DOD, DSD and DSeD on Various
Human Cancer and Normal Cellsa)
IC50 (mg/ml)
Cell lines
DOD
ꢁ400
371.0ꢂ40.3
ꢁ400
NE
NE
ꢁ400
DSD
DSeD
A375
115.0ꢂ8.4
236.6ꢂ17.4
190.1ꢂ25.9
ꢁ400
343.0ꢂ3.2
174.3ꢂ23.6
83.3ꢂ9.9
HepG2
MCF-7
HeLa229
Neuro-2a
PC-3NE
109.8ꢂ10.7
123.5ꢂ14.1
343.0ꢂ3.2
103.5ꢂ4.4
HK-2
HS68
ꢁ400
ꢁ400
ꢁ400
ꢁ400
353.7ꢂ1.6
ꢁ400
NE: no effect. a) Cells were treated with various concentrations of tested com-
pounds for 72 h. Cell viability was determined by MTT assay and IC50 values were cal-
culated as described in Experimental. Each value represents the meanꢂS.D. of three
independent experiments.
In the present study, we have synthesized three hetero-
cycles, 1,4-difuranyl-1,4-diketone (DOD), 1,4-dithienyl-1,4-
diketone (DSD) and 1,4-diselenophene-1,4-diketone (DSeD),
and compared their in vitro anticancer activities. The results
showed that substitution of sulfur and oxygen with Se signifi-
cantly enhanced the apoptosis-inducing activities of thio-
phenes and furans against human melanoma cells. Further
investigation on the intracellular mechanisms showed that
DSeD triggered caspase-dependent and ROS-independent
apoptosis in A375 cells through induction of mitochondrial
dysfunction.
Fig. 1. Cell Growth Inhibition Induced by DSeD in A375 Cells
Cells were treated with different concentrations of DSeD for 24, 48, and 72 h. Cell
viability was determined by MTT assay.
Results and Disscussion
DSeD, DSD and DOD were synthesized by double
Friedel–Crafts reaction,19) and characterized by HNMR, elec-
trospray ionization (ESI)-MS and IR. The in vitro anticancer
activities of DSeD, DSD and DOD were firstly screened
against a panel of six human cancer cell lines, including
A375, MCF-7, HepG2, HeLa299, Neuro-2a and PC-3 cells
by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
Fig. 2. Cell Cycle Analysis of A375 Cells Exposed to DSeD
The cells treated with DSeD for 72 h were collected and stained with PI after fixa-
tion. Each value represents the mean of three independent experiments.
mide (MTT) assay after 72 h treatment. As shown in Table 1, of apoptosis or cell cycle arrest or a combination of these
DSeD exhibited broad inhibition on the tested cancer cells two modes. Therefore, we performed propidium iodide (PI)-
with IC50 values significantly lower than those of its analogs, flow cytometric analysis to determine whether apoptosis was
DOD and DSD, indicating the higher cytotoxic effects of involved in the cell death induced by DSeD in the most sus-
DSeD on cancer cells. Moreover, to investigate the kinetics ceptible A375 human melanoma cells. Figure 2 showed the
of the antiproliferative effects of DSeD, we treated A375 representative DNA histograms obtained after PI staining of
cells with different doses of DSeD for various periods of permeabilized cells, which revealed that exposure of A375
time and analyzed the cell viability by MTT assay. Data in cells to different concentrations of DSeD for 72 h leaded to
Fig. 1 showed that DSeD treatment resulted in time- and dose-dependent increases in the proportion of apoptotic cells
dose-dependent decrease in cell viability. Despite this po- as reflected by the Sub-G1 cell population. Moreover, no sig-
tency, DSeD was relatively nontoxic toward human normal nificant change in cell cycle distribution was observed in
cells, including HS68 fibroblasts and HK-2 kidney cells DSeD-treated cells. Therefore, cell death induced by DSeD
(Table 1). Over 70% of HS68 and HK-2 cells remained vi- was primarily attributed to induction of apoptosis. This find-
able after 72-h incubation with the presence of 400 mg/ml ing was further confirmed by DNA fragmentation and nu-
DSeD (data not shown). These results suggest that DSeD clear condensation as detected by terminal deoxynucleotidyl
possesses great selectivity between cancer and normal cells.
transferase dUTP nick end labeling (TUNEL) enzymatic la-
We next investigated the underlying mechanism of DSeD- beling and 4ꢀ,6-diamidino-2-phenyindole (DAPI) co-staining
induced cell death. Inhibition of proliferation in cancer cells assay. DNA fragmentation is an important biochemical hall-
treated with anticancer drugs could be the result of induction mark of cell apoptosis. TUNEL assay can be used to detect

October 2011
1229
early stage of DNA fragmentation in apoptotic cells prior to downstream apoptotic signal. To determine whether caspase
changes in morphology. As shown in Fig. 3, dose-dependent activation was involved in the DSeD-induced apoptosis, the
increase in DNA fragmentation and nuclear condensation activity of an important effector caspase (caspase-3) and two
were observed in A375 cells exposed to 40 and 80 mg/ml of initiator caspases (caspase-8 and caspase-9) were measured
DSeD.
by fluorometric assays. As shown in Fig. 4A, treatments of
Apoptosis may occur via two crucial pathways, including A375 cells with DSeD activated caspase-3, -8 and -9 in a
death receptor-mediated (extrinsic) and the mitochondria- dose-dependent manner. Activities of caspase-9 and caspase-
mediated (intrinsic) pathways. The former is triggered by ac- 3 increased for 1.3—1.8 and 1.2—1.6 folds, respectively, in
tivation of death receptors, such as Fas and tumor nephrosis cells exposed to 20—80 mg/ml of DSeD by comparing with
factor related apoptosis inducing ligand (TRAIL) receptors control. In contrast, only slight increase in activity of cas-
(DR4, DR5), and the subsequent cleavage of caspase-8/10.20) pases-8 (1.1—1.2 fold) in response to DSeD treatment was
The mitochondrial (intrinsic) pathway is mediated by Bcl-2 observed. These results suggest that the contribution of ex-
family proteins, a group of anti-apoptotic and pro-apoptotic trinsic pathway to DSeD-induced apoptosis is likely to be
proteins that regulate the release of apoptogenic factors from insignificant.
the mitochondria into cytosol, such as cytochrome c,
In order to further evaluate the roles of caspases in DSeD-
Smac/Diablo and AIF, which then activate the caspase-de- induced apoptosis, we examined the effects of various cas-
pendent or -independent apoptotic pathways.21) Activation of pase inhibitors, including general caspase inhibitor z-VAD-
caspase-9 during this process will cleave and trigger the fmk, caspase-8 inhibitor z-IETD-fmk and caspase-9 inhibitor
z-LEHD-fmk, on cell apoptosis. As shown in Fig. 4B, DSeD-
induced apoptotic cell death was remarkably suppressed by
pretreatment of cells with 40 mM z-VAD-fmk as measured by
flow cytometric analysis. These results indicate that DSeD-
induced apoptosis mainly occurs in a caspase-dependent
manner. Moreover, caspase-9 inhibitor z-LEHD-fmk signifi-
cantly attenuated the DSeD-induced apoptotic cell death, but
caspase-8 inhibitor z-IETD-fmk failed to do so. Based on
these results, it can be concluded that mitochondria-mediated
apoptotic pathway play the major role in DSeD-induced
apoptosis in A375 cells. This conclusion was further verified
by the observation of mitochondrial dysfunction in response
to DSeD treatment.
Mitochondria play a critical role in the regulation of apo-
ptosis. The intermembrane space contains several apopto-
genic factors, such as cytochrome c, apoptosis-inducing fac-
tor, SMAC/Diablo and endonuclease G, which are liberated
into cytosol during the disruption of DYm.22) The intrinsic
and extrinsic apoptosis pathways can converge at the mito-
Fig. 3. Representative Photomicrographs of DNA Fragmentation and Nu-
chondria level and trigger mitochondria membrane perme-
abilization.23) Many studies have showed that permeabiliza-
tion of the outer mitochondria membrane and the subsequent
clear Condensation in Response to DSeD Treatment as Detected by TUNEL
Assay and DAPI Staining. A375 Cells Were Treated with 40 and 80 mg/ml
DSeD for 24 h.
Fig. 4. DSeD Induces Caspase-Dependent Apoptosis in A375 Cells
(A) Analysis of caspase activation in DSeD-induced apoptosis in A375 cells. Caspase activities were measured using synthetic fluorescent substrates for caspase-3, caspase-8
and caspase-9 as described in Results and Discussion. (B) Effects of various caspase inhibitors (40 mM) on apoptosis induced by DSeD. Cells were pre-treated with caspase
inhibitors for 2 h followed by co-incubation with DSeD for 24 h. Apoptotic cells were determined by flow cytometric analysis. All results were obtained from three independent
experiments. Significant difference between treatment and control groups is indicated at ∗ pꢃ0.05 level.

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Vol. 59, No. 10
release of pro-apoptotic proteins from the intermembrane been reported as an important regulator of cell apoptosis in-
space play an essential role in both caspase-dependent and duced by various chemopreventive and chemotherapeutic
caspase-independent apoptosis pathways.24) JC-1 is a cationic agents.26) Several studies have clarified that change in intra-
dye that exhibits potential-dependent accumulation in mito- cellular ROS level could induce cancer cell apoptosis, and in-
chondria. During the loss of DYm, the fluorescence of JC-1 hibit the invasion and metastasis.27) A mass of apoptotic
dye shifts from red to green. The increase in green fluores- stimuli cause cancer cell apoptosis through triggering cy-
cence indicates the loss of DYm in the treated cells. There- tochrome c release and overproduction of ROS.28) Excess
fore, we studied the status of DYm in DSeD-treated A375 ROS could attack various components of DNA, leading to
cells by JC-1 flow cytometric analysis. As shown in Fig. 5, the generation of a variety of ROS-mediated modified prod-
DSeD treatment induced a dose-dependent increase in deple- ucts, including oxidized bases, DNA strand breaks, DNA
tion of DYm. The percentage of depolarized mitochondria in intra-strand adducts, and DNA–protein crosslinks.29) The de-
cells exposed to 20, 40 and 80 mg/ml of DSeD increased tection of mitochondrial dysfunction by JC-1 probe led us to
from 1.2% (control) to 11.4%, 30.0% and 44.4%, respec- examine the role of ROS in DSeD-induced apoptosis. The in-
tively.
tracellular ROS generation in A375 cells treated by DSeD
Mitochondria are the major intracellular source of ROS, was measured by DCF-flow cytometry. This assay is based
especially superoxide and hydrogen peroxide.25) ROS has on the cellular uptake of a non-fluorescent probe DCFH-DA,
Fig. 5. DSeD Induces the Depletion of Mitochondrial Membrane Potential (DYm)
Cells treated with DSeD were harvested and stained with the mitochondria-selective dye JC-1 and then analyzed by flow cytometry. The number in the right region of each dot
plot represents the percentage of cells that emit green fluorescence due to the depletion of DYm.
Fig. 6. ROS Production in A375 Cells Exposed to DSeD (A), in Vitro Antioxidant Activities of DSeD (B) and the Effects of NAC (2 mM) on DSeD-
Induced Cell Apoptosis (C) and Cell Viability (D)
(A) Cells were exposed to the indicated concentrations of DSeD for 3 h and the levels of the intracellular ROS were analyzed by DCFH-DA fluorescence intensity. (B) In vitro
antioxidant activity of DSeD as determined by DPPH free radical scavenging assays. (C, D) Cells were exposed to 80 mg/ml DSeD for 24 h with or without the pretreatment of
NAC for 4 h. Apoptotic cell death was determined by flow cytometric analysis. Values expressed are meansꢂS.D. of triplicates. Significant difference between treatment and con-
trol groups is indicated at ∗ pꢃ0.05 or ∗∗ pꢃ0.01 level.

October 2011
1231
American Type Culture Collection (Manassas, VA, U.S.A.). The cells were
maintained in RPMI 1640 or Dulbecco’s modified Eagle’s medium (DMEM)
medium, which was supplemented with penicillin (100 units/ml), 10% fetal
bovine serum and streptomycin (50 units/ml) at 37 °C in a humidified incu-
bator with 5% CO2 atmosphere.
MTT Assay Cell viability was examined by measuring the ability of
cells to metabolize MTT to a purple formazan dye.34) Cells were seeded in
96-well tissue culture plates for 24 h and then incubated with the tested com-
pounds at different concentrations for different periods of time. After incu-
bation, 20 ml/well of MTT solution (5 mg/ml phosphate buffered saline
(PBS)) was added and incubated for 5 h. The medium was aspirated and re-
placed with 150 ml/well dimethyl sulfoxide (DMSO) to dissolve the for-
mazan salt formed. The color intensity of the formazan solution, which re-
flects the cell growth condition, was measured at 570 nm using a microplate
spectrophotometer (VSERSA Max).
which is subsequently hydrolyzed by intracellular esterase to
form dichlorofluorescein, DCFH. The non-fluorescent sub-
strate is oxidized by the intracellular free radicals, producing
a fluorescent product DCF.30) The treatment of DSeD led to a
rapid disruption of DYm in A375 cells (Fig. 5). Therefore,
we examined the intracellular ROS generation in A375 cells
by measuring the DCF fluorescence intensity to test whether
ROS was implicated in DSeD-induced apoptosis. As shown
in Fig. 6A, DSeD treatments for 3 h resulted in dose-depen-
dent decrease in DCF fluorescence intensity, indicating the
down-regulation of intracellular ROS generation by DSeD.
The total antioxidant activity of DSeD was thus evaluated by
1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scaveng-
ing assay. The results showed that DSeD dramatically inhib-
Flow Cytometric Analysis The cell cycle distribution was examined by
flow cytometry according to Li et al.35) Briefly, the cells cultured with or
ited the formation of DPPH free radicals in a dose-dependent without DSeD were trypsinized, washed with PBS and fixed with 70%
ethanol overnight at ꢅ20 °C. The fixed cells were washed with PBS and
stained with PI working solution (1.21 mg/ml Tris, 700 U/ml RNase,
50.1 mg/ml PI, pH 8.0) for 4 h in darkness. The stained cells were analyzed
with Epics XL-MCL flow cytometer (Beckman Coulter, Miami, FL, U.S.A.).
manner (Fig. 6B), demonstrating the potent antioxidant activ-
ity of DSeD. Moreover, a thiol-reducing antioxidant N-
acetylcysteine (NAC) failed to prevent the cells from DSeD-
induced apoptosis (Fig. 6C) and growth inhibition (Fig. 6D).
These results indicate that DSeD-induced apoptosis in A375
cells is independent of ROS generation.
Cell cycle distribution was analyzed using MultiCycle software (Phoenix
Flow Systems, San Diego, CA, U.S.A.). The proportion of cells in G0/G1, S,
G2/M phases was represented as DNA histograms. Apoptotic cells with hy-
podiploid DNA content were measured by quantifying the sub-G1 peak in
the cell cycle pattern. For each experiment, 10000 events per sample were
recorded.
TUNEL Assay and DAPI Staining Apoptotic DNA fragmentation in-
duced by ASDO was examined by using an in situ cell death detection kit
following the manufacturer’s protocol. Briefly, cells cultured in chamber
slides were fixed with 3.7% formaldehyde for 10 min and permeabilized
with 0.1% triton X-100 in PBS. After then, the cells were incubated with
100 ml/well of TUNEL reaction mixture at 37 °C for 1 h. The nuclei of the
cells were double stained with 1 mg/ml of DAPI for 15 min. Stained cells
were examined on a fluorescence microscope (Nikon Eclipse 80i).
Caspase Activity Assay Harvested cell pellets were suspended in cell
lysis buffer and incubated on ice for 1 h. After centrifugation at 11000ꢇg
Experimental
Materials Furan (AR, Aldrich), thiophene (AR, Aldrich), selenophene
(AR, Sigma), AlCl3 (AR, Aldrich) and succinyl chloride (AR, Sigma) were
purchased commercially. DPPH, thiazolyl blue tetrazolium bromide (MTT),
DAPI, propidium iodide (PI) and NAC were obtained from Sigma. Caspase-
3 substrate (Ac-DEVD-AFC), caspase-8 substrate (Ac-IETD-AFC) and Cas-
pase-9 substrate (Ac-LEHD-AFC) were purchased from Calbiochem. The
general caspase inhibitor (z-VAD-fmk) was purchased from Calbiochem.
The caspase-8 inhibitor (z-IETD-fmk) and caspase-9 inhibitor (z-LEHD-
fmk) were obtained from Merck. Air-sensitive reagents were manipulated in
an argon atmosphere. All solvents were dried and purified by standard pro-
cedures.
Synthesis and Characterization The schematic route for the synthesis for 30 min, supernatants were collected and immediately measured for pro-
of DOD, DSD and DSeD was showed in Chart 1.
Synthesis of DOD A CH2Cl2 solution containing furan (5 ml) and suc-
tein concentration and caspase activities. The cell lysates were placed in
96-well plates and then specific caspase substrates (Ac-DEVD-AFC for
cinyl chloride (1 g) was added dropwise to an anhydrous CH2Cl2 solution caspase-3, IETD-AFC for caspase-8 and Ac-LEHD-AFC for caspase-9
(100 ml) containing AlCl3 (5 g) under Ar2 at 0 °C. The reaction mixture was substrate) were added. Plates were incubated at 37 °C for 1 h and caspase
stirred at 0 °C for 2 h, slowly warmed to room temperature, and stirred for
12 h. Then the mixture was poured into a beaker containing ice. Ethyl ac-
etate was added and the organic layer was in a separatory funnel. After
removing the solvent under vacuum, the residue crystallised with
activity was determined by fluorescence intensity with the excitation and
emission wavelengths set at 380 and 440 nm, respectively.
Evaluation of Mitochondrial Membrane Potential (DYm) Cells in
6-well plates were trypsinized and resuspended in 0.5 ml of PBS buffer
hexanes : ethyl acetate (7 : 3). Yields, 25%.31) 1H-NMR (CDCl3) d: 7.61 (2H, containing 10 mg/ml of JC-1. After incubation for 10 min at 37 °C in the in-
dd, Jꢄ1.7 Hz, 5-H), 7.25 (2H, dd, Jꢄ3.6 Hz, 3-H), 6.55 (2H, dd, Jꢄ3.6 Hz,
4-H) and 3.30 (4H, s, CH2); IR (KBr) cmꢅ1: 1661 (CꢄO), 1651, 1574, 1496,
1324 and 1036; MS (EI) m/z: 218 [MꢆH]ꢆ.
cubator, cells were immediately centrifuged to remove the supernatant. Cell
pellets were suspended in PBS and then analyzed by flow cytometry. The
percentage of the green fluorescence from JC-1 monomers was used to
represent the cells that lost DYm.
Synthesis of DSD DSD was prepared in a method similar to that of
DOD, except, the residue crystallized from hexanes. Yields, 50%.32) 1H-
Measurement of ROS Generation The effects of DSeD on ROS-initi-
NMR (CDCl3) d: 3.41 (4H, s, COCH2), 7.16 (2H, dd, Jꢄ3.8, 1.1 Hz, H- ated intracellular oxidation were evaluated by DCF fluorescence assay.16)
4,4ꢀ), 7.66 (2H, dd, Jꢄ3.8, 5.0 Hz, H-3,3ꢀ), 7.83 (2H, dd, Jꢄ1.1, 5.0 Hz H- Briefly, collected cells were incubated with DCFH-DA at a final concentra-
5,5ꢀ); IR (KBr) cmꢅ1: nꢄ3100, 2920, 1648 ; MS (EI) m/z: 250 [MꢆH]ꢆ.
Synthesis of DSeD DSeD was synthesized in an approach similar to
that of DOD, with the crude product was purified by crystallizing from pe-
troleum. Yields, 25%.33) 1H-NMR (CDCl3) d: 8.36 (2H, dd, Jꢄ5.2, 0.8 Hz),
8.02 (H, dd, Jꢄ4, 1.2 Hz), 7.40 (2H, dd, Jꢄ5.6, 4.4 Hz), 3.39 (4H, s); IR
(KBr) cmꢅ1: 1652 (CꢄO); MS (EI) m/z: 345.9 [MꢆH]ꢆ.
tion of 10 mM at 37 °C for 30 min. The loaded cells were then washed twice
with PBS and ROS level was determined by measuring the fluorescence in-
tensity on a Tecan SAFIRE fluorescence reader.
DPPH Free Radical Scavenging Assays DPPH free radical scavenging
activity of DSeD was measured according to the method as previously de-
scribed.36) Briefly, 20 ml of test samples at different concentrations was
mixed with 180 ml of DPPH solution (60 mM in methanol) for 30 min in the
Cell Culture The cell lines used in this study, including human
melanoma A375 cells, human liver cancer HepG2 cells, human breast carci- dark, and then, the change in absorbance at 517 nm was measured. DMSO
noma MCF-7 cells, human cerical cancer HeLa299 cells, mouse neuroblas-
toma Neuro-2a cells, human Prostate cancer PC-3 cells, human proximal tu-
bular epithelial HK-2 cells and human HS68 fibroblast were obtained from
was used as a negative control.
Statistical Analysis Experiments were carried out at least in triplicate
and results were expressed as meanꢂS.D. Statistical analysis was performed
using SPSS statistical program version 13 (SPSS Inc., Chicago, IL, U.S.A.).
Difference with ∗pꢃ0.05 or ∗∗ pꢃ0.01 was considered statistically significant.
Acknowledgements This work was supported by Natural Science Foun-
dation of China and Guangdong Province, Key Project of Science and Tech-
nology Department of Guangdong Province, the Fundamental Research
Funds for the Central Universities and Program for New Century Excellent
Talents in University.
Chart 1. Schematic Route for Synthesis of DOD, DSD and DSeD

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Vol. 59, No. 10
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