State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, PR China
2’,4’-Dihydroxy-6’-methoxy-3’,5’-dimethylchalcone inhibits apoptosis of
MIN6 cells via improving mitochondrial function
Yingdi Luo, Yanhua Lu
Received December 16, 2011, accepted January 13, 2012
Dr. Yan-hua Lu, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology,
Box 283#, 130 Meilong Road, Shanghai 200237, PR China
Pharmazie 67: 798–803 (2012)doi: 10.1691/ph.2012.1848
Mitochondrial dysfunction due to oxidative stress and concomitant ?-cell apoptosis may play a key role in
type 2 diabetes. Inhibiting ?-cell apoptosis through ameliorating oxidative mitochondrial dysfunction with
specific natural products may have preventive or therapeutic potential. In this study, the anti-apoptotic effect
of 2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethylchalcone (DMC), a isolated chalcone from the buds of Cleisto-
calyx operculatus, on H2O2induced MIN6 cells damage was investigated. Exposure to H2O2at 250?M
for 3h, the viability of MIN6 cells was significantly decreased and the apoptosis apparently occurred. A
pre-treatment with DMC at the concentrations of 12.5–25?M, before H2O2addition, reduced nucleus frag-
potential (MMP), and consequently, inhibited apoptosis. Furthermore, decreased activities of caspase-3
and caspase-9 were observed. These results clearly demonstrated DMC protected MIN6 cells against
apoptosis due to its highly protective effect on mitochondria, and thus, it has great potential as a candidate
drug for the diabetes care.
Both forms of diabetes mellitus, type I and type II. Both are
to be the main form of ?-cell death in both types (Evans et al.
In type I diabetes, members of the Bcl-2 protein family on the
mitochondria membrane regulate the release of cytochrome c
and caspase-3 and execute cell death (Friedlander 2003). While
in type II, mitochondrial oxidant production associated with
hyperlipemia and hyperglycemia disrupts glucose-stimulated
insulin secretion and causes cellular damage through activating
JNK/SAPK, PKC, AGE/RAGE, and others (Cnop et al. 2005;
Chandra et al. 2001).
Medicinal herbs have been used for diabetes treatment for a
long history in many countries (Chan et al. 2009; Modak et al.
bioactive compounds from natural origin. Modern pharmaco-
logical researches have revealed that many herbal extracts have
great potential in diabetes care. Among the pharmacologically
active natural products accounting for this effect are flavonoids.
rats 8Sorenson et al. 1994). The isoflavone purarin could inhibit
the H2O2induced apoptosis in rat islet cells with an increase in
the activities of antioxidant enzymes (Linag et al. 2006). Narin-
genin, a citrus flavonoid, increases muscle cell glucose uptake
via AMPK (Zygmunt et al. 2010).
Cleistocalyx operculatus (Roxb.) Merr. et Perry (Myrtaceae) is
a well known medicinal plant whose buds are commonly used
as an ingredient for tonic drinks in Southern China (Ye et al.
Fig. 1: Structure of 2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethylchalcone
2004a, b). Its main bioactive compounds comprise a variety
dimethylchalcone (DMC) (Fig. 1) was found to be the most
abundant (Ye et al. 2004 a,b). Accumulative evidence demon-
strates that DMC exerts anti-inflammatory effects through
blocking NF-?B activation and effectively reverses multi-drug
resistance in resistant human hepatocellular carcinoma cell line
BEL-7402/5-FU (Kim et al. 2010; Huang et al. 2011). Hep-
atoprotective effects of DMC against CCl4-induced acute liver
injury in mice have recently been found as well (Lu et al. 2011).
In addition, DMC has been reported to have anti-diabetic (Ma
et al. 2005), anti-bacterial (Gafner et al. 1996), spasmolytic
(Amor et al. 2005) and anti-tumoral (Ye et al. 2005) proper-
ties. Although DMC has beneficial effects on various cells or
tissues, it is unclear whether it plays a role in preventing ?-cell
failure. Therefore, in the present study we examined the anti-
apoptotic effect of DMC on H2O2induced MIN6 cell damage,
and the cellular mechanisms related to this effect.
2. Investigations and results
2.1. Cell viability assay
To determine cell viability, the MTT method was used. MIN6
cell viability was significantly decreased by 41.82±1.89%
798Pharmazie 67 (2012)
Fig. 2: Cell protective effect of DMC on H2O2induced cytotoxicity on MIN6 cells.
Cells were pre-treated with 25?M rhGLP-1 or DMC (6.25∼25?M) for 48h,
then incubated in the presence of 250?M H2O2for 3h. The viability of
control cells was defined as 100%. Data were presented as means±SD and
were representative of an average of three independent experiments per
concentrations. *P<0.05, **P<0.01 compared to H2O2cells. #P<0.05,
##P<0.01 compared to control cells
when exposed to 250?M H2O2 for 3h (Fig. 2). Long term
pre-treatment (48h) of DMC and rhGLP-1 protected the MIN6
cells from H2O2induced toxicity. Pre-treatment with DMC at
25?M increased cell viability by 16.41%, while rhGLP-1 at
with DMC had no cytotoxicity between 6.25 and 25?M (data
2.2. Hoechst nuclear staining
exposed to the pro-apoptotic agents H2O2. Control cells were
regular and their fluorescence was well-distributed throughout
the cell nuclei (Fig. 3A). In contrast, the nuclei of MIN6 cells
exposed to 250?M H2O2exhibited cell shrinkage, chromatin
condensation and fragmented bodies (Fig. 3B). Obviously, cells
(Fig. 3D) had less apoptotic cells than those exposed to H2O2
(Fig. 3B), and the protective effect of DMC was better than
rhGLP-1(7∼36). The treatment with DMC and rhGLP-1 before
2.3. FACS analysis of apoptotic cells
That H2O2-induced apoptosis was further documented using
annexin-V and PI staining. Apoptotic cells were analyzed by
a flow cytometer. Cells in the lower right quadrant indicate
annexin-positive, early apoptotic cells (LR). The cells in the
upper right quadrant indicate annexin-positive/PI-positive, late
apoptotic cells (UR). The cells in the lower left quadrant indi-
cate non-apoptotic (LL). The apoptosis was calculated by UR
cells and LR cells.
The result of annexin-V and PI double staining is shown in
Fig. 4. Long term pretreatment (48h) with different concentra-
H2O2increased cell apoptosis by 70%, whereas DMC at 25?M
rescued theses cells from H2O2induced apoptosis by 32.27%,
which was better than the positive control rhGLP-1 at the same
2.4. Measurement of endogenous reactive oxygen species
Intracellular ROS formation was determined by using DCFH-
DA, a probe which was oxidized to green fluorescent DCF by
reactive oxygen and nitrogen species. The fluorescent intensity
was measured by a FACS. As shown in Fig. 5, the exposure of
Fig. 3: Effect of DMC on the morphology of nuclear chromatin stained by Hoechst33342. Cells were pretreated with 25?M rhGLP-1 and 25?M DMC for 48h, then exposed to
250?M H2O2for 3h. After fixing the cells, they were stained with 10?g/ml Hoechst 33342. Morphological changes of nuclear chromatin were then viewed under a
fluorescence microscope. A. MIN6 cells cultured in regular medium; B. MIN6 cells exposed to 250?M H2O2for 3h. C. MIN6 cells pretreated with 25?M rhGLP-1 for
48h, then exposed to 250?M H2O2for 3h. D.MIN6 Cells pretreated with 25?M DMC for 48h, then exposed to 250?M H2O2for 3h
Pharmazie 67 (2012)799
Fig. 4: Inhibitory effect of DMC on H2O2induced apoptosis in MIN6 cells. FACS
analysis via Annexin V-FITC/PI staining was used to observe the induction of
apoptosis. (A), cells cultured in regular medium; (B), cells exposed to H2O2
(250?M for 3h); (C), cells pretreated with 25?M rhGLP-1. (D)–(F), cells
pretreated with DMC (6.25∼25?M for 48h). Cells in the lower right
quadrant indicate Annexin-positive, early apoptotic cells (LR). The cells in
the upper right quadrant indicate Annexin-positive/PI-positive, late apoptotic
cells (UR). Data were presented as means±SD and were representative of an
average of three independent experiments per concentrations. *P<0.05,
**P<0.01 compared to H2O2cells. #P<0.05, ##P<0.01 compared to
of both 6.25∼25?M DMC and 25?M rhGLP-1 inhibited the
H2O2induced ROS formation in MIN6 cells. The ROS level
was decreased gradually with the raise of the concentration of
2.5. Evaluation of mitochondrial membrane potential
Exposure of MIN6 cells to 250?M H2O2 leaded to 60%
decrease of MMP. DMC not only prevented this decrease but
also improved the MMP in a dose-dependent manner (Fig. 6).
in MMP both at 12.5?M and 25?M (P<0.01 vs H2O2cells)
as well as rhGLP-1 at 25?M. DMC at 25?M was better than
Fig. 5: Effect of DMC on reactive oxygen species in H2O2-induced MIN6 cells using
ROS kit. Cells were pre-treated with 25?M rhGLP-1 or DMC (6.25∼25?M)
for 48h, then incubated in the presence of 250?M H2O2for 3h. After that,
loaded with DCFH-DA probe, and then analyzed by flow cytometry. Data
were presented as means±SD and were representative of an average of three
independent experiments per concentrations. *P<0.05, **P<0.01 compared
to H2O2cells. #P<0.05, ##P<0.01 compared to control cells
induced apoptotic cells.
2.6. Caspase activity
To evaluate whether specific caspase subtypes contributed to
?-cell death, we measured caspase-3 and caspase-9 activities.
H2O2raised the caspase-3 and caspase-9 activities by 109.33%
and 49.33% respectively both compared with control cells
Fig. 6: Changes in mitochondrial membrane potential in MIN6 cells. Cells were
pre-treated with 25?M rhGLP-1 or DMC (6.25∼25?M) for 48h, then
incubated in the presence of 250?M H2O2for 3h. After that, the
mitochondrial membrane potential was analyzed by flow cytometer using
fluorescentdye Rho 123. Data were presented as means±SD and were
representative of an average of three independent experiments per
concentrations. *P<0.05, **P<0.01 compared to H2O2cells. #P<0.05,
##P<0.01 compared to control cells
800Pharmazie 67 (2012)
Fig. 7: DMC reduces caspase-3 and caspase-9 activities in H2O2-induced MIN6
cells apoptosis. Cells were pre-treated with 25?M rhGLP-1 or DMC
(6.25∼25?M) for 48h, then incubated in the presence of 250?M H2O2for
3h. After that, the caspase-3 and caspase-9 activities in MIN6 cells were
detected by caspase-3 and caspase-9 kits. Data were presented as
means±SD and were representative of an average of three independent
experiments per concentrations. *P, #P<0.05, **P, ##P<0.01 compared to
H2O2cells in caspase-3, caspase-9 activity analysis respectively. &&P,
$$P<0.01 compared to control cells
(Fig. 7). DMC at 6.25∼25?M decreased the caspase-3 activity
as well as caspase-9 (compared with H2O2cells). The results
suggested that activation of caspase-3, 9 possibly contributed to
DMC, the main compound from buds of C. operculatus, exerts
various biological actions. One of the effects is its significant
ability to lower the blood glucose in alloxan-diabetic mice.
In an oral glucose tolerance test, at a dosage of 1.0mg/20g
mice, DMC can lower the blood glucose levels in glucose-
hyperglycaemic mice when administered 15min after a glucose
load (Ma et al. 2005). The in vitro study proved that DMC
owns the peroxisome proliferator-activated receptor-? (PPAR-
?) ligand-binding activity, and can promote glucose uptake in
contribute to its anti-diabetic activity. However, a direct effect
of DMC on ?-cells has not been reported.
In the present study, ?-cell damage was induced by H2O2. It is
generally believed that H2O2as a cytotoxic molecule plays an
important role attacking pancreatic ?-cells leading to alteration
of function and eventually ?-cell destruction. It is released from
doses of H2O2can cause cell necrosis; while in low doses, it
induces apoptosis in many cultured cell lines (Hui et al. 2003;
Hampton and Orrenius 1997). We exposed MIN6 cells to H2O2
in different concentrations and incubation times to induce apo-
annexin-V/PI, internucleosomal DNA fragmentation and acti-
vation of caspase-3. Based on these data, exposure to H2O2at
250?M for 3h was chosen to be our research model. The addi-
tion of rhGLP-1 to cells exposed to H2O2could not increase
their survival rate, while the pre-treatment of DMC at 25?M
before the exposure to H2O2had a significant improvement of
Mitochondria are the principal energy sources of the cell
that convert nutrients into energy through cellular respiration
(Wallace 2005). They are key organelles for ?-cell function
survival (Maechler and Wohlheim 2001). However, mitochon-
dria also play a key role in triggering apoptosis (Newmeyer and
chain failure and mitochondrial membrane potential loss rise
in the cell and alter the permeability of their membranes until
the mitochondrial membrane permeabilization is achieved, the
and cytochrome c. Active caspase-9, in turn, activates caspase-
3, leading to activation of the caspase-3 cascade and apoptosis
(Sampson et al. 2010). The caspases represent a novel class of
cytoplasmic cysteine proteases mediating apoptosis. Caspase-3
malian cells. Our data showed that H2O2caused a significant
damage to MIN6 cells. Many cells characterized by cell shrink-
age, chromatin condensation or fragmented bodies were clearly
observed among H2O2treated cells by Hoechst 33342. After
pretreatment with DMC, fluorescent staining analysis revealed
that the cell nucleus was nearly to the normal cells and less
apoptotic cells were observed. The FACS analysis also showed
DMC inhibited cell apoptosis. Furthermore, DMC significantly
(P<0.01) attenuated the caspase-3, 9 activities in MIN6 cells
of DMC was associated with the attenuation of caspase-3, 9
The protective effect of DMC against H2O2induced apoptosis
was also supported by higher MMP and lower ROS. The major
site of ROS production in the cell is the mitochondrial respi-
ratory chain. Mitochondrial radical production associated with
hyperlipidemia and hyperglycemia disrupts glucose-stimulated
insulin secretion and causes cellular damage (Cnop et al. 2005).
its lower antioxidant enzymes level (Lenzen et al. 1996). Hence
strategies to decrease mitochondrial free radical production and
oxidative damage may have therapeutic potential (Green et al.
2004). Pretreatment with resveratrol, a natural polyphenol with
antioxidative properties, for 12h at a concentration of 30?M,
inhibits streptozotocin-induced apoptosis of rat islets (Ku et al.
2011). Curcumin, a potent antioxidant/radical scavenging com-
pound, shows significant inhibition of nitric oxide generation
as compared to streptozotocin-induced islets without curcumin
function at 10?M without affecting the normal function of
these cellular structures (Meghana et al. 2007). Our studies also
showed that DMC pretreatment at 12.5?M and 25?M atten-
uated H2O2induced ROS production which is responsible for
the mitochondrial dysfunction characterized by a decrease in
membrane potential and prevented the loss of MMP.
H2O2induced cell apoptosis by improving mitochondrial func-
still unclear. In summary, the present study provides evidence
pancreatic ?-cells by protecting mitochondria function. There-
fore, the results reported here support the potential of DMC to
be used as an effective anti-apoptosis drug in diabetes therapy.
DMC was isolated from C. operculatus in our lab as described by Ye
et al. (2004 a,b), and its purity was above 98% (HPLC analysis). Recom-
Pharmazie 67 (2012)801
bined human Glucagon-like peptide-1(7∼36) (rhGLP-1(7∼36)) was kindly
given from Huay bio-lab Co. Ltd (Shanghai, China). Dulbecco’s modified
bovine serum (FBS) was obtained from Hyclone (Losan, UT, US); Hoechst
33342 was purchased from Biyuntian institute of Biotechnology (Haimen,
China); Caspase-3,Caspase-9 activity kits and Annexin V-FITC/PI staining
kit were from Keygen (Nanjin, China); 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazoliumbromide (MTT) was from Amersco (Solon, OH, US);
2’,7’-Dichlorofluorescein diacetate (DCFH-DA) was obtained from Sigma
(St. Louis, MO); The other regents were analytic reagents (AR).
4.2. Cell lines and cell culture
Mouse insulinoma cell line, MIN6, was given from Huay bio-lab Co.
Ltd (Shanghai, China) and cultured in DMEM with 10% FBS, 10mM
HEPES, 2mM l-glutamine, 1mM sodium pyruvate, 100 U/ml penicillin
and 100?g/ml streptomycin, at 37◦C under humidified condition of 5%
DMC was dissolved in dimethylsulfoxide (DMSO) and stored at −20◦C
before use. When needed, the stock solution was diluted with cell culture
media for the required concentrations. The final DMSO concentration must
be below 0.1% (v/v) which would not be harmful to cellular activity in
previous experiments. The rhGLP-1(7∼36), taken as positive control, was
obtained in the way of genetic engineering. During the gene recombination,
4.3.1. Cell viability analysis
MIN6 cells were seeded into 96-well plates as 2×104cells/well and pre-
incubated for 4h. After that, the cells were pre-treated at 37 for 48h with or
without DMC (6.25∼25?M) or rhGLP-1(7∼36) (25?M), Then they were
of MTT in the dark, the MTT medium was replaced with DMSO. The
absorbance of the dissolved formazan was measured at 570 and 630nm as
test and reference wavelengths, respectively in an Automated Microplate
Reader (model3550-UV, Bio-Rad, US).
4.3.2. Hoechst nuclear staining
Hoechst 33342 was used to distinguish normal cell nuclei and apoptotic
pre-incubated for 6h. They were pretreated with DMC and rhGLP-1(7∼36)
the control. After treatment, they were washed with PBS (pH7.3) twice and
fixed with 0.5ml 1% paraformaldehyde for 20min at room temperature.
After a remove of the fixing solution, the cells were washed twice with
PBS (pH 7.3) and stained with 10?g/ml Hoechst 33342 for 5min at room
temperature. Then, they were washed in PBS twice. The fluorescence of
Hoechst was visualized under a fluorescence microscope (Olympus XB-51,
4.3.3. Flow cytometric assay of apoptosis
MIN6 cells were seeded into six well plates as 2×104cells/well and pre-
without DMC (6.25∼25?M) or rhGLP-1(7∼36) (25?M), Then they were
exposed to 250?M H2O2for 3h. Cells were then harvested and suspended
(Becton Dickinson calibur, US) using the annexin V-FITC/PI staining kit.
4.3.4. Measurement of endogenous reactive oxygen species (ROS)
DCFH-DA was used to measure intracellular oxidative activity by flow
cytometric analysis. After treatment, cells were harvested and incubated
in 10?M DCFH-DA for 30min at 37◦C in dark condition. Then they were
washed in PBS for several times, the endogenous ROS production were
measured by flow cytometer (Becton Dickinson calibur, US).
4.3.5. Evaluation of mitochondrial membrane potential (MMP)
MMP was monitored by rhodamine123 fluorescence in MIN6 cells. Briefly,
Cells were loaded with 10?g/ml rhodamine123 for 30min at 37◦C in dark
place. After centrifugation, the cells were washed twice and resuspended
with 500?l PBS, followed by analysis on flow cytometer (Becton Dickin-
son calibur, US) with an excitation wavelength of 480nm and an emission
wavelength of 530nm.
4.3.6. Caspase activity
Activation of caspases was measured by caspase-3 and caspase-9 activity
assay kits. Cells were collected by centrifugation, placed on ice after adding
lysis buffer for 40min. The supernatant from lysed cells was mixed with
reaction buffer and substrate for 4h at 37◦C in dark place. The substrates of
caspase-3 and caspase-9 were Ac-DEVD-pNA and Ac-LEHD-pNA respec-
tively. The absorbance of both caspase-3 and caspase-9 was measured at
4.4. Statistical analysis of data
Data were presented as the mean±s.e.m. Statistical significance was tested
with an unpaired two-tailed Student t-test. Statistical data were from at
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