2. Materials and methods
2.8. Quantitative real-time PCR
Each real-time PCR was carried out in triplicate in a 25 μl volume using SYBR
Green qPCR Master Mix (Fermentas Life Sciences,St. Leon-Rot, Germany)
according to the manufacturer’s protocol. Primers sequences were as follows:
survivin 5’-ATTCGTCCGGTTGCGCTTTCC-3’ and
5’-CACGGCGCACTTTCTTCGCAG-3’; β-Actin:
5’-GCGAGAAGATGACCCAGATC-3’ and 5’-GGATAGCAACGCCTGGATAG-3’.
The PCR conditions were for 10 min at 95 °C for initial denaturation, followed by 40
cycles of 95 °C for 15 s and 60 °C for 1 min in the ABI Prism 7000 Sequence
Detection System (Applied Biosystems, Foster City, CA). Relative gene expression
quantifications were calculated according to the comparative Ct method using β-actin
as an internal standard. The fold amplification of genes was respectively detected by
calculating the 2-∆∆Ct of the genes.
2.9. Immunoprecipitation
The PureproteomeTM Protein G Magnetic Beads (Millipore, Bedford, MA) were
mixed so that all of the beads are uniformly resuspended. The beads were plased into
a 1.5 ml microcentrifuge tubes, then tubes were removed into the Magna GrIP Rack
(Millipore). Then the storage buffer was removed with a pipette. The beads were
washed by adding 500 μl of PBS containing 0.1 % Tween® 20 surfactant and
vortexing vigorously for 10 seconds. The tubes were returned to the magnetic rack and allow the beads to adhere to the side. The buffer was removed with a pipette. The
washed beads were resuspend in 350 μl of PBS containing 0.1 % Tween 20 surfactant.
The survivin antibody was added to the resuspended beads with incubation at room
temperature for 30 minutes. Then the tubes were placed into the magnetic rack, and
then the buffer was removed with a pipette. The beads were washed 3 times with 500
μl of PBS containing 0.1 % Tween 20 surfactant. After the last wash, the tubes were
removed from the rack and the cell lysates were added. According to the relative
protein expression of survivin in control and baicalein-treated samples, the total protein lysates were adjusted in immunoprecipitation analysis for equal survivin protein expression of control and baicalein-treated sample. Then samples were
immobilized survivin antibody at 2–8 °C with continuous mixing overnight. The
tubes were placed into the magnetic rack, and then removed the sample with a pipette.
The beads were washed 3 times with 500 μl of PBS containing 0.1 % Tween 20
surfactant. After the last wash, the tube were removed from the magnetic rack and
added the sample buffer for western blot analysis.
2.10. Immunofluorescence staining and confocal microscopy
To view the protein expression of survivin and ubiquitin after baicalein treatment,
the cells were subjected to immunofluorescence staining and confocal microscopy.
After fixation with 4 % paraformaldehyde solution, the cells were washed three times
with PBS, and non-specific binding sites were blocked in PBS containing 10 % FBS
and 0.3 % Triton X-100 for 1 h at 37 °C. Thereafter, the cells were separately
incubated with mouse anti-survivin (1:200) antibody and rabbit anti-ubiquitin (1:400)
antibody in PBS containing 10 % FBS for 1 h at 37 °C, and washed three times with
0.3 % Triton X-100 in PBS. Then the cells were individually incubated with goat
anti-mouse Cy3 (1:200) and anti-rabbit FITC in PBS containing 10 % FBS for 1 h at
37 °C. The nuclei were stained with Hoechst 33258. The samples were examined
under a confocal microscope Fluoview 300 (Olympus, Tokyo, Japan).
2.11. Statistical analysis
Data were analyzed using Student’s t test, and a p value of <0.05 was considered
as statistically significant in the experiments.
3. Results
3.1. Baicalein induces cytotoxicity and proliferation inhibition in bladder cancer cells
To examine the cytotoxicity and proliferation following baicalein treatment in
BFTC905 bladder cancer cells, the cells were analyzed by MTT assay. Treatment
with 20–100 μM baicalein for 24 h significantly reduced the cell viability via a
concentration-dependent manner in BFTC905 cells (Fig. 1). The value of IC50 (the
concentration of 50 % inhibition of cell viability) was around 30 μM. Moreover,
baicalein inhibited cell proliferation and induced cell death that can be observed by
time-lapse living cell morphology observation alteration. The arrows show that
baicalein induced the cell death at 24 h observation (Fig. 2). However, the untreated
cells clearly displayed the increase of cell proliferation and cell number at 24 h
observation (Fig. 2).
3.2. Baicalein inhibits survivin protein expression but not altered gene expression in bladder cancer cells
To study the effect of survivin protein expression by baicalein in BFTC905
bladder cancer cells, the baicalein-treated cells were analyzed by Western blot. The
protein levels of survivin were decreased by 20–80 μM baicalein for 24 h in
BFTC905 cells (Fig. 3A and 3B). The quantified data also shows that baicalein
significantly reduced survivin protein expression in BFTC905 cells (Fig. 3B).
Moreover, we have further investigated the survivin expression on transcriptional
levels by reverse transcription-PCR and real-time PCR. The qualities of total RNA
extracts were presented by the contents of 28S rRNA and 18S rRNA (Fig. 4A).
However, baicalein did not alter the survivin mRNA expression (Fig. 4B and 4C).
The survivin mRNA expression in baicalein-treated cells was compared to the control
for 24 h by real-time PCR (Fig. 5A). The mRNA level of survvin was not statistically
altered by treatment with baicalein (Fig. 5B).
3.3. Baicalein reduces the protein stability of survivin in bladder cancer cells
To further determine the effect of baicalein on the protein stability and half-life
of survivin proteins, a protein synthesis inhibitor (cycloheximide, CHX) was
examined on the effect of survivin protein expression. Treatment with 10 μg/ml CHX
for 24 h reduced around the half of total amount of survivin proteins (Fig. 6A).
However, treatment with CHX and baicalein, the survivin protein levels were
decreased more quickly than CHX alone at 6–24 h (Fig. 6B). Co-treatment of CHX
and baicalein almost completely blocked the survivin protein expression after 12 h
period (Fig. 6B).
3.4. Proteasome inhibitor decreases baicalein-induced survivin protein degradation in bladder cancer cells
To investigate the role of proteasome on baicalein-induced down-regulation of
survivin protein expression, MG132 (a proteasome inhibitor) was utilized in this
study. Treatment with 60 μM baicalein for 24 h significantly reduced survivin protein
expression (Fig. 7). Pre-treatment of BFTC905 cells with 5 μM MG132 potentially
reversed survivin protein level in the baicalein-treated cells (Fig. 7). In annevin V/PI
apoptosis assay, baicalein increased higher apoptosis level than untreated sample in
BFTC905 cells (Fig. 8). Moreover, pre-treatment of MG132 potentially inhibited
apoptosis in the baicalein-treated BFTC905 cells (Fig. 8).
3.5. Baicalein treatment increases ubiquitination of survivin
We have determined the effect of baicalein on the protein ubiquitination of
survivin. As shown in Fig. 9, baicalein induced ubiquitinated survivin levels in
BFTC905 cells. To further confirm the ubiquitination of survivin proteins, the
baicalein-treated cells were subjected to immunofluorescence staining and confocal
microscopy. Baicalein reduced the intensity of red fluorescence (Cy3) of survivin
proteins. However, baicalein induced high intensity of green fluorescence of ubiquitin
proteins. The yellow color indicated that co-locolization of survivin and ubiquitin
(Fig. 9, arrows).
3.6. Co-treatment of baicalein and oxaliplatin enhances the
cytotoxicity and survivin protein inhibition in bladder cancer cells
As shown in Fig. 11, co-treatment with 10–50 μM baicalein and 1 μM
oxaliplatin for 24 h enhanced the cytotoxicity in BFTC905 cells. To study the
combination effect of baicalein and oxaliplatin on the survivin protein inhibition, the
cells were co-treated with baicalein and oxaliplatin followed by Western blot analysis.
Both of baicalein and oxaliplatin significantly reduced survivin protein expression
(Fig. 12A and 12B). Co-treatment with baicalein and oxaliplatin for 24 h enhanced
the decrease of the survivin protein expression (Fig. 12A and 12B).
4. Discussion
Various types of flavonoids display anticancer effects on growth inhibition and
apoptosis (Brusselmans et al., 2005; Lee et al., 2005a; Psahoulia et al., 2007; Spencer
et al., 2003; Yin et al., 2001). In this study, baicalein significantly inhibited cell
viability in the human BFTC905 bladder cancer cells. We also found that baicalein
induced growth inhibition and cell death by time-lapse observation in BFTC905 cells.
Survivin has been demonstrated to inhibit apoptosis and to promote mitotic
progression in cancer cells (Ambrosini et al., 1997; Li et al., 1998). Interestingly,
baicalein significantly reduced survivin protein expression in BFTC905 cells.
However, baicalein did not influence the survivin mRNA expression by RT-PCR and
real-time PCR assays. As a consequence, we suggest that baicalein inhibits the
survivin expression on the alteration of protein level but not gene expression.
Ubiquitin-proteasome pathway is an essential mechanism participating in
cellular process. Proteasome degradation has also been shown to play an important
role in regulation of apoptosis and cell proliferation by indomethacin (Chiou and
Mandayam, 2007). Furthermore, kaempferol and quercetin enhanced apoptosis by
degradation of survivin in glioma cells (Siegelin et al., 2008; Siegelin et al., 2009). It
has been shown that survivin can be degraded via the ubiquitin-proteasome pathway
in a cell cycle-dependent manner (Zhao et al., 2000). We have shown that baicalein
inhibited survivin protein expression (Chao et al., 2007); however, suppression of
survivin on the post-translational level by baicalein has not been shown previously.
We have further determined the half-life of survivin by baicalein. Baicalein enhanced
the survivin protein degradation when the cells were co-treated with CHX. The data
indicates that baicalein induces the survivin protein instability in bladder cancer cells.
Furthermore, MG132 proteasome inhibitor prevented survivin protein degradation in
the baicalein-treated cells. In addition, MG132 can reduce the baicalein-induced
apoptosis. Baicalein also activated ubiquitination of survivin in bladder cancer cells.
Accordingly, our findings suggest that baicalein inhibits survivin protein expression
through the ubiquitin-proteasome pathway in human bladder cancer cells. We provide
a model of baicalein-induced down-regulation of survivin as shown in Fig. 13.
The stability of survivin resulted from the protein phosphorylation at Thr34 by
the mitotic kinase complex CDK1/cyclin B1 (O'Connor et al., 2000a; Wall et al.,
2003). Quercetin increases the survivin protein expression, which correlates with
raising the protein levels of cyclin B1 and phospho-CDK1 (Kuo et al., 2004). The
levels of cyclin B1 and CDK1 were reduced for inducing G2/M arrest by baicalein in
bladder cancer cells (Chao et al., 2007). The role of CDK1/cyclin B1 on the
regulation of survivin protein should be further investigated. It has been shown that
XAF1-XIAP complex enhanced degradation of survivin protein (Arora et al., 2007).
Therefore, further investigations are required to determine the roles of XAF1-XIAP
and CDK1/cyclinB1 on the regulation of survivin protein expression following
baicalein in bladder cancer cells.
Combination of various anticancer agents may increase the efficiency of cancer
therapy (Hochster et al., 2003; Ramanathan et al., 2003; Rathkopf et al., 2009).
Previously, Oxaliplatin reduced survivin protein expression and induced apoptosis in
cancer cells (Lin et al., 2005). In this study, we have further examined the anticancer
effects of combination of baicalein and oxaliplatin on the cell viability and survivin
expression in human bladder cancer cells. Both baicalein and oxaliplatin significantly
induced cell death in BFTC905 cells. Interestingly, co-treatment of baicalein and
oxaliplatin additively decreased the levels of survivin proteins and increased
cytotoxicity in BFTC905 cells. Accordingly, the combination of baicalein and
oxaliplatin may increase anticancer effects on survivin inhibition and cancer cell
death, providing important strategy for cancer therapy.
5. Conclusion
We have summerized that the down-regulation of survivin by baicalein is
mediated ubiquitin-proteasome degradation pathway in human bladder cancer cells
(Fig. 13). Our findings indicate that the blockage of survivin by baicalein may
provide the novel strategies for elevating the efficiency of cancer therapy in bladder
cancer.
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