Celecoxib and acetylbritannilactone interact synergistically to suppress breast
cancer cell growth via COX-2-dependent and -independent mechanisms
B Liu1,2, JK Wen1, BH Li3, XM Fang1, JJ Wang1, YP Zhang1, CJ Shi1, DQ Zhang4 and M Han*,1
1
Department of Biochemistry and Molecular Biology, Institute of Basic Medicine, Key Laboratory
of Neural and Vascular Biology, Ministry of Education, Hebei Medical University, Shijiazhuang,
PR China;
2
Hebei Provincial Crops Hospital of Chinese People’s Armed Police Force,
Shijiazhuang, PR China; 3Department of Surgery, Fourth Hospital, Hebei Medical University,
Shijiazhuang, PR China and 4College of Pharmacy, Hebei Medical University, Shijiazhuang, PR
China
Supplementary Figures S1-S7
Supplementary Figure S1. The combination treatment of celecoxib and ABL on
MDA-MB-231 cells. (a) Western blot analysis for COX-2 expression in MDA-MB-231,
MDA-MB-468 and MCF-7 cells. MDA-MB-231 cells showed high level of constitutive COX-2
protein expression, MDA-MB-468 and MCF-7 expressed no constitutive COX-2 expression. (b)
After exposure to the indicated doses of ABL with celecoxib (2.5 or 5.0 μM) in MDA-MB-231 cells
for 48 hours, cell growth inhibition was determined. Results were expressed as mean ± S.E.M. from
at least three independent experiments. (c) MDA-MB-468 and MCF-7 cells were treated with
celecoxib (2.5-10 μM) plus ABL (12.5-100 μM) at a fixed ratio of 1:5 or 1:10, and then the
combination index (CI) was calculated.
Supplementary Figure S2. Effects of celecoxib, ABL or their combinations on the apoptosis
of breast cancer cells. (a) The percentage of apoptotic cells was determined by DAPI staining using
fluorescence microscopy. *, P < 0.05, compared with vehicle-treated cells. (b) The percentage of
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apoptotic cells was determined by DAPI staining in MDA-MB-468 cells. (c) PARP, caspase-3 and
caspase-9 proteins were analyzed by Western blot.
Supplementary Figure S3. Exogenous PGE2 prevented the combination-induced apoptosis in
MDA-MB-231 and MCF-7-COX-2 cells. (a) Results of RT-PCR (left) and Western blots (right) for
COX-2 expression in Control and KD cells. PGE2 production in Control and KD cells was
determined by ELISA. Control, MDA-MB-231 cells transfected with a general negative sequence
siRNA; KD, COX-2 knocked down MDA-MB-231 cells. (b) COX-2 expression was analyzed by
Western blot in the MCF-7-mock and MCF-7-COX-2 cells. (c) MDA-MB-231 and MCF-7-COX-2
cells were exposed to the combination and varying amounts of exogenous PGE2 for 48 hours.
Apoptotic cells were detected and results were expressed as mean ± S.E.M.
Supplementary Figure S4. Effects of the combination of celecoxib and ABL on COX-2
expression and activity in breast cancer cells. (a) MDA-MB-231 cells were treated for 48 hours,
COX-2 and COX-1 were immunostained with anti-COX-2 or anti-COX-1 antibodies (green).
Nuclei were stained with PI (red). (b) After transfected with COX-2-Luc reporter, MCF-7 cells
were treated with TPA and the combination for 24 hours. Luciferase activity was measured. Results
were expressed as mean ± S.E.M. *, P < 0.05, compared with TPA-treatment group only. (c)
MCF-7 cells were cotreated with TPA and the combination for 48 hours. COX-2 and COX-1
expression were detected. Densitometric analysis of COX-2 protein expression, normalized to actin
levels, was provided.
Supplementary Figure S5. Effects of the combination of celecoxib and ABL on G0/G1 cell
phase. (a) Cells were exposed to the combination and varying amounts of exogenous PGE2 for 24
hours. The rates of G0/G1 phase cells (for Control, KD, MCF-7-COX-2, MCF-7-mock, and
MDA-MB-468 cells, respectively) were detected. (b) Expression of p21 was tested in the cells by
Western blot. Densitometric analysis of p21 expression, normalized to actin levels, was provided.
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Supplementary Figure S6. Enhanced Akt and p38 activities restored COX-2 activity and
transcription after combination treatment. (a) MCF-7 cells were transfected with the COX-2-Luc
plasmid along with Ca-Akt or MKK6b vector and then treated with the combination of celecoxib
and ABL for 24 hours. COX-2 promoter activity was measured. Results were expressed as mean ±
S.E.M. (b) The transfected cells were treated with TAP and the combination for 24 hours. COX-2
mRNA expression was examined using RT-PCR. Quantification of COX-2 mRNA, normalized to
actin levels, was provided.
Supplementary Figure S7. Effects of celecoxib and ABL individually and in combination on
MDA-MB-231 Xenograft model. (a) Immunohistochemistry with COX-2 antibody was performed
in paraffin-included sections of xenograft specimens, and the semiquantification of the differences
in the COX-2 expression was presented as a bar graph. (b) The level of PGE2 in mice serum was
measured using ELISA kit. Results were expressed as mean ± S.E.M. obtained from at least three
independent experiments. *, P < 0.05, compared with vehicle group.
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