SCC carried out the most of the experiments and drafted the manuscript. MJW, HHY, YLC, SZL
participated in the design and coordination of the study. SPC carried out the statistical analysis. SYH
carried out the immunostaining. HJH and CYP conceived of the study, participated in its design and
coordination, and drafted the manuscript. All authors read and approved the final manuscript.
Acknowledgements
This work was partly supported by grants from the National Science Council, Taiwan
(NSC-93-2320-B-303-004, NSC-94-2320-B-303-005 and NSC-96-2113-M-303-001), and Buddhist
Tzu-Chi General Hospital, Hualien, Taiwan (TCSP-01-02). We thank Dr. Tony Jer Fu Lee (PhD,
Dean of College of Life Sciences, Tzu Chi University, Hualien, Taiwan) for reviewing the
manuscript.
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Figure Legends
Figure 1 - Morphological changes and anti-proliferation effects after isochaihulactone
treatment of prostate cancer cells. (A) Human prostate cancer cell lines DU-145, PC-3, LNCaP
were treated with isochaihulactone from 6.25 to 50 µM at 48 h and analyzed with the MTT assay.
LNCaP cells were treated with 0.2% DMSO as a control (B) or 20 µM isochaihulactone (C) for 24 h.
LNCaP cells were treated with increasing concentration of isochaihulactone from 3.125 to 50 µM at
various times from 24 to 72 h and analyzed with the MTT assay (D). The data represent the means ±
S.D. from three independent experiments. **, P <0.01 versus vehicle.
Figure 2 - Isochaihulactone apparently induces G2/M phase arrest and changes the
expression profiles of G2/M regulatory proteins. (A) Isochaihulactone induced cell cycle arrest at
G2/M in LNCaP cells. For the cell cycle analysis, cells were seeded at 5x105 per 5-cm plate in
triplicates and treated with 20 µM isochaihulactone for 12-48 h. The data represent the means ±
S.D. from three different experiments. *, P <0.05; **, P <0.01; versus control. (B) Cells were treated
with 20 µM isochaihulactone for 6-72 h. Western blot analysis of p53, p21, cdc25c, cyclin B1 and
cdc2 was performed. β-actin was used as an internal control.
Figure 3 - Isochaihulactone induces cell death and initiates Bcl-2 phosphorylation and
caspase activation in LNCaP cells. LNCaP cells were treated with 0.2% DMSO (A) or 20 µM
stained with DAPI. LNCaP cells were treated with 0.2% DMSO (C) or 20 µM isochaihulactone (D)
for 60 h and then were fixed and stained with the TUNEL assay. Nuclei were stained with DAPI. (E)
Isochaihulactone induced caspase-9 activation, followed by Bcl-2 phosphorylation and then
caspase-3 activation. Cells were treated with 20 µM isochaihulactone for the indicated time and
analysis by Western blotting. Membranes were probed with caspase-8, phosphor-Asp330 caspase-9,
phosphor-Ser70 Bcl-2, cleaved-caspase-3, PARP antibodies. β-actin was used as an internal control.
Figure 4 - Growth inhibition of LNCaP cells induced by isochaihulactone is partially rescued
by JNK1/2 inhibitor. (A) MTT assay of LNCaP cells pretreated with p38 inhibitor SB203580 (10 or
20 µM), the JNK1/2 inhibitor SP600125 (10 or 20 µM) or the ERK1/2 inhibitor PD98059 (25 or 50
µM) for 1 h and then treated with 20 µM of isochaihulactone for 48 h. The values are the mean ±
S.D. from three independent experiments performed in duplicate. (B) Cells were treated with 20 µM
isochaihulactone for the indicated times. Phospho-ERK1/2, total-ERK1/2, phospho-JNK, total-JNK,
phospho-38, total-p38 were detected by western blotting. (C) Cells were treated with 20 µM
isochaihulactone for 48 h in the presence or absence of JNK1/2 inhibitor SP600125 (20 µM). Cell
cycle analysis was done as described in Methods. Isochaihulactone-induced sub-G1 population
(20.51%) was decreased by JNK1/2 inhibitor SP600125 pre-treatment (7.54%). The data represent
the means ± S.D. from three independent experiments. **, P <0.01; ***, P <0.001 versus vehicle.
Figure 5 - Isochaihulactone induces NAG-1 expression via JNK1/2 activation, and
isochaihulactone-induced cell death can be rescued by NAG-1 siRNA transfection. (A)
Expression of EGR-1 and NAG-1 after treatment of LNCaP cells with 20 µM isochaihulactone for
the indicated times. (B) NAG-1 expression of LNCaP cells pretreated with the p38 inhibitor
SB203580 (20 µM), the JNK1/2 inhibitor SP600125 (20 µM), or the MEK1/2 inhibitor PD98059 (50
µM) for 1 h and then treated with 20 µM isochaihulactone for 24 h. (C) Suppression of
isochaihulactone-induced NAG-1 expression in LNCaP cells by NAG-1 siRNA transfection. LNCaP
cells were transfected with scramble siRNA (#) or 20 nM, 40 nM NAG-1 siRNA for 48 h using the
RNAifect transfection reagent followed by treatment with 20 µM isochaihulactone for 48 or 72 h.
Western blot analysis was performed for NAG-1. (D) Isochaihulactone-induced anti-proliferative
activity was measured with the MTT assay in LNCaP cells transfected with scramble (#) or NAG-1
siRNA for 48 h and then treated with 20 µM isochaihulactone for 48 h. The data represent the means
± S.D. from three independent experiments. ***, P <0.001 versus vehicle.