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http://jpet.aspetjournals.org/content/suppl/2014/11/04/jpet.114.218792.DC1
1521-0103/352/1/175–184$25.00 http://dx.doi.org/10.1124/jpet.114.218792
THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 352:175–184, January 2015
Copyright ª 2014 by The American Society for Pharmacology and Experimental Therapeutics
Seong-Hoon Kim, Ha-Na Lyu, Ye Seul Kim, Yong Hyun Jeon, Wanil Kim, Sangjune Kim,
Jong-Kwan Lim, Ho Won Lee, Nam-In Baek, Kwan-Yong Choi, Jaetae Lee, and
Kyong-Tai Kim
Department of Life Sciences (S.-H.K., H.-N.L., Y.S.K., W.K., S.K., K.-T.K.), Division of Integrative Biosciences and Biotechnology
(J.-K.L., K.-Y.C., K.-T.K.), Pohang University of Science and Technology, Pohang, South Korea; Graduate School of
Biotechnology and Plant Metabolism Research Center, Kyung-Hee University, Suwon, South Korea (N.-I.B.); and Department of
ABSTRACT
To date, many anticancer drugs have been developed by directly and demonstrated that it inhibited BAF phosphorylation in vitro
or indirectly targeting microtubules, which are involved in cell and in vivo. Moreover, we demonstrated direct binding between
division. Although this approach has yielded many anticancer brazilin and BAF. The inhibition of BAF phosphorylation induced
drugs, these drugs produce undesirable side effects. An abnormal nuclear envelope reassembly and cell death, indicating
alternative strategy is needed, and targeting mitotic exit may be that perturbation of nuclear envelope reassembly could be a novel
one alternative approach. Localization of phosphorylated barrier- approach to anticancer therapy. We propose that brazilin isolated
to-autointegration factor (BAF) to the chromosomal core region is from C. sappan may be a new anticancer drug candidate that
essential for nuclear envelope compartment relocalization. In this induces cell death by inhibiting vaccinia-related kinase 1–mediated
study, we isolated brazilin from Caesalpinia sappan Leguminosae BAF phosphorylation.
Introduction Maiato, 2004; Brito and Rieder, 2006). Thus, rather than
targeting spindle assembly, an alternative strategy of target-
Drugs that depolymerize and stabilize microtubules, in- ing mitotic exit has been proposed (Manchado et al., 2012). In
cluding vinca alkaloids (vinblastine and vincristine) and contrast to strategies that rely on microtubule disruption,
taxanes (paclitaxel/taxol and docetaxel), have long been used directly targeting mitotic exit does not promote adaptation or
to treat cancer (Jordan and Wilson, 2004; Dumontet and mitotic slippage. Therefore, if cancer cells are damaged at
Jordan, 2010). In addition to their anticancer effects, however, mitotic exit, they cannot escape death. To date, however,
these drugs cause various side effects. To avoid this problem, drugs that target mitotic exit have not been commonly used.
researchers have attempted to develop drugs that indirectly To complete mitotic exit, nuclear membrane reformation
target microtubules via microtubule regulator proteins, such and cytokinesis must take place. Nuclear envelope reassem-
as polo-like kinase 1 (Plk1), Aurora A and B, and Eg5 (Blangy bly is a phenomenon in which the nuclear membrane and
et al., 1995; Carmena and Earnshaw, 2003; Barr et al., 2004). associated proteins, such as barrier-to-autointegration factor
However, some cancer cells that are treated with these drugs (BAF), LEM-domain proteins (LAP2, emerin, and MAN1),
skip mitotic arrest and survive as a result of adaptation or lamin, and nuclear pore complex, relocate around separated
mitotic slippage in which the spindle assembly checkpoint chromosomes during telophase (Haraguchi et al., 2001;
does not activate cell cycle arrest after chromosomal segrega- Dechat et al., 2004). BAF is the first of the nuclear
tion, resulting in the maintenance of aneuploidy (Rieder and membrane–associated proteins to be recruited to the core
region, the central region of the assembling nuclear rim near
spindle microtubules during nuclear envelope reformation
This work was supported by grants from the National Research Foundation
of Korea (NRF) [Grant 2013-056085, 2014R1A2A2A01002931]; the Next-
(Haraguchi et al., 2008). Relocalization of BAF into the core
Generation BioGreen 21 Program [No. PJ00950301] funded by the Korean region of chromosomes leads to recruitment of other nuclear
Rural Development Administration; and the BK21 Plus program [Grant membrane–anchored proteins and nuclear envelope trans-
10Z20130012243] funded by the Korean Ministry of Education.
dx.doi.org/10.1124/jpet.114.218792. membrane proteins. Therefore, BAF is key in controlling the
s This article has supplemental material available at jpet.aspetjournals.org. escape of dividing cells from mitotic exit. Phosphorylated
ABBREVIATIONS: BAF, barrier-to-autointegration factor; DMSO, dimethylsulfoxide; DsRed, Discosoma fluorescent red; EtOAc, ethyl acetate;
GFP, green fluorescent protein; IR, infrared; MS, magnetic spectrometry; PBS, phosphate-buffered saline; TUNEL, terminal deoxynucleotidyl
transferase–mediated digoxigenin-deoxyuridine nick-end labeling; VRK1, vaccinia-related kinase 1.
175
176 Kim et al.
residues of vaccinia-related kinase 1 (VRK1), a unique Santa Clara, CA). The [1H]NMR, [13C]NMR, distortionless enhance-
upstream kinase of BAF, are involved in the dissociation of ment by polarization transfer, correlation spectroscopy, heteronuclear
chromosomes from the nuclear envelope at mitosis onset and multiple-bond correlation, and heteronuclear multiple-quantum corre-
nuclear envelope reformation at the end of mitosis (Nichols lation NMR spectra were obtained with a Bruker DRX-500 Fourier
transform-NMR spectrometer (Bruker Corporation, Bruker, Billerica,
et al., 2006; Haraguchi et al., 2008). In Caenorhabditis elegans
MA). The internal standard, methanol-d4 with trimethoxysilane, was
embryos, depletion of the VRK1 homolog abolishes targeting purchased from Sigma-Aldrich (St. Louis, MO). Merck silica gel 60
of the BAF-1 homolog to the core-like region and prevents (63–200 mm) was used for column chromatography. Analytical thin-
nuclear envelope reformation (Gorjanacz et al., 2007). Indeed, layer chromatography was performed using Merck silica gel 60 F-254
BAF phosphorylation by VRK1 is required for nuclear and silica gel 60 RP-18 F-254 thin-layer chromatography plates. Spots
envelope reassembly, although the process is so complex that were detected by UV light (254 and 365 nm) and by spraying with 10%
the detailed molecular mechanism remains elusive. H2SO4 and heating on a hot plate.
We hypothesized that inhibition of VRK1-mediated BAF
phosphorylation might trigger severe cell cycle defects
because BAF phosphorylation is essential for nuclear enve- Reagents
lope reassembly. To identify novel inhibitors of BAF phos- Lamin B, glyceraldehyde 3-phosphate dehydrogenase, and gluta-
phorylation, we screened a natural herb library. To date, thione S-transferase antibody were purchased from Santa Cruz
natural products have been used to develop new treatments Biotechnology (Santa Cruz, CA) and histone H3 phospho-Ser10
for many diseases. Plants contain a variety of metabolites for antibody was purchased from Abcam (Cambridge, UK) and those
et al., 2006). During the flow period, the intensity of the relative to control cells (Fig. 4B). These data show that control
interaction between brazilin and BAF was assessed via signal cells progress normally from M phase to interphase, whereas
alterations. The dissociation constant (KD) was 38.35 mM, nuclear envelope reformation is disrupted in brazilin-treated
suggesting a good affinity between BAF and brazilin (Table 1). cells. Similarly, in siRNA-vrk-1 C. elegans embryos, the nuclear
Brazilin Disrupts Nuclear Envelope Reassembly by rim signals mAb414 and Nup96 disappear from the nuclear
Inhibiting BAF Phosphorylation. BAF phosphorylation periphery (Gorjanacz et al., 2007).
is a prerequisite for nuclear envelope reassembly during Because nuclear envelope reformation is a final step in
telophase (Haraguchi et al., 2008). Phosphorylation of BAF mitosis, failure of the nuclear envelope to reassemble could
leads to its localization at the core region of the dividing delay mitotic exit. To determine whether brazilin-mediated
chromosome, inducing the recruitment of other nuclear inhibition of lamin reformation delays mitotic progression,
envelope-associated proteins, including LEM-domain pro- we observed the cell cycle distribution of A549 cells using
teins, lamin, and nuclear pore complex. To determine flow cytometry. Treatment with brazilin increased the
whether brazilin perturbs recruitment of nuclear envelope- proportion of G2/M cells 1.5-fold relative to control cells
associated proteins by blocking VRK1-mediated BAF phos- (Fig. 4, C and D).
phorylation, we employed immunocytochemistry to observe G2/M arrest induced by brazilin in multiple myeloma U266
the location of lamin during telophase. In cells treated with cells has also been observed (Kim et al., 2012). These results
brazilin, lamin dispersed outside the nucleus, whereas in imply that brazilin-induced disruption of lamin localization
control cells it was located around the rim of the nucleus, as perturbs normal cell cycle progression. In particular, brazilin
previously reported (Fig. 4A). The number of cells with lamin disrupts recruitment of lamin to the anaphase chromatin
localization defects was 6-fold higher in brazilin-treated cells surface, delaying mitotic progression.
180 Kim et al.
TABLE 1
Kinetic parameters of the binding of brazilin to BAF
Ka Kd KD
M21S21 S21 mM
BAF/brazilin 4.873 1.869e24 38.35
benefits over targeting other mitotic kinases or microtubules. inhibitors investigated as a substitute for microtubule blockers
To test our hypothesis, we attempted to identify a specific have displayed weak anticancer effects (Komlodi-Pasztor et al.,
inhibitor of BAF phosphorylation. We screened a library of 2012; Mitchison, 2012).
natural compounds and isolated brazilin, a BAF inhibitor We and other researchers expected that mitotic exit might
from C. sappan. We confirmed that brazilin isolated from represent an alternative target, because it would not allow
C. sappan inhibits VRK1-mediated BAF phosphorylation in cancer cells to survive via mitotic slippage or adaptation. An
vitro and in vivo. In addition, we determined that brazilin- example in support of this hypothesis is that tumor regression
induced BAF inhibition blocks nuclear envelope reformation is greater in response to a treatment ablating Cdc20, which
during telophase, inducing cell death in cancer cells. activates anaphase promoting complex, than in response to
Brazilin has several merits as an anticancer drug. First, other mitotic drugs currently used in cancer therapy (Manchado
BAF is an excellent target compared with other cancer drug et al., 2010).
targets, such as microtubules or microtubule regulatory We selected brazilin, a BAF inhibitor, to target the end of
proteins. Despite reliable anticancer activity, microtubule mitosis. BAF plays a role in recruiting nuclear envelope-
inhibitors have severe side effects. And contrary to expectation, associated proteins, including lamin, emerin, MAN1, and
at tolerable concentrations, microtubule regulatory protein LAP2, to the chromosomal core region (Haraguchi et al.,
182 Kim et al.
2008), supporting nuclear envelope reformation at mitotic exit diagnosis of cancers (Fischer et al., 2004; Bussolati, 2008;
(Haraguchi et al., 2001; Molitor and Traktman, 2014). Phos- Bussolati et al., 2008; Petersen et al., 2009; Gorjanacz, 2014).
phorylation by VRK1, a novel BAF kinase, determines BAF Cancer cells are so susceptible to nuclear envelope alterations
localization during nuclear envelope reassembly (Haraguchi that drugs that disturb nuclear envelope distribution or
et al., 2008; Molitor and Traktman, 2014). Therefore, inhibition reformation could easily induce cell death. Therefore, a treat-
of VRK1-mediated BAF phosphorylation deserves attention in ment that weakens the nuclear envelope might display
the field of cancer treatment. synthetic lethality (Gorjanacz, 2014). As BAF supports
Recently, it was reported that aberrant alterations of nuclear envelope structures, the depletion or altered expres-
nuclear envelope components, including lamin, emerin, sion of BAF results in nuclear envelope alterations (Molitor
LAP2, and nucleoporin, are connected to cancer development and Traktman, 2014). Therefore, targeting BAF could trigger
(Chow et al., 2012). Cancer cells often have altered nuclear synthetic lethality by destroying the weak nuclear envelope of
structures, including nuclear envelope invaginations and cancer cells. Because depletion of VRK1 disrupts nuclear
multilobulation; in fact, these features are used in the clinical envelope morphology—consistent with the BAF mutation
Brazilin as BAF Inhibitor 183
phenotype (Molitor and Traktman, 2014)—targeting VRK1 Byun S, Lee KW, Jung SK, Lee EJ, Hwang MK, Lim SH, Bode AM, Lee HJ, and Dong
Z (2010) Luteolin inhibits protein kinase C(epsilon) and c-Src activities and UVB-
may induce synthetic lethality as well. induced skin cancer. Cancer Res 70:2415–2423.
For these reasons, several researchers have considered the Carmena M and Earnshaw WC (2003) The cellular geography of aurora kinases. Nat
Rev Mol Cell Biol 4:842–854.
possibility of developing BAF phosphorylation inhibitors as Choi SY and Moon CK (1997) Effects of brazilin on the altered immune functions in
anticancer drugs (Gorjanacz, 2014). We have reported that the early phase of halothane intoxication of C57BL/6 mice. Planta Med 63:400–404.
Chow KH, Factor RE, and Ullman KS (2012) The nuclear envelope environment and
Obtusilactone B, an inhibitor of BAF phosphorylation, its cancer connections. Nat Rev Cancer 12:196–209.
perturbs detachment of chromosomes from the nuclear Cragg GM, Grothaus PG, and Newman DJ (2009) Impact of natural products on
envelope at S phase and M phase entry, thus displaying developing new anti-cancer agents. Chem Rev 109:3012–3043.
Dechat T, Gajewski A, Korbei B, Gerlich D, Daigle N, Haraguchi T, Furukawa K,
anticancer activity (Kim et al., 2013). However, treatment Ellenberg J, and Foisner R (2004) LAP2alpha and BAF transiently localize to
with Obtusilactone B did not result in obstruction of telo- telomeres and specific regions on chromatin during nuclear assembly. J Cell Sci
117:6117–6128.
phase, which would be more effective. In this article, we Dumontet C and Jordan MA (2010) Microtubule-binding agents: a dynamic field of
propose that blocking mitotic exit via brazilin-induced BAF cancer therapeutics. Nat Rev Drug Discov 9:790–803.
Fischer AH, Bardarov S, Jr, and Jiang Z (2004) Molecular aspects of diagnostic
inhibition may be a good target that results in cancer cell nucleolar and nuclear envelope changes in prostate cancer. J Cell Biochem 91:
death. 170–184.
Gordaliza M (2007) Natural products as leads to anticancer drugs. Clin Transl Oncol
The mechanism by which brazilin binds specifically to BAF 9:767–776.
and impairs only telophase progression, but not S phase, Gorjánácz M (2014) Nuclear assembly as a target for anti-cancer therapies. Nucleus
5:47–55.
remains elusive. Brazilin may directly bind the Thr-2, Thr-3, Gorjánácz M, Klerkx EP, Galy V, Santarella R, López-Iglesias C, Askjaer P,
and Ser-4 residues of BAF, which are known VRK1-mediated and Mattaj IW (2007) Caenorhabditis elegans BAF-1 and its kinase VRK-1 par-
Exome sequencing and functional analysis identifies BANF1 mutation as the cause Shimi T, Koujin T, Segura-Totten M, Wilson KL, Haraguchi T, and Hiraoka Y (2004)
of a hereditary progeroid syndrome. Am J Hum Genet 88:650–656. Dynamic interaction between BAF and emerin revealed by FRAP, FLIP, and FRET
Rieder CL and Maiato H (2004) Stuck in division or passing through: what happens analyses in living HeLa cells. J Struct Biol 147:31–41.
when cells cannot satisfy the spindle assembly checkpoint. Dev Cell 7:637–651. Weaver BA and Cleveland DW (2005) Decoding the links between mitosis, cancer, and
Segura-Totten M, Kowalski AK, Craigie R, and Wilson KL (2002) Barrier-to- chemotherapy: the mitotic checkpoint, adaptation, and cell death. Cancer Cell 8:7–12.
autointegration factor: major roles in chromatin decondensation and nuclear as- Won HS, Lee J, Khil LY, Chae SH, Ahn MY, Lee BH, Chung JH, Kim YC, and Moon
sembly. J Cell Biol 158:475–485. CK (2004) Mechanism of action of Brazilin on gluconeogenesis in isolated rat
Sevilla A, Santos CR, Barcia R, Vega FM, and Lazo PA (2004a) c-Jun phosphoryla- hepatocytes. Planta Med 70:740–744.
tion by the human vaccinia-related kinase 1 (VRK1) and its cooperation with the
N-terminal kinase of c-Jun (JNK). Oncogene 23:8950–8958.
Sevilla A, Santos CR, Vega FM, and Lazo PA (2004b) Human vaccinia-related Address correspondence to: Kyong-Tai Kim, Division of Integrative
kinase 1 (VRK1) activates the ATF2 transcriptional activity by novel phos- Biosciences and Biotechnology, POSTECH, Hyojadong, Pohang, Gyeongbuk,
phorylation on Thr-73 and Ser-62 and cooperates with JNK. J Biol Chem 279: South Korea. E-mail: ktk@postech.ac.kr
27458–27465.
Brazilin isolated from Caesalpinia. sappan suppresses nuclear envelope reassembly by inhibiting BAF phosphorylation
1 Supplemental Figure 1
1 2 3 4 5 6 7 8 9 10(M)
9
silica gel C.C.
10 n-hexnae-EtOAc=2:3→1:2→ EtOAc
11
1 2 3 4 5 6 7 8 9 10 11 12 13(M)
(200 mg) (96 mg) (129 mg)
12
ODS C.C. ODS C.C.
MeOH-H2O=1:2→2:3→ MeOH MeOH-H2O=1:2→2:3→ MeOH
13
silica gel C.C.
n-hexnae-EtOAc=2:3→1:2
14
5 7 2+3 2+3
15 (26.5 mg) (39.1 mg) (46.2 mg) (63.2 mg)
16
4-O-Methylsappanol 4-O-Methylepisappanol brazilin
17
18
19
20
21
1
Kim et al. The Journal of Pharmacology and Experimental Therapeutics
Brazilin isolated from Caesalpinia. sappan suppresses nuclear envelope reassembly by inhibiting BAF phosphorylation
23 Supplemental Figure 2
24
25
26
100kDa
70kDa
27
28 15kDa
29
15kDa
30
31
32
33
34
35
36
37
38
39
40
41
43 In vitro kinase assay in which VRK1 and H3 were exposed to increasing concentrations of brazilin
44 (0.0, 10, 25, 50 and 100μM). Immunoblotting was performed by using indicated antibodies.
45
2
Kim et al. The Journal of Pharmacology and Experimental Therapeutics
Brazilin isolated from Caesalpinia. sappan suppresses nuclear envelope reassembly by inhibiting BAF phosphorylation
46 Supplemental Figure 3
47
48 A
49
50
51
52
53
54
55
56
B
57
58
IB : α-p-BAF
59
10 kDa
60
IB : α-GAPDH
35 kDa
61
62
63
65 (A) Images of A549 cells treated with brazilin or DMSO. Cells were transfected with GFP-BAF or
66 DsRed-VRK1 and stained with Hoechst. Images were acquired by using confocal microscopy. Scale
67 bars, 40μm. (B) Immunoblotting of A549 cell lysate treated with brazilin (0.0, 10, and 25μM).
68 Immunoblotting was performed with GAPDH as the loading control.
69
3
Kim et al. The Journal of Pharmacology and Experimental Therapeutics
Brazilin isolated from Caesalpinia. sappan suppresses nuclear envelope reassembly by inhibiting BAF phosphorylation
70 Supplemental Figure 4
71
72
73 A
74 2500000
2000000
75 A549 A549-FIG
1500000
RLU
76
1000000
77
500000
78
0
media 5x104 1x105 2x105
79 Cell number
80 B
81 3.5
82 2.5
O.D 450nm
2
83
1.5
84 1
0.5
85
0
A549 A549-FIG
86
87
88 Supplemental Figure 4. Luciferase assay and cell proliferation assay in A549-FIG cells.
89 (A) Luciferase assay in A549 cells infected with or without lenti-Fluc-IRES-GFP. (B) Cell count kit-8
90 assay in A549 cell infected with or without lenti-Fluc-IRES-GFP. Optical density was detected in
91 450nm.