A new isoquinolinium derivative, cadein1, preferentially induces apoptosis in p53-defective cancer cells with functional mismatch repair via a p38-dependent pathway

Article abstract of DOI:10.1074/jbc.M109.070466

We screened a protoberberine backbone derivative library for compounds with anti-proliferative effects on p53-defective cancer cells. A compound identified from this small molecule library, cadein1 (cancer-selective death inducer 1), an isoquinolinium derivative, effectively leads to a G₂/M delay and caspase-dependent apoptosis in various carcinoma cells with nonfunctional p53. The ability of cadein1 to induce apoptosis in p53-defective colon cancer cells was tightly linked to the presence ofa functionalDNAmismatch repair(MMR)system, which is an important determinant in chemosensitivity. Cadein1 was very effective in MMR⁺/p53^- cells, whereas it was not effective in p53⁺ cells regardless of theMMRstatus. Consistently, when the function ofMMRwas blocked with short hairpinRNAinSW620 (MMR⁺/p53 ^-) cells, cadein1 was no longer effective in inducing apoptosis. Besides, the inhibition of p53 increased the pro-apoptotic effect of cadein1 in HEK293(MMR⁺/p53⁺) cells, whereas it did not affect the response to cadein1 in RKO (MMR^-/p53⁺) cells. The apoptotic effects of cadein1 depended on the activation of p38 but not on the activation of Chk2 or other stress-activated kinases in p53-defective cells. Taken together, our results show that cadein1 may have a potential to be an anti-cancer chemotherapeutic agent that is preferentially effective on p53-mutant colon cancer cells with functional MMR.

Full text of DOI:10.1074/jbc.M109.070466

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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 5, pp. 2986–2995, January 29, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
A New Isoquinolinium Derivative, Cadein1, Preferentially
Induces Apoptosis in p53-defective Cancer Cells with
Functional Mismatch Repair via a p38-dependent Pathway*
□
S
Received for publication, September 25, 2009, and in revised form, November 25, 2009 Published, JBC Papers in Press, November 30, 2009, DOI 10.1074/jbc.M109.070466
Eun Ryoung Jang‡1, Minsook Ryu§, Jeong Eun Park‡, Jung-Ho Kim¶, Jong-Soo Lee§, and Kiwon Song‡2
From the ‡Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, the
§Department of Molecular Science and Technology, College of Natural Sciences, Ajou University, Suwon 443-749, and the
¶Hanwha Chemical Research and Development Center, Taejeon 305-345, Korea
We screened a protoberberine backbone derivative library for
compounds with anti-proliferative effects on p53-defective can-
cer cells. A compound identified from this small molecule
library, cadein1 (cancer-selective death inducer 1), an isoquino-
linium derivative, effectively leads to a G2/M delay and caspase-
dependent apoptosis in various carcinoma cells with non-
functional p53. The ability of cadein1 to induce apoptosis in
p53-defective colon cancer cells was tightly linked to the presence
ofa functional DNA mismatch repair (MMR) system, which is an
important determinant in chemosensitivity. Cadein1 was very
genes involved in cell death, including BAX, PUMA, NOXA,
and Fas (2, 3). Because p53 activation inhibits cell proliferation,
mutation of the p53 gene or disruption of pathways that lead to
p53 activation has been frequently observed in most types of
human cancer (4).
The p53-dependent induction of apoptosis in response to
genotoxic damages is an important aspect of tumor suppres-
sion. Thus, the loss of p53 in human cancers not only contrib-
utes to aggressive tumor behavior but often leads to resistance
to radiation and chemotherapeutic drugs. For example, treat-
؉
؊
ϩ/ϩ
effective in MMR /p53 cells, whereas it was not effective in
ment of p53
mouse thymocytes with radiation results in
؉
p53 cells regardless of the MMR status. Consistently, when the
Ϫ/Ϫ ϩ/ϩ
apoptosis, whereas p53
thymocytes are resistant. p53
function of MMR was blocked with short hairpin RNA in SW620
mouse embryonic fibroblasts transformed by adenoviral E1A
؉
؊
(MMR /p53 ) cells, cadein1 was no longer effective in inducing
apoptosis. Besides, the inhibition of p53 increased the pro-apo-
protein and Ha-ras oncogene undergo apoptosis in response to
Ϫ/Ϫ
␥-irradiation or chemotherapeutic agents, whereas p53
؉
؉
ptotic effect of cadein1 in HEK293 (MMR /p53 ) cells, whereas
fibroblasts are resistant to these anti-cancer therapies (5). In
addition, some p53 mutations in cancers suppress the function
of p73, which induces apoptosis through a p53-independent
mechanism (6). Thus, the common loss of p53 function in can-
cer cells presents a major limitation for anti-cancer therapies.
DNA mismatch repair (MMR)3 is a post-replicative DNA
repair process that corrects single-base mismatches and small
mismatched loops in the daughter strand of newly replicated
DNA. Loss of MMR by mutation of MSH2 or MLH1 is respon-
sible for the majority of cases of hereditary nonpolyposis colon
cancer and is also common in a variety of sporadic cancers
including endometrial, ovarian, breast, prostate, lung, and pan-
creatic cancer (7–9). In addition to an increased rate of muta-
tion throughout the genome, the loss of MMR often alters the
sensitivity to some therapeutic DNA damaging agents. MMR
deficiency results in strong resistance to the base analog, anti-
metabolite 6-thioguanine, moderate resistance to methylating
agents, such as N-methyl-NЈ-nitro-N-nitrosoguanidine and
temozolomide, and weak resistance to cisplatin and carboplatin
(10–13). In the case of cisplatin, the loss of both MMR and p53
leads to a cooperative increase in resistance and limits the ther-
apeutic potential of cisplatin for colon cancer (14). Although
the mechanism is not clear, the decreased sensitivity to DNA
damaging agents caused by loss of MMR is likely to be due to
؊
؉
it did not affect the response to cadein1 in RKO (MMR /p53 )
cells. The apoptotic effects of cadein1 depended on the activa-
tion of p38 but not on the activation of Chk2 or other stress-
activated kinases in p53-defective cells. Taken together, our
results show that cadein1 may have a potential to be an anti-
cancer chemotherapeutic agent that is preferentially effective
on p53-mutant colon cancer cells with functional MMR.
The p53 tumor suppressor is essential for maintaining
genomic stability in mammals. When cells are subjected to
stress signals such as hypoxia, radiation, or chemotherapeutic
drugs, p53 is activated, and its ubiquitin-dependent degrada-
tion is blocked leading to an accumulation of active p53 tran-
scription factor (1). Activated p53 regulates most downstream
signals for cell cycle arrest and apoptosis by affecting the
expression of its target genes. Transcriptional targets of p53
include the cyclin-dependent kinase inhibitor, p21/WAF1, and
* This work was supported by National Research and Development Program
for Cancer Control, Ministry of Health and Welfare, Republic of Korea Grant
NCCR-0920300 and in part by Korea Health Industry Development Insti-
tute (Grant B05-0016-AM0815-07A3-00030B).
□S
The on-line version of this article (available at http://www.jbc.org) contains
supplemental Figs. 1–3.
1 Supported by Korea Research Foundation Grant KRF-2006-8-1284) funded 3 The abbreviations used are: MMR, DNA mismatch repair; PARP, poly(ADP-
by the Korean Government Ministry of Education (Ministry of Education
and Human Resources Development).
ribose)polymerase; ATM, ataxia-telangiectasia mutated; MAPK, mitogen-
activated protein kinase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide; JNK, c-Jun N-terminal kinase; ERK, extracellular sig-
nal-regulated kinase.
2 To whom correspondence should be addressed. Tel.: 82-2-2123-2705; Fax:
82-2-362-9897; E-mail: bc5012@yonsei.ac.kr.
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A Selective Anti-carcinoma Effect of Cadein1
decreased levels of apoptosis resulting from impaired detection morphology of the cells was observed with a phase contrast
of DNA adducts or a failure to initiate repair (11).
microscope (Fig. 1B, Model CK2, Olympus Optical Co.).
Chemical Treatment and Cell Cycle Assay—A stock solution
In this study we prepared a small library based on the back-
bone of protoberberine and screened for compounds with of cadein1 (1 mg/ml) was prepared in 70% (v/v) ethanol. Stock
effective anti-proliferative activity. In particular, we searched solutions of berberine (1 mg/ml) and SB203580 (10 mM, AG
for an anti-proliferative compound selective to cells without Scientific) were prepared by dissolving these compounds in di-
wild type p53 function, as loss of p53 function is a common methyl sulfoxide (DMSO) and diluted to the final concentra-
feature of most cancer cells and often accompanies resistance tion in media. Unless indicated otherwise, cells were grown for
to anti-cancer therapeutics. A new isoquinolinium derivative, 1 day to 60–70% confluency and treated with various concen-
cadein1 (cancer-selective death inducer 1), was identified in the trations of cadein1. Cells were synchronized at G1/S by a mod-
screen, which induces apoptosis most effectively in p53-defec- ified double thymidine block (15). For fluorescence-activated
tive carcinoma cells with functional MMR. The apoptotic effect cell sorter analysis, cells were trypsinized, fixed with cold 70%
of cadein1 in cancer cells depends on the activation of p38. Given ethanol (Ϫ20 °C), treated with 100 ␮g/ml RNase A (Sigma), and
thatdecreasedsensitivitytochemotherapiesinhumancancercells stained with 100 ␮g/ml propidium iodide (Sigma). The DNA
without functional p53 is a major drawback of cancer treatment, content of cells was measured by flow cytometry (BD Bio-
cadein1 may be a potent anti-cancer agent against human cancers, sciences) and analyzed using Cell Quest software.
specifically p53-deficient cancers with functional MMR.
Cell Viability Assay—Cell proliferation was evaluated by
spectrophotometric measurement of the mitochondrial dehy-
drogenase activity using 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
EXPERIMENTAL PROCEDURES
Synthesis of the Modified Isoquinolinium Derivative Cadein1— phenyl tetrazolium bromide (MTT) (16). Cells (5 ϫ 103 cells/
Cadein1 (C33H48ClF3NO2, Mr 564.20), an isoquinolinium salt, ml) were plated in triplicate in 96-well plates for 1 day and
was synthesized as follows. Briefly, 3,4-dimethoxyphenylethyl- incubated with varying concentrations of cadein1 (1, 3, 4, 5, 6,
amine (Aldrich) was acylated with long chain acid chloride or 9 ␮M) for 24 h. After incubation with MTT (5 mg/ml), the
(CH3(CH2)14COCl, triethylamine/ethylene dichloride, room dissolved formazan product was measured on a microplate
temperature, 95%) to yield amide. Amide was subsequently reader (SOFTmax PRO 4.0).
treated with phosphorus oxychloride (POCl3, CH3CN, reflux,
Immunoblotting—Cells were lysed in a buffer containing 17
82%) to give isoquinoline, which was further reacted with 2, mM Tris, pH 8.0, 50 mM NaCl, 0.3% Triton X-100, and protease
6-difluorobenzyllbenzyl
chloride
(2,6-F2C6H4CH2Cl, inhibitors. Specific proteins were detected using the following
CH3CN, reflux, 62%) to yield the desired isoquinolinium salt as primary antibodies: phospho-p38, p38, p53 (Santa Cruz Bio-
a yellow solid. The isoquinolinium salt compound was verified technology, Santa Cruz, CA); cleaved caspase-3, cleaved PARP,
by spectroscopic analysis (1H NMR, 13C NMR, high resolution phospho-Chk2 (Thr-68), phospho-p38 (Thr-180/Tyr-182),
mass spectrometry).
p38, PARP, phospho-stress-activated protein kinase/JNK,
Cell Culture and Screening of Isoquinolinium Derivatives— p-ERK1/2 (Cell Signaling Technology, Inc., Beverly, MA); cyclin
WI-38 human normal lung fibroblast cells were purchased A, cyclin B1, cyclin E (NeoMarkers, Fremonts, CA); MLH1, p53
from the American Type Culture Collection (Manassas, VA) (BD Pharmingen); ␥-H2AX(Ser-139) (Upstate biotechnology,
and cultured in Eagle’s minimum essential medium with 10% NY). Immunoreactive bands were normalized to those of control
fetal bovine serum (all from HyClone, Logan, UT). Human nor- actin (Santa Cruz Biotechnology) or ␣-tubulin (Calbiochem).
mal lung fibroblast IMR90, human cervical carcinoma HeLa,
Transfection—Wild type p53 in pcDNA3.1 and pSuper-Neo-
and human embryonic kidney HEK293 cells were grown in Dul- p53 were kindly provided by Dr. H. W. Lee (Yonsei University,
becco’s modified Eagle’s medium with 10% fetal bovine serum. Seoul, Korea) and Dr. J. Shin (Sungkyunkwan University,
SW620 and RKO human colon cancer cells were grown in Lei- Suwon, Korea), respectively. shp53-pLKO.1 puro vector was
bovitz’s L-15 medium and minimum Eagle’s medium with 10% purchased from Addgene (Cambridge, MA). pSuperRetro-
fetal bovine serum, respectively. The four sublines of HCT116 shMLH1 was kindly provided by Dr. A. Zhitkovich (Brown Uni-
human colorectal adenocarcinoma, HCT116, HCT116-Ch3, versity). The cells were transfected with those constructs using
HCT116-E6, and HCT116-Ch3/E6, human colorectal cell lines LipofectamineTM 2000 (Invitrogen) according to the manufac-
(HT29, DLD-1, and SW480), and H1299 human lung cancer turer’s protocol. Transient transfection of wild type p38 and
cells were maintained in Roswell Park Memorial Institute dominant-negative p38 in HeLa cells was performed with the
(RPMI) 1640 with 10% fetal bovine serum. The chromosome- Effectene kit (Qiagen, Valencia, CA), according to the manu-
complemented lines (HCT116-Ch3 and HCT116-Ch3/E6) facturer’s instructions.
were maintained in medium containing 400 ␮g/ml G418, and
RESULTS
the cell lines expressing Papillomavirus E6 (HCT116-E6 and
HCT116-Ch3/E6) were cultured in medium supplemented
A Novel Isoquinolinium Derivative Cadein1 Efficiently Blocks
with 80 ␮g/ml hygromycin B. All cells were cultured in 5% CO2 Proliferation of p53-deficient Carcinoma Cells—The use of phy-
in a medium with penicillin and streptomycin at 37 °C. For tochemicals as anti-cancer chemotherapeutics has attracted con-
screening of 80 isoquinolinium derivatives, cells were grown for siderable interest. Among 121 prescription drugs in use for can-
1 day to 60–70% confluence and treated with various concen- cer therapy, 90 are derived from natural plant sources (17, 18).
trations of derivatives. To determine the anti-proliferative Protoberberine-derived alkaloids, such as berberine, have a
effects of isoquinolinium derivatives including cadein1, the wide range of pharmacological activities, including cytotoxicity
JANUARY 29, 2010•VOLUME 285•NUMBER 5
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A Selective Anti-carcinoma Effect of Cadein1
against several carcinomas in vitro (19, 20). Thus, we generated results confirm that cadein1-induced apoptosis in p53-defec-
tive cancer cells is mediated by activation of caspase-3.
a privileged small scale chemical library by modifying one of the
two isoquinoline rings of protoberberine (Fig. 1A and Ref. 21)
and screened the anti-proliferative effects of 80 isoquinolinium
derivatives in several carcinoma cell lines and non-cancerous
cells (Fig. 1B).
To better assess the hypersensitivity of p53-negative cells to
cadein1, the DNA content of cells was analyzed after incubation
with 9 ␮M cadein1 for 24 h. As shown in Fig. 2D, the sub-G1
fraction, which presumably contains dying cells, was highly
increased in HeLa cells (9 ␮M, 50.27%), whereas a negligible
sub-G1 fraction was observed in IMR90 cells (9 ␮M, 2.11%).
These observations showed that, at low concentrations,
cadein1 efficiently induces cell death in HeLa cells but not in
IMR90 cells, supporting that p53-defective cancer cells are
more sensitive to cadein1 than p53-positive normal cells.
To investigate the effect of cadein1 on cell cycle progression,
we analyzed the cell cycle profiles of cadein1-treated HeLa cells.
After synchronization at G1 via a double thymidine block, HeLa
cells were released from the G1 block in the presence of 4 ␮M
cadein1. Cadein1 induced a transient G2/M delay in HeLa cells
as compared with non-treated cells (Fig. 2E). In addition, cyclin
A degradation was delayed, and cyclin B persisted in the
cadein1-treated cells over 24 h (Fig. 2E), suggesting that a cell
cycle delay at G2/M occurs in response to cadein1 treatment.
However, cyclin E was not detectable in these cells due to the
G2/M delay (Fig. 2E).
Given that decreased sensitivity to anti-cancer agents in p53-
deficient human cancer cells is the major limitation to cancer
therapeutics, our search was focused on identifying an anti-
proliferative compound selective to cells without wild type p53
function (Fig. 1B). Thus, in the screen we compared the anti-
proliferative effect of each compound in p53-negative cancer
cells, such as HeLa and HCT116-Ch3/E6, and in p53-positive
HEK293 cells and IMR90 primary cells. Among these com-
pounds, one new isoquinolinium derivative efficiently blocked
the proliferation of p53-negative carcinoma cells but not of
normal cells (data not shown). We designated this isoquino-
linium derivative, cadein1 (cancer-specific death inducer 1).
The anti-proliferative effect of cadein1 in p53-negative car-
cinoma cells was very strong compared with the lead com-
pound berberine (Fig. 1C). As shown in Fig. 1C, cadein1 signif-
icantly inhibited the proliferation of HeLa cells at 6 ␮M, whereas
the lead compound, berberine, did not affect HeLa cells at low
concentrations (15 ␮M or less).
Cadein1 Sensitively Inhibits Proliferation of p53-deficient
Colon Cancer Cells with Functional MMR—Because p53 coop-
erates with the MMR to induce cell death in response to some
chemotherapeutic compounds in colon cancer cells (14), the
effect of an MMR deficiency on cadein1-induced cell death of
p53-negative cells was examined. We assessed the cytotoxic
effect of cadein1 across a panel of sublines of the MMR-defi-
cient HCT116 human colorectal adenocarcinoma cells because
each HCT116 subline contains different combinations of p53
and MMR functions in the same genetic background. Four sub-
lines of HCT116 colon cancer cells were used: HCT116
Cadein1 Efficiently Induces Apoptosis in p53-defective Can-
cer Cells via the Caspase Pathway and Transient G2/M Delay—
To determine the optimal conditions for cadein1 treatment, we
first performed MTT assays to assess the effective concentra-
tion range for inhibition of cell growth in HeLa, IMR90, and
WI-38 cells. As observed in the screen, HeLa cells with non-
functional p53 were more sensitive to cadein1 than were p53-
positive IMR90 and WI-38 primary cells (Fig. 2A). The viability
of HeLa cells was 18.6% at 6 ␮M cadein1, whereas IMR90 and
WI-38 cells were 61.55 and 74.13% viable, respectively (Fig. 2A).
Additionally, in low concentrations (9 ␮M or less) of cadein1, a
strong cytotoxic effect was observed in other p53-defective car-
cinoma cells including human colorectal HT29, SW480,
SW620, and HCT116-Ch3/E6 cells and human lung cancer
H1229 cells (data not shown). These observations strongly sug-
gest that cadein1 is able to inhibit the proliferation of p53-de-
ficient cancer cells.
Ϫ
ϩ
ϩ
ϩ
(MMR /p53 ), HCT116-Ch3 (MMR /p53 ), HCT116-E6
Ϫ
Ϫ
ϩ
Ϫ
(MMR /p53 ), and HCT116-Ch3/E6 (MMR /p53 ). HCT116
contains a hemizygous mutation in hMLH1 resulting in a trun-
cated, nonfunctional protein (22). The chromosome 3-comple-
mented cells (identified here as HCT116-Ch3 and HCT116-
Ch3/E6) are competent in DNA mismatch repair, as MMR
function has been restored by transfer of a copy of MLH1 on
chromosome 3 (14, 23). In HCT116-E6 and HCT116-Ch3/E6
cell lines, constitutive high level expression of the human Pap-
illomavirus type-16 E6 gene disrupts p53 function, as it pro-
motes the ubiquitin-dependent degradation of p53 (14). The
HCT116 sublines were exposed to a range of cadein1 (0–4 ␮M)
for 24 h, and the anti-proliferative effect of cadein1 on the
HCT116 sublines was measured by an MTT assay. Exposure to
cadein1 caused a dose-dependent loss of cell viability (Fig. 3A).
To examine whether cadein1-induced cell death is the result
of apoptosis, we evaluated the expression of proteins in the
apoptotic pathway after cadein1 treatment. Because the cleav-
age of PARP (substrate of caspase-3, poly(ADP-ribose)poly-
merase) is a hallmark of apoptotic cell death, whole cell lysates
from HeLa and WI-38 were subjected to Western blotting
using the antibody against cleaved-PARP. When incubated
with 6 ␮M cadein1 for 12 h, activated PARP was significantly
detected in HeLa cells, but no cleaved PARP was observed in
WI-38 cells (Fig. 2B).
ϩ
The percentages of cell viability in HCT116-Ch3/E6 (MMR /
Ϫ
Ϫ
Ϫ
ϩ
p53 ), HCT116-E6 (MMR /p53 ), HCT116-Ch3 (MMR /
ϩ
Ϫ
ϩ
p53 ), and HCT116 (MMR /p53 ) cells treated with 4 ␮M
cadein1 were 17.7, 49, 55.4, and 68.6%, respectively (Fig. 3A).
We analyzed the cadein1-induced apoptotic cell death by
examining the expression of caspase-3 (effecter caspase) and
PARP in HeLa and IMR90 cells after treatment with cadein1.
When cells were treated with 6 ␮M cadein1, as expected,
cleaved caspase-3 and PARP were detected in a time-depen-
ϩ
Ϫ
The HCT116-Ch3/E6 (MMR /p53 ) cells displayed the most
significant sensitivity to cadein1 among the four HCT116 sub-
lines. Although this line does not have functional p53, the
Ϫ
Ϫ
HCT116-E6 (MMR /p53 ) cells had decreased sensitivity to
dent manner in HeLa cells but not in IMR90 cells (Fig. 2C). These cadein1 relative to HCT116-Ch3/E6 cells (Fig. 3A). Interest-
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A Selective Anti-carcinoma Effect of Cadein1
FIGURE 1. Cadein1, a novel isoquinolinium derivative, was identified by its potent cytotoxic effects on p53-mutant cancer cells. A, shown is a scheme
for the synthesis of isoquinolinium derivatives including cadein1. B, shown is a scheme for screening the anti-proliferative effects of isoquinolinium derivatives
in p53-negative HeLa and HCT116-Ch3/E6 cancer cells and in p53-positive HEK293 and IMR90 cells. The cells were treated with a range of different concen-
trations of derivatives for 24 h. Cell morphology was assessed using a phase contrast microscope. C, the anti-proliferative effect of cadein1 was measured by
MTT assays and compared across a range of different concentrations of the lead compound berberine for 24 h in HeLa cells. Three independent experiments
were performed at each point, and the mean value is plotted with S.D.
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