Investigating isoquinoline derivatives for inhibition of inhibitor of apoptosis proteins for ovarian cancer treatment

Article abstract of DOI:10.2147/DDDT.S137608

Objective: To discover novel isoquinoline derivatives for inhibition of inhibitor of apoptosis proteins (IAP) for the treatment of ovarian cancer. Methods: We first synthesized 533 isoquinoline derivatives, and screened them using CCK-8 to measure their antiproliferative activity. These compounds were further tested by Hoechst staining and flow cytometric analysis to assess proapoptotic activity. The in vivo antitumor efficacy and safety of the screened compounds were evaluated on the xenograft mouse model. Ki-67 staining and TUNEL assay were used to evaluate proliferation and apoptosis in the resected tumors, respectively. Western blot and polymerase chain reaction (PCR) were conducted to evaluate the levels of proliferating cell nuclear antigen (PCNA), caspase-3, PARP, and IAP in resected tumors. Results: Compound B01002 and C26001 displayed antiproliferative and proapoptotic activity on SKOV3 ovarian cancer with an IC50 of 7.65 and 11.68 μg/mL, respectively. Both compounds inhibited tumor growth in a xenografted mouse model with good safety profiles, and tumor growth inhibition (TGI) of B01002 and C26001 was 99.53% and 84.23%, respectively. Resected tumors showed that both compounds inhibited tumor cell proliferation and induced apoptosis in vivo. Caspase-3 and PARP were activated, whereas IAP proteins were downregulated at the protein level. Conclusion: Compound B01002 and C26001 could inhibit ovarian tumor growth and promote tumor apoptosis, partly by downregulating the IAPs, and, thus, might be promising candidates for treatment of ovarian cancer.

Full text of DOI:10.2147/DDDT.S137608

Drug Design, Development and erapy
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Open access FullText article
O r i g i n a l r e s e a r c h
investigating isoquinoline derivatives for inhibition
of inhibitor of apoptosis proteins for ovarian
cancer treatment
This article was published in the following Dove Press journal:
Drug Design, Development andTherapy
11 September 2017
Number of times this article has been viewed
chen chen1,*
Jie Wu2,*
Objective: To discover novel isoquinoline derivatives for inhibition of inhibitor of apoptosis
proteins (IAP) for the treatment of ovarian cancer.
Pengfei Zhu3
congjian Xu1
liangqing Yao1
Methods: We first synthesized 533 isoquinoline derivatives, and screened them using CCK-8 to
measure their antiproliferative activity. These compounds were further tested by Hoechst stain-
ing and flow cytometric analysis to assess proapoptotic activity. The in vivo antitumor efficacy
and safety of the screened compounds were evaluated on the xenograft mouse model. Ki-67
staining and TUNEL assay were used to evaluate proliferation and apoptosis in the resected
tumors, respectively. Western blot and polymerase chain reaction (PCR) were conducted to
evaluate the levels of proliferating cell nuclear antigen (PCNA), caspase-3, PARP, and IAP in
resected tumors.
1Obstetrics and gynecology hospital
and shanghai Key laboratory of
Female reproductive endocrine
related Diseases, 2Department of
chemistry, Fudan University, shanghai,
3Department of Obstetrics and
gynecology, shangyu city hospital,
shangyu, Zhejiang Province, People’s
republic of china
Results: Compound B01002 and C26001 displayed antiproliferative and proapoptotic activity
on SKOV3 ovarian cancer with an IC50 of 7.65 and 11.68 µg/mL, respectively. Both com-
pounds inhibited tumor growth in a xenografted mouse model with good safety profiles, and
tumor growth inhibition (TGI) of B01002 and C26001 was 99.53% and 84.23%, respectively.
Resected tumors showed that both compounds inhibited tumor cell proliferation and induced
apoptosis in vivo. Caspase-3 and PARP were activated, whereas IAP proteins were downregu-
lated at the protein level.
*These authors contributed equally
to this work
Conclusion: Compound B01002 and C26001 could inhibit ovarian tumor growth and promote
tumor apoptosis, partly by downregulating the IAPs, and, thus, might be promising candidates
for treatment of ovarian cancer.
Keywords: isoquinoline derivatives, target-oriented syntheses, IAP inhibitor, chemotherapy,
ovarian cancer
Introduction
Malignant tumors could fend off anticancer therapies in several ways. One strategy
involves the evasion of apoptosis by upregulating the inhibitors of apoptosis proteins
(IAP).1 IAP comprise a family of functionally and structurally related proteins that share
the presence of one or more baculoviral IAP repeat (BIR) domains.2 The property of
apoptosis inhibition has been described for at least four IAP family members – cIAP1,
cIAP2, XIAP, and survivin.3–5 IAP subverts apoptosis through binding and inactivating
caspases via one or more BIR domains,6 which are approximately 80-amino acid zinc-
binding domains and are essential for the antiapoptotic functions of the IAP. For example,
the second BIR domain (BIR2) of XIAP is essential for potent inhibition of caspase-3.
Elevated IAP have been identified in numerous neoplasms, and have been proved
to be correlated with poor prognosis. In several recent reports, it has been demonstrated
correspondence: liangqing Yao
Obstetrics and gynecology hospital
and shanghai Key laboratory of Female
reproductive endocrine related
Diseases, Fudan University, 419 Fangxie
road, shanghai 200011, People’s republic
of china
Tel +86 139 1664 4444
email yaoliangqing@126.com
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that IAP play an active role in promoting tumor maintenance chemical groups, and then evaluated their antitumor activities
through the inhibition of cellular death, thus enhancing on ovarian cancer cells in vivo. Two compounds screened out
tumor’s resistance to radiotherapy and chemotherapy.7,8 were further evaluated on the nude mouse xenograft model,
Furthermore, IAP participate in signaling pathways asso- and showed good antitumor efficacy and in vivo safety. They
ciated with tumor proliferation, invasion, metastasis, and inhibited tumor cell proliferation mainly in an apoptotic way,
angiogenesis.9–11 Therefore, agents targeting IAP might be as caspase-3 and PARP were activated, whereas IAP were
an effective approach for the treatment of cancer.
downregulated at the protein level.
Different IAP-targeting therapies have been evaluated
in preclinical and clinical studies in a variety of tumors
recently.12 Among those therapeutic strategies, one appeal- synthesis
ing approach is based on the occupation of the IAP–BIR We synthesized 533 molecules with an isoquinoline skeleton
peptide-binding pocket by certain reagents and, thus, interfer- as described previously.15,16
Materials and methods
ence with the IAP–caspases interactions.13 An endogenous
Unless otherwise stated, all commercial reagents were
inhibitor of the BIR peptide, the four amino-acid N-terminus used as received. All solvents were dried and distilled accord-
of the second mitochondrial activator of caspases (Smac; ing to standard procedures. Flash column chromatography
AVPI), can antagonize IAPs, with high binding affinity was performed using silica gel (60-Å pore size, 32–63 µm,
against the BIR2/3 domains of XIAP, the BIR3 domains of standard grade). Analytical thin-layer chromatography was
cIAP1/2, and the single BIR domain of survivin. This AVPI undertaken using glass plates pre-coated with 0.25 mm
tetrapeptide sequence has been regarded as the lead structure 230–400 mesh silica gel impregnated with a fluorescent
for designing small-molecule BIR inhibitors for antagonizing indicator (254 nm). Thin-layer chromatography plates were
IAPs by many researchers. Moreover, efforts to design new visualized by exposure to ultraviolet light. Organic solu-
compounds capable of inhibiting BIR domains have focused tions were concentrated on rotary evaporators at ~20 Torr
on high-throughput-screening bioactive Smac mimetics that at 25°C–35°C. Nuclear magnetic resonance (NMR) spectra
are based on those AVPI tetrapeptides, and subsequently are recorded in parts per million from internal tetramethyl-
evaluate their anti-cancer activity and underlying mechanism. silane on the δ scale.
Preclinical studies have demonstrated the capacity of those
cell culture and cell growth inhibition
small-molecule BIR inhibitors to inhibit tumor growth and
to sensitize cells to conventional therapeutic agents, such as assay
radiation or cisplatin, in multiple solid tumors. To date, at The human ovarian cancer SKOV3 cells (purchased from
least six BIR inhibitors have entered human clinical trials. Sigma-Aldrich Co., St Louis, MO, USA) were seeded in
Preliminary data indicate good tolerance and predicted 96-well flat-bottomed cell culture plates at a density of
biomarker modulation, such as cIAP1 downregulation and 1×103 cells/well and grown overnight.17 Then, 5 mg compound
increased levels of processed caspase-3. However, it appears stock in 100% dimethylsulfoxide (DMSO) was diluted in
that those reported BIR inhibitors either have low potency medium at concentrations of 0, 1, 2.5, 5, 7.5, and 10 µg/mL
or poor metabolic stability. For instance, results from phase separately, filtered, serially added into a 96-well plate, and
I clinical trials indicated that AT406 (Ascenta Therapeutics) continuously incubated for 24 h. Viable cells were quantified
did not show equivalent efficacy as previously tested in by a CCK-8 assay in which the absorbance of the samples was
mice, and HGS1029 (Aegera Therapeutics/Human Genome measured at 450 nm.18 IC50wascalculatedaccordingtothecell
Sciences) required intravenous administration owing to large growth curves. Experiments were repeated at least three times.
molecular size.12,14 Therefore, efforts need to be made to
develop more efficient BIR inhibitors with improved potency apoptosis assay
and better pharmacokinetics.
Apoptotic nuclear changes were assessed by Hoechst stain-
Molecules with an isoquinoline skeleton have been ing, followed by fluorescent microscopic examination.19
reported to have the capacity to antagonize IAP with a high SKOV3 cells were seeded in dish slides. After incubation
binding affinity to the BIR2 domain, highlighting their utility with testing agents (5 µg/mL compounds and 1% DMSO;
as a potential drug lead.11 Compounds with an isoquinoline 5 µg/mL cisplatin [DDP] as positive control; phosphate
skeleton are reported to be capable of antagonizing XIAP. buffered-saline [PBS] as negative control) for 24 h, cells were
In this study, we firstly designed and synthesized series of stained with 20 mg/mL Hoechst at 37°C for 20 min, washed
small compounds, with an isoquinoline skeleton and different three times with PBS, and fixed with 4% formaldehyde for
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new isoquinoline derivatives show antitumor effects with iaP inhibition
15 min at room temperature. Morphological changes of on blood parameters in mice was determined in the collected
nuclei were observed under a fluorescent microscope. The blood samples. Serum aspartate aminotransferase (AST),
percentage of apoptotic cells was calculated for each field alanine transaminase (ALT), alkaline phosphatase (ALP),
and averaged for the treatment group. The cytotoxicity of blood urea nitrogen (BUN), and creatinine (Cr) levels were
cisplatin was also evaluated under the same experimental determined using enzyme-linked immunosorbent assay kits
conditions for comparison. In each slide, at least 120 cells (Shanghai Institute of Biological Products Co.).
were assessed in three contiguous fields.
TUNEL staining and immunofluorescence
SKOV-3 cells were treated with different concentra-
analysis
tions of each compound (5 µg/mL of compounds and 1%
DMSO; 10 µg/mL of compounds and 1% DMSO) or PBS
for 24 h. Cells were washed and resuspended in binding
buffer at a concentration of 1×106 cells/mL; 100 µL of the
solution (1×105 cells) was transferred to a 5 mL culture tube.
Then, 5 µL of recombinant human annexin V-FITC conju-
gate (ANNEXINV01, Invitrogen) and 5 µL of propidium
iodide solution (P4864, Sigma-Aldrich) were added, and
cells were gently vortexed and incubated for 15 min at room
temperature (25°C) in the dark. Thereafter, 400 µL binding
buffer was added to each tube. Finally, the harvested cells
were analyzed by flow cytometry.
TUNEL assay was conducted using the In Situ Cell Death
Detection Kit (Roche) according to the kit protocol. Briefly,
the dissected tumors were fixed in 4% paraformaldehyde.
Then, TUNEL staining was conducted in fixed tumor sections
(5 µM) that were counterstained with 4,6-diamidino-2-
phenylindole (DAPI). The numbers of TUNEL-positive cells
and the total cells in tissue sections were counted.21
immunohistochemical assessment of
Ki-67
Moreover, 5 µm-thick sections were cut from the tissue
blocks, and then dewaxed and rehydrated. MIB1 antibody
(Abcam) at a dilution of 1:100 was used for Ki-67
immunostaining.22 Harris hematoxylin was used to coun-
terstain Ki-67–immunostained sections. All sections were
assessed blindly and independently by two observers.
Preceding this, the percentage of positively staining cells
was recorded and used as a quality control measure in our
statistical analysis.
Tumor xenograft study
This study was approved by the Animal Ethics Committee
of Fudan University (approval number: 20140823), and all
animals were maintained and used in accordance with the
guidelinesoftheInstitutionalAnimalCareandUseCommittee
of Fudan University. SKOV3 cells were suspended in PBS,
and then implanted subcutaneously into the right oxter of 40
female nude mice (age 6–8 weeks).20 Tumor volumes were
evaluated using the formula V = 0.5 × a × b2, where a and b
are the largest and smallest perpendicular tumor diameters,
respectively. Sixmicewiththeappropriatemeantumorvolume
were assigned randomly to each of the four groups. The mean
tumor volume for all four groups was 189±3 mm3 at the initia-
tion of treatment (day 0). Mice (six per group) were treated
with compound B01002, C26001, and DDP by intraperitoneal
injection at a dose of 4 mg/kg every other day, with PBS as
the control. Tumor volumes and mice body weights were mea-
sured every other day. Percent tumor growth inhibition (TGI)
was calculated using the formula % TGI = 100 * (1 - tumor
volumedose/tumor volumePBS). At the end of the experiment, the
mice were sacrificed and the tumor tissues were harvested and
weighed; the liver, spleen, small bowel, kidneys, and lungs
were excised; and blood samples were collected.
Western blot analysis
The resected tumors were washed with cold PBS, and pre-
pared by homogenization in RIPA buffer (1% Nonidet P-40,
0.5% sodium deoxycholate, and 0.1% sodium dodecylsul-
fate in PBS) supplemented with protease inhibitor cocktail.
Then, 30 mg protein was resolved by sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and immu-
noblotted with the respective antibodies.23
gene expression analysis
We isolated RNA with the RNeasy Lipid Tissue kit
(QIAGEN) and DNase digestion, synthesized cDNA with
the High-capacity cDNA Reverse Transcription kit, and
conducted quantitative real-time PCR (RT-PCR) with goTaq
qPCR Master Mix in a Bio-Rad CDX96 Real-time PCR
system. We calculated relative gene expression levels by the
Ct
∆∆ method using cyclophilin A as the internal control.24
hematological and biochemical study
At the end of the experimental period, blood samples were statistical analysis
collected from all animals from the retro-orbital venous All data were presented as mean ± standard error. For relative
plexus for safety evaluation. The effect of these compounds gene expression, the mean value of the control group was
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defined as 100%. Two-tailed unpaired Student’s t-test and position of isoquinoline), 7.44 (t, J=7.6 Hz, 1H) (Ar-H, the
ANOVA were used for statistical evaluation of the data.24 seventh position of isoquinoline), 7.10 (s, 1H) (Ar-H, the
The Sigma stat statistical analysis program was used for data fourth position of isoquinoline), 6.93 (t, J=6.0 Hz, 1H)
analysis. P,0.05 was considered significant.
(Ar-H, the fifth position of pyridinyl), 2.06–2.11 (m, 1H)
(CH, cyclopropyl), 1.11–1.18 (m, 2H), (CH2, cyclopropyl),
0.97–0.99 (m, 2H) (CH2, cyclopropyl); 13C NMR
Results
isoquinoline derivatives were synthesized (100 MHz, CDCl3) δ 154.00, 153.80, 150.75, 147.92,
147.55, 138.04, 130.20, 126.84, 125.66, 121.57, 117.56,
to inhibit iaP
117.26, 113.13, 110.62, 17.40 (CH, cyclopropyl), 9.01
(CH2, cyclopropyl); IR (cm-1): 3446.23, 3048.23, 2927.25,
2852.63; HRMS (ESI) calculated for C17H15N3: 262.1344
(M + H+), found: 262.1396.
The synthetic routes for isoquinolines 1-2 are shown in
Scheme 1. Isoquinoline 1 (B01002) was prepared by a silver
triflate-catalyzed reaction of 2-alkynylbenzaldoxime with
amine. Isoquinoline 2 (C26001) was synthesized by a silver
triflate-catalyzed reaction of 2-alkynylbenzaldehyde with
2-isocyanoacetate.
Spectroscopic data for C26001 are as follows: 1H NMR
(400 MHz, CDCl3) δ 8.38 (s, 1H) (Ar-H, the fourth position
of isoquinoline), 8.00 (d, J=8.4 Hz, 1H) (Ar-H, the eighth
position of isoquinoline), 7.67 (s, 1H) (Ar-H, the fifth
position of isoquinoline), 7.40 (d, J=8.4 Hz, 1H) (Ar-H,
the seventh position of isoquinoline), 7.22–7.12 (m, 5H)
(Ar-H, phenyl), 4.73 (s, 2H) (CH2, Ar-CH2-Ph), 4.53–4.49
(m, 2H) (CH2, -COOCH2CH3), 2.49 (s, 3H) (CH3, Ar-CH3),
1.47 (t, J=6.8 Hz, 3H) (CH3, -COOCH2CH3); 13C NMR
(100 MHz, CDCl3 ) δ 166.2 (carbonyl or the first position
of isoquinoline), 160.4 (carbonyl or the first position of
isoquinoline), 141.0, 139.3, 136.6, 131.7, 128.6, 128.5,
127.7, 127.0, 126.8, 126.3, 126.0, 125.0, 61.7 (-0-CH2-),
42.5 (Ar-CH2-Ph), 21.8 (CH3, Ar-CH3), 14.5 (CH3, -CH2-
CH3); HRMS (ESI) calculated for C20H19NO2: 306.1494
(M + H+), found: 306.1525.
To gain insights into the underlying mechanisms, apop-
totic nuclear changes were assessed by Hoechst staining.
Figure 1B demonstrated that B01002 and C26001, both at
a concentration of 5 µg/mL significantly induced apoptosis
of SKOV3 cells. Then, a flow cytometric analysis with
annexin V–FITC and PI staining was further performed.
The level of apoptotic cells in SKOV3 was assessed by the
percentage of annexin V-positive/PI-negative cells present
after exposure of SKOV3 cells to different concentra-
tions (5 or 10 µg/mL) of B01002 and C26001 for 24 h, as
illustrated in Figure 1C. At the concentration of 5 µg/mL,
B01002 and C26001 induced apoptosis, as indicated by a
21% and 9% increase in apoptotic cells (annexin V-positive/
PI-negative), respectively. At a concentration of 10 µg/mL,
B01002 and C26001 promoted cell death via both necrosis
and apoptosis. For B01002, 46% of cells were necrotic
(annexin V-positive/PI-positive), and 5% of cells were
apoptotic, whereas for C26001, 20% cells were necrotic
and 46% were apoptotic. As a comparison, 4% cells were
apoptotic in the control group.
isoquinoline derivatives inhibit
proliferation and promote apoptosis
Molecular structures of isoquinoline derivatives are shown
in Supplementary materials. Firstly, we tested the antipro-
liferative activity of each compound in vitro on human
SKOV3 ovarian cancer cells and, as shown in Figure 1A,
CCK-8 assays revealed that two compounds – denoted as
B01002 and C26001 – inhibited SKOV3 cell proliferation in
a concentration-dependent manner with an IC50 of 7.65 and
11.68 µg/mL, respectively. The screened results of all 533
compounds are shown in Supplementary materials.
Spectroscopic data for B01002 are as follows: 1H NMR
(400 MHz, CDCl3) δ 8.57 (d, J=8.4 Hz, 1H) (Ar-H, the
eighth position of isoquinoline), 8.28 (dd, J=1.2, 5.2 Hz,
1H) (Ar-H, the sixth position of pyridinyl), 8.08 (s, 1H)
(ArNHAr’), 7.94 (d, J=8.4 Hz, 1H) (Ar-H, the fifth position
of isoquinoline), 7.71 (t, J=8.0 Hz, 1H) (Ar-H, the fourth
position of pyridinyl), 7.65 (d, J=8.0 Hz, 1H) (Ar-H, the third
position of pyridinyl), 7.58 (t, J=8.0 Hz, 1H) (Ar-H, the sixth
1
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Scheme 1 syntheses of isoquinoline 1 and 2.
Notes: reagents and conditions: (a) agOTf, PyBrOP, iPr2etn, 1,4-dioxane, 70°c;
(b) agOTf, DBU, ch3cn, 80°c.
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new isoquinoline derivatives show antitumor effects with iaP inhibition
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Figure 1 B01002 and c26001 inhibited sKOV3 cell proliferation and promoted apoptosis in vitro. sKOV3 cells were treated with B01002 and c26001 for 24 h. (A) cell
viability inhibition curve of B01002 and c26001 by ccK-8 assay. (B) representative images of hoechst staining of cells treated by B01002 and c26001. arrows: apoptotic
nuclear changes. scale bar: 100 µm. (C) Apoptosis of cells treated by different concentrations of B01002 and C26001 evaluated by flow cytometry analysis using V-FITC and
Pi staining.
Abbreviation: ctr, control.
Blood samples were tested for blood count (RBC, WBC,
in vivo dosage experiment proved the
safety of isoquinoline derivatives
B01002 and C26001 were selected for in vivo assays. The
hemoglobin, and platelets), and hepatorenal function (AST,
ALT, ALP, BUN, and Cr). Supplementary materials show
that the counts of blood components in mice treated with
compounds B01002 and C26001 were injected into six
these two compounds were not altered significantly compared
animals, and DDP was administered as positive control,
with the control. WBCs were slightly reduced by C26001, by
with PBS as negative control. Daily observation of clinical
12%. Supplementary materials also show mean AST, ALT,
signs, physical examination, body weight, and food con-
ALP, BUN, and Cr values in animals treated with B01002
sumption with no abnormalities suggested good tolerance.
and C26001 were largely unchanged.
A general observation of these organs treated with B01002
and C26001 showed no abnormal changes, whereas there
isoquinoline derivatives decreased tumor
size in a xenograft mouse model
The in vivo antitumor activity of B01002 and C26001 was
determined in nude mice bearing ovarian xenografted tumors.
were some lesions in the liver of the mice treated with DDP.
Histopathological studies were carried out subsequently, and
were also consistent. Abnormal findings were observed in
the organs of the mice treated with DDP; no lesions were
found in the organs of mice treated with B01002 or C26001. The two compounds, as well as DDP and PBS, were slowly
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Figure 2 B01002 and c26001 inhibited tumor growth on sKOV3 xenograft mouse model. (A) Tumor growth curves of different treatment groups of PBs, DDP, B01002,
and c26001. (B) Mean tumor growth inhibition (Tgi) was calculated at the end of the treatment. (C) Pictures of resected tumors of four groups. **P,0.05.
Abbreviations: ctr, control; DDP, cisplatin; PBs, phosphate buffered-saline.
injected intraperitoneally at the dosages and the time inter- significantly decreased after treatment with C26001 (13.1%).
vals described earlier (Figure 2A). Both compounds induced However, PCNA was not decreased in tumors treated with
tumor growth delay for a longer period of time when com- B01002. Additionally, quantitative real-time PCR was con-
pared with PBS controls. Furthermore, B01002 and C26001 ducted to assess mRNA levels of PCNA in resected tumors.
revealed a clear tumor reduction of 99.53% and 84.23%, Treatment with C26001 resulted in an evident rise (33%) in
respectively, compared with PBS controls (Figure 2B). In the mRNA levels of PCNA (Figure 3C). These results suggest
summary, these preliminary data demonstrate the potential of that the antiproliferative potential of C26001 is associated
B01002 and C26001 for the treatment of ovarian cancer.
with the elevation of mRNA levels of PCNA. Moreover,
B01002 and C26001 caused tumor cell apoptosis (16.5%
and 37.2%, respectively) in vitro as revealed by TUNEL
assay (Figure 4), suggesting that these two compounds may
predominantly kill cells via the apoptosis pathway.
isoquinoline derivatives inhibited tumor
cell proliferation mainly in an apoptotic
way
To study the antiproliferative effects of B01002 and C26001
on xenografted tumors, we immunostained the resected
tumors with a Ki-67 antibody and immunoblotted the resected
isoquinoline derivatives inhibited iaP at
the protein level and showed antagonistic
tumor with a proliferating cell nuclear antigen (PCNA) effect through the caspase–iaP pathway
antibody, respectively. The proliferation index was deter- Levels of IAP including XIAP, cIAP, and survivin in resected
mined according to the Ki-67 expression level. We found tumors were measured using Western blotting assay, and the
that tumors treated with B01002 and C26001 had a signifi- results were shown in Figure 5A. XIAP, cIAP-1, and sur-
cantly lower percentage of Ki-67 positivity, compared with vivin were all reduced in tumors treated with B01002 and
tumors treated with PBS (33.2% and 68.5%, respectively; C26001, compared with tumors treated with DDP or PBS.
Figure 3A). To further verify these findings, we examined mRNA levels of XIAP, cIAP-1, and survivin were examined
the expression level of a typical marker of cell proliferation, by quantitative RT-PCR (Figure 5B). For mRNA levels of
PCNA, which is abundant in SKOV3 cell lines. Figure 3B XIAP, no significant differences were found between each
shows that PCNA (a typical marker of cell proliferation) was group. However, an increased mRNA level of cIAP-1 in the
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new isoquinoline derivatives show antitumor effects with iaP inhibition
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Figure 3 B01002 and c26001 inhibited tumor proliferation in vivo. (A) left, representative Ki-67 staining images of tumors resected from tumor-bearing mice treated with
B01002, C26001, DDP, or PBS, magnification ×100; Right, the ratio of positive Ki-67 nuclear staining to total number of nuclei was measured for each field, and the cumulative
results are summarized as bar graphs. (B) changes in Pcna expression of xenografted tumors following 14-day treatment. (C) mrna levels of Pcna in different groups
determined by qrT-Pcr. *P.0.05; **P,0.05.
Abbreviations: ctr, control; DDP, cisplatin; PBs, phosphate buffered-saline; Pcna, proliferating cell nuclear antigen; qrT-Pcr, quantitative real-time Pcr.
B01002 group and an increased mRNA level of survivin in could inhibit tumor cell proliferation. Hoechst staining, as well
C26001 group were found.
as fluorescence-activated cell sorting (FACS), results displayed
that the two isoquinoline derivatives induced tumor cell apop-
tosis but not necrosis. Moreover, both of these new compounds
wereprovedcapableofdecreasingtumorgrowthandhavegood
safety profiles in the xenograft mouse model. Further analysis
of the resected tumors revealed that the two compounds
downregulated several IAP, including XIAP, cIAP-1, and
survivin, thus promoting caspase-induced apoptosis, which
may be one of the mechanisms of their antitumor activity.
As the incidence of ovarian cancer keeps rising and
chemoresistance becomes increasingly common in the clinic,
exploring new chemotherapeutic agents has become an
important and urgent task, especially for patients with locally
advanced, platinum-resistant ovarian cancer.25 Modern drug
discovery often involves screening small molecules for their
ability to bind to a preselected protein target. Drug discovery
can also involve screening small molecules for their ability
to modulate a biological pathway in cells or organisms,
without regard for any particular protein target.26–28 In this
Cleaved PARP and cleaved caspase-3 were also tested
by Western blotting assay (Figure 5A). Cleaved PARP was
elevated in cells treated by C26001, whereas both cleaved
PARP and cleaved caspase-3 were elevated in cells treated
by B01002, indicating that both of the compounds acti-
vated apoptosis through the caspase–IAP pathway. However,
it is notable that the protein level of cleaved caspase-3 was
correlated with that of XIAP (Pearson’s correlation 0.911,
P,0.05). mRNA levels of PARP in the B01002 and C26001
group were decreased as compared with the control group.
However, there were no significant differences in mRNA
levels of caspase-3 between groups; instead, mRNA levels
of all treatment groups were decreased as compared with
the control group.
Discussion
In this study, we synthesized 533 new isoquinoline derivatives
in the hope of inhibiting IAP and killing ovarian cancer. The
CCK-8 assay showed that two of the isoquinoline derivatives study, we synthesized a collection of 533 molecules with an
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Figure 4 B01002 and c26001 induced apoptosis in vivo. TUnel detection of apoptotic cells of tumors resected from tumor-bearing mice from different groups (red). nuclei
are stained with DAPI (blue). Top, representative image of DAPI and TUNEL staining. Arrows, cells exhibiting typical apoptotic morphology. Magnification ×100. Bottom,
percentages of TUnel-positive cells determined by cell counting. **P,0.05.
Abbreviations: ctr, control; DDP, cisplatin; DaPi, 4,6-diamidino-2-phenylindole.
isoquinoline skeleton, aiming at perturbing the caspase–IAP efforts have been made over the past decade to develop strate-
pathway to induce apoptosis and kill cancer cells.
gies to neutralize IAP, including antisense oligonucleotides
IAP block programmed cell death and are expressed at and small-molecule inhibitors.35,36 Smac is a mitochondrial
high levels in various human cancers. Moreover, IAP have protein that is released into the cytosol upon the induction
been implied in the regulation of additional signaling cas- of programmed cell death and promotes apoptosis by neu-
cades, such as mitogen-activated protein kinase (MAPK) tralizing IAP.37 On this basis, a variety of small-molecule
pathway,29 TGFb signaling,30,31 as well as innate and adaptive inhibitors have been developed that mimic the binding
immunity signaling pathways32,33 – making them attractive domain of the native Smac protein to IAP. So far, various
targets for cancer drug development.34 Therefore, many Smac mimetics (monovalent or bivalent) are proved to inhibit
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new isoquinoline derivatives show antitumor effects with iaP inhibition
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Figure 5 B01002 and c26001 inhibited inhibitor of apoptosis proteins (iaP). (A) Western blot of iaPs and apoptosis-related proteins; β-actin was used as loading control.
(B) mrna levels of iaP and apoptosis-related proteins by qrT-Pcr. **P,0.05 compared with the control group.
Abbreviations: ctr, control; DDP, cisplatin; iaP, inhibitor of apoptosis proteins; qrT-Pcr, quantitative real-time Pcr.
Smac tetrapeptide binding to recombinant IAP, rescue IAP- are expected to have very low oral bioavailability and will
bound caspase-3 activity, and show antiproliferative activities have to be administered intravenously – a potential disadvan-
against malignant human cancer cells.38,39 Monovalent Smac tage if the drug must be administered frequently.41 Therefore,
mimetics are less potent than their corresponding bivalent smaller molecules with higher efficacy are still needed.
Smac mimetics.40 However, because bivalent Smac mimetics
Isoquinoline derivatives have been reported to show high
have molecular weights exceeding 1,000, such compounds affinity for the BIR2 domain and demonstrated potent IAP
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inhibitory activities in biochemical and cellular assays.42 Acknowledgment
Therefore, we screened the 533 isoquinoline derivatives syn- This study was supported by Science and Technology
thesized earlier to obtain compounds with effective antitumor Commission of Shanghai Municipality (124119a2900,
activity on ovarian cancer.
114119a2100 to L Yao), and New Hundred Talents Program
Firstly, we tested their cytotoxicity in vitro using the of Health and Family Planning Commission of Shanghai
SKOV3 ovarian cancer cell line, and found that both B01002 (13B122 to L Yao).
and C26001 could inhibit tumor cell proliferation effectively
(IC50 =7.65 and 11.68 µg/mL, respectively). Moreover, Disclosure
we found that both compounds could induce apoptosis of The authors declare that there are no conflicts of interest in
tumor cell, which seems the main mechanism of the toxicity, this work.
as a very small percentage of necrotic cells was observed.
To evaluate the translational potential of these new agents, References
1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation.
Cell. 2011;144(5):646–674.
we tested their in vivo efficacy and safety using a xeno-
graft mouse model. Consistent with the in vitro results, our
study showed that both compounds decreased the size of
xenografted tumors. By Ki-67 staining and PCNA level, we
showed that both compounds inhibited the tumor prolifera-
tion in vivo. By measuring body weight, food consumption,
observing clinical signs, physical examination, blood routine
testing, and biochemical tests, as well as necropsy of major
organs, the mice showed good tolerance in various dosages
to both compounds, indicating their clinical translational
potential in future applications.
2. Fulda S, Vucic D. Targeting IAP proteins for therapeutic intervention
in cancer. Nat Rev Drug Discov. 2012;11(2):109–124.
3. Gill C, Dowling C, O’Neill AJ, Watson RW. Effects of cIAP-1, cIAP-2
and XIAP triple knockdown on prostate cancer cell susceptibility to
apoptosis, cell survival and proliferation. Mol Cancer. 2009;8:39.
4. LaCasse EC, Mahoney DJ, Cheung HH, Plenchette S, Baird S,
Korneluk RG. IAP-targeted therapies for cancer. Oncogene. 2008;
27(48):6252–6275.
5. Song Z, Yao X, Wu M. Direct interaction between survivin and Smac/
DIABLO is essential for the anti-apoptotic activity of survivin during
taxol-induced apoptosis. J Biol Chem. 2003;278(25):23130–23140.
6. Fulda S. Regulation of cell migration, invasion and metastasis by IAP
proteins and their antagonists. Oncogene. 2014;33(6):671–676.
7. Fulda S. Inhibitor of apoptosis (IAP) proteins as therapeutic targets for
radiosensitization of human cancers. Cancer Treat Rev. 2012;38(6):
760–766.
8. Wen X, Lin ZQ, Liu B, Wei YQ. Caspase-mediated programmed cell
death pathways as potential therapeutic targets in cancer. Cell Prolif.
2012;45(3):217–224.
9. Vucic D, Fairbrother WJ. The inhibitor of apoptosis proteins as thera-
peutic targets in cancer. Clin Cancer Res. 2007;13(20):5995–6000.
10. Yang X, Xing H, Gao Q, et al. Regulation of HtrA2/Omi by X-linked
inhibitor of apoptosis protein in chemoresistance in human ovarian
cancer cells. Gynecol Oncol. 2005;97(2):413–421.
Our data also revealed that these two new isoquinoline
derivatives can downregulate XIAP, cIAP-1, and survivin at
the protein level, but not at the mRNA level, suggesting that
they work on the translational or post-translational process.
As IAP block apoptosis by neutralizing caspases, it is not
surprising that our new isoquinoline derivatives exert a pro-
apoptosis effect, as also evidenced by TUNEL staining. The
fact that both compounds upregulated the cleaved caspase-3
and partly upregulated cleaved PARP at the protein level
further verified that the two compounds inhibited the tumor
cells through the IAP–caspases pathway.
11. MehrotraS,LanguinoLR,RaskettCM,MercurioAM,DohiT,AltieriDC.
IAP regulation of metastasis. Cancer Cell. 2010;17(1):53–64.
12. González-López M, Welsh K, Finlay D, et al. Design, synthesis and
evaluation of monovalent Smac mimetics that bind to the BIR2 domain
of the anti-apoptotic protein XIAP. Bioorg Med Chem Lett. 2011;
21(14):4332–4336.
In addition, it is worth noting that isoquinoline-skeleton
compounds are reported to participate in several signaling
ways associated with tumor proliferation, invasion, metastasis,
and angiogenesis, indicating that the antitumor effect of isoqui-
noline derivatives is not only limited to downregulating IAP.43
We hope to explore more biological functions of these new
compounds to fully elaborate their antitumor mechanisms.
In conclusion, we have synthesized two new isoquinoline
derivatives that significantly inhibit ovarian tumor growth
and promote tumor apoptosis, both in vivo and in vitro, partly
by downregulating the IAP. Combined with their good safety
profile, this makes it worthwhile to evaluate their efficacies
in a clinical setting in the future.
13. Boulahjar R, Ouach A, Matteo C, et al. Novel tetrahydropyrido[1,2-a]
isoindolone derivatives (valmerins): potent cyclin-dependent kinase/
glycogen synthase kinase 3 inhibitors with antiproliferative activities
and antitumor effects in human tumor xenografts. J Med Chem. 2012;
55(22):9589–9606.
14. Morris GF, Mathews MB. Regulation of proliferating cell nuclear anti-
gen during the cell cycle. J Biol Chem. 1989;264(23):13856–13864.
15. Zheng D, Chen Z, Liu J, Wu J. An efficient route to 1-aminoisoquinolines
via AgOTf-catalyzed reaction of 2-alkynylbenzaldoxime with amine.
Org Biomol Chem. 2011;9(13):4763–4765.
16. Zheng D, Li S, Wu J. An unexpected silver triflate catalyzed reaction
of 2-alkynylbenzaldehyde with 2-isocyanoacetate. Org Lett. 2012;
14(11):2655–2657.
17. Fani S, Kamalidehghan B, Lo KM, Hashim NM, Chow KM,
Ahmadipour F. Synthesis, structural characterization, and anticancer
activity of a monobenzyltin compound against MCF-7 breast cancer
cells. Drug Des Devel Ther. 2015;9:6191–6201.
submit your manuscript | www.dovepress.com
Drug Design, Development andTherapy 2017:11
2706
Dovepress

Dovepress
new isoquinoline derivatives show antitumor effects with iaP inhibition
18. Yao Z, Sun B, Hong Q, et al. PACE4 regulates apoptosis in human
prostate cancer cells via endoplasmic reticulum stress and mitochondrial
signaling pathways. Drug Des Devel Ther. 2015;9:5911–5923.
19. Jin R, Xia Y, Chen Q, et al. Da0324, an inhibitor of nuclear factor-
kappaB activation, demonstrates selective antitumor activity on human
gastric cancer cells. Drug Des Devel Ther. 2016;10:979–995.
20. Jin Z, Niu H, Wang X, Zhang L, Wang Q, Yang A. Preclinical study
of CC223 as a potential anti-ovarian cancer agent. Oncotarget. Epub
2017 May 10.
31. Hofer-Warbinek R, Schmid JA, Stehlik C, Binder BR, Lipp J, de
Martin R. Activation of NF-kappa B by XIAP, the X chromosome-
linked inhibitor of apoptosis, in endothelial cells involves TAK1. J Biol
Chem. 2000;275(29):22064–22068.
32. Beug ST, Cheung HH, LaCasse EC, Korneluk RG. Modulation of
immune signalling by inhibitors of apoptosis. Trends Immunol. 2012;
33(11):535–545.
33. Lopez J, Meier P. To fight or die – inhibitor of apoptosis proteins at the
crossroad of innate immunity and death. Curr Opin Cell Biol. 2010;
22(6):872–881.
34. Fulda S. Promises and challenges of Smac mimetics as cancer thera-
peutics. Clin Cancer Res. 2015;21(22):5030–5036.
21. Jiang S, Chen X. HMGB1 siRNA can reduce damage to retinal cells
induced by high glucose in vitro and in vivo. Drug Des Devel Ther.
2017;11:783–795.
22. Pala Ş, Atilgan R, Kuloğlu T, et al. Protective effects of vitamin C and
vitamin E against hysterosalpingography-induced epithelial degen-
eration and proliferation in rat endometrium. Drug Des Devel Ther.
2016;10:4079–4089.
35. LaCasse EC. Pulling the plug on a cancer cell by eliminating XIAP
with AEG35156. Cancer Lett. 2013;332(2):215–224.
36. Straub CS. Targeting IAPs as an approach to anti-cancer therapy.
Curr Top Med Chem. 2011;11(3):291–316.
23. Pandey S. Aldose reductase inhibitor fidarestat as a promising drug
targeting autophagy in colorectal carcinoma: a pilot study. Asian Pac
J Cancer Prev. 2015;16(12):4981–4985.
24. Kadivar A, Kamalidehghan B, Akbari Javar H, Karimi B, Sedghi R,
Noordin MI. Antiproliferation effect of imatinib mesylate on MCF7,
T-47D tumorigenic and MCF 10A nontumorigenic breast cell lines via
PDGFR-beta, PDGF-BB, c-Kit and SCF genes. Drug Des Devel Ther.
2017;11:469–481.
37. Fulda S. Molecular pathways: targeting death receptors and smac
mimetics. Clin Cancer Res. 2014;20(15):3915–3920.
38. Talbott RL, Borzilleri RM, Chaudhry C, et al. Pharmacology of smac
mimetics; chemotype differentiation based on physical association
with caspase regulators and cellular transport. Exp Cell Res. 2015;
338(2):251–260.
39. Wang S. Design of small-molecule Smac mimetics as IAP antagonists.
Curr Top Microbiol Immunol. 2011;348:89–113.
25. Ren F, Shen J, Shi H, Hornicek FJ, Kan Q, Duan Z. Novel mechanisms
and approaches to overcome multidrug resistance in the treatment of
ovarian cancer. Biochim Biophys Acta. 2016;1866(2):266–275.
26. Huang R, Leung IK. Protein-directed dynamic combinatorial chemistry:
a guide to protein ligand and inhibitor discovery. Molecules. 2016;21(7).
pii:E910.
27. Narayana KKC, Aswathanarayan JB, Vittal RR. Endophytic peptides –
a source of therapeutic agents. Curr Protein Pept Sci. 2017;18(3):
284–290.
28. Schreiber SL. Target-oriented and diversity-oriented organic synthesis
in drug discovery. Science. 2000;287(5460):1964–1969.
29. Varfolomeev E, Goncharov T, Maecker H, et al. Cellular inhibitors
of apoptosis are global regulators of NF-kappaB and MAPK activa-
tion by members of the TNF family of receptors. Sci Signal. 2012;
5(216):ra22.
40. Li L, Thomas RM, Suzuki H, De Brabander JK, Wang X, Harran PG.
A small molecule Smac mimic potentiates TRAIL- and TNFalpha-
mediated cell death. Science. 2004;305(5689):1471–1474.
41. Sun H, Nikolovska-Coleska Z, Lu J, et al. Design, synthesis, and
characterization of a potent, nonpeptide, cell-permeable, bivalent Smac
mimetic that concurrently targets both the BIR2 and BIR3 domains in
XIAP. J Am Chem Soc. 2007;129(49):15279–15294.
42. KimKS,ZhangL,WilliamsD,etal.Discoveryoftetrahydroisoquinoline-
based bivalent heterodimeric IAP antagonists. Bioorg Med Chem Lett.
2014;24(21):5022–5029.
43. Pandey MK, Prasad S, Tyagi AK, et al. Targeting cell survival proteins
for cancer cell death. Pharmaceuticals (Basel). 2016;9(1). pii:E11.
30. Birkey Reffey S, Wurthner JU, Parks WT, Roberts AB, Duckett CS.
X-linked inhibitor of apoptosis protein functions as a cofactor in
transforming growth factor-beta signaling. J Biol Chem. 2001;276(28):
26542–26549.
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