Indenoisoquinoline derivatives as topoisomerase I inhibitors that suppress angiogenesis by affecting the HIF signaling pathway

Biomedicine & Pharmacotherapy 67 (2013) 715–722

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Original article

Indenoisoquinoline derivatives as topoisomerase I inhibitors that suppress angiogenesis by affecting the HIF signaling pathway Xiaoli Xu a,b, Fang Liu a,b, Shengmiao Zhang a,b, Jianmin Jia a,b, Zhiyu Li c, Xiaoke Guo a,b, Yong Yang d, Haopeng Sun a,b,c,*, Qidong You a,b,*,1 a

State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China Jiang Su Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, Nanjing 210009, China Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China d Department of Physiology, School of Pharmacy, China Pharmaceutical University, Nanjing, China b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 19 May 2013 Accepted 17 June 2013

Both Topoisomerase I and angiogenesis factors have caught pharmaceutical chemists’ attention in antitumor chemotherapy field. A series of indenoisoquinoline derivatives with high Top I inhibitory from our previous work were evaluated for their anti-angiogenesis property using classic in vitro and vivo models. The results demonstrated that all the compounds could significantly decrease the proliferation of endothelial cells in a concentration-dependent manner. Besides, compound 1 exerted marked inhibition of angiogenesis in vivo and in vitro models. The HIF signaling pathway in HUVECs was affected by compound 1 in a time-dependent manner. These data suggest that the tested compound 1 could serve as promising lead compound for further development and optimization. ß 2013 Elsevier Masson SAS. All rights reserved.

Keywords: Indenoisoquinoline derivatives Anti-angiogenesis HIF-1a

1. Introduction Tumor development is characterized by a sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis [1]. Among this, angiogenesis which is the recruitment of new blood vessels from pre-existing vasculature is a prerequisite event in several solid tumors and haematological malignancies [2]. The vessels could support tumor in the form of nutrients and oxygen and evacuate metabolic wastes and carbon dioxide and then tumor deteriorate beyond a critical size or metastasize to other organs. Inhibition of angiogenesis is now an important therapeutic strategy for cancer patients. Both the tumor cells and the tumor microenvironment could modulate the tumor angiogenesis by regulating a balance of stimulatory and inhibitory factors. Among the stimulatory factors, the entire HIF family of oxygen-sensitive transcription factors plays a critical key role in hypoxia-mediated angiogenesis [3]. Hif-1a is a transcriptionally active heterodimeric complex comprised of two subunits, the constitutively expressed Hif-1b and the highly regulated and the most commonly studied member Hif-1a. Under normoxic

* Corresponding authors. China Pharmaceutical University, Tongjiaxiang 24, Nanjing 210009, China. Tel.:/fax: +86 25 83271216. E-mail addresses: [email protected] (H. Sun), [email protected] (Q. You). 1 Tel./fax: +86 25 83271351. 0753-3322/$ – see front matter ß 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.biopha.2013.06.004

conditions, the expression of HIF-1a is maintained at a very low level owning to the rapid proteasomal degradation. However, the tumor surrounding microenvironment is hypoxia, HIF-1a is no longer modified, and thus is stabilized. HIF-1a are accumulated and then translocated to the nucleus where it forms a dimeric complex with the constitutively expressed HIF-1b protein. The complex regulates a series of genes that participate in angiogenesis, iron metabolism, glucose metabolism, and cell proliferation [4]. As a result, the HIF pathway has been exploited for the development of new cancer therapies, including the development of small molecule inhibitors targeting HIF-1a [5]. The highly supercoiled DNA needs to be relaxed prior to cellular processes such as replication and transcription. Topoisomerase I (Top I) is a ubiquitous and essential enzyme that could effectively solve the DNA topological problems so it is crucial during basic cellular processes. Besides, over-expression of this enzyme in several types of cancers, e.g., lung, colorectal, and ovarian cancers [6] makes it a validated target for cancer chemotherapy [7]. Since the pentacyclic antitumor alkaloid camptothecin (CPT) was originally isolated from the Chinese tree Camptotheca acuminate in 1966, it had been developed as a specific used inhibitor of Topo I. Subsequently, topotecan [8] and irinotecan [9], derivatives of camptothecin, were approved by FDA for the treatment of recurrent ovarian cancer and second-line small-cell lung cancer. Top I has been a validated target for cancer chemotherapy [7]. In 1978, the first of indenoisoquinolines were synthesized for in vitro screen TOP I inhibitor [10]. Dozens of years after the identification

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Table 1 Inhibitory effects of indenoisoquinoline derivatives on the proliferation of endothelial cells (ECs).

No.

R3

ECs (IC50) mM

R1

R2

1

NO2

F

0.36  0.09

2

NO2

F

1.80  0.12

3

NO2

F

2.70  1.00

4

NO2

F

5

NO2

Cl

29.19  3.10

6

NO2

Cl

5.28  1.30

7

NO2

Cl

7.71  0.90

8

NO2

Cl

3.69  0.35

3.74  2.30

9

NO2

Cl

10 11

NO2 NO2

OCH3 OCH3

12

NO2

OCH3

5.58  0.24

13

NO2

OCH3

0.87  0.12

14

NO2

OCH3

8.60  0.91

15 16

NH2 NH2

F F

17

NH2

F

2.21  0.06

18

NH2

F

2.36  0.12

19

NH2

F

8.08  0.90

20

NH2

Cl

28.03  1.60

21 22

NH2 NH2

OCH3 OCH3

23

NH2

OCH3

25.79  1.90

24

NH2

OCH3

5.52  0.50

of the first indenoisoquinoline as a Top I inhibitor, two derivatives, NSC 724776 and 725998, are poised for clinical trials [11]. Indenoisoquinolines, compared with camptothecins, are hydrolytically stable but alternatively suffered from intrinsically low biological activity. As a result, considerable numbers of efforts have been devoted to overcome the disadvantage. One of the trails is our

3.42  0.80 Cl

Cl

Cl

29.19  0.87 1.34  0.15

133.4  5.40 1.907  0.14

78.27  3.4 29.7  2.5

previous study [12] leading to the discovery of compounds 1–24 with high inhibition activity. Tumor-endothelial cells could highly express Top I [13,14] and several researches have focused on the role of Top I inhibitors in the anti-angiogenic procedures. In 1999, Clements et al. had demonstrated topotecan inhibited angiogenic growth in the in vivo

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experiment. The final DMSO concentration did not exceed 0.1% throughout the study. In our study, all the control groups were given DMSO at 0.1% final concentration. 2.2. Cell culture

Scheme 1. Diagram of signaling pathways for compound 1 mediated antiangiogenesis.

rat disc angiogenesis model and O’Leary et al. reported the parallel results in a mouse cornea angiogenesis model in vivo in the same year. Furthermore, in neuroblastoma cells, topotecan was able to decrease the transactivation and secretion of VEGF by inhibiting the accumulation of Hif-1a and Hif-2a [15]. Metronomic chemotherapy with cytotoxic agents such as topoisomerase inhibitors irinotecan and mitoxantrone has been shown able to inhibit angiogenesis and, consequently, tumor growth by targeting vascular endothelial cells (ECs) [16]. It has been deemed that cytotoxic chemotherapy could inhibit the tumor angiogenesis by impacting the tumor vasculature such as killing of ECs, decreasing levels of angiogenesis stimulators and increasing the levels of endogenous angiogenesis inhibitors [17]. Chemotherapy targeting ECs has the advantage that the circulating drugs could rapidly attack the ECs which are less prone to mutate and acquire drug resistance because of the more stable genes than other tumor cells. The anti-proliferative properties of compounds 1–24 against different tumor cell lines and their Top I toxicities have been tested before and they showed prominent biological activity. The present work was to further study their anti-angiogenic activity and mechanism. These compounds can be divided into two series. The first series, the nitro substituent on the isoquinoline ring and the indenone ring with the electron-donating methoxy substituent and the electron-withdrawing substituent fluorine and chlorone, the other series of analogues was isoquinoline ring with aniline in an effort to further explore the effect of the nitro group (Table 1). Then both in vitro and in vivo angiogenesis model was applied to study the mechanism of compound 1 which showed most inhibitory activity on angiogenesis. The mechanism of indenoisoquinoline derivatives on angiogenesis may represent an advance in comparison to individual chemotherapy treatment and will serve as a basis to improve cancer therapy (Scheme 1).

Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cord veins by collagenase treatment as described previously [18]. The harvested cells were grown in medium RPMI-1640 containing20% FBS, 0.1 U/L penicillin, 0.1 mg/ L streptomycin. The cells were incubated in a humidified atmosphere of 95% air + 5% CO2 at 37 8C. After three to five passages, HUVECs were collected for use in experiments. Cells were maintained at 37 8C in a humidified incubator containing 21% O2 and 5% CO2 in air (referred to as normoxic conditions). Hypoxic conditions (1% O2) were achieved by culture in an anaerobic workstation incubator (Thermo)flushed with a gas mixture of 1% O2, 5% CO2, and 94% N2. The cells were obtained from consenting donors, as approved by the Research Ethics Board at the Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, with consideration for human ethics. 2.3. Cell proliferation assay In order to measure the number of viable cells, the 3-[4,5dimethylthiazol-2yl]-2,5-dimethyltetrazolium bromide (MTT) assay was used, as previously described [19]. In the cell proliferation assay, cells were plated in triplicate on 96-well plates (4000 cells/well) and incubated for 24 h. The cells were then incubated in culture medium that contained various concentrations of tested compounds, 0.1 mM/L, 1 mM/L, 10 mM/L, 100 mM/L, each dissolved in less than 0.1% DMSO (100 mL/well), and they were incubated for 48 h. The medium was then removed and the 3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay was performed by adding 20 mL MTT (5 mg/ mL) at 37 8C for four hours. Light absorbance of the solution was measured with a reader (Varioskan Flash Thermo Scientific) at 490 nm and expressed as absorbance. 2.4. Wound-healing assay (cell migration) Cell migration was carried out as previously described [20] with minor modifications. Endothelial cells were cultured in a six-well plate. Monolayers of EC (90% confluent) were starved overnight in serum-free medium and wounded using a 200 mL tip. The debris was removed by washing with serum-free medium and compound or vehicle was added in RPIM-1640 with 50 ng/mL VEGF (vascular endothelial growth factor) and 2% FBS. After 24 h incubation, the cells that migrated into the wounded area or that protruded from the border of the wound were visualized, photographed and quantified under an inverted microscope. 2.5. Chicken chorioallantoic membrane (CAM) assay

2. Materials and methods 2.1. Reagents Medium, supplements, and fetal calf serum were purchased from Gibco (Invitrogen, America). Antibodies (anti-HIF-1a) were supplied by R&D (America). Recombinant human vascular endothelial cell growth factor was purchased from Sigma (America). The synthesis of the indenoisoquinoline derivatives were reported in our previous manuscript [10]. They were dissolved at a concentration of 10 mM in 100% DMSO as a stock solution, stored at –20 8C, and diluted with medium before each

Anti-angiogenic activity of compound 1 on CAM was assayed as described previously [21]. Briefly, fertilized chicken eggs were incubated at 37 8C for 9 days. After this incubation, a small hole was punched on the broad side of the egg, and a window was carefully created through the eggshell. Sterilized filter paper disks (5  5 mm) saturated with vehicle or with compound 1 (2, 20 and 200 ng/egg) were placed on the CAMs. The eggs were then incubated at 37 8C for another 3 days. Lastly, an appropriate volume of 10% fat emulsion (Intralipose, 10%) was injected into the embryo chorioallanto to observe the density and length of vessels toward the CAM face, and then the CAMs were photographed.

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Three eggs were used per group, and the number of newly formed vessels was counted. 2.6. Rat aortic ring assay Rat aortic ring assay was performed as described previously [22]. The thoracic aorta was dissected from 6-week-old SpragueDawley rats and cut into 1-mm-longrings, and then these rings were placed in 24-well plates. The rings were treated as described previously. Clotting media contained RPIM-1640 plus 0.3% fibrinogen and 0.5%e-amino-n-caproic acid (ACA, Sigma). Growth media consisted of RPIM-1640 with 20% FBS and 0.5%ACA. Then the growth media was added to the wells with or without different concentrations of compound 1. Plates were then stored in incubator at 37 8C and 5% CO2. After 7 days, the microvessel growth was quantified by manually counting the number of microvessels from the rat aortic rings, with three rings used as a group. The use of animals was in accordance with the guidelines established by the National Science Council of Republic China, with adherence to the ethical guidelines for the care and use of animals. 2.7. Tube formation assay A 96-well tissue culture plate was pre-chilled at –20 8C and carefully coated with Matrigel (100 mL/well; Becton Dickinson, Bedford, MA, USA) avoiding bubbles. The plate was incubated at

37 8C for 30 minutes to allow the Matrigel to solidify. HUVECs were incubated in medium containing 1% FBS in the absence or presence of different concentrations compound for 4 h. These cells were harvested after trypsin treatment and suspended in medium containing 1% FBS before seeding and plating onto matrigel and then VEGF (10 ng/mL) was added. After 8 h, tubular structures were quantified by manual counting of the tube numbers, and three randomly chosen fields were analyzed for each well. 2.8. Western blotting HUVECs were pretreated with various concentrations of compound 1 under hypoxia. After stimulation, cells were collected; lysed in lysis buffer [50 mM Tris-Cl, pH 7.6, 150 mM NaCl, 1 mM EDTA, 1% (m/v) Nonidet P-40 (NP-40), 0.2 mM Phenylmethanesulfonylfluoride (PMSF), 0.1 mM NaF and 1.0mMdithiothreitol (DTT)], and lysates were centrifuged at 4 8C for 15 min at 13,000  g. The concentration of protein in the supernatants was measured by the BCA (bicinchoninic acid) assay with a Varioskan multimode microplate spectrophotometer (Thermo, Waltham, MA, USA). Then equal amounts of protein (50 mg) were separated by 8% or 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto the PVDF membranes (Millipore). The blots were incubated with specific antibodies against the indicated primary antibodies overnight at 4 8C followed by IRDyeTM800-conjugated secondary antibody for

Fig. 1. A. The wound-healing assay was used to evaluate motility of HUVECs after treating with compounds 1, 2, 11, 13 for 24 h. B. Quantity of migrated cells presents an average from three experiments independently. Data were represented as means  SEM.

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Fig. 2. Effect of compound 1 on tube formation of HUVECs. HUVECs were incubated with compound 1 for 6 h, and then transferred to the matrigel with 50 ng/mL VEGF. A– D. The tube formation groups with compound 1 treatment at concentrations of 0.1%DMSO, 0.1, 1.0 and 10 mM. E. Quantitation of the number of branches formed in the tube formation assay. Compound 1 exerted a concentration-dependent inhibition of VEGF-stimulated tube formation of HUVECs. Data were represented as means  SEM. * P < 0.05, ***P < 0.001 vs. the control group.

1 h at 37 8C. Detection was performed by the Odyssey Infrared Imaging System (LI-COR; Lincoln, NE, USA). All blots were stripped and incubated with polyclonal anti-b-actin antibody to ascertain equal loading of proteins. 2.9. Real-time qRT-PCR EC (5  105) cultured in 6-well cell culture plates were incubated for the indicated times with or without compound 1 under normoxic (21% O2) or hypoxic (1% O2) conditions. The RT was completed with a Clontech Advantage RT-for-PCR kit using hexamer random primers. The expression of each target gene was normalized to the relative expression of GAPDH as an internal efficiency control. The values are expressed as fold of the control. Primers are shown as follows: VEGF, forward primer 50 -TACCTCCACCATGCCAGGTG-30 , reverse primer 50 -AAGATGTCCACCAGGGTCTC-30 ; HIF-1aforward primer 50 -CTCAAAGTCGGACAGCCTCA-30 , reverse primer 50 -CCCTGCAGTAGGTTTCTGCT-30 ; AMD, forward primer 50 -CGCAGTTCCGAAAGAAGTGG-30 , reverse primer 50 -CCAGTTGTGTTCTGCTCGTCC-30 ; Glut1, forward primer 50 -GAGGAGCTCTTCCACCCTCT-30 , reverse primer 50 -TCCTCCTGGACTTCACTGCT-30 ; GAPDH (internal control), forward primer 50 -AGGTCGGAGTCAACGGATTTG-30 , reverse primer 50 -GTGATGGCATGGACTGTGGT-30 . 2.10. Statistical analysis All data were obtained from at least three independent experiments and expressed as mean  SEM. Comparisons of the different groups were performed with Student’s t-test. Significant differences comparing more than two groups were analyzed by 1- or 2-factor ANOVA and Bonferroni’s test for multiple comparisons, using Graphpad prism.

activity of indenoisoquinoline derivatives against HUVEC cell line, among which compounds 1, 2, 11, 13 were more effective than others. Among the tested compounds, compound 1 exhibited the lowest IC50 (0.36 mM) and was found to be the most potent, causing its effect at significantly lower concentrations compared with all the other compounds, whose potencies were significantly different. 3.2. Indenoisoquinoline derivatives reduces VEGF-stimulated HUVEC migration As is known, cell migration is critical for endothelial cells to form blood vessels in angiogenesis and thus is necessary in tumor growth and metastasis [23]. Compounds 1, 2, 11, 13 were tested in the healing migration assays because of their relevant high inhibitory (IC50 range, 0.36–1.8 mM) of endothelial cells growth. In this experiment, the wounds could be gradually ‘healed’ by cell migration with VEGF as a stimulator. Representative photographs of cells migrating into scratch wounds in ECs monolayers are shown in Fig. 1A. The wounds were gradually ‘healed’ by cell migration during 0–24 h and VEGF potentiated cell migration into the wound area. As shown in Fig. 1A, compared with the VEGF alone group, all indenoisoquinoline derivatives tested caused a decrease in the number of migrated ECs, with compound 1 being the most effective. Compound 1 suppressed the VEGF-induced migration in a concentration-dependent manner over the controls at 0.4, 1, and 2.5 mM, respectively, suggesting that it significantly inhibited the migration properties of endothelial cells at very low concentrations (nmol/L) (Fig. 1A). These results demonstrate that compound 1 has an ability to reduce VEGF-induced migration of ECs. 3.3. Compound 1 suppresses tube formation of endothelial cells

3. Results 3.1. Effect of indenoisoquinoline derivatives on the number of HUVEC in vitro Angiogenesis is characterized by the local proliferation of endothelial cells. We studied the ability of all the compounds to inhibit the growth of endothelial cells. All indenoisoquinoline derivatives tested for the first time caused a concentrationdependent decrease in the number of HUVEC cells. Table 1 lists

It has been known that tube formation of endothelial cells is the key step in angiogenesis although angiogenesis is a complex procedure of several kinds of cells [24]. The angiogeneic factor VEGF can stimulate ECs to seed on matrigel to differentiate and form capillary-like tubes [25]. To further study the effect of compound 1 on angiogenesis in vitro endothelial cells, tube formation assay was conducted. After 8 to 12 hours of incubation, the ability of endothelial cells to form a mesh of tubes was assessed with an inverted photomicroscope. The tube structures were

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Fig. 3. Effect of compound 1 on rat aortic ring microvessel sprouting angiogenesis in CAM model. A–D. The rat aortic ring groups with compound 1 treatment at concentrations of 0.1% DMSO, 0.4, 2.0 and 10 mM. Compound 1 inhibited neovessel formation from the rat aortic rings, the inhibitory percentages of concentrations at 0.4, 2.0 and 10 mM were 45.2%, 63.9% and 82.2%, respectively (E). F–I. The CAM groups with compound 1 treatment at concentrations 2 ng, 20 ng, 200 ng/egg for 72 h. The quantitative data were summarized and indicated that compound 1 obviously decreased the number of the blood vessels (J). Data were represented as means  SEM. **P < 0.01, ***P < 0.001 vs. the control group.

concentration-dependently disrupted when cells were exposed to compound 1. ECs treated with lower concentrations of compound 1 (0.1,1 mM) differentiated into short tubes but were unable to form meshes, whereas those treated with higher concentration of compound 1 (10 mM) remained dotted on the matrigel. Their dose-response curves are shown in Fig. 2E. It can be seen the inhibitory gradient of tube forming corresponded directly with the one of cell migration, further vertifying the crucial significance of cell migration. These results show that compound 1 has the ability to block VEGF-induced tube formation of ECs. 3.4. Compound 1 inhibits angiogenesis in vitro In order to examine the inhibitory effect of compound 1 on angiogenesis, the aorta sprout outgrowth assay in vitro was performed using isolated aortas from mice. This assay mimics many steps in angiogenesis, such as, endothelial cell proliferation, migration, and tube formation. After being 7 days of incubation, the numbers of microvessel growth of the aortic rings were quantified and compared. As can be seen from the results in Fig. 3, Compound 1 dramatically decreased the number of sprouting vessels in the rat aorta ring. On the final study day, the observed growth inhibition was 45.2% with 0.2 mM, 63.9% with 2 mM and 82.2% with 10 mM (Fig. 3A–E). It is suggested that compound 1 dramatically inhibited angiogenesis in vitro.

3.5. Compound 1 shows an in vivo anti-angiogenic effect in the CAM assay The CAM assay provides a characteristic model for estimating the effect of anti-angiogenic agents on the process of blood vessel formation, and so it has always been used to model angiogenesis in vivo. To further investigate the effect of compound 1 on angiogenesis in vivo, we applied the CAM model. We treated CAM at different dosages to evaluate the effect of compound 1 on angiogenesis. As shown in Fig. 3F–J, branches of blood vessels normally formed after 3 days incubation in the vehicle treated control group, whereas vessel sprouting on the surface of the CAM was significantly reduced in the presence of compound 1. Quantitative analysis revealed that Compound 1 at 2, 20, and 200 ng/egg caused gradually reduction in the number of new blood vessels, respectively. These results demonstrate that compound 1 could suppress angiogenesis in CAM models. 3.6. Effects of compound 1 on HIFa signaling pathway In this part, we performed Western-blot analysis and real-time PCR to evaluate the molecular mechanism by which compound 1 antagonizes hypoxia-stimulated angiogenesis in HUVECs. The effect of compound 1 on the expression of HIFa was tested over a range of concentration in the hypoxia-stimulated HUVECs for 6 h. Expression levels of HIFa proteins were detected by western

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Fig. 4. A. Effect of compound 1 on HIF-1a protein expression in human EC under hypoxic conditions (1% O2). B. Effect of compound 1 (0.2 mM) on mRNA expression of HIF and target genes in human ECs.

blotting. Hypoxia-induced HIF-1a protein accumulation was dosedependently attenuated by compound 1. As shown in Fig. 4A, the protein levels of the HIF-1a was significantly reduced at a concentration of 0.1 mM and barely detected at 0.2 mM concentration. However, compound 1 didn’t eliminate the expression of HIF-1a completely, which may attribute to the complexicity in angiogenesis. The down-regulation of HIF-1a was also confirmed in transcriptional levels of mRNA using Real-time PCR as can be seen in Fig. 4B. As expected, the mRNA levels of ADM, VEGF and Glut1 were reduced since they are all the targets of HIF-1a protein. Angiogenesis is a highly regulated process and is largely under the control of the VEGF gene and so on [1]. The hypoxic induction of ADM, VEGF and Glut1mRNA levels, both target genes of HIF-1a, as well as HIF-1a, were substantially repressed in the presence of compound 1. As expected, hypoxia (1% O2) strongly induced ADM, VEGF and Glut1 mRNA expression (Fig. 4B). Taken together, these results suggest that compound 1 may attenuate HIF-1a gene expression at a transcriptional level, thereby affecting HIF1dependent cellular adaptive responses. 4. Discussion Topoisomerase I, a significant protein in promoting cell proliferation, has become a successful therapeutic target for several anticancer agents [26]. Topoisomerase I inhibitors, such as the most well known ones camptothecin and topotecan, showed an ability to suppress tumor angiogenesis in vitro and in vivo. They were identified as HIF-1a inhibitors in a high throughput cellbased HRE-luciferase reporter screen [5] It was demonstrated that topotecan inhibits HIF-1a, leading to a remarkable decrease in angiogenesis and significant tumor growth inhibition [15,27,28]. In our previous work, a series of indenoisoquinoline derivatives were

synthesized and evaluated for anti-angiogenesis activity. The potent 24 compounds presented significant inhibitory effect on Top I. In the present work, we studied the effect of these topoisomerase I inhibitors on the activity of anti-angiogenesis. The data revealed that some tested compounds have potent inhibitory effect on angiogenesis in vivo and in vitro. The present findings demonstrated that HIF-1a was suppressed by compound 1 under hypoxic conditions, which is accompanied by attenuating relevant target genes such as VEGF, ADM, and Glut1. Concomitantly, the decreased HIF-1a protein expression induced by compound 1 reduced angiogenic responses in ECs. Angiogenesis, the regulated process leading to the formation of new blood vessels, contains tightly multiplex steps. The initial step begins when the balance between pro-angiogenic cytokines and anti-angiogenic cytokines is broken, resulting the receptors for the pro-angiogenic regulators exposing on the surface of vascular endothelial cells in abundance. Therefore during the process of tumor angiogenesis, endothelial cell proliferation and migrate are the most important steps [1]. We tested the anti-proliferative and anti-angiogenic effects of indenoisoquinoline derivatives by using in vivo and in vitro models. From the result of antiproliferation assay on ECs presented in Table 1, the substitution pattern at the indenoisoquinoline 3-position, 9-position, and the lactam side chain has a pronounced effect on the biological activities of the molecules. Comparing compounds 1 and 16 (IC50, 0.36 mM, 1.9 mM) with compounds 3 and 19 (IC50, 2.7 mM, 8.08 mM) led up to the conclusion that compounds with a nitro substituent on the isoquinoline ring were more efficiently than that with an aminogroup. It accorded with previous investigation that the biological activity of indenoisoquinolines was improved by introducing a nitro substituent on the isoquinoline ring [29]. These compounds showed weaker inhibitory activity against ECs compared with

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tumor cells which added the selectivity of compounds. Compounds 1, 2, 11, 13 with better ECs inhibitory activity were chosen to test the ability for preventing the migration of ECs. The results displayed that compound 1 showed the most effective in the wound-healing assay in Fig. 1A. Compound 11 performed better than compound 13, meanwhile, worse than compound 1, suggesting that modification of a methoxy group at the 9-position to the electron-withdrawing fluorine atom led to enhancement of angiogenesis inhibition, especially combined with a methylpiperazinyl substituted lactam nitrogen. Above all, compound 1 was acting as an effective Top I inhibitor. These data further support the previously described conclusion that topoisomerase I inhibitor could suppress the tumor angiogenesis in vitro and in vivo [1,16]. In our following study, both in vitro and in vivo approaches were used to investigate the potential anti-angiogenic activity of compound 1. Compound 1 inhibited the formation of capillary-like chords in response to VEGF stimulation in vitro. Furthermore, it suppressed the microvessel sprouting from vascular tissues of rat aorta. In addition, it prominently decreased new vessel formation and vascular network in the CAM model. These results implied that compound 1 could act as a probable anti-angiogenic compound. HIF-1a is a key transcriptional activator of genes overexpressed in broad range of tumor types, and is needed for angiogenesis, glycolytic metabolism, cell survival and invasion [30]. Since upregulated HIF expression promotes tumor angiogenesis, inhibition of HIF activity is a promising approach to suppress tumor angiogenesis. Here, we provided evidence that compound 1 with anti-angiogenesis activity markedly interrupted the hypoxiainduced HIF-1a expression. Many genes, such as Glut 1 and ADM, VEGF is critical for adaptation to a hypoxia environment and is primarily regulated by HIF-1a under oxygen-deprived conditions. It was observed that compound 1 had an ability to reduce the expression of VEGF, ADM, and Glut 1 mRNA levels induced by hypoxia. This inhibitory effect of compound 1 on HIF-1a expression and its signal transduction may be at least in part contribute to its potent anti-angiogenic activity. In conclusion, a series of indenoisoquinoline derivatives were screened as inhibitors of anti-angiogenesis by using a rat ring model, a wound-healing model, a tube formation model and CAM assay. Compound 1 significantly inhibited angiogenesis in vitro and in vivo. It exerted inhibition by interfering with angiogenesis at various steps and through interfering with HIF-1a signal pathway. These data suggest that compound 1 could be investigated for their usefulness in the treatment of angiogenesis-related pathologies. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements The authors express their deepest appreciation to Professor Zhiyu Li for his compounds and to Professors Yong Yang by the availability of the laboratory, respectively. References [1] Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011;144:646–74. [2] Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000;407:249–57.

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