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Novel Ligustrazine–Based Analogs of Piperlongumine Potently Suppress Proliferation and Metastasis of Colorectal Cancer Cells in Vitro and in Vivo Yu Zou, Di Zhao, Chang Yan, Yanpeng Ji, Jin Liu, Jinyi Xu, Yisheng Lai, Jide Tian, Yihua Zhang, and Zhangjian Huang J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01096 • Publication Date (Web): 09 Feb 2018 Downloaded from http://pubs.acs.org on February 10, 2018

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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Medicinal Chemistry

Novel Ligustrazine–Based Analogs of Piperlongumine Potently Suppress Proliferation and Metastasis of Colorectal Cancer Cells in Vitro and in Vivo Yu Zou,a,b,d,# Di Zhao,a,c,# Chang Yan,a,b Yanpeng Ji, a,b Jin Liu,a,b Jinyi Xu,a Yisheng Lai,a,b Jide Tian,e Yihua Zhang*,a,b and Zhangjian Huang,*,a,b a

State Key Laboratory of Natural Medicines, China Pharmaceutical University,

Nanjing 210009, P.R. China. b

Jiangsu Key Laboratory of Drug Discovery for Metabolic Diseases, China

Pharmaceutical University, Nanjing 210009, P.R. China.

c

Clinical Pharmacokinetics Laboratory, Department of Clinical Pharmacy, School of

Basic Medicine and Clinical Pharmacy, China Pharmaceutical University, Nanjing 211198, P.R. China. d

Department of Pharmacy, College of Medicine, Wuhan University of Science and

Technology, Wuhan, Hubei Province 430065, P.R. China. e

Department of Molecular and Medical Pharmacology, University of California, Los

Angeles, California 90095, United States.

ACS Paragon Plus Environment

Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Piperlongumine 1 increases reactive oxygen species (ROS) levels and preferably induces cancer cell apoptosis by triggering different pathways. However, the poor solubility of 1 limits its intensive investigation and clinical application. Ligustrazine possesses a water-soluble pyrazine skeleton and can inhibit proliferation and metastasis of cancer cells. We synthesized compound 3 by replacement of the trimethoxyphenyl of 1 with ligustrazine moiety, and further introduced 2-Cl, -Br, and -I to 3 for synthesis of 4-6, respectively. Compound 4 possessed 14-fold greater aqueous solubility than 1 and increased ROS levels in colorectal cancer HCT-116 cells. Additionally, 4 preferably inhibited proliferation, migration, invasion, and hetero-adhesion of HCT-116 cells. Treatment with 4 suppressed tumor growth and lung metastasis in vivo and prolonged the survival of tumor-bearing mice. Furthermore, 4 mitigated TGF-β1-induced epithelial-mesenchymal transition and Wnt/β-catenin activation by inhibiting the Akt and GSK-3β phosphorylation in HCT-116 cells. Collectively, 4 displayed significant anti-proliferation and anti-metastasis activities, superior to 1.

INTRODUCTION Accumulating evidence reveals that increased levels of reactive oxygen species (ROS) by elevating ROS production or reducing ROS-scavenging capacity is often associated with promoting cancer cell growth.1 Moderate levels of oxidative stress can enhance proliferation, angiogenesis, and metastasis of cancer cells.2 In contrast, high levels of ROS can irreversibly damage DNA and lipids, and induce cancer cell apoptosis.3 In addition, some therapeutic agents and high levels of oxidative stress can


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synergistically damage cancer cells.4,5 Therefore, manipulation of ROS levels is an important strategy to selectively kill cancer cells. Actually, there have been many efforts to increase the levels of ROS specifically in cancer cells, which is known as “oxidation therapy”.6-8 One strategy for the oxidation therapy is to directly deliver ROS-promoting agents, such as piperlongumine (1, Figure 1),9 arsenictrioxide (As2O3),10 or glucose oxidase11 to tumor tissues. Piperlongumine 1, an alkaloid in the fruit of long pepper, is a naturally occurring small molecule product with multiple pharmacological activities.12-16 Recent studies have documented that it selectively increases ROS in tumor cells, inhibits proliferation, invasion and migration, and induces apoptosis of the cells.17-19 However, the poor solubility of 1 limits its intensive investigation and clinical application.20,21 Although a great number of derivatives and analogs of 1 have been synthesized in order to get a more promising anticancer drug,22-26 the solubility currently remains challenging. Additionally, the derivatives and analogs of 1 with both anti-proliferative and anti-metastatic activities have so far not been documented. In this regard, the development of new anticancer analogs of 1 with improved water solubility as well as potent anti-proliferative and anti-metastatic effects should be of great importance. Our previous study indicated that replacing the trimethoxyphenyl of 1 by a pyrazine or pyridine ring remarkably improved aqueous solubility and displayed stronger anticancer activity in vitro.27 In addition, the 2-halogenated 1 reportedly showed more potent in vitro anticancer activity than 1 due to its higher reactivity of C2-C3 Michael acceptor.26,27 It is notable that ligustrazine (2) with a water-soluble pyrazine skeleton



is a Chinese traditional medicine for treatment of cardiovascular and cerebrovascular diseases.28 Recently, 2 has been reported to inhibit the proliferation and metastasis of tumor cells, increase intracellular ROS accumulation, and induce cancer cell apoptosis.29,30 Accordingly, we hypothesized that hybrids of 1 with 2 via replacement of the trimethoxyphenyl of 1 by ligustrazine moiety and with or without introduction of a 2-halogen could produce a hitherto unknown class of analogs of 1 possessing better solubility and more potent anti-proliferative and anti-metastasis activities against cancer cells. To test the hypothesis, we synthesized novel hybrids 3-6 (Figure 1) and investigated their bioactivity in vitro and in vivo, and the potential mechanisms.

Figure 1. The structure-based hybridization and scaffold hopping of novel piperlongumine−ligustrazine hybrids 3-6.

RESULTS AND DISCUSSION Chemistry The synthetic routes of target compounds 3-6 are depicted in Scheme 1. 2-Hydroxymethyl-3,5,6-trimethylpyrazine 9 was prepared by the Boekelheide


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reaction starting from hydrochloride of 2 via a four-step reaction sequence as described previously.31 The compound 9 was oxidized by 2-iodoxybenzoic acid (IBX) to give aldehyde 10, which underwent Wittig-Horner reaction to furnish ester 11. Hydrolysis of 11 led to free acid 12, which was converted to acylchloride, without isolation

and

purification

followed

by

reacting

with

corresponding

5,6-dihydropyridin-2(1H)-one 17, as well as with 6-substituted-5,6-dihydro-pyridin2(1H)-ones 19 and 21 to provide compounds 3-5, respectively. Compound 6 was obtained by iodination at position 2 of 3. Compound 13, an analogue of 3 without C2-C3 double bond, was synthesized by treatment of 12 with the commercially available piperidin-2-one 14 for biological evaluation. As key intermediates, 17, 19 and 21 were prepared via two- or three-step reactions from 14 (Scheme 1B).23 Compound 17 was obtained by treatment of 14 with trimethyl chlorosilane (TMSCl), phenylselenyl chloride, and hydrogen peroxide, successively. Treatment of 14 with PCl5 yielded 2,2-dichloropiperidin-2-one 18, followed by dehydrochlorination in the presence of Li2CO3 to offer 6-chloro-5,6-dihydropyridin2(1H)-one 19. 6-Bromo-5,6-dihydro-pyridin- 2(1H)-one 21 was similarly prepared as 19. The structures of all hybrids were fully characterized by 1H-NMR, 13C-NMR, MS, and HRMS, and the 7,8-olefinic bond in the hybrids was identified as E configuration. All target compounds with a purity of >95%, determined by high performance liquid chromatography (HPLC) analysis, were used for subsequent experiments.



B

O

O

O

a

HN

14

Cl

HN

O f

HN

20

Si

c

NH

17

O Cl

e

Cl

HN

19

18

O

O N

16

O

14

14

Se

b

15

d

HN

Si

N

O HN

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Br

O Br

g

Br

HN

21

Scheme 1. A. The synthetic route of compound 3-6. Reagents and conditions: a. NaOH, H2O, 1-2 h, 96%; b. 30% H2O2, AcOH, 70 ºC, 8 h, 85%; c. Ac2O, reflux for 2 h, 87%; d. 20% NaOH, 90%; e. IBX, DMSO, rt, 0.5 h, 92%; f. Wittig-Hornor reaction: toluene, NaH, triethyl phosphonoacetate, rt, under dark, 8-10 h, 76%; g. KOH, 16 h, 89%; h. anhydrous DCM, TEA, pivaloyl chloride, one drop of DMF; then n-butyllithium, 17，19 or 21 for 3 (65%), 4 (61%) or 5 (59%). i. iodine, Py, THF, rt, overnight for 6, 74%; j. (1). anhydrous DCM, pivaloyl chloride, one drop of DMF;


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(2). anhydrous THF, TEA, n-butyllithium and 14 for 13, 67%. B. The synthesis route of 17, 19 and 21. Reagents and conditions: a. toluene, TEA, TMSCl, 0 ºC, 4 h; b. THF, phenylselenyl chloride, LDA, -50 ºC; c. THF, H2O2, 77% (a-c); d. PCl5, chloroform, rt; e. Li2CO3, LiCl, anhydrous DMF, 130 ºC, 7 h, 75% (d-e); f. (i). PCl5, DCM, 0 ºC; (ii). ZnI, Br2, rt, overnight; g. Li2CO3, LiCl, anhydrous DMF, 130 ºC, 7 h, 68% (f-g).

The solubility of compound 1, 3-6 was determined in both pure water and phosphate buffer (50 mM, pH 7.4) at 20 °C by HPLC. 32 It was found that all hybrids were more soluble than 1. Among them, solubility of 3 (42.63 µg/mL) and 4 (22.84 µg/mL) was approximately 26- and 14-fold greater than 1 (1.63 µg/mL), respectively. The improvement of the aqueous solubility is probably attributable to the ligustrazine moiety, which is more water-soluble than thrimethoxyphenyl ring in 1.

Table 1. The solubility (µg/mL) of compounds 1 and 3-6. compound

1

3

4

5

6

In water

1.63

42.63

22.84

20.17

14.50

In PBS (pH 7.4)

0.32

328.16

74.62

67.28

49.85

Biological evaluations Assessment of in vitro anti-proliferative activity. Compounds 3-6 were tested for their anti-proliferative activity against human glioma U87MG, colorectal cancer HCT-116, lung cancer A549 and leukemic K562 by MTT assay.



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As shown in Table 2, all hybrids showed a more potent activity in inhibiting the proliferation of cancer cells than 1 alone or combination of 1 with 2. The most active compound 4 exhibited a strong cytotoxicity against human colorectal cancer HCT-116 cells with an IC50 of 0.30 ± 0.03 µM, which was 27-fold stronger than 1 (IC50 = 8.13 ± 0.51 µM). Notably, compounds 1, 3-6 more strongly inhibited the proliferation of HCT-116 and K562 cells than that of U87MG and A549. Probably, the former cells are more sensitive to the ROS elevation induced by the tested compounds than the latter ones. Interestingly, compound 13 without C2-C3 double bond failed to inhibit the proliferation of cancer cells tested, suggesting that the C2-C3 double bond may be essential for the inhibitory activity of piperlongumine-ligustrazine hybrids.

Table 2. The IC50 (µM) of 3-6 against human cancer cells a Compound

U87MG

HCT-116

A549

K562

3

8.73 ± 0.40

1.67 ± 0.30

4.86 ± 0.21

1.13 ± 0.01

4

3.43 ± 0.31

0.30 ± 0.03

2.21 ± 1.19

0.25 ± 0.01

5

4.28 ± 0.87

0.46 ± 0.03

2.48 ± 0.65

0.48 ± 0.02

6

7.26 ± 0.83

0.76 ± 0.05

3.19 ± 0.48

0.64 ± 0.01

13

>50.00

>50.00

>50.00

>50.00

1

17.34 ± 0.23

8.13 ± 0.51

15.28 ± 0.19

5.05 ± 0.02

1+2

16.15 ± 0.81

8.17 ± 0.26

15.22 ± 1.20

5.09 ± 0.10

a

Cells were treated with indicated compounds in triplicate for 72 hours and the cell

viability was determined using MTT assay, respectively.


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Further tests demonstrated that 4 was also powerful against other colorectal cancer cells with IC50 values ranging from 0.54-1.21 µM, which were much less than that of 1 (Table 3). In contrast, 4 showed much lower inhibitory activity against non-tumor colon CCD-841 cells (IC50 = 51.55 µM), suggesting that 4 may selectively inhibit the proliferation of colorectal cancer cells. Thus, we selected it for further investigations.

Table 3. The IC50 (µM) of 4 against colorectal cancer cells and non-tumor colon cellsa

a

Cell lines

HT-29

SW620

HCT-8

HCT-116

CCD-841

1

2.65 ± 0.17 4.62 ± 0.22 4.10 ± 0.20 8.13 ± 0.11 44.32 ± 1.77

4

0.54 ± 0.04 0.87 ± 0.06 1.21 ± 0.08 0.30 ± 0.03 51.55 ± 2.08

Cells were treated with indicated compounds in triplicate for 72 hours and the cell

viability was determined using MTT assay, respectively.

Comparison of the Chemical Reactivity of the Two Olefins in 4 It has been reported that the C2-C3 olefin is more reactive than the C7-C8 olefin in compound 1,22 and that the C2-C3 olefin on the ring A is more reactive than C9-C11 olefin on the ring C in 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester (CDDO-Me).33 Structurally, compound 4 possesses two Michael acceptors, i.e. C2-C3 and C7-C8 olefins, that may influence its actions on cells. Therefore, it was of interest to examine the chemical reactivity of C2-C3 and C7-C8 olefins in 4 on a representative thiol-containig nucleophile, methyl thioglycolate. It could be seen from Figure S1, the proton signal and integral value of C3-H (designated as Hc) in 4 at δ



7.37 ppm was time-dependently decreased, and two new proton signals at δ 3.60 and 5.08 ppm appeared, with the integral value gradually increased, which was closely related to C3-H. Thus, in combination of other relative variations in the 1H NMR spectra (Figure S1, Tables S1 and S2), two protons could be unambiguously assigned as Hi and Hj of compound 22 (Scheme S1), respectively. Interestingly, no addition reaction was observed at C8, even in the presence of 3.0 equivalents of methyl thioglycolate (Figure S2, Tables S3 and S4). And this selective reaction on C2-C3 olefin is consistent with the previous reports on compound 1 and CDDO-Me.22,33 Pharmacokinetics of 4 in rats. The pharmacokinetics of 4 was evaluated in rats in comparison with 1. Compound 1 or 4 at 20 mg/kg was intravenously administered to rats (n = 3), blood samples were taken from suborbital vein at 0.033, 0.083, 0.25, 0.5, 0.75, 1, 2, 4 h, and analyzed by LC-MS-MS. The corresponding blood concentration–time profiles of 1 and 4 were shown in Figure S5, and pharmacokinetic parameters in Table S6. AUC0–∞ (area under concentration–time curve from time zero to infinite), T1/2 (half-life), and Cmax (maximum plasma concentration) of 1 and 4 were 273.0 ± 40.1 ng·h/mL, 2.87 ± 0.22 h, and 563.8 ± 129.6 ng/mL, as well as 70.2 ± 16.9 ng·h/mL, 2.17 ± 0.95 h, and 44.3 ± 4.8 ng/mL, respectively. The CL (clearance rates) and Vss (distribution volumes) of 1 and 4 were 0.074 ± 0.012 mL/h/kg and 0.31 ± 0.07 mL/kg, as well as 0.297 ± 0.077 mL/h/kg and 0.903 ± 0.4 mL/kg, respectively. Considering the fact that the 2-halogenated derivatives or analogs of 1 display higher reactivity of C2-C3 Michael acceptor than 1,26,27 it would be expected that 4 would possess a faster metabolic behavior relative to 1, leading to the lower values of


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AUC0-∞, t1/2, and Cmax as well as higher values of CL and Vss of 4 as compared to those of 1. In addition, the stability of 4 in HCT-116 cell lysis, simulated gastric fluid (pH 1.4) and intestinal fluid (pH 6.8) were investigated. As shown in Figures S6-8, 4 bearing a basic ligustrazine moiety was much more stable in simulated gastric fluid than in HCT-116 cell lysis and intestinal fluid.

Compound 4 selectively promotes more ROS accumulation than 1 in HCT-116 cells. It is known that the inhibition of glutathione S-transferase P1 (GSTP1) activity by 1 disturbs glutathione (GSH) balance, leading to intracellular ROS accumulation in cancer cells.34 Accordingly, we examined whether and how 4 impacted ROS levels in HCT-116 and CCD-841 cells. HCT-116 and CCD-841 cells were treated in triplicate with 1 and 4 at 1, 5, and 10 µM for 1, 2 and 3 h, respectively. The levels of intracellular ROS were detected with 2',7'-dichlorodihydrofluoresceine diacetate (DCFH-DA) by using a fluorescent microplate reader to measure the fluorescent signals. As shown in Figure 2A-C, treatment with 4 rapidly and significantly increased the levels of intracellular ROS in HCT-116 cells while the same treatment only increased moderate levels of ROS in CCD-841 cells. The selectivity of 4 was obviously greater than that of 1, indicating that 4 more preferably promoted intracellular ROS accumulation relative to 1 in the cancer cells. Fluorescence microscopy analysis indicated that treatment with 1 and 4 at 10 µM for 3 h resulted in green fluorescent positive HCT-116 and CCD-841 cells (Figure



2D-I). The images (Figures 2E and 2F) further supported that 4 more selectively increased the levels of ROS in the cancer cells than 1.

Figure 2. Compound 4 selectively promotes the accumulation of intracellular ROS in colorectal cancer cells. HCT-116 and CCD-841 cells were treated in triplicate with 1 and 4 at 1, 5, and 10 µM for 1, 2, 3 h, respectively. The intracellular ROS were stained with DCFH-DA and measured by a fluorescent spectrometry and microscope. Data are representative images or expressed as the means of individual groups of cells (relative to DMSO control) from three separate experiments. A-C. the levels of ROS in HCT-116 and CCD-841 cells (relative to DMSO control). D-I. Fluorescence images of HCT-116 cells (D-F), fluorescent images of CCD-841 cells (G-I), and both types of cells were treated with, or without, 1 and 4 at 10 µM for 3 h, respectively.

Importantly, pretreatment with GSH not only reduced the ROS levels (data not shown), but also diminished the inhibition of 4 on the proliferation of HCT-116 cells


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from 82.9% to 36.1% (Figure 3). A similar result was observed in the case of compound 1 (Figure 3), which is reportedly a ROS-promoting agent.9 These results suggest that 4 may inhibit the proliferation of colorectal cancer cells by selective promotion of ROS accumulation, at least in part, through disturbing the GSH balance.

Figure 3. Pretreatment with GSH diminishes the inhibition of 4 or 1 on the proliferation of HCT-116 cells. HCT-116 cells were pretreated in the presence or absence of GSH (10 mM) for 1 h then washed, and treated with 4 (1.52 µM) or 1 (8.0 µM) for 72 h. The viability of cells was determined by MTT and inhibitory rates were calculated. Data are expressed as the mean ± SD of individual groups of cells from three independent experiments. ***P < 0.001.

Compound 4 inhibits the growth of HCT-116 xenograft tumors in mice. To investigate the safety profile of the hybrids, the acute toxicity of 4 was determined in ICR mice at doses of 50, 100, 150, 200, 300 and 400 mg/kg (ip, n = 10 per group) for 14 days. As shown in Table 4, treatment with 4 at the lowest dose (50 mg/kg) did not



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cause any death in mice. However, treatment with 4 at 400 mg/kg killed all the mice. Finally, the median lethal dose (LD50) value of 4 was calculated to be 147.54 mg/kg. To evaluate in vivo anti-colon cancer activity of 4, BALB/c nude mice were inoculated subcutaneously with colorectal cancer HCT-116 cells. After the establishment of solid tumors, the mice were randomized and treated intraperitoneally with oxaliplatin (5 mg/kg), 1 (5 mg/kg) or 4 (2.5 or 5 mg/kg) daily for 21 consecutive days (Figure 4), respectively. As shown in Figure 4 and Table S5, treatment with 4 at 5 mg/kg inhibited the growth of the colon cancer cells in the mice by 71.4% (w/w), which was more potent than 1 (60.6 %) and positive control oxaliplatin (65.3%) at the same does. In addition, treatment with 4 significantly reduced the volumes and size of colorectal tumors in mice. Moreover, there was no obvious abnormality in the liver, kidney, lung and heart in terms of the size and morphology in both controls and 4-treated mice except for a slight, but not significant reduction in body weight as compared with each other (Figure 4 and Table S5).

Table 4. Acute toxicity of 4 in mice dose （ mg/kg no. of mice ）

total mortality

survival (%)

400

10

10

0

300

10

9

10

200

10

7

30

150

10

6

40

100

10

2

80


LD50a（mg/kg）

147.54

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50 a

10

0

100

The 95% confidence limits: 114.88-181.16 mg/kg

Figure 4. Treatment with 4 inhibits the growth of HCT-116 cells in vivo. Nude mice were inoculated with HCT-116 cells and after establishment of solid tumors, the mice were treated intraperitoneally with vehicle, oxaliplatin (5 mg/kg), 1 (5 mg/kg) or 4 (2.5 and 5 mg/kg) daily. The tumor volumes and body weights of individual groups of mice were monitored and the tumor sizes and weights were measured at the end of experiment. Data are expressed as the mean ± SD of individual groups of mice (n=8 per group). A. Image of all tumors. B. The body weights of mice. C. The volumes of tumors. ***P < 0.001 vs control at the last measurement. D. The weights of tumors. ***P < 0.001 vs control, #P < 0.05.

Compound 4 mitigates the TGF-β1-mediated migration and invasion of HCT-116 cells



To verify that the hybrid 4 could inhibit cancer metastasis, a series of migration and invasion assays were conducted in vitro. Transforming growth factor-β1 (TGF-β1) can stimulate tumor cell migration and invasion.35,36 Accordingly, we firstly examined the cytotoxic effects of TGF-β1 (10 ng/mL) or combination of TGF-β1 (10 ng/mL) with different concentrations (2, 10 or 50 nM) of 4 on HCT-116 cells by MTT assay using 1 and oxaliplatin as controls. The results indicated that treatment with TGF-β1 or treatment in combination of TGF-β1 with either 4 or 1 at three concentrations for 24 and 48 h did not affect the viability of HCT-116 cells, although treatment with 4 at 50 nM for 72 h reduced the viability of HCT-116 cells independent of TGF-β1 treatment (Figure S3). Accordingly, the anti-migration and anti-invasion effects of 4 were further determined by transwell migration (Figures 5A and 5D), invasion (Figures 5B and 5E) and lateral migration (Figures 5C and 5F) assays for maximum incubation time for 48h. It was found that treatment with TGF-β1 significantly promoted migration and invasion of HCT-116 cells while treatment with 4 minimized the TGF-β1-stimulated migration and invasion in a dose- and time-dependent manner. The anti-migration and anti-invasion effects of 4 were significantly stronger than that of 1 and slightly potent or similar to that of oxaliplatin in HCT-116 cells. Therefore, compound 4 had a potent activity against the migration and invasion of HCT-116 cells in vitro.


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400

#

*** 200

***

***

***

300

100

**

200

#

***

***

***

***

100 0

60

# #

###

** #

40

*

* * ***

20

* ** ***

Control TGF-β (10 ng/ml) TGF-β + 4 (2 nM) TGF-β + 4 (10 nM) TGF-β + 4 (50 nM) TGF-β + 1 (50 nM) TGF-β + oxaliplatin (50 nM)

β

Co nt ro (1 l 0 TG ng /m Fβ+ l) TG 4 (2 FnM β+ 4 ) TG (1 0 FnM β+ TG ) 4 TG ( 50 FFβ+ nM β+ ox ) 1 al (5 ip 0 la nM tin ) (5 0 nM )

0

TG F-

(1 0

C on tro l TG ng /m Fβ l) TG +4 (2 FnM β+ ) TG 4 (1 0 FnM β+ TG ) 4 TG (5 F0 Fβ+ nM β+ ox 1 ) al (5 ip 0 la nM tin ) (5 0 nM )

0

###

80

48 h

###

600

F ###

24 h

400

Healed rate (%)

E No. of cells in per field

D 800

TG Fβ

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


No. of cells in per field

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Figure 5. Effects of the tested compounds on transwell migration (A, D), invasion (B, E) and lateral migration (C, F) of HCT-116 cells. A. The HCT-116 cells were seeded on chambers and incubated with the compounds for 48 h. Cells that migrated through the chambers were stained with crystal violet, and representative images were captured. B. The HCT-116 cells were seeded on chambers and incubated with the indicated factors for 48 h. Cells that migrated through the matrigel-coated chambers were stained with crystal violet, and representative images were captured. C. The HCT-116 cells were seeded on 48-well plates. After 24 or 48 h incubation with the indicated factors, representative images of wound were captured. D. The cells that migrated through the chambers were counted from three independent experiments. E. The cells that migrated through the matrigel-coated chambers were counted from



three independent experiments. F. The rate of lateral migration is presented. Experiment was confirmed for three independent times. All the data in D-F were expressed as the means ± SD of each group of cells. *P < 0.05, **P < 0.01, ***P < 0.001 vs respective TGF-β group; #P < 0.05, ###P < 0.001.

Compound 4 inhibits adhesion of HCT-116 cells to HUVECs. Adhesion of cancer cells, especially for circulating tumor cells (CTCs), to vascular endothelium is crucial for invasion and metastasis of cancers.35,36 Therefore, we tested the impact of 1 and 4 on adhesion of HCT-116 cells to human umbilical vein endothelial cells (HUVECs).35 Monolayer of HUVECs were pretreated with, or without, interleukin 1 beta (IL-1β, 1 ng/mL) for 4 h and were co-incubated with fluorescence labeled HCT-116 in the presence or absence of 1 (50 nM) and 4 (2, 10, 50 nM) for 1 h. After being washed, the percentages of HCT-116 cells that had adhered to the HUVECs were determined (Figure 6). The results indicated that while treatment with IL-1β significantly enhanced the adhesion of HCT-116 cells to HUVECs, treatment with 4 markedly decreased the percentages of adhered HCT-116 to HUVECs in a dose-dependent manner, which was much more potent than 1 at the same dose.


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Figure 6. Compound 4 inhibits adhesion of HCT-116 cells to HUVECs induced by IL-1β. HUVEC monolayers were pretreated with IL-1β (1 ng/mL) for 4 h, and then were co-incubated with Rhodamine 123-labeled HCT-116 cells with or without 4 and 1 at indicated concentrations for 1h. The adhered HCT-116 cells to HUVECs were captured and the percentages of adhered HCT-116 cells were determined. Data are representative images or expressed as the mean ± SD of each group of cells from three separate experiments. A. Fluorescent images of HCT-116 cells (green) adhered to the HUVECs. B. Quantitative analysis of the percentages of adhered HCT-116 cells. ** P < 0.01, ***P < 0.001 vs IL-1β group; #P < 0.05, ###P < 0.001.

Compound 4 suppresses in vivo lung metastasis of colorectal cancer. We further examined whether treatment with 4 could modulate the metastasis of colorectal cancer. We established a lung metastasis model of human colon cancer HCT-116 cells in BALB/c nude mice to examine its in vivo anti-colon cancer metastatic activity. The mice were intraperitoneally administrated 4 at 1, 2 or 4 mg/kg. Compound 1 and oxaliplatin (4 mg/kg) were employed as positive controls. The test proceeded three times a week for six consecutive weeks. Their body weights and survival in individual groups of mice were monitored. At the end of experiment, the lung metastatic tumors of colon cancers were examined by histology (Figure 7). In comparison with the control mice, treatment with 4, like treatment with 1 or oxaliplatin, prevented the loss of body weights in tumor-bearing mice and treatment with 4 at 4 mg/kg increased the body weights of tumor-bearing mice (Figure 7A). Similarly, treatment with 4 at 2 or 4



mg/kg, like treatment with 1 or oxaliplatin, prevented the death of tumor-bearing mice (Figure 7B). In addition, treatment with 4 appeared to reduce the size of lung metastatic tumors and significantly decreased the numbers of lung metastatic nodules (Figures 7C and D). The therapeutic effect of 4 was significantly stronger than that of 1 and oxaliplatin. Collectively, treatment with 4 significantly inhibited the lung metastasis of colorectal cancers.

Figure 7. Compound 4 inhibits the lung metastasis of colorectal cancer in mice. HCT-116 cells in the logarithmic growth phase were prepared in 5 × 106 / mL cell suspension with serum-free medium under sterile conditions and injected with 0.2 mL into the tail vein of nude mice. The mice were randomized and treated intraperitoneally with 4, 1 or oxaliplatin three times a week for 6 weeks. The body weight of mice was recorded every week after injection and all the nude mice were sacrificed after 40 days. At the end of experiment, the lung metastatic tumors of colon cancers were examined by histology. Data are representative images or expressed as


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the mean ± SD of individual groups of mice. (A) The body weights. (B) The survival rates (n = 6). Four groups where rates were treated with 4 at 2 or 4 mg/kg, 1 at 4 mg/kg, and oxaliplatin at 4 mg/kg have 100% survival rate. (C) Representative images of lung metastatic tumors (n = 3, scale bar: 100 µm). (D) The number of lung metastatic nodules in mice (n = 4-6 per group).

*P < 0.05, **P < 0.01 vs the control

group; #P < 0.05, ##P < 0.01.

Compound 4 reverses the TGF-β1-induced process of epithelial-mesenchymal transition in HCT-116 cells. The process of epithelial-mesenchymal transition (EMT) is important for tumor metastasis and cancer cells undergoing the EMT process usually have the following characteristics37-40: i) Reduction or loss of adhesion molecule E-cadherin; ii) Up-regulation expression of vimentin, one of the main mesenchymal cell markers and transcription factors, Snail and Twist; iii) Variation in the compositions of extracellular matrix, which facilitate tumor invasion and metastasis; and accompanied by changes in the multiple signal pathways. The upregulated Snail can promote the expression of matrix metalloproteinase (MMP), but repress E-cadherin expression while up-regulated Twist expression not only enhances N-cadherin expression, but also reduces E-cadherin expression as well as activates the PI3K/AKT and Wnt/β-catenin signaling.37 Moreover, TGF-β1 can enhance the EMT process and promote migration and invasion of cancers.41 Accordingly, we tested the effect of treatment with 4 on the TGF-β1-induced EMT process in HCT-116 cells.



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As shown in Figure 8, treatment with TGF-β1 (10 ng/mL) for 48 h significantly reduced the levels of E-cadherin expression (p

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