Rhein inhibits angiogenesis and the viability of hormone-dependent and -independent cancer cells under normoxic or hypoxic conditions in vitro

Chemico-Biological Interactions 192 (2011) 220–232

Contents lists available at ScienceDirect

Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint

Rhein inhibits angiogenesis and the viability of hormone-dependent and -independent cancer cells under normoxic or hypoxic conditions in vitro Vivian E. Fernand a, Jack N. Losso b,⇑, Robert E. Truax c, Emily E. Villar a, David K. Bwambok a, Sayo O. Fakayode d, Mark Lowry a, Isiah M. Warner a a

Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803, United States Department of Food Science, Louisiana State University, Baton Rouge, LA 70803, United States Biotechnology Laboratories, Louisiana State University AgCenter, Baton Rouge, LA 70803, United States d Department of Chemistry, Winston-Salem State University, Winston-Salem, NC 27110, United States b c

a r t i c l e

i n f o

Article history: Received 22 December 2010 Received in revised form 15 March 2011 Accepted 21 March 2011 Available online 30 March 2011 Keywords: Rhein Natural products Anthraquinones Non-invasive breast cancer Invasive breast cancer Hypoxia tumor angiogenesis

a b s t r a c t Hypoxia is a hallmark of solid tumors, including breast cancer, and the extent of tumor hypoxia is associated with treatment resistance and poor prognosis. Considering the limited treatment of hypoxic tumor cells and hence a poor prognosis of breast cancer, the investigation of natural products as potential chemopreventive anti-angiogenic agents is of paramount interest. Rhein (4,5-dihydroxyanthraquinone-2carboxylic acid), the primary anthraquinone in the roots of Cassia alata L., is a naturally occurring quinone which exhibits a variety of biologic activities including anti-cancer activity. However, the effect of rhein on endothelial or cancer cells under hypoxic conditions has never been delineated. Therefore, the aim of this study was to investigate whether rhein inhibits angiogenesis and the viability of hormone-dependent (MCF-7) or -independent (MDA-MB-435s) breast cancer cells in vitro under normoxic or hypoxic conditions. Rhein inhibited vascular endothelial growth factor (VEGF165)-stimulated human umbilical vein endothelial cell (HUVEC) tube formation, proliferation and migration under normoxic and hypoxic conditions. In addition, rhein inhibited in vitro angiogenesis by suppressing the activation of phosphatidylinositol 3-kinase (PI3K), phosphorylated-AKT (p-AKT) and phosphorylated extracellular signal-regulated kinase (p-ERK) but showed no inhibitory effects on total AKT or ERK. Rhein dose-dependently inhibited the viability of MCF-7 and MDA-MB-435s breast cancer cells under normoxic or hypoxic conditions, and inhibited cell cycle in both cell lines. Furthermore, Western blotting demonstrated that rhein inhibited heat shock protein 90alpha (Hsp90a) activity to induce degradation of Hsp90 client proteins including nuclear factor-kappa B (NF-jB), COX-2, and HER-2. Rhein also inhibited the expression of hypoxia-inducible factor-1 alpha (HIF-1a), vascular endothelial growth factor (VEGF165), epidermal growth factor (EGF), and the phosphorylation of inhibitor of NF-jB (I-jB) under normoxic or hypoxic conditions. Taken together, these data indicate that rhein is a promising anti-angiogenic compound for breast cancer cell viability and growth. Therefore, further studies including in vivo and pre-clinical need to be performed. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Breast cancer is one of the most prevalent cancers and second leading cause of cancer death in women, after lung cancer in the Western world [1]. Breast cancer as a heterogeneous disease is characterized by activation of multiple signal pathways which stimulate tumor growth, proliferation, inhibit apoptosis, promote the formation of new blood vessels, as well as invasion and ⇑ Corresponding author. Address: 111 Food Science Building, Louisiana State University, Baton Rouge, LA 70803, United States. Tel.: +1 225 578 3883; fax: +1 225 578 5300. E-mail address: [email protected] (J.N. Losso). 0009-2797/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2011.03.013

metastasis [2]. Thus far, the survival rate of breast cancer has been increasing since 1990 due to earlier detection and more effective chemotherapy [1,3]. Although most cancer cells initially respond well to standard therapies a subset of cells become refractory to treatment, partly due to a hypoxic tumor environment [4]. Inadequate oxygen supply to growing tumor cells, mainly because of insufficient blood supply and increased distance between the main blood vessels and the oxygen consuming tumor cells leads to hypoxia [5]. Hypoxia, a decrease in tissue oxygen levels (<1% O2), has been identified as the major physiologic stimulus for vascular endothelial growth factor (VEGF), which plays a critical role in tumor angiogenesis of many cancers, including breast cancer [5–7]. Several studies

V.E. Fernand et al. / Chemico-Biological Interactions 192 (2011) 220–232

have shown that hypoxia can lead to advanced disease stages with poor prognosis [5,8]. The transcription of the VEGF gene is primarily modulated by hypoxia inducible factor-1 (HIF-1) [9], which is a heterodimer consisting of two bHLH-PAS proteins: HIF-1a and HIF-1b (or aryl hydrocarbon nuclear translocator, ARNT) [10]. The expression of HIF-1a, the oxygen-dependent subunit, is strongly regulated by hypoxia, whereas the subunit HIF-1b is constitutively expressed [10,11]. Several studies have shown that HIF-1a is over-expressed in many human tumor cells including pre-invasive and invasive breast cancer [12,13]. In addition, HIF-1a is a client protein of heat shock protein 90 (Hsp90). Hsp90 is a molecular chaperone which mediates the folding, assembly, and maturation of many client proteins such as the human epidermal growth factor receptor-2 (HER2), Akt, Raf-1, Cdk4, and mutated p53, which are directly involved in the malignancy [14]. For instance, over-expression of HER-2 protein is present in 20–25% of breast cancer cases identified in the US [15,16]. Many anticancer drugs presently used in clinical practice are natural products (such as vinca alkaloids and taxoids) or derivatives of natural products (such as ectoposides) [17]. The investigation of natural products as potential inhibitors of tumor angiogenesis has shown to be promising in the discovery and development of new anti-cancer drugs. For instance, the flavonoids luteolin and apigenin have been reported to inhibit angiogenesis through blocking VEGF expression by suppression of HIF-1a [18,19]. In addition, some dietary substances and plant-derived compounds such as conjugated linoleic acid [20] and epigallocatechin gallate [21] have proven to be potential inhibitors of tumor angiogenesis and/or reduce the growth of various cancer cells. Rhein (4,5-dihydroxyanthraquinone-2-carboxylic acid) is the primary anthraquinone in the roots of the medicinal herb Cassia alata L. (Fabaceae), indigenous to South America [22,23]. It is a laxative, relieves pain and fever, and inhibits inflammation [24,25]. Rhein also suppresses cell proliferation in human breast, central nervous system, colon, and lung cancer cells [26], as well as induces apoptosis in various human cancer cell lines [27,28]. Furthermore, in vivo studies have proven that rhein inhibits the glycolysis of Ehrlich ascites tumor cells by reducing their glucose uptake, which in turn lowers the energy metabolism of the neoplastic cells [29]. Rhein’s breast cancer cell proliferation property has been suggested by Nair and coworkers [26]. However, the mechanisms by which rhein inhibits breast cancer cell viability is not well characterized. Therefore, in the present study we investigated the effect of rhein on VEGF165-stimulated HUVEC in vitro angiogenesis and the viability of hormone-dependent (MCF-7) and –independent (MDA-MB-435s) breast cancer cells. In addition, we characterized the molecular mechanism of rhein’s bioactivity by focusing on hypoxia, apoptosis inhibitor protein NF-jB, and heat shock protein 90 expression.

221

tained from Chemicon (Temecula, CA). Rabbit anti- p-Akt, Akt, pERK, and ERK were purchased from Cell Signaling Technology (Danvers, MA). Bovine serum albumin (BSA) standard was obtained from Bio-Rad (Hercules, CA). Recombinant human VEGF165 standard was purchased from PeproTech Inc. (Rocky Hill, NJ). 2.2. Cell culture Human umbilical vein endothelial cells (HUVECs) were obtained from Clontech (Mountain View, CA) and cultured according to manufacturer’s instructions. Human breast cancer cells (MCF-7 and MDA-MB-435s) and normal human breast cells (Hs578 Bst) were purchased from ATCC (Manassas, VA), and cultured as recommended by the provider. In the context of this in vitro study, CoCl2 (100 lM)-treated cells were considered as hypoxic whereas untreated cells were considered as normoxic. All incubations were performed at 37 °C in 5% CO2 employing a humidified incubator. 2.3. Endothelial cell proliferation assay Cell proliferation assay was performed using the CellTiter96Ò AQueous One Solution Cell Proliferation Assay (Promega, Madison, WI) following the manufacturer’s instructions. Briefly, HUVEC (1  104 cells/100 ll) were incubated with 0–200 lM rhein, 10 ng/ml of VEGF165 in the presence or absence of 100 lM CoCl2 for 24 h. 2.4. Endothelial cell migration and invasion assay The endothelial cell migration assay (Cell Biolabs, San Diego, CA) was conducted as described previously [30]. HUVEC (1.0  106 cells) were pretreated for 24 h with 50 lM of rhein or DMSO (vehicle). Similarly, endothelial cell invasion was assayed using the fluorometric format CytoSelectTM 96-well Cell Invasion Assay (Cell Biolabs, Inc. San Diego, CA) following the manufacturer’s instructions. HUVEC (2.0  106 cells) in 100 ll were treated with vehicle or 50 lM of rhein for 24 h. The assay was conducted as described by Chintalapati et al. [30]. The plates of both assays were read by use of a Perkin Elmer LS 50B fluorescence spectrometer at 480/520 nm using 530 nm cutoff. 2.5. Endothelial tube formation assay

2. Materials and methods

The endothelial tube formation assay was performed with an in vitro angiogenesis assay kit from Cell Biolabs, Inc. (San Diego, CA) using a 96-well microplate. HUVEC (1.0  104 cells in 100 ll) were seeded according to manufacture’s instructions onto a gel matrix alone or mixed with rhein (0–100 lM) under normoxic or hypoxic conditions in the presence of VEGF165 (10 ng/ml) and incubated for 6 h. After incubation, tube formation was examined in each well and photographed using a Leitz phase contrast inverted microscope.

2.1. Reagents

2.6. Cell viability assay for normal breast and breast cancer cells

Rhein, dimethyl sulfoxide (DMSO), cobaltous chloride (CoCl2), and rabbit anti-b-actin were purchased from Sigma–Aldrich (St. Louis, MO). Stock solutions of 10 and 20 mM rhein were prepared in DMSO and stored at 20 °C for up to 1 month. Mouse monoclonal antibody against Hsp90b was obtained from Invitrogen (Carlsbad, CA), mouse monoclonal anti-COX-2 was purchased from Cayman Chemical Company (Ann Arbor, MI), mouse monoclonal anti-HER-2 was from Thermo Fisher Scientific (Fremont, CA), and rabbit polyclonal antibody against Hsp90a was purchased from Stressgen Bioreagents (Ann Arbor, MI). Rabbit anti-PI3K was ob-

Cell viability was assessed using the CellTiter96Ò AQueous One Solution Cell Proliferation assay (Promega, Madison, WI) as described previously [30]. Normal breast and breast cancer cells were seeded in 96-well plates at a density of 5  103 cells/well and incubated overnight. Subsequently, cells were treated with rhein (12.5–200 lM) or vehicle (<0.1% DMSO) in triplicate under normoxic or hypoxic conditions. Replicate plates were incubated for 24 or 48 h and read as per manufacturer’s instructions. The percentage of viable cells was determined by normalizing cell viability to the levels of the control.

222

V.E. Fernand et al. / Chemico-Biological Interactions 192 (2011) 220–232

2.7. Migration and invasion of MDA-MB-435s cells Cell migration and invasion assays were each conducted by using the Quantitative Cell migration QCM™ assay (Chemicon, Temecula, CA) and CytoSelect™ 96-well cell invasion assay (Cell Biolabs, Inc. San Diego, CA) following the manufacturer’s instructions, respectively. Cell migration was performed by chemotaxis as follows. MDA-MB-435s cancer cells (1.0  106 cells in 100 ll) treated with vehicle (<0.1% DMSO) or rhein (50 lM) were seeded in the top of the insert in serum-free media while VEGF165 (10 ng/ml) as chemoattractant was placed underneath the membrane. In invasion assays, MDA-MB-435s cancer cells (2.0  106 cells in 100 ll) treated with vehicle (<0.1% DMSO) or rhein (50 lM) were seeded in the top of the insert pre-coated with matrigel in serum-free media while the chemoattractant (10 ng/ml VEGF165) was placed underneath the membrane. After incubation, the migration chamber was handled as suggested by the manufacturer. The fluorescence of the lysed cells was read at 480/520 nm using a Perkin Elmer LS 50B spectrofluorometer.

2.8. Cell cycle analysis by flow cytometry Breast cancer cells were seeded at a density of 2.5  106 cells in T-25 flasks overnight, and incubated with vehicle or rhein at 50, 100, or 200 lM for 48 h. Cells were harvested, trypsinized, stained using the Beckman–Coulter DNA Prep Reagent kit (cat# 6607055) following the manufacturer’s instructions. The stained cells were read on a Beckman–Coulter Epics FC500 flow cytometer using CXP software for the initial acquisition. The data were then modeled using ModFit LT v3.2 software from Verity Software House (Topsham, ME).

2.9. Cell death ELISA A Cell Death Detection ELISA assay (Roche Applied Science, Indianapolis, IN) was performed according to manufacturer’s instructions to determine the amount of cytoplasmic histone-related DNA fragments (nucleosomes) in MCF-7 or MDA-MB-435s cells after induction of cell death. In brief, 1  106 cells/well were plated in a 12-well plate, treated with rhein for 48 h and lysed to produce nucleosomes. Apoptotic cell death was detected in the sample using a Benchmark Plus microplate reader at 405 nm, with a reference wavelength at 490 nm (Bio-Rad, Hercules, CA).

2.10. Protein extraction, immunoprecipitation, and Western blot Cytoplasmic and nuclear fractions from control or rhein-treated MCF-7 or MDA-MB-435s cells were isolated using the Nuclear Extraction kit (Active Motif, Carlsbad, CA). The protein concentration in cytoplasmic and nuclear extracts was determined employing the DC Protein Assay (Bio-Rad, Hercules, CA). Immunoprecipitation (IP) of cytoplasmic extracts was performed using the IP protocol (Santa Cruz Biotechnology, Santa Cruz, CA). SDS–PAGE of protein extracts (50 lg) was carried out on 4–12% Bis–Tris gels (Invitrogen, Carlsbad, CA). Western blotting was performed using the WesternbreezeÒ Immunodetection Kit (Invitrogen, Carlsbad, CA) as per manufacturer instructions. Antibodies of interest were diluted as indicated by their respective providers. Proteins of interest were identified, after chromogenic detection, using controls and molecular protein markers. Blots were imaged with a Kodak Gel Logic 200 Imaging System and the relative pixel density was measured by use of Kodak Molecular Imaging Software (version 4).

2.11. Quantitation of VEGF165, EGF, and HIF-1a levels in MCF-7 or MDA-MB-435s The levels of VEGF165 or EGF secreted by MCF-7 or MDA-MB435s cells, untreated or treated with rhein under normoxic or hypoxic conditions for 48 h, were determined using the human VEGF165 Sandwich ELISA kit (Chemicon International, Temecula, CA) or the Quantikine human EGF ELISA kit (R&D Systems, Minneapolis, MN) as per manufacturer’s instructions, respectively. The levels of HIF-1a in normalized nuclear extracts of control and rhein-treated breast cancer cells, after 48 h incubation under hypoxic conditions, were analyzed using the TransAM HIF-1 ELISA kit (Active Motif, Carlsbad, CA). 2.12. Determination of I-jB and NF-jB p50, p65 in MCF-7 or MDAMB-435s cells The levels of Ij-B in whole cell extracts of control and rheintreated MCF-7 or MDA-MB-435s cells were determined by use of the Function ELISA I-jB kit (Active Motif, Carlsbad, CA). The nuclear factor-jB (NF-jB) DNA-binding activity in normalized nuclear extracts of control and rhein-treated MCF-7 or MDA-MB-435s cells was determined using the ELISATransAM NF-jB p50 and NF-jB p65 kit (Active Motif, Carlsbad, CA). All assays were performed under normoxic or hypoxic conditions in the presence of 10 ng/ml TNF-a according to provider’s instructions. 2.13. Statistical analysis All experiments were conducted in triplicate. Statistical analysis was performed using one-way analysis of variance (ANOVA). Duncan’s new multiple range test was performed to determine the significant difference between treatments within the 95% confidence interval using SPSS 16.0 software (SPSS Inc, Chicago, IL, USA).

3. Results 3.1. Rhein inhibits endothelial cell proliferation, migration, invasion, and tube formation To determine the effect of rhein on endothelial cell proliferation, we first performed a concentration-dependent study of cell proliferation induced by 10 ng/ml VEGF165 and 100 lM CoCl2. A 24-hour treatment with rhein inhibited the proliferation of HUVEC in a concentration-dependent manner with an IC50 value around 50 lM (Fig. 1A). Rhein equally blocked endothelial cell proliferation induced by VEGF165 in absence of CoCl2 (Fig. 1B). Rhein was more effective against VEGF165 alone than a combination of VEGF165 and CoCl2. Endothelial cell migration and invasion are important steps of angiogenesis. Therefore, we performed these assays to determine the effect of rhein on endothelial cell migration and invasion. Rhein at 25 and 50 lM inhibited VEGF165-induced HUVEC migration and invasion in the presence of 100 lM CoCl2 (Fig. 1C and D). To ascertain the inhibitory effect of rhein on angiogenesis, we performed the in vitro angiogenesis assay and examined the ability of endothelial cells to form tubes in absence or presence of rhein (0–100 lM). VEGF165-stimulated tube formation of HUVECs was completely disrupted at 50 lM rhein within 6 h (Fig. 1E). Additionally, to investigate the molecular mechanism of rhein-induced inhibition on VEGF-dependent angiogenesis, we examined the effects of rhein on VEGF-dependent PI3K, AKT and ERK activation. Results in Fig. 2 indicate that rhein at 50 lM suppressed VEGF-dependent PI3K, p-AKT and p-ERK activation but had no effect on the levels of total AKT or ERK.

223

V.E. Fernand et al. / Chemico-Biological Interactions 192 (2011) 220–232

B

A a

a

ab

b

b

a

c c

d d

e e

f

CoCl2

-

+

-

+

+

+

+

+

VEGF

-

-

+

+

+

+

+

+

Rhein (μ M)

-

-

-

-

25

50

100

200

C

f

VEGF

-

+

+

+

+

+

Rhein (μM)

-

-

25

50

100

200

D c

c d

d b

b e

e a

a

CoCl2

-

-

+

+

+

VEGF

-

+

+

+

+

Rhein (μ M)

-

-

-

25

50

E

Control

VEGF165

CoCl2

-

-

+

+

+

VEGF

-

+

+

+

+

Rhein (μM)

-

-

-

25

50

VEGF165+Rhein (N: 50 µM) VEGF165+Rhein (H: 50µM)

Fig. 1. Rhein inhibits endothelial cell proliferation, migration and invasion, and tube formation under normoxic or hypoxic conditions. (A) Cell proliferation was performed, using 1  104 HUVEC/100 ll, 0–200 lM rhein, and 10 ng/ml of VEGF165 in presence or absence of 100 lM CoCl2 for 24 h, by the MTS assay as indicated in Section 2.3. (B) Effect of rhein on HUVEC proliferation in the presence of VEGF165 under normoxic condition (same conditions as under Fig. 1A). (C) Cell migration was conducted by treating HUVEC (1  106 cells) with or without rhein (0–50 lM). The assay was performed as specified in Section 2.4. (D) Cell invasion was performed by treating HUVEC (2.0  106 cells) with or without rhein (0–50 lM). The assay was carried out as indicated in Section 2.4. (E) Effect of rhein on VEGF-induced tube formation under normoxic (N) and hypoxic (H) conditions. Cells were treated with rhein (0–100 lM) in the absence (control) or presence of VEGF165 (10 ng/ml). After 6 h, disruption of endothelial tube formation was detected at 50 lM rhein (N and H) and photographs were taken (100). All incubations were at 37 °C in 5% CO2 humidified incubator. Results are mean ± SD of three determinations. Means without a common letter differ, p < 0.05.

3.2. Rhein inhibits MCF-7 and MDA-MB-435s cell viability Rhein had no effect on the viability of Hs578 Bst normal breast cells (Fig. 3). However, rhein significantly inhibited the viability of MCF-7 (Fig. 3A and B) and MDA-MB-435s (Fig. 3C and D) in a time

and dose-dependent manner under normoxic and hypoxic conditions. Values from normoxic and hypoxic conditions were statistically compared. Results show that there was no significant difference on rhein-mediated reduction in MCF-7 cell viability between normoxia and hypoxia at 24 or 48 h treatment (Fig. 3A and

224

V.E. Fernand et al. / Chemico-Biological Interactions 192 (2011) 220–232

Normoxic conditions

Hypoxic conditions Biomarkers 1

6.4

2.8

2.0

HIF-1α 1

1.4

0.8

0.6

1

1.8

0.7

0.5

1

1.5

0.9 0.7

1

2.1

0.8

0.1

1

1.3

0.9 0.9

1

1

1

0.9

1

1.1

0.9 0.6

1

2.1

1.4

0.6

1

1.4

1.2 1.1

1

1

1.2

0.6

1

1

1

1

1

1

1.2

Size (kDa) 120

PI3K

85

p-Akt

60

Akt

60

p-ERK

44, 42

ERK

44, 42

β-actin β

45

1.1

CoCl2

-

-

-

-

-

+

+

+

μ Rhein (μM)

-

-

25

50

-

-

25

50

Fig. 2. Effect of rhein on hypoxia inducible factor-1a and PI3K/Akt/ERK pathway in HUVEC. Endothelial cells were incubated with rhein (0–50 lM) under normoxic or hypoxic conditions. Cells were lysed with lysis buffer. Equal amounts of protein (50 lg) were separated by electrophoresis. The expression of PI3K, p-Akt, total Akt, p-ERK, total ERK under normoxic conditions and HIF-1a, PI3K, p-Akt, total Akt, p-ERK, total ERK under hypoxic conditions were determined by Western blot. Gels shown are representative from at least three separations. The intensity of the control was normalized to 1 and the intensity of each band from rhein-treated cells is compared to the control.

B). After 48 h treatment, the effect of hypoxia on rhein-mediated reduction of MDA-MB-435s cell viability was significantly different from normoxic conditions at 25, 50, or 100 lM. Rhein treatment was more toxic to MDA-MB-435s cells in normoxic conditions compared to hypoxia (Fig. 3D). Our results also show that under normoxic conditions at concentration of 25–200 lM there was a significant difference between rhein-mediated reduction of MCF7 and MDA-MB-435s cell viability; with rhein being more toxic to the invasive cells (Fig. 3B and D). However, under hypoxic conditions rhein treatment was more effective against MDA-MB-435s cells only at 12.5 lM, while at 100 lM it was more toxic to the MCF-7 cells. At all other concentration investigated there was no significant difference between MCF-7 and MDA-MB-435s cells on the effect of hypoxia on rhein. The IC50 value of rhein after 48 h incubation under normoxic or hypoxic conditions for MCF-7 cells was 81.3 or 71.3 lM and for MDA-MB-435s cells 52.1 or 127.3 lM, respectively. Based on the obtained results, we selected for further studies the 48 h incubation with 50, 100, and 200 lM of rhein.

tion of both, we performed flow cytometry to analyze the DNA content of the cells using propidium iodide staining (Fig. 5). Two cellular populations were separated into diploid and aneuploid MCF-7. The DNA index of MCF-7 was less than 1.50, which indicates that the genomically stable aneuploid MCF-7 was composed of tumor cells with aneuploid DNA. Rhein dose-dependently increased MCF-7 cells under normoxic and hypoxic conditions into S-phase (Fig. 5A). MDA-MB-435s cells treated with rhein under normoxic or hypoxic conditions accumulated in G2/M- and Sphases. However, as shown in Fig. 5B, there were no cells in subG1 phase indicating no sign of apoptosis. Studies performed with human hepatoblastoma G2 (Hep G2) cells demonstrated that rhein induced cell cycle arrest at G1-phase and led to the accumulation of hepatocellular BEL-7402 cells in S-phase [31,32] while in A549 lung cancer cells rhein-induced cell cycle arrest in G0/G1-phase [27]. These results and ours suggest that the effect of rhein on cancer cell cycle may be cell type specific.

3.3. Rhein inhibits cell migration and invasion of MDA-MB-435s In this study, we also examined the effect of rhein on the migration and invasion of MDA-MB-435s cells. Rhein at 50 lM inhibited after 48 h VEGF165-induced MDA-MB-435s cell migration. Our results in Fig. 4A indicate that rhein suppressed cell migration under normoxic and hypoxic conditions to 60% and 42%, respectively. In addition, rhein inhibited the invasion of MDA-MB-435s cells in the presence of VEGF165 as chemoattractant. Our data suggest that rhein inhibited cell invasion by 47% both under normoxic and hypoxic conditions (Fig. 4B). Statistically, there was no significant difference between normoxia and hypoxia on the effect of rhein on migration or invasion of MDA-MB-435s cells.

To further evaluate this cell-specific apoptotic process in MCF-7 cells, we determined the amount of nucleosomes during nuclear DNA denaturation after 48 h incubation with rhein using cell death ELISA. Our results indicate that the nucleosome production in MCF7 cells was significantly increased only at 200 lM rhein, suggesting that cell death occurred by apoptosis, both under normoxic and hypoxic conditions (Fig. 6). In addition, there was no significant difference between normoxic and hypoxic treatment on the effect of rhein on MCF-7 cell apoptosis. However, in agreement with the data in Fig. 5B, MDA-MB-435s cells treated with rhein did not induce apoptotic cell death under normoxic or hypoxic conditions as there were no significant changes in the enrichment factor between the control and rhein-treated cells (results not shown).

3.4. Rhein induces MCF-7 or MDA-MB-435s cell cycle arrest

3.6. Western blot analysis

To determine whether the decrease in MCF-7 or MDA-MB-435s cell viability was the result of cycle arrest, apoptosis, or a combina-

Cytoplasmic extracts of MCF-7 and MDA-MB-435s cells were analyzed using Western Blot to evaluate the effect of rhein on

3.5. Rhein induces apoptosis in MCF-7 but not in MDA-MB-435s

225

V.E. Fernand et al. / Chemico-Biological Interactions 192 (2011) 220–232

A

B

Hs 578 Bst

Hs578 Bst MCF-7 Normoxic

MCF-7 Normoxic

120

MCF-7 Hypoxic

Cell Viability (% of Control)

100 80

**

60

**

40 20

Cell Viability (% of Control)

MCF-7 Hypoxic 120

100

**

80 60

**

40

**

20 0

0 0

12.5

25

50

100

0

200

12.5

25

50

Rhein (μM)

Rhein (μM)

24 hours

48 hours

C

100

D

Hs578 Bst

200

Hs578 Bst

MDA Normoxic

MDA Normoxic

MDA Hypoxic

MDA Hypoxic

100

**

80

***

*

60 40

**

20 0 0

12.5

25

50

100

20 0

Cell Viability (% of Control)

Cell Viability (% of Control)

120

120 100 80

*

*

60

*

*

40

*

20

**

0 0

12.5

25

50

Rhein (μM)

Rhein (μM)

24 hours

48 hours

100

200

Fig. 3. Effect of rhein on cell viability of normal breast and breast cancer cells under normoxic or hypoxic conditions. The inhibitory activity of rhein (12.5–200 lM) was measured after 24 or 48 h using the CellTiter 96 Aqueous One Solution Cell Proliferation assay as described in Section 2.6. Measurements were performed in triplicate in two different cultures. Results are expressed as the mean% of MTS absorbance. ⁄Indicates significant difference at p < 0.05, compared with the control. (A) Effect of rhein on Hs578 Bst, MCF-7 normoxic conditions, and MCF-7 hypoxic conditions after 24 h incubation. (B) Effect of rhein on Hs578 Bst, MCF-7 normoxic conditions, and MCF-7 hypoxic conditions after 48 h incubation. (C) Effect of rhein on Hs578 Bst, MDA-MB-435s normoxic conditions, and MDA-MB-435s hypoxic conditions after 24 h incubation. (D) Effect of rhein on Hs578 Bst, MDA-MB-435s normoxic conditions, and MDA-MB-435s hypoxic conditions after 48 h incubation.

some key proteins (i.e. Hsp90a, Hsp90b, COX-2, and HER-2) involved in the regulation of angiogenesis, invasion, and proliferation of breast cancer. Treatment of MCF-7 cells with 100 or 200 lM rhein under normoxic or HIF-1a-induced conditions decreased Hsp90a, COX-2, and HER-2 levels (Fig. 7A: I, III, and IV), while it had no significant effect on the expression of Hsp90b (Fig. 7A: II). Within MCF-7 cells, there was no significant difference between normoxia and hypoxia. The relative densities compared with the control of these four biomarkers in rhein-treated MCF-7 cells are reported in Fig. 7A: V and VI. The treatment of MDA-MB-435s cells with 100 or 200 lM rhein under normoxic conditions had various effects on the investigated proteins. Levels of Hsp90a were decreased only after treatment with 200 lM rhein (Fig. 7B: I). In addition, after treatment with 100 or 200 lM rhein, COX-2 expression (Fig. 7B: III) was not significantly reduced, while the HER-2 levels were decreased in a concentration-dependent manner (Fig. 7B: IV). On the contrary, under hypoxic conditions, the levels of Hsp90a, COX-2, and HER2 in rhein-treated cancer cells were significantly decreased (Fig. 7B: I, III, and IV). There was no significant difference between normoxia and hypoxia. Our results also show that rhein, both under normoxic and hypoxic conditions, had no significant effect on Hsp90b levels in MDA-MB-435s cells (Fig. 7B: II). In Fig. 7B: V and VI are shown the relative densities compared with the control of Hsp90a, Hsp90b, COX-2, and HER-2. There was no significance

between MCF-7 and MDA-MB-435s cells except at 200 lM under normoxia for Hsp 90a. Our results also show no significant different between MCF-7 and MDA-MB-435s at all concentration under both conditions for COX-2. In addition, we found no significant difference between MCF-7 and MDA-MB-435s cells at all concentrations except for cells treated with 100 lM rhein under hypoxic conditions for HER-2. 3.7. Rhein inhibits VEGF165, EGF, and HIF-1a in MCF-7 and MDA-MB435s cells In order to assess the effect of rhein on the inhibition of the angiogenic stimulator VEGF165, supernatants from control or rhein-treated MCF-7 and MDA-MB-453s cells were analyzed using ELISA. Rhein dose-dependently decreased the levels of VEGF165 in MCF-7 cells or MDA-MB-435s cells under either normoxic or hypoxic conditions (Fig. 8A and B). There was no significant difference between normoxic and hypoxic conditions in either MCF-7 or MDA-MB-435s cells. Similarly, there was no significance difference between the effect of rhein on MCF-7 and MDA-MB-435s. The effect of rhein on MCF-7 or MDA-MB-435s was not cell specific for VEGF. Furthermore, was examined (using ELISA) the effect of rhein on the EGF, a growth factor involved in cancer metastasis (i.e. cell proliferation and survival, angiogenesis, tissue invasion, and metasta-

V.E. Fernand et al. / Chemico-Biological Interactions 192 (2011) 220–232

Relative Inhibition (% of Control) Invasion

Relative Inhibition (% of Control) Migration

226

120

A

100 80

*

60

*

40 20 0

Control

Normoxic

Hypoxic

B 120 100 80 60 40

*

*

Normoxic

Hypoxic

20 0

Control

Fig. 4. Effect of rhein on MDA-MB-435s (A) cell migration and (B) cell invasion under normoxic or hypoxic conditions. MDA-MB-435s breast cancer cells (1.0  106 cells for cell migration and 2  106 cells for invasion) were treated with vehicle (<0.1% DMSO) or rhein (50 lM). The assay was performed as indicated in Section 2.7. Data were collected using fluorescence spectroscopy. Results are expressed as the mean ± S.E.M. ⁄p < 0.05, compared with the control.

sis). Our results indicate that rhein under normoxic or hypoxic conditions, decreased EGF levels in MCF-7 or MDA-MB-435s cell lines (Fig. 8C and D). Our MCF-7 results indicate that there was no significant difference between normoxia and hypoxia. With regard to MDA-MB-435s cells, there was no significant difference between normoxia and hypoxia except for rhein treatment at 50 lM. Similarly to VEGF, the effect of rhein on MCF-7 or MDA-MB-435s was not cell specific for EGF. To ascertain the effect of rhein as potential inhibitor of HIF-1a activity in hypoxia-induced tumor angiogenesis of breast cancer cells, nuclear extracts of control and rhein-treated MCF-7 and MDA-MB-435s cells were analyzed for HIF-1a using ELISA. Our results show that rhein dose-dependently and significantly inhibited CoCl2-stabilized HIF-1a expression in MCF-7 and MDA-MB-435s cells (Fig. 8E). At 50 lM rhein treatment, there was a significant difference between MCF-7 and MDA-MB-435s cells. However, at 100 or 200 lM rhein treatment, there was no significant difference on the effect of rhein on HIF-1a levels in both cell lines. 3.8. Rhein inhibits I-jB and NF-jB p50, p65 Another transcription factor that is stimulated by hypoxia is NF-

jB, which is implicated in the regulation of several genes involved in oncogenesis and angiogenesis. The NF-jB exists in an inactive form in the cytoplasm, where it is composed of three major subunits: I-jB (inhibitory subunit), bound to the p50–p65 heterodimer [33,34]. Therefore, we assessed the effect of rhein using ELISA on TNF-a stimulated I-jB phosphorylation and degradation, as well as NF-jB p50 and p65 nuclear translocation in MCF-7 or MDA-MB-435s cells after 48 h incubation under normoxic or hypoxic conditions. Rhein dose-dependently decreased the phosphorylation of I-jB in MCF-7 cells (Fig. 9A). Our data indicate that there was no significant difference between normoxia and hypoxia. According to our MDA-MB-435s cell study, rhein dose-dependently reduced I-jB phosphorylation and degradation both under normoxic and hypoxic conditions (Fig. 9B). Our results from this

study also show that there was no significant difference between normoxia and hypoxia at all rhein concentrations investigated. In addition, there was no significant difference between MCF-7 and MDA-MB-435s except for rhein treatment at 50 lM under normoxic conditions. The relative activity of NF-jB p50 (Fig. 10A) and NF-jB p65 (Fig. 10B) in MCF-7 cells treated with rhein was also significantly decreased in a dose-dependent way under normoxic or hypoxic conditions. In addition, there was no significant difference between normoxia and hypoxia on the effect of rhein on NF-jB p50. However, our NF-jB p65 data indicate that only at 100 lM there was no significant difference between normoxia and hypoxia. Furthermore, our results show that the nuclear translocation of NF-jB p50 (Fig. 10C) and NF-jB p65 (Fig. 10D) in rhein-treated MDA-MB-435s cells was significantly suppressed under both conditions. Our data also indicate that for NF-jB p50 there was no significant difference between normoxia and hypoxia except at 200 lM rhein treatment. The inhibition effect of rhein on the nuclear translocation of NF-jB p65 in MDA-MB-435s cells was dose-dependently more effective under hypoxic than normoxic conditions and statistically different (Fig. 10D). Statistically, rhein treatment was cell specific except at 200 lM under normoxic conditions for NF-jB p50. Rhein appeared to be more toxic to MDA-MB-435s than MCF-7 cells. The results of NF-jB p65 indicate that there was a significant difference between MCF-7 and MDA-MB-435s cells at all concentrations except at 50 and 100 lM rhein treatment under hypoxic conditions. For our NF-jB p65 study, rhein treatment appeared to be more toxic to MCF-7 cells than MDA-MB-435s cells except at 200 lM under hypoxic conditions.

4. Discussion The roots of C. alata, a medicinal plant from Suriname, have been used for generations to treat various diseases [23]. In an early study, we found that rhein is one of the major bioactive compounds in the roots of C. alata [22]. Rhein has been investigated as potential inhibitor of cancer cell viability and the mechanisms by which rhein inhibits cancer cell viability have been reported to include induction of apoptosis [35], inhibition of matrix metalloproteinase-2, and -9, inhibition of urokinase plasminogen activator [36], and inhibition of MAPkinase and p-AKT activation [37]. Herein this study, we suggest additional mechanisms by which rhein may be beneficial against angiogenesis and breast cancer cell viability. Cell migration and invasion, as well as tube formation are important processes during angiogenesis. Furthermore, angiogenesis is regulated by hypoxia through the stimulation of VEGF. Therefore, in this study, we examined the anti-angiogenic effects of rhein using HUVEC under normoxic or hypoxic conditions. Exposing HUVEC to increasing concentrations of rhein under normoxic or both normoxic and CoCl2 conditions in the presence of VEGF165 showed a significant decrease in cell proliferation, migration, and tube formation. We found that 50 lM rhein suppressed the stimulatory effect of VEGF165 (10 ng/ml) on endothelial cell tube formation under normoxic or hypoxic conditions; and hence, disrupted the tube formation. We also evaluated the effect of rhein on Akt and ERK. The former protein, Akt, is involved in cell cycle, proliferation, and apoptosis [38], whereas the latter, ERK, is involved in cell proliferation, survival, and migration [39]. The results of our Western blot analyses indicate that rhein inhibited VEGF165-induced angiogenesis under normoxic or hypoxic conditions by suppressing PI3K, p-Akt, and p-ERK, or HIF-1a, PI3K, p-Akt, and p-ERK, respectively. Our results also show that rhein did not decrease the amount of total Akt or total ERK in HUVECs. To our knowledge, this is the first report that shows that rhein inhibits angiogenesis by

227

V.E. Fernand et al. / Chemico-Biological Interactions 192 (2011) 220–232

A

MCF-7 control

MCF-7 Normoxic 50

MCF-7 Hypoxic 50

MCF-7 Normoxic 100

Diploid: 70.64 % Dip G1: 72.34% Dip G2: 3.62% Dip S: 24.04 % Aneuploid: 29.36 % An1 G1: 74.97 % An1 G2: 25.03 %

Diploid: 80.09 % Dip G1: 71.18 % Dip G2: 3.51 % Dip S: 25.32 % Aneuploid: 19.91 % An1 G1: 55.66 % An1 G2: 20.78 % An1 S: 23.56 %

Diploid: 80.81 % Dip G1: 37.20 % Dip G2: 12.09 % Dip S: 50.71 % Aneuploid: 19.19 % An1 G1: 9.71 % An1 G2: 53.01 % An1 S: 37.27 %

Diploid: 75.86 % Dip G1: 37.79 % Dip G2: 13.77 % Dip S: 48.44 % Aneuploid: 24.14 % An1 G1: 43.60 % An1 G2: 33.97 % An1 S: 22.43 %

An1 S: 0.00 %

B

MCF-7 Hypoxic 100

MCF-7 Normoxic 200

Diploid: 87. 87 % Dip G1: 36.12 % Dip G2: 14. 70 % Dip S: 49.18 % Aneuploid: 12.13 % An1 G1: 29.51 % An1 G2: 67.76 % An1 S: 3.33 %

Diploid: 73.44 % Dip G1: 42.91 % Dip G2: 9.68 % Dip S: 47.40 % Aneuploid 1: 26.56 % An1 G1: 37.48 % An1 G2: 11.60 %

MDA control Dip G1: 82.63 % Dip G2: 5.79 % Dip S: 11.58 %

An1 S: 50.92

%

MCF-7 Hypoxic 200 Diploid: 84.48 % Dip G1: 35.28 % Dip G2: 12.06 % Dip S: 52.67 % Aneuploid 1: 15.52 % An1 G1: 35.64 % An1 G2: 57.06 % An1 S: 7.30 %

MDA normoxic 50

MDA hypoxic 50

Dip G1: 50.04 % Dip G2: 15.87 % Dip S: 34.09 %

Dip G1: 45.66 % Dip G2: 16.68 % Dip S: 37.66 %

MDA normoxic 100

MDA hypoxic 100

Dip G1: 47.18 % Dip G2: 15.84 % Dip S: 36.97 %

Dip G1: 41.83 % Dip G2: 15.18 % Dip S: 42.99 %

MDA normoxic 200

MDA hypoxic 200

Dip G1: 41.42 % Dip G2: 16.79 % Dip S: 41.57 %

Dip G1: 40.00 % Dip G2: 18.43 % Dip S: 41.79 %

Fig. 5. Effect of rhein on (A) MCF-7 or and (B) MDA-MB-435s cell cycle. Cells (2.5  106 cells in T-25 flasks) were treated with rhein (0–200 lM) under normoxic or hypoxic conditions for 48 h. Cell cycle assay was performed as indicated in Section 2.8. The data were modeled using ModFit LT v3.2 software from Verity Software House (Topsham, ME). Data are a representative from 3 independent experiments.

down-regulating PI3K/Akt/ERK pathway and HIF-1a. The link between PI3K/Akt pathway and angiogenesis makes this pathway an attractive target for anti-angiogenic therapies. Hence, several natural and synthetic compounds have emerged recently as potential inhibitors of PI3K/Akt pathway. For example, wortmannin, a

furanosteroid metabolite of the fungi Penicillium funiculosum inhibits angiogenesis through the PI3k/Akt pathway [40]. LY294002, a morpholine derivative of quercetin inhibits angiogenesis through the PI3k/Akt pathway [41]. Nevertheless, these compounds are toxic in vivo or poorly soluble in aqueous solution [42,43].

228

V.E. Fernand et al. / Chemico-Biological Interactions 192 (2011) 220–232

Normoxic Hypoxic

Enrichment Factor

3.00

*

2.50

*

2.00 1.50 1.00 0.50 0.00 Control

50

100

200

Rhein (μM) Fig. 6. Effect of rhein on cell death of MCF-7 breast cancer cells. Cells were incubated with rhein (50, 100, 200 lM) or vehicle (<0.1% DMSO) in culture medium under normoxic or hypoxic conditions for 48 h. Apoptosis was detected by use of cell death ELISA. Results are expressed as the enrichment factor (mean ± S.E.M.). The enrichment factor is the ratio of the absorbance of the sample (dying/dead cells) and the absorbance of the control (viable cells). All experiments were performed in triplicate with two replicates each. ⁄Indicates significant difference at p < 0.05, compared with the control.

Many naturally occurring bioactive compounds including quinones with good bioavailability index and less toxicity have been identified as inhibitors of angiogenesis [44,45]. Our results showing that rhein, an anthraquinone from the roots of C. alata or rhubarb, inhibited PI3k/Akt and hypoxia-induced angiogenesis is promising. Therefore, in the present study, we also evaluated the anticancer effect of rhein against hormone-dependent or -independent cancer cells. Rhein treatment under hypoxic or normoxic conditions significantly induced anti-proliferative effect against MCF-7 and MDA-MB-435s cells. According to our cell cycle results the significant decrease in cell viability in MCF-7 cells after treatment with low concentrations of rhein (50 and 100 lM) was not correlated to apoptotic cell death, but most possibly to cell cycle arrest or activation of another cell death mechanism [46]. However, at high concentration of rhein (200 lM rhein) the decrease in viability was correlated to apoptotic cell death due to an increase of cells at the sub-G1 level, 3.12% of cells under normoxic and 1.79% under hypoxic conditions. All these findings for the rhein-treated MCF-7 cells were confirmed by our cell death study. Interestingly, MDAMB-435s cells accumulated in the G2/M- and S-phase. Our cell cycle data also indicate that using 50–200 lM rhein does not result in cells at the sub-G1 levels. Therefore, these data suggest that the decreased cell viability at 50, 100, or 200 lM was not related to apoptotic cell death, but most probably to inhibition of the cell cycle or activation of another cell death mechanism. There is also a particular interest in compounds that target Hsp90 as treatment modality for cancer because Hsp90 appears to be at the crossroad of multiple signaling pathways associated with tumor viability, angiogenesis, and survival. Hsp90 is highly expressed in tumor cells and chaperones more than one hundred oncogenic proteins. Inhibiting Hsp90 in cancer has emerged as a target for developing anticancer compounds since inhibitors of Hsp90 have higher affinity with Hsp90 present in tumor cells than Hsp90 in normal cells [47]. Several naturally occurring or synthetic Hsp90 inhibitors have been discovered and investigated in vitro and in vivo. For instance, geldanamycin (17-(Allylamino)-17demethoxygeldanamycin or 17AAG) is a benzoquinone that binds and inhibits Hsp90 in vitro and in vivo. However, because of insolubility and toxicity, derivatives of geldanamycin with improved solubility and bioavailability such as alvespimycin or 17-DMAG have been designed and investigated in vitro and in vivo [48]. Herein this study, we hypothesized that rhein, an anthraquinone, inhibits Hsp90. We focused on Hsp90a, Hsp90b, HIF-1a, NF-jB, VEGF165, HER-2, and COX-2 relationship because (i) Hsp90 chaper-

ones most of these proteins, and (ii) HIF-1a and NF-jB are critical to VEGF regulation and these markers predict disease aggressiveness. Our results show that rhein down-regulated the levels of Hsp90a and its client proteins including HIF-1a, NF-jB, COX-2, HER-2, VEGF165, and EGF in both MCF-7 and MDA-MB-435s cells. However, it is not known whether Hsp90a down-regulation was through an ATP-dependent or -independent mechanism. Activation of NF-jB is crucial for the induction of COX-2. In addition, activation of NF-jB typically involves the phosphorylation of I-jB by I-jB kinase (IKK) complex, I-jB degradation, release of and nuclear translocation of NF-jB. To further characterize the mechanism by which rhein reduces the viability of MCF-7 or MDA-MB-435s cells, we examined the effect of rhein on the constitutive phosphorylation of I-jB in both cell lines. Our results show that rhein dose-dependently decreased the constitutive phosphorylation of I-jB in both cell lines under normoxic or hypoxic conditions. Rhein inhibited NF-jB p50 and p65 activity in both MCF-7 and MDA-MB-435s cells. In breast cancer NF-jB over-expression is associated with poor prognosis. Because of the central role of NF-jB in apoptosis, its inhibition by rhein is very interesting as it resulted in effective MCF-7 or MDA-MB-435s growth inhibition. COX-2 is a biomarker of breast cancer. It is conceivable that COX-2 down-regulation was associated with the inhibition of NFjB because the promoter sequence of COX-2 contains binding sites for NF-jB. Down-regulation of HIF-1a and NF-jB was associated with decreased secretion of VEGF165 in the growth media. The oncoprotein HER/neu, belongs to the epidermal growth factor family and may trigger activation of PI3K/Akt in breast cancer cell [49,50]. In this study, we also examined whether or not rhein-mediated down-regulation of HER2/neu. The results show that rhein decreased the levels of HER2/neu under both normoxic and hypoxic conditions. Finally, we investigated the effect of rhein on VEGF165 and EGF levels in MCF-7 or MDA-MB-435s cells under normoxic or hypoxic conditions. Our results indicate a down regulation of both growth factors in both cell lines. These findings were in good correlation with the decreased levels of HIF-1a after treatment with rhein, which indicates the direct association that exists between HIF1a, VEGF, and EGF [21]. EGF triggers angiogenesis, tumor growth, invasion, and metastasis. Our data show that rhein dose-dependently inhibited EGF, suggesting that rhein may also inhibit tumor cell growth, invasion, and metastasis. In summary, the major finding of this study is that rhein inhibits angiogenesis in HUVEC under normoxic conditions through the PI3K/Akt/ERk pathway and hypoxia-induced angiogenesis in HUVEC through the PI3K/Akt/ERk pathway and HIF1a. Rhein was not cytotoxic to Hs578 Bst normal breast cells; however, it down-regulated Hsp90a in both MCF-7 and MDAMB-435s cells without affecting the levels of Hsp90b. The inhibition of Hsp90 led to attenuation of several client proteins of Hsp90 and resulted in inhibition of several pathways involved in MCF-7 or MDA-MB-435s cell viability. In addition, rhein inhibited the expression of HIF-1a in both cell lines. The anti-proliferative activity of rhein in MCF-7 and MDA-MB-435s cells could be due to the inhibition of NF-jB and non-apoptotic cell death under normoxic or hypoxic conditions. The inhibitory activity of rhein on MCF-7 or MDA-MB-435s was not cell specific. Rhein treatment indicated similar inhibitor activity against MCF-7 or MDA-MB-435s cells under normoxic and hypoxic conditions. Our results reveal novel in vitro anti-angiogenic and anti-cancer activities of rhein (a natural anthraquinone) on the proliferation of MCF-7 and MDA-MB-435s cells. Hence, rhein is a potential chemopreventive natural compound. To the best of our knowledge, this is the first report that identifies rhein as inhibitor of hypoxia-induced angiogenesis, Hsp90a, and HIF-1a. However,

229

V.E. Fernand et al. / Chemico-Biological Interactions 192 (2011) 220–232

-

100

Hypoxic

200

-

100 200 (μM) μ

I

Hsp 90α

II

Hsp 90β

III

COX-2

IV

HER-2

120 100 80 60

*

*

*

**

Control 100 μM 200 μM

40 20 0 Hsp 90α

Hsp 90β

COX-2

HER-2

VI 120 Relative Intensity (% of control)

Normoxic

Relative Intensity (% of control)

V

A

100 80

*

**

60

*

Control

*

100 μM 200 μM

40 20 0 Hsp 90α

B

-

100 200

Hypoxic -

100 200 (μM) μ

I

Hsp 90α

II

Hsp 90β

III

COX-2

IV

HER-2

COX-2

HER-2

120 100 80

*

*

60

*

Control 100 μM 200 μM

40 20 0 Hsp 90α

Hsp 90β

COX-2

HER-2

VI 120 Relative Inhibition (% of control)

Normoxic

Relative Inhibition (% of control)

V

Hsp 90β

100 80

**

**

**

Control 100 μM

60

200 μM 40 20 0 Hsp 90α

Hsp 90β

COX-2

HER-2

Fig. 7. Effect of rhein on (A) MCF-7 and (B) MDA-MB-435s cell expressions of (I) Hsp90a, (II) Hsp90b, (III) COX-2, and (IV) HER-2. Cells were untreated (control) or treated with 100 or 200 lM rhein under normoxic and hypoxic conditions for 48 h. Cell extracts were prepared and subjected to Western Blot analysis to investigate Hsp90a, Hsp90b, COX-2, and HER-2 protein level. Blots are a representative of three separate experiments. (V or VI) Relative band intensity of the expression factors Hsp90a, Hsp90b, COX-2, and HER-2 was determined by use of densitometry under normoxic or hypoxic conditions, respectively. Bars represent the mean ± S.E.M. from three independent experiments. ⁄p < 0.05, compared with the control.

while we have demonstrated that rhein inhibits angiogenesis in vitro it remains to demonstrate that rhein is also effective

against angiogenesis in vivo and work is in progress to determine whether rhein can inhibit angiogenesis in vivo.

230

V.E. Fernand et al. / Chemico-Biological Interactions 192 (2011) 220–232

B

120

Normoxic

100

VEGF (% of Control)

VEGF (% of Control)

A

Hypoxic

80 60

* *

40

* *

20 0

120

Normoxic

100

Hypoxic

80

* *

60

* *

40 20 0

0

50

100

200

0

50

100

D

Normoxic

120

Hypoxic

100 80

*

*

*

*

60

EGF (% of Control)

EGF (% of Control)

C

200

Rhein (μM)

Rhein (μM)

*

40 20

140

Normoxic

120

Hypoxic

100

*

80

* *

*

60

*

40 20

0

0 0

50

100

200

0

50

HIF-1α levels (% of control)

E

100

200

Rhein (μM)

Rhein (μM)

120

MCF-7

100

MDA-MB-435s

80

60

*

40

* *

20

*

* *

0 50 μM

Control

100 μM

200 μM

Fig. 8. Rhein inhibits growth factors in supernatant (VEGF165, EGF) and in nuclear extract (HIF-1a). Rhein’s inhibitory effect on VEGF165 protein levels in (A) MCF-7 and (B) MDA-MB-435s cells. Cells were treated with rhein (50, 100, and 200 lM) for 48 h under normoxic or hypoxic conditions, supernatants were collected and assayed for VEGF165. Inhibition effect of rhein on EGF protein levels in (C) MCF-7 and (D) MDA-MB-435s cells. Breast cancer cells were treated with rhein (50–200 lM) for 48 h under normoxic or hypoxic conditions, supernatants were collected and analyzed for EGF. (E) Rhein inhibits HIF-1a expression in MCF-7 and MDA-MB-435s cells. Cells were untreated (control) or treated with 50, 100, and 200 lM rhein under hypoxic conditions for 48 h before harvesting. Normalized nuclear extracts were subjected to HIF-1a analysis. Pretreatment of cells with rhein significantly diminished the translocation of HIF-1a. Experiments were performed in triplicate. Bars represent the mean ± S.E.M. ⁄ p < 0.05 versus control.

B

120.00

Normoxic 100.00

Hypoxic

80.00

*

60.00

*

40.00

* *

20.00 0.00 0

50

100 Rhein (μM)

200

Phospho-IkB (% of Control)

Phospho-IKB (% of Control)

A

120

Normoxic 100

Hypoxic

80

*

*

60

*

*

40

* *

20 0 0

50

100

200

Rhein (μM)

Fig. 9. Rhein inhibits Ij-B phosphorylation and degradation in (A) MCF-7 and (B) MDA-MB-435s cells. Breast cancer cells were incubated with rhein (50–200 lM) for 48 h under normoxic or hypoxic conditions in the presence of TNF-a (10 ng/ml). Whole cell extracts were acquired as earlier indicated in Section 2.10. Normalized extracts of control and rhein-treated MCF-7 cells were assayed for Ij-B levels using ELISA. Values are means ± S.E.M. of triplicate results. ⁄p < 0.05 compared with control.

231

V.E. Fernand et al. / Chemico-Biological Interactions 192 (2011) 220–232

120

B

Normoxic

120

Hypoxic

100

* *

80 60 40

* *

20

* *

Relative activity of NF-kB p65 (% of control)

Relative activity of NF-kB p50 (% of control)

A

0

Normoxic Hypoxic

100 80

*

*

60 40 20

* *

100

200

0

Control

50

100

Control

200

50

Rhein (μM)

Rhein (μM)

C

D

Normoxic

120

Hypoxic

100 80

* *

60 40

* *

20 0 Control

50

100

*

* 200

Rhein (μM)

Relative activity of NF-kB p65 (% of control)

Relative activity of NF-kB p50 (% of control)

* *

Normoxic

120

Hypoxic 100

*

80

*

60

*

40

*

20

*

*

0 Control

50

100

200

Rhein (μM)

Fig. 10. Rhein inhibits NF-jB DNA-binding activity in MCF-7 and MDA-MB-435s cells. Breast cancer cells were incubated with rhein for 48 h under normoxic or hypoxic conditions in the presence of TNF-a (10 ng/ml). Normalized nuclear extracts of control and rhein-treated breast cancer cells were assayed for NF-jB DNA-binding activity using ELISA. (A) Rhein inhibits NF-jB p50 activity in nuclear extracts of MCF-7 cells under normoxic or hypoxic conditions. (B) Inhibition of NF-jB p65 activity under normoxic or hypoxic conditions in nuclear extracts of MCF-7 cells by rhein. (C) Inhibition effect of rhein under normoxic or hypoxic conditions on NF-jB p50 activity in nuclear extracts of MDA-MB-435s cells. (D) Rhein inhibits NF-jB p65 activity in nuclear extracts of MDA-MB-435s cells under normoxic or hypoxic conditions. Results are expressed as the mean ± S.E.M. ⁄p < 0.05 compared with control.

5. Conflict of interest statement None declared. Acknowledgements This work was supported by the National Science Foundation (CHE0616824) and the National Institutes for Health (1R01GM079670-01A2). References [1] A. Jemal, R. Siegel, J. Xu, E. Ward, Cancer statistics, CA Cancer J. Clin. (2010), doi:10.1002/CAAC20073. [2] D. Coradini, M.G. Daidone, Biomolecular prognostic factors in breast cancer, Curr. Opin. Obstet. Gynecol. 16 (2004) 49–55. [3] K.C. Chu, R.E. Tarone, L.G. Kessler, L.A. Ries, B.F. Hankey, B.A. Miller, B.K. Edwards, Recent trends in US breast cancer incidence, survival, and mortality rates, J. Natl. Cancer Inst. 88 (1996) 1571–1579. [4] P. Vaupel, O. Thews, M. Hoeckel, Treatment resistance of solid tumors: role of hypoxia and anemia, Med. Oncol. 18 (2001) 243–259. [5] A.L. Harris, Hypoxia – a key regulatory factor in tumor growth, Nature Rev. Cancer 2 (2002) 38–47. [6] J. Folkman, Tumor angiogenesis: therapeutic implications, New Engl. J. Med. 285 (1971) 1182–1186. [7] J.D. Carpini, A.K. Karam, L. Montgomery, Vascular endothelial growth factor and its relationship to the prognosis and treatment of breast, ovarian, and cervical cancer, Angiogenesis 13 (2010) 43–58. [8] K. Lundgren, C. Holm, G. Landberg, Hypoxia and breast cancer: prognostic and therapeutic implications, Cell. Mol. Life Sci. 64 (2007) 3233–3247. [9] J.A. Forsythe, B.H. Jiang, N.V. Iyer, F. Agani, S.W. Leung, R.D. Koos, G.L. Semenza, Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1, Mol. Cell. Biol. 16 (1996) 4604–4613. [10] G.L. Wang, B.-H. Jiang, E.A. Rue, G.L. Semenza, Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension, Proc. Natl. Acad. Sci. USA 92 (1995) 5510–5514.

[11] P. Jaakkola, D.R. Mole, Y.-M. Tian, M.I. Wilson, J. Gielbert, S.J. Gaskell, A. von Kriegsheim, H.F. Hebestreit, M. Mukherji, C.J. Schofield, P.H. Maxwell, C.W. Pugh, P.J. Ratcliffe, Targeting of HIF-alpha to the von Hippel-Lindau ubiquitylation complex by O2-regulated prolyl hydroxylation, Science 292 (2001) 468–472. [12] R. Bos, H. Zhong, C.F. Hanrahan, E.C. Mommers, G.L. Semenza, H.M. Pinedo, M.D. Abeloff, J.W. Simons, P.J. van Diest, E. van der Wall, Levels of hypoxiainducible factor-1 alpha during breast carcinogenesis, J. Natl. Cancer Inst. 93 (2001) 309–314. [13] R. Bos, P. van der Groep, E. Greijer Astrid, A. Shvarts, S. Meijer, M. Pinedo Herbert, L. Semenza Gregg, J. van Diest Paul, E. van der Wall, Levels of hypoxia-inducible factor-1alpha independently predict prognosis in patients with lymph node negative breast carcinoma, Cancer 97 (2003) 1573–1581. [14] M.G. Rowlands, Y.M. Newbatt, C. Prodromou, L.H. Pearl, P. Workman, W. Aherne, High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity, Anal. Biochem. 327 (2004) 176–183. [15] D.J. Slamon, G.M. Clark, S.G. Wong, W.J. Levin, A. Ullrich, W.L. McGuire, Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene, Science 235 (1987) 177–182. [16] H.R. Chang, Trastuzumab-based neoadjuvant therapy in patients with HER2positive breast cancer, Cancer 116 (2010) 2856–2867. [17] D.J. Newman, G.M. Cragg, Natural products as sources of new drugs over the last 25 years, J. Natl. Prod. 70 (2007) 461–477. [18] Y. Hasebe, K. Egawa, Y. Yamazaki, S. Kunimoto, Y. Hirai, Y. Ida, K. Nose, Specific inhibition of hypoxia-inducible factor (HIF)-1 alpha activation and of vascular endothelial growth factor (VEGF) production by flavonoids, Biol. Pharm. Bull. 26 (2003) 1379–1383. [19] J. Fang, C. Xia, Z. Cao, J.Z. Zheng, E. Reed, B.-H. Jiang, Apigenin inhibits VEGF and HIF-1 expression via PI3K/AKT/p70s6K1 and HDM2/p53 pathways, Faseb. J. 19 (2005) 342–353. [20] C. Bocca, F. Bozzo, S. Cannito, S. Colombatto, A. Miglietta, CLA reduces breast cancer cell growth and invasion through ERa and PI3K/Akt pathway, Chem. Biol. Interact. 183 (2010) 187–193. [21] M. Shimizu, Y. Shirakami, H. Sakai, Y. Yasuda, M. Kubota, S. Adachi, H. Tsurumi, Y. Hara, H. Moiwaki, ( )-Epigallocatechin gallate inhibits growth and activation of the VEGF/VEGFR axis in human colorectal cancer cells, Chem. Biol. Interact. 185 (2010) 247–252. [22] V.E. Fernand, D.T. Dinh, S.J. Washington, S.O. Fakayode, J.N. Losso, R.O. van Ravenswaay, I.M. Warner, Determination of pharmacologically active

232

[23] [24] [25]

[26]

[27]

[28]

[29]

[30]

[31] [32]

[33]

[34] [35]

[36]

V.E. Fernand et al. / Chemico-Biological Interactions 192 (2011) 220–232 compounds in root extracts of Cassia alata L. by use of high performance liquid chromatography, Talanta 74 (2008) 896–902. H. Heyde, Medicinal Plants in Suriname (Medicijn Planten in Suriname), third ed., Stichting Gezondheidsplanten Informatie, Paramaribo, 1990. C.M. Spencer, M.I. Wilde, Diacerein, Drugs 53 (1997) 98–106. S. Franchi-Micheli, L. Lavacchi, C.A. Friedmann, L. Zilletti, The influence of rhein on biosynthesis of prostaglandin-like substances in-vitro, J. Pharm. Pharmacol. 35 (1982) 262–264. R.H. Cichewicz, Y. Zhang, N.P. Seeram, M.G. Nair, Inhibition of human tumor cell proliferation by novel anthraquinones from daylilies, Life Sci. 74 (2004) 1791–1799. T.C. Hsia, J.S. Yang, G.W. Chen, T.H. Chiu, H.F. Lu, M.D. Yang, F.S. Yu, K.C. Liu, K.C. Lai KC, C.C. Lin, J.G. Chung, The roles of endoplasmic reticulum stress and Ca2+ on rhein-induced apoptosis in A-549 human lung cancer cells, Anticancer Res. 29 (2009) 309–318. W.W. Lai, J.S. Yang, K.C. Lai, C.L. Kuo, C.K. Hsu, C.K. Wang, C.Y. Chang, J.J. Lin, N.Y. Tang, P.Y. Chen, W.W. Huang, J.G. Chung, Rhein induced apoptosis through the endoplasmic reticulum stress, caspase- and mitochondria-dependent pathways in SCC-4 human tongue squamous cancer cells, In Vivo 23 (2009) 309–316. A. Floridi, S. Castiglione, C. Bianchi, A. Mancin, Effect of rhein on the glucose metabolism of Ehrlich ascites tumor cells, Biochem. Pharmacol. 40 (1990) 217–222. M. Chintalapati, R. Truax, R. Stout, R. Portier, J.N. Losso, In vitro and in vivo anti-angiogenic activities and inhibition of hormone-dependent and independent breast cancer cells by ceramide methylaminoethylphosphonate, J. Agric. Food Chem. 57 (2009) 5201–5210. P.L. Kuo, Y.L. Hsu, L.T. Ng, C.C. Lin, Rhein inhibits the growth and induces the apoptosis of Hep G2 cells, Planta Med. 70 (2004) 12–16. P. Shi, Z. Huang, G. Chen, Rhein induces apoptosis and cell cycle arrest in human hepatocellular carcinoma BEL-7402 cells, Am. J Chin. Med. 36 (2008) 805–813. P.A. Baeuerle, D. Baltimore, The physiology of the NF-kB transcription factor, in: P. Cohen, J.G. Foulkes (Eds.), Molecular Aspects of Cellular Regulation, vol. 6, Elsevier, Amsterdam, 1991, pp. 423–446. F. Pacifico, A. Leonardi, NF-kB in solid tumors, Biochem. Pharmacol. 72 (2006) 1142–1152. M.L. Lin, S.S. Chen, Y.C. Lu, R.Y. Liang, Y.T. Ho, C.Y. Yang, J.G. Chung, Rhein induces apoptosis through induction of endoplasmic reticulum stress and Ca2+dependent mitochondrial death pathway in human nasopharyngeal carcinoma cells, Anticancer Res. 27 (2007) 3313–3322. Y.Y. Cheng, S.Y. Chiang, J.G. Lin, Y.S. Ma, C.L. Liao, S.W. Weng, T.Y. Lai, J.G. Chung, Emodin, aloe-emodin and rhein inhibit migration and invasion in human tongue cancer SCC-4 cells through the inhibition of gene expression of matrix metalloproteinase-9, Int. J. Oncol. 36 (2010) 1113–1120.

[37] G. Aviello, I. Rowland, C.I. Gill, A.M. Acquaviva, F. Capasso, M. McCann, R. Capasso, A.A. Izzo, F.J. Borrelli, Anti-proliferative effect of rhein, an anthraquinone isolated from Cassia species, on Caco-2 human adenocarcinoma cells, J. Cell. Mol. Med. 14 (2010) 2006–2014. [38] B.D. Manning, L.C. Cantley, AKT/PKB signaling: navigating downstream, Cell 129 (2007) 1261–1274. [39] D.A. Murphy, S. Makonnen, W. Lassoued, M.D. Feldman, C. Carter, W.M. Lee, Inhibition of tumor endothelial ERK activation, angiogenesis, and tumor growth by sorafenib (BAY43-9006), Am. J. Pathol. 169 (2006) 1875–1885. [40] G.D. Thakker, D.P. Hajjar, W.A. Muller, T.K. Rosengart, The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling, J. Biol. Chem. 274 (1999) 10002–10007. [41] A. Nakashio, N. Fujita, T. Tsuruo, Topotecan inhibits VEGF- and bFGF-induced vascular endothelial cell migration via downregulation of the PI3K-Akt signaling pathway, Int. J. Cancer 98 (2002) 36–41. [42] H.K. Abbas, C.J. Mirocha, Isolation and purification of a hemorrhagic factor (wortmannin) from Fusarium oxysporum (N17B), Appl. Environ. Microbiol. 54 (1988) 1268–1274. [43] G.J. Downing, S. Kim, S. Nakanishi, K.J. Catt, T. Balla, Characterization of a soluble adrenal phosphatidylinositol 4-kinase reveals wortmannin sensitivity of type III phosphatidylinositol kinases, Biochemistry 35 (1996) 3587–3594. [44] Y. Lu, J. Zhang, J. Qian, The effect of emodin on VEGF receptors in human colon cancer cells, Cancer Biother. Radiopharm. 23 (2008) 222–228. [45] T. Kaneshiro, T. Morioka, M. Inamine, T. Kinjo, J. Arakaki, I. Chiba, N. Sunagawa, M. Suzui, N. Yoshimi, Anthraquinone derivative emodin inhibits tumorassociated angiogenesis through inhibition of extracellular signal-regulated kinase 1/2 phosphorylation, Eur. J. Pharmacol. 553 (2006) 46–53. [46] E. Pozo-Guisado, A. Alvarez-Barrientos, S. Mulero-Navarro, B. Santiago-Josefat, P.M. Fernandez-Salguero, The antiproliferative activity of resveratrol results in apoptosis in MCF-7 but not in MDA-MB-231 human breast cancer cells: cellspecific alteration of the cell cycle, Biochem. Pharmacol. 64 (2002) 1375–1386. [47] A. Kamal, L. Thao, J. Sensintaffar, L. Zhang, M.F. Boehm, L.C. Fritz, F.J. Burrows, A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors, Nature 425 (2003) 407–410. [48] M. Hollingshead, M. Alley, A.M. Burger, S. Borgel, C. Pacula-Cox, H.H. Fiebig, E.A. Sausville, In vivo antitumor efficacy of 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride), a water-soluble geldanamycin derivative, Cancer Chemother. Pharmacol. 56 (2005) 115–125. [49] J.Y. Jang, Y.K. Jeon, C.W. Kim, Degradation of HER2/neu by ANT2 shRNA suppresses migration and invasiveness of breast cancer cells, BMC Cancer 10 (2010) 391–400. [50] V. Wells, L. Mallucci, Phosphoinositide 3-kinase targeting by the beta galactoside binding protein cytokine negates akt gene expression and leads aggressive breast cancer cells to apoptotic death, Breast Cancer Res. 11 (2009) R2, doi:10.1186/bcr2217.