Clin Exp Med DOI 10.1007/s10238-015-0336-7

ORIGINAL ARTICLE

Curcumin inhibits LPA-induced invasion by attenuating RhoA/ROCK/MMPs pathway in MCF7 breast cancer cells Kai Sun • Xiaoyi Duan • Hui Cai • Xiaohong Liu Ya Yang • Min Li • Xiaoyun Zhang • Jiansheng Wang

•

Received: 15 October 2014 / Accepted: 8 January 2015 Ó Springer-Verlag Italia 2015

Abstract Breast cancer generally shows poor prognosis because of its invasion and metastasis. Lysophosphatidic acid (LPA) induces and aggravates cancer invasion and metastasis by activating its downstream signal pathways. RhoA/ROCK/ MMP signaling was found one of the LPA-induced pathways, which may be involved in invasion of breast cancer. Furthermore, we investigated whether this pathway was involved in curcumin’s effect against LPA-induced invasion. LPA incubation was used to enhance invasion of MCF-7 breast cancer cells. RhoA expression was knocked-down by siRNA technique. MTT assay was used to evaluate the proliferation. Transwell assay was utilized to investigate the invasion ability of MCF-7 cells. Real-time PCR and Western blotting were used to assess the expressions of RhoA, ROCK1, ROCK2, MMP2 and MMP9 at both translational and transcriptional levels. The RhoA and ROCK activities were also evaluated.

LPA incubation significantly boosted invasion rate of MCF-7. RhoA silencing by siRNA dramatically inhibited LPAenhanced invasion. Concurrently, RhoA and ROCK activities and expression levels of RhoA, ROCK1, ROCK2, MMP2 and MMP9 were down-regulated by RhoA siRNA transfection. In order to avoid influence of cytotoxicity of curcumin, concentrations below 45 lmol/L were selected to further investigate the mechanism of curcumin’s anti-invasion effect. Invasion of LPA-incubated MCF-7 cells was impaired by curcumin in a concentration-dependent manner. Concurrently, RhoA and ROCK activities and expression levels of RhoA, ROCK1, ROCK2, MMP2 and MMP9 were downregulated by curcumin in a concentration-dependent manner. In conclusion, RhoA/ROCK/MMPs pathway activation is involved in LPA-induced invasion in MCF-7 cells; curcumin inhibited LPA-induced invasion in MCF-7 cells by attenuating RhoA/ROCK/MMPs pathway.

K. Sun  H. Cai  Y. Yang  M. Li  J. Wang (&) The Second Department of Thoracic Surgery, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, Shaanxi, China e-mail: [email protected]

Keywords Breast cancer  Lysophosphatidic acid  Invasion  Curcumin

Introduction X. Duan Department of Nuclear Medicine, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, China H. Cai Department of Anesthesia, First Affiliated Hospital of Xi’an Jiaotong University, Xi’an, Shaanxi, China X. Liu Department of General Surgery, Second Affiliated Hospital of Lanzhou University, Lanzhou, Gansu, China X. Zhang Department of Pathology, Lanzhou University Medical School, Lanzhou, Gansu, China

Breast cancer has been one of the major diseases, which are threatening women’s health and life worldwide for many decades [1]. As a malignant tumor, breast cancer is among the leading causes of cancer-related deaths [2]. Though current therapies such as surgeries, chemotherapy and radiotherapy are proved effective in reducing primary tumor, the 5-year survival rate of breast cancer is still poor mainly due to its intensive tendency of invasion and metastasis [3]. Understanding the biological mechanisms of invasion and metastasis is required for identifying novel therapeutic targets and drug action mechanisms.

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Although the molecular structure is relatively simple, the natural phospholipid lysophosphatidic acid (LPA) is considered highly bioactive [4]. It was found that LPA played important roles in regulating proliferation, migration and invasion in cancer cells. Thus, secreted by activated platelets in cancer patients, LPA could be treated as one of the serum molecular markers of cancer [5]. Furthermore, LPA was reported and correlated with the invasive and metastatic behaviors of several malignant cancers including breast cancer [6]. For instance, LPArelated pathways, such as PI3K/PAK1/ERK [7], CD24 [8], RaP1 and IQGAP1 [9], were found associated with cell migration and invasion in breast cancer cells. The biological activities of LPA are conducted by several signaling pathways after interacting with LPA receptors. Under elevated LPA stimulation, several heterotrimeric G proteins, including Gi/o, Gq/11 and G12/13, are activated to stimulate the activation of multiple downstream signaling pathways [10]. These G proteins could further interact with protein containing PDZ domains such as PDZ-RhoGEF [11]. Recent emerging evidences indicated that LPA could activate effecter molecule Ras homolog gene family member A (RhoA) by mediating the interaction between RhoGEFs and G12/13 [12]. The upregulation of RhoA expression was well documented positively related with cancer invasion and metastasis [13]. Over-expression of RhoA promoted the invasive behavior of colon cancer cells by inducing the expression of matrix metalloproteinases (MMPs) [14]. Curcumin, also referred as diferuloylmethane, is one of the main bioactive components of turmeric (Curcuma Longa). Curcumin has been used as food additives, spice and colorant for decades. In traditional Chinese medicine (TCM), curcumin has been used in treatment of cardiovascular and digestive disease from the ancient time [15]. In the modern biomedical studies, curcumin was reported effectively impaired proliferation and invasion of many human cancers including breast cancer [16]. Curcumin was proved to induce apoptosis of breast cancer cells both in vitro and in vivo [17]. Multiple molecular mechanisms including NF-kappa B and HER2 activation [18], E-cadherin/beta-catenin modifying [19] and PTEN/AKT/p53 modulation [20] were involved. Notably, curcumin could inhibit invasion of malignant cells by reducing expression of MMPs [21]. However, the exact mechanisms are still unclear. In this study, in order to determine whether LPA/RhoA/MMP was involved in invasion of breast cancer, the small interfering RNA (siRNA) technique was introduced to disturb the RhoA signaling in LPA-incubated MCF-7 breast cancer cells. We also investigated the role of RhoA in curcumin’s inhibitory effects on LPA-stimulated invasion of MCF-7 cells.

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Materials and methods Cell culture and treatment The breast cancer cell line MCF-7 was provided by Sagene Inc (Guangzhou, China). Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, New York, USA) supplemented with fetal bovine serum (5 %, Gibco, New York, USA), L-glutamine (200 mmol/L, Sigma-Aldrich, St.Louis, USA), penicillin–streptomycin mix (100 U/mL penicillin and 100 lg/mL streptomycin, Sigma-Aldrich, St.Louis, USA) and nonessential amino acids (1009, Invitrogen, Carlsbad, USA) in culture flasks in a humidified incubator filled by fresh air with 5 %CO2 at 37 °C. Subsequent experiments were implemented when cells reached confluence over 80 %. LPA (Avanti, Alabaster, USA) at concentration of 1 lmol/L was used to incubate MCF-7 cells for 24 h [22]. Small interfering RNA (siRNA) transfection Specific siRNA and scramble siRNA sequences against RhoA were designed and synthesized by TaKaRa (Tokyo, Japan). The RhoA siRNA sequence was 50 -CCUUAUA GUUACUGUGUAATT-30 [23]. The scramble sequence was 50 -GCAATTCTAGTCTTGTTATTA-30 . 1 9 106 cells were seeded into culturing dishes and incubated at 37 °C for 48 h. 25 mg/dish siRNA was transfected into the cells by using HiPerFect transfection kit (Qiagen, Shanghai, China) per manufacturer’s instructions. After 48-hour incubation under standard condition, cells were harvested for subsequent experiments. Cell proliferation assay The proliferation of MCF-7 cells was assessed by colorimetric 3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay. Cells were planted into the wells on a 96-well plate, and MTT (Sigma-Aldrich, St.Louis, USA) solution (5 mg/mL) was used to incubate the cells at 37 °C for 4 h. Then, the cells were washed by PBS before 150 lL dimethylsulfoxide (DMSO, Sigma-Aldrich, St.Louis, USA) was added into each well. The absorbance at 540 nm (A540) was detected by a plate reader. The formula ‘‘inhibition rate = [1 - A540 (experimental well)/A540(control well)] 9 100 %’’ was used to calculate the inhibition rate. Cell Matrigel invasion assay The BD BioCoat Matrigel Invasion Chambers System (New Jersey, USA) was used in this study to evaluate the invasive ability of MCF-7 cells according to manufacturer’s protocol.

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Briefly, 5 9 104 cells were seeded into the top chamber, which was filled with basal medium (only DMEM). The lower chamber was filled with nutrient-rich medium (DMEM supplemented with fetal bovine serum) to perform chemoattraction. After 24-h incubation at standard condition, invaded cells (the cells passed through Matrigel) were fixed by methanol and stained by crystal violet. The inhibition rate was calculated after cell count was determined by light microscopic (Motic, Xiamen, China) observation. Real-time PCR The total RNA in MCF-7 cells was extracted by using TriReagent (MRC, Huston, USA) according to the manufacturer’s instructions. The possible DNA contamination was eliminated by Turbo DNA-Free kit (Ambion, Alabaster, USA). The reverse transcription and cDNA synthesis were performed by using SuperScript III Reverse Transcriptase (Invitrogen, Alabaster, USA). The quantitative real-time PCR was implemented by using All-in-one TM qPCR kit (Gene Copoeia, Rockville, USA) according to the protocol provided by the manufacturer. Each cycle contained (1) denaturation at 95 °C for 15 s; (2) annealing at 69 °C for 15 s and (3) extension at 72 °C for 30 s. GAPDH was introduced as the internal reference. Gene primers for RhoA, ROCK1, ROCK2, MMP2, MMP9 and GAPDH were designed and provided by TaKaRa. The primers for each gene are listed in Table 1. Relative expression levels of each target mRNA were calculated by Dcycle threshold method (DCt = CtTarget-CtGAPDH) when GAPDH was introduced as internal reference.

manufacturers’ instructions. Before vertical electrophoresis was performed, protein concentration was detected by a BCA kit (Thermo, Pittsburg, USA). Fifty microgram of protein sample was loaded and separated by vertical electrophoresis. Then, the separated protein was transferred to poly vinylidene difluoride (PVDF, Thermo, Pittsburg, USA) membranes, which were then incubated with blocking buffer (5 % defatted milk, Tris-buffered saline and 0.1 % Tween 20) for 1 h at 37 °C to eliminate nonspecific binding. After washing, specific antibodies against RhoA (1:5,000, Abcam, Cambridge, USA), ROCK1 (1:5,000, Abcam, Cambridge, USA), ROCK2 (1:5,000, Abcam, Cambridge, USA), MMP2 (1:2,000, Santa Cruz, Dallas, USA), MMP9 (1:5,000, Abcam, Cambridge, USA) and GAPDH (1:5,000, Santa Cruz, Dallas, USA) at 4 °C for 12 h. Immunoblots were then detected by SuperSignal West Pico kit (Thermo Scientific, Pittsburg, USA). The relative protein expressions were analyzed by software ImageJ2x (NIH). RhoA and ROCK activity assay Enzyme-linked immunosorbent (ELISA)-based method Equal amount MCF-7 cells was collected and lyzed. The lysates were collected and further centrifuged at 1,000g for 10 min at 4 °C. The resulted supernatant was used for RhoA and ROCK activity assay. RhoA activity was measured by using RhoA Activity Assay kit (NewEast Bioscience, Wuhan, China), while ROCK activity was measured by ROCK Activity Assay kit (Cell Biolabs, San Diego, USA) according to the manufacturers’ instructions.

Western blotting Western blotting-based method The cell lysates of MCF-7 cells were acquired after lyzed by RIPA lysis buffer system (Santa Cruz, Dallas, USA) on ice, and the total protein was isolated by using Protein Extraction kit (Beyotime, Shanghai, China) according to Table 1 Primers for real-time PCR

The supernatant described above was used and analyzed by immunoblotting. As the down-stream targets, the expression levels of RhoA GTP and phosphorylated MYPT1Thr696

Gene name

Type

Sequences

RhoA

F

50 -TGGAAAGACATGCTTGCTCAT-30

ROCK1 ROCK2 MMP2

0

Size (bp) 22 0

R

5 -GCCTCAGGCGATCATAATCTTC-3

F

50 -AAAGCCTTACTGTCGATTGCC-30

21

R

50 -AGGGTAATGCAACTTCCACTG-30

21

F

50 -AGTCAATCTCACCAGCACATA-30

21

R

50 -AATAGCGTCCAAAGCCAACAC-30

21

F

50 -CAGGGAATGAGTACTGGGTCTATT-30 0

21

0

24

R

5 -ACTCCAGTTAAAGGCAGCATCTAC-3

MMP9

F R

50 -AATCTCTTCTAGAGACTGGGAAGGAG-30 50 -AGCTGATTGACTAAAGTAGCTGGA-30

26 24

GAPDH

F

50 -CTTTTGCGTCGCCAGCCGAG-30

20

R

50 -GCGCCCAATACGACCAAATCCG-30

20

24

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(p-MYPT1) were considered the indictors for the activities of RhoA and ROCK, respectively. The Western blotting procedure was similar to the contents described above by using the anti-RhoA GTP (Santa Cruz, Dallas, USA) and anti-phosphoMYPT1 (Abcam, Cambridge, USA) antibodies. GAPDH was still introduced as the internal reference.

Fig. 1 RhoA siRNA transfection inhibited invasion of LPA-incubated MCF-7 cells. Ctrl refers to untreated MCF-7 cells; Ctrl?Scramble refers to MCF-7 cells transfected by scramble siRNA; RhoA-/refers to MCF-7 cells transfected by RhoA siRNA; LPA refers to MCF-7 cells incubated with LPA (1 lmol/L for 24 h); LPA?Scramble refers to scramble siRNA transfected MCF-7 cells incubated with LPA (1 lmol/L for 24 h); LPA?RhoA-/- refers to MCF7 cells transfected by RhoA siRNA and then incubated with LPA. Left part of this figure demonstrated that the captured images of results of Transwell Matrigel invasion assay in MCF-7 cells invaded through Matrigel were fixed and stained by crystal violet. Columns on the right part of this figure indicated invasion rate of MCF-7 from Ctrl, RhoA-/-, LPA and LPA?RhoA-/-, respectively. Values were presented as (mean ± SD). a Differences were significant from Ctrl; bdifferences were significant from LPA?Scramble; cdifferences were significant from RhoA-/-; d differences were significant from LPA?Scramble; e differences were significant from LPA

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Statistics The result values were expressed as mean ± standard deviation and further analyzed by software SPSS (ver.16.0, SPSS). The significance of differences among groups was analyzed by ANOVA followed by Bonferroni post hoc

Clin Exp Med

tests or when appropriate Student’s t tests. P \ 0.05 was considered to be statistically significant.

Results RhoA siRNA transfection inhibited invasion of LPAincubated MCF-7 cells Matrigel invasion assay was used to evaluate the invasive ability of MCF-7 cells in this study. As demonstrated in Fig. 1, the invasion rate of MCF-7 cells was significantly elevated after exposed to LPA (81.85 ± 3.86 %) when compared with control (65.80 ± 2.94 %, P = 0.0046), RhoA-/- (65.46 ± 2.91 %, P = 0.0042) and control treated with scramble siRNA (65.40 ± 2.40 %,

Fig. 2 RhoA siRNA transfection reduced activities of RhoA and ROCK in LPA-incubated MCF-7 cells. Ctrl refers to untreated MCF-7 cells; RhoA-/- refers to MCF-7 cells transfected by RhoA siRNA; LPA refers to MCF-7 cells incubated with LPA (1 lmol/L for 24 h); LPA?RhoA-/- refers to MCF-7 cells transfected by RhoA siRNA and then incubated with LPA. The enzymatic activities of RhoA and ROCK were assessed by both ELISA-based and Western blottingbased methods. a Demonstrated the immunoblots of RhoA GTP and

P = 0.0033). Furthermore, the siRNA technique was employed in the present study to investigate the role of RhoA in LPA-enhanced invasion. Also, as shown in Fig. 1, the LPA incubation increased invasion rate of MCF-7 cells significantly. However, RhoA silence decreased invasion rate of LPA-incubated MCF-7 cells dramatically (28.88 ± 3.37, P = 0.0001). RhoA siRNA transfection reduced RhoA/ROCK/MMP activation in LPA-incubated MCF-7 cells Real-time PCR, Western blotting and enzymatic activity assay were utilized to assess the activation of RhoA/ ROCK/MMP signaling pathway in MCF-7 cells. As shown in Figs. 2 and 3, in MCF-7 cells without LPA incubation, the RhoA/ROCK/MMP signaling remained deactivated.

p-MYPT1. Columns on (b) and (d) indicate the relative expression levels of RhoA GTP and p-MYPT1 in Ctrl, RhoA-/-, LPA and LPA?RhoA-/-, respectively. Columns on (c) and (e) indicate the enzymatic activities of RhoA and ROCK detected by ELISA-based method in Ctrl, RhoA-/-, LPA and LPA?RhoA-/-, respectively. Values were presented as (mean ± SD). aDifferences were significant from Ctrl; bdifferences were significant from RhoA-/-; cdifferences were significant from LPA

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However, LPA incubation significantly stimulated the activation of RhoA/ROCK/MMP signaling in MCF-7 cells (All P \ 0.05). Also, as shown in Figs. 2 and 3, we further found that the RhoA silence did not influence the molecular expressions in RhoA/ROCK/MMP pathway when MCF-7 was not incubated with LPA. Nevertheless, in LPA-incubated MCF-7 cells, RhoA silence significantly down-regulated RhoA signaling, resulting in decreased expressions of ROCK1, ROCK2, MMP2 and MMP9 (All P \ 0.05). Curcumin inhibited proliferation of MCF-7 cells in a concentration-dependent manner As demonstrated in Fig. 4a, after LPA incubation, when further incubated with serial diluted curcumin, the proliferation of MCF-7 was inhibited in a concentrationdependent manner (All P \ 0.05). We found that

Fig. 3 RhoA siRNA transfection reduced RhoA/ROCK/MMP activation in LPA-incubated MCF-7 cells. Ctrl refers to untreated MCF-7 cells; RhoA-/- refers to MCF-7 cells transfected by RhoA siRNA; LPA refers to MCF-7 cells incubated with LPA (1 lmol/L for 24 h); LPA?RhoA-/- refers to MCF-7 cells transfected by RhoA siRNA and then incubated with LPA. Activation of RhoA/ROCK/MMPs pathway was assessed by real-time PCR and Western blotting. Left part of this figure shows the immunoblots of RhoA, ROCK1, ROCK2,

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curcumin began to inhibit proliferation of LPA-incubated MCF-7 cells dramatically at concentration of 60 lmol/L (P \ 0.05 when compared with lower concentrations). Thus, curcumin at concentrations of 0, 15, 30 and 45 lmol/L was selected in the subsequent experiments concerning the anti-invasion effect of curcumin. Curcumin impaired invasive behavior of LPAincubated MCF-7 cells in a concentration-dependent manner Figure 4b demonstrates the anti-invasion effect of curcumin in LPA-incubated MCF-7 cells, which was detected by Matrigel invasion assay. As the concentration increased, curcumin inhibited cell invasion significantly in a concentration-dependent manner (All P \ 0.05).

MMP2, MMP9 and the internal reference GAPDH. Columns in the right part of this figure represented the relative expression levels of mRNA (top) and protein (bottom) of RhoA, ROCK1, ROCK2, MMP2 and MMP9 in Ctrl, RhoA-/-, LPA and LPA?RhoA-/-, respectively, in a (mean ± SD) manner. aDifferences were significant from Ctrl; b differences were significant from RhoA-/-; cdifferences were significant from LPA

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Curcumin inhibited the activation of RhoA/ROCK/ MMP signaling in LPA-incubated MCF-7 cells in a concentration-dependent manner Figures 5 and 6 show the relative expression levels of mRNA of RhoA, ROCK1, ROCK2, MMP2 and MMP9 in curcumintreated LPA-incubated MCF-7 cells. Figure 5 shows the activity assessment of RhoA and ROCK, while Fig. 6 shows the immunoblots of RhoA, ROCK1, ROCK2, MMP2 and MMP9 in LPA-incubated MCF-7 cells, which were then treated by serial concentrations of curcumin. We found that curcumin inhibited the activities of RhoA and ROCK, expressions of RhoA, and subsequently down-regulated the expressions of ROCK1, ROCK2, MMP2 and MMP9 at both transcriptional and translational levels (All P \ 0.05).

Fig. 4 a Curcumin inhibited proliferation of MCF-7 cells in a concentration-dependent manner. After LPA incubation (1 lmol/L, 24 h), the proliferation of curcumin-incubated MCF-7 cells was evaluated by MTT assay. The line chart in Fig. 3 demonstrated the calculated proliferation inhibition rate MCF-7 incubated with serial diluted curcumin (0, 15, 30, 45, 60 and 75 lmol/L) in a (mean ± SD) manner. Asterisk differences were significant from previous concentration b Curcumin impaired invasive behavior of LPA-incubated MCF-7 cells in a concentration-dependent manner. After incubated

Discussion It is now generally accepted that as one of the critical molecular adaptors, LPA mediates multiple cell responses. It was found potent of stimulating malignancy in many human cancer cells including breast cancer [24]. RhoA was found one of the down-stream effectors of LPA signaling [12]. In other studies, RhoA signaling was found to initiate expressions of MMPs in cancer [23]. Thus, we hypothesized that RhoA was the key molecule mediating LPA-induced invasion in breast cancer. Previously, curcumin showed its potent therapeutic effect against invasion in many cancer cells including breast cancer [25]. Few study investigated whether curcumin could inhibit LPA-enhanced invasion, and the related

with LPA, MCF-7 cells were treated by serial diluted curcumin (0, 15, 30, 45 lmol/L). Left part of this figure demonstrated the captured images of results of Transwell Matrigel invasion assay in MCF-7 cells invaded through Matrigel were fixed and stained by crystal violet. Columns on the right part of this figure indicated invasion rate of MCF-7 in a (mean ± SD) manner. aDifferences were significant from 0 lmol/L; bdifferences were significant from 15 lmol/L; cdifferences were significant from 30 lmol/L

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Clin Exp Med Fig. 5 Curcumin reduced the activities of RhoA and ROCK in LPA-incubated MCF-7 cells in a concentration-dependent manner. a Demonstrated the immunoblots of RhoA GTP and p-MYPT1. Columns on (b) and (d) indicate the relative expression levels of RhoA GTP and p-MYPT1 in MCF-7 cells treated by serial diluted curcumin (0, 15, 30, 45 lmol/ L). Columns on (b) and (d) indicate the relative expression levels of RhoA GTP and p-MYPT1 in MCF-7 cells treated by serial diluted curcumin (0, 15, 30, 45 lmol/ L). Columns on (c) and (e) indicate the enzymatic activities of RhoA and ROCK detected by ELISA-based method in MCF-7 cells treated by serial diluted curcumin (0, 15, 30, 45 lmol/L). a Differences were significant from 0 lmol/L; bdifferences were significant from 15 lmol/ L; cdifferences were significant from 30 lmol/L

mechanism still remained ambiguous. In the present study, we also investigated whether RhoA signaling was involved in curcumin’s inhibitory effect on LPA-induced invasion. There were two major findings that we acquired in this study. Firstly, by using siRNA technique, we found that RhoA signaling was essential in mediating enhanced invasion in LPA-incubated breast cancer cells. Secondly, curcumin inhibited invasion of LPA-incubated breast cancer cells by alleviating activation of RhoA/ROCK/ MMP signaling. As other malignant tumors, invasive behavior contributed significantly to the mortality and declined quality of life in patients with breast cancer [26]. Cancer cells could infiltrate to surrounding tissues and invade through blood and vessel walls. Via blood and lymph fluid, breast cancer cells planted to other organs. The distant metastasis made surgeries inadequate in treating breast cancer [27]. It was believed that external stimuli-triggered sequential modules could lead to the migration and invasion of cancer cells. Certain stimuli, LPA for instance, play positive roles in increasing cell mobility, deformability and invasive features [28].

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LPA is a natural phospholipid with very simple molecular structure presented in blood serum exerting wide range of biological activities [29]. It was believed that after being released from activated platelets, lysophospholipids were converted to LPA by lysophospholipase in serum [30]. Various biological processes including cAMP accumulation, intracellular calcium homeostasis regulation and cell mobility were described related with LPA [31]. It was reported that LPA has a positive role in promoting oncogenesis and accelerating progression of several cancers such as ovarian cancer, colon cancer, gastric cancer and breast cancer [5, 32]. In this study, we also identified the phenomenon that LPA incubation dramatically strengthened the invasive ability of MCF-7 cells, which was evidenced by the Matrigel invasion assay. The above malignancy-promoting effect of LPA was considered mediated by signals conducted by LPA receptors (LPARs) [28]. Till now, there were six subtypes of LPARs, namely LPAR1–LPAR6, have been identified and cloned. Among which, LPAR1–LPAR3 belong to the endothelial differentiation gene family of G protein-coupled receptors, while LPAR4 and LPAR5 were thought

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Fig. 6 Curcumin inhibited the activation of RhoA/ROCK/MMP signaling in LPA-incubated MCF-7 cells in a concentration-dependent manner. After incubated with LPA, MCF-7 cells were treated by serial diluted curcumin (0, 15, 30, 45 lmol/L). Left part of this figure shows the immunoblots of RhoA, ROCK1, ROCK2, MMP2, MMP9 and the internal reference GAPDH. Columns in the right part of this

figure represented the relative expression levels of mRNA (top) and protein (bottom) of RhoA, ROCK1, ROCK2, MMP2 and MMP9 in MCF-7 cells incubated with curcumin in a (mean ± SD) manner. a Differences were significant from 0 lmol/L; bdifferences were significant from 15 lmol/L; cdifferences were significant from 30 lmol/L

related to the purinergic G protein-coupled receptor family [33]. Previous studies indicated that different subtypes of LPARs were involved in different kinds of cancers. For instance, rather than LPAR2 and LPAR3, LPAR1 was indispensible in LPA-induced migration in MDA-MB-231 and 4T1 breast cancer cell lines [22]. All LPARs were coupling to three heterotrimeric GTP-binding proteins, which are Gq/11, Gi/0 and G12/13. These GTP-binding proteins trigger distinct downstream pathways to engage various biological processes including cancer invasion and migration [34]. It was evidenced that after coupled, G12/13 could activate the small GTPase RhoA [35]. In other published literatures, ROCK activation was described LPAR dependent in cancer cells incubated with LPA [35]. ROCK plays an important role in enhancing cancer cell invasion and motility to contribute to the progression and metastasis of cancers, especially in advanced cancers [36]. ROCK is also considered one of the canonical downstream effecters of RhoA. Thus, it was reasonable for us to

speculate that the RhoA/ROCK pathway was activated in LPA-incubated cancer cells. Indeed, in this study, we found RhoA/ROCK signaling pathway was activated after LPA incubation, along with increased invasion ability in MCF-7 cells. MMPs that is produced and secreted by cancer cells take responsibility for invasion and metastasis of malignant cancers. By degrading extracellular matrix (ECM), cancer cells could infiltrate through surrounding tissues to generate new lesions [37]. More importantly, MMPs could break down base membrane of blood and lymphatic vessels, facilitating cancer cells invade through vessel wall and resulting in distant organ and lymph node metastasis. MMP2 and MMP9 showing enzymatic activity of collagenase are typical members of MMPs family and generally considered as biomarkers for cancers [38]. Several studies indicated that the expression of MMPs was associated with RhoA/ ROCK signaling [39, 40]. In this study, we found that LPA incubation induced activation of RhoA/ROCK/MMPs

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signaling; RhoA silencing dramatically down-regulated ROCK expression and subsequently expressions of MMP2 and MMP9 in LPA-incubated MCF-7 cells. This result indicated that RhoA/ROCK/MMPs pathway was involved in LPA-induced invasion. Curcumin is considered one of the major bioactive components of Curcuma logna. Previous studies revealed curcumin’s inhibitory effects against proliferation, invasion and metastasis of various human cancers [41]. In this study, in LPA-incubated MCF-7 cells, curcumin still exerted anticancer pharmacological activity. Specifically, we found that curcumin at concentrations above 45 lmol/L inhibited proliferation of LPA-incubated MCF-7 cells. Furthermore, we also found that curcumin impaired LPA-induced invasion of MCF-7 cells in a concentration-dependent manner. Concurrently, curcumin inhibited activation of RhoA/ ROCK/MMPs pathway in a concentration-dependent manner. These results suggested that inhibition of RhoA/ ROCK/MMPs pathway was one of the mechanisms of curcumin effect against LPA-induced invasion.

6.

7.

8.

9.

10.

11.

12.

Conclusions 13.

1.

RhoA/ROCK/MMPs pathway activation is involved in LPA-induced invasion in MCF-7 cells. Curcumin inhibited LPA-induced invasion in MCF-7 cells by attenuating RhoA/ROCK/MMPs pathway.

14.

Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant No. 81171397).

15.

The authors declare that they have no conflict

16.

2.

Conflict of interest of interest.

17.

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