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 Table of Contents  
ORIGINAL ARTICLE
Year : 2014  |  Volume : 10  |  Issue : 4  |  Page : 991-997

Carvedilol suppresses migration and invasion of malignant breast cells by inactivating Src involving cAMP/PKA and PKCδ signaling pathway


Department of Breast Surgery, The Second Hospital of Shandong University, Jinan, Shandong, China

Date of Web Publication9-Jan-2015

Correspondence Address:
Fu Qinye
The Department of Breast Surgery, The Second Hospital of Shandong University, Jinan, Shandong 250033
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.137664

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 > Abstract 

Context: Carvedilol (CAR) can inhibit cell growth and induce cell apoptosis in breast cancer in vitro. But it is still not known whether CAR affects the migration and invasion of breast cancer cells.
Aims: To investigate the effects of CAR on migration and invasion of breast cancer cells and its corresponding signal pathways.
Settings and Design: Firstly, the invasive potential of breast cancer cells were investigated after incubation with CAR and/or norepinephrine (NE). If the invasive potential of breast cancer cells were inhibited by CAR, then the signal pathways related to migration and invasion were detected, such as Src, cyclic adenosine monohposphate (cAMP)/protein kinase A (PKA), etc.
Subjects and Methods: Membrane invasion culture system (MICS) chamber was used to measure the invasive and migratory potential of breast cancer cells. Western blot analysis and small interfering RNA (siRNA) transfection experiment were employed to determine the signal pathway adopted by CAR in suppressing migration and invasion of MDA-MB-231 and MCF-7 cells. cAMP-Glo and PKCδ kinase activity assay kit were used to measure cAMP and PKCδ activity, respectively, according to the manufacturer's instructions.
Statistical analysis used: Statistical differences between the mean values of control and experimental groups were determined using two-tailed, unpaired Student's t-tests.
Results: CAR significantly decreased the potential of migration and invasion of breast cancer cells. CAR inhibited Src activation in MDA-MB-231 and MCF-7 cells through blocking beta or alpha adrenergic receptor (ADR), respectively. Furthermore, CAR suppressed the Src activation through different signaling pathways. Under treatment of CAR, cAMP/PKA-Src pathway was inhibited in MDA-MB-231 cells; but in MCF-7 cells, CAR mainly inhibited the PKCδ-Src pathway.
Conclusions: CAR was an anti-metastatic agent, which targets Src involving cAMP/PKA or PKCδ pathway in malignant breast cells.

 > Abstract in Chinese 

卡维地洛通过阻止Src相关cAMP/PKA和PKCδ信号通路来抑制恶性乳腺癌细胞的侵袭和迁移

摘要

背景:卡维地洛(CAR)在体外可抑制乳腺癌细胞生长并诱导细胞凋亡。但它是否影响乳腺癌细胞的侵袭和迁移仍是未知的。


目的:研究卡维地洛对乳腺癌细胞的侵袭和迁移及其相应的信号通路的影响。

背景设计:首先,评估了卡维地洛和/或去甲肾上腺素(NE)孵育后的乳腺癌细胞的侵袭能力。如果乳腺癌细胞的侵袭能力被卡维地洛抑制,就对迁移和侵袭相关的信号通路进行检测,如Src,环磷酸腺苷(cAMP)/蛋白激酶A(PKA)等。


对象和方法:膜侵袭培养系统(MIC)室用来测定乳腺癌细胞的侵袭和迁移的潜力。蛋白质印迹分析和小干扰RNA(siRNA)转染实验被用来确定卡维地洛采用的抑制MDA-MB-231和MCF-7细胞侵袭的信号通路。根据制造商的说明,用cAMP-Glo和PKCδ激酶活性检测试剂盒分别测定cAMP及PKCδ活性。


统计分析:采用双尾法测定对照组和实验组的平均值之间的差异,非配对t检验。


结果:卡维地洛明显降低乳腺癌细胞的侵袭和迁移的潜力。卡维地洛抑制MDA-MB-231和MCF-7细胞中Src的激活分别通过阻断β或α-肾上腺素能受体(ADR)。此外,卡维地洛抑制Src通过激活不同的信号通路。在卡维地洛治疗下,MDA-MB-231细胞的cAMP/PKA-Src通路被抑制;但在MCF- 7细胞,卡维地洛主要抑制PKCδ-Src通路。


结论:卡维地洛是一种抗肿瘤药,主要作用于恶性乳腺癌Src相关的cAMP/PKA 或 PKCδ信号通路。



关键词:卡维地洛,恶性乳腺细胞,迁移,信号通路,SRC


Keywords: Carvedilol, malignant breast cell, migration, signal pathway, Src


How to cite this article:
Dezong G, Zhongbing M, Qinye F, Zhigang Y. Carvedilol suppresses migration and invasion of malignant breast cells by inactivating Src involving cAMP/PKA and PKCδ signaling pathway. J Can Res Ther 2014;10:991-7

How to cite this URL:
Dezong G, Zhongbing M, Qinye F, Zhigang Y. Carvedilol suppresses migration and invasion of malignant breast cells by inactivating Src involving cAMP/PKA and PKCδ signaling pathway. J Can Res Ther [serial online] 2014 [cited 2019 Nov 20];10:991-7. Available from: http://www.cancerjournal.net/text.asp?2014/10/4/991/137664


 > Introduition Top


Neurotransmitters play important role in promoting tumor growth and progression. [1],[2] Psychological factors such as stress and chronic depression activate sympathetic nerve fibers to release neurotransmitter, especially norepinephrine (NE), into the local tissue microenvironment. The tumor microenvironment is an important determinant of cancer progression, and NE can modulate multiple cellular functions important for cancer progression.

Breast cancer cells express alpha and/or beta adrenergic receptors (ADRs), and excessive NE might promote proliferation and metastasis of breast cancer cells, but ADR blocker can inhibit such promoting effect. [3],[4] As a whole, breast cancer tissue is a complex of different types of breast cancer cells which express different level of alpha or beta ADR. So it is impossible for selective adrenergic blocker (for example, propranolol) to act on all types of breast cancer cells. [3],[5] Carvedilol (CAR), a nonselective adrenergic blocker, was primarily designed to treat cardiovascular disease by blocking alpha and beta ADRs. [6] Preclinical study indicated that CAR could inhibit cell growth and induce cell apoptosis in some cancer in vitro. [7],[8] But it is still not known whether CAR affects the migration and invasion of breast cancer cells.

Src, a membrane-associated non-receptor tyrosine kinase, plays a crucial role in malignant cell invasion and migration. [9],[10] Deregulation and increased activity of Src have been observed in multiple human malignancies. In vitro, inhibitors of Src decreased phosphorylation of multiple Src substrates that affects migration and invasion of malignant breast cells. [11] Recent researches indicate that beta adrenergic activation leads to Src phosphorylation through beta adrenergic/cyclic adenosine monohposphate (cAMP)/protein kinase A (PKA) signal pathway. [12] Activation of alpha adrenergic could regulate Stat3 activity through Src phosphorylation, but the role of cAMP/PKA signal pathway in Src phosphorylation mediated by alpha adrenergic is not well known. [13]

Although CAR can block alpha and beta ADRs to treat cardiovascular disease, and also inhibit cancer cell proliferation in vitro, [7],[8] it is not well-understood whether CAR can inhibit Src phosphorylation through blocking alpha and beta ADRs, leading to inhibition of migration and invasion of breast cancer.

In this study, we intended to detect whether CAR could suppress migration and invasion of breast cancer, and if this is indeed the case, what role does the adrenergic/cAMP/PKA/Src signal pathway play? We performed a series of experiments to testify the infiltration-inhibiting effect of CAR and its signal pathway. Our results show that CAR inhibits these properties of malignant breast cells involved targeting cAMP/PKA- and PKCδ-Src pathways.


 > Subjects and methods Top


Cells and drugs

Human breast cancer cell lines, MDA-MB-231 and MCF-7 were purchased from Shanghai Institute of Cell Biology (Shanghai, China) and were cultured with Dulbecco's modified Eagle's medium (DMEM). CAR and NE were purchased from Sigma (St Louis, MO). These agents were prepared as 50 μM stock solutions stored at 4°C in double distilled water.

Cell culture

MCF-7 and MDA-MB-231 cell lines were routinely cultured in phenol red free DMEM. The culture media were supplemented with 10% fetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The two cell lines were routinely maintained in culture at 37°C and 5% CO 2 and regularly screened to ensure the absence of mycoplasma contamination. Cells in culture plate were harvested using 0.05% trypsin (Sigma Chemical Co.) and centrifuged after the addition of DMEM for trypsin inactivation and then resuspended in culture medium. Following trypan blue exclusion assay, breast cancer cells were planted in six-well culture plates at a concentration of 5 × 10 5 cells/well with 100% vitality.

Reagents

DMEM, fetal bovine serum, glutamine, antibiotics, and the Lipofectamine plus™ reagent and Lipofectamine™ 2000 reagent were purchased from Invitrogen (Carlsbad, CA). Dibutyryl-cAMP (dbcAMP), trypan blue, crystal violet, and Protein A-Sepharose were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies against Src, SrcS 17 ,alpha1 and beta2 ADR, and PKCδ were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary horseradish peroxidase-conjugated antibodies were purchased from BioSource International (Camarillo, CA). Fluorescein Isothiocyanate (FITC)-conjugated rabbit anti-mouse IgG antibody was obtained from Dako (Dako A/S, Glostrup, Denmark). The enhanced chemiluminescence (ECL) kit was from GE Amersham Biosciences. cAMP-Glo Assay kit and PKCδ kinase activity kit were from Promega Co (Madison, WI).

Migration and invasion assay

In this study, the membrane invasion culture system (MICS) chamber was used to measure the invasive and migratory potential of breast cancer cells. When required, CAR and/or NE were added at the start of the experiment. The MICS assay was performed as previously described. [14] Briefly, single cell suspension was seeded into the upper chamber at a concentration of 1 × 10 5 cells per chamber. After incubation with CAR and/or NE for indicated time, cells were collected, fixed, stained, and counted by light microscopy. For invasion assay, the upper chamber of hanging cell culture insert was precoated with Matrigel (BD Biosciences, USA). The following procedures were the same as that of migration assay.

Cell cycle analysis

The effects of drugs on MCF-7 and MDA-MB-231 cell cycle were examined using a DNA analysis kit (BD Pharmingen, USA) according to the manufacturer's instructions. Briefly, breast cancer cells were incubated at a cell density of 5 × 10 5 cells/ml in the presence of CAR/NE applied separately or in combination for 48 h. Cells were then harvested, centrifuged, washed, and resuspended in buffer for 5 min at room temperature. Trypsin was added and samples were incubated for 20 min at room temperature. After the addition of trypsin inhibitor in buffer, cells were incubated with propidium iodide (PI), in the dark, for 20 min at 4°C. Lastly, flow cytometric analysis was performed using a FACScan flow cytometer (FACS Diva, Beckton-Dickinson, USA) and the fluorescence intensity data were detected using the instruments' operating software (CellQuest; BD Pharmingen). The percentages of breast cancer cell population in G 0 /G 1 , S, and G2/M phases were determined by the ModFit cell cycle analysis program.

Apoptotic cell death analysis

Externalization of phosphotidylserine (the manifestation of apoptosis) was studied by the Annexin V-binding assay. Briefly, cells were washed twice with phosphate-buffered saline (PBS) and resuspended by binding buffer containing 0.01 M HEPES, 0.14 mM NaCl, and 2.5 mM CaCl 2 . A cell suspension (1 × 10 5 cells/100 μl) in binding buffer was incubated with 5 μl of FITC-labeled Annexin V (BD Pharmingen) dye, and PI for 15 min, in the dark, at room temperature. After incubation, the PI fluorescence and Annexin V were measured in a BD FACSCalibur and analyzed with the instruments' operating software (CellQuest; BD Pharmingen). Data collection and analysis were processed with CellQuest and WinMDI programs.

Western blot analysis

Cell lysates were prepared by washing cells with PBS and incubating them for 10 min at 4°C in modified radioimmunoprecipitation assay lysis buffer. Cells were scraped from plates and centrifuged for 20 min at 4°C, and the supernatant was collected. Protein concentrations were determined using a bicinchoninic acid (BCA) reagent kit (Pierce), and 40 μg of whole cell lysates were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples were transferred to a nitrocellulose membrane by wet electrophoresis (BioRad), blocked with 5% fat-free dry milk in PBS for 1 h at room temperature, and incubated with corresponding primary antibody overnight at 4°C. Primary antibody was detected with anti-rabbit immunoglobulin G (IgG; Amersham Biosciences, Piscataway, NJ). The used antibodies include anti-Src S17 ( 1:400, BD), anti-Src (1:400, BD), anti-alpha1 (1:100 Santa Cruz), and anti-beta2 (1:100 Santa Cruz). Following three washes with PBS, the membrane was incubated with corresponding secondary antibodies and visualized with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, IL).

Small interfering RNA (siRNA)

Chemically synthesized, double-stranded, ON-TARGETplus SMARTpool siRNAs targeting beta2-ADR, alpha1-ADR, and PKCδ were purchased from Dharmacon (Chicago, IL). ON-TARGETplus non-targeting siRNAs were used as a control. MDA-MB-231 and MCF-7 cells were transfected with Lipofectamine 2000 according to manufacturer's instructions using 100 nm siRNA targeting beta2 ADR, alpha1 ADR, and PKCδ using Dharmafect-1 transfection reagent diluted in antibiotic-free culture medium. Cells were exposed to treatments, 48 h later. Transfection efficacy was verified by Western blotting analysis of beta2 ADR, alpha1 ADR, and PKCδ expression.

Determination of cAMP synthesis and PKCδ activity

After treatment with CAR or NE, cellular concentration of cAMP was measured by cAMP-Glo Assay (Promega) according to the manufacturer's instructions. PKCδ activity was assessed using a PKCδ kinase activity assay kit (Assay Designs, Inc. MI, USA). The kinase activity kits are based on a solid-phase enzyme-linked immunosorbent assay (ELISA) that uses a specific synthetic peptide as substrate for PKCδ, and a polyclonal antibody that recognizes the phosphorylated form of the substrate.

Statistical analysis

Analyses were performed with the Statistical Package for Social Sciences (SPSS)/PC version 16.0 statistical packages. Data are presented as the mean ± standard deviation (SD). Statistical differences between the mean values of control and experimental groups were determined using two-tailed, unpaired Student's t-tests. Data were considered to be statistically significant when P < 0.05 (*) and P < 0.01 (**).


 > Results Top


CAR suppressed migration and invasion induced by NE in breast cancer cells

We employed MICS chamber assays to investigate whether cell migration and invasion induced by NE were affected by CAR. We exposed cells to 10 μM of NE for 24 h, compared with untreated cells, NE treatment significantly increased migration and invasion of MDA-MB-231 cell. However, under 24 h treatment of NE (10 μM) and CAR at the indicated concentrations (0.1, 1.0, and 5.0 μmol/L), fewer MDA-MB-231 cells were found to infiltrate the Matrigel-precoated membranes than that of NE treated cells [Figure 1] and [Figure 2]. Interestingly, CAR also inhibited migratory and invasive properties of MCF-7 cells induced by NE under 24 h treatment of CAR (0.1, 1.0, and 5.0 μmol/L) [Figure 1] and [Figure 2]. The invasion-inhibiting effect of CAR on MDA-MB-231 and MCF-7 cells were time- and concentration-dependent [Figure 3].
Figure 1: Inhibitory effect of CAR on migration and invasion of MDA-MB-231 and MCF-7 cells. (a) 0.1, 1.0, or 5.0 μM CAR was added to the culture of MDA-MB-231 and MCF-7 cells for 24 h with or without 10 μM of NE; the number of cells that passed through the membranes was counted in three separate fields (×100). The average values were shown with ±SD. Compared with control, *P<0.05 and **P<0.01. (b) The experiments were performed in the same conditions except for the use of matrigel-precoated membranes instead of uncoated ones. Compared with control, *P<0.05 and **P<0.01. CAR = Carvedilol, NE = norepinephrine, SD = standard deviation

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Figure 2: 1 × 105 breast cancer cells were seeded into the upper chamber of 24-well culture plate, and infiltrating cells were photographed when cultivated with indicated concentrations of CAR for 24 h with or without 10 μM of NE

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Figure 3: A 1.0 μM of CAR was added to the cell culture for 12, 24, 36, and 48 h with or without 10 μM of NE, the number of cells that passed through the membranes was counted in three separate fields (×100). The average values were shown with ±SD. Compared with 12 h, *P<0.05 and **P<0.01

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In order to exclude the effect of cytotoxicity caused by CAR on invasive properties of MDA-MB-231 and MCF-7 cells, we measured the cell proliferation index, cell cycle distributions, and apoptotic cell percentages after CAR treatment. We found that CAR showed no significant cytotoxicity, apoptosis-stimulating activity, or cell cycle arrest effect on the two cell lines at the above concentrations.

These data demonstrated that, within a dose range where apoptotic and cell cycle arrest event did not occur, CAR was able to suppress the migration and invasion of breast cancer cells in a dose- and time-dependent manner.

CAR-suppressed Src activation in breast cancer cells

Src can be activated in response to adrenergic signaling and Src phosphorylation is essential for cAMP signaling in cancer cells. [12],[15] Given pSrc S17 levels increased markedly following exposure to NE, and Src s17 phosphorylation plays an essential role in the migration and invasion of breast cancer cells, [12],[16] we used Western blot analysis to determine whether CAR affected NE-mediated Src activation in breast cancer cells. The results revealed that MDA-MB-231 cell cultivated with NE (10 μM) showed elevated level of Src S17 phosphorylation (at 12 h, nearly three-fold), compared with control cells. When cultivated with NE (10 μM) for 24 h, level of Src S17 phosphorylation in MDA-MB-231 cells was lower than that for 12 h. When 1.0 μM of CAR was added to the cultivation media, Src S17 phosphorylation level induced by NE was suppressed by greater than 40% at 12 h cultivation [Figure 4]a. In accordance with the results in MDA-MB-231 cells, similar results were also found in MCF-7 cells [Figure 4]b, but the Src S17 phosphorylation level was less than that in MDA-MB-231 cell under the same cultivating time and drug concentration.
Figure 4: CAR suppress SrcS17 phosphorylation induced by NE on breast cancer cells. Breast cancer cells were treated with 10 μM NE/ CAR (1.0 μM) for 6, 12, and 24 h, and Western blot analysis was employed to probe pSrcS17 expression (top panel), and GAPDH was selected as endogenous control. Quantitative analysis of pSrcS17 band intensity was performed with densitometry (bottom panel). (a) MDA-MB-231 and (b) MCF-7. The average values were shown with ± SD. Compared with NE group in the same incubation time, *P<0.05 and **P<0.01. GAPDH = Glyceraldehyde 3-phosphate dehydrogenase

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CAR inhibited Src activation by blocking alpha1/beta2 ADR in MCF-7 and MDA-MB-231 cells

Considering the fact that different types of breast cancer cells express different levels of alpha or beta ADRs. [3],[4],[5] It is necessary to examine the specificity of ADRs in Src inactivation mediated by CAR. In accordance with the results of literature, [3],[4],[5] we found that MCF-7 cells expressed mainly alpha ADR (especially alfa1), and MDA-MB-231 cells mainly beta ADR (especially beta2). We utilized beta2 ADR-targeted siRNA to transfect MDA-MB-231 cells. After confirming no beta2 ADR expression in MDA-MB-231 cells, we treated these cells with NE alone or followed with CAR for indicated time. The results showed that NE treatment marginally increased Src S17 phosphorylation in MDA-MB-231 cells, but treatment with NE followed CAR completely inhibited Src S17 phosphorylation, as compared with untransfected cells [Figure 5]a. Similar results were also found in MCF-7 cells after treatment of NE or followed with CAR, which transfected with alfa1 ADR-targeted siRNA [Figure 5]b. These data suggest that CAR can inhibit Src S17 phosphorylation in MDA-MB-231 and MCF-7 cells through blocking both alpha and beta ADR.
Figure 5: CAR inhibited Src activation by blocking alpha/beta ADR in breast cancer cells. The data are presented as mean ± SD of three independent experiments. Compared with pcDNA group, *P<0.05. ADR = Adrenergic receptor

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CAR suppressed the Src activation through different signaling pathway in MDA-MB-231 and MCF-7 cells

The cAMP-dependent PKA induced the phosphorylation of Src had been reported in multiple cancer cell types. [17],[18],[19],[20] We next performed a series of experiments to interpret whether cAMP/PKA was involved in the signaling pathway of CAR-mediated Src inactivation. cAMP-Glo Assay indicated that, compared with untreated cells, cAMP activity increased significantly in MDA-MB-231 cells treated with NE for 6 h. While MDA-MB-231 cells were incubated with CAR and NE simultaneously for 6 h, the cAMP activity was not found to exceed that of untreated cells [Figure 6]a. However, when 50 μM dbcAMP (PKA agonist) was added into the cultivation of CAR and NE, rapid Src s17 phosphorylation was shown in MDA-MB-231 cells [Figure 6]c.
Figure 6: NE-mediated cAMP/PKA and PKCδ signaling activation was suppressed by CAR in breast cancer cells. cAMP = Cyclic adenosine monohposphate, PKA = Protein kinase A

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However, cAMP activity was not found to change in MCF-7 cells after treatment with NE or CAR, and addition of dbcAMP in the cultivation had no effect on Src s17 phosphorylation [Figure 6]b. Obviously, Src inactivity mediated by CAR is not via cAMP/PKA signal pathway in MCF-7 cells. The classical mechanism to mediate alpha ADR effect in different cells is that activation of alpha ADR by catecholamine leads to activation of phospholipase C, which mediates the activation of protein kinase C (PKC). [21] Activated PKC is capable of interacting with and activating several substrates including SRC/janus kinase (JAK) and PI-3K/AKT. [22],[23] Recent research showed that PKCδ played important role in Src phosphorylation in malignant cells. [24],[25] So, we employed PKCδ kinase activity kit to detect PKCδ activity in MCF-7 cells treated by NE and CAR. Our data in this study documented that PKCδ activity increased in MCF-7 cells treated by NE (nearly two-fold), but CAR could inhibit PKCδ activity induced by NE [Figure 6]d. PKCδ-targeted siRNA experiment also confirmed that PKCδ plays important role in Src S17 phosphorylation in MCF-7 cell [Figure 6]e.

These results strongly indicate that Src is a downstream target of cAMP/PKA and PKCδ signaling pathway, and CAR suppressed Src activation involving cAMP/PKA or PKCδ signal pathway in MDA-MB-231 or MCF-7 cells.


 > Discussion Top


0 Clinical and animal studies now support the notion that psychological factors might promote tumor growth and progression. [26],[27] Psychological factors such as stress and chronic depression activate sympathetic nerve fibers to release neurotransmitters, especially norepinephrine (NE). Neurotransmitters play important role in promoting malignant growth and progression. [28] Recent studies indicated that neurotransmitter receptors, such as alpha and beta ADRs exist in many tumor cells including breast cancer. Excessive secretion of NE might promote invasion and metastasis in breast cancer cells. [29]

CAR is a cardiovascular drug licensed for the treatment of chronic heart failure. As a nonselective adrenergic blocker, CAR can block simultaneously alpha and beta ADRs in cardiovascular system. In addition, CAR has the potential of anti-neoplastic effect in breast cancer and glioma. [7],[8] Considering the inhomogeneity of ADR subtypes in breast cancer, [3],[4],[5] in our experiment, we applied CAR to treat MCF-7 and MDA-MB-231 cell lines in which the expression of alpha and beta ADR subtypes is discrepant. In this study, we originally provided evidences that CAR could inhibit migration and invasion of the two breast cancer cell lines. We selected 0.1, 1.0, and 5.0 μM CAR to perform the experiments, because higher dose of CAR (50 μM) showed cytotoxicity to breast cancer cells. [7] Our data demonstrated that, within a dose range where inhibition of cell proliferation did not occur, CAR was able to suppress the migration and invasion of breast cancer cells even at a low dose.

Src, a membrane-associated non-receptor tyrosine kinase, plays a prominent role in a variety of cellular signal transduction pathways that are involved in cell motility, adhesion, and invasion. [30],[31] Studies using an inducible dominant-negative Src demonstrated that Src suppression significantly reduced migration, attachment, and spread in breast cancer cells. Src activity is increased in invasive compared with noninvasive breast cancer cell, and Src inhibitor could decrease cell motility and invasiveness. [16],[32] Now Src has become a novel therapeutic target in treatment of breast cancer metastases. [33],[34] To investigate whether Src plays a role in migration inhibition mediated by CAR in breast cancer cells, we employed Western blot to determine Src phosphorylation level. The results showed that NE elevated level of Src S17 phosphorylation and CAR suppressed Src S17 phosphorylation in both MDA-MB-231 and MCF-7 cells. But the Src S17 phosphorylation level in MCF-7 cell was less than that in MDA-MB-231 cell. These data support the important role of Src in CAR-mediated inhibition of migration and invasion of breast cancer cells.

CAR has the ability of suppressing Src S17 phosphorylation in both MDA-MB-231 and MCF-7 cells, but these two cell lines express different levels of alpha and beta ADR. [3],[4] To examine the specificity of ADRs in mediating CAR-induced Src S17 phosphorylation, we utilized siRNA experiments to clarify the receptor types involved in CAR-mediated Src inactivation. We found that NE treatment marginally increased Src S17 phosphorylation in both MDA-MB-231 and MCF-7 cells that transfected beta2-ADR or alfa1-ADR siRNA, and treatment with NE followed CAR completely inhibited Src S17 phosphorylation in the two transfected cell lines. These data suggest that NE can activate alpha ADR in beta ADR siRNA transfected MDA-MB-231 cells, and resulting in Src S17 phosphorylation marginally. Because of weak expression of alpha1-ADR in MDA-MB-231 cells, Src S17 phosphorylation mediated by NE was unconspicuous. The result was similar in alpha1-ADR siRNA transfected MCF-7 cells. This means CAR can inhibit migration and invasion of MDA-MB-231 and MCF-7 cells through blocking both alpha and beta ADR.

Ligation of beta receptors by norepinephrine stimulates adenylyl cyclase synthesis of cAMP, and cAMP regulates a diverse array of cellular processes via activating PKA. PKA regulates a wide variety of cellular processes, including activate Src. [12],[17] To test whether cAMP/PKA was involved in CAR-mediated Src inactivation, we applied cAMP-Glo assay to investigate the cAMP activity. The data revealed that cAMP activity increased in MDA-MB-231 cells treated by NE, but CAR inhibited the cAMP activity mediated by NE. Src S17 phosphorylation was not altered when MDA-MB-231 cells were incubated with CAR and NE, simultaneously. However, when dbcAMP was added in the cultivation, rapid Src S17 phosphorylation was found. These results strongly indicate that Src is a downstream target of cAMP/PKA signaling pathway, and CAR suppressed Src activation involving cAMP/PKA signaling pathway in MDA-MB-231 cells.

As to MCF-7 cell, cAMP activity did not increase after treatment with NE. Even with addition of dbcAMP in the cultivation, we did not find increase in Src S17 phosphorylation. It means that NE takes no effect on PKA activation in MCF-7 cell. Given the important role of PKCδ in Src phosphorylation in malignant cells, [24],[25] we employed PKCδ kinase activity kit to detect PKCδ activity in MCF-7 cells after treatment with NE and CAR. It showed in our study that PKCδ activity increased in MCF-7 cells treated by NE, and PKCδ siRNA or CAR could inhibit NE-mediated PKCδ activation, resulting in decreased Src S17 phosphorylation. This suggests that the signal pathway of Src S17 phosphorylation was different in MCF-7 and MDA-231 cells.

Taken together, we designed and performed experiments to prove that CAR-mediated suppression of migration and invasion of breast cancer cells involved inactivating cAMP/PKA-Src and PKCδ-Src signaling pathways. But further experiments are necessary to identify the possible down-stream molecules affected by Src that carry out the migration-inhibiting action mediated by CAR, such as STAT3, CRE, phosphatidylinositol 3 kinase (PI3-kinase), focal adhesion kinase (FAK), etc. [13],[35],[36] Before the CAR became a promising agent for breast cancer therapy and applied for clinical treatment, it is necessary to conduct experiments to verify the efficiency of CAR in vivo.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]



 

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