HER-2/EGFR, the major targets for anti-metastasis effect of tetraarsenic oxide on SKBR3 breast cancer cells   
Qiuyu Liu1,2, Illju Bae2, Linlin Qian2, Zenglin Lian3*
1. School of Pharmaceutical Sciences, Beijing University of Chinese Medicine, Beijing 100102, China
2. Chonjisan Biological Medicine Technology Development (Beijing) Co., Ltd., Beijing 100080, China
3. Institute of Biological Chinese Medicine, Beijing 101111, China      

 
Abstract: Breast cancer is one of the most common female malignant tumors in the world. Although many therapeutic methods for HER-2 positive breast cancer have been developed, the drug resistance and distant metastasis still remain. Tetraarsenic oxide (As4O6) has been demonstrated with an anticancer effect on squamous cell carcinoma and cervical cancer. However, there is no report about the relationship between As4O6 and HER-2 positive breast cancer. In the present study, we detected the inhibitory efficacy and mechanism of As4O6 on the migration and invasion of SKBR3 breast cancer cells using molecular biological methods. The wound-healing assay, matrigel migration assay, transwell invasion assay and cell adhesion assay were used to assess the migration, invasion and adhesion of SKBR3 cells intervened by As4O6. Meanwhile, the reverse transcription-PCR and western blotting were performed to investigate the mechanism of As4O6 on the migration and invasion of SKBR3 breast cancer cells. The results demonstrated that As4O6 could efficiently inhibit the migration and invasion of SKBR3 cells, the HER-2 positive breast cancer cells, and the adhesion of SKBR3 cells was decreased after As4O6 treatment. The mechanism revealed that As4O6 anticancer efficacy was related to HER-2/EGFR pathways. As4O6 exerted its inhibitory effects on migration and invasion in HER-2 positive breast cancer cells by regulating the factors (EGFR, HER-2, Akt, MMP-9) in HER2/ EGFR signaling pathway and other key molecules. In conclusion, the present study indicated that As4O6 inhibited the invasion and migration process of HER-2 positive breast cancer SKBR3 cells by negatively regulating the HER-2/EGFR-mediated signaling pathway. These data provided evidence that As4O6 might serve as potential anti-metastasis drug for clinical treatment of breast cancer.        
Keywords: HER-2 positive breast cancer; Tetraarsenic oxide; Migration; Invasion; Adhesion; Signaling pathway   
CLC number:R979.1                Document code: A                 Article ID: 10031057(2017)28708
 
 
1. Introduction
As a highly metastatic cancer, breast cancer accounts for high mortality rate every year[1,2]. The breast cancer cells have lower adhesiveness due to the loss of characteristics of normal cells, and they can spread the whole body through the blood or lymph system to form metastases[3]. Although many therapeutic methods has been developed for HER-2 positive breast cancer, such as chemotherapy with trastuzumab[4], the drug resistance and distant metastasis can not be avoided. Besides, the intravenous injection has side effects and security hidden danger. Therefore, it is vital to find a reliable and better therapeutic agent for the treatment of HER-2 positive breast cancer.
It has been shown that epidermal growth factor receptor (EGFR) and HER-2 play an important role in the development of the clinical target therapy[5]. Many strategies for targeting EGFR and HER-2 have been developed, but only several such strategies are really successful in clinical use. Therefore, it is necessary to explore a novel efficacious therapeutic agent targeting EGFR and HER-2 for the treatment of breast cancer.
As an arsenic compound, tetraarsenic oxide (As4O6) is used not only in China but also in Korea as an anti-tumorsubstance for malignancies by external or internal application. In Korea, As4O6 has been used in folk remedy for cancer management since the late 1980s. The beneficial impact of As4O6 on radiotherapy sensitivity and combination therapy has been noted[6,7]. Besides, As4O6 regulates a variety of signaling events, including NF-κB signaling pathway and caspase-dependent apoptosis[8,9]. Only one report has shown that As4O6 enhances TNF-α-induced anticancer effects on MCF-7 human breast cancer cells in vitro by inhibiting NF-κB signaling pathway[10]. However,the mechanism of As4O6 on invasion and migration of HER-2 positive breast cancer cells remains largely unexplored. Therefore, we aimed to evaluate the inhibitory effect of As4O6 on HER-2/EGFR-mediated invasion and migration in SKBR3 breast cancer cells and elucidate the molecular mechanism involved in the present study.
2. Experimental
2.1. Materials and reagents
SKBR3 human breast cancer cells were purchased from the American Type Culture Collection (Manassas,VA, USA). SKBR3 cells were cultured in DMEM medium (Gibco, Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco-BRL, Grand Island, NY, USA), penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37 ºC in a humidified atmosphere of 95% air and 5% CO2. As4O6 was provided by the Chonjisan Institute (Seoul, Korea), and its purity was 99.99%. Rabbit monoclonal antibodies against β-actin, MMP-9, mTOR, p-mTOR, EGFR, p-EGFR, Akt, p-Akt, HER2 and p-HER-2 were supplied from Cell signaling Technology Inc. (Beverly, MA, USA).
2.2. Cell proliferation assay
Cell proliferation assay was performed to assess cell proliferation viability by MTT assay. SKBR3 cells wereseeded in 96-well plates at a density of 4×103 cells/well and treated with As4O6 at indicated concentrations for 48 h. Then, 20 µL of MTT (0.5 mg/mL) was added to each well and incubated at 37 ºC for 4 h. After incubation, the culture medium was discarded, 100 µL of dimethyl sulfoxide was added to dissolve the formazan crystals, then the absorption was determined at a wavelength of 570 nm.
2.3. Wound healing assay
Cells were plated in 6-well plates at a density of 5×105 cells/well overnight. Subsequently, the wound was created by scratching the cells with a sterile 10-µL pipette tip. Then, cells were washed three times with PBS and incubated with serum-free medium or serum-free medium containing As4O6 (0, 0.5, 1, 1.5, 2, 3 µmol/L) for 48 h. The inverted microscope was used to take photos at 0, 24, 48 and 72 h to observe cell migration, respectively.
2.4. Transwell invasion assay
Transwell invasion assays were performed using SKBR3 cells after As4O6 treatment. Cells were plated in 6-well plates at a density of 5×105 cells/well and incubated with serum-free medium containing As4O6 (0, 0.5, 1, 1.5, 2, 3 µmol/L) for 48 h. Cells were washed three times with PBS and digested into single cell suspension (1×105 cells/mL) containing 1% BSA. The top chamber was coated with matrigel (SKBR3: 30 µL serum-free medium, 1:7) before adding cell suspension. After incubation at 37 ºC for 48 h in the incubator, cells on the upper surface of membrane were wiped off with cotton swab. The migratory cells attaching to the outside bottom surface of the membranewere fixed with methanol and stained with 0.1% crystal violet for 15–20 min, respectively. Cells on the bottom surface of the membrane filter were counted in five randomly selected visual fields under an inverted microscope at 100× magnification. Data were presented as the average number of cells adhering to the bottom surface.  
2.5. Matrigel migration assay
Matrigel migration assays were performed using SKBR3 cells after As4O6 treatment. Cells were incubated in serum-free medium containing As4O6 (0, 0.5, 1, 1.5, 2, 3 µmol/L) for 48 h, washed three times with PBS and digested into single cell suspension (1×105 cells/mL) containing 1% BSA with trypsin. After incubation at 37 ºC for 48 h in the incubator, cells on the upper surface of membrane were wiped off with cotton swab. The migratory cells attaching to the outside bottom surface of the membrane were fixed with methanol and stained with 0.1% crystal violet for 15–20 min, respectively. Cells on the bottom surface of the membrane filter were counted in five randomly selected visual fields under an inverted microscope at 100× magnification. Data were presented as the average number of cells adhering to the bottom surface.
2.6. Cell adhesion assay
SKBR3 cells were plated in 6-well plates at a density of 5×105 cells /well. After overnight incubation, serum-free medium containing As4O6 (0, 0.5, 1, 1.5, 2, 3 µmol/L) was addedto the well, and cells were incubated for 48 h. Then, cells were washed three times with PBS and digested into single cell suspension (1×105 cells/mL), 0.5 mL cell suspension was added into 24-well plates per well. About 4 h later, cells were washed three times with PBS, fixed with methanol for 20 min, stained with 0.1% crystal violet for 15–20 min, washed three times with water and then air dried. Cells adhering to the 24-well plates were counted under the inverted microscope.
2.7. Reverse transcription-PCR (RT-PCR)
SKBR3 cells were plated in 6-well plates at a density of 5×105 cells /well. After overnight incubation, cells were incubated with medium containing As4O6 (0, 0.5, 1, 1.5, 2, 3 µmol/L) for 48 h. Then, cells were washed three times with PBS and prepared for RNA extraction. Total RNA was isolated using Trizol reagent (TaKaRa, Japan). The concentration and integrity of RNA were determined by absorbance at 260 nm and the values of OD260/OD280. Subsequently, 500 ng total RNA was reversely transcribed into cDNA following AMV reverse transcription kit instruction (TaKaRa, Japan). After reverse transcription reaction, 5 µL of the final cDNA was used for PCR reaction using Taq DNA polymerase (TaKaRa, Japan). The annealing temperature andcycles were as follows: 58 ºC, 30 cycles (β-actin); 58.3 ºC, 38 cycles (HER-2); 58.3 ºC, 38 cycles (EGFR); 58 ºC, 35 cycles (ICAM); 60.5 ºC, 45 cycles (MMP-9). β-Actin was used as the housekeeping gene. The amplicons were resolved on 1.5% agarose gels, followed by ethidium bromide staining. The image was observed and photographed using Gel Imaging System (ChampGel 5000, Beijing, China). The PCR primers were shown in Table 1. 

Table 1. Primer sequences. 


2.8. Western blotting
SKBR3 cells were washed three times with ice-cold PBS, and then lysed in RAPI lysis solution containing a phosphorylation and non-phosphorylation protease inhibitor mixture. Protein concentration was determinedwith BCA method. The amounts of lysates were resolved on sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. Subsequently, the membrane was blocked with 1× TBS containing 0.05% Tween-20 and 2% BSA at room temperature (25 ºC) for 1 h. After blocking, the membranes were incubated overnight at 4 ºC with the respective primary antibodies (ICAM-1, MMP-9, mTOR, p-mTOR, EGFR, p-EGFR, Akt, p-Akt, HER-2, p-HER2). The membranes were washed with 1× PBS and incubated with green fluorescencesecondary antibodies in the dark for 1 h at room temperature, and then the membranes were detected at 800 nm.
2.9. Statistical analysis
The experiments were conducted in triplicate, and SPSS 22.0 software was used for statistical analyses. The comparison between two groups was evaluated using Students t-test. The results were expressed as means±SD. Values of P<0.05 were considered statistically significant.
3. Results and discussion
3.1. The inhibitory effects of As4O6 on SKBR3 cell proliferation
To determinate the anti-tumor activity of As4O6 in SKBR3 breast cancer cells, the cells were treated with various concentrations of As4O6 (0–128 µmol/L) for 48 h. The inhibitory rate was significantly increased and OD values were significantly decreased with the concentration of As4O6. When it was 3 µmol/L, the inhibitory rate could reach 20%–30%. The results showed that cell growth was significantly inhibited by As4O6 in a dose-dependent manner (Fig. 1). 

   
Figure 1.
As4O6 suppresses SKBR3 cell proliferation. OD values (A) and cell inhibitory rate (B) were shown. 

3.2. Suppression of As4O6 on migration and invasion in SKBR3 breast cancer cells
To determine the effects of As4O6 on invasion and migration in SKBR3 cells, we detected the effects by the wound healing assay, transwell migration and invasion assay. When it was 24 h, the wound healing was not obvious. The wound healing rate was significantly increased in SKBR3 cells in a dose-and time-dependent manner when it was 48 h or 72 h (Fig. 2). Compared with the control group, As4O6 treatment exhibited obvious inhibitory effects on the migration of SKBR3 cells. In order to further identify the migration of SKBR3cells, we performed the transwell migration assay (Fig. 3A). As the As4O6 concentration was increased, the number of cells passing through the membrane was decreased. Moreover, 3 µmol/L of As4O6 significantly reduced the migration of SKBR3 cells from the upper chamber to the outside lower chamber compared with the control group (Fig. 3B). 


Figure 2. As4O6 suppresses SKBR3 cell migration. The wound was created by scratching the cells with a sterile 10-µL pipette tip and incubated with As4O6 (0, 0.5, 1, 1.5, 2, 3 µmol/L) for 72 h. **P<0.01, compared with the 0 µmol/L group.


Figure 3.
As4O6 significantly decreases cell migration and invasion in SKBR3 breast cancer cells. The ability of migration (B) and invasion (C) in SKBR3 cells were detected by the transwell migration and invasion assay after As4O(0, 0.5, 1, 1.5, 2, 3 µmol/L) treatment. *P<0.05, **P<0.01, compared with the 0 µmol/L group. 

   The invasion of SKBR3 cells before and after treatme
nt was assessed by the transwell assay (Fig. 3A). We found that As4O6 had an inhibitory effect, and there was a significant trend that 3 µmol/L of As4O6 performed efficiency on SKBR3 cells (Fig. 3C). These results indicated that As4O6 played a vital role in inhibiting invasion and migration of SKBR3 cells.  
3.3. The inhibitory effect of As4O6 on adhesion of SKBR3 breast cancer cells
Loss of adhesion of the malignant cells is one of the fundamental pathways to promote tumor cell migration and invasion. To identify the adhesion of SKBR3 cells after As4O6 treatment, we seeded the cells into 6-well plates and treated by As4O6 for 48 h. Then the starved cells were digested into single cell suspension, seeded into 24-well plates and incubated for 4 h. The cell adhesion was observed under the inverted microscope. Data showed that As4O6 lowered cells adhesion of breast cancer cells in a dose-dependent manner (Fig. 4A). The number of adhering SKBR3 cells treated with 3 µmol/L of As4O6 was 2-fold higher compared with the control group (Fig. 4B). The resultssuggested that As4O6 had a better inhibitory effect on adhesion of SKBR3 breast cancer cells.


Figure 4.
The inhibitory effect of As4O6 on the adhesion of SKBR3 cells. The SKBR3 cells were incubated with As4O6 (0, 0.5, 1, 1.5, 2, 3 µmol/L) for 48 h, digested into single cell suspension and then incubated in 24-well plates for 4 h. **P<0.01, compared with the 0 µmol/L group.  
 
3.4. The signaling pathway is regulated by As4O6 in SKBR3 breast cancer cells
Metastasis is the important factor associated with poor prognosis after breast cancer treatment. To identifythe underlying mechanism of As4O6-inhibited migration, invasion and adhesion of SKBR3 breast cancer cells, we determined the expressions of MMP-9 and ICAM-1 at the transcription level. The data showed that As4O6 could decrease the transcriptional levels of EGFR, HER-2, MMP-9 and ICAM-1 in SKBR3 breast cancer cells. Especially when As4O6 was 3 µmol/L (Fig. 5), the inhibitory effect was obvious. The results demonstratedthat As4O6 could exert an inhibitory effect on the transcription of the key factors in the HER2/EGFR pathway. 


Figure 5.
Effect of As4O6 on the transcriptional levels of EGFR, HER-2, MMP-9 and ICAM-1 in SKBR3 breast cancer cells. The SKBR3 cells were incubated with As4O6 (0, 0.5, 1, 1.5, 2, 3 µmol/L) for 48 h, then total RNA was extracted from SKBR3 breast cancer cells, and RT-PCR reaction was immediately conducted. 

   To further clarify the mechanism, we utilized western
blotting to determine the expressions of associated proteins. Data showed that As4O6 could significantly decrease the protein expression of ICAM-1 when As4O6 was 3 µmol/L. However, MMP-9 expression was not be detected, which could be explained by the low expression of MMP-9 caused. Moreover, we detected the upstream targets of MMP-9[1113], including the protein expressions of phosphorylated Akt (p-Akt) and phosphorylated mTOR (p-mTOR). However, the protein level of p-mTOR remained barely changed with the increase in the concentration of As4O6, and p-Akt appeared an increasing trend compared with the control (Fig. 6). Considering the importance of EGFR and HER-2, the vital and upstream targets in HER-2 positive breast cancer, we next determined the protein expressions of phosphorylated EGFR and phosphorylated HER-2, and both of them were decreased. Combined with electrophoresis results, it indicated that As4O6 had an effect on HER-2/EGFR signaling pathway though regulating the upstream targets, EGFR, HER-2, andthe downstream targets, MMP-9 and ICAM-1. However, the signaling pathway was not regulated by mTOR, while it was mainly regulated though HER-2/EGFR-Akt-MMP-9. As4O6 might be a potential agent in the inhibitory process of migration and invasion in breast cancer clinic therapy. 


Figure 6.
As4O6 afects the protein levels of phospho-HER-2, phospho-EGFR, phospho-mTOR, phospho-Akt, MMP-9 and ICAM-1 in SKBR3 breast cancer cells. We used western blotting to detect the expressions of proteins related to the HER2/ EGFR signaling pathway, migration, invasion and adhesion.  
4. Conclusions
In this study, we demonstrated that As4O6 could effectively, at least partially, reduce the expression of invasion- and migration-associated proteins and inhibited metastasis by negatively regulating the HER-2/EGFR-Akt-MMP-9 signaling pathway and other associated key factors. However, there were certain limitations in the present study. Firstly, we did not evaluate animal models, so the further study should consider the function of As4O6 on animal models. Secondly, the study only determined the anti-metastatic effect of As4O6 on HER-2 positive breast cancer cells by negatively regulating EGFR/HER-2-Akt-MMP-9 signaling pathway, and we did not assess other alternative signaling pathways. Hence, it was necessary to resolve these problems in further studies. To the best of our knowledge, this study provided the first evidence that As4O6 inhibited metastasis of HER-2 positive SKBR3 breast cancer cells by repressing EGFR/HER2-Akt-MMP-9 signaling pathway. Based on all above-mentioned data, As4O6might serve as a potential inhibitor of tumor metastasisin future clinical therapy for the breast cancer, especiallyfor HER-2 positive patients. In addition, our data could provide a basis for the further clinic research on breast cancer.  
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HER-2/EGFR,六氧化四砷干预HER2阳性乳腺癌细胞SKBR3侵袭转移的重要靶点
刘秋雨1,2, 裴日周2, 钱林林2, 连增林3*
1. 北京中医药大学 中药学院,北京 100102
2. 天地散生物医药科技开发(北京)有限公司,北京 100080
3. 北京亦创生物技术产业研究院生物中药研究所, 北京101111       
摘要:乳腺癌一直女性最常见恶性肿瘤的研究焦点。虽然现在对HER-2阳性乳腺癌的治疗方法颇多, 但是肿瘤耐药和癌细胞远处转移仍是无法避免的难题。六氧化四砷(As4O6)已经被证实对鳞癌和子宫颈癌有一定的抗肿瘤效,但是对HER-2阳性乳腺癌的研究未见报道。本研究旨在运用分子生物学方法探究As4O6HER-2阳性乳腺癌SKBR3细胞的侵袭及迁移能力的抑制作用机制。通过As4O6干预SKBR3细胞, 采用划痕实验、细胞迁移实验、Transwell侵袭实验和细胞黏附实验检测其对SKBR3细胞的迁移、侵袭及黏附能力的影响, 同时, 采用RT-PCRWestern blotting进一步阐As4O6对乳腺癌SKBR3细胞侵袭转移的分子作用机制。结果显示, As4O6能有效抑制HER-2阳性乳腺癌SKBR3细胞的侵袭和迁移, 并且SKBR3细胞的黏附能力在As4O6的干预下也有所减弱。实验结果表明, As4O6抗肿瘤效果与HER-2/EGFR信号通路有关, 通过调节在HER-2/EGFR信号通路中的细胞因子(EGFR, HER-2, Akt, MMP-9)和其他关键分子, 实现对HER-2性乳腺癌细胞迁移和侵袭的抑制作用。总之, As4O6抑制HER-2阳性乳腺癌SKBR3细胞的侵袭和迁移能力是通过HER-2/EGFR信号通路的负调节实现的。因此, As4O6可作为潜在的抗肿瘤转移药物和分子靶点抑制剂在乳腺癌的临床治疗中使用 
关键词: HER-2阳性乳腺癌; 六氧化四砷; 迁移; 侵袭; 黏附; 信号通路
 
  
Received: 2016-11-15, Revised: 2016-12-28, Accepted: 2017-01-10.
*Corresponding author. Tel.: +86-18910068158, E-mail: zenglinlian@aliyun.com    
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