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Arsenic trioxide suppresses cell growth and migration via inhibition of miR-27a in breast cancer cells
Biochemical and Biophysical Research Communications 469 (2016) 55e61
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Arsenic trioxide suppresses cell growth and migration via inhibition of miR-27a in breast cancer cells Shunhua Zhang a, 1, Cong Ma b, 1, Haijie Pang b, 1, Fanpeng Zeng b, Long Cheng b, Binbin Fang b, Jia Ma c, Ying Shi c, Haiyu Hong d, Jianyan Chen e, *, Zhiwei Wang c, f, **, Jun Xia c, *** a
Department of Medical Imaging, Bengbu Medical College, Anhui, 233030, China Research Center of Clinical Laboratory Science, Bengbu Medical College, Anhui, 233030, China Department of Biochemistry and Molecular Biology, Bengbu Medical College, Anhui, 233030, China d Department of Surgery, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, 519000, China e Department of Anesthesiology, Shenzhen Baoan Hospital Affiliated to Southern Medical University, Guangdong, 518100, China f Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, 02215, MA, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 November 2015 Accepted 16 November 2015 Available online 22 November 2015
Accumulating evidence has demonstrated that arsenic trioxide (ATO) exhibits its anti-cancer activities in a variety of human malignancies. Recent studies have revealed that ATO regulated multiple microRNAs (miRNAs) in human cancers. However, the exact mechanism of ATO-mediated tumor suppressive function has not been fully elucidated. In the present study, we explore whether ATO governed oncogenic miR-27a in breast cancer cells by multiple methods such as MTT assay, RT-PCR, Wound healing assay, Western blotting analysis, migration, Transwell invasion assay, and transfection. Our results showed that ATO inhibited cell growth, migration, invasion, and induced cell apoptosis in breast cancer cells. Further molecular analysis dissected that ATO inhibited miR-27a expression in breast cancer cells. Moreover, inhibition of miR-27a suppressed cell growth, migration, invasion, and trigged cell apoptosis, whereas overexpression of miR-27a enhanced cell growth, motility, and inhibited apoptosis in breast cancer cells. Notably, we found that miR-27a inhibitor treatment potentiates ATO-induced breast cancer cell growth inhibition, apoptosis and motility inhibition. However, overexpression of miR-27a partly abrogated ATOmediated anti-tumor activity. Our findings provide a novel anti-tumor mechanism of ATO involved in miR-27a for the treatment of breast cancer. © 2015 Elsevier Inc. All rights reserved.
Keywords: Arsenic trioxide miR-27a Apoptosis Cell growth Breast cancer
1. Introduction Breast cancer is the most commonly diagnosed malignancy and the leading cause of cancer-related mortality in women in the United States [1]. In 2015, an estimated 231, 840 women will be diagnosed with breast cancer, and 40,290 will die from this disease. Specifically, breast cancer alone is expected to account for approximately 30% of all new cancers in women [1]. Although the rapid uptake of mammography screening and multiple treatment
* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (J. Chen), [email protected]. edu (Z. Wang), [email protected] (J. Xia). 1 Zhang S, Ma C and Pang H contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2015.11.071 0006-291X/© 2015 Elsevier Inc. All rights reserved.
strategies have been used to detect and treat breast cancer, this disease is still the second cause of cancer death in women after lung cancer [2,3]. Therefore, it is important to discover new agents for the better treatment of breast cancer. Emerging evidence has demonstrated that breast cancer is a biologically heterogeneous disease and dysregulated cellular pathways have been discovered [4,5]. Clinically, breast cancer patients have different sensitivities to treatment due to different activated pathways [6]. Thus, targeting these pathways could be a useful approach for breast cancer treatment. It has been known that arsenic trioxide (ATO), a clinically effective reagent for APL (acute promyelocytic leukemia), exerted its anti-tumor activities via targeting multiple pathways in various types of human cancers including breast cancer [7e10]. For example, it has been found that ATO induced cell growth arrest involved in FOXO3a and IkB kinase b expression and localization in breast cancer cells [11]. Zhang et al.
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reported that ATO re-sensitizes ERa-negative breast cancer cells to endocrine therapy through restoring ERa expression [12]. Additionally, ATO was found to overcome rapamycin-induced feedback activation of Akt and ERK signaling, resulting in anti-tumor effects in breast cancer [13]. Although these findings suggest that ATO could exert its anti-tumor activities by targeting multiple pathways, further investigations are required to explore the comprehensive molecular mechanisms of ATO-mediated ant-cancer functions. Recent studies have identified that microRNAs (miRNAs) play a critical role in cell growth, apoptosis, invasion, and metastasis in human breast cancer development and progression [14]. It is clear that miRNAs, small non-coding RNAs, negatively govern gene expression via post-transcription [15]. Interestingly, miRNAs can function as oncogenes or tumor suppressors due to their different targets [16]. For example, miR-27a has been validated as an oncomiRNA and valuable marker in breast cancer. High expression of miR-27a is associated with poor overall survival in breast cancer patients [17]. Further study discovered that miR-27a regulated endothelial differentiation of breast cancer stem like cells [18]. These reports indicated that miR-27a could be a promising therapeutic target in human cancers. In the present study, we explored the anti-cancer potential of ATO and function of miR-27a in breast cancer cells. We further determined whether ATO could reduce the expression of miR-27a in breast cancer cells. Moreover, we investigated whether ATO inhibited cell growth, induced apoptosis, retarded cell migration and invasion via targeting miR-27a. We found that ATO-mediated anti-cancer activity is partly through inhibition of miR-27a. Our findings revealed that inhibition of miR-27a could enhance the efficacy and safety to ATO for the treatment of breast cancer by a decrease in dose.
8 mM ATO or miR-27a inhibitor or miR-27a mimics or combination for 16 h. Photographic images were taken from each well at 0 h and 24 h. 2.5. Invasion assay The invasion assay was conducted in MDA-MB-231 and SK-BR-3 cells treated with 8 mM ATO or miR-27a inhibitor or miR-27a mimics or the combination by Transwell inserts with Matrigel. After incubation for 24 h, the invading cells were fixed and stained with Giemsa solution. The stained invasive cells were photographed. 2.6. miRNA real-time reverse transcriptase-PCR To explore whether ATO treatment regulated the expression of miR-27a in MDA-MB-231 and SK-BR-3 cells, we performed miR-27a real-time reverse transcriptase-PCR (RT-PCR) assay as described previously [19]. 2.7. miRNA-27a inhibitor or mimics tranfection MDA-MB-231 and SK-BR-3 cells were seeded in six-well plates and transfected with antisense miR-27a olignucleotide or miR-27a mimics or the nonspecific control as described previously [21]. 2.8. Western blotting assay
2. Materials and methods
To determine the expression of Fbw7 in MDA-MB-231 and SKBR-3 cells treated with 8 mM ATO or miR-27a inhibitor or the combination for 72 h, we conducted Western blotting analysis. The cells were lysed in the cold lysis buffer and the proteins were separated on SDS-PAGE and immunoblotted with indicated antibodies as described previously [22].
2.1. Cell culture and reagents
2.9. Statistical analysis
MDA-MB-231 and SK-BR-3 were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37 C and 5% CO2. ATO and MTT [3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] are bought from Sigma (St. Louis, Mo). AntiFbw7, anti-b-actin, and the secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
All statistical analyses were performed using GraphPad Prism 4.0 (Graph pad Software, La Jolla, CA) by Student's test. P < 0.05 was considered as statistically significant.
2.2. MTT assay The MDA-MB-231 and SK-BR-3 cells (5 103) were seeded in a 96-well culture plate for overnight and then treated with different concentrations of ATO or miR-27a inhibitor or miR-27a mimics or the combination for 72 h. Cell proliferation was measured by MTT assay as described previously [19].
3. Results 3.1. ATO suppressed cell growth in a dose-dependent manner To explore whether ATO treatment inhibited cell growth, we conducted MTT assay in MDA-MB-231 and SK-BR-3 cells treated with different concentrations of ATO (0, 2, 4, 6, 8, 10, 12, 14 mM). We observed that ATO significantly inhibited cell growth in a dosedependent manner in both MDA-MB-231 and SK-BR-3 cells (Fig. 1A). Our results further revealed that about 8 mM ATO led to 50e60% cell growth inhibition in these two cell lines. Therefore, we used 8 mM ATO for following further studies.
2.3. Apoptosis analyses 3.2. ATO inhibited cell migration and invasion The MDA-MB-231 and SK-BR-3 cells were cultured in 6-well plate overnight and treated with different concentration of ATO or miR-27a inhibitor or miR-27a mimics or the combination for 48 h, respectively. Then, Annexin V-FITC/PI staining and FACS were used to detect the apoptosis as described before [20]. 2.4. Wound healing assay The MDA-MB-231 and SK-BR-3 cells were cultured in 6-well plate for 48 h. After the cells are confluent, the straight scratch wound was made using a pipette tip. Then, cells were treated with
To determine whether ATO could suppress cell migration in breast cancer cells, a scratch wound-healing assay was conducted in MDA-MB-231 and SK-BR-3 cells treated with ATO. We found that ATO inhibited cell migration in a dose-dependent manner in both cell lines (Fig. 1B and Supplementary Fig 1A). To further confirm whether ATO has inhibitory effect on cell invasion, we performed cell invasion assay using Transwell inserts with Matrigel. Our invasion assay results demonstrated that ATO inhibited cell invasion in a dose-dependent manner (Fig. 1C and Supplementary Fig 1B). These findings indicated that ATO inhibit cell motility activity.
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Fig. 1. Effect of ATO on cell growth, migration, invasion, and apoptosis. A, MTT assay was conducted to detect cell proliferation in MDA-MB-231 and SK-BR-3 cells after ATO treatment for 72 h. B, Cell migration was detected using Wound-healing assay in breast cancer cells after ATO treatment. C, Cell invasion was measured using Transwell inserts with Matrigel in breast cancer cells after ATO treatment for 24 h. D, Apoptotic cell death was measured using Annexin V-FITC/PI method in MDA-MB-231 cells after ATO treatment for 48 h.
3.3. ATO induced cell apoptotic death To further determine whether ATO induced cell apoptosis, Annexin V-FITC/PI and FACS were performed in MDA-MB-231 and SK-BR-3 cells with different concentrations of ATO treatment. As expected, we found the percentage of apoptotic cells was significantly increased in ATO-treated MDA-MB-231 (Fig. 1D) and SK-BR-3 cells (Data not shown). Notably, ATO treatment led to cell apoptosis in a dose-dependent manner in MDA-MB-231 breast cancer cells (Fig. 1D). 3.4. ATO treatment decreased miR-27a expression Studies have suggested that miR-27a plays an oncogenic role in breast cancer progression [17]. Therefore, we detected the miR-27a expression in MDA-MB-231 and SK-BR-3 cells treated with 8 mM ATO. As shown in Fig. 2A, ATO inhibited the expression of miR-27a in these two breast cancer cells. This result defines that ATO may be a potential inhibitor of oncomiR-27a.
(anti-miR-27a) for 72 h. Our MTT assay revealed that inhibition of miR-27a led to cell growth inhibition in MDA-MB-231 and SK-BR-3 cells (Fig. 2B and Supplementary fig 2A). More importantly, downregulation of miR-27a by its inhibitor attenuated cell growth inhibition induced by ATO (Fig. 2B). In line with this, overexpression of miR-27a enhanced cell growth in MDA-MB-231 cells and partly abrogated ATO-mediated cell growth inhibition (Fig. 2B). 3.6. Inhibition of miR-27a suppressed cell motility To validate the role of miR-27a in cell migration, a scratch wound-healing assay was used to detect the migratory capacity in MDA-MB-231 and SK-BR-3 cells treated with miR-27a inhibitor. Our data demonstrated that inhibition of miR-27a suppressed cell migration in both breast cancer cells (Fig. 2C and Supplementary fig 2B). Our findings further showed that miR-27a inhibitor plus ATO treatment led to a lower level of migration compared with ATO treatment alone or miR-27a inhibitor alone (Fig. 2C). Moreover, miR-27a mimics treatment enhanced cell migration in breast cancer cells (Fig. 2D).
3.5. Inhibition of miR-27a retarded cell growth 3.7. Inhibition of miR-27a suppressed cell invasion To study the function of miR-27a in breast cancer cell growth, we treated MDA-MB-231 and SK-BR-3 cells with miR-27a inhibitor
We also performed the Matrigel invasion chamber assay to
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Fig. 2. Effect of miR-27a inhibitor on cell growth and migration. A, The expression of miR-27a was measured by real-time RT-PCR in MDA-MB-231 and SK-BR-3 cells after ATO treatment for 72 h. B, MTT assay was conducted in MDA-MB-231 cells after ATO treatment or miR-27a inhibitor or miR-27a mimics or the combination. C, Cell migration was measured using Wound-healing assay in MDA-MB-231 cells after ATO treatment or miR-27a inhibitor or the combination. D, Cell migration was conducted by Wound-healing assay in MDA-MB-231 cells treated with ATO or miR-27a mimics or the combination.
measure whether miR-27a suppressed invasion in MDA-MB-231 and SK-BR-3 cells treated with miR-27a inhibitor. We found that down-regulation of miR-27a inhibited cell invasion in MDA-MB231 and SK-BR-3 cells (Fig. 3A and Supplementary fig 3Ae3B). Remarkably, our results showed that miR-27a inhibitor in combination with ATO treatment caused a lower level of invasion compared with ATO treatment alone or miR-27a inhibitor alone (Fig. 3A, C). Moreover, overexpression of miR-27a partly abrogated ATO-induced cell invasion inhibition (Fig. 3B, C).
3.8. Inhibition of miR-27a induced apoptosis Next, we measured the cell apoptosis in MDA-MB-231 and SKBR-3 cells after miR-27a inhibitor treatment. We found that down-regulation of miR-27a led to increased percentage of apoptotic cells in both breast cancer cells (Fig. 3D). Notably, miR27a inhibitor plus with ATO treatment caused higher percentage of apoptotic cells compared with miR-27a inhibitor alone or ATO alone (Fig. 3D). Furthermore, miR-27a mimics abrogated ATO-
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Fig. 3. Effect of miR-27a on cell invasion and apoptosis. A, The cell invasion was measured using Transwell inserts with Matrigel in breast cancer cells after ATO treatment for 24 h. B, The cell invasion was performed in MDA-MB-231 cells treated with ATO or miR-27a mimics or the combination. C, Quantitative results are illustrated for panel A and B. D, Cell apoptosis was detected by Annexin V-FITC/PI method in MDA-MB-231 and SK-BR-3 cells after ATO treatment or miR-27a inhibitor or the combination.
induced apoptosis in MDA-MB-231 cells (Supplementary fig 4). 3.9. Inhibition of miR-27a increased the Fbw7 expression It has been known that Fbw7 is a potential target gene of miR27a. Therefore, we sought to explore whether ATO could upregulate Fbw7 expression via inhibition of miR-27a in MDA-MB-
231 and SK-BR-3 cells. We found that down-regulation of miR27a or ATO treatment in two breast cancer cells led to upregulation of Fbw7 (Fig. 4A and Supplementary fig 5). Moreover, ATO in combination with miR-27a inhibitor caused to a greater degree compared with ATO treatment alone or miR-27a inhibitor alone (Fig. 4A). Consistently, overexpression of miR-27a inhibited Fbw7 expression and subsequently increased Cyclin E level (Fig. 4A).
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These results demonstrated that ATO increased Fbw7 partly due to down-regulation of miR-27a. 4. Discussion A line of evidence suggests that ATO exhibits therapeutic effects in a variety of human cancers. ATO impedes cancer development and progression through targeting cellular pathways, leading to inhibition of cell proliferation and invasion, and promoting apoptosis. Recent studies have revealed that ATO exerts its tumor suppressive function through regulation of miRNAs. For instance, Cao et al. reported that ATO down-regulated the expression of miR19a in bladder cancer cells, leading to cell growth inhibition and apoptosis [23]. Similarly, ATO induced apoptosis via governing expression of miR-376a in retinoblastoma cells [24]. Another study identified that ATO targeted miR-125b in glioma cells [25]. In line with this, ATO attenuated the invasion potential of human liver cancer cells via targeting miR-491 [26]. Notably, ATO inhibited cell growth partly through targeting miR-328/hERG (human ether-ago-go-related gene) pathway in breast cancer cells [27]. In addition, ATO induced the mesenchymal to epithelial transition (MET) via up-regulation of miR-200c through demethylation in breast cancer cells, leading to anti-migration/invasion effects [28]. These studies suggest the critical role of ATO in regulation of miRNAs in human cancers. Multiple studies have unraveled that miR-27a plays an oncogenic role in the development and progression of human breast cancers. It has been reported that miR-27a modulated radiosensitivity through targeting Cdc27 in triple negative breast cancer cells [29]. Further studies validated miR-27a could be a novel diagnostic
and prognostic biomarker in triple negative breast cancers [30]. Consistently, miR-27a was validated as an independent predictor of overall survival in patients with breast cancer [17]. Therefore, inhibition of miR-27a could represent a promising approach to treat breast cancer. To achieve this goal, several compounds have been discovered to target miR-27a. For example, genistein downregulated miR-27a expression and subsequently blocked ovarial cancer cell growth and migration [31]. Grape seed proanthocyanidins extract inhibited the expression of miR-27a and suppressed pancreatic cancer cell growth [32]. One study revealed that pomegranate polyphenolics decreased the miR-27a expression and inhibited cell survival in breast cancer cells [33]. In the current study, we found that ATO inhibited the expression of miR-27a in breast cancer cells, suggesting that ATO could be an inhibitor of oncomiR-27a. A number of evidence has demonstrated that Fbw7, a putative tumor suppressor, is a target of miR-27a in cancer cells [34e36]. Specifically, miR-27a controlled Fbw7-dependent Cyclin E degradation and cell cycle progression [34]. It has been known that Fbw7 is critically involved in cell growth, apoptosis, drug resistance, and cancer stem cells [37]. Consistent with these findings, we observed that inhibition of miR-27a upregulated the expression of Fbw7 and down-regulated Cyclin E level. Strikingly, our results suggest that miR-27a inhibitor sensitized breast cancer cells to ATO by inducing cell growth inhibition and apoptosis as well as invasion inhibition partly due to its up-regulation of Fbw7 and down-regulation of Cyclin E protein level. In line with this finding, overexpression of miR-27a partly abrogated ATO-mediated anti-tumor activity via targeting Fbw7. In summary, our experimental evidence supports the tumor suppressive function of ATO in human breast cancer. Mechanistically, we propose that ATO down-regulated the expression of miR-27a and subsequent upregulation of its downstream gene Fbw7, leading to inhibition of cell proliferation, invasion and induction of apoptosis in breast cancer cells (Fig. 4B). Furthermore, our findings indicate that inhibition of miR-27a enhances the efficacy to ATO for the treatment of breast cancer by a low dose. The combination of ATO and miR-27a inhibitor presents a novel therapeutic remedy for breast cancer. Without a doubt, further in-depth exploring the molecular physiological properties of ATO is required for successful treatment of breast cancer. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work is supported by grant from National Natural Science Foundation of China (NSFC 81172087). This work is also supported in part by Natural Science Foundation of Anhui Province (1508085SMH232) and Natural Science Research key Project of Education Office of Anhui Province (KJ2014A153) and the program for graduate research of Bengbu Medical College (Byycx1315). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.11.071. References
Fig. 4. Effect of ATO on the expression of Fbw7 and cyclin E. A, The expression of Fbw7 and cyclin E was measured by Western blotting analysis in MDA-MB-231 cells after ATO treatment or miR-27a inhibitor or miR-27a mimics or the combination. B, Schematic illustration of molecular pathways that affected by ATO in breast cancer cells.
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Arsenic trioxide suppresses cell growth and migration via inhibition of miR-27a in breast cancer cells Shunhua Zhang a, 1, Cong Ma b, 1, Haijie Pang b, 1, Fanpeng Zeng b, Long Cheng b, Binbin Fang b, Jia Ma c, Ying Shi c, Haiyu Hong d, Jianyan Chen e, *, Zhiwei Wang c, f, **, Jun Xia c, *** a
Department of Medical Imaging, Bengbu Medical College, Anhui, 233030, China Research Center of Clinical Laboratory Science, Bengbu Medical College, Anhui, 233030, China Department of Biochemistry and Molecular Biology, Bengbu Medical College, Anhui, 233030, China d Department of Surgery, The Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai, 519000, China e Department of Anesthesiology, Shenzhen Baoan Hospital Affiliated to Southern Medical University, Guangdong, 518100, China f Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, 02215, MA, USA b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 November 2015 Accepted 16 November 2015 Available online 22 November 2015
Accumulating evidence has demonstrated that arsenic trioxide (ATO) exhibits its anti-cancer activities in a variety of human malignancies. Recent studies have revealed that ATO regulated multiple microRNAs (miRNAs) in human cancers. However, the exact mechanism of ATO-mediated tumor suppressive function has not been fully elucidated. In the present study, we explore whether ATO governed oncogenic miR-27a in breast cancer cells by multiple methods such as MTT assay, RT-PCR, Wound healing assay, Western blotting analysis, migration, Transwell invasion assay, and transfection. Our results showed that ATO inhibited cell growth, migration, invasion, and induced cell apoptosis in breast cancer cells. Further molecular analysis dissected that ATO inhibited miR-27a expression in breast cancer cells. Moreover, inhibition of miR-27a suppressed cell growth, migration, invasion, and trigged cell apoptosis, whereas overexpression of miR-27a enhanced cell growth, motility, and inhibited apoptosis in breast cancer cells. Notably, we found that miR-27a inhibitor treatment potentiates ATO-induced breast cancer cell growth inhibition, apoptosis and motility inhibition. However, overexpression of miR-27a partly abrogated ATOmediated anti-tumor activity. Our findings provide a novel anti-tumor mechanism of ATO involved in miR-27a for the treatment of breast cancer. © 2015 Elsevier Inc. All rights reserved.
Keywords: Arsenic trioxide miR-27a Apoptosis Cell growth Breast cancer
1. Introduction Breast cancer is the most commonly diagnosed malignancy and the leading cause of cancer-related mortality in women in the United States [1]. In 2015, an estimated 231, 840 women will be diagnosed with breast cancer, and 40,290 will die from this disease. Specifically, breast cancer alone is expected to account for approximately 30% of all new cancers in women [1]. Although the rapid uptake of mammography screening and multiple treatment
* Corresponding author. ** Corresponding author. *** Corresponding author. E-mail addresses: [email protected] (J. Chen), [email protected]. edu (Z. Wang), [email protected] (J. Xia). 1 Zhang S, Ma C and Pang H contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2015.11.071 0006-291X/© 2015 Elsevier Inc. All rights reserved.
strategies have been used to detect and treat breast cancer, this disease is still the second cause of cancer death in women after lung cancer [2,3]. Therefore, it is important to discover new agents for the better treatment of breast cancer. Emerging evidence has demonstrated that breast cancer is a biologically heterogeneous disease and dysregulated cellular pathways have been discovered [4,5]. Clinically, breast cancer patients have different sensitivities to treatment due to different activated pathways [6]. Thus, targeting these pathways could be a useful approach for breast cancer treatment. It has been known that arsenic trioxide (ATO), a clinically effective reagent for APL (acute promyelocytic leukemia), exerted its anti-tumor activities via targeting multiple pathways in various types of human cancers including breast cancer [7e10]. For example, it has been found that ATO induced cell growth arrest involved in FOXO3a and IkB kinase b expression and localization in breast cancer cells [11]. Zhang et al.
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reported that ATO re-sensitizes ERa-negative breast cancer cells to endocrine therapy through restoring ERa expression [12]. Additionally, ATO was found to overcome rapamycin-induced feedback activation of Akt and ERK signaling, resulting in anti-tumor effects in breast cancer [13]. Although these findings suggest that ATO could exert its anti-tumor activities by targeting multiple pathways, further investigations are required to explore the comprehensive molecular mechanisms of ATO-mediated ant-cancer functions. Recent studies have identified that microRNAs (miRNAs) play a critical role in cell growth, apoptosis, invasion, and metastasis in human breast cancer development and progression [14]. It is clear that miRNAs, small non-coding RNAs, negatively govern gene expression via post-transcription [15]. Interestingly, miRNAs can function as oncogenes or tumor suppressors due to their different targets [16]. For example, miR-27a has been validated as an oncomiRNA and valuable marker in breast cancer. High expression of miR-27a is associated with poor overall survival in breast cancer patients [17]. Further study discovered that miR-27a regulated endothelial differentiation of breast cancer stem like cells [18]. These reports indicated that miR-27a could be a promising therapeutic target in human cancers. In the present study, we explored the anti-cancer potential of ATO and function of miR-27a in breast cancer cells. We further determined whether ATO could reduce the expression of miR-27a in breast cancer cells. Moreover, we investigated whether ATO inhibited cell growth, induced apoptosis, retarded cell migration and invasion via targeting miR-27a. We found that ATO-mediated anti-cancer activity is partly through inhibition of miR-27a. Our findings revealed that inhibition of miR-27a could enhance the efficacy and safety to ATO for the treatment of breast cancer by a decrease in dose.
8 mM ATO or miR-27a inhibitor or miR-27a mimics or combination for 16 h. Photographic images were taken from each well at 0 h and 24 h. 2.5. Invasion assay The invasion assay was conducted in MDA-MB-231 and SK-BR-3 cells treated with 8 mM ATO or miR-27a inhibitor or miR-27a mimics or the combination by Transwell inserts with Matrigel. After incubation for 24 h, the invading cells were fixed and stained with Giemsa solution. The stained invasive cells were photographed. 2.6. miRNA real-time reverse transcriptase-PCR To explore whether ATO treatment regulated the expression of miR-27a in MDA-MB-231 and SK-BR-3 cells, we performed miR-27a real-time reverse transcriptase-PCR (RT-PCR) assay as described previously [19]. 2.7. miRNA-27a inhibitor or mimics tranfection MDA-MB-231 and SK-BR-3 cells were seeded in six-well plates and transfected with antisense miR-27a olignucleotide or miR-27a mimics or the nonspecific control as described previously [21]. 2.8. Western blotting assay
2. Materials and methods
To determine the expression of Fbw7 in MDA-MB-231 and SKBR-3 cells treated with 8 mM ATO or miR-27a inhibitor or the combination for 72 h, we conducted Western blotting analysis. The cells were lysed in the cold lysis buffer and the proteins were separated on SDS-PAGE and immunoblotted with indicated antibodies as described previously [22].
2.1. Cell culture and reagents
2.9. Statistical analysis
MDA-MB-231 and SK-BR-3 were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum at 37 C and 5% CO2. ATO and MTT [3-(4,5-dimethythiazol- 2-yl)-2,5-diphenyl tetrazolium bromide] are bought from Sigma (St. Louis, Mo). AntiFbw7, anti-b-actin, and the secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
All statistical analyses were performed using GraphPad Prism 4.0 (Graph pad Software, La Jolla, CA) by Student's test. P < 0.05 was considered as statistically significant.
2.2. MTT assay The MDA-MB-231 and SK-BR-3 cells (5 103) were seeded in a 96-well culture plate for overnight and then treated with different concentrations of ATO or miR-27a inhibitor or miR-27a mimics or the combination for 72 h. Cell proliferation was measured by MTT assay as described previously [19].
3. Results 3.1. ATO suppressed cell growth in a dose-dependent manner To explore whether ATO treatment inhibited cell growth, we conducted MTT assay in MDA-MB-231 and SK-BR-3 cells treated with different concentrations of ATO (0, 2, 4, 6, 8, 10, 12, 14 mM). We observed that ATO significantly inhibited cell growth in a dosedependent manner in both MDA-MB-231 and SK-BR-3 cells (Fig. 1A). Our results further revealed that about 8 mM ATO led to 50e60% cell growth inhibition in these two cell lines. Therefore, we used 8 mM ATO for following further studies.
2.3. Apoptosis analyses 3.2. ATO inhibited cell migration and invasion The MDA-MB-231 and SK-BR-3 cells were cultured in 6-well plate overnight and treated with different concentration of ATO or miR-27a inhibitor or miR-27a mimics or the combination for 48 h, respectively. Then, Annexin V-FITC/PI staining and FACS were used to detect the apoptosis as described before [20]. 2.4. Wound healing assay The MDA-MB-231 and SK-BR-3 cells were cultured in 6-well plate for 48 h. After the cells are confluent, the straight scratch wound was made using a pipette tip. Then, cells were treated with
To determine whether ATO could suppress cell migration in breast cancer cells, a scratch wound-healing assay was conducted in MDA-MB-231 and SK-BR-3 cells treated with ATO. We found that ATO inhibited cell migration in a dose-dependent manner in both cell lines (Fig. 1B and Supplementary Fig 1A). To further confirm whether ATO has inhibitory effect on cell invasion, we performed cell invasion assay using Transwell inserts with Matrigel. Our invasion assay results demonstrated that ATO inhibited cell invasion in a dose-dependent manner (Fig. 1C and Supplementary Fig 1B). These findings indicated that ATO inhibit cell motility activity.
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Fig. 1. Effect of ATO on cell growth, migration, invasion, and apoptosis. A, MTT assay was conducted to detect cell proliferation in MDA-MB-231 and SK-BR-3 cells after ATO treatment for 72 h. B, Cell migration was detected using Wound-healing assay in breast cancer cells after ATO treatment. C, Cell invasion was measured using Transwell inserts with Matrigel in breast cancer cells after ATO treatment for 24 h. D, Apoptotic cell death was measured using Annexin V-FITC/PI method in MDA-MB-231 cells after ATO treatment for 48 h.
3.3. ATO induced cell apoptotic death To further determine whether ATO induced cell apoptosis, Annexin V-FITC/PI and FACS were performed in MDA-MB-231 and SK-BR-3 cells with different concentrations of ATO treatment. As expected, we found the percentage of apoptotic cells was significantly increased in ATO-treated MDA-MB-231 (Fig. 1D) and SK-BR-3 cells (Data not shown). Notably, ATO treatment led to cell apoptosis in a dose-dependent manner in MDA-MB-231 breast cancer cells (Fig. 1D). 3.4. ATO treatment decreased miR-27a expression Studies have suggested that miR-27a plays an oncogenic role in breast cancer progression [17]. Therefore, we detected the miR-27a expression in MDA-MB-231 and SK-BR-3 cells treated with 8 mM ATO. As shown in Fig. 2A, ATO inhibited the expression of miR-27a in these two breast cancer cells. This result defines that ATO may be a potential inhibitor of oncomiR-27a.
(anti-miR-27a) for 72 h. Our MTT assay revealed that inhibition of miR-27a led to cell growth inhibition in MDA-MB-231 and SK-BR-3 cells (Fig. 2B and Supplementary fig 2A). More importantly, downregulation of miR-27a by its inhibitor attenuated cell growth inhibition induced by ATO (Fig. 2B). In line with this, overexpression of miR-27a enhanced cell growth in MDA-MB-231 cells and partly abrogated ATO-mediated cell growth inhibition (Fig. 2B). 3.6. Inhibition of miR-27a suppressed cell motility To validate the role of miR-27a in cell migration, a scratch wound-healing assay was used to detect the migratory capacity in MDA-MB-231 and SK-BR-3 cells treated with miR-27a inhibitor. Our data demonstrated that inhibition of miR-27a suppressed cell migration in both breast cancer cells (Fig. 2C and Supplementary fig 2B). Our findings further showed that miR-27a inhibitor plus ATO treatment led to a lower level of migration compared with ATO treatment alone or miR-27a inhibitor alone (Fig. 2C). Moreover, miR-27a mimics treatment enhanced cell migration in breast cancer cells (Fig. 2D).
3.5. Inhibition of miR-27a retarded cell growth 3.7. Inhibition of miR-27a suppressed cell invasion To study the function of miR-27a in breast cancer cell growth, we treated MDA-MB-231 and SK-BR-3 cells with miR-27a inhibitor
We also performed the Matrigel invasion chamber assay to
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Fig. 2. Effect of miR-27a inhibitor on cell growth and migration. A, The expression of miR-27a was measured by real-time RT-PCR in MDA-MB-231 and SK-BR-3 cells after ATO treatment for 72 h. B, MTT assay was conducted in MDA-MB-231 cells after ATO treatment or miR-27a inhibitor or miR-27a mimics or the combination. C, Cell migration was measured using Wound-healing assay in MDA-MB-231 cells after ATO treatment or miR-27a inhibitor or the combination. D, Cell migration was conducted by Wound-healing assay in MDA-MB-231 cells treated with ATO or miR-27a mimics or the combination.
measure whether miR-27a suppressed invasion in MDA-MB-231 and SK-BR-3 cells treated with miR-27a inhibitor. We found that down-regulation of miR-27a inhibited cell invasion in MDA-MB231 and SK-BR-3 cells (Fig. 3A and Supplementary fig 3Ae3B). Remarkably, our results showed that miR-27a inhibitor in combination with ATO treatment caused a lower level of invasion compared with ATO treatment alone or miR-27a inhibitor alone (Fig. 3A, C). Moreover, overexpression of miR-27a partly abrogated ATO-induced cell invasion inhibition (Fig. 3B, C).
3.8. Inhibition of miR-27a induced apoptosis Next, we measured the cell apoptosis in MDA-MB-231 and SKBR-3 cells after miR-27a inhibitor treatment. We found that down-regulation of miR-27a led to increased percentage of apoptotic cells in both breast cancer cells (Fig. 3D). Notably, miR27a inhibitor plus with ATO treatment caused higher percentage of apoptotic cells compared with miR-27a inhibitor alone or ATO alone (Fig. 3D). Furthermore, miR-27a mimics abrogated ATO-
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Fig. 3. Effect of miR-27a on cell invasion and apoptosis. A, The cell invasion was measured using Transwell inserts with Matrigel in breast cancer cells after ATO treatment for 24 h. B, The cell invasion was performed in MDA-MB-231 cells treated with ATO or miR-27a mimics or the combination. C, Quantitative results are illustrated for panel A and B. D, Cell apoptosis was detected by Annexin V-FITC/PI method in MDA-MB-231 and SK-BR-3 cells after ATO treatment or miR-27a inhibitor or the combination.
induced apoptosis in MDA-MB-231 cells (Supplementary fig 4). 3.9. Inhibition of miR-27a increased the Fbw7 expression It has been known that Fbw7 is a potential target gene of miR27a. Therefore, we sought to explore whether ATO could upregulate Fbw7 expression via inhibition of miR-27a in MDA-MB-
231 and SK-BR-3 cells. We found that down-regulation of miR27a or ATO treatment in two breast cancer cells led to upregulation of Fbw7 (Fig. 4A and Supplementary fig 5). Moreover, ATO in combination with miR-27a inhibitor caused to a greater degree compared with ATO treatment alone or miR-27a inhibitor alone (Fig. 4A). Consistently, overexpression of miR-27a inhibited Fbw7 expression and subsequently increased Cyclin E level (Fig. 4A).
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These results demonstrated that ATO increased Fbw7 partly due to down-regulation of miR-27a. 4. Discussion A line of evidence suggests that ATO exhibits therapeutic effects in a variety of human cancers. ATO impedes cancer development and progression through targeting cellular pathways, leading to inhibition of cell proliferation and invasion, and promoting apoptosis. Recent studies have revealed that ATO exerts its tumor suppressive function through regulation of miRNAs. For instance, Cao et al. reported that ATO down-regulated the expression of miR19a in bladder cancer cells, leading to cell growth inhibition and apoptosis [23]. Similarly, ATO induced apoptosis via governing expression of miR-376a in retinoblastoma cells [24]. Another study identified that ATO targeted miR-125b in glioma cells [25]. In line with this, ATO attenuated the invasion potential of human liver cancer cells via targeting miR-491 [26]. Notably, ATO inhibited cell growth partly through targeting miR-328/hERG (human ether-ago-go-related gene) pathway in breast cancer cells [27]. In addition, ATO induced the mesenchymal to epithelial transition (MET) via up-regulation of miR-200c through demethylation in breast cancer cells, leading to anti-migration/invasion effects [28]. These studies suggest the critical role of ATO in regulation of miRNAs in human cancers. Multiple studies have unraveled that miR-27a plays an oncogenic role in the development and progression of human breast cancers. It has been reported that miR-27a modulated radiosensitivity through targeting Cdc27 in triple negative breast cancer cells [29]. Further studies validated miR-27a could be a novel diagnostic
and prognostic biomarker in triple negative breast cancers [30]. Consistently, miR-27a was validated as an independent predictor of overall survival in patients with breast cancer [17]. Therefore, inhibition of miR-27a could represent a promising approach to treat breast cancer. To achieve this goal, several compounds have been discovered to target miR-27a. For example, genistein downregulated miR-27a expression and subsequently blocked ovarial cancer cell growth and migration [31]. Grape seed proanthocyanidins extract inhibited the expression of miR-27a and suppressed pancreatic cancer cell growth [32]. One study revealed that pomegranate polyphenolics decreased the miR-27a expression and inhibited cell survival in breast cancer cells [33]. In the current study, we found that ATO inhibited the expression of miR-27a in breast cancer cells, suggesting that ATO could be an inhibitor of oncomiR-27a. A number of evidence has demonstrated that Fbw7, a putative tumor suppressor, is a target of miR-27a in cancer cells [34e36]. Specifically, miR-27a controlled Fbw7-dependent Cyclin E degradation and cell cycle progression [34]. It has been known that Fbw7 is critically involved in cell growth, apoptosis, drug resistance, and cancer stem cells [37]. Consistent with these findings, we observed that inhibition of miR-27a upregulated the expression of Fbw7 and down-regulated Cyclin E level. Strikingly, our results suggest that miR-27a inhibitor sensitized breast cancer cells to ATO by inducing cell growth inhibition and apoptosis as well as invasion inhibition partly due to its up-regulation of Fbw7 and down-regulation of Cyclin E protein level. In line with this finding, overexpression of miR-27a partly abrogated ATO-mediated anti-tumor activity via targeting Fbw7. In summary, our experimental evidence supports the tumor suppressive function of ATO in human breast cancer. Mechanistically, we propose that ATO down-regulated the expression of miR-27a and subsequent upregulation of its downstream gene Fbw7, leading to inhibition of cell proliferation, invasion and induction of apoptosis in breast cancer cells (Fig. 4B). Furthermore, our findings indicate that inhibition of miR-27a enhances the efficacy to ATO for the treatment of breast cancer by a low dose. The combination of ATO and miR-27a inhibitor presents a novel therapeutic remedy for breast cancer. Without a doubt, further in-depth exploring the molecular physiological properties of ATO is required for successful treatment of breast cancer. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work is supported by grant from National Natural Science Foundation of China (NSFC 81172087). This work is also supported in part by Natural Science Foundation of Anhui Province (1508085SMH232) and Natural Science Research key Project of Education Office of Anhui Province (KJ2014A153) and the program for graduate research of Bengbu Medical College (Byycx1315). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.11.071. References
Fig. 4. Effect of ATO on the expression of Fbw7 and cyclin E. A, The expression of Fbw7 and cyclin E was measured by Western blotting analysis in MDA-MB-231 cells after ATO treatment or miR-27a inhibitor or miR-27a mimics or the combination. B, Schematic illustration of molecular pathways that affected by ATO in breast cancer cells.
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