A Novel All-trans Retinoid Acid Derivative Induces Apoptosis in MDA-MB-231 Breast Cancer Cells

Breast cancer is one of the most common cancer around the world, and remains the second leading cause of cancer-related deaths in women (Ma et al., 2013). Breast cancer incidence and death rates increase with age, approximately 95% of new cases occur in women 40 years of age and older (DeSantis et al., 2011), whereas women at age 40 or younger have a poorer prognosis than their older counterparts and bear a disproportionate degree of physical and psychological morbidity (Andres et al., 2009). Therefore, it is important to find a drug to induce the apoptosis of breast cancer cells. All trans-retinoic acid (ATRA) induces apoptosis and differentiation in solid tumors, including breast cancer, and has become a therapeutic tool in this disease (Marchetti et al., 2011). Apoptosis, the most common form of programmed cell death in vertebrate, defines a type of regulated cell death associated with various morphological features that include cell shrinkage, nuclear/cytoplasmic fragmentation, and formation of dense bodies. Many studies have


Introduction
Breast cancer is one of the most common cancer around the world, and remains the second leading cause of cancer-related deaths in women (Ma et al., 2013).Breast cancer incidence and death rates increase with age, approximately 95% of new cases occur in women 40 years of age and older (DeSantis et al., 2011), whereas women at age 40 or younger have a poorer prognosis than their older counterparts and bear a disproportionate degree of physical and psychological morbidity (Andres et al., 2009).Therefore, it is important to find a drug to induce the apoptosis of breast cancer cells.All trans-retinoic acid (ATRA) induces apoptosis and differentiation in solid tumors, including breast cancer, and has become a therapeutic tool in this disease (Marchetti et al., 2011).Apoptosis, the most common form of programmed cell death in vertebrate, defines a type of regulated cell death associated with various morphological features that include cell shrinkage, nuclear/cytoplasmic fragmentation, and formation of dense bodies.Many studies have

A Novel All-trans Retinoid Acid Derivative Induces Apoptosis in MDA-MB-231 Breast Cancer Cells
Bei Wang 1,2 *, Yun-Wen Yan 2 , Qing Zhou 2 , Shu-Yu Gui 3 , Fei-Hu Chen 4 , Yuan Wang 2,3 * indicated that crucial apoptotic modulators are deregulated in metastatic cancer cells and supported the hypothesis that suppression of apoptosis has a vital role during the metastatic process (Korzeniewska et al., 2007).Recently reported that caspase-Mediated Pathway involved in Apoptosis in breast cancer cells (Zhou et al., 2013;Zhang et al., 2014).ER stress-induced apoptosis has been implicated in the pathogenesis of several conformational diseases.Studies performed with a number of cell lines and in vivo model have shown that survival and death decisions during UPR are mediated via the proapoptotic CHOP (C/EBP-homologous protein transcription factor also known as GADD 153 growth arrest and DNA damage inducible protein) and the anti-apoptotic BIP (GRP78: a Ca 2+ dependent, ER associated transmembrane chaperone), respectively (Price et al., 2010).The Ras-mitogen-activatd protein kinase (MAPK) pathway participates in the control of many important cellular processes including survival, proliferation, apoptosis and differentiation.Recent studies have shown that MAPK pathway could be as an apoptosis enhancer in melanoma (Haydn et al., 2014).
Retinoids regulate various biological activities by binding RARs and RXRs.Ligand binding to the receptor causes conformational changes that modulate receptor complex function.In addition, these receptor complexes have a range of additional co-activators and co-repressors that modulate receptor activity.The composition of receptor complex is complicated by the presence of distinct sub-types and isoforms.Both RAR and RXR have three sub-types α, β and γ, each with different isoforms.RARα and RARγ both have two isoforms 1 (RARα1, RARγ1) and 2 (RARα2, RARγ2) whereas RARβ have five isoforms (RARβ1-4, and 11).In the case of RXR, all sub-types have two isoforms 1 and 2 (Das et al., 2014).The RARs bind ATRA, whereas the RXRs bind 9 Cis-RA selectively.
Since 1989, when ATRA was introduced as a targeted therapy against acute promyelocytic leukemia (APL), it has been used extensively as an anti-tumor agent for many types of tumors (Yung et al., 1989).However, as we know, the toxicity of retinoids has limited their general use for cancer prevention.Therefore, the present work is aimed at developing new synthetic retinoids which are less toxic and more effective as chemoprevention agents.ATPR is synthesized by school of Pharmacy, Anhui Medical University and is a novel ATRA derivatives.Recently, a serious of studies associated with ATPR about migration of lung cancer were published (Wang et al., 2013;Fang et al., 2014).In a previous study, we have demonstrated that ATPR plays a more important role in inducing the differentiation and inhibiting the proliferation and migration of MDA-MB-231 cells than ATRA at the same dose and treatment time (Wang et al., 2013).So far, whether ATPR could induce apoptosis of MDA-MB-231 cells and the precise mechanism are unknown, although ATPR has anti-tumor effects such as inhibiting the proliferation and arresting the cell cycle in human breast cancer cell line MCF-7 (Wang et al., 2013).Therefore, the present study was undertaken to investigate the effect of ATPR inducing apoptosis of Breast cancer MDA-MB-231 cells and explore their mechanism whether ATPR induces apoptosis via ER stress involving MAPK signal pathway and binding which one of retinoic acid receptors.Finally, the mechanisms underlying anti-neoplastic potential of ATPR in breast cancer could be partly elucidated.

Cell lines and cell culture
Breast cancer MDA-MB-231 cells were maintained in DMEM medium supplemented with 10% fetal bovine serum and 100 U/ml penicillin, 100U/ml streptomycin in humidified 5% CO 2 at 37℃.The media were changed every two or three days.

Cell viability assay
Cell viability was measured using the MTT assay.Breast cancer MDA-MB-231 cells (5x10 3 cells/well) were seeded into 96-well plates and cultured.The cells were treated at different times (24hr, 48hr, 72hr) and different concentration of ATRA and ATPR (10,12.5,15,17.5,20 μmol/L), equivalent DMSO was added as the control group, then incubation with MTT solution for 4h.Finally, the cells were exposed to an MTT-formazan dissolving solution (DMSO) for 30 minutes.The optical density (OD) was measured using an absorbance microplate reader (Bio-Tek, ELX800) at a wavelength of 490nm.The cell viability was expressed as a percentage of the OD value of the control cultures.The IC 50 value was measured using a sigmoidal equilibrium model regression by XLfit version 4.3.2(ID Business Solutions Ltd), and defined as the concentration of ATPR required for a 50% reduction in growth.

Mircoscopy observation of Breast cancer MDA-MB-231 cells
Breast cancer cells were seeded into 6-well plates and cultured, then cells were treated by ATRA and ATPR (15 μmol/L) for 48 h.DMSO was added in the control group.Images of cells were captured using the microscope .

Assessment of cell cycle distribution
After MDA-MB-231 cells were treated with ATPR and ATRA, the cell cycle analysis and cell apoptotic rate were determined by flow cytometry analysis.MDA-MB-231 were incubated in decanter at a density of 1×10 5 .After 48 h, cells were treated with ATRA and ATPR at the same concentration of 15 μmol/L.Equivalent DMSO was added as the control group.After treatment, Cells were harvested by trypsinization and washed twice with ice-cold PBS, fixed with 70% alcohol overnight, and stained with PI (1mg/ml) in the presence of 1% RNase A for at least 30 minutes before analysis by flow cytometry (Becton Dickinson, USA).GO/G1, S, and G2/M cells were gated out as appropriate.Data were analyzed with Modfit software.

Statistical analysis
Three or more separate experiments were performed independently for each experiment.Statistical analysis was performed by Student's test or ANOVE.Data are presented as means±standard deviation.Significance was noted at p<0.05

Effect of ATPR on proliferation of MDA-MB-231 cells
In previous published article (Wang et al., 2013), we have already chosen a concentration of ATRA and ATPR (15umol/l) as a treated concentration (Table 1).

Effect of ATPR on the morphology of MDA-MB-231 cells
Breast cancer MDA-MB-231 cells were treated with ATRA (15 μmol/L) and ATPR (15 μmol/L) and DMSO (1.5%), the morphology of breast cancer MDA-MB-231 treated by ATPR changed from spindle to round, and the number of cell reduced.(Figure 1) Bright field images were taken at the indicated time points.Magnification, 100×

Apoptosis analysis by flow cytometry
The percentage of cells with the sub-G1 DNA content reflects the apoptotic rate of the cell population.The ratio of apoptosis was 33.23% after breast cancer MDA-MB-231 cells were treated by ATPR (15 μmol/L) whereas ATRA (15 μmol/L) had no apoptotic effect on MDA-MB-231 cells.Figure 2 also showed that the cell cycles

Effect of ATPR on ER stress in MDA-MB-231 cells
To catch out the effect of ATPR on ER stress, the expression of BiP and CHOP were detected at the protein level by Western blot analysis as BiP and CHOP were the markers of ER stress.The expression of BiP decreased while the expression of CHOP increased after the breast cancer MDA-MB-231cells were treated by ATPR (Figure 3).

Effect of ATPR on MAPK signal pathway in MDA-MB-231 cells
To find out the whether MAPK signal pathway involved in apoptosis of breast cancer MDA-MB-231 cells, western blot was used to investigate the expression of phosphorylation of ERK, JNK and p38.Interesting to find the expression of phosphorylation of ERK had a significant decrease when the breast cancer MDA-MB-231 cells were treated by ATPR.In contrast, no significant decreases can be observed, to our surprise, minor increases can be found in comparison to control (Figure 4).

Effect of ATPR on retinoid receptor in MDA-MB-231 cells
To check which one of all retinoid-receptors binds to ATPR in breast cancer MDA-MB-231 cells, the expression of RXR and RAR were detected by western bolt.ATRA and ATPR both reduced the expression of RXRα.In compared with ATRA, data revealed that ATPR significantly decreased the expression of RARβ and RXRβ.By comparison with DMSO group, the protein level of RARγ and RXRγ exhibited no significant decrease (Figure 5).

Discussion
In this research, we studied the anti-tumor effect of A Novel All-trans Retinoid Acid Derivative Induces Apoptosis in MDA-MB-231 Breast Cancer Cells emanates from the ER.Proteins must be folded into proper conformations, so as to realize their cellular functions.Unfolded or misfolded proteins are extremely harmful to cells because cell survival can be threatened (Kopito et al., 2000).The UPR enables cells to reduce unfolded protein folding, secretion and degradation (Zhao et al., 2006).In many tumors, BiP is highly expressed and is crucial for tumor cells survival (Dudek et al., 2009).BiP, a chaperone predominantly in the ER lumen interacts with unfolded proteins through its C-terminal substrate-binding domain, which is tightly regulated by a conformational change depending on ATP occupation of its NBD (nucleotidebinding domain) (Mayer et al., 2005).As a negative regulator of UPR, BiP sequesters PERK, ATF6, and IRE1 on the ER membrane via binding to their luminal domains.BiP dissociating from PERK, ATF6, and IRE1 leads to downstream UPR signaling.ATF6 is synthetized as an inactive precursor coding for a bZIP transcription factor in the cytoplasmic domain.Under ER stress, ATF6 traffics to Golgi apparatus where S1P and S2P proteases cleave the cytosolic and transmembrane domains, releasing a fragment named ATF6f (Haze et al., 1999).ATF6 translocates to the nucleus regulating the transcription of genes involved in ER homeostasis, such as ER chaperones and ERAD components (Asada et al., 2011).PERK is a transmembrane protein kinase that under ER stress conditions dimerizes and autophosphorylates, favoring the phosphorylation of eIF2α (eukaryotic translation initiation factor 2α). Phosphorylated eIF2α causes a global translational arrest as a fast adaptive reaction (Harding et al., 2000) and favors the selective translation of ATF4 (activating transcription factor 4) (Vattem et al., 2004), regulating the expression genes involved in folding oxidative stress and amino acid metabolism (Harding et al., 2003).
The loss of BIP, which means the lack of a key player in ER stress, triggered by DNA damage could accelerate cell death (Takayanagi et al., 2013).As mentioned above, BiP expression decreased after MDA-MB-231 cells were treated by ATPR leading to unfolded proteins accumulate , as a result of activating ER stress.At the same time, the expression of CHOP increased.CHOP is a 29KDa protein with 169 (human) or 168 (rodents) amino-acid residues.CHOP protein was first identified to be a member of the CCAAT/enhancer binding proteins (C/EBPs) that serves as a dominant negative inhibitor of C/EBPs.CHOP is also known as growth arrest-and DNA damage-inducible gene 153 (GADD153) and DNA-damage-inducible transcript 3 (DDIT3) (Oyadomari et al., 2003).In general, clearance of misfolded ER proteins, along with proapoptotic outputs such as CHOP production.
Vitamin A has been demonstrated to inhibit the induction and retard the growth of experimental tumors.The effect of vitamin A varies in different animals and is also dependent on the site of application (Alizadeh et al., 2014).Isomerization of all-trans RA was done under experimental and physiological conditions.Different isomers activate different receptors, which lead to different biological effects.Retinoids that specifically bind to RXR are called rexinoids and have been effective in cancer treatment.Retinoids are comprised of three units: a bulky hydrophobic region, a linker unit and a polar terminus, which is usually a carboxylic acid.Modification of each unit has generated many more compounds.Active retinoid-receptors consist of RAR/RXR heterodimers, which bind to Retinoic-acid-responsive-elements (RAREs) in retinoid-responsive genes (Baumrucker et al., 2006).In the present study, after cells were treated by ATPR, ATPR and ATRA both reduced the expression of RXRα and the expression of RARβ and RXRβ both decreased while other Retinoic-acid receptors had no significant decrease.Therefore, we suppose that ATPR induced the apoptosis of MDA-MB-231 cells through binding RARβ and RXRβ, and RARβ forms heterodimers with RXRβ.Because of antibody of RARα, the protein expression of RAR α was not detected.
MAPK are a group of protein serine/threonine kinases, which are activated in response to various extra-cellular stimuli including the extracellular signal-regulated kinases (ERKs), the c-Jun N-termial kinases (JNKs) and the p38 MAPK kinases (Zhang et al., 2014).MAPK super-family consists of three serine/threonine kinase cascades.ERKs responsed to growth factors or other external mitogenic signals by promoting cell proliferation and opposing cell death signal.The other two pathway-p38 MAPK and JNK pathways are typically described as stress-activated kinase that promote inflammation, or, in certain cases, programmed cell death (Olson et al., 2004).We found that the ATPR inhibited phosphorylation of JNK, p38, and ERK within 48 h.We previously reported that ATPR could inhibit phosphorylation of p38, JNK and ERK within 2 h.We further extended the time in order to determine which time of ATPR could inhibit phosphorylation of MAPK signal pathway significantly.As a result, ATPR inhibited phosphorylation of p38 after cells were treated by ATPR for 2h.The level of phosphorylation of ERK and JNK reduced continuously within 48h.These data indicated that ATPR induced apoptosis of breast cancer MDA-MB-231 cells involving MAPK signal pathway.In the following work, inhibitors of various MAPK signal pathway would be required to use in treating MDA-MB-231, for instance, SB203580, an inhibitor of p38, PD98059, an inhibitor of ERK and SP600125, an inhibitor of JNK.These inhibitors alone and in combination can further explore the mechanism of MAPK signal pathway in inducing the apoptosis of MDA-MB-231 cells treated by ATPR.
It is not clear whether MAPKs and ER stress are involved in ATPR induced cell death of Breast cancer MDA-MB-231 cells and Little is known about the relevance between ATPR and RARs and RXRs in inducing the apoptosis of breast cancer MDA-MB-231 cells.In the present study, we demonstrated, for the first time, ATPR could induce apoptosis of MDA-MB-231 cell through ER stress and associated with MAPK signal through binding RARβ/RXRβ heterodimer.These data qualify ATPR as a potent anti-tumor drug to better understand the mechanisms that induces the apoptosis of breast cancer cells.So far, we only did the preliminary research, and future further investigation are needed to clearly define the mechanism of apoptosis of ATPR on breast cancer MDA-MB-231.For example, to further confirm the relation about ATPR and ER stress, MAPK signal pathway and retinoic acid receptor, inhibitors of MAPK signal pathway need to be added to the MDA-MB-231 cells.In addition, ER-stress sensors IRE1, PERK, and ATF6 needed to be detected in order to further confirm that ATPR induced apoptosis involving ER stress.
In summary, newly synthesized retinoic acid derivative ATPR could inhibit the growth and induce the apoptosis of MDA-MB-231 cells.The possible mechanism of apoptosis of MDA-MB-231 cells treated by ATPR could be through binding to RARβ/RXRβ heterodimers, then activated ER stress involving MAPK pathway.The mechanisms of apoptosis of breast cancer MDA-MB-231 cells induced by ATPR need to be further studied.These findings support that the potential application of ATPR in the treatment of breast cancer.

Table 1 . Effects of ATRA and ATPR on the Proliferation of MDA-MB-231 Cells Detected by MTT
*p<0.05 vs the DMSO control