- Email: [email protected]
Role of eIF3a in 4-amino-2-trifluoromethyl-phenyl retinate-induced cell differentiation in human chronic myeloid leukemia K562 cells
Accepted Manuscript Role of eIF3a in 4-amino-2-trifluoromethyl-phenyl retinateinduced cell differentiation in human chronic myeloid leukemia K562 cells
Ge Li, Ke Wang, Yue Li, Jinging Ruan, Cong Wang, Yuejiao Qian, Shengqin Zu, Beibei Dai, Yao Meng, Renpeng Zhou, Jingfang Ge, Feihu Chen PII: DOI: Reference:
S0378-1119(18)31069-2 doi:10.1016/j.gene.2018.10.035 GENE 43291
To appear in:
Gene
Received date: Revised date: Accepted date:
18 April 2018 8 October 2018 11 October 2018
Please cite this article as: Ge Li, Ke Wang, Yue Li, Jinging Ruan, Cong Wang, Yuejiao Qian, Shengqin Zu, Beibei Dai, Yao Meng, Renpeng Zhou, Jingfang Ge, Feihu Chen , Role of eIF3a in 4-amino-2-trifluoromethyl-phenyl retinate-induced cell differentiation in human chronic myeloid leukemia K562 cells. Gene (2018), doi:10.1016/ j.gene.2018.10.035
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Role of eIF3a in 4-amino-2-trifluoromethyl-phenyl retinate-induced cell differentiation in human chronic
T
myeloid leukemia K562 cells
IP
Ge Li a,b, Ke Wang a,b, Yue Li a,b, Jinging Ruan a,b, Cong Wang a,b, Yuejiao Qian a,b, Shengqin Zu a,b,
CR
Beibei Dai a,b, Yao Meng a,b, Renpeng Zhou a,b, Jingfang Ge a,b, Feihu Chen a,b *.
a. Anhui Key Laboratory of Bioactivity of Natural Products, School of Pharmacy, Anhui Medical
US
University, Hefei 230032, China; b. The Key Laboratory of Anti-inflammatory and Immune medicine,
AN
Anhui Medical University, Ministry of Education, Hefei 230032, China.
ED
M
*Correspondence: Professor. Feihu Chen, [email protected]; Tel. /fax: +86-551 65161116.
Abstract
PT
4-amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative
CE
designed and synthesized by our team, has been demonstrated its anti-tumor effect through inducing differentiation and inhibiting proliferation. Eukaryotic initiation factor 3a (eIF3a) plays a critical role in
AC
affecting tumor cell proliferation and differentiation. However, whether eIF3a is implicated in chronic myeloid leukemia cells differentiation remains unclear. Our results demonstrated that eIF3a could be suppressed by ATPR in K562 cells. The results also confirmed that ATPR could arrest cell cycle in G0/G1 phase and induced differentiation. Moreover, over-expression of eIF3a promoted not only protein expression of c-myc and cyclin D1, but also prevented the expression of p-Raf-1, p-ERK and the myeloid differentiation markers CD11b and CD14 and had an influence on inducing the morphologic mature. However, silencing eIF3a expression by small interfering RNA could have an adverse effect on K562 cells. In addition, PD98059 (a MEK inhibitor) could block cell differentiation of CML cells and contributed to
ACCEPTED MANUSCRIPT the expression of c-myc and cyclin D1. In conclusion, these results indicated that eIF3a played an important role in ATPR-induced cell differentiation in K562 cells, its mechanism might be related to its ability in regulating the activation of ERK1/2 signaling pathway in vitro.
AC
CE
PT
ED
M
AN
US
CR
IP
T
Keywords: ATPR; CML; eIF3a; Differentiation; ERK
ACCEPTED MANUSCRIPT 1 Introduction
Chronic myeloid leukemia (CML) is malignant clonal hematopoietic stem cell disease and arises from the appearance of the abnormal Philadelphia chromosome(Harrington, Kizilors, & de Lavallade, 2017). Typically, CML starts with a chronic phase (CP) to accelerated phase (AP) then ending in a terminal
T
phase (TP) called blast crisis (BC), which can be detected by the number and maturation of
IP
leukocytes(Horwitz, Wang, Liu, Wu, Chu, Wang, et al., 2014). Highly proliferating with an undifferentiated state is a common feature of CML tumorigenesis, and targeting at the normal
CR
differentiation of leukemia cells is considered as a novel therapeutic strategy for multiple hematological malignancies (Harris, 2005). As an anti-cancer reagent, ATRA is frequently used to induce maturation of
US
APL cells in clinical treatment(Avvisati & Tallman, 2003; Bobba & Doll, 2012; Saletta, Suryo Rahmanto, & Richardson, 2010), however, ATRA also have a clear effect on cell proliferation, differentiation and
AN
apoptosis of CML cells(Kumar, Khanduja, Verma, Verma, Avti, & Pathak, 2008; Marley, Davidson,
M
Goldman, & Gordon, 2002; Nilsson, Olofsson, & Olsson, 1984). But the adverse reactions of ATRA including severe impacts on teratogenesis and liver toxicity limited its clinical application(Burnett, Wetzler,
ED
& Lowenberg, 2011). Therefore, structural modification of ATRA may provide some new possibilities in reducing its side reaction and improving its function. 4-amino-2-trifluoromethyl-phenyl retinate (ATPR,
PT
patent number: CN101591280, CN102008443A) (Figure 1A) is a reactive derivative of ATRA designed by our team. Results of our previous studies have demonstrated its strong effects of proliferation inhibition and
CE
differentiation induction on tumor cells(H. Liu, Chen, Zhang, Zhou, Gui, & Wang, 2016; N. Wang, Ge, Pan, Peng, Chen, Wang, et al., 2013). However, the underlying mechanism of anti-cancer function of ATPR
AC
remains unclear.
Eukaryotic initiation factors (eIFs) play critical roles in the initiation step of protein translation. Among them, eIF3a, the largest and most core subunit of eIF3, has received more attentions depend on its close relationship with cell development and cancer(Saletta, Suryo Rahmanto, & Richardson, 2010). Apart from that, it has been reported that eIF3a was overexpressed in cancer tissues including lung, breast, colon and bladder(Z. Liu, Dong, Yang, Chen, Pan, Yang, et al., 2007; Pincheira, Chen, & Zhang, 2001; Spilka, Ernst, Bergler, Rainer, Flechsig, Vogetseder, et al., 2014; Yin, Meng, Qian, Li, Chen, Zheng, et al., 2015).
ACCEPTED MANUSCRIPT It has been demonstrated that eIF3a could regulate cell proliferation(C. Fang, Chen, Wu, Yin, Li, Huang, et al., 2017) and negatively related to cell differentiation(Z. Liu, et al., 2007). A previous study confirmed that eIF3a has a marked decline in L-Mimosine blocking mammalian cells at late G1 phase and affecting translation of mRNAs(Dong & Zhang, 2003). In our previous study(Meng, Zhang, Xia, Ge, Chen, 2016), proteomics analysis was used to investigate the changes of protein expression in ATPR-treated K562 cells
T
and the results showed that some key differentiation-related proteins including eIF3a might played crucial
CR
IP
roles in the anti-tumor effect of ATPR.
To investigate the relationship between eIF3a and ATPR-induced differentiation of CML cell, K562
US
cells were cultured and the expression of eIF3a were measured in this study. Moreover, the effects of eIF3a
over-expression or silencing on ATPR-induced cell differentiation were analyzed in K562 cells and
M
AN
the result also showed that ERK signaling pathway might involve in its mechanism.
ED
2 Materials and methods
PT
2.1 Materials
ATPR (purity: 99.66%) was synthesized by our laboratory (School of Pharmacy, Anhui Medical
CE
University). ATRA was purchased from Sigma (USA). Both ATRA and ATPR were dissolved in absolute
AC
alcohol of 10-2 mol/L and kept at -20°C.
Monoclonal rabbit eIF3a antibody, monoclonal rabbit p-Raf-1 (Ser338) antibody, monoclonal rabbit Raf-1, monoclonal rabbit cyclin D1 antibody and monoclonal rabbit c-myc antibody were purchased from Cell Signaling Technology. Monoclonal rabbit p-ERK1/2 antibody and monoclonal rabbit ERK1/2 were purchased from abcam (USA). PD98059 (S1177) was obtained from Selleck (Shanghai, China).
2.2 Cell cultures
ACCEPTED MANUSCRIPT Human chronic myeloid leukemia cell line K562 cells was purchased from KeyGEN BioTECH (Nanjing, China). The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and in humidified atmosphere of 5% CO2 at 37°C.
2.3 Western blot analysis
T
Total protein was collected from cells in RIPA lysis buffer, separated by sodium dodecyl
IP
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then blotted onto PVDF membrane (Millipore
CR
Corp, Billerica, MA, USA). The membranes were incubated in solution with 5% non-fat milk (dissolved in TBST) at 37°C for 2h and probed with primary antibodies eIF3a(1:300)，p-Raf-1 (1:1000)，Raf-1 (1:1000), c-myc(1:1000) and β-actin (1:500) for 4°C
US
p-ERK1/2(1:1000), ERK1/2(1:1000), cyclin D1(1:1000),
overnight. After washing with TBST, the membrane was incubated with secondary antibody. Finally,
AN
protein signals were visualized using the ECL-chemiluminescent kit (ECL-plus; Thermo Fisher Scientific,
ED
2.4 siRNA and Plasmid Transfection
M
Waltham, NH, USA) and then exposed to X-ray film.
The cells were transfected with 5ug of eIF3a siRNA or pEGFP-N1-eIF3a and eIF3a negative control
PT
in Opti-MEM (Gibco) medium by using Lipofectamine 2000 reagent (Invitrogen, Co). After cells were harvested at 6-8h, the cells were resuspended in a complete medium for further analysis. Small interfering
CE
RNA (siRNA) against eIF3a gene (GenePharma, Shanghai, China) has a sequence of sense: 5’-CCAUGAUAUUGCCCAGCAATT-3’,
antisense:
5’-UUGCUGGGCAAUAUCAUGGTT-3’.
The
AC
overexpression eIF3a plasmid (pEGFP-N1-eIF3a) was purchased from Personalbio (Shanghai, China).
2.5 Morphological assessment
The K562 cells morphology changes were evaluated by conventional light-field microscopy using the Olympus (Japan) optical microscopy after cells stained with Wright-Giemsa staining solution (Jiancheng Biology, China).
2.6 Cell cycle analysis
ACCEPTED MANUSCRIPT Cells were harvested after treatment and washed with PBS, then collected by centrifugation and fixed in ice-cold 70% ethanol at least 2h. Then cells were washed with PBS and stained with PI (1mg/mL) in the presence of 1% RNase A and incubated at 37°C for 30 min and analyzed using a flow cytometer (BD Biosciences, USA).
T
2.7 Differentiation marker analysis
IP
The expression of CD11b and CD14 antigen (BioLegend, USA) on the surface of K562 cells were
CR
measured by flow cytometry. After treatment, cells were washed with PBS twice, then incubated with respective monoclonal antibody CD11b (CD11b-APC/Cy7) or CD14 (CD14-APC) for 30 min. The cells
US
were then washed twice with PBS and finally resuspended in 400μL PBS for measurement. CD11b and
AN
CD14 expression levels were measured using flow cytometry (BD Biosciences, USA).
2.8 Statistical analysis
M
All data were expressed as the mean ± SD. One-way analysis of variance (ANOVA) and unpaired
ED
Student’s t-test were performed for the group comparison. P<0.05 and P<0.01 were considered statistically
PT
significant.
CE
3 Results
AC
3.1 ATPR inhibits eIF3a expression and affects cell cycle distribution in K562 cells
To study whether ATPR has an impact on the expression level of eIF3a in CML cells, the protein expression of eIF3a treated with ATPR was measured using western blot (Fig. 1B and 1C). The results revealed that the expression of eIF3a was decreased distinctly after treatment with 10-6 M ATPR lasting from 48 h to 72h. Therefore, we pick 10-6 M and 72h as ATPR best stimulation concentration and time point. These results suggest that ATPR could suppress the expression of eIF3a in K562 cells in vitro. And ATPR had a stronger effect on suppressing the expression of eIF3a than ATRA.
ACCEPTED MANUSCRIPT According to Fig. 1D, the cell in the G0/G1 phase was increased in the ATPR or ATRA treated groups, while the cell in S phase was decreased. The result prompted that ATPR could affect cell cycle distribution
AC
CE
PT
ED
M
AN
US
CR
IP
T
and caused G0/G1 arrest in K562 cells.
ACCEPTED MANUSCRIPT Figure 1. ATPR inhibits the expression of eIF3a and cellular proliferation activity in K562 cells. (A) Structure of 4-amino-2-trifloromethyl-phenyl retinate (ATPR). (B) K562 cells were treated with an ATPR concentration gradient (10-5, 10-6, 10-7, 10-8, 10-9 M) or ATRA (10-6 M) for 72 h. (C) K562 cells were treated with ATPR at different points (0, 24, 48, 72 h) or ATRA (10-6M) for 72h. The protein expression of eIF3a was assessed by western blot. (D) K562 cells were treated with ATPR or ATRA at 10-6 M, 72h for analysis of cell cycle distribution. All the data are expressed in *P<0.05, **P<0.01, versus control group.
IP
T
mean ± SD of three independent experiments.
CR
3.2 eIF3a overexpression reduces ATPR-induced differentiation in K562 cells
US
As shown in Fig. 2A, a significant increase of eIF3a could be detected after transfected with pEGFP-N1-eIF3a in K562 cells. The protein expression of cyclin D1 and c-myc were decreased and the
AN
expression of phosphorylated Raf-1 and phosphorylated ERK were increased after ATRA or ATPR treated alone (Fig. 2B). However, These changes were reversed after transfection of pEGFP-N1-eIF3a. The
M
morphologic changes of K562 cells were shown in Fig. 2C. Compared with the negative control, the matured granulocytes in ATPR-treated group had smaller or irregular nucleus and a decreased ratio of
ED
nuclear area to cytoplasmic area. But after infecting pEGFP-N1-eIF3a, the characteristics of matured
PT
granulocytes induced by ATPR or ATRA could be reduced. Moreover, flow cytometric analysis demonstrated that the expression of CD11b (a granulocytic differentiation marker) was significantly
CE
elevated after treatment with ATPR or ATRA, but the result showed an apparently decreasing percentage of CD11b-positive cells after transfected pEGFP-N1-eIF3a (Fig. 2D). Similarly, after overexpression of
AC
eIF3a, ATRA or ATPR-induced stable expression of CD14 (a monocyte differentiation marker) was decreased in K562 cells(Fig. 2E). All these data exposed that ATPR might have a strong effect on inducing K562 cells differentiation, and up-regulation of eIF3a could suppress it.
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
Figure 2. Overexpression of eIF3a inhibits ATPR-induced differentiation in K562 cells. After transfection with eIF3a negative control or pEGFP-N1-eIF3a, K562 cells were treated with ATPR (10-6 M) or ATRA (10-6 M) for 72h. After that, (A and B) the protein expression of eIF3a, p-Raf-1, p-ERK, cyclin D1 and c-myc were determined by western blot analysis. (C) Wright–Giemsa staining results showed cell morphological changes in K562 cells and it could be detected that the rate of natured cells with irregular nuclear and decreased nuclear/cytoplasm ratio was climbing from 11.1% to over 63.5% or 60.5% after ATPR or ATRA treated. The rates had an apparent decline when transfecting eIF3a. Cell differentiation markers CD11b (D) and CD14 (E) expressions were measured by flow
ACCEPTED MANUSCRIPT cytometry. Data were presented as mean ± SD of three independent experiments. *P<0.05, **P<0.01 versus negative control or ATPR, #P<0.05, ##P<0.01 versus ATRA group.
3.3 eIF3a silencing could influences ATPR-induced differentiation in K562 cells.
In order to further confirmed preceding results, eIF3a siRNAs was used to silence the expression of
T
eIF3a. As shown in Fig. 3A, the expression of eIF3a was successfully suppressed in K562 cells after
IP
transfected with eIF3a siRNA. The results also showed that cyclin D1 and c-myc expression were declined
CR
in ATPR or ATRA-treated groups, and the expression of phosphorylated Raf-1 and phosphorylated ERK are the opposites (Fig. 3B). These changes were more significant after transfected with eIF3a siRNA. In
US
K562 cells, ATPR or ATRA-treated cells displayed matured granulocytes with smaller or irregular nucleus and decreased nuclear/cytoplasm ratio. After using eIF3a siRNA, more significantly matured appearances
AN
with kidney-shape nucleus and decreased nuclear/cytoplasm ratio could be detected (in Fig. 3C). The relative proportions of CD11b and CD14 in K562 cells have a clear increase after ATRA or ATPR treatment,
M
and it presented more meaningful increasing both in CD11b and in CD14 expression after transfected with eIF3a siRNA (Fig. 3D and 3E). Therefore, the studies further indicated that silence of eIF3a could
ED
decelerate the proliferation process of K562 cells and accelerated its differentiation. Take together, these results showed that ATPR could induce K562 cells differentiation and eIF3a is negatively correlated with
AC
CE
PT
ATPR-induced cell differentiation.
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
Figure 3. Transfection of K562 cells with eIF3a siRNA facilitates ATPR-induced differentiation. After transfection with negative siRNA or eIF3a siRNA, K562 cells were treated with ATPR (10-6 M) or ATRA (10-6 M) for 72h. After that, (A and B) the protein expression of eIF3a, p-Raf-1, p-ERK, cyclin D1 and c-myc were detected by western blot analysis. (C) Cell morphological assays were assessed by Wright–Giemsa staining. Cell differentiation status was measured by the expression of CD11b (D) and CD14 (E). Data were presented as mean ± SD of three independent experiments. *P< 0.05, **P<0.01, versus negative control or ATPR group, #P<0.05, ##P<0.01 versus ATRA.
ACCEPTED MANUSCRIPT 3.4 ATPR induces K562 cells differentiation by activating the eIF3a-dependent ERK pathway.
In order to verify whether ERK signaling pathway plays a crucial role in ATPR-induced differentiation, we use PD98059, a MEK inhibitor to arrest ERK signaling pathway. The expression of eIF3a, cyclin D1, c-myc, p-ERK and p-Raf-1 were measured by western blot analysis. Compared with
T
negative group, the accelerated expression of cyclin D1 and c-myc could be detected (Fig. 4A) and the
IP
protein level of p-ERK was decelerated (Fig. 4B) after PD98059 pretreatment. But the protein expression of p-Raf-1 and eIF3a had no clear changes after PD98059 incubated. Morphological analysis in Fig. 4C
CR
fully exposed the greater nuclear/cytoplasm ratio and less mature granulocytes when pretreated with PD98059. The levels of both CD11b and CD14 surface antigens diminished apparently in K562 cells after
US
treated with PD98059 (Fig. 4D and 4E). We can draw a conclusion derived from the above results that a blockade of ERK signaling pathway by PD98059 could attenuate ATPR-induced cell differentiation in
AC
CE
PT
ED
M
AN
K562 cells.
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
Figure 4. eIF3a blocks ATPR-induced K562 cells differentiation via ERK1/2 signaling pathway. After
AC
pretreatment with PD98059 (10uM) for 24h, K562 cells were incubated with ATPR (10 -6 M) and ATPR (10-6 M), (A and B) the expressions of eIF3a, p-Raf-1, p-ERK, cyclin D1 and c-myc were determined by western blot analysis. (C) Cell morphological assays was assessed by Wright–Giemsa staining. CD11b (D) and CD14 (E) were detected by flow cytometry. Data are expressed in mean ± SD of three independent experiments and indicates statistical significance versus negative control or ATPR at *P<0.05, **P<0.01, versus ATRA group at #P<0.05, ##P<0.01.
3.6 ATPR could induce cell differentiation in NB4 cells
ACCEPTED MANUSCRIPT The APL cell line NB4 cells was used to demonstrate the general effect and function of ATPR on other leukemia cells. As shown in Fig. 5A, western blot result showed that ATPR could facilitate the accumulation of eIF3a expression. Furthermore, Wright–Giemsa staining assay detected that ATPR could also induce NB4 cells differentiation. Obviously, after induction for 72 hours with ATPR (10-6 M) or ATRA (10-6 M), a greater fraction of mature cells with smaller nucleus were revealed in Fig. 5B. Flow
T
cytometry results showed that ATPR promoted the expression of CD11b in NB4 cells (Fig. 5C). Moreover,
AC
CE
PT
ED
M
AN
US
CR
IP
10-6 M ATPR treatment led to a significant increase of the CD14 antigens levels in Fig. 5D.
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
Figure 5. ATPR accelerates myeloid differentiation in NB4 cells. NB4 cells were treated with 10-6 M ATPR or ATRA for 72 hours. (A) The expression of eIF3a was detected by western blot analysis. (B) The cell morphological changes were observed by Wright–Giemsa staining assays. The expressions of CD11b (C) and CD14 (D) were evaluated by flow cytometric analysis. The results represent the mean ± SD of data from three independent experiments. *P<0.05, **P<0.01 versus control group.
ACCEPTED MANUSCRIPT 4 Discussions
In this study, we investigated the role of eIF3a in the anti-tumor effect of ATPR on CML and APL cells. Our results showed that the expression of eIF3a in K562 and NB4 cells were decreased after being treated by ATPR, and ATPR could induce a decline of cyclin D1 and c-myc expression, but accelerated the
T
expression of phosphorylated ERK, phosphorylated Raf-1 and myeloid differentiation markers CD11b and
IP
CD14 and also have an influence on the morphology changes. Moreover, overexpression of eIF3a could induce the expression of cyclin D1 and c-myc, decelerated the expression of p-ERK and p-Raf-1 and
CR
suppressed ATPR-induced K562 cells differentiation. However, ATPR-induced differentiation could be impelled when using eIF3a siRNA to down-regulate eIF3a expression. Furthermore, PD98059, a MEK
US
inhibitor, could restrain ATPR-induced differentiation in K562 cells. These results indicated that eIF3a
AN
played an important role in ATPR-induced cell differentiation in K562 cells, which might be involved with its ability in regulating the activation of ERK signaling pathway in vitro. What’s more, this study proved
M
that ATPR could also induce NB4 cells differentiation.
ED
The evidence revealed that ATRA could effectively block acquisition of resistance in CML cells on imatinib treatment and induced cell differentiation.(Horwitz, et al., 2014) As a derivative of all-trans
PT
retinoic acid, ATPR also showed its significant effect on inhibiting proliferation and inducing differentiation in our previous studies(Fan, Cheng, Wang, Gui, Chen, Zhou, et al., 2014; Hu, Pan, Chen,
CE
Qin, Wu, Zhu, et al., 2014; H. Wang, Gui, Chen, Zhou, & Wang, 2013; N. Wang, et al., 2013), with a preferable solubility and stability to ATRA. It has been demonstrated that ATPR could inhibit proliferation
AC
and induced differentiation in human gastric carcinoma xenografts via up-regulating retinoic acid receptor β(Ju, Wang, Wang, & Chen, 2015). ATPR also causes cell cycle arrest in human hepatocellular carcinoma and gastric cancer in vitro(H. Liu, Chen, Zhang, Zhou, Gui, & Wang, 2016; Xia, Zhao, Wang, Qiao, Zhang, Yin, et al., 2017). Recently, our studies first confirmed that ATPR could modulate NB4 cells differentiation by inducing autophagy(Li, Li, Wang, Xie, Zhou, Meng, et al., 2017). Congruously, our results demonstrated that ATPR could attenuate the expression of cyclin D1 and c-myc and caused G0/G1 phase arrest in K562 cells. Moreover, ATPR accelerated the expression of CD11b and CD14 and had a positive impact on cell morphological changes both in CML and APL cell lines. These results indicated the
ACCEPTED MANUSCRIPT anti-tumor effect of ATPR again. In our previous study (Meng, Zhang, Xia, Ge, Chen, 2016), preliminary proteomic analysis was used to investigate the potential molecular mechanisms and specific targets underlining the effect of ATPR in K562 cells. The result showed that eIF family were extremely likely to be core proteins in ATPR-inducing differentiation in K562 cells, especially eIF3a. This study further
T
revealed that the protein expression of eIF3a was down-regulate after ATPR-treated.
IP
As a eukaryotic translation initiation factor, eIF3a has been revealed that could interact complicatedly with multiple factors and played a unique role in modulating tumor. Increasing researches have uncovered
CR
a strong connection between eIF3a and cancer for the extremely high frequency of gene occurs in biological process, such as cell cycle, growth, proliferation, differentiation and division(Saletta, Suryo
US
Rahmanto, & Richardson, 2010). What’s more, knockdown of eIF3a caused significant decrease in cell proliferation and motility ability in pancreatic cancer cells by depleting clonogenic abilities, cell growth rates
AN
and invasion abilities(S. Q. Wang, Liu, Yao, & Jin, 2016). L-mimosine, a plant amino acid, could reversibly
M
block mammalian cells at late G1 phase and accelerated functional differentiation by affecting translation of mRNAs of eIF3a(Dong, Arnold, Yang, Park, Hrncirova, Mechref, et al., 2005). Dong.et.al verified that
ED
reducing eIF3a expression oscillated with cell cycle and peaks in S phase and also reduced cell proliferation rate by elongating cell cycle, but the cell cycle distribution had no changes(Dong, Liu, Cui,
PT
Pincheira, Yang, Liu, et al., 2009). Yu Zhang et al. have proposed that the increased expression of eIF3a improves cisplatin sensitivity in ovarian cancer probably by regulating p27 Kip1 and XPC translation(Zhang,
CE
Yu, Tian, Li, Zhang, Zhang, et al., 2015). However, it remains unclear whether eIF3a has a similar function in hematological diseases. Our current data first clarified that ATPR (10-6 M) could suppress the protein
AC
expression of eIF3a. In order to prove eIF3a involved in ATPR-induced differentiation, K562 cells were transfected with pEGFP-N1-eIF3a to up-regulating eIF3a. Results confirmed that cyclin D1 and c-myc were increased significantly and ATPR-induced ERK activation and the myeloid differentiation had an obvious lessening after transfected. Moreover, ATPR-induced cell differentiation could be further promoted by using eIF3a siRNA. Hence, these results demonstrated that eIF3a negatively correlated with ATPR-induced K562 cells differentiation.
ACCEPTED MANUSCRIPT ERK pathway is activated by receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR). After the formation of complex with guanine nucleotide exchange factor (GEF) SOS, the adaptor proteins SHC and Grb2, Ras, an upstream activator of the ERK pathway, could be stimulated by SOS/SHC/Grb2 complex binding to specific phosphotyrosines at the EGFR and have a high concentration around with Ras. Then Raf is recruited to the plasma membrane and activated by an interaction with
T
Ras(Shaul & Seger, 2007). The Raf gene has three subunits, A-Raf, B-Raf and Raf-1 (c-Raf), which have
IP
individual properties after being triggered. Its dephosphorylated at S259 site and phosphorylated at S338
CR
site are requisite for Raf-1 excitation(Dhillon, Meikle, Yazici, Eulitz, & Kolch, 2002; Dhillon, von Kriegsheim, Grindlay, & Kolch, 2007). Activated Raf-1 could phosphorylates MEK, immediately, ERK
US
would be activated at downstream and then induce diversified biological effects. Prior publications showed that ERK signaling pathway significantly support cell proliferation(Yang, Ding, Guo, Zheng, Wang, Sun, et
al.,
2017)
and
tumor
AN
al., 2017), differentiation (Shao, Liu, Li, Xian, Zhou, Yang, et al., 2016; Tang, Chai, Ye, Yu, Cao, Yang, et formation(Quintero
Barceinas,
Garcia-Regalado,
Arechaga-Ocampo,
M
Villegas-Sepulveda, & Gonzalez-De la Rosa, 2015). Moreover, the expression of p-ERK occurs
ED
progressively as cell differentiation is induced by dasatinib, but MEK inhibitors PD98059 and U0216 can block the myeloid differentiation(Y. Fang, Zhong, Lin, Zhou, Jing, Ying, et al., 2013). Our previous studies
PT
found that ATPR inhibited phosphorylation of ERK within 48h, but showed a slight increase at 48h(B. Wang, Yan, Zhou, Gui, Chen, & Wang, 2014). In this report, we clarified that enhanced phosphorylation of
CE
ERK could be detected after cells were treated by ATPR for 72h. It has been reported that the rapid and sustained activation of MEK/ERK may be required for cell differentiation, whereas not benefit Barceinas,
Garcia-Regalado,
Arechaga-Ocampo,
Villegas-Sepulveda,
&
AC
proliferation(Quintero
Gonzalez-De la Rosa, 2015). It means that the discrepant observations are probably related to the ways ERK was activated. This phenomenon may explain why activation of ERK could have a promoting effect both on cell proliferation and differentiation. In this study, we detected that ATPR could activate ERK signaling and promoted K562 cells differentiation. What’s more, attenuation of the ERK activation blocked not only the cell differentiation but also the increased protein expression of cyclin D1 and c-myc. It indicated that MEK/ERK signaling pathway might positively modulate ATPR-induced differentiation by regulating the protein expression of cyclin D1 and c-myc. Interestingly, it is displayed that eIF3a regulates
ACCEPTED MANUSCRIPT ERK signaling and its biological effects via binding to Raf-1(Xu, Lu, Romano, Pitt, Houslay, Milligan, et al., 2012). Our findings testified similar results that Raf-1 was increased and eIF3a was decreased after ATPR treatment, with no significant differences after PD98059 pretreatment. This phenomenon indicated that eIF3a regulated the ERK signaling pathway by inhibiting Raf-1 and eIF3a might involve in
T
ATPR-induced differentiation via affecting the activation of ERK signaling.
IP
In conclusion, our data confirmed that ATPR could down-regulate eIF3a expression and eIF3a plays a important role in ATPR promoted cell differentiation in K562 cells in vitro, which might be involved the
CR
activation of ERK signaling pathway. The data presented here warrant that ATPR may lead to a new
AN
US
probability for CML and other leukemia therapies.
M
Acknowledgments
ED
This work was supported by the National Major Scientific and Technological Special Project for “Significant New Drugs Development” (No 2011ZX09401). Thanks are due to School of Basic Medical
AC
CE
PT
Sciences from Anhui Medical University for providing technical support for flow cytometry.
ACCEPTED MANUSCRIPT References
AC
CE
PT
ED
M
AN
US
CR
IP
T
Avvisati, G., & Tallman, M. S. (2003). All-trans retinoic acid in acute promyelocytic leukaemia. Best Pract Res Clin Haematol, 16(3), 419-432. Bobba, R. K., & Doll, D. C. (2012). Promyelocytic blast crisis of chronic myelogenous leukemia. Blood, 120(19), 3873. Burnett, A., Wetzler, M., & Lowenberg, B. (2011). Therapeutic advances in acute myeloid leukemia. J Clin Oncol, 29(5), 487-494. Dhillon, A. S., Meikle, S., Yazici, Z., Eulitz, M., & Kolch, W. (2002). Regulation of Raf-1 activation and signalling by dephosphorylation. EMBO J, 21(1-2), 64-71. Dhillon, A. S., von Kriegsheim, A., Grindlay, J., & Kolch, W. (2007). Phosphatase and feedback regulation of Raf-1 signaling. Cell Cycle, 6(1), 3-7. Dong, Z., Arnold, R. J., Yang, Y., Park, M. H., Hrncirova, P., Mechref, Y., Novotny, M. V., & Zhang, J. T. (2005). Modulation of differentiation-related gene 1 expression by cell cycle blocker mimosine, revealed by proteomic analysis. Mol Cell Proteomics, 4(7), 993-1001. Dong, Z., Liu, Z., Cui, P., Pincheira, R., Yang, Y., Liu, J., & Zhang, J. T. (2009). Role of eIF3a in regulating cell cycle progression. Exp Cell Res, 315(11), 1889-1894. Dong, Z., & Zhang, J. T. (2003). EIF3 p170, a mediator of mimosine effect on protein synthesis and cell cycle progression. Mol Biol Cell, 14(9), 3942-3951. Fan, T. T., Cheng, Y., Wang, Y. F., Gui, S. Y., Chen, F. H., Zhou, Q., & Wang, Y. (2014). A novel all-trans retinoid acid derivative N-(3-trifluoromethyl- phenyl)- retinamide inhibits lung adenocarcinoma A549 cell migration through down-regulating expression of myosin light chain kinase. Asian Pac J Cancer Prev, 15(18), 7687-7692. Fang, C., Chen, Y. X., Wu, N. Y., Yin, J. Y., Li, X. P., Huang, H. S., Zhang, W., Zhou, H. H., & Liu, Z. Q. (2017). MiR-488 inhibits proliferation and cisplatin sensibility in non-small-cell lung cancer (NSCLC) cells by activating the eIF3a-mediated NER signaling pathway. Sci Rep, 7, 40384. Fang, Y., Zhong, L., Lin, M., Zhou, X., Jing, H., Ying, M., Luo, P., Yang, B., & He, Q. (2013). MEK/ERK dependent activation of STAT1 mediates dasatinib-induced differentiation of acute myeloid leukemia. PLoS One, 8(6), e66915. Harrington, P., Kizilors, A., & de Lavallade, H. (2017). The Role of Early Molecular Response in the Management of Chronic Phase CML. Curr Hematol Malig Rep, 12(2), 79-84. Harris, H. (2005). A long view of fashions in cancer research. Bioessays, 27(8), 833-838. Horwitz, M. S., Wang, Z., Liu, Z., Wu, X., Chu, S., Wang, J., Yuan, H., Roth, M., Yuan, Y.-C., Bhatia, R., & Chen, W. (2014). ATRA-Induced Cellular Differentiation and CD38 Expression Inhibits Acquisition of BCR-ABL Mutations for CML Acquired Resistance. PLoS Genetics, 10(6), e1004414. Hu, K. W., Pan, X. H., Chen, F. H., Qin, R., Wu, L. M., Zhu, H. G., Wu, F. R., Ge, J. F., Han, W. X., Yin, C. L., & Li, H. J. (2014). A novel retinoic acid analog, 4-amino-2-trifluoromethyl-phenyl retinate, inhibits gastric cancer cell growth. Int J Mol Med, 33(2), 415-422. Ju, J., Wang, N., Wang, X., & Chen, F. (2015). A novel all-trans retinoic acid derivative inhibits proliferation and induces differentiation of human gastric carcinoma xenografts via up-regulating retinoic acid receptor beta. Am J Transl Res, 7(5), 856-865. Kumar, S., Khanduja, K. L., Verma, N., Verma, S. C., Avti, P. K., & Pathak, C. M. (2008). ATRA promotes alpha tocopherol succinate-induced apoptosis in freshly isolated leukemic cells from chronic myeloid leukemic patients. Mol Cell Biochem, 307(1-2), 109-119. Li, Y., Li, G., Wang, K., Xie, Y. Y., Zhou, R. P., Meng, Y., Ding, R., Ge, J. F., & Chen, F. H. (2017). Autophagy contributes to 4-Amino-2-Trifluoromethyl-Phenyl Retinate-induced differentiation in human acute promyelocytic leukemia NB4 cells. Toxicol Appl Pharmacol, 319, 1-11. Liu, H., Chen, F., Zhang, L., Zhou, Q., Gui, S., & Wang, Y. (2016). A novel all-trans retinoic acid derivative 4-amino2trifluoromethyl-phenyl retinate inhibits the proliferation of human hepatocellular carcinoma HepG2 cells by inducing G0/G1 cell cycle arrest and apoptosis via upregulation of p53 and ASPP1 and downregulation of iASPP. Oncol Rep, 36(1), 333-341.
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
Liu, Z., Dong, Z., Yang, Z., Chen, Q., Pan, Y., Yang, Y., Cui, P., Zhang, X., & Zhang, J. T. (2007). Role of eIF3a (eIF3 p170) in intestinal cell differentiation and its association with early development. Differentiation, 75(7), 652-661. Marley, S. B., Davidson, R. J., Goldman, J. M., & Gordon, M. Y. (2002). Effects of combinations of therapeutic agents on the proliferation of progenitor cells in chronic myeloid leukaemia. Br J Haematol, 116(1), 162-165. Nilsson, B., Olofsson, T., & Olsson, I. (1984). Myeloid differentiation in liquid cultures of cells from patients with chronic myeloid leukemia: effects of retinoic acid and indomethacin. Exp Hematol, 12(2), 91-99. Pincheira, R., Chen, Q., & Zhang, J. T. (2001). Identification of a 170-kDa protein over-expressed in lung cancers. Br J Cancer, 84(11), 1520-1527. Quintero Barceinas, R. S., Garcia-Regalado, A., Arechaga-Ocampo, E., Villegas-Sepulveda, N., & Gonzalez-De la Rosa, C. H. (2015). All-Trans Retinoic Acid Induces Proliferation, Survival, and Migration in A549 Lung Cancer Cells by Activating the ERK Signaling Pathway through a Transcription-Independent Mechanism. Biomed Res Int, 2015, 404368. Saletta, F., Suryo Rahmanto, Y., & Richardson, D. R. (2010). The translational regulator eIF3a: the tricky eIF3 subunit! Biochim Biophys Acta, 1806(2), 275-286. Shao, X., Liu, Y., Li, Y., Xian, M., Zhou, Q., Yang, B., Ying, M., & He, Q. (2016). The HER2 inhibitor TAK165 Sensitizes Human Acute Myeloid Leukemia Cells to Retinoic Acid-Induced Myeloid Differentiation by activating MEK/ERK mediated RARalpha/STAT1 axis. Sci Rep, 6, 24589. Shaul, Y. D., & Seger, R. (2007). The MEK/ERK cascade: from signaling specificity to diverse functions. Biochim Biophys Acta, 1773(8), 1213-1226. Spilka, R., Ernst, C., Bergler, H., Rainer, J., Flechsig, S., Vogetseder, A., Lederer, E., Benesch, M., Brunner, A., Geley, S., Eger, A., Bachmann, F., Doppler, W., Obrist, P., & Haybaeck, J. (2014). eIF3a is over-expressed in urinary bladder cancer and influences its phenotype independent of translation initiation. Cell Oncol (Dordr), 37(4), 253-267. Tang, L., Chai, W., Ye, F., Yu, Y., Cao, L., Yang, M., Xie, M., & Yang, L. (2017). HMGB1 promotes differentiation syndrome by inducing hyperinflammation via MEK/ERK signaling in acute promyelocytic leukemia cells. Oncotarget, 8(16), 27314-27327. Wang, B., Yan, Y. W., Zhou, Q., Gui, S. Y., Chen, F. H., & Wang, Y. (2014). A novel all-trans retinoid acid derivative induces apoptosis in MDA-MB-231 breast cancer cells. Asian Pac J Cancer Prev, 15(24), 10819-10824. Wang, H., Gui, S. Y., Chen, F. H., Zhou, Q., & Wang, Y. (2013). New insights into 4-amino-2-tri-fluoromethyl-phenyl ester inhibition of cell growth and migration in the A549 lung adenocarcinoma cell line. Asian Pac J Cancer Prev, 14(12), 7265-7270. Wang, N., Ge, J. F., Pan, C. X., Peng, X. Q., Chen, H. H., Wang, X. Q., Tang, J., Hu, W., & Chen, F. H. (2013). Anti-tumor effect of 4-Amino-2-Trifluoromethyl-Phenyl Retinate on human breast cancer MCF-7 cells via up-regulation of retinoid receptor-induced gene-1. Biomed Pharmacother, 67(8), 687-692. Wang, S. Q., Liu, Y., Yao, M. Y., & Jin, J. (2016). Eukaryotic Translation Initiation Factor 3a (eIF3a) Promotes Cell Proliferation and Motility in Pancreatic Cancer. J Korean Med Sci, 31(10), 1586-1594. Xia, Q., Zhao, Y., Wang, J., Qiao, W., Zhang, D., Yin, H., Xu, D., & Chen, F. (2017). Proteomic analysis of cell cycle arrest and differentiation induction caused by ATPR, a derivative of all-trans retinoic acid, in human gastric cancer SGC-7901 cells. Proteomics Clin Appl, 11(7-8). Xu, T. R., Lu, R. F., Romano, D., Pitt, A., Houslay, M. D., Milligan, G., & Kolch, W. (2012). Eukaryotic translation initiation factor 3, subunit a, regulates the extracellular signal-regulated kinase pathway. Mol Cell Biol, 32(1), 88-95. Yang, Y., Ding, L., Guo, Z. K., Zheng, X. L., Wang, L. S., Sun, H. Y., Jin, Z. G., & Wang, H. X. (2017). The epigenetically-regulated miR-34a targeting c-SRC suppresses RAF/MEK/ERK signaling pathway in K-562 cells. Leuk Res, 55, 91-96. Yin, J. Y., Meng, X. G., Qian, C. Y., Li, X. P., Chen, J., Zheng, Y., Liu, R., Zhou, H. H., & Liu, Z. Q. (2015). Association of positively selected eIF3a polymorphisms with toxicity of platinum-based chemotherapy in NSCLC patients. Acta Pharmacol Sin, 36(3), 375-384.
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
Zhang, Y., Yu, J. J., Tian, Y., Li, Z. Z., Zhang, C. Y., Zhang, S. F., Cao, L. Q., Zhang, Y., Qian, C. Y., Zhang, W., Zhou, H. H., Yin, J. Y., & Liu, Z. Q. (2015). eIF3a improve cisplatin sensitivity in ovarian cancer by regulating XPC and p27Kip1 translation. Oncotarget, 6(28), 25441-25451. Meng, Y.; Zhang, D.; Xia, Q.; Ge, J.; Chen, F. (2016). The proteomics research of 4-amino-2-trifluoromethyl-phenylretinate on human leukemia K562 cells. Chinese Pharmacological Bulletin. 32(1), 27-32.(in Chinese)
ACCEPTED MANUSCRIPT Abbreviations list Full name
ATRA
All-trans retinoic acid
ATPR
4-Amino-2-Trifluoromethyl-Phenyl Retinate
AP
Ammonium persulphate
APL
Acute promyelocytic leukemia
CML
Chronic myeloid leukemia
DEPC
Diethyl pyrocarbonate
DMSO
Dimethyl sμlfoxide
EGFR
Epidermal growth factor receptor
ERK
Extracellular regulated protein kinases
eIF3a
Eukaryotic initiation factors
FBS
Fetal Bovine Serum
IP
CR
US
AN
M Gram
ED
G GEF
Guanine nucleotide exchange factor
PT
Gly
CE
H IRE
AC
M mA
T
Abbreviations
Glycine Hour Iron-responsive element mol/L Milliampere
MEK
Mitogen-activated protein kinase kinase
Mg
Milligramme
mRNA
Messenger ribnucleic acid
Min
Minue
ml
Milliliter
mM
mmol/L
ACCEPTED MANUSCRIPT
nmol/L
PAGE
Polyacrylamide gel electrophoresis
PBS
Phosphate buffered saline
PD98059
MEK inhibitor
PML
Promyelocytic leukemia gene
PMSF
Phenylmethyl sμlfonylfluoride
PVDF
PolyVinylidene difiuoride
RAR
Retinoic acid receptor
rpm
Round per minute
RXR
Retinoic X receptor
s
Second
SDS
Sodium dobecyl sμlphate
siRNA
Small interfering RNA
M
AN
US
CR
IP
T
nM
N’N’N’N-Tetramethylethylenediamine
ED
TEMED Tris
Transmission electron microscopy Voltage
PT
V
AC
CE
μM
μmol/L
ACCEPTED MANUSCRIPT Research highlights < eIF3a could be suppressed by 4-amino-2-trifluoromethyl-phenyl retinate (ATPR) in CML cell line K562 cells > < eIF3a involved in ATPR-induced differentiation in K562 cells >
AC
CE
PT
ED
M
AN
US
CR
IP
T
ATPR-induced cell differentiation in K562 cells >
Figure 1
Figure 2abc
Figure 2d
Figure 2e
Figure 3ac
Figure 3d
Figure 3e
Figure 4abc
Figure 4d
Figure 4e
Figure 5ab
Figure 5cd
Ge Li, Ke Wang, Yue Li, Jinging Ruan, Cong Wang, Yuejiao Qian, Shengqin Zu, Beibei Dai, Yao Meng, Renpeng Zhou, Jingfang Ge, Feihu Chen PII: DOI: Reference:
S0378-1119(18)31069-2 doi:10.1016/j.gene.2018.10.035 GENE 43291
To appear in:
Gene
Received date: Revised date: Accepted date:
18 April 2018 8 October 2018 11 October 2018
Please cite this article as: Ge Li, Ke Wang, Yue Li, Jinging Ruan, Cong Wang, Yuejiao Qian, Shengqin Zu, Beibei Dai, Yao Meng, Renpeng Zhou, Jingfang Ge, Feihu Chen , Role of eIF3a in 4-amino-2-trifluoromethyl-phenyl retinate-induced cell differentiation in human chronic myeloid leukemia K562 cells. Gene (2018), doi:10.1016/ j.gene.2018.10.035
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Role of eIF3a in 4-amino-2-trifluoromethyl-phenyl retinate-induced cell differentiation in human chronic
T
myeloid leukemia K562 cells
IP
Ge Li a,b, Ke Wang a,b, Yue Li a,b, Jinging Ruan a,b, Cong Wang a,b, Yuejiao Qian a,b, Shengqin Zu a,b,
CR
Beibei Dai a,b, Yao Meng a,b, Renpeng Zhou a,b, Jingfang Ge a,b, Feihu Chen a,b *.
a. Anhui Key Laboratory of Bioactivity of Natural Products, School of Pharmacy, Anhui Medical
US
University, Hefei 230032, China; b. The Key Laboratory of Anti-inflammatory and Immune medicine,
AN
Anhui Medical University, Ministry of Education, Hefei 230032, China.
ED
M
*Correspondence: Professor. Feihu Chen, [email protected]; Tel. /fax: +86-551 65161116.
Abstract
PT
4-amino-2-trifluoromethyl-phenyl retinate (ATPR), a novel all-trans retinoic acid (ATRA) derivative
CE
designed and synthesized by our team, has been demonstrated its anti-tumor effect through inducing differentiation and inhibiting proliferation. Eukaryotic initiation factor 3a (eIF3a) plays a critical role in
AC
affecting tumor cell proliferation and differentiation. However, whether eIF3a is implicated in chronic myeloid leukemia cells differentiation remains unclear. Our results demonstrated that eIF3a could be suppressed by ATPR in K562 cells. The results also confirmed that ATPR could arrest cell cycle in G0/G1 phase and induced differentiation. Moreover, over-expression of eIF3a promoted not only protein expression of c-myc and cyclin D1, but also prevented the expression of p-Raf-1, p-ERK and the myeloid differentiation markers CD11b and CD14 and had an influence on inducing the morphologic mature. However, silencing eIF3a expression by small interfering RNA could have an adverse effect on K562 cells. In addition, PD98059 (a MEK inhibitor) could block cell differentiation of CML cells and contributed to
ACCEPTED MANUSCRIPT the expression of c-myc and cyclin D1. In conclusion, these results indicated that eIF3a played an important role in ATPR-induced cell differentiation in K562 cells, its mechanism might be related to its ability in regulating the activation of ERK1/2 signaling pathway in vitro.
AC
CE
PT
ED
M
AN
US
CR
IP
T
Keywords: ATPR; CML; eIF3a; Differentiation; ERK
ACCEPTED MANUSCRIPT 1 Introduction
Chronic myeloid leukemia (CML) is malignant clonal hematopoietic stem cell disease and arises from the appearance of the abnormal Philadelphia chromosome(Harrington, Kizilors, & de Lavallade, 2017). Typically, CML starts with a chronic phase (CP) to accelerated phase (AP) then ending in a terminal
T
phase (TP) called blast crisis (BC), which can be detected by the number and maturation of
IP
leukocytes(Horwitz, Wang, Liu, Wu, Chu, Wang, et al., 2014). Highly proliferating with an undifferentiated state is a common feature of CML tumorigenesis, and targeting at the normal
CR
differentiation of leukemia cells is considered as a novel therapeutic strategy for multiple hematological malignancies (Harris, 2005). As an anti-cancer reagent, ATRA is frequently used to induce maturation of
US
APL cells in clinical treatment(Avvisati & Tallman, 2003; Bobba & Doll, 2012; Saletta, Suryo Rahmanto, & Richardson, 2010), however, ATRA also have a clear effect on cell proliferation, differentiation and
AN
apoptosis of CML cells(Kumar, Khanduja, Verma, Verma, Avti, & Pathak, 2008; Marley, Davidson,
M
Goldman, & Gordon, 2002; Nilsson, Olofsson, & Olsson, 1984). But the adverse reactions of ATRA including severe impacts on teratogenesis and liver toxicity limited its clinical application(Burnett, Wetzler,
ED
& Lowenberg, 2011). Therefore, structural modification of ATRA may provide some new possibilities in reducing its side reaction and improving its function. 4-amino-2-trifluoromethyl-phenyl retinate (ATPR,
PT
patent number: CN101591280, CN102008443A) (Figure 1A) is a reactive derivative of ATRA designed by our team. Results of our previous studies have demonstrated its strong effects of proliferation inhibition and
CE
differentiation induction on tumor cells(H. Liu, Chen, Zhang, Zhou, Gui, & Wang, 2016; N. Wang, Ge, Pan, Peng, Chen, Wang, et al., 2013). However, the underlying mechanism of anti-cancer function of ATPR
AC
remains unclear.
Eukaryotic initiation factors (eIFs) play critical roles in the initiation step of protein translation. Among them, eIF3a, the largest and most core subunit of eIF3, has received more attentions depend on its close relationship with cell development and cancer(Saletta, Suryo Rahmanto, & Richardson, 2010). Apart from that, it has been reported that eIF3a was overexpressed in cancer tissues including lung, breast, colon and bladder(Z. Liu, Dong, Yang, Chen, Pan, Yang, et al., 2007; Pincheira, Chen, & Zhang, 2001; Spilka, Ernst, Bergler, Rainer, Flechsig, Vogetseder, et al., 2014; Yin, Meng, Qian, Li, Chen, Zheng, et al., 2015).
ACCEPTED MANUSCRIPT It has been demonstrated that eIF3a could regulate cell proliferation(C. Fang, Chen, Wu, Yin, Li, Huang, et al., 2017) and negatively related to cell differentiation(Z. Liu, et al., 2007). A previous study confirmed that eIF3a has a marked decline in L-Mimosine blocking mammalian cells at late G1 phase and affecting translation of mRNAs(Dong & Zhang, 2003). In our previous study(Meng, Zhang, Xia, Ge, Chen, 2016), proteomics analysis was used to investigate the changes of protein expression in ATPR-treated K562 cells
T
and the results showed that some key differentiation-related proteins including eIF3a might played crucial
CR
IP
roles in the anti-tumor effect of ATPR.
To investigate the relationship between eIF3a and ATPR-induced differentiation of CML cell, K562
US
cells were cultured and the expression of eIF3a were measured in this study. Moreover, the effects of eIF3a
over-expression or silencing on ATPR-induced cell differentiation were analyzed in K562 cells and
M
AN
the result also showed that ERK signaling pathway might involve in its mechanism.
ED
2 Materials and methods
PT
2.1 Materials
ATPR (purity: 99.66%) was synthesized by our laboratory (School of Pharmacy, Anhui Medical
CE
University). ATRA was purchased from Sigma (USA). Both ATRA and ATPR were dissolved in absolute
AC
alcohol of 10-2 mol/L and kept at -20°C.
Monoclonal rabbit eIF3a antibody, monoclonal rabbit p-Raf-1 (Ser338) antibody, monoclonal rabbit Raf-1, monoclonal rabbit cyclin D1 antibody and monoclonal rabbit c-myc antibody were purchased from Cell Signaling Technology. Monoclonal rabbit p-ERK1/2 antibody and monoclonal rabbit ERK1/2 were purchased from abcam (USA). PD98059 (S1177) was obtained from Selleck (Shanghai, China).
2.2 Cell cultures
ACCEPTED MANUSCRIPT Human chronic myeloid leukemia cell line K562 cells was purchased from KeyGEN BioTECH (Nanjing, China). The cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and in humidified atmosphere of 5% CO2 at 37°C.
2.3 Western blot analysis
T
Total protein was collected from cells in RIPA lysis buffer, separated by sodium dodecyl
IP
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then blotted onto PVDF membrane (Millipore
CR
Corp, Billerica, MA, USA). The membranes were incubated in solution with 5% non-fat milk (dissolved in TBST) at 37°C for 2h and probed with primary antibodies eIF3a(1:300)，p-Raf-1 (1:1000)，Raf-1 (1:1000), c-myc(1:1000) and β-actin (1:500) for 4°C
US
p-ERK1/2(1:1000), ERK1/2(1:1000), cyclin D1(1:1000),
overnight. After washing with TBST, the membrane was incubated with secondary antibody. Finally,
AN
protein signals were visualized using the ECL-chemiluminescent kit (ECL-plus; Thermo Fisher Scientific,
ED
2.4 siRNA and Plasmid Transfection
M
Waltham, NH, USA) and then exposed to X-ray film.
The cells were transfected with 5ug of eIF3a siRNA or pEGFP-N1-eIF3a and eIF3a negative control
PT
in Opti-MEM (Gibco) medium by using Lipofectamine 2000 reagent (Invitrogen, Co). After cells were harvested at 6-8h, the cells were resuspended in a complete medium for further analysis. Small interfering
CE
RNA (siRNA) against eIF3a gene (GenePharma, Shanghai, China) has a sequence of sense: 5’-CCAUGAUAUUGCCCAGCAATT-3’,
antisense:
5’-UUGCUGGGCAAUAUCAUGGTT-3’.
The
AC
overexpression eIF3a plasmid (pEGFP-N1-eIF3a) was purchased from Personalbio (Shanghai, China).
2.5 Morphological assessment
The K562 cells morphology changes were evaluated by conventional light-field microscopy using the Olympus (Japan) optical microscopy after cells stained with Wright-Giemsa staining solution (Jiancheng Biology, China).
2.6 Cell cycle analysis
ACCEPTED MANUSCRIPT Cells were harvested after treatment and washed with PBS, then collected by centrifugation and fixed in ice-cold 70% ethanol at least 2h. Then cells were washed with PBS and stained with PI (1mg/mL) in the presence of 1% RNase A and incubated at 37°C for 30 min and analyzed using a flow cytometer (BD Biosciences, USA).
T
2.7 Differentiation marker analysis
IP
The expression of CD11b and CD14 antigen (BioLegend, USA) on the surface of K562 cells were
CR
measured by flow cytometry. After treatment, cells were washed with PBS twice, then incubated with respective monoclonal antibody CD11b (CD11b-APC/Cy7) or CD14 (CD14-APC) for 30 min. The cells
US
were then washed twice with PBS and finally resuspended in 400μL PBS for measurement. CD11b and
AN
CD14 expression levels were measured using flow cytometry (BD Biosciences, USA).
2.8 Statistical analysis
M
All data were expressed as the mean ± SD. One-way analysis of variance (ANOVA) and unpaired
ED
Student’s t-test were performed for the group comparison. P<0.05 and P<0.01 were considered statistically
PT
significant.
CE
3 Results
AC
3.1 ATPR inhibits eIF3a expression and affects cell cycle distribution in K562 cells
To study whether ATPR has an impact on the expression level of eIF3a in CML cells, the protein expression of eIF3a treated with ATPR was measured using western blot (Fig. 1B and 1C). The results revealed that the expression of eIF3a was decreased distinctly after treatment with 10-6 M ATPR lasting from 48 h to 72h. Therefore, we pick 10-6 M and 72h as ATPR best stimulation concentration and time point. These results suggest that ATPR could suppress the expression of eIF3a in K562 cells in vitro. And ATPR had a stronger effect on suppressing the expression of eIF3a than ATRA.
ACCEPTED MANUSCRIPT According to Fig. 1D, the cell in the G0/G1 phase was increased in the ATPR or ATRA treated groups, while the cell in S phase was decreased. The result prompted that ATPR could affect cell cycle distribution
AC
CE
PT
ED
M
AN
US
CR
IP
T
and caused G0/G1 arrest in K562 cells.
ACCEPTED MANUSCRIPT Figure 1. ATPR inhibits the expression of eIF3a and cellular proliferation activity in K562 cells. (A) Structure of 4-amino-2-trifloromethyl-phenyl retinate (ATPR). (B) K562 cells were treated with an ATPR concentration gradient (10-5, 10-6, 10-7, 10-8, 10-9 M) or ATRA (10-6 M) for 72 h. (C) K562 cells were treated with ATPR at different points (0, 24, 48, 72 h) or ATRA (10-6M) for 72h. The protein expression of eIF3a was assessed by western blot. (D) K562 cells were treated with ATPR or ATRA at 10-6 M, 72h for analysis of cell cycle distribution. All the data are expressed in *P<0.05, **P<0.01, versus control group.
IP
T
mean ± SD of three independent experiments.
CR
3.2 eIF3a overexpression reduces ATPR-induced differentiation in K562 cells
US
As shown in Fig. 2A, a significant increase of eIF3a could be detected after transfected with pEGFP-N1-eIF3a in K562 cells. The protein expression of cyclin D1 and c-myc were decreased and the
AN
expression of phosphorylated Raf-1 and phosphorylated ERK were increased after ATRA or ATPR treated alone (Fig. 2B). However, These changes were reversed after transfection of pEGFP-N1-eIF3a. The
M
morphologic changes of K562 cells were shown in Fig. 2C. Compared with the negative control, the matured granulocytes in ATPR-treated group had smaller or irregular nucleus and a decreased ratio of
ED
nuclear area to cytoplasmic area. But after infecting pEGFP-N1-eIF3a, the characteristics of matured
PT
granulocytes induced by ATPR or ATRA could be reduced. Moreover, flow cytometric analysis demonstrated that the expression of CD11b (a granulocytic differentiation marker) was significantly
CE
elevated after treatment with ATPR or ATRA, but the result showed an apparently decreasing percentage of CD11b-positive cells after transfected pEGFP-N1-eIF3a (Fig. 2D). Similarly, after overexpression of
AC
eIF3a, ATRA or ATPR-induced stable expression of CD14 (a monocyte differentiation marker) was decreased in K562 cells(Fig. 2E). All these data exposed that ATPR might have a strong effect on inducing K562 cells differentiation, and up-regulation of eIF3a could suppress it.
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
Figure 2. Overexpression of eIF3a inhibits ATPR-induced differentiation in K562 cells. After transfection with eIF3a negative control or pEGFP-N1-eIF3a, K562 cells were treated with ATPR (10-6 M) or ATRA (10-6 M) for 72h. After that, (A and B) the protein expression of eIF3a, p-Raf-1, p-ERK, cyclin D1 and c-myc were determined by western blot analysis. (C) Wright–Giemsa staining results showed cell morphological changes in K562 cells and it could be detected that the rate of natured cells with irregular nuclear and decreased nuclear/cytoplasm ratio was climbing from 11.1% to over 63.5% or 60.5% after ATPR or ATRA treated. The rates had an apparent decline when transfecting eIF3a. Cell differentiation markers CD11b (D) and CD14 (E) expressions were measured by flow
ACCEPTED MANUSCRIPT cytometry. Data were presented as mean ± SD of three independent experiments. *P<0.05, **P<0.01 versus negative control or ATPR, #P<0.05, ##P<0.01 versus ATRA group.
3.3 eIF3a silencing could influences ATPR-induced differentiation in K562 cells.
In order to further confirmed preceding results, eIF3a siRNAs was used to silence the expression of
T
eIF3a. As shown in Fig. 3A, the expression of eIF3a was successfully suppressed in K562 cells after
IP
transfected with eIF3a siRNA. The results also showed that cyclin D1 and c-myc expression were declined
CR
in ATPR or ATRA-treated groups, and the expression of phosphorylated Raf-1 and phosphorylated ERK are the opposites (Fig. 3B). These changes were more significant after transfected with eIF3a siRNA. In
US
K562 cells, ATPR or ATRA-treated cells displayed matured granulocytes with smaller or irregular nucleus and decreased nuclear/cytoplasm ratio. After using eIF3a siRNA, more significantly matured appearances
AN
with kidney-shape nucleus and decreased nuclear/cytoplasm ratio could be detected (in Fig. 3C). The relative proportions of CD11b and CD14 in K562 cells have a clear increase after ATRA or ATPR treatment,
M
and it presented more meaningful increasing both in CD11b and in CD14 expression after transfected with eIF3a siRNA (Fig. 3D and 3E). Therefore, the studies further indicated that silence of eIF3a could
ED
decelerate the proliferation process of K562 cells and accelerated its differentiation. Take together, these results showed that ATPR could induce K562 cells differentiation and eIF3a is negatively correlated with
AC
CE
PT
ATPR-induced cell differentiation.
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
Figure 3. Transfection of K562 cells with eIF3a siRNA facilitates ATPR-induced differentiation. After transfection with negative siRNA or eIF3a siRNA, K562 cells were treated with ATPR (10-6 M) or ATRA (10-6 M) for 72h. After that, (A and B) the protein expression of eIF3a, p-Raf-1, p-ERK, cyclin D1 and c-myc were detected by western blot analysis. (C) Cell morphological assays were assessed by Wright–Giemsa staining. Cell differentiation status was measured by the expression of CD11b (D) and CD14 (E). Data were presented as mean ± SD of three independent experiments. *P< 0.05, **P<0.01, versus negative control or ATPR group, #P<0.05, ##P<0.01 versus ATRA.
ACCEPTED MANUSCRIPT 3.4 ATPR induces K562 cells differentiation by activating the eIF3a-dependent ERK pathway.
In order to verify whether ERK signaling pathway plays a crucial role in ATPR-induced differentiation, we use PD98059, a MEK inhibitor to arrest ERK signaling pathway. The expression of eIF3a, cyclin D1, c-myc, p-ERK and p-Raf-1 were measured by western blot analysis. Compared with
T
negative group, the accelerated expression of cyclin D1 and c-myc could be detected (Fig. 4A) and the
IP
protein level of p-ERK was decelerated (Fig. 4B) after PD98059 pretreatment. But the protein expression of p-Raf-1 and eIF3a had no clear changes after PD98059 incubated. Morphological analysis in Fig. 4C
CR
fully exposed the greater nuclear/cytoplasm ratio and less mature granulocytes when pretreated with PD98059. The levels of both CD11b and CD14 surface antigens diminished apparently in K562 cells after
US
treated with PD98059 (Fig. 4D and 4E). We can draw a conclusion derived from the above results that a blockade of ERK signaling pathway by PD98059 could attenuate ATPR-induced cell differentiation in
AC
CE
PT
ED
M
AN
K562 cells.
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
Figure 4. eIF3a blocks ATPR-induced K562 cells differentiation via ERK1/2 signaling pathway. After
AC
pretreatment with PD98059 (10uM) for 24h, K562 cells were incubated with ATPR (10 -6 M) and ATPR (10-6 M), (A and B) the expressions of eIF3a, p-Raf-1, p-ERK, cyclin D1 and c-myc were determined by western blot analysis. (C) Cell morphological assays was assessed by Wright–Giemsa staining. CD11b (D) and CD14 (E) were detected by flow cytometry. Data are expressed in mean ± SD of three independent experiments and indicates statistical significance versus negative control or ATPR at *P<0.05, **P<0.01, versus ATRA group at #P<0.05, ##P<0.01.
3.6 ATPR could induce cell differentiation in NB4 cells
ACCEPTED MANUSCRIPT The APL cell line NB4 cells was used to demonstrate the general effect and function of ATPR on other leukemia cells. As shown in Fig. 5A, western blot result showed that ATPR could facilitate the accumulation of eIF3a expression. Furthermore, Wright–Giemsa staining assay detected that ATPR could also induce NB4 cells differentiation. Obviously, after induction for 72 hours with ATPR (10-6 M) or ATRA (10-6 M), a greater fraction of mature cells with smaller nucleus were revealed in Fig. 5B. Flow
T
cytometry results showed that ATPR promoted the expression of CD11b in NB4 cells (Fig. 5C). Moreover,
AC
CE
PT
ED
M
AN
US
CR
IP
10-6 M ATPR treatment led to a significant increase of the CD14 antigens levels in Fig. 5D.
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
ACCEPTED MANUSCRIPT
Figure 5. ATPR accelerates myeloid differentiation in NB4 cells. NB4 cells were treated with 10-6 M ATPR or ATRA for 72 hours. (A) The expression of eIF3a was detected by western blot analysis. (B) The cell morphological changes were observed by Wright–Giemsa staining assays. The expressions of CD11b (C) and CD14 (D) were evaluated by flow cytometric analysis. The results represent the mean ± SD of data from three independent experiments. *P<0.05, **P<0.01 versus control group.
ACCEPTED MANUSCRIPT 4 Discussions
In this study, we investigated the role of eIF3a in the anti-tumor effect of ATPR on CML and APL cells. Our results showed that the expression of eIF3a in K562 and NB4 cells were decreased after being treated by ATPR, and ATPR could induce a decline of cyclin D1 and c-myc expression, but accelerated the
T
expression of phosphorylated ERK, phosphorylated Raf-1 and myeloid differentiation markers CD11b and
IP
CD14 and also have an influence on the morphology changes. Moreover, overexpression of eIF3a could induce the expression of cyclin D1 and c-myc, decelerated the expression of p-ERK and p-Raf-1 and
CR
suppressed ATPR-induced K562 cells differentiation. However, ATPR-induced differentiation could be impelled when using eIF3a siRNA to down-regulate eIF3a expression. Furthermore, PD98059, a MEK
US
inhibitor, could restrain ATPR-induced differentiation in K562 cells. These results indicated that eIF3a
AN
played an important role in ATPR-induced cell differentiation in K562 cells, which might be involved with its ability in regulating the activation of ERK signaling pathway in vitro. What’s more, this study proved
M
that ATPR could also induce NB4 cells differentiation.
ED
The evidence revealed that ATRA could effectively block acquisition of resistance in CML cells on imatinib treatment and induced cell differentiation.(Horwitz, et al., 2014) As a derivative of all-trans
PT
retinoic acid, ATPR also showed its significant effect on inhibiting proliferation and inducing differentiation in our previous studies(Fan, Cheng, Wang, Gui, Chen, Zhou, et al., 2014; Hu, Pan, Chen,
CE
Qin, Wu, Zhu, et al., 2014; H. Wang, Gui, Chen, Zhou, & Wang, 2013; N. Wang, et al., 2013), with a preferable solubility and stability to ATRA. It has been demonstrated that ATPR could inhibit proliferation
AC
and induced differentiation in human gastric carcinoma xenografts via up-regulating retinoic acid receptor β(Ju, Wang, Wang, & Chen, 2015). ATPR also causes cell cycle arrest in human hepatocellular carcinoma and gastric cancer in vitro(H. Liu, Chen, Zhang, Zhou, Gui, & Wang, 2016; Xia, Zhao, Wang, Qiao, Zhang, Yin, et al., 2017). Recently, our studies first confirmed that ATPR could modulate NB4 cells differentiation by inducing autophagy(Li, Li, Wang, Xie, Zhou, Meng, et al., 2017). Congruously, our results demonstrated that ATPR could attenuate the expression of cyclin D1 and c-myc and caused G0/G1 phase arrest in K562 cells. Moreover, ATPR accelerated the expression of CD11b and CD14 and had a positive impact on cell morphological changes both in CML and APL cell lines. These results indicated the
ACCEPTED MANUSCRIPT anti-tumor effect of ATPR again. In our previous study (Meng, Zhang, Xia, Ge, Chen, 2016), preliminary proteomic analysis was used to investigate the potential molecular mechanisms and specific targets underlining the effect of ATPR in K562 cells. The result showed that eIF family were extremely likely to be core proteins in ATPR-inducing differentiation in K562 cells, especially eIF3a. This study further
T
revealed that the protein expression of eIF3a was down-regulate after ATPR-treated.
IP
As a eukaryotic translation initiation factor, eIF3a has been revealed that could interact complicatedly with multiple factors and played a unique role in modulating tumor. Increasing researches have uncovered
CR
a strong connection between eIF3a and cancer for the extremely high frequency of gene occurs in biological process, such as cell cycle, growth, proliferation, differentiation and division(Saletta, Suryo
US
Rahmanto, & Richardson, 2010). What’s more, knockdown of eIF3a caused significant decrease in cell proliferation and motility ability in pancreatic cancer cells by depleting clonogenic abilities, cell growth rates
AN
and invasion abilities(S. Q. Wang, Liu, Yao, & Jin, 2016). L-mimosine, a plant amino acid, could reversibly
M
block mammalian cells at late G1 phase and accelerated functional differentiation by affecting translation of mRNAs of eIF3a(Dong, Arnold, Yang, Park, Hrncirova, Mechref, et al., 2005). Dong.et.al verified that
ED
reducing eIF3a expression oscillated with cell cycle and peaks in S phase and also reduced cell proliferation rate by elongating cell cycle, but the cell cycle distribution had no changes(Dong, Liu, Cui,
PT
Pincheira, Yang, Liu, et al., 2009). Yu Zhang et al. have proposed that the increased expression of eIF3a improves cisplatin sensitivity in ovarian cancer probably by regulating p27 Kip1 and XPC translation(Zhang,
CE
Yu, Tian, Li, Zhang, Zhang, et al., 2015). However, it remains unclear whether eIF3a has a similar function in hematological diseases. Our current data first clarified that ATPR (10-6 M) could suppress the protein
AC
expression of eIF3a. In order to prove eIF3a involved in ATPR-induced differentiation, K562 cells were transfected with pEGFP-N1-eIF3a to up-regulating eIF3a. Results confirmed that cyclin D1 and c-myc were increased significantly and ATPR-induced ERK activation and the myeloid differentiation had an obvious lessening after transfected. Moreover, ATPR-induced cell differentiation could be further promoted by using eIF3a siRNA. Hence, these results demonstrated that eIF3a negatively correlated with ATPR-induced K562 cells differentiation.
ACCEPTED MANUSCRIPT ERK pathway is activated by receptor tyrosine kinases, such as the epidermal growth factor receptor (EGFR). After the formation of complex with guanine nucleotide exchange factor (GEF) SOS, the adaptor proteins SHC and Grb2, Ras, an upstream activator of the ERK pathway, could be stimulated by SOS/SHC/Grb2 complex binding to specific phosphotyrosines at the EGFR and have a high concentration around with Ras. Then Raf is recruited to the plasma membrane and activated by an interaction with
T
Ras(Shaul & Seger, 2007). The Raf gene has three subunits, A-Raf, B-Raf and Raf-1 (c-Raf), which have
IP
individual properties after being triggered. Its dephosphorylated at S259 site and phosphorylated at S338
CR
site are requisite for Raf-1 excitation(Dhillon, Meikle, Yazici, Eulitz, & Kolch, 2002; Dhillon, von Kriegsheim, Grindlay, & Kolch, 2007). Activated Raf-1 could phosphorylates MEK, immediately, ERK
US
would be activated at downstream and then induce diversified biological effects. Prior publications showed that ERK signaling pathway significantly support cell proliferation(Yang, Ding, Guo, Zheng, Wang, Sun, et
al.,
2017)
and
tumor
AN
al., 2017), differentiation (Shao, Liu, Li, Xian, Zhou, Yang, et al., 2016; Tang, Chai, Ye, Yu, Cao, Yang, et formation(Quintero
Barceinas,
Garcia-Regalado,
Arechaga-Ocampo,
M
Villegas-Sepulveda, & Gonzalez-De la Rosa, 2015). Moreover, the expression of p-ERK occurs
ED
progressively as cell differentiation is induced by dasatinib, but MEK inhibitors PD98059 and U0216 can block the myeloid differentiation(Y. Fang, Zhong, Lin, Zhou, Jing, Ying, et al., 2013). Our previous studies
PT
found that ATPR inhibited phosphorylation of ERK within 48h, but showed a slight increase at 48h(B. Wang, Yan, Zhou, Gui, Chen, & Wang, 2014). In this report, we clarified that enhanced phosphorylation of
CE
ERK could be detected after cells were treated by ATPR for 72h. It has been reported that the rapid and sustained activation of MEK/ERK may be required for cell differentiation, whereas not benefit Barceinas,
Garcia-Regalado,
Arechaga-Ocampo,
Villegas-Sepulveda,
&
AC
proliferation(Quintero
Gonzalez-De la Rosa, 2015). It means that the discrepant observations are probably related to the ways ERK was activated. This phenomenon may explain why activation of ERK could have a promoting effect both on cell proliferation and differentiation. In this study, we detected that ATPR could activate ERK signaling and promoted K562 cells differentiation. What’s more, attenuation of the ERK activation blocked not only the cell differentiation but also the increased protein expression of cyclin D1 and c-myc. It indicated that MEK/ERK signaling pathway might positively modulate ATPR-induced differentiation by regulating the protein expression of cyclin D1 and c-myc. Interestingly, it is displayed that eIF3a regulates
ACCEPTED MANUSCRIPT ERK signaling and its biological effects via binding to Raf-1(Xu, Lu, Romano, Pitt, Houslay, Milligan, et al., 2012). Our findings testified similar results that Raf-1 was increased and eIF3a was decreased after ATPR treatment, with no significant differences after PD98059 pretreatment. This phenomenon indicated that eIF3a regulated the ERK signaling pathway by inhibiting Raf-1 and eIF3a might involve in
T
ATPR-induced differentiation via affecting the activation of ERK signaling.
IP
In conclusion, our data confirmed that ATPR could down-regulate eIF3a expression and eIF3a plays a important role in ATPR promoted cell differentiation in K562 cells in vitro, which might be involved the
CR
activation of ERK signaling pathway. The data presented here warrant that ATPR may lead to a new
AN
US
probability for CML and other leukemia therapies.
M
Acknowledgments
ED
This work was supported by the National Major Scientific and Technological Special Project for “Significant New Drugs Development” (No 2011ZX09401). Thanks are due to School of Basic Medical
AC
CE
PT
Sciences from Anhui Medical University for providing technical support for flow cytometry.
ACCEPTED MANUSCRIPT References
AC
CE
PT
ED
M
AN
US
CR
IP
T
Avvisati, G., & Tallman, M. S. (2003). All-trans retinoic acid in acute promyelocytic leukaemia. Best Pract Res Clin Haematol, 16(3), 419-432. Bobba, R. K., & Doll, D. C. (2012). Promyelocytic blast crisis of chronic myelogenous leukemia. Blood, 120(19), 3873. Burnett, A., Wetzler, M., & Lowenberg, B. (2011). Therapeutic advances in acute myeloid leukemia. J Clin Oncol, 29(5), 487-494. Dhillon, A. S., Meikle, S., Yazici, Z., Eulitz, M., & Kolch, W. (2002). Regulation of Raf-1 activation and signalling by dephosphorylation. EMBO J, 21(1-2), 64-71. Dhillon, A. S., von Kriegsheim, A., Grindlay, J., & Kolch, W. (2007). Phosphatase and feedback regulation of Raf-1 signaling. Cell Cycle, 6(1), 3-7. Dong, Z., Arnold, R. J., Yang, Y., Park, M. H., Hrncirova, P., Mechref, Y., Novotny, M. V., & Zhang, J. T. (2005). Modulation of differentiation-related gene 1 expression by cell cycle blocker mimosine, revealed by proteomic analysis. Mol Cell Proteomics, 4(7), 993-1001. Dong, Z., Liu, Z., Cui, P., Pincheira, R., Yang, Y., Liu, J., & Zhang, J. T. (2009). Role of eIF3a in regulating cell cycle progression. Exp Cell Res, 315(11), 1889-1894. Dong, Z., & Zhang, J. T. (2003). EIF3 p170, a mediator of mimosine effect on protein synthesis and cell cycle progression. Mol Biol Cell, 14(9), 3942-3951. Fan, T. T., Cheng, Y., Wang, Y. F., Gui, S. Y., Chen, F. H., Zhou, Q., & Wang, Y. (2014). A novel all-trans retinoid acid derivative N-(3-trifluoromethyl- phenyl)- retinamide inhibits lung adenocarcinoma A549 cell migration through down-regulating expression of myosin light chain kinase. Asian Pac J Cancer Prev, 15(18), 7687-7692. Fang, C., Chen, Y. X., Wu, N. Y., Yin, J. Y., Li, X. P., Huang, H. S., Zhang, W., Zhou, H. H., & Liu, Z. Q. (2017). MiR-488 inhibits proliferation and cisplatin sensibility in non-small-cell lung cancer (NSCLC) cells by activating the eIF3a-mediated NER signaling pathway. Sci Rep, 7, 40384. Fang, Y., Zhong, L., Lin, M., Zhou, X., Jing, H., Ying, M., Luo, P., Yang, B., & He, Q. (2013). MEK/ERK dependent activation of STAT1 mediates dasatinib-induced differentiation of acute myeloid leukemia. PLoS One, 8(6), e66915. Harrington, P., Kizilors, A., & de Lavallade, H. (2017). The Role of Early Molecular Response in the Management of Chronic Phase CML. Curr Hematol Malig Rep, 12(2), 79-84. Harris, H. (2005). A long view of fashions in cancer research. Bioessays, 27(8), 833-838. Horwitz, M. S., Wang, Z., Liu, Z., Wu, X., Chu, S., Wang, J., Yuan, H., Roth, M., Yuan, Y.-C., Bhatia, R., & Chen, W. (2014). ATRA-Induced Cellular Differentiation and CD38 Expression Inhibits Acquisition of BCR-ABL Mutations for CML Acquired Resistance. PLoS Genetics, 10(6), e1004414. Hu, K. W., Pan, X. H., Chen, F. H., Qin, R., Wu, L. M., Zhu, H. G., Wu, F. R., Ge, J. F., Han, W. X., Yin, C. L., & Li, H. J. (2014). A novel retinoic acid analog, 4-amino-2-trifluoromethyl-phenyl retinate, inhibits gastric cancer cell growth. Int J Mol Med, 33(2), 415-422. Ju, J., Wang, N., Wang, X., & Chen, F. (2015). A novel all-trans retinoic acid derivative inhibits proliferation and induces differentiation of human gastric carcinoma xenografts via up-regulating retinoic acid receptor beta. Am J Transl Res, 7(5), 856-865. Kumar, S., Khanduja, K. L., Verma, N., Verma, S. C., Avti, P. K., & Pathak, C. M. (2008). ATRA promotes alpha tocopherol succinate-induced apoptosis in freshly isolated leukemic cells from chronic myeloid leukemic patients. Mol Cell Biochem, 307(1-2), 109-119. Li, Y., Li, G., Wang, K., Xie, Y. Y., Zhou, R. P., Meng, Y., Ding, R., Ge, J. F., & Chen, F. H. (2017). Autophagy contributes to 4-Amino-2-Trifluoromethyl-Phenyl Retinate-induced differentiation in human acute promyelocytic leukemia NB4 cells. Toxicol Appl Pharmacol, 319, 1-11. Liu, H., Chen, F., Zhang, L., Zhou, Q., Gui, S., & Wang, Y. (2016). A novel all-trans retinoic acid derivative 4-amino2trifluoromethyl-phenyl retinate inhibits the proliferation of human hepatocellular carcinoma HepG2 cells by inducing G0/G1 cell cycle arrest and apoptosis via upregulation of p53 and ASPP1 and downregulation of iASPP. Oncol Rep, 36(1), 333-341.
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
Liu, Z., Dong, Z., Yang, Z., Chen, Q., Pan, Y., Yang, Y., Cui, P., Zhang, X., & Zhang, J. T. (2007). Role of eIF3a (eIF3 p170) in intestinal cell differentiation and its association with early development. Differentiation, 75(7), 652-661. Marley, S. B., Davidson, R. J., Goldman, J. M., & Gordon, M. Y. (2002). Effects of combinations of therapeutic agents on the proliferation of progenitor cells in chronic myeloid leukaemia. Br J Haematol, 116(1), 162-165. Nilsson, B., Olofsson, T., & Olsson, I. (1984). Myeloid differentiation in liquid cultures of cells from patients with chronic myeloid leukemia: effects of retinoic acid and indomethacin. Exp Hematol, 12(2), 91-99. Pincheira, R., Chen, Q., & Zhang, J. T. (2001). Identification of a 170-kDa protein over-expressed in lung cancers. Br J Cancer, 84(11), 1520-1527. Quintero Barceinas, R. S., Garcia-Regalado, A., Arechaga-Ocampo, E., Villegas-Sepulveda, N., & Gonzalez-De la Rosa, C. H. (2015). All-Trans Retinoic Acid Induces Proliferation, Survival, and Migration in A549 Lung Cancer Cells by Activating the ERK Signaling Pathway through a Transcription-Independent Mechanism. Biomed Res Int, 2015, 404368. Saletta, F., Suryo Rahmanto, Y., & Richardson, D. R. (2010). The translational regulator eIF3a: the tricky eIF3 subunit! Biochim Biophys Acta, 1806(2), 275-286. Shao, X., Liu, Y., Li, Y., Xian, M., Zhou, Q., Yang, B., Ying, M., & He, Q. (2016). The HER2 inhibitor TAK165 Sensitizes Human Acute Myeloid Leukemia Cells to Retinoic Acid-Induced Myeloid Differentiation by activating MEK/ERK mediated RARalpha/STAT1 axis. Sci Rep, 6, 24589. Shaul, Y. D., & Seger, R. (2007). The MEK/ERK cascade: from signaling specificity to diverse functions. Biochim Biophys Acta, 1773(8), 1213-1226. Spilka, R., Ernst, C., Bergler, H., Rainer, J., Flechsig, S., Vogetseder, A., Lederer, E., Benesch, M., Brunner, A., Geley, S., Eger, A., Bachmann, F., Doppler, W., Obrist, P., & Haybaeck, J. (2014). eIF3a is over-expressed in urinary bladder cancer and influences its phenotype independent of translation initiation. Cell Oncol (Dordr), 37(4), 253-267. Tang, L., Chai, W., Ye, F., Yu, Y., Cao, L., Yang, M., Xie, M., & Yang, L. (2017). HMGB1 promotes differentiation syndrome by inducing hyperinflammation via MEK/ERK signaling in acute promyelocytic leukemia cells. Oncotarget, 8(16), 27314-27327. Wang, B., Yan, Y. W., Zhou, Q., Gui, S. Y., Chen, F. H., & Wang, Y. (2014). A novel all-trans retinoid acid derivative induces apoptosis in MDA-MB-231 breast cancer cells. Asian Pac J Cancer Prev, 15(24), 10819-10824. Wang, H., Gui, S. Y., Chen, F. H., Zhou, Q., & Wang, Y. (2013). New insights into 4-amino-2-tri-fluoromethyl-phenyl ester inhibition of cell growth and migration in the A549 lung adenocarcinoma cell line. Asian Pac J Cancer Prev, 14(12), 7265-7270. Wang, N., Ge, J. F., Pan, C. X., Peng, X. Q., Chen, H. H., Wang, X. Q., Tang, J., Hu, W., & Chen, F. H. (2013). Anti-tumor effect of 4-Amino-2-Trifluoromethyl-Phenyl Retinate on human breast cancer MCF-7 cells via up-regulation of retinoid receptor-induced gene-1. Biomed Pharmacother, 67(8), 687-692. Wang, S. Q., Liu, Y., Yao, M. Y., & Jin, J. (2016). Eukaryotic Translation Initiation Factor 3a (eIF3a) Promotes Cell Proliferation and Motility in Pancreatic Cancer. J Korean Med Sci, 31(10), 1586-1594. Xia, Q., Zhao, Y., Wang, J., Qiao, W., Zhang, D., Yin, H., Xu, D., & Chen, F. (2017). Proteomic analysis of cell cycle arrest and differentiation induction caused by ATPR, a derivative of all-trans retinoic acid, in human gastric cancer SGC-7901 cells. Proteomics Clin Appl, 11(7-8). Xu, T. R., Lu, R. F., Romano, D., Pitt, A., Houslay, M. D., Milligan, G., & Kolch, W. (2012). Eukaryotic translation initiation factor 3, subunit a, regulates the extracellular signal-regulated kinase pathway. Mol Cell Biol, 32(1), 88-95. Yang, Y., Ding, L., Guo, Z. K., Zheng, X. L., Wang, L. S., Sun, H. Y., Jin, Z. G., & Wang, H. X. (2017). The epigenetically-regulated miR-34a targeting c-SRC suppresses RAF/MEK/ERK signaling pathway in K-562 cells. Leuk Res, 55, 91-96. Yin, J. Y., Meng, X. G., Qian, C. Y., Li, X. P., Chen, J., Zheng, Y., Liu, R., Zhou, H. H., & Liu, Z. Q. (2015). Association of positively selected eIF3a polymorphisms with toxicity of platinum-based chemotherapy in NSCLC patients. Acta Pharmacol Sin, 36(3), 375-384.
ACCEPTED MANUSCRIPT
AC
CE
PT
ED
M
AN
US
CR
IP
T
Zhang, Y., Yu, J. J., Tian, Y., Li, Z. Z., Zhang, C. Y., Zhang, S. F., Cao, L. Q., Zhang, Y., Qian, C. Y., Zhang, W., Zhou, H. H., Yin, J. Y., & Liu, Z. Q. (2015). eIF3a improve cisplatin sensitivity in ovarian cancer by regulating XPC and p27Kip1 translation. Oncotarget, 6(28), 25441-25451. Meng, Y.; Zhang, D.; Xia, Q.; Ge, J.; Chen, F. (2016). The proteomics research of 4-amino-2-trifluoromethyl-phenylretinate on human leukemia K562 cells. Chinese Pharmacological Bulletin. 32(1), 27-32.(in Chinese)
ACCEPTED MANUSCRIPT Abbreviations list Full name
ATRA
All-trans retinoic acid
ATPR
4-Amino-2-Trifluoromethyl-Phenyl Retinate
AP
Ammonium persulphate
APL
Acute promyelocytic leukemia
CML
Chronic myeloid leukemia
DEPC
Diethyl pyrocarbonate
DMSO
Dimethyl sμlfoxide
EGFR
Epidermal growth factor receptor
ERK
Extracellular regulated protein kinases
eIF3a
Eukaryotic initiation factors
FBS
Fetal Bovine Serum
IP
CR
US
AN
M Gram
ED
G GEF
Guanine nucleotide exchange factor
PT
Gly
CE
H IRE
AC
M mA
T
Abbreviations
Glycine Hour Iron-responsive element mol/L Milliampere
MEK
Mitogen-activated protein kinase kinase
Mg
Milligramme
mRNA
Messenger ribnucleic acid
Min
Minue
ml
Milliliter
mM
mmol/L
ACCEPTED MANUSCRIPT
nmol/L
PAGE
Polyacrylamide gel electrophoresis
PBS
Phosphate buffered saline
PD98059
MEK inhibitor
PML
Promyelocytic leukemia gene
PMSF
Phenylmethyl sμlfonylfluoride
PVDF
PolyVinylidene difiuoride
RAR
Retinoic acid receptor
rpm
Round per minute
RXR
Retinoic X receptor
s
Second
SDS
Sodium dobecyl sμlphate
siRNA
Small interfering RNA
M
AN
US
CR
IP
T
nM
N’N’N’N-Tetramethylethylenediamine
ED
TEMED Tris
Transmission electron microscopy Voltage
PT
V
AC
CE
μM
μmol/L
ACCEPTED MANUSCRIPT Research highlights < eIF3a could be suppressed by 4-amino-2-trifluoromethyl-phenyl retinate (ATPR) in CML cell line K562 cells > < eIF3a involved in ATPR-induced differentiation in K562 cells >
AC
CE
PT
ED
M
AN
US
CR
IP
T
ATPR-induced cell differentiation in K562 cells >
Figure 1
Figure 2abc
Figure 2d
Figure 2e
Figure 3ac
Figure 3d
Figure 3e
Figure 4abc
Figure 4d
Figure 4e
Figure 5ab
Figure 5cd