original article upregulating mir-146a by physcion ... · upregulating mir-146a by physcion...

Am J Cancer Res 2016;6(11):2547-2560www.ajcr.us /ISSN:2156-6976/ajcr0035745

Original Article Upregulating miR-146a by physcion reverses multidrug resistance in human chronic myelogenous leukemia K562/ADM cells

Wenjun Liu, Juan He, Yiling Yang, Qulian Guo, Fei Gao

Department of Pediatrics, Affiliated Hospital of Southwest Medical University, Luzhou 646000, Sichuan, China

Received July 13, 2016; Accepted July 28, 2016; Epub November 1, 2016; Published November 15, 2016

Abstract: The aim of this study was to evaluate the role of miR-146a in the drug resistance of chronic myelogenous leukemia (CML) cells (K562/ADM) and to investigate the reversal effect of physcion, a natural compound, on the multidrug-resistance in CML. Our results showed that miR-146a was significantly downregulated in drug-resistant K562 cells and the overexpression of miR-146a in K562/ADM cells could restore the sensitivity to adriamycin (ADM). In addition, our results showed that the downregulation of miR-146a was associated with increase in CXCR4 expression, which was a direct target of miR-146a. Moreover, our findings also provided experimental evidence that physcion could enhance the anti-proliferative effect of ADM in K562/ADM cells by upregulating miR-146a. In conclu-sion, this present study showed that miR-146a conferred ADM resistance in CML cells and physcion could improve the sensitivity of K562/ADM cells by enhancing apoptosis via upregulating miR-146a.

Keywords: miR-146a, CXCR4, drug resistance, physcion, chronic myeloid leukemia

Introduction

Chronic myeloid leukemia (CML), a malignant transformation of hematopoietic stem cells, accounts for 20% of adult leukemia with an incidence rate of 1-2/100,000 [1]. CML is asso-ciated with reciprocal t(9,22)(q34;q11) chromo-somal translocation, leading to BCR-ABL fusion gene lesion, which encodes a leukemia-specific oncoprotein involved in CML pathogenesis, BCR-ABL [2, 3]. The constitutive activation of BCR-ABL, which is a potent tyrosine kinase, leads to the constitutive proliferation and sur-vival through activating the growth factors-independent proliferation signaling and stimu-lating the downstream molecules including STAT5, CRKL, and ERK [4]. Because of the acti-vation of survival signaling, the clinical stages of CML progress from a chronic phase charac-terized by excessive proliferation with capacity for differentiation to the second accelerated phase featured with rapid progression after 4-6 years following the chronic phase, to the third phase called blast crisis presented as a fatal acute leukemia [5]. More importantly, CML presents with increasing resistance to

chemotherapeutics along with the disease pro-gression and failure to respond to chemothera-py is considered the key contributing factor for the high mortality associated with CML [6]. Therefore, discovery of novel agents that can enhance the therapeutic efficacy and overcome the drug resistance are of great interest to scientists.

MicroRNAs (miRNAs), an abundant class of 18-5 nucleotides evolutionarily highly con-served endogenous non-coding small RNAs, are aberrantly expressed in cancer and act as tumor suppressors or oncogenes by suppress-ing mRNA translation or cleaving target mRNAs to induce their degradation by binding to the 3’-UTR of target mRNA [7]. Besides solid tumors, aberrant expression patterns of miRNAs have also been displayed in a variety of many hematological malignancies including such as chronic lymphoid leukemia, acute myeloid leu-kemia and myeloproliferative neoplasm [8, 9]. As regards to CML, Rokah et al have reported downregulation of miR-31, miR-34a, miR-155, and miR-564 in K562 cells compared to control cells and suggested a potential role of miRNAs

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Physcion reverses multidrug resistance in CML

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in CML pathogenesis [10]. A recent study has also showed that BCR-ABL kinase can modu-late the expression of apoptomiRs and apop-tomiRs that present a distinct expression pat-tern in different CML phases, also suggesting that microRNAs may be considered as novel therapeutic targets in CML [11].

Physcion, an active ingredient in the medicinal plant Radix et Rhizoma Rhei [12], has been used as a laxative, is hepatoprotective, and has anti-inflammatory and anti-microbial properties [12-15]. Recently, physcion has been found to be able to interfere in the physiological activi-ties of cancer cells including apoptosis [16-19], cell cycle progression [17], and metastasis [20]. However, the role of physcion on hemato-logical malignancies has not been elucidated. In the present study, we evaluated the role of miR-146a in the drug resistance of CML cells (K562/ADM) and also investigated the reversal effect of physcion, a natural compound, on multidrug-resistance in CML.

Materials and methods

Cell culture

Human CML cell line K562 cells and adriamycin (ADM)-resistant K562/ADM cells, obtained from the Cell Bank of the Shanghai Institute of Biochemistry and Cell Biology, were maintained at a density of 1 × 105 cells in RPMI-1640 medi-um (Gibco BRL Co. Ltd. Gaithersburg, MD) con-taining 10% heat-inactivated fetal bovine serum (FBS; Hyclone, Logan, UT), 100 IU/ml penicillin, and 100 mg/ml streptomycin (Sigma-Aldrich, St. Lious, MO). The cells were kept in a CO2 incubator at 37°C and the culture condi- tion was maintained as 90% humidity and 5% CO2. To maintain drug resistance, ADM (5 mg/ml) was added in the culture medium of K562/ADM cells until at least 2 weeks before the experiments.

Cell viability assay

Cells were plated at a density of 5.0 × 103 cells/well in 96-well culture plates for 24 hours before being challenged by different concentra-tions of physcion or ADM alone, or in combina-tion for 24, 48, and 72 hours. The cell viability was determined by using a CCK-8 kit (Beyotime, Shanghai, China) according to the instructions of the manufacturer. The absorbance at 450

nm (A) was measured and inhibition rate (IR) of cell proliferation was calculated according to the following formula: IR = A value of the control group-A value of the experimental group) / (A value of the control group-A value of the back-ground group) × 100%. Half inhibition concen-tration (IC50) was calculated by mid-efficacy analysis (LOGIT method) with SPSS 11.0 soft-ware (Chicago, IL) and the reversal index (RI) was calculated by dividing the IC50 value in the blank control group by the IC50 value in the reversed group.

Colony formation assay

K562/ADM cells suspended in RPMI-1640 aga-rose medium containing physcion at different concentration were seeded in each well of a 6-well plate over a bottom layer of solidified RPMI-1640 agarose medium. Cultures were maintained for 14 days without fresh medium feeding at 37°C in a humidified atmosphere of 95% air and 5.0% CO2. Then cell colonies with over 50 cells were enumerated and stained with violet crystal before being photographed using a digital camera (Nikon DXM1200, Tokyo, Japan).

Cell apoptosis assay

Cell apoptosis was assessed using FITC Ann- exin V Apoptosis Detection Kit (BD Biosciences Pharmingen, San Diego, CA) according to the manufacturer’s instructions. Briefly, K562/ADM cells were treated with various concentra-tions of physcion for 48 hours, collected, and washed with ice-cold PBS, and then suspended in 500 μl of annexin V binding buffer. 100 μl aliquot was taken, 2 μl of annexin V-FITC and 2 μl of PI were added, and the mixture was incu-bated for 5 minutes at room temperature in the dark. After the addition of 400 μl of binding buffer, 1 × 104 cells were analyzed on a FACSCalibur (BD Biosciences, San Diego, CA) by using CellQuest software. Annexin V-FITC positive cells were considered to be undergoing apoptosis and those negative for FITC were considered to be alive.

Apoptosis detection by morphological changes using Hoechst staining

Hoechst 33258 (Sigma-Aldrich, MO, USA) was also performed to detect apoptotic cells. Apoptosis was indicated by the presence of


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condensed or fragmented nuclei which bind Hoechst 33258 with high affinity. Following treatment, K562/ADM cells were washed with PBS and fixed with pre-cooled methanol at 500 μl/well for 10 minutes. Afterwards, the cells were stained with 1 μM Hoechst 33258 for 10 minutes and analyzed on a Leica fluorescence microscope. Two hundred cells in three ran-domly selected fields were counted and scored for the incidence of apoptotic chromatin.

Quantitative RT-PCR (qRT-PCR)

For miRNA detection, total miRNA was extract-ed using a commercial kit (Applied Biosystems, Carlsbad, CA) and miR-146a was quantified by real time PCR with a TaqMan Probe (Applied Biosystems, Carlsbad, CA). For mRNA detec-tion, RNA simple Total RNA Kit (TIANGEN Co., Beijing, China) was used to extract total RNA from cells. High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA, USA) was used to convert RNA into cDNA. The primers for CXCR4 expression were synthesized according to the published sequence by Sangog (Shanghai, China) [21]. PCR reaction solution included a master mix that including SYBR GREEN mastermix (Solarbio Co., Beijing, China), forward primer, reverse primer, and 10 ng tem-plate cDNA. GADPH was used as internal con-trol to normalize PCR results. PCR results were analyzed using the comparative ΔCt method (ABPrism software, Applied Biosystems, Foster City, CA).

miR-146a knockdown or overexpression

The lentiviral constructs of miR-146a mimic and miR-con, and miR-146a inhibitor were obtained from Qiagen (Dusseldorf, Germany). K562/ADM or K562 cells were seeded into each well of a 96-well plate, incubated over-night, and then, were transfected with a miR-27a mimic, miR-27a inhibitor, or control miR-con according to the manufacturer’s instruc-tions (Lipofectamine-2000, Invitrogen, Carls- bad, CA). The transfection efficiency was con-firmed by qPCR analysis.

Knockdown of CXCR4 using siRNA

The siRNA oligos for CXCR4 were synthesized based on published sequence [21] by Sangon Biotech (Shanghai, China) and a scramble sequence was used as control. Cells in logarith-

mic growth phase were seeded in 6-well plates at a density of 3 × 105 cells per well and incu-bated overnight and then transfected with siRNA or scramble siRNA, respectively, using Lipofectamine 2000 (Invitrogen, Grand Island, NY) according to the manufacturer’s protocol. Transfected cells were incubated for another 48 hours and the knockdown were verified by western blot analysis.

Surface CXCR4 expression

To examine the cell-surface CXCR4 expression, the cells were first stained with anti-mouse CXCR4-PE (eBioscience, San Diego, CA) anti-body before being fixed using the Perme- abilization kit (BD Biosciences, San Jose, CA). Then the cells were stained a second time with the same antibodies. Samples were analyzed using the FACSCalibur (BD Biosciences, San Diego, CA) and FlowJo Software (Tree Star, Ashland, OR). MFI results were normalized to the MFI of the corresponding isotype control.

Western blot

Western blot was performed following standard protocol. Briefly, following treatment, K562/ADM cells were harvested from flasks, and lysed with ice-cold lysis buffer (Beyotime, Shanghai, China) for 30 minutes on ice. Then the collected proteins were subjected to SDS-PAGE gels and transferred onto PVDF mem-branes (Millipore, MA). Proteins were probed with specific primary antibodies and a rabbit polyclonal antibody to β-actin used as a gel loading control. Specific primary antibodies against CXCR4 and β-actin were purchased from Abcam (Shanghai, China). After another wash with TBST, a secondary antibody (HRP-conjugated secondary antibody, Boster, Wuhan, China) was added for blots detection. Signals were detected using chemiluminescent sub-strate (KPL, Guildford, UK) and the blot intensi-ty was quantified using BandScan software (Glyko, Novato, CA).

Construction of reporter plasmids and lucifer-ase assay

The reporter plasmid was constructed as previ-ously described [22]. A fragment containing CXCR4 3’UTR was amplified by PCR from human genomic DNA utilizing specific primers (sense sequence: 5’-GCTCTAGACACAGATGTA-


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AAAGAC-3’; antisense sequence: 5’-GCTCTAG- ACCACTGGTACAAAATCTTTATGTAAG-3’) and in- serted into a pGL3 vector (Promega, Madison, WI) downstream to the stop codon of firefly-luciferase reporter gene, thus resulting in the pGL3-3’UTR/CXCR4 construct. For luciferase assay, 293T recipient cells were transiently co-transfected with 0.2 μg of pGL3-3’UTR/CXCR4 constructs, 0.02 μg of pRL-TK-Renilla luciferase reporter plasmids (Promega, Madison, WI) con-taining the Renilla-luciferase for normalization, and with 5 pmol of miR-146a overexpression construct or control. 24 hours after transfec-tion, the cells were lysed and the luciferase activity was measured with a luminometer using the dual-luciferase reporter assay sys-tem, according to the manufacturer’s instructions.

Migration analysis

Transwell chamber was used in the in vitro migration assay to examine K562/ADM chemo-taxis (Corning Costar, Cambridge, MA). K562/ADM cells were treated with physcion at differ-ent concentrations for 48 hours before being harvested and suspended at a concentration of 5 × 105 cells/ml in the RMPI-1640 medium. Cell suspensions were placed in the upper chamber with the presence or absence of 100 ng/ml CXCL12 in the lower chamber. Following incubation for 8 hours, non-migrated cells on the upper surface were removed and the migrated cells were stained for counting.

Animal experiments

The animal study was carried out according to the regulations of the State Food and Drug Administration (SFDA) of China on Animal Care and the study protocol was approved by Medi- cal Ethics Committee of our hospital. Female

5-week-old BALB/c nude mice were bought from the Experimental Animal Center of Southwest Medical University (Luzhou, Sichuan, China). K562/ADM cells (5 × 106) at exponen-tial phase were suspended in PBS and subcu- taneously injected on the right flank. Five days after injection, the mice were randomly allocat-ed to four groups (n = 6, per group). Tumour vol-ume (V) was calculated as:

V (mm3) = 1/2 × Length × Width2

Tumour measurements and body weight of the mice were recorded every other day. On day 16, all mice were sacrificed.

Statistical analysis

The data are presented as mean ± SD (Standard Deviation) and represent the results of three separate experiments each conducted in qua-druplicate unless otherwise stated. All statisti-cal analysis was performed using SPSS11.0 software (Chicago, IL). Statistical comparisons were performed by one-way ANOVA followed by Dunnett’s t-test. The difference with a P value less than 0.05 was defined as statistically significant.

Results

miR-146a is downregulated in ADM-resistant K562/ADM cells

Based on CCK-8 analysis, the IC50 value of K562 and K562/ADM to ADM at 48 hours was 0.15 μM and 3.5 μM, indicating that K562/ADM was 23-fold resistant to ADM compared to parental K562 cells (Table 1). To examine whether miR-146a was involved in the acquired resistance of K562 cells to ADM, miR-146a lev-els was determined by RT-PCR analysis. As shown in Figure 1A, the expression level of miR-146a in K562/ADM was significantly lower relative to K562 cells (P<0.01), leading to the postulation that miR-146a might play a role in the resistance of K562 cells to ADM.

Knockdown of miR-146a render the K562 cells resistant to ADM

To demonstrate the role of miR-146a in acquired resistance to ADM, a miR-146a inhibitor

Table 1. IC50 value at 48 hours

Cell line IC50 (μM)

K562/ADM 3.5±0.3K562 0.15±0.06K562+miR-146a inhibitor 2.8±0.4K562/ADM+miR-146a mimic 0.42±0.05K562/ADM+physcion (2 μM) 1.7±0.3K562/ADM+physcion (5 μM) 0.68±0.12


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was used to knockdown the miR-146a expres-sion in K562 cells. As shown in Figure 1B, the miR-146a level was significantly suppressed by the transfection of the miR-146a inhibitor. Cell viability assay showed that K562 cells with miR-146a knockdown had a significantly higher survival rate than K562 cells, and the K562 cells transfected with negative control (NC) (IC50 values were 2.8 μM, 0.15 μM, and 0.18 μM, respectively, Table 1). In addition, the flow cytometric analysis also showed that knock-

down of miR-146a rendered the K562 cells sig-nificantly more resistant to ADM-induced apop-tosis (Figure 1C). Collectively, these results suggested the association of miR-146a with ADM resistance in K562 cells.

Upregulation of mi-R146a enhances the sensi-tivity of K562/ADM cells to ADM

The role of miR-146a was further investigated with a miR-146a mimic. As shown in Figure 1D,

Figure 1. MiR-146a confers to ADM resistance in K562 cells. A. MiR-146a is significantly downregulated in K562/ADM cells. B. Expression of miR-146a in K562 cells is significantly suppressed using miR-146a inhibitor. C. Knock-down of miR-146a renders K562 cells more resistant to ADM-induced apoptosis. D. Expression of miR-146a in K562/ADM cells is significantly elevated using miR-146a mimic. E. Overexpression of miR-146a makes K562/ADM cells significantly more sensitive to ADM-induced apoptosis. **P<0.01.


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the expression of miR-146a was significantly increased by the miR-146a mimic compared with K562/ADM cells or K562/ADM cells trans-fected with NC (P<0.01). CCK-8 assay showed that K562/ADM cells transfected with miR-146a mimic were significantly more sensitive to ADM compared with K562/ADM cells and K562/ADM cells transfected with NC (IC50 value was 0.42 μM, 3.5 μM and 3.2 μM, respectively,

Table 1). Correspondingly, upregulation of miR-146a also rendered K562/ADM cells more sen-sitive to ADM-induced apoptosis (Figure 1E).

miR-146a confers ADM resistance in K562 cells mainly by targeting CXCR4

P-gp, MRP-1, and CXCR4 are known effector molecules involved in the resistance of K562

Figure 2. CXCR4 3’-UTR is directly targeted by miR-146a. A. Knockdown of miR-146a in K562 cells is associated with upregulation of CXCR4 mRNA. B. Overexpression of miR-146a in K562/ADM cells is associated with downregulation of CXCR4 mRNA. C. Surface expression of CXCR4 in K562 or K562/ADM cells is inversely correlated with miR-146a levels. D. Protein expression of CXCR4 in K562 or K562/ADM cells is inversely correlated with miR-146a levels. E. MiR-146a significantly decreases the luciferase reporter activity. **P<0.01.


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cells to ADM [21, 23]. Therefore, we examined whether miR-146a could modulate the expres-sion of these molecules. As shown in Figure 2A and 2B, knockdown of miR-146a in K562 cells or overexpression miR-146a in K562/ADM cells did not cause significant change in mRNA expression of P-gp or MRP-1. In contrast, our results showed that miR-146a expression inversely correlated with CXCR4 mRNA expres-sion. Moreover, flow cytometric and western blot analysis also showed that miR-146a expression inversely correlated with repression of CXCR4 expression (Figure 2C and 2D). Given the fact that miRNAs usually exert their func-tions by negatively regulating the expression of their target genes, we conducted luciferase activity assay to determine whether CXCR4 was a direct target of miR-146a. As shown in Figure 2E, miR-146a significantly reduced firefly lucif-erase activity compared to miRNA control, indi-cating that miR-146a directly target the 3’UTR

of CXCR4, thus being able to repress the expression of the CXCR4. To further verify that miR-146a conferred ADM resistance in K562 cells mainly by targeting CXCR4, rescue experi-ments through knockdown of CXCR4 in K562 cells infected with lentivirus expressing miR-146a inhibitor was done. As shown in Figure 3A and 3B, RT-PCR and western blot results indi-cated that CXCR4 siRNA alleviated the increase in CXCR4 expression induced by the miR-146a inhibitor. Moreover, knockdown of CXCR4 could significantly attenuate the ADM resistance of K562 cells induced by the miR-146a inhibitor (Figure 3C and 3D), suggested that miR-146a played its function in drug-resistance of K562 by targeting CXCR4.

Physcion enhances the sensitivity of K562/ADM to ADM

First, we examined the anti-proliferative effect on physcion on K562/ADM cells. As shown in

Figure 3. Knockdown of miR-146a reduces the sensitivity of K562 cells to ADM through upregulating CXCR4. A. The mRNA expression of CXCR4 in K562 cells transfected with miR-146a inhibitor is significantly decreased by CXCR4 siRNA. B. The protein expression of CXCR4 in K562 cells transfected with miR-146a inhibitor is significantly decreased by CXCR4 siRNA. C. Knockdown of CXCR4 in K562 cells transfected with miR-146a inhibitor restores the sensitivity to ADM. D. Knockdown of CXCR4 in K562 cells transfected with miR-146a inhibitor restores the sensitiv-ity to ADM-induced apoptosis. **P<0.01.


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Figure 4A, physcion, in a dose-dependent man-ner, reduced the viability of K562/ADM cells. When K562/ADM cells were challenged with same dosage of physcion, the anti-proliferative effect was more profound at 48 hours incuba-tion than 24 hours. However, when the treat-ment of physcion was prolonged to 72 hours, no marked further suppression on cell growth was observed (P>0.05 vs. 48 hours, Table 1). Given that physcion treatment at 2 and 5 μM for 48 hours did not show significant cytotoxic-ity to K562/ADM cells (growth inhibition less than 10%), this dosage was used to examine whether physcion could reverse the drug-resis-tance in K562/ADM cells. CCK-8 analysis showed that physcion treatment at 2 and 5 μM decreased IC50 value of ADM in K562/ADM cells from 3.5 μM to 1.7 μM and 0.68 μM, respectively. Our results indicated that phy-scion enhanced the sensitivity of K562/ADM cells to ADM by 2.06- and 5.3-fold at 2 and 5

μM, respectively (Table 1). The resistance reversal was also confirmed by colony forma-tion assay. As shown in Figure 4B, when K562/ADM cells were treated with physcion in combi-nation with ADM, the colony number and size was significantly decreased, compared with cells treatment with ADM alone (P<0.05), sup-porting that physcion increased the sensitivity of K562/ADM cells to ADM. Next, the effect of physcion on ADM-induced apoptosis in K562/ADM cells was assessed. As shown in Figure 4C, flow cytometric analysis showed that phy-scion at both 2 and 5 μM was able to signifi-cantly increase the apoptotic cell population in K562/ADM cells, compared with cells treated with only ADM (P<0.05). In addition, Hoechst staining was also conducted to ascertain the enhanced ADM-induced apoptosis by physcion. As shown in Figure 4D, though physcion by itself could not induce apoptosis, it did signifi-cantly caused more profound apoptosis in

Figure 4. Physcion reverses the resistance of K562/ADM cells to ADM. A. The effect of physcion on cell viability was determined by CCK-8 assay. B. Combination of physcion and ADM exerted more profound anti-proliferative effect on K562/ADM cells. C. Physcion renders K562/ADM cells more sensitive to adriamycin-induced apoptosis. D. Combi-nation of physcion and ADM results in significantly more apoptosis in K562/ADM cells. **P<0.01.


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K562/ADM cells, as demonstrated by clear nuclear condensation.

Physcion reverses drug-resistance by upregu-lating miR-146a and interfering CXCL12/CXCR4 signaling

A number of anthraquinone compounds exert anti-tumor effect through modulating miRNAs [24, 25], thus, we examined whether physcion could influence the expression of miR-146a. As shown in Figure 5A, our results showed that

physcion treatment can lead to a dose-depen-dent increase in the level of miR-146a in K562/ADM cells. Moreover, our results also showed that the sensitizing effect of physcion was almost completely abrogated by miR-146a inhibitor, indicating that physcion reversed the drug resistance in K562/ADM cells by modulat-ing miR-146a (Figure 5B and 5C). Correspond- ing to the effect of physcion on miR-146a, phy-scion treatment resulted in a dose-dependent decrease in the expression of CXCR4 in K562/ADM cells (Figure 5D and 5E). In addition, we

Figure 5. Physcion reverses the resistance of K562/ADM cells through upregulating miR-146a and interfering CXC12/CXCR4 signaling. A. Physcion dose-dependently increases the expression of miR-146a in K562/ADM cells. B. Knockdown of miR-146a attenuates the reversal effect of physcion on drug-resistance in K562/ADM cells. C. Knockdown of miR-146a attenuates the enhancing effect of physcion on adriamycin-induced apoptosis in K562/ADM cells. D. Physcion dose-dependently decreases the mRNA expression of CXCR4 in K562/ADM cells. E. Phy-scion dose-dependently decreases the protein expression of CXCR4 in K562/ADM cells. F. Physcion dose-depend-ently interferes with the binding between CXC12 and CXCR4. **P<0.01.


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conducted migration assay to evaluate the effect of physcion on CXCR12 binding to CXCR4 in K562/ADM cells. Our results showed that the migration was significantly impaired by phy-scion treatment (Figure 5F). Taken together, our results showed that physcion reversed drug resistance by both modulating CXCR4 expres-sion through upregulating miR-146a and inter-fering binding between CXCR12 and CXCR4.

Physcion combined with ADM exerts anti-tumor effect in vivo

A xenograft-nude mouse model was used to evaluate the in vivo reversal effect of physcion on drug-resistance. As shown in Figure 6A, the K562/ADM-bearing mice did not present marked weight loss in the treatment course, suggesting that physcion has a good safety pro-file. The in vivo anti-tumor effect was assessed

by comparing the RTV. As shown in Figure 6B, ADM alone showed slight effect on the drug-resistant K562/ADM tumor. In contrast, phy-scion at dosage of 5 mg/kg/day significantly potentiated the anti-tumour activity of ADM in K562/ADM xenografts (P<0.01 vs. control). Moreover, miR-146a expression was signifi-cantly increased while CXCR4 expression was significantly suppressed in tumor tissue from mice model treated with combination of phy-scion and ADM (P<0.01 vs. control) (Figure 6C and 6D). Collectively, our results showed that the therapeutic regimen of physcion and ADM was effective in terms of inhibition of drug-resistant tumor growth.

Discussion

As a group of highly conserved non-protein-coding RNAs, miRNAs can regulate the expres-

Figure 6. Physcion potentiates the anti-tumor effect of ADM in vivo. A. The body weight of animal model is not signifi-cantly changed by treatment. B. Combination of physcion and ADM shows more significant inhibiting effect on tumor growth. C. Inhibition of tumor growth is associated with upregulation of miR-146a and downregulation of CXCR4 mRNA in vivo. D. Inhibition of tumor growth is associated with downregulation of CXCR4 protein in vivo. **P<0.01.


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sion of a variety of target genes in eukaryotic cells [26], thus playing a role in cell prolifera-tion, apoptosis, development, and differentia-tion [27]. MiR-146a, was first found to be involved in the innate immune and inflammato-ry response to microbial infection [28], and has now been found to play a role in carcinogenesis and the development and metastasis of solid tumors [29, 30]. As regards to hematological malignancies, it has been found that aged knockout mice develop myeloid and lymphoid malignancies [31, 32]. Garzon et al have found that miR-146a is expressed at relatively high levels in bone marrow CD34+ progenitors from healthy donors whereas at low levels in acute myeloid leukemia (AML) patients and even lower levels in monocytes, granulocytes, eryth-rocytes, and megakaryocytes from the periph-eral blood or bone marrow of healthy donors [33]. Xu et al have also reported that miR-146a functions as an oncogene in acute promyelo-cytic leukemia (APL) and contributes to miR-146a development by repressing Smad4 [34]. However, the role of miR-146a in CML remains to be elucidated. In the present study, our results showed that miR-146a expression is lower in drug-resistant K562/ADM cells com-pared with sensitive K562 cells. Moreover, our study has provided evidence that upregulating miR-146a could restore the sensitivity of K562/ADM cells to ADM by repressing CXCR4 expres-sion and a natural agent, physcion, could sensi-tize K562/ADM cells by modulating miR-146a and thus interfering CXC12/CXCR4 signaling.

Mounting evidence suggests that miRNAs are involved in drug resistance to chemotherapeu-tics in cancer treatment due to their ability to regulate genes associated with drug-resistance [35]. The correlation between miR-146a and drug-resistance of cancer cells has also been described in a few studies. Significant upregu-lation of miR-146a was found in cisplatin-resis-tant breast cancer MCF-7 cells compared with sensitive MCF-7 cells [36]. A study in hepatocel-lular carcinoma cells found that miR-146a was significantly higher expressed in IFN-α-resistant clones and decreased the sensitivity to IFN-α through the suppression of apoptosis [37]. Another study in hepatocellular carcinoma cells by Zhuo et al also reported that miR-146a was upregulated in drug-resistant cell lines [38]. In colorectal cancer, high expression of miR-146a is correlated with cetuximab resistance and a

poorer prognosis [39]. However, conflicting results have also been reported. MiR-146a was found to be expressed in a significantly lower level in cisplatin-resistant ovarian cancer A2780 cells, although miR-146a inhibitor was not able to establish resistance in A2780 cells [40]. In non-small cell lung cancer cells, low expression of miR-146a was correlated with advanced clinical TNM stages and distant metastasis despite chemotherapy [41]. More- over, a recent study showed that pharmacologi-cal induction of miR-146a sensitized human glioblastoma cells to temozolomide-induced apoptosis [42]. In the current study, we found that miR-146a expression was significantly lower in ADM resistant K262 cells. Furthermore, transfection of miR-146a inhibitor in sensitive K562 cells presented resistance to ADM-induced apoptosis while transfection of miR-146a mimic in K562/ADM restored the sensi-tivity to ADM. Our findings here suggest that miR-146a confers resistance of K562 cells to ADM. Taking into account already existing results from previous studies, it is reasonable to believe that the role of miR-146a in drug-resistance is specific to distinct tumor type. Given that miR-146a targets more than one sig-naling pathway in cancerous cells, whether miR-146a is pro- or anti-resistant may depends on the key signaling pathway it regulates in the specific cancer cells.

CXCR4, a chemokine receptor expressed in hematopoietic and epithelial cancer cells, has been found to mediate contact between leuke-mia cells and stromal cells and thus protects leukemia cells from spontaneous and chemo-therapy-induced cell death [43]. Therefore, CXCR4 is regarded as a crucial factor for sur-vival and drug resistance in leukemia with a major prognostic impact in acute myeloid leu-kemia [44, 45]. In the present study, our results showed that miR-146a expression inversely correlated with CXCR4 expression. In addition, we found that resistance to ADM in K562 cells established by knockdown of miR-146a could be significantly attenuated by CXCR4 siRNA, suggesting that CXCR4 was the key factor that mediated the ADM resistance resulted from miR-146a downregulation. Moreover, we found that miR-146a directly targeted the 3’-UTR of CXCR4 mRNA and repressed its expression, which was consistent with a recent study [46]. Interestingly, a recent study reported that over-


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expression of CXCR4 3’UTR inhibited the activ-ity of miR-146a, thus elevating the expression of CXCR4 in breast cancer MCF-7 cells, implying the regulation of CXCR4 by miR-146a might not be as simple as we thought [22]. Nevertheless, the presence of a more complex relationship between miR-146a and CXCR4 and its role in drug-resistance of K562 cells needs to be vali-dated by further studies.

In 2005, physcion, as an active ingredient iso-lated from the culture of Pleospora sp. IFB-E006, has been found to have cytotoxic effect in colon cancer and leukemia cells [47]. Later studies found that physcion was able to induce cell cycle arrest and apoptosis in human breast cancer cells and cervical carcinoma cells, respectively [17, 18]. Recently, EMMPRIN and SOX2 were identified as molecule targets of physcion, which explained its apoptosis-induc-ing and anti-metastatic effect in colorectal can-cer cells [19, 20]. Moreover, a study by Elf et al reported that physcion suppressed cancer cell proliferation and tumour growth in nude mice xenografts through inhibiting 6-phosphogluco-nate dehydrogenase [48]. In the present study, we provide the first evidence for the modulating effect of physcion on miR-146a and its capabil-ity of reversing drug resistance, supporting the concept that natural compounds can target multiple signaling pathways to exert anti-tumor effect.

In conclusion, our results in the present study demonstrated that the downregulation of miR-146 and upregulation of CXCR4 might correlate with the drug resistance of K562/ADM cells. In addition, physcion could reverse the K562/ADM resistance to ADM by inducing miR-146a expression and thus inhibiting CXCL12/CXCR4 signaling. Given the low toxicity of physcion on normal cells, physcion can be considered as a potential candidate of adjunctive therapeutic agent for CML.

Acknowledgements

This work was supported by Basic Research project of Sichuan Province (No. 14JC0193).

Disclosure of conflict of interest

None.

Address correspondence to: Wenjun Liu, Depart- ment of Pediatrics, Affiliated Hospital of Southwest

Medical University, Luzhou 646000, Sichuan, China. Tel: 8608303165590; E-mail: [email protected]

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