All-trans retinoic acids induce differentiation and sensitize a radioresistant breast cancer cells to chemotherapy

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1 Yn et l. BMC Complementry nd Alterntive Medicine (216) 16:113 DOI /s y RESEARCH ARTICLE All-trns retinoic cids induce differentition nd sensitize rdioresistnt rest cncer cells to chemotherpy Yunwen Yn 1,2, Zhen Li 3, Xing Xu 3,4, Clrk Chen 3, Wei Wei 1, Ming Fn 5, Xufeng Chen 3, Jin Jin Li 5, Yun Wng 2 nd Jioti Hung 3 Open Access Astrct Bckground: Rdiotherpy is of criticl importnce in the tretment of rest cncer. However, not ll ptients derive therpeutic enefit nd some rest cncers re resistnt to the tretment, nd re thus evidenced with prospective distnt metsttic spred nd locl recurrence. In this study, we investigted the potentil therpeutic effects of ll-trns retinoic cid () on rdition-resistnt rest cncer cells nd the ssocited invsiveness. Methods: The cells with gined rdition resistnce fter long term tretment with frctionted ionizing rdition were derived from humn rest cncer cell line, nd re enriched with cells expressing puttive rest cncer stem cell iomrker CD44 + /CD24 -/low /ALDH +. The enhnced invsiveness nd the cquired resistnces to chemotherpeutic tretments of cells were mesured, nd potentil effects of ll-trns retinoic cid () on the induction of differentition, invsion nd migrtion, nd on the sensitivities to chemotherpies in cells were investigted. Results: cells re with enrichment of cncer stem-cell like cells with positive stining of CD44 + /CD24 -/low, OCT3/4 nd NANOG. cells showed n incresed tumoregensis potentil nd enhnced ggressiveness of invsion nd migrtion. Tretment with induces the differentition in cells, resulting in reduced invsiveness nd migrtion, nd incresed sensitivity to Epiruincin tretment. Conclusion: Our study suggests potentil clinic impct for s chemotherpeutic gent for tretment of therpy-resistnt rest cncer especilly for the metsttic lesions. The study lso provides rtionle for s sensitizer of Epiruincin, first-line tretment option for rest cncer ptients. Keywords: Brest cncer, Rdition resistnce, Cncer stem cell, Bckground Brest cncer is the leding cncer dignosed in women nd is second only to lung cncer in terms of cncer deth, cusing extensive moridity nd psychologicl distress to millions glolly [1, 2]. Despite the tremendous efforts nd progress in rest cncer reserch nd erly dignosis, clinicl outcome for rest cncer ptients is still disppointing. Resistnces to current therpeutic regimen, nd s much s 4 % of relpses with recurrent nd/or metsttic disese Correspondence: wngyun@hmu.edu.cn 2 Deprtment of Biochemistry, Lortory of Moleculr Biology, Anhui Medicl University, Hefei, Chin Full list of uthor informtion is ville t the end of the rticle remin to e gret chllenges in clinicl mngement for rest cncer ptients [3 5]. It is lso needed to e indicted tht, while overll rest cncer mortlity rtes hve decresed over the lst severl decdes [6], the survivl rtes for metsttic rest cncer re currently estimted t less thn 25 % for 5-yer nd 5 1 % for 1-yer [3, 7 9]. Rdition therpy continues to e n importnt prt of conditioning regimens for rest cncer tretment. Rdition therpy given fter surgery in erly stge rest cncer ptients hs shown significnt effects of incresing the proility of oth locl control nd survivl [1]. The most recent met-nlysis including 1,81 women in 17 clinicl trils of rdition or no rdition 216 Yn et l. Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Yn et l. BMC Complementry nd Alterntive Medicine (216) 16:113 Pge 2 of 11 fter lumpectomy showed tht rdition reduced the 1- yer risk of ny recurrence in lymph node-negtive women from 31 to 15.6 % nd reduced the 15-yer risk of deth from rest cncer from 2.5 to 17.2 % [11]. However, the rte of totlly control of tumor growth y rdiotherpy remins uncceptle low, nd studies hve indicted tht rest cncer ptients my fil to rdition therpy nd cncer cells in these ptients ecome resistnt to the tretment [12 15]. Elucidtion of mechnism cusing tumor rdioresistnce nd definition of effective therpeutic trgets to enhnce tumor response, especilly for the most resistnt nd ggressive cncer cells in the recurrent nd metsttic lesions, re thus urgently needed. In our previous studies, we oserved rest cncer cell popultion (MCF + FIR) tht could survive fter course of clinicl frctionted doses of rdition nd showed enhnced rdioresistnce compred to the wild type prentl cells [16, 17]. With sucloning, different clones with vried rdiosensitivity were isolted from this rdioresistnt popultion [18]. Cells expressing the iomrkers of rest cncer stem cells (BCSCs; e,g., CD44 + /CD24 -/low /ALDH + ) were further sorted nd confirmed in one of these clones () [19], indicting tht BCSCs cn survive long-term frctionted rdition nd e responsile tumor repopultion with rdition resistnce. In supporting this oservtion, other studies lso demonstrted the enrichment of cncer stem cells during the course of frctionted rdition [2, 21]. In ddition, rdition is lso shown to e le to reprogrm the differentited rest cncer cells into induced rest cncer stem cells (BCSCs) [22]. These nd other results provide the evidence indicting tht, while some ptients with erly-stge rest cncer cn enefit from rdition therpy, others my gin resistnce to rdiotherpy with potentil of incresed recurrence nd/or distnt metstsis due to the enrichment of BCSCs [23]. Thus, trgeting BCSCs in ptients with rdition resistnt rest cncer my impede n importnt clinicl impct for decresing cncer metsttic potentil in these ptients. In this study, we used the MCF + FIR cellulr model to investigte the roles of BCSCs in enhnced cpility of cncer cell invsion nd the cquired resistnces to chemotherpy of rest cncer. The potentil therpeutic effects of ll-trns retinoic cid (), which hs een used in the mngement of certin hemtologic mlignncies nd solid tumors, including rest cncer [24], on the induction of differentition of enriched BCSCS, inhiition of ggressive growth nd sensitiztion to chemotherpeutic gent in MCF/C6 cells. The results indicte tht is promising cndidte to trget rdioresistnt rest cncer cells with enrichment of BCSCs. Methods Regents ws purchsed from Sigm Aldrich (St. Louis, MO), nd ws dissolved in dimethylsulphoxide () s stock solution. Primry ntiodies for Involucrin, Sydencn-3, nd E-Cdherin were purchsed from Snt Cruz Biotechnology (Snt Cruz, CA). Anti-CD44 ntiody ws from ABGENT (Sn Diego, CA). Anti-β-ctin ntiody ws from Cell Signling Technology (Beverly, MA). sirna oligos for CD44 nd control sirna-a were lso from Snt Cruz Biotech. Inc. Enzymes I-SceI ws from New Englnd Biols (Ipswich, MA). Cell culture Humn rest cncer cells were from Americn Type Culture Collection (ATCC, Mnsss, VA). Rditionresistnt cells were generted from MCF-7 cells y exposure to frctionized ionizing irrdition (FIR) with totl dose of 6 Gy of γ-irrdition (2 Gy per frction, five times per week for 6 weeks) s previously descried [17]. nd cells were mintined in ATCCformulted RPMI-164 medium supplemented with 1 % Fetl ovine serum (FBS), 5 % sodium pyruvte, 5 % nonessentil mino cid, 1 U/mL of penicillin, nd 1 mg/ml streptomycin in 37 C incutor (5 % CO 2 ). To mintin the rdition-resistnt phenotype, cells were lso frequently exposed to irrdition (IR) t 2Gy for five times per week nd rdioresistnce ws vlidted efore ech designted experiment. Clonogenic survivl ssy Cells in log-phse were plted nd then immeditely treted with indicted tretment. 24 h lter, cells were wshed twice with pre-wrmed medium, nd were then mintined in corresponding medium for 1 14 dys nd stined with crystl violet. Colonies consisting >5 cells were considered s survivl colonies nd directly scored using n inverted microscope. Averge numers for survivl colonies were plotted versus untreted control to determine the survivl frctions. When pretretment pplied, cells were treted 1 μm for 72 h. Cells were then re-plted nd treted with indicted chemodrugs for 24 h, nd mintined in corresponding medium for colony formtions s descried ove. Assys for invsion, migrtion nd wound heling nd cells in log-phse were trypsinized, nd cells in growth medium contining 1 % FBS were re-seeded in 1 BME (Trevigen, Githersurg, MD) coted 8.-μm pore size cell culture inserts (for 24- well plte, Millipore, Dnvers, MA). Complete growth medium contining 1 % FBS ws plced outside the chmers, nd cells were llowed to invde towrd the

3 Yn et l. BMC Complementry nd Alterntive Medicine (216) 16:113 Pge 3 of 11 ttrctnt of full-serum medium. Chmer filter processing nd visuliztion/quntittion of invsion were performed, s previously descried [25]. Cells migrted to ottom chmer were lso visulized/quntified for migrtion nlysis. For wound heling nlysis, cells were grown in monolyers in triplicte in 24-well pltes for 72 h. The confluent monolyer ws then scrped with sterile tip. The migrtion into the wounded monolyer ws ssessed y microscopy. When sirna trnsfection pplied, cells were trnsiently trnsfected with SiRNA- CD44 or SiRNA-Control-A, nd then mintined in complete medium for 72 h until confluent monolyer formed for wound heling nlysis, or re-seeded in BME-precoted cell culture inserts for invsion/migrtion ssys. Flow cytometry nlysis After tretments, cells were detched y using stempro ccutse (Life Technologies, Grnd Islnd, NY), nd wshed twice with PBS. Cells were then stined with PE-conjugted nti-sox2, nti-oct3/4, nd nti- NANOG ntiodies, or co-stined with PE-conjugted nti-cd24 nd FITC-conjugted nti-cd44 ntiodies (BD Biosciences, Sn Jose, CA). In the process for stining of Sox 2, Oct3/4 nd Nnog, BD Perm/ WshTM uffer ws lso used per mnufcture s instruction. PE- or FITC-positive cells were quntified y flow cytometric nlysis on Flow Cytometer LSRII (BD Biosciences, Sn Jose, CA). Up to cells were counted during flow cytometry nlysis. For cell cycle nlysis, cells were collected nd fixed with 75 % ethnol, stined with propidium iodide nd nlyzed y flow cytometry with events counting per run, s descried previously [26]. The percentge of cells in the G 1, S, nd G 2 /M phses of the cell cycle were determined y using Flowjo softwre (Flowjo dt nlysis softwre, OR). Immunolot ssy Cell lystes were prepred in RIPA uffer with mild soniction, nd sujected to SDS-PAGE gel for immunolot ssys. β-ctin ws included to determine equivlent protein loding. in vivo end-joining ssy in vivo end-joining ssy ws sed on the rectivtion of linerized plsmid s previously reported [27]. Briefly, cells were treted with 1 μm of, or s control, for 72 h, cells were then co-trnsfected with 1.2 μg linerized EJ5-GFP sustrtes (linerized with I-SceI) nd.5 μg circulr pdsred-express2-n1 (s trnsfection control) y using electroportion (Gene Pulse Xcell, Bio-Rd, Hercules, CA). After trnsfection, cells were plted nd cultured in fresh complete medium for 72 h. In experiment, 1 μm ws dded into culture medium fter trnsfections nd ws included s control. Flow cytometry nlysis ws performed with Fortess Flow Cytometer (Fluofrm, Princeton, NJ). Up to cells were counted. The rtio of GFP-positive cells to DsRed-positive cells ws used s mesure of endjoining efficiencies. Tumor inititing test Tumor inititing test ws conducted following the descried methods [19, 28] nd the protocol ws reviewed nd pproved y the Chncellor s Animl Reserch Committee (ARC) t the University of Cliforni Los Angeles (ARC # ). Six weeks old femle NOD/SCID mice (Jckson L, Br Hror, ME) were pretreted for 5 dys with estrogen pellets (Innovtive Reserch of Americ, Srsot FL) nd freshly prepred nd cells were resuspended in serumfree PBS/Mtrigel mixture (1:1 V/V), nd 1 13 cells were inoculted sucutneously to ilterl frnks of sme niml. Tumorigenesis ws ssessed twice week with plption. Tumor sizes were determined from cliper mesurements of tumor length (L) nd width (W) ccording to the formul (LxW2)/2. Sttisticl nlyses Sttisticl nlyses were performed using the Student s t-test. A p vlue <.5 ws considered s significnt (). Results Enhnced cncer cell invsiveness nd migrtion of rdition-resistnt cells Rdition in cncer tretment is intended to destroy cncer cells y dmging their DNA, nd the resistnce of cells to IR is thus modulted y three intimtely relted cellulr processes, including DNA dmge repir [29]. In this study, we first verified the rdioresistnce of cells. We found tht the clonogenic survivl rte ws enhnced in cells to out 12-fold when compred to tht of wild type cells (Fig. 1). Using in vivo end-joining ssy, we detected the DNA repir cpcity in versus wild type cells nd the results showed tht NHEJ (non-homologous end-joining) DNA repir efficiency ws out two-folds in cells compred to the wild type cells (Fig. 1). In greement with NHEJ eing n indictor of intrinsic DNA dmge repir cpcity [29, 3], these results indicte tht DNA repir ccpicity plys role in signling the rdioresistnt phenotype of cells. It hs een previously shown tht HER2-positive cells in were with incresed invsiveness

4 Yn et l. BMC Complementry nd Alterntive Medicine (216) 16:113 Pge 4 of 11 IR (Gy): Survivl Frction (1%) c Invsion Migrtion Reltive NHEJ efficiency hr 48 hr d Rtio (%) Rtio (%) Migrtion reltive Distnce Rtio (%) Wound Heling hr e E-Cdeherin β-actin 72 hr Fig. 1 Rdition-resistnt cells re more invsive cncer cells. Incresed rdioresistnce mesured y clonogenic survivls of nd cells. NHEJ efficiency mesured y in vivo EJ ssy. Cells were co-trnsfected with linerized EJ5-GFP plsmid nd control pdsred, nd were then treted with 2 Gy of IR. Re-circulted EJ5-GFP ws counted y flow cytometry nlysis 72 h fter trnsfection. c Representtive imges for trnswell invsion ssy nd wound-heling ssys (top: invsion ssy; middle: migrtion ssy; ottom: wound heling ssy). d Reltive quntittion of cellulr invsiveness, migrtion nd wound heling ility in cells compred with the wild type cells. e Western lots of E-Cdeherin inndcells.β-ctin ws included for equivlent protein loding. Dt represent the verge from t lest three independent experiments. Indictes sttisticl significnce (p <.5) [19]. In n ttempt to test whether cells hve overll chnges in cncer cell invsiveness nd migrtion, we performed the ssys in nd cells. We oserved tht the cpilities of cncer cell invsion/migrtion were drmticlly enhnced in cells versus prentl cells. cells lso showed incresed ility for wound heling (Fig. 1c, d). In ddition, sustntil mount of E-cdherin, protein prominently ssocited with tumor invsiveness nd metsttic dissemintion [31], ws found to e reduced in the cells (Fig. 1e). Enrichment of stem cell-like cncer cells in cells We next exmined the potentil enrichment of stem cell-like cncer cells, or cncer stem cells (CSCs), in cells. Our previous study hs reveled the enrichment of HER2 + /CD44 + /CD24 -/low cncer stem cell popultion in cells. In this study, we used cncer stem cell surfce mrker CD44 + /CD24 -/low, first descried mrker for BCSCs [32, 33], nd emryonic stem cell mrkers Oct3/4 [34], Sox II [35] nd Nnog [36] to determine the puttive cncer stem cells. Flow cytometry nlyses showed significnt increses of cell popultions with positive stining of CD44 + /CD24 -/low (from 1.26 ±.52 to 35.8 ± 3.41), Oct3/4 (2.78 ±.87 to 23.7 ± 4.66) nd Nnog (from 47.6 ± 2.33 to 74.1 ± 4.27) in cells (Fig. 2, c). In ddition, we lso detected increse of CD44-positive popultion, determinnt cell memrne protein in cell migrtion nd invsion [37], in cells, which ws further confirmed y western lot nlysis (Fig. 2, c). In NOD/ SCID mouse, we found tht ll the sites inoculted with cells (1 cells/injection) developed tumors (4/4) with n verge volume of 259 mm 3 t dy 35; wheres three of four sites inoculted with the sme numer of cells showed detectle tumors with n verge volume of 2 mm 3 (Fig. 2d nd Additionl file 1: Figure S1). cells lso showed shorter ltency for forming tumors when compred to cells (16 ± 5 dys versus 26 ± 2 dys). Thus, the results of tumor inititing test suggested tht rdioresistnt cells re more tumorigenic thn prentl cells.

5 Yn et l. BMC Complementry nd Alterntive Medicine (216) 16:113 Pge 5 of 11 CD44 + /CD24 -/Low SOX2 OCT3/4 NANOG c Percentge (%) CD44 + /CD24 -/low OCT3/ Percentge (%) Nnog + 8 SOX CD44+ CD44+ d Percentge (%) CD44 + CD44 β-ctin Tumor Volume (cm 3 ) Fig. 2 Enrichment of BCSCs in cells. Flow cytometry nlysis for different stem-cell surfce mrkers in nd cells (left); Incresed CD44 expression in cells compring to prentl cells. Top: flow cytometry nlysis of CD44 expression; Bottom left: digrm showing the percentges of cell popultions with CD44 expression; Bottom right: Western lot nlysis for CD44 protein expression. Dt represent the verge from t lest three independent experiments. c Digrm (right) showing the chnges of the cell frctions with corresponding positive stem cell mrkers. d Tumorogenesis of cells verses cells. Top: imges for collected tumors from Tumor inititing test; ottom: digrm showing the verge of tumor volumes. Indictes sttisticl significnce (p <.5) Knocking-down CD44 expression inhiited the ggressive growth of /FIR C6 cells Memers of the CD44 fmily of trnsmemrne glycoproteins, in prticulrly CD44v6 isoforms, were shown to e metsttic determinnts of tumor cells, nd the expression of severl CD44 proteins correltes with ggressive stges of vrious humn cncers. Thus, CD44 hs een considered s therpeutic trget for metstsizing tumors [38 4]. In CD44- overexpressed cells, sirna-medited CD44 inhiition led to reduction in cell invsiveness nd migrtion y ner 7 % (Fig. 3). The gp filling rtes were lso reduced (ner 8-folds) y knocking-down of CD44 in cells. These results indicte tht CD44-expressing BCSCs re indeed enriched in the rdioresistnt popultion, nd CD44 cn e used s n effective therpeutic trget to tret rdioresistnt rest cncer. induces differentition nd inhiits cncer cell invsion in MCF-7/C6 cells is routinely used s therpeutic gent to induce differentition of leukemic stem cells in cute promyelocytic leukemi [41]. hs lso een reported to induce differentition in cncer stem cells, including BCSCs [42 44]. Given the evidences ove showing tht rdioresistnt cells re with enrichment of CSCs, we thus tested the potentil effects of on differentition of popultion. Our results showed tht tretment with 1 μm of for 72 h significntly reduced the percentges of cell frctions of CD44 + /CD24 -/low -positive (from 28.1 ± 2.38 to 4.27 ±.51) nd NANOG-positive (from 72.8 ± 4.88 to 5.2 ± 3.79), nd slightly reduced the percentge of cells tht were OCT3/4-positive (from 16.6 ± 1.52 to 12.9 ± 2.33). Exposure to lso incresed the expressions of differentition mrkers, involucrin nd syndecn 3 [45, 46],

6 SiRNACD44 SiRNAControl Pge 6 of 11 SiRNACD44 SiRNAControl Yn et l. BMC Complementry nd Alterntive Medicine (216) 16:113 CD44 β-ctin SiRNA-CD44 hrs Wound Heling 48 hrs SiRNA/ CD44l 48 hrs SiRNA/ Control Migrtion 48 hrs Rtio (%) Invsion c Rtio (%) SiRNA-Control Reltive Migrtion Distnce Rtio (%) Fig. 3 CD44 inhiition reduced invsiveness, migrtion nd the ility of wound heling in cells. sirna trnsfection knocks down CD44 expression in cells. Left: Western lot showing the inhiition of CD44 in cells fter trnsfection of sirna-cd44 for 48 h; Right: Flow cytometry nlysis showing the decrese of cell frctions with CD44-positive expression in cells with trnsfection of sirna-cd44. Representtive imges for cncer cell invsion, migrtion nd wound-heling ssys in cells with or without CD44 inhiition. c Quntittion of invsiveness, migrtion nd wound heling ility in cells with CD44 inhiition. Dt represent the verge from t lest three independent experiments. Indictes sttisticl significnce (p <.5) in cells (Fig. 4 nd, nd Additionl file 1: Figure S1B). In ddition, cell cycle nlysis showed tht tretment cused increses of S phse in / C6 cells fter exposure for 24 h when compred to tht of untreted control (from percentge of ± 1.63 to ± 1.82) or to tht of tretment (from percentge of ± 2.14 to ± 1.82), which support the concept tht tht could induce cell prolifertions in quiescent BCSCs popultion [47]. As expected, we lso found tht tretment reduced the percentges of invsive cells in MCF-7/C6 cells (Fig. 4d nd Additionl file 1: Figure S1C). enhnces sensitivity of FIR cells to rdition tretment To further evlute the potentil therpeutic impcts of on rest cncers cells with cquired rdition resistnce, we tested whether tretment could chnge the sensitivities of cells to rdition nd chemotherpeutic tretments. For this, we first exmined the direct cytotoxic effect of on / C6 cells nd results indicte tht tretment with t the concentrtion rnging up to 5 μm induced dosedependent inhiition on clonogenic survivl (Fig. 5). We next exmined the effects of on cellulr cpility of DNA dmge repir nd rdition sensitivity in cells. As shown in Fig. 5, tretment with 1 μm of for three dys reduced end-joining ctivity with sttisticl significnce (from.192 ±.23 to.132 ±.18, p =.352). In ddition, pretretment with t 1 μm for 72 h sensitized cells to rdition tretment, s determined with clonogenic survivl (from percentge of ± 5.3 to ± 4.83, p =.162, (Fig. 5 nd c). enhnces sensitivity of cells to chemotherpy With clonogenic ssys, we lso oserved tht pretretment with enhnced clonogenic cell killing effects of epiruincin nd 5-Fu on cells. However, it

7 Yn et l. BMC Complementry nd Alterntive Medicine (216) 16:113 Pge 7 of 11 Sydencn-3 NANOG Involucrin β-ctin SOX II c G/G1 = 43.5 S = G2/M = G/G1 = 4.33 S = 2.69 G2/M = 21.1 G/G1 = S = G2/M = G/G1 = S = G2/M = OCT3/4 d hours CD24 low / CD44 + Fig. 4 induces differentition of cells. Flow cytometric results for stem-cell surfce mrkers in -treted cells. cells were treted with 1. μm of for 72 h, nd were then nlyzed y flow cytometry ssy. Westen lots showing tht tretment with 1. μm for 72 h induces expressions of differentition mrker involucrin nd Syndecn 3 proteins in cells. β-ctin ws included for equivlent protein loding. c The effect of on cell cycle progress in cells. Cells were treted with 1. μm, nd then nlyzed y flow cytometry. d Representtive imges showing tretment reduces the invsiveness of cells. Cells were pretreted with 1. μm of for 72 h, nd invsion ssy ws performed s descried in Mterils did not ffect the clonogenic survivl of cells treted with 1 nm of Doxetxel (Fig. 6). We further noticed decrese of G 2 /M distriution of cells in epiruincintreted cells when cells were pretreted with, suggesting tht the pretretmentenhnced cell killing effect of epiruincin in cells my occur in G 2 /M phse of cell cycle. Discussion Rdioresistnce of cncer cells my rise from self-repir mechnisms (minly DNA dmge repir) or repopultion of rdioresistnt cncer stem cells, or oth [48]. Dt presented here indicte tht the rdioresistnt popultion derived from long-term frctionted doses of rdition is with enrichment of BCSCs nd enhnced cpility of NHEJ repir. Compred to prentl cells, cells re ggressive with incresed cpcity of invsiveness nd migrtion, nd inhiition of CD44 expression could effectively reduce cncer cell invsiveness nd migrtion in cells. Most importnt, our dt demonstrted tht tretment with cn induce differentition of the enriched BCSCs in cell popultion nd sensitized them to chemotherpeutic gent epiruincin. More thn 6 % cncer ptients worldwide use rdiotherpy for the control of tumor growth during the course of their disese. However, in spite of significnt dvncements in tumor imging nd precise of tumor dose clcultion nd delivery, the rte of totl tumor growth control y rdiotherpy remins disppointing.

8 Yn et l. BMC Complementry nd Alterntive Medicine (216) 16:113 Pge 8 of 11 Survivl Frction (1%) µm 3 µm 5 µm µm c Survivl Frction (1%) μM Percentge (%) W/O IR IR Fig. 5 exposure increses the sensitivities of cells to rdition tretment. Clonogenic survivl of cells exposed to tretment. Cells were plted nd treted with indicted doses of for 24 h, nd cells were then cultured for colony formtion. exposure reduces NHEJ ctivity in MCF 7 FIR cells, nd increses rdiosensitivity. Cells were co-trnsfected with control pdsred nd linerized EJ5-GFP plsmid, nd were then treted with 1. μm of for 72 h. in vivo EJ5 ctivity ws mesured s descried in Mterils. Left: in vivo EJ5 ssy; Right: digrm showing the inhiition of EJ5 reunion ility in -treted cells; c Clonogenic survivl ssy ws performed to determine the chnges of sensitivity in cells treted with 2Gy ionizing rdition. Cells were pretreted with 1. μm of, or s control, for 72 h, nd 5 cells were then plted nd irrdited with 2 Gy of IR. Left: tretment reduced clonogenic survivl of irrdited cells. Right: demonstrtive imges for colony survivl of irrdited cells. Dt represent the verge from t lest three independent experiments. Indictes sttisticl significnce (p <.5) No IR IR Although rdition therpy cn decrese the risk of locl cncer recurrence nd improves survivl, clinicl evidence hs shown the detrimentl effect of tretment interruptions on tumor control in rest cncer ptients [49]. Interestingly, rdition cn lso induce BCSC phenotype in differentited rest cncer cells [21, 22], nd CSCs-medited tumor innte resistnce to cytotoxic gents thus ecome mjor clinicl chllenges towrds the complete erdiction of miniml residul disese in cncer ptients [5]. CSCs re lso likely to ply essentil roles in the metsttic spred of primry tumors ecuse of their self-renewl cpility nd their potentil to give rise to differentited progenies tht cn dpt to different trget orgn microenvironments [51 54]. Preclinicl study hs suggested differentition therpy to e one of the promising strtegies for trgeting BCSCs in rest cncer [55]. Thus, trgeting these enriched puttive BCSCs in rest cncer cells fter sulethl doses of rdition tretment my hve importnt clinicl impct for rest cncer ptients. To this setting, rdioresistnt used in this study is useful experimentl model to mimic the rdioresistnt lesions in the clinic, especilly for the therpy-resistnt phenotype of metsttic tumors. cells were derived from cells fter frctionized ionizing rdition nd re with developed rdition resistnce [16, 19, 56]. Chrcteriztion nd elucidtion of the mechnistic insights nd potentil therpeutic trget to this unique rdioresistnt, BCSCs-enriched popultion which is highly relevnt to the clinic recurrent/metsttic lesions, will generte informtive dt for the enefit of rest cncer ptients. Our present work demonstrtes the increses of puttive CSCs popultions in cells. Compred to prentl cells, cells lso exhiited enhnced cpilities for cncer cell invsion nd migrtion, indicting incresed potentil for metstsis. Thus, rdioresistnt with BCSCs enrichment is useful experimentl model to mimic the rdioresistnt lesions in the clinic, especilly for the therpy-resistnt phenotype of metsttic tumors. Preclinicl study hs suggested differentition therpy to e one of the promising strtegies for trgeting BCSCs in rest cncer [55]. Our dt lso showed tht inhiition of CD44 expression could effectively reduce cncer cell invsiveness nd migrtion in / C6 cells (Fig. 3). In this study, we demonstrted the potentil therpeutic effects of on cells. Retinoids nd its derivtives such s re promising ntineoplstic gents endowed with oth therpeutic nd chemo-preventive potentil ecuse they re le to regulte cell growth, differentition nd poptosis

9 Yn et l. BMC Complementry nd Alterntive Medicine (216) 16:113 Pge 9 of 11 Survivl Frction (1%) Control Epiruincin 2 nm Epiruicin +Epiruicin Epiruicin: 1 2 3nM G/G1=37.31 S=3.96 G2/M=22.44 G/G1=48.2 S=19.6 G2/M=32.57 Control Epiruincin 2 nm G/G1=33.19 S=32.35 G2/M=22.32 G/G1=47.14 S=23.41 G2/M= Control 5-Fu Control Control Doxtxel -1µM Control Doxtxel Fig. 6 exposure increses the sensitivities of cells to chemotherpeutic tretments. Effect of on clonogenic survivl nd cell cycle distriution in epiruincin-treted cells. Cells were pretreted with 1. μm, or s control, for 72 h, nd 5 cells were then plted nd treted with indicted concentrtions of Epiruicin. 24 h lter, cells were wshed with fresh medium nd were then mintined for colony formtion ssy, or collected for cell cycle nlysis. Top left: Survivl curve for colony formtion; Top right: demonstrtive imges for colony survivl of epiruincin-treted cells. Bottom: -induced cell cycle chnges in epiruincin-treted cells. Effect of on responses of cells to tretments of 5-Fu nd Doxetxel. Cells were pretreted with s descried ove, nd were then treted with 1. μg/ml of 5-Fu or.5 nm of Doxetxel for 24 h. Colony formtion experiments were then performed. Top left: Digrm showing the chnge of colony formtion in cells exposed to 5-Fu tretment; top right: demonstrtive imges for colony survivl of 5-Fu-treted cells; Bottom left: Digrm showing the chnge of colony formtion in cells exposed to Doxetxel tretment; Bottom right: demonstrtive imges for colony survivl of 5-Fu-treted cells. Dt represent the verge from t lest three independent experiments. Indictes sttisticl significnce (p <.5) Survivl Frction (1%) Survivl Frction (1%) 5-Fu [57 59]. We previously hve showed the inhiitory effects of on prolifertion nd cncer cell migrtion of rest cncer cells [6]. hs lso een recently demonstrted of the ility to induce cncer stem cell differentition [42]. We showed here tht cn induce differentition of enriched BCSCs in cells, nd inhiit cncer cell invsiveness/migrtion nd increse the sensitivities of cells to rdition tretment nd to the tretments of epiruincin nd 5-Fu of this cell popultion. These results thus not only indicte potentil clinic impcts of differentition tretment with s single gent for BCSCs in therpy-resistnt rest cncers, ut lso suggest pproches with comintion of nd epiruincin, or other stndrd-nti-rest cncer chemotherpy, s novel therpeutic strtegy for clinic mngement iming to minimize the risk of recurrent/metstsis, the mjor life-thretening tumors in mny cncer ptients [61]. Conclusions Our study suggests potentil clinic impct of s chemotherpeutic gent for tretment of rditionresistnt rest cncer. The study lso provides rtionle for s sensitizer of Epiruincin, firstline tretment option for rest cncer ptients. Avilility of dt nd mterils Not pplicle. Additionl file Additionl file 1: Figure S1. A. Representtive imges for H.E stining of the tumors formed in NOD/SCID mouse in tumor inititive test. B. Digrms showing the effects of on percentges of cell popultions with positive stining of stem cell mrkers Nnog, OCT3/ 4ndCD44 + /CD24 -/low in cells. C. Digrms showing the rtios of cncer cell invsiveness cells treted with s shown in Fig. 4d. Dt represent the verge from t lest three independent experiments. Indictes sttisticl significnce (p <.5). (PDF 159 k) Competing interests The uthors declre tht they hve no competing interests. Authors contriutions YY, XC, JLi, YW nd JH conceived, ccomplished nd designed the study; YY, XC, ZL, XX, CC, WW nd MF conducted the nlysis nd mnged dt collection; YY, XC, ZL nd JL contriuted to the writing of the mnuscript drfts; ll uthors red nd pproved the finl mnuscript.

10 Yn et l. BMC Complementry nd Alterntive Medicine (216) 16:113 Pge 1 of 11 Acknowledgments This work ws supported y Ntionl Nturl Science Foundtion of Chin (No ) to YW, nd y Ntionl Institutes of Helth RO1 Grnts CA to JL. The funders hd no role in study design, dt collection nd nlysis, decision to pulish, or preprtion of the mnuscript. Author detils 1 Institute of Clinicl Phrmcology, Anhui Medicl University, Hefei, Chin. 2 Deprtment of Biochemistry, Lortory of Moleculr Biology, Anhui Medicl University, Hefei, Chin. 3 Deprtment of Pthology nd Lortory Medicine, University of Cliforni, Los Angeles, USA. 4 School of Life Sciences, Anhui University, Hefei, Chin. 5 Deprtment of Rdition Oncology, University of Cliforni, Dvis, USA. Received: 23 Septemer 215 Accepted: 19 Mrch 216 References 1. Edwrds BK, Noone AM, Mriotto AB, Simrd EP, Boscoe FP, et l. 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