Combination of metformin and 5-aminosalicylic acid cooperates to decrease proliferation and induce apoptosis in colorectal cancer cell lines

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1 Sber et l. BMC Cncer (2016) 16:126 DOI /s RESEARCH ARTICLE Open Access Combintion of metformin nd 5-minoslicylic cid coopertes to decrese prolifertion nd induce poptosis in colorectl cncer cell lines Mon M. Sber 1, My A. Gll 1, Aff A. Ain-Shok 1 nd Smi A. Shoumn 2* Abstrct Bckground: The link between inflmmtion nd cncer hs been confirmed by the use of nti-inflmmtory therpies in cncer prevention nd tretment. 5-minoslicylic cid (5-ASA) ws shown to decrese the growth nd survivl of colorectl cncer (CRC) cells. Studies lso reveled tht metformin induced poptosis in severl cncer cell lines. Methods: We investigted the combintory effect of 5-ASA nd metformin on nd Cco-2 CRC cell lines. Apoptotic mrkers were determined using western blotting. Expression of pro-inflmmtory cytokines ws determined by RT-PCR. Inflmmtory trnscription fctors nd metsttic mrkers were mesured by ELISA. Results: enhnced CRC cell deth induced by 5-ASA through significnt increse in oxidtive stress nd ctivtion of poptotic mchinery. Moreover, metformin enhnced the nti-inflmmtory effect of 5-ASA by decresing the gene expression of IL-1β, IL-6, COX-2 nd TNF-α nd its receptors; TNF-R1 nd TNF-R2. Significnt inhibition of ctivtion of NF-κB nd STAT3 trnscription fctors, nd their downstrem trgets ws lso observed. lso enhnced the inhibitory effect of 5-ASA on MMP-2 nd MMP-9 enzyme ctivity, indicting decrese in metstsis. Conclusion: The current dt demonstrte tht metformin potentites the ntitumor effect of 5-ASA on CRC cells suggesting their potentil use s n djuvnt tretment in CRC. Keywords: 5-ASA, Inflmmtion, CRC, NF-κB, STAT3, Bckground Colorectl cncer (CRC) is the third most common cncer with lifetime risk of 5 %. A functionl link between chronic inflmmtion nd cncer hs long been suspected but the complete underlying moleculr pthwys remin unknown [1]. Inflmmtory bowel diseses (IBD), including ulcertive colitis (UC) nd Crohn s disese (CD) re chronic inflmmtory disorders of the gstrointestinl trct tht led to impirment of the gstrointestinl structure nd function [2 4]. Ptients suffering from IBD re t n incresed risk of developing CRC, this depends on disese durtion, s well s, the extent nd severity of inflmmtion [4]. IBD-ssocited CRC ccounts for 1 2 % of ll CRC cses, however, IBD * Correspondence: smi.shoumn@nci.cu.edu.eg 2 Prmcology Unit,Cncer Biology Deprtment, Ntionl Cncer Institute, Ciro University, Ciro 11796, Egypt Full list of uthor informtion is vilble t the end of the rticle ptients re six times more likely to die from CRC thn the generl popultion [4, 5]. Although crcinogenesis in IBD follows different sequence of genetic ltertions thn tht observed in spordic CRC, ptients with spordic CRC hve elevted inflmmtory cytokine levels indictive of subclinicl inflmmtory disese [6 8]. Meslmine [5-minoslicylic cid (5-ASA)] is the drug of choice in IBD, minly UC, for mintennce of remission nd tretment of mild relpses. It is wek COX nd LOX inhibitor s it is structurlly relted to NSAIDs, but unlike these compounds it hs low systemic resorption nd very few side effects even t high doses for long time periods [9]. Epidemiologicl investigtions suggested tht long term 5-ASA consumption decreses the risk of developing CRC in IBD ptients [10, 11]. In ddition, severl experimentl studies showed tht 5-ASA decreses growth nd survivl of CRC cells [12 15]. The ntiprolifertive nd pro-poptotic effects of 5-ASA on severl 2016 Sber et l. Open Access This rticle is distributed under the terms of the Cretive Commons Attribution 4.0 Interntionl License ( which permits unrestricted use, distribution, 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 Public Domin Dediction wiver ( pplies to the dt mde vilble in this rticle, unless otherwise stted.

2 Sber et l. BMC Cncer (2016) 16:126 Pge 2 of 12 tumor-derived cell lines hve been previously reported nd different mechnisms hve been proposed nmely, inhibition of; Wnt/β-ctenin pthwy [16], EGFR ctivtion [17 19], NF-κB [20], nd COX-2 expression [21]. Moreover, Rousseux et l. showed tht meslmine ctivtes PPAR-y nd enhnces its expression in CRC cells [22]. Type 2 DM prevlence is estimted to be 8 18 % in newly dignosed cncer ptients, [23]. Evidence indictes tht type 2 DM is positively ssocited with incidence nd mortlity of CRC with 30 % incresed reltive risk compred with non-dibetic individuls [24]. The ssocited hyperglycemi nd hyperinsulinemi result in direct stimultion of cell growth nd DNA synthesis long with n increse in pro-inflmmtory cytokines production [25]., bigunide derivtive, is n orl ntidibetic drug used s the first line phrmcologicl tretment in type 2 DM. It cts minly by inhibiting heptic glucose production nd decresing peripherl tissue resistnce to insulin. This reduces the circulting glucose nd insulin levels thus reducing the incidence of dibetes-relted complictions [26]. is sfe drug with the most frequent dverse effects being gstrointestinl symptoms but they re usully mild nd trnsient [27]. Mny studies reveled the beneficil effect of metformin in decresing CRC risk [28, 29]. Moreover, it ws found to synergisticlly increse poptosis of CRC cells in-vitro when combined with other chemotherpy drugs [30, 31]. Since inflmmtion nd hyperglycemi re ssocited with incresed risks of cncer s well s of the mjor cuses of cncer progression, the im of the present study ws to evlute the effect of combining 5-ASA nd metformin on CRC cell lines. Methods Drugs ws obtined from CID Co. (Ciro, Egypt). It ws freshly dissolved in culture medium, Roswell Prk Memoril Institute 1640 (RPMI-1640), s 80 mm stock solution. 5-ASA ws kindly provided by Minphrm (Ciro, Egypt) nd dissolved in phosphte buffered sline (PBS) just before use. It ws dded to the medium with the finl mximum concentrtion of PBS 0.1 % v/v, nd experiments crried out protected from light. Chemicls nd ntibodies PBS, RPMI-1640 medium, nd sulphorodmine-b (SRB) were ll purchsed from Sigm Aldrich (St Louis, Missouri, USA). Polyclonl nti-humn Bx nd Bcl-2 ntibodies were obtined from Invitrogen (Crlsbd, CA, USA). Monoclonl nti-humn β-ctin ws obtined from Sigm-Aldrich. All other chemicls were of regent grde nd used without further purifiction. Cell culture The two vilble humn colorectl cncer cell lines, Cco-2 nd, were obtined from the Americn Type Culture Collection (Mnsss, USA). They were mintined nd grown t the Egyptin Ntionl Cncer Institute (Ciro, Egypt) in RPMI-1640 supplemented with 10 % fetl bovine serum, 2 mm L-glutmine, g/l sodium bicrbonte nd 1 % penicillin/streptomycin. Cells were cultured in humidified incubtor t 37 C in 5 % crbon dioxide (CO 2 ). No ethicl pprovl ws required for ny spect of this study. Cytotoxicity ssy Cytotoxicity ws evluted using the SRB ssy. Briefly, exponentilly growing cells were seeded in 96-well microtitre pltes t n initil density of /well. After 24 h, metformin nd 5-ASA were dded with vrious concentrtions nd incubted t 37 C for 48 h to determine their IC 50 s (the concentrtion of the drug required to produce 50 % inhibition of cell growth). Cells were fixed with 10 % trichlorocetic cid for 1 h t 4 C nd stined with 0.4 % SRB for 30 min., wells were then wshed four times with 1 % cetic cid nd ir-dried. The dye ws solubilized with 10 mm Tris bse (ph 1) nd the opticl density (O.D.) ws mesured spectrophotometriclly t 570 nm with the microplte reder (Tecn SunriseTM, Männedorf, Switzerlnd). The percentge of cell survivl ws clculted s follows: survivl frction = O.D. (treted cells)/o.d. (control cells). The IC 50 vlues of the two cncer cell lines fter 48 h tretment were clculted using sigmoidl dose response curve-fitting models (Grphpd Prism Softwre, version 5.03, Inc. Avendi de l Ply L Joll, USA). Cco-2 nd cells were then treted with combintion of subic 50 concentrtions of metformin nd different concentrtions of 5-ASA to determine the concentrtion t which 5-ASA would give significnt difference thn metformin lone. Oxidtive stress mrkers Cco-2 nd cells were grown in 75 cm 2 flsks nd llowed to dhere for 24 h. Cells were treted with metformin, 5-ASA or the combintion of both substnces for 48 h then collected by trypsinistion. The cell pellet ws wshed twice with PBS. To the cell suspension 1 ml of 20 % (w/v) trichlorocetic cid (TCA; Sigm, USA) contining 0.8 % (w/v) thiobrbituric cid (TBA; Sigm, USA) ws dded nd mixed well. MDA (decomposition product of the lipid peroxidtion process) level ws determined colorimetriclly by mesuring the pink pigment product resulting from the rection of one molecule of MDA with two molecules of TBA t 535 nm. Protein ws mesured by the method of Brdford nd MDA is expressed in nmol MDA per mg protein. For determintion of totl SH group, protein precipittion

3 Sber et l. BMC Cncer (2016) 16:126 Pge 3 of 12 ws crried out using 10 % TCA then smples were centrifuged t 3000 rpm for 10 min t 4 C. The resultnt superntnt ws mixed with phosphte buffer nd Ellmn s regent (Sigm-Aldrich, Miln, Itly). The method depends on the reduction of thiol regent; Ellmn s regent by the sulfhydryl SH group in GSH to form the yellow chromophore; 5-thionitrobenzoic cid, mesured spectrophotometriclly t 412 nm. GSH is expressed in nmol GSH per mg protein. Rel-time PCR nlysis Anlysis of COX-2, IL-1β, IL-6, TNF-α, TNF-R1, nd TNF-R2 RNA expression ws performed by rel-time PCR. Cco-2 nd cells, treted with metformin nd 5-ASA, were collected by trypsynistion. Totl RNA ws extrcted from cells using TRIzol regent (Invitrogen, Miln, Itly), ccording to the mnufcturer s instructions. Concentrtion nd purity of the RNA ws checked by A 260 /A 280 opticl density rtio. RNA (1 μg/ smple) ws retro-trnscribed into complementry DNA (cdna) nd 1 μl of cdna/smple ws then mplified using the following conditions: denturtion 1 min t 95 C, nneling 30s t 60 C for COX-2, TNF-R2, IL-1β, nd β-ctin or 30s t 57 C for TNF-α, TNF-R1 nd IL- 6, followed by 30s of extension t 72 C. Primers sequence ws s shown in Tble 1 nd they were obtined from Invitrogen (Miln, Itly). RT-PCR ws performed using the IQ SYBER Green Supermix (Bio-Rd Lbortories, Miln, Itly). mrna levels were clculted reltive to β-ctin, which ws unffected by metformin nd 5-ASA tretment. Western blot nlysis Aliquots of protein superntnts contining equl mounts of protein nd sodium dodecyl sulphte (SDS) reducing buffer were boiled for 5 min. They were then electrophoresed on SDS-polycrylmide gels nd trnsferred to polyvinyldiene difluoride membrnes. The membrnes were blocked with 5 % non-ft dry milk nd probed with specific primry ntibodies of monoclonl nti-bx nd Bcl2 ntibodies followed by incubtion with peroxidse-conjugted secondry ntibodies. The blots were developed with Amershm ECL western blotting kit (GE Helthcre, Amershm Plce, Little Chlfont, U.K) ccording to the mnufcturer s instructions.theblotswerequntifiedbychemidocxrs (Bio-Rd Lbortories Inc., Hercules, CA, USA.) softwre nd protein loding ws corrected for β-ctin s loding control. ELISA techniques The different proteins were determined in both cell lines ccording to the kit mnufcturer s instructions. For NFκB the Kmiy Biomedicl ssy kit (Settle, USA) ws used, while the RyBiotech (Georgi, USA) ws used for TNF-α, IL-6, STAT3, MMP-2 nd 9. The ssy employs the quntittive sndwich enzyme immunossy technique. A monoclonl ntibody specific for humn NF-κB, TNF-α, IL-6, STAT3, MMP-2 or 9 hs been pre-coted onto microplte. Smples were pipetted into the wells nd the mesured humn biomrkers present in the solutions were bound by the immobilized ntibody. A yellow color is developed which is proportionl to the mount of NF-κB/ TNF-α/IL-6/ STAT3/ MMP-2/MMP-9 bound. The intensity of the color is mesured t 450 nm. Cspse-3 ctivity ws mesured bsed on spectrophotometric detection of the chromophore p-nitroniline (pna) t 405 nm fter clevge from its lbelled substrte DVD-pNA. Protein concentrtion of the smples ws nlyzed nd normlized in lysis buffer to equl protein concentrtions. Colorimetric ssy (Cspse-3/CPP32, BioVision, Milpits, USA) ws used ccording to the mnufcturer s instructions. Scrtch wound heling ssy Cco-2 nd cells were grown in 6 well pltes nd llowed to dhere for 24 h. Gently nd slowly the monolyer ws scrtched in one direction with new 1 ml pipette tip cross the center of the well. The resulted gp distnce therefore equls to the outer dimeter of the end of the tip. The wells were then wshed twice with medium to remove the detched cells. Cells were treted with metformin, 5-ASA or the combintion of both substnces for 48 h. Cells were wshed twice with 1x PBS, then fix the Tble 1 Primer sequences Gene COX-2 IL-1β IL-6 TNF-α TNF-R1 TNF-R2 β-ctin Primer FWD: 5 -CCC TTC CTT CGA AAT GCA AT-3 REV: 5 -CAT TTG AAT CAG GAA GCT GC-3 FWD: 5 -GGA CAA GCT GAG GAA GAT GC-3 REV: 5 -TTT TTT GCT GTG AGT CCC GG-3 FWD: 5 -GAG ACT TGC CTG GTG AAA AT-3 REV: 5 -CAG GGG TGG TTA TTG CAT CT-3 FWD: 5 ACA AGC CTG TAG CCC ATG TT-3 REV: 5 AAA GTA GAC CTG CCC AGA CT-3 FWD: 5 -CGC TTC AGA AAA CCA CCT CAG AC-3 REV: 5 -CCA AAG AAA ATG ACC AGG GGC-3 FWD: 5 -GCT CTG ACC AGG TGG AAA CTC AAG-3 REV: 5 -GGA TGA AGT CGT GTT GGA GAA CG-3 FWD: 5 -TCT GGC ACC ACA CCT TCT ACA ATG-3 REV: 5 -AGC ACA GCC TGG ATA GCA ACG-3

4 Sber et l. BMC Cncer (2016) 16:126 Pge 4 of 12 cells with 3.7 % prformldehye for 30 min nd they were stined with 1 % crystl violet in 2 % ethnol for 30 min. Photos were tken for the stined monolyer on microscope. The gp distnce ws mesured using the Leic Qwin-Plus softwre (Leic Microsystems, UK). Sttisticl nlysis All the dt re expressed s men ± SD from three different experiments nd comprisons between mens were crried out using one wy nlysis of vrince (ANOVA) followed by Tukey-Krmer multiple comprisons test. A probbility level of less thn 5 ws ccepted s being significnt in ll types of sttisticl tests. All sttisticl nlysis ws performed using GrphPd InStt, version 5.0 (GrphPd, Sn Diego, Cliforni, USA). Results Co-incubtion of metformin enhnced 5-ASA-medited inhibition of cell counts in Cco-2 nd cells Cco-2 cells were treted with 13 mm metformin, 2.5 mm 5-ASA, or the combintion of both for 48 h. Similrly, cells were treted with 8 mm metformin, 3 mm 5-ASA, or the combintion of both for 48 h. Significnt inhibition of cell prolifertion were seen in ll tretment groups with the combintion group showing the highest inhibition reching 55 % of the control compred to nerly 40 % reched by the solo tretments in both cell lines Fig. 1. Exggerted increse in oxidtive stress upon combined tretment with metformin nd 5-ASA Combintion of subic 50 concentrtions of both drugs produced pronounced increse in MDA level (Fig. 2) nd greter decrese in the intrcellulr GSH level thn ech drug lone (Fig. 2b). In Cco-2 cells the combintion of metformin nd 5-ASA resulted in significnt increse in MDA level of 3 folds compred to, 1.9 nd 1.75 folds produced by either of the tretments. In ddition, reduction of GSH exceeding 85 % ws observed compred to nerly 50 % decrese produced by ech drug lone. A similr effect ws produced when both drugs were dded to the cells where MDA level showed n elevtion mounted to 2.5 folds compred to nerly 1.7 fold increse produced by solo tretments. Moreover, intrcellulr GSH depletion reched 65 %, while only 35 % decrese in GSH levels ws observed fter dding metformin or 5-ASA lone. Surviving Frction () IC50=4mM IC50=3.5mM Cco-2 Surviving Frction (b) IC50=10mM IC50=15mM Cco Concentrtion of 5-ASA (mm) Concentrtion of (mm) (c) Cco-2 Surviving Frction bc bc Fig. 1 Effect of different concentrtions of () 5-ASAnd(b) on surviving frctions of Cco-2 nd cells treted for 48 h. c Surviving frction of Cco-2 nd cells fter tretment with subic 50 concentrtions of metformin, 5-ASA nd combintion of both for 48 h. All dt re expressed s men ± SD of 3 seprte experiments performed in triplictes. The sttisticl significnce of the results ws nlyzed using one wy ANOVA followed by Tukey-Krmer multiple comprison test. Significntly different from control, b from metformin nd c from 5-ASA (P <5)

5 Sber et l. BMC Cncer (2016) 16:126 Pge 5 of 12 () 4 Cco-2 bc (b) Cco-2 in Fold chnge MDA level bc in Fold chnge GSH level bc bc 0 (c) 4 Cco-2 bc bc Cspse-3 ctivity (Fold chnge) Fig. 2 Effect of tretment of Cco-2 nd cells for 48 h with subic 50 concentrtions of metformin, 5-ASA or the combintion of both drugs on oxidtive stress mrkers mesured s () MDA level(b) GSH level nd on (c) cspse-3 ctivity. Dt re indictive of 3 seprte experiments. The sttisticl significnce of the results ws nlyzed using one wy ANOVA followed by Tukey-Krmer multiple comprison test. Significntly different from control, b from metformin nd c from 5-ASA (P <5) Co-incubtion of metformin enhnces 5-ASA-induced poptosis The ddition of metformin to 5-ASA succeeded in ctivting the cspse-3 enzyme more thn the individul tretments in both cell lines reching 3 fold the control group (Fig. 2c). Solo tretments in Cco-2 nd cells incresed ctive cspse-3 to levels rnging from folds. The increse in cspse-3 ctivity ws ccompnied by incresed poptotic Bx levels nd decresed expression of the nti-poptotic Bcl-2 protein in both cell lines (Fig. 3), following tretment with 5-ASA, metformin or their combintion fter 48 h. The combintion group showed the most significnt chnge in Bx levels, s compred to individul tretments where it produced 16 nd 13 folds increse in Cco-2 nd cells, respectively. On the other hnd, the nti-poptotic Bcl-2 expression decresed by 85 % nd 80 % in Cco-2 nd cells fter combintion tretment. Combintion of metformin nd 5-ASA downregultes TNF-α, TNF-α receptors (TNF-R1 nd TNF-R2), nd IL-1β nd inhibits the ctivtion of NF-κB All tretment groups produced prominent decrese in the expression of TNF-α nd its receptors (Fig. 4). In Cco-2 cells, the combintion group did not significntly differ from the 5-ASA group lthough it produced 85 % decrese in TNF-α expression while 5-ASA produced 80 % decrese. However, in cells the combintion of both drugs downregulted TNF-α expression by 80 % compred to 60%decreseproducedbyeitherdrug.Theseresultswere in greement with the protein levels of TNF-α tht decresed significntly fter the combintion tretment compred to either of the drugs lone in both cell lines s shown in Fig. (4e). Moreover, following tretment of Cco-2 cells with the two drugs, there ws downregultion of both TNF-α receptors tht ws greter nd sttisticlly significnt compred to the solo drug tretments where TNF-R1 nd TNF-R2 gene expression levels decresed by 95 % nd 90 % compred to the control group. In ddition, the combintion group in cells decresed TNF-R2 expression level reching solely 10 % compred to the untreted cells. Concerning IL-1β gene expression level, fter tretment of Cco-2 nd cells with combintion of metformin nd 5-ASA, IL-1β levels decresed by 55 % compred to the control (Fig. 4d). This decrese ws sttisticlly significnt from the metformin group but not the 5-ASA group. On the other hnd, 75 % decrese ws observed in the HCT- 116 cells tht ws significnt from ll tretment groups. In prllel with the previous results, NF-κB levels (Fig. 5c) showed significnt decrese to 29 % nd 51 %

6 Sber et l. BMC Cncer (2016) 16:126 Pge 6 of 12 () (b) (c) Fold chnge in Bcl2/B-ctin rtio Cco-2 bc bc (d) Fold chnge in Bx/B-ctin rtio 8 Cco-2 bc bc 0 Fig. 3 Western blot for Bcl-2 nd Bx levels in () Cco-2 nd (b) cells fter tretment with metformin, 5-ASA or their combintion. One representtive western blot of three independent experiments is shown. Effect of tretment of Cco-2 nd cells for 48 h with subic 50 concentrtions of metformin, 5-ASA or the combintion of both drugs on (c) Bcl-2 level (d) Bx level. All dt re expressed s men ± SD of 3 seprte experiments. The sttisticl significnce of the results ws nlyzed using one wy ANOVA followed by Tukey-Krmer multiple comprison test. Significntly different from control, b from metformin nd c from 5-ASA (P <5) in Cco-2 nd cells upon the ddition of metformin to 5-ASA. Intensified inhibition of IL-6 gene expression nd inhibition of STAT3 ctivtion upon ddition of metformin to 5-ASA The expression of IL-6 (Fig. 5) fter exposure to metformin or 5-ASA ws decresed significntly by 30 % for both drugs in the Cco-2 cell line. On cells, metformin nd 5-ASA produced 65 % nd 50 % decrese in IL-6 expression, compred to the control. Furthermore, the combintion resulted in n exggerted decrese of 80 % in IL-6 expression compred to either drug lone in both cell lines. Confirming the gene expression results, the IL-6 protein level showed significnt decrese fter tretment in both cell lines thn the solo tretments s shown in Fig. (4f). As illustrted in Fig. (5d) the gretest inhibition of STAT3 ctivity ws observed in the combintion group reching 29 % nd 50 % in Cco-2 nd cell lines. exggertes the 5-ASA-induced inhibition of COX-2 enzyme gene expression The expression of COX-2 gene, fter tretment of Cco-2 cells with metformin decresed by 75 % compred to control, while tretment with the nti-inflmmtory gent 5- ASA cused further downregultion in the gene expression by 85 % (Fig. 5b). The COX-2 gene expression reched its mximum inhibition (90 %) when the Cco-2 cells were treted by both gents. The sme pttern ws reflected on the cells, where the COX-2 gene expression ws downregulted by metformin, 5-ASA nd their combintion in this descending order by 50 %, 60 % nd 90 %, compred with the control group. Pronounced suppression of mtrix metlloproteinse-2 nd 9 nd inhibition of cell migrtion fter combining metformin nd 5-ASA The ddition of metformin to 5-ASA in Cco-2 cells significntly lowered MMP-2 nd 9 levels to 33%nd 35%respectively (Fig. 6). Similrly, tretment of the cells with combintion of both drugs decresed MMP-2 nd 9 enzyme levels to 34 % nd 21 % compred to the control. The results of the scrtch wound heling ssy were in ccordnce with the MMP levels s the combintion of both drugs resulted in 25 % decrese from control t zero time in Cco-2 cells nd 20 % inhibition in cells. Moreover the results of this ssy confirm the fct tht cells re highly tumorigenic nd metsttic Fig. 7.

7 Sber et l. BMC Cncer (2016) 16:126 Pge 7 of 12 () Cco-2 (b) Cco-2 Fold chnge in TNF-lph mrna levels b bc Fold chnge in TNF-R1 mrna levels bc (c) Cco-2 (d) Cco-2 Fold chnge in TNF-R2 mrna levels bc bc Fold chnge in IL-1B mrna levels b bc (e) Fold chnge in TNF-lph protein levels Cco-2 bc bc (f) Fold chnge in IL-6 protein levels 5-ASA (2.5mM) Cco-2 bc 5-ASA (3mM) Fig. 4 Effect of tretment of Cco-2 nd cells for 48 h with subic 50 concentrtions of metformin, 5-ASA or the combintion of both drugs on () TNF-α (b) TNF-R1 (c) TNF-R2 nd (d) IL-1β gene expression levels nd on (e) TNF-α nd (f) IL-6 protein levels. All dt re expressed s men ± SD of 3 seprte experiments. The sttisticl significnce of the results ws nlyzed using one wy ANOVA followed by Tukey-Krmer multiple comprison test. Significntly different from control, b from metformin nd c from 5-ASA (P <5) bc Discussion In the current study, we used 2 colorectl cncer cell lines Cco-2 nd HCT116. Cco-2 cell line is colorectl denocrcinom cells with intermedite chrcteristics, s it is spontneously differentiting tumor in norml culture, it is tumorigenic but hsn t metsttic behvior in vivo.. Tretment with very low drug concentrtions of metformin, 5-ASA nd their combintion ws coupled by significnt decrese in the surviving frction of CRC cells. The incresed cell deth could be explined by intrcellulr GSH depletion nd increse in MDA level, indicting tht cell deth could be initited or underwent under oxidtive stress originting from modultion of the intrcellulr redox system. Our results re in ccordnce with previous studies showing tht 5- ASA or metformin cn increse oxidtive stress in certin types of cncer thus leding to incresed poptosis [12, 32]. Another explntion for the incresed poptosis is the inhibition of inflmmtion. The centrl plyers involved in inflmmtion-medited tumor progression include IL-1β, IL-6ndTNF-α [33]. Dt from severl inflmmtion-ssocited cncer models implicte these inflmmtory cytokines in being the bridge between inflmmtion nd tumorigenesis [34]. On CRC cells, ddition of metformin to 5-ASA significntly reduced thegeneexpressionoftnf-α nd its receptors, TNF- R1 nd TNF-R2. Altered expression of the genes coding for TNF-α nd its receptors ws observed in neoplstic diseses [35]. In CRC, mlignnt cell derived TNF-α, enhnces the growth nd metstsis of the tumor s evidenced from niml models

8 Sber et l. BMC Cncer (2016) 16:126 Pge 8 of 12 () Cco-2 (b) Cco-2 Fold chnge in IL-6 mrna levels bc bc Fold chnge in COX-2 mrna levels bc (c) in NF-KB level Fold chnge Cco-2 bc bc (d) level in STAT3 Fold chnge 5-ASA (2.5mM) 5-ASA (3mM) Cco-2 bc Fig. 5 Effect of tretment of Cco-2 nd cells for 48 h with subic 50 concentrtions of metformin, 5-ASA or the combintion of both drugs on () IL-6 nd (b) COX-2 gene expression levels. Fold chnge in levels of (c) NF-κB nd (d) STAT3 trnscription fctors in Cco-2 nd HCT- 116 cells fter 48 h of tretment with metformin, 5-ASA or their combintion. All dt re expressed s men ± SD of 3 seprte experiments. The sttisticl significnce of the results ws nlyzed using one wy ANOVA followed by Tukey-Krmer multiple comprison test. Significntly different from control, b from metformin nd c from 5-ASA (P <5) bc [36]. Chronic TNF-α production by mlignnt or host cells or both my directly contribute to oncogene ctivtion, DNA dmge nd metstsis [37]. The min receptor mediting TNF-α effects is TNF-R1 which when stimulted, induces ctivtion of NF-κB fter series of intrcellulr events [38]. On the other hnd, the incresed TNF-α level could be due to the continuous ctivtion of NF-κB [39]. NF- κb ctivtion hs long been known to suppress poptosis s it promotes trnscription of nti-poptotic genes, one of which is Bcl-2 [40]. The decline in TNF-α or TNF- () Fold chnge in MMP-2 level Cco-2 bc bc (b) Fold chnge in MMP-9 level Cco-2 bc bc Fig. 6 Fold chnge in () MMP-2nd(b) MMP-9 enzyme levels fter 48 h of tretment of Cco-2 nd cells with metformin, 5-ASA or their combintion. All dt re expressed s men ± SD of 3 seprte experiments. The sttisticl significnce of the results ws nlyzed using one wy ANOVA followed by Tukey-Krmer multiple comprison test. Significntly different from control, b from metformin c from 5-ASA (P <5)

9 Sber et l. BMC Cncer (2016) 16:126 Pge 9 of 12 Cco-2 5-ASA Combintion 5-ASA Combintion Fig. 7 Photos of the stined monolyer of Cco-2 nd cells without tretment nd fter tretment for 48 h with subic 50 concentrtions of metformin, 5-ASA or the combintion of both drugs R1 genetic expression will therefore decrese NF-κB ctivtion nd thus increse poptosis. In our study, combintion of 5-ASA nd metformin resulted in the gretest inhibition in the TNF-α nd TNF-R1 gene expression, nd NF- κb protein level with subsequent increse in poptosis, evident by decresed level of Bcl-2 protein expression. Although TNF-α medites its ction minly through TNF-R1 ctivtion, TNF-R2 hs been linked to incresed colon cncer cell growth nd prolifertion [41, 42]. It ws found tht both IL-6 nd TNF-α interct to induce TNF-R2 expression nd function in colon cncer cells, suggesting tht specific microenvironment of multiple cytokines is

10 Sber et l. BMC Cncer (2016) 16:126 Pge 10 of 12 required to induce TNF-R2, s found in IBD or IBD ssocited CRC. Our study reveled pronounced down-regultion of TNF-R2 gene expression in Cco-2 nd cell lines in ll tretment groups with the gretest effect being tht of the combintion group. There were no previous studies conducted on 5-ASA or metformin tht ssessed their effect on TNF-R2 expression. In this study evidence of the effect of both drugs either lone or in combintion on TNF-R2 expression levels ws demonstrted. Together with TNF-α, IL-1β lso increses NF-κB trnscription thus incresing tumor dhesiveness, invsion nd ngiogenesis [43]. Likewise, ctivtion of NF-κB results in n increse in IL-1β [39]. Polymorphisms of IL-1β re ssocited with incresed cncer risk [44]. Cco 2 cells expresses ctive COX 2 nd epiderml growth fctor receptor (EGFR). On the other hnd cell line re undifferentited colorectl crcinom cells tht don t express COX2 nd re positive for trnsforming growth fctor bet 1(TGFbet1)ndbet2(TGFbet2)expression.Inthe current study, combining metformin with 5-ASA showed remrkble decrese in IL-1β gene expression in both cell lines respectively. The decrese in NF-κB protein level, cell prolifertion, increse in poptosis nd decrese in MMP-2 nd 9 expression, ll support the inhibition of the downstrem signling pthwy of IL-1β. Adding to the pnoply of molecules, IL-6 is one of the best chrcterized pro-tumorigenic cytokines. IL-6 is not only produced by immune cells but lso by epithelil nd mlignnt cells. Its production is induced by vriety of stimuli one of which is NF-κB. Therefore, IL-6 production cn be stimulted indirectly by TNF-α or IL- 1β tht ctivte the NF-κB or directly by PGE2 [45]. IL-6 promotes colon cncer cell prolifertion, survivl, migrtion, invsion, metstsis, ngiogenesis nd inflmmtion [46]. These effects re result of the ctivtion of the downstrem trget of IL-6, the STAT3 trnscription fctor. Persistent ctivtion of STAT3 hs been reported in vriety of humn tumors, including the colon [47]. This persistent ctivtion cn lso be the reson of incresed interleukins levels [48]. The current study reveled tht ddition of metformin to 5-ASA resulted in prominent decrese in IL-6 mrna levels tht ws ccompnied by decrese in STAT3 level nd therefore incresed cell deth. It is demonstrted herein tht the combintion group expressed the lowest Bcl-2 nd highest Bx protein levels. This ws ssocited with incresed cspse-3 ctivity nd poptosis in both cell lines. This effect my be ttributed in prt to the suppression of IL-6 nd STAT3. COX-2 is nother importnt pro-inflmmtory meditor tht is implicted in the process of crcinogenesis. It ws shown to be upregulted erly in CRC nd plys mjor role in its progression [49] by regulting the process of prolifertion, ngiogenesis nd metstsis [50]. It ws previously demonstrted tht one of the mechnisms of 5-ASA to decrese prolifertion ws by inhibiting COX-2 enzyme [21]. s well, hs been reported to inhibit inflmmtory responses nd COX-2 expression [51], however, its effect on COX-2 expression in CRC cells hs not been clrified. Our dt show tht combintion of both drugs resulted in n exggerted inhibition of COX-2 expression thn tht produced by the solo tretments suggesting synergistic effect. This explins the inhibition of MMP s level tht ws observed in the combintion group since COX-2 overexpression increses invsiveness of CRC cells by inducing MMP expression [52]. It ws reported tht COX- 2 inhibitors decrese MMP-2 nd 9 expression [53]. Therefore, the decrese in MMP-2 nd 9 level could be due to inhibition of COX-2 nd the NF-κB nd STAT3 signling pthwys. Severl studies showed tht metformin or 5-ASA cn inhibit MMP s expression [54 56]. Conclusions Our dt (Fig. 8) clrify tht tretment of CRC cells with combintion of 5-ASA nd metformin increses cell deth thn either drug lone. This increse in poptosis my be due to inhibition of the STAT3 nd NF-κB signling pthwys. The decresed level of cytokines tht stimulte those pthwys (TNF-α, IL-1β nd IL-6), lowered protein levels of the ctivted trnscription fctors (STAT3 nd NF-κB) nd decresed protein or expression Fig. 8 Addition of 5-ASA nd metformin on CRC cells inhibit the NF-κB nd STAT3 signling pthwys. Inhibition of gene expression of IL-1β nd TNF-α decreses ctivtion of NF-κB resulting in inhibition of trget gene expression. Moreover, decrese in IL-6 mrna levels reduces STAT3 ctivtion nd thus decresing trget gene trnscription. The trget genes for both pthwys include COX-2, Bcl-2, IL-6 nd MMP s. Low levels of COX-2 nd IL-6 gene expression nd Bcl-2, MMP-2 nd 9 protein levels confirm the inhibition of both the signling pthwys

11 Sber et l. BMC Cncer (2016) 16:126 Pge 11 of 12 levels of their trget genes (TNF-α, IL-6, COX-2, MMP- 2, MMP-9 nd Bcl-2) confirm the inhibition of the key pthwys in inflmmtion-medited tumor promotion nd progression. Abbrevitions COX: cyclooxygense; CRC: colorectl cncer; DM: dibetes mellitus; EGFR: Epiderml growth fctor receptor; GSH: glutthione; IBD: inflmmtory bowel disese; IL-1β: Interleukin-1β; IL-6: Interleukin-6; LOX: Lypooxygense; MDA: mlondildehyde; MMP: mtrix metlloproteinse; NF-κB: nucler fctor kpp B; NSAIDs: Non-steroidl nti-inflmmtory drugs; O.D.: opticl density; PPAR-y: peroxisome-prolifertor-ctivted receptor gmm; RPMI- 1640: Roswell Prk Memoril Institute 1640; STAT3: signl trnsducer nd ctivtor of trnscription 3; TNF-R: Tumor necrosis fctor receptor; TNF-α: Tumor necrosis fctor-α. Competing interests The uthors declre tht they hve no competing interests. Authors contributions MM crried out the prcticl work except western blotting nd drfted the mnuscript. MG did the sttisticl nlysis, crried western blot nd drfted the mnuscript. AA-S prticipted in the design of the work nd revised the mnuscript. SS offered tissue culture cncer lb nd shred with A A-S in designing nd reviewing the mnuscript. All uthors red nd pproved the finl mnuscript. Acknowledgements This work ws supported nd in prt by Phrmcology nd Toxicology Deprtment, Fculty of Phrmcy nd Cncer Biology Deprtment, Ntionl Cncer Institute, Ciro University. Author detils 1 Phrmcology nd Toxicolgy Deprtment, Fculty of Phrmcy, Ciro University, Ciro 11562, Egypt. 2 Prmcology Unit,Cncer Biology Deprtment, Ntionl Cncer Institute, Ciro University, Ciro 11796, Egypt. Received: 3 September 2015 Accepted: 10 Februry 2016 References 1. Coussens LM, Werb Z. Inflmmtion nd cncer. Nture. 2002;420(6917): Boum G, Strober W. The immunologicl nd genetic bsis of inflmmtory bowel disese. Nt Rev Immunol. 2003;3(7): Eden J. Review rticle: colorectl crcinom nd inflmmtory bowel disese. 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