Quercetin and Sorafenib as a Novel and Effective Couple in Programmed Cell Death Induction in Human Gliomas

Transcription

1 Neurotox Res (2014) 26:64 77 DOI /s x ORIGINAL ARTICLE Quercetin nd Sorfeni s Novel nd Effective Couple in Progrmmed Cell Deth Induction in Humn Glioms Jonn Jkuowicz-Gil Ew Lngner Dorot Bądziul Iwon Wertel Wojciech Rzeski Received: 31 Octoer 2013 / Accepted: 16 Decemer 2013 / Pulished online: 24 Decemer 2013 Ó The Author(s) This rticle is pulished with open ccess t Springerlink.com Astrct The im of the present study ws to investigte the effect of sorfeni nd quercetin on the induction of poptosis nd utophgy in humn nplstic strocytom (MOGGCCM) nd gliolstom multiforme (T98G) cell lines. In MOGGCCM cells, sorfeni initited minly poptosis, medited y the mitochondril pthwy with mitochondril memrne permeiliztion, cytochrome c relese to the cytoplsm, nd ctivtion of cspse 9 nd 3. Additionl incution with quercetin potentited the propoptotic properties of sorfeni. In T98G cells, utophgy ws oserved most frequently fter the sorfeni tretment. It ws ccompnied y incresed eclin 1 nd LC3II expression. Administrtion of quercetin fter the sorfeni tretment resulted in n incresed numer of utophgic cells. After simultneous drug ppliction, the level of utophgy ws lower in fvour of poptosis. Inhiition of J. Jkuowicz-Gil (&) D. Bądziul Deprtment of Comprtive Antomy nd Anthropology, Mri Curie-Skłodowsk University, Akdemick 19, Lulin, Polnd e-mil: jjgil@poczt.umcs.lulin.pl; dorot.dziul@wp.pl E. Lngner W. Rzeski Deprtment of Medicl Biology, Institute of Agriculturl Medicine, Jczewskiego 2, Lulin, Polnd e-mil: ew.lngner@gmil.com W. Rzeski e-mil: rzeskiw@hektor.umcs.lulin.pl I. Wertel 1st Deprtment of Gynecology, University School of Medicine, Stszic 16, Lulin, Polnd e-mil: iwonwertel@wp.pl W. Rzeski Deprtment of Immunology nd Virology, Mri Curie- Skłodowsk University, Akdemick 19, Lulin, Polnd het shock proteins expression y specific smll interfering RNA significntly incresed the sensitivity of oth the cell lines to induction of poptosis, ut not utophgy. We demonstrted for the first time tht sorfeni nd quercetin re very effective progrmmed cell deth inducers in T98G nd MOGGCCM cells, especilly in cells with locked expression of het shock proteins. Keywords Sorfeni Quercetin Apoptosis Autophgy Glioms Het shock proteins Introduction Mlignnt glioms re the most prevlent primry rin tumours in dults, exhiiting high rte of cell prolifertion nd migrtion ctivities. The mjor group is represented y nplstic strocytom (AA, WHO grde III) nd gliolstom multiforme (GBM, WHO grde IV). Despite tremendous efforts in improvement of therpeutics, such s surgery, rdiotherpy nd chemotherpy, the clinicl outcome of glioms remins dismying. The medin survivl in ptients with nplstic strocytom is out 30 months, while with gliolstom multiforme it is less thn 15 months under stndrd cre tretment. Therefore, there is n urgent need for new tretments sed on etter understnding of the moleculr sis of mlignnt progression of glioms (Kleihues et l. 2002; Ohgki nd Kleihues 2005; Omuro et l. 2007). It hs een proven tht upregultion of the Rs Rf MEK ERK pthwy tkes prt in mplifiction of mitogenic stimuli nd promotion of cellulr prolifertion of mlignnt glioms. Therefore, downregultion of this signl trnsmission my e vlule therpy for gliom ptients leding to poptosis or utophgy induction (Lo

2 Neurotox Res (2014) 26: ). A promising cndidte for such n ction is sorfeni smll-molecule multikinse inhiitor tht ws originlly developed s n inhiitor of Rf kinse, n essentil serine/threonine kinse constituent of the mitogen-ctivted protein kinse (MAPK) pthwy. Intrcrnil ppliction of sorfeni cused inhiition of cell prolifertion, reduction of ngiogenesis, nd induction of utophgy nd poptosis of gliom cells. Systemic dministrtion of sorfeni ws well tolerted nd the drug crossed the lood rin rrier effectively (Hhn nd Stdler 2006; Sieglin et l. 2010; Wilhelm et l. 2004). Unfortuntely, its use in the clinicl tretment of ptients with mlignnt glioms hs yielded disppointing results in some cses (Nors et l. 2011; Sieglin et l. 2010; Yng et l. 2010). Nevertheless, the ility of sorfeni to inhiit tumour cell prolifertion suggests tht it my e useful in comintion with other therpeutic gents. It is well known tht nturl ctive compounds my ct in synergy with drugs used in clinicl pplictions. One of them is quercetin (3,3 0,4 0,5,7- penthydroxyflvone), nturl flvonoid found in rod rnge of fruits nd vegetles. It hs multiple iologicl, phrmcologicl nd medicl pplictions nd is one of the most potent ntioxidnts. Quercetin fcilittes poptosis of tumour cells y cspse 3 nd cspse 9 ctivtion nd cytochrome c relese. It is well-known inhiitor of expression of het shock proteins (Hsps) (Brgnhol et l. 2006; Hosokw et l. 1992; Rmos 2007; Russo 2007; Schültke et l. 2005). Therefore, the im of our studies ws to investigte the effect of sorfeni pplied lone or in comintion with quercetin on induction of poptosis nd utophgy in humn gliolstom multiforme T98G nd nplstic strocytom MOGGCCM cell lines. We nlysed the typicl morphology for progrmmed cell deth s well s the moleculr mechnism underlying these processes sed on the expression of Hsp27, Hsp72, cytochrome c, LC3, eclin 1, nd Rs nd Rf proteins nd the ctivity of cspse 3, cspse 8 nd cspse 9. Additionlly, we studied the sensitivity of MOGGCCM nd T98G cell lines with locked Hsp27 nd Hsp72 expression to progrmmed cell deth induction upon comined drug tretment. Mterils nd Methods Cells nd Culture Conditions Humn gliolstom multiforme cells (T98G, Europen Collection of Cell Cultures) nd humn nplstic strocytom cells (MOGGCCM, Europen Collection of Cell Cultures) were grown in 3:1 mixture of Dulecco s Modified Egle Medium (DMEM) nd Hm s nutrient mixture F-12 (Sigm) supplemented with 10 % foetl ovine serum (Sigm), penicillin (100 units/ml) (Sigm) nd streptomycin (100 lg/ml) (Sigm). The cultures were kept t 37 C in humidified tmosphere of 95 % ir nd 5%CO 2. Drug Tretment Sorfeni (Nexvr, BAY ) t the finl concentrtions 0.25, 0.5, 0.75, 1, 5 lm nd quercetin (3,3 0,4 0,5,7- penthydroxyflvone) (Sigm) t the finl concentrtions 30 lm in the cse of MOGGCCM nd 50 lm in the cse of T98G were used in the experiments. The quercetin concentrtions were chosen on the sis of our erlier studies conducted on oth the cell lines (Jkuowicz-Gil et l. 2010, 2011, 2013). The drugs were dissolved in dimethyl sulfoxide (DMSO, Sigm). The finl concentrtion of DMSO in the culture medium did not exceed 0.01 %, which, s indicted in preliminry experiments, did not influence cell viility nd the expression of the proteins studied. Two vrints of drug tretment were performed. In the first one, the cells were incuted only with sorfeni for 24 or 48 h. In the second one, quercetin nd sorfeni were dded to the culture medium t the sme time nd incuted for 24 or 48 h. The control cells were incuted with 0.01 % of DMSO. Detection of Apoptosis nd Necrosis with Fluorochromes For identifiction of poptosis nd necrosis, the cells were stined with fluorescent dyes Hoechst (Sigm) nd propidium iodide (Sigm), respectively (Jnkowsk et l. 1997). The morphologicl nlysis ws performed under fluorescence microscope (Nikon E 800). Cells exhiiting lue fluorescent nuclei (frgmented nd/or with condensed chromtin) were interpreted s poptotic. Cells exhiiting pink fluorescent nuclei were interpreted s necrotic. At lest 1,000 cells in rndomly selected microscopic fields were counted under the microscope. Ech experiment ws performed in triplicte. Detection of Acidic Vesiculr Orgnelles with Acridine Ornge Autophgy is process of sequestrting cytoplsmic proteins into the lytic comprtment nd is chrcterized y formtion nd promotion of cidic vesiculr orgnelles (AVOs). Vitl stining with 1 lg/ml of cridine ornge ws performed for 15 min to detect AVOs in cells treted with quercetin nd/or sorfeni (Tkeuchi et l. 2005). Typicl cridine ornge-positive cells exhiiting grnulr discretion of AVOs in the cytoplsm were interpreted s utophgic. The morphologicl nlysis ws performed

3 66 Neurotox Res (2014) 26:64 77 under fluorescence microscope (Nikon E 800). At lest 1,000 cells in rndomly selected microscopic fields were counted. Ech experiment ws performed in triplicte. The percentge of utophgic cells ws clculted s the numer of cells with AVOs versus the totl numer of stined cells. Detection of Apoptosis nd Necrosis y Flow Cytometry The Annexin V-FITC poptosis detection kit (Sigm) ws used for detection of poptosis nd necrosis y flow cytometry. The smples were prepred ccording to the mnufcturer s protocol. Briefly, the control nd drugtreted cells were incuted with 5 ll of Annexin V-FITC nd 10 ll of propidium iodide for 10 min. Immeditely fter stining, the cells were nlysed with the FcsCnto instrument (Becton Dickinson, Sn Jose, CA, USA). In totl, 30,000 events were cquired nd nlysed using FcsDiv softwre. Cells tht were in the erly poptotic process were stined only y Annexin V-FITC; lte poptotic cells were stined y oth the fluorochromes. Cells stined only y propidium iodide were interpreted s necrotic. Live cells showed no stining y either propidium iodide or Annexin V-FITC. Ech experiment ws performed in triplicte. Mitochondril memrne potentil Fluorochrome 3,3 0 -dihexyloxcrocynine iodide [DiOC 6 (3)] ws used for studying the chnges in the mitochondril memrne potentil (Dw m ) in cells incuted with quercetin nd sorfeni. At low concentrtions, the stin ccumultes in mitochondri. Loss of DiOC 6 (3) stining indictes disruption of the mitochondril inner trnsmemrne potentil (Kim et l. 2008). The control nd drug-treted cells were incuted with fluorochrome t the finl concentrtion 50 nm for 20 min t 37 C in the drk, wshed three times with phosphte uffered sline (PBS), nd nlysed with the FcsCnto instrument (Becton Dickinson, Sn Jose, CA, USA). In totl, 30,000 events were cquired nd nlysed using FcsDiv softwre. Ech experiment ws performed in triplicte. The results were expressed s reltive intensity of DiOC 6 (3) rightness. Mitochondril Frction Isoltion A commercil isoltion kit ws used (Sigm) for isoltion of mitochondri. The smples were prepred ccording to the mnufcturer s protocol. Briefly, fter quercetin or/nd sorfeni tretment t the density of out , the cells were trypsinised, centrifuged for 5 min (6009g) nd wshed twice with PBS. The cell pellet ws resuspended in lysis uffer (Sigm), incuted for 5 min on ice nd centrifuged for 10 min (6009g). The superntnt ws trnsferred into fresh tue nd centrifuged t 11,0009g for 10 min. The pellet ws resuspended in cell lysis uffer nd used for electrophoresis. Isoltion of the Cytosolic Frction After the quercetin nd/or sorfeni tretment, the cells were lysed in hot luryl sulphte (SDS)-loding uffer (125 mm Tris HCl ph 6.8; 4 % SDS; 10 % glycerol; 100 mm dithiothreitol), oiled in wter th for 10 min nd centrifuged t 10,0009g for 10 min; next, the superntnts were collected. The protein concentrtion ws determined y the Brdford method (Brdford 1976) nd smples of the superntnts contining 80 lg of proteins were used for electrophoresis. Immunolotting The cytoplsmic nd mitochondril smples were seprted y 10 % SDS-polycrylmide gel electrophoresis (Lemmli 1970) nd susequently trnsferred onto n Immmoilon P memrne (Sigm). Following the trnsfer, the memrne ws locked with 3 % low-ft milk in PBS for 1 h nd incuted overnight with mouse nti-hsp72 monoclonl ntiody (SPA 810, StressGen) t the concentrtion 0.2 lg/ ml, nti-hsp27 (SPA 800, StressGen) t the concentrtion 0.1 lg/ml, rit nti-lc3 (Sigm) t the concentrtion 2 lg/ ml, nti-eclin 1 ntiody (Sigm) t the concentrtion 3 lg/ ml, nti-rs (Snt Cruz Biotechnology) t the concentrtion 0.5 lg/ml, nti-rf (Snt Cruz Biotechnology) t the concentrtion 0.5 lg/ml, nd sheep nti-cytochrome c ntiody (Sigm) t the concentrtion 0.2 lg/ml. The memrnes were wshed three times with PBS contining 0.05 % Triton X-100 (Sigm) for 10 min nd incuted for 2 h with lkline phosphtse-conjugted got nti-mouse, nti-sheep or ntirit secondry ntiodies (Sigm). The memrnes were visulised with n lkline phosphtse sustrte (5-romo-4- chloro-3-indolylphosphte nd nitro-lue tetrzolium, Sigm) in colour development uffer N,N-dimethylformmide (DMF, Sigm). The dt were normlised reltive to - ctin (detected y mouse nti--ctin ntiodies t the concentrtion 0.5 lg/ml, Sigm). The levels of protein expression were determined using the Bio-Profil Bio-1D Windows Appliction V progrmme. The vlue ws expressed s reltive intensity of nd thickness, width nd colour depth. Three independent experiments were performed. Cspse Activity Assy Cspses re cysteine proteses tht exist in norml conditions s inctive pro-forms or zymogens. They re

4 Neurotox Res (2014) 26: cleved to the ctive form following induction of poptosis. The ctivity of cspses 3, 8 nd 9 ws estimted using SensoLyte Ò AMC Cspse Sustrte Smpler Kit (An- Spec) in the control nd drug-treted cells. Smple preprtion nd the enzymtic rection were performed ccording to the mnufcturer s protocol. The fluorescence of 7-minocoumrin (AMC) ws monitored t E x /E m = 354/442 nm in 96-well lck micropltes using 2030 Multilel Reder Victor TM x4 (Perkin Elmer). Cell Trnsfection The MOGGCCM nd T98G cells t density of were seeded in six-well tissue culture plte nd incuted t 37 C inco 2 incutor for 24 h until confluence reched %. The cells were wshed with 3:1 DMEM:Hm s F-12 mixture without serum nd ntiiotics, nd spirted. For ech trnsfection, proes contining 2 ll of specific nti-hsp27 or nti-hsp72 smll interfering RNA (sirna) duplex (Snt Cruz Biotech) nd 2 ll of Trnsfection Regent (Snt Cruz Biotech) were prepred, overlid onto wshed cells nd incuted for 5 h t 37 C inco 2 incutor. After this time, the medium ws enriched with growth medium contining 20 % of foetl ovine serum nd 200 lg/ ml of ntiiotics without removing the trnsfection mixture nd the cells were incuted for dditionl 18 h. Then the medium ws replced y fresh norml growth medium nd the trnsfected cells were used for further experiments. The effectiveness of gene silencing ws nlysed t the protein level y the immunolotting technique. Indirect Immunofluorescence After the tretment with quercetin nd/or sorfeni, the cells were wshed three times with PBS nd fixed in 3.7 % prformldehyde (Sigm) in PBS for 10 min. After extensive wshing with PBS, the cells were treted with 0.2 % Triton X-100 (Sigm) for 7 min nd then wshed three times with PBS, ll t room temperture. Susequently, locking step of 30-min incution in 5 % low-ft milk t room temperture ws included. Next, the cells were incuted with rit ntiody nti-eclin 1(Sigm) t concentrtion of 30 lg/ml. The primry ntiodies were detected with fluorescein (FITC)-conjugted nti-rit ntiodies (Sigm). Protein loclistion in the cells ws nlysed using the scnning hed PASCAL5 (Zeiss). The fluorescent chnnel ws k = 488 nm. Three independent experiments were performed. Over 100 cells were nlysed in ech experimentl vrint. Sttisticl Anlysis The dt re presented s men ± stndrd devition (SD). The sttisticl evlution ws performed with one-wy Anov test followed y Dunnett s multiple comprison test. P \ 0.05 ws tken s the criterion of significnce. Results Sensitivity of T98G nd MOGGCCM Cells to Sorfeni Tretment To estimte the sensitivity of T98G nd MOGGCCM cells to the sorfeni tretment, stining method with dyes specific for poptosis, necrosis nd utophgy; i.e. Hoechst 33342, propidium iodide nd cridine ornge, respectively, ws employed (Fig. 1). Microscopic oservtions reveled tht sorfeni pplied t the concentrtions 0 5 lm to the MOGGCCM culture medium for 24 h (Fig. 1) hd no considerle effect on cell deth. A significnt increse in the numer of poptotic cells ws oserved fter 48 h of incution, reching mximum (5.6 %) t the concentrtion 5 lm (Fig. 1c). Besides poptosis, sorfeni initited necrosis t level exceeding the percentge of poptotic cells. In the cse of T98G, sorfeni pplied to the culture medium for 24 (Fig. 1) nd 48 h (Fig. 1d, h) ppered to e potent utophgy inducer. In the cse of 24-h long incution, sorfeni t the concentrtion 1 lm initited the process in 16 % of the cells. The longer incution time ws even more effective. Aout 40 % of cells treted with 0.75 or 1 lm of sorfeni exhiited right red vesicles t the edge of cells oserved fter stining with cridine ornge (Fig. 1h). The indirect immunofluorescence technique reveled tht the utophgy mrker eclin1 ws loclised t these vesicles (Fig. 1g). The drug hd no significnt effect on induction of poptosis nd necrosis in T98G cells. Sensitivity of T98G nd MOGGCCM Cells to Comined Sorfeni nd Quercetin Tretment Bsed on the results descried erlier, to exmine the comined effect of sorfeni with quercetin, the drug t the concentrtion 0.75 lm pplied to MOGGCCM cells for 24 h nd T98G cells for 48 h ws chosen. The incution of T98G cells with sorfeni t the concentrtion 0.75 lm with incresing quercetin concentrtions (0 50 lm) for 48 h reveled tht such comintion minly induced progrmmed cell deth. When the cells were treted simultneously with the comintion of 50 lm of quercetin nd 0.75 lm of sorfeni, poptosis ws oserved in 20 % of the cells, which ws ccompnied y utophgy in 25 % of the cells (Fig. 1f). When the MOGGCCM cells were incuted with sorfeni (0.75) nd quercetin (30 lm) for 24 h, poptosis ws the most frequent (35 %) (Fig. 1e). Co-incution with sorfeni nd flvonoid t the

5 68 Neurotox Res (2014) 26:64 77 Fig. 1 The level of poptosis, necrosis nd utophgy induction in the nplstic strocytom MOGGCCM (, c, e) nd the gliolstom multiforme T98G (, d, f h) cell lines treted only with sorfeni (0 5 lm) for 24 h (, ) or 48 h(c, d) nd sorfeni in comintion with quercetin (e, f) stined with Hoechst 33342, propidium iodide nd cridine ornge, C-control cells (without sorfeni or quercetin), *P \ 0.05 compred to control g loclistion of the utophgy mrker protein eclin 1 in T98G cells fter tretment with sorfeni (0.75 lm) for 48 h (pointed y rrows) h utophgic vcuoles in T98G cells stined with cridine ornge fter 48-h-long incution with sorfeni (0.75 lm) (pointed y rrows) c e d f g h concentrtion 50 lm resulted in severe necrosis. The level of utophgy ws not significnt. Evlution of Cell Deth nd Mitochondril Trnsmemrne Potentil y Flow Cytometry To confirm the reliility of the results otined through the microscopic oservtions, T98G cells treted with quercetin (50 lm) nd sorfeni (0.75 lm) for 48 h nd MOGGCCM cells treted with quercetin (30 lm) nd sorfeni (0.75 lm) for 24 h seprtely s well s in comintion of oth the drugs were nlysed y flow cytometry ccording to the type of cell deth (poptosis or necrosis) nd the mitochondril memrne potentil (Blsurmnin nd Schroit 2003; Ghoril et l. 2005). The incution of the cells with quercetin confirmed tht the flvonoid induced minly necrosis in the MOGGCCM (Tle 1) nd hd no such effect on the T98G cells (Tle 1). Sorfeni lone induced minly lte poptosis in oth the cell lines t the level of 6 %. When quercetin nd sorfeni were dded to the culture medium together, lte poptosis ws frequently oserved in the MOGGCCM cells (6.9 %). In the cse of the T98G cells, erly poptosis ws predominnt nd reched out 6.3 %. It is known tht decresed mitochondril memrne potentil is good indictor of poptotic cell deth

6 Neurotox Res (2014) 26: Tle 1 The effect of quercetin (Q) nd sorfeni (S) on poptosis nd necrosis induction in MOGGCCM () nd T98G () cells nlysed y flow cytometry with Annexin V-FITC detection kit Necrosis (%) Erly poptosis (%) Lte poptosis (%) Control 0.2 ± ± ± 0.07 Q (30 lm) 48.8 ± 5.3* 0.5 ± ± 0.07 S (0.75 lm) 0.7 ± ± ± 0.35* SQ 0.5 ± ± ± 0.29* Control 0.3 ± ± ± 0.07 Q (50 lm) 2.7 ± ± 1.2* 8.6 ± 0.07* S (0.75 lm) 0.1 ± ± ± 0.29* SQ 0.3 ± ± 0.77* 4.8 ± 0.4* * P \ 0.05 (Ghoril et l. 2005). In our experiments, oth quercetin nd sorfeni pplied lone or in comintion decresed this potentil in the T98G nd MOGGCCM cells (Fig. 2). In the gliolstom multiforme cells, the lowermost vlue ws oserved fter simultneous drug tretment. In the cse of MOGGCCM cells, sorfeni lone or in comintion with quercetin decresed the mitochondril memrne potentil most effectively, in comprison to the control. An increse in this prmeter ws oserved only fter incution with quercetin. The Effect of Sorfeni nd Quercetin on Expression of Mrker Proteins Using the immunolot technique, we studied the effect of sorfeni nd quercetin, pplied seprtely or in comintions, on the level of the pro-poptotic cytochrome c expression (in the cytoplsmic nd mitochondril frction), nti-poptotic moleculr chperones Hsp27 nd Hsp72, pro-survivl Rs, Rf, nd pro-utophgic eclin1nd LC3 in the MOGGCCM (Fig. 3) nd T98G (Fig. 4) cells. Additionlly, we nlysed fluorimetriclly the ctivity of cspses 3, 8 nd 9. Quntittive nd qulittive nlyses of the immunolots reveled tht quercetin nd sorfeni pplied lone or in comintion inhiited the expression of Hsp72, Hsp27, Rs nd Rf in ll the experimentl vrints in oth the cell lines. The drugs were lso very effective in decresing the level of cytochrome c in the mitochondril frction, which ws ccompnied y incresed ccumultion of the protein in the cytoplsm. In the cse of eclin 1, quercetin nd sorfeni hd no significnt effect on the protein expression in the MOGGCCM cells nd its level ws similr to the control one in ll the experimentl vrints. In the T98G cells, overexpression of eclin 1 ws oserved fter seprte sorfeni tretment nd fter sorfeni with quercetin. In other experimentl vrints, the level of eclin 1 ws similr to the control. Conversion of LC3I into its smller form LC3II is the hllmrk of utophgy. Similr to eclin1, incresed level of LC3II ws oserved only in T98G cells fter seprte sorfeni tretment nd in comintion with quercetin. In the cse of cspses, quercetin nd sorfeni pplied in comintion (ut not in the seprte ppliction) incresed the ctivity of cspse 3 nd cspse 9 in the MOGGCCM cells. In the T98G cells, elevted ctivity of the enzymes ws oserved fter seprte quercetin tretment nd when oth the drugs were dded t the sme time. Sorfeni nd quercetin pplied lone or in comintion hd no effect on cspse 8 ctivity in the MOGGCCM nd T98G cells. Blocking the Hsp27 nd Hsp72 Expression in T98G nd MOGGCCM Cells To lock the expression of Hsp27 nd Hsp72, the T98G nd MOGGCCM cells were trnsfected with specific sir- NA. Western lot nlysis reveled tht the silencing ws very effective t the protein level (Fig. 5) nd no expression of Hsps ws oserved even fter susequent quercetin nd/or sorfeni tretment. Incution of the cells either with only the trnsfection regent or with only sirnas hd no effect on the expression of het shock proteins. Apoptosis, Autophgy nd Necrosis Induction in Trnsfected Cells Fig. 2 The mitochondril memrne potentil in MOGGCCM nd T98G cells stined with DiOC 6 (3), incuted seprtely with quercetin (Q) nd sorfeni (S) or in comintion of oth the drugs (SQ) for 24 h (MOGGCCM) or 48 h (T98G) nd nlysed y flow cytometry, C control cells, *P \ 0.05 compred to control The effect of sorfeni nd quercetin on cell deth induction in the T98G nd MOGGCCM (Fig. 6) cells with locked Hsp expression ws studied y mens of microscopic oservtions (Fig. 6, ) nd flow cytometry (Fig. 6c, d). Both the methods reveled tht quercetin nd sorfeni were extremely effective in cell deth induction in trnsfected MOGGCCM nd T98G cells, especilly

7 70 Neurotox Res (2014) 26:64 77 Fig. 3 The level of Hsp27 (), Hsp72 (), cytochrome c (c cytoplsmic, d mitochondril frction), eclin 1 (e), LC3 (f), Rs (g) nd Rf (h) expression with representtive lots nd the ctivity of cspse 3, 8, 9 (i) fter sorfeni (S) nd quercetin (Q) tretment for 24 h in MOGGCCM. The dt were normlised reltive to -ctin (not shown). C control cells, SQ simultneous drug tretment, * P \ 0.05 compred to control c d e f g h i when oth the drugs were dded to the culture medium simultneously. Microscopic oservtions of T98G cells with diminished Hsp27 expression showed over 60 % of poptotic cells fter the quercetin tretment, 15 % fter the sorfeni incution nd 40 % fter the simultneous drug ppliction. In the cse of reduced Hsp72 expression, sorfeni pplied in comintion with the flvonoid ppered to e the most effective in poptosis induction, s it initited progrmmed cell deth in out 50 % of cells. Sorfeni itself ppered to e the lest effective in induction of poptosis (19 %) ut still the result ws significnt. In the MOGGCCM cells, locking of the Hsp27 or Hsp72 expression resulted in n incresed numer of poptotic cells fter the simultneous sorfeni nd quercetin tretment (out 30 %). Quercetin ws less effective ut still the effect ws significnt. Interestingly, locking of the Hsp27 or Hsp72 expression did not increse the sensitivity of the cell line to poptosis induction fter the sorfeni tretment.

8 Neurotox Res (2014) 26: c d e f g h i Fig. 4 The level of Hsp27 (), Hsp72 (), cytochrome c (c cytoplsmic, d mitochondril frction), eclin 1 (e), LC3 (f), Rs (g) nd Rf (h) expression with representtive lots nd the ctivity of cspse 3, 8, 9 (i) fter sorfeni (S) nd quercetin (Q) tretment for 48 h in In comprison to the microscopic oservtions, flow cytometry ppered to e more sensitive method for detection of poptosis in MOGGCCM nd T98G cells trnsfected with specific sirna. Apoptosis ws oserved in over 90 % of the cells, irrespective of the type of cells, type of locked protein nd susequent drug tretment. The T98G. The dt were normlised reltive to -ctin (not shown). C control cells, SQ simultneous drug tretment, *P \ 0.05 compred to control method lso llowed distinguishing etween erly nd lte poptosis. As shown in Fig. 6c nd d, poptosis fter the quercetin nd/or sorfeni tretment ws detected minly in the erly stge of the process in oth the cell lines. Our results indicte tht inhiition of Hsp72 or Hsp27 expression hd no significnt effect on necrosis or

9 72 Neurotox Res (2014) 26:64 77 Fig. 5 The level of Hsp27 nd Hsp72 expression in T98G (, c) nd MOGGCCM (, d) cells fter trnsfection with specific nti- Hsp27 (, ) or nti-hsp72 (c, d) sirna (si27 nd si72, respectively) nd susequent quercetin (Q) nd sorfeni (S) tretment, estimted y immunolotting. T98G cells were incuted with 50 lm of quercetin nd 0.75 lm sorfeni, while MOGGCCM cells with 30 lm (Q30) of quercetin nd 0.75 lm of sorfeni. Protein level ws normlised ccording to -ctin expression. C control, TR trnsfection regent, TRsi trnsfection regent with specific sirna, SQ simultneous quercetin nd sorfeni tretment, *P \ 0.05 compred to control c d Fig. 6 The effect of quercetin (Q) nd sorfeni (S) on poptosis, necrosis nd utophgy induction in MOGGCCM (, c, e) nd T98G (, d, f) cells trnsfected with specific sirna nti-hsp27 (si27) nd nti-hsp72 (si72). C control, TR trnsfection regent,, cell deth estimted y microscopic oservtion of cells stined with Hoechst 33342, propidium iodide, cridine ornge, c, d poptosis nd necrosis induction estimted y flow cytometry with the Annexin V-FITC detection kit, e, f the mitochondril memrne potentil studied y flow cytometry fter stining with DiOC 6 (3), *P \ 0.05 compred to control c d e f

10 Neurotox Res (2014) 26: Fig. 7 The level of cytochrome c ( cytoplsmic, mitochondril frction), Rs (c), Rf (d), LC3 (e) nd eclin 1(f) expression with representtive lots nd the ctivity of cspse 3, 8, 9 (f) fter sorfeni (S) nd quercetin (Q) tretment for 24 h in MOGGCCM cells trnsfected with specific sirna nti-hsp27 (si27) nd nti-hsp72 (si72). The dt were normlised reltive to -ctin (not shown). C control cells, SQ simultneous drug tretment, si27 or si72 specific sirna locking Hsp27 or Hsp72 expression, TR trnsfection regent, TRsi trnsfection regent with specific sirna *P \ 0.05 compred to control c d e f g utophgy induction upon the quercetin nd sorfeni tretment. The Effect of Quercetin nd Sorfeni on the Mitochondril Memrne Potentil in Trnsfected Cells The flow cytometry nlysis of DiOC 6 (3)-stined MOG- GCCM nd T98G cells with locked Hsp27 nd Hsp72 expression reveled tht quercetin nd sorfeni pplied lone or in comintion significntly decresed the mitochondril memrne potentil. As shown in Fig. 6e, such n inhiitory effect in the MOGGCCM cells ws the most efficient fter the quercetin tretment, while in the T98G cells (Fig. 6f) fter the simultneous drug incution. The Effect of Sorfeni nd Quercetin on Mrker Protein Expression in Trnsfected Cells Quntittive nd qulittive nlyses of immunolots with proteins isolted from the MOGGCCM (Fig. 7) nd T98G (Fig. 8) cells with locked Hsp27 or Hsp72 expression reveled tht quercetin nd sorfeni pplied lone or in comintion incresed the level of pro-poptotic cytochrome c in the cytoplsm, which ws

11 74 Neurotox Res (2014) 26:64 77 c d e f g Fig. 8 The level of cytochrome c ( cytoplsmic, mitochondril frction), Rs (c), Rf (d), LC3 (e) nd eclin 1 (f) expression with representtive lots nd the ctivity of cspse 3, 8, 9 (g) fter sorfeni (S) nd quercetin (Q) tretment for 48 h in T98G cells trnsfected with specific sirna nti-hsp27 (si27) nd nti-hsp72 ccompnied y decresed level of the protein in the mitochondrion. Neither of the drugs hd n effect on the eclin 1 expression nd the level of the protein ws similr to the control. No conversion of LC3I into its smller form LC3II ws lso oserved. Quercetin nd sorfeni pplied lone or in comintion inhiited the (si72). The dt were normlised reltive to -ctin (not shown). C control cells, SQ simultneous drug tretment, si27 or si72 specific sirna locking Hsp27 or Hsp72 expression, TR trnsfection regent, TRsi trnsfection regent with specific sirna, *P \ 0.05 compred to control expression of Rs nd Rf in oth the cell lines. In the cse of cspses, oth the drugs pplied lone or in comintion incresed the ctivity of cpse 3 nd cspse 9 in T98G nd MOGGCCM cells with locked Hsp expression. Sorfeni nd quercetin did not chnge the ctivity of cspse 8 in either cell line.

12 Neurotox Res (2014) 26: Discussion In mlignnt glioms, severl mechnisms responsile for progrmmed cell deth induction, like poptosis or utophgy, re locked while moleculr chperones promoting cell survivl re overexpressed (Ghoril et l. 2005;Ohgki nd Kleihues 2005; Omuro et l. 2007). Recent pulictions indicte tht mjority of glioms disply upregulted Rf kinse (the first effector kinse downstrem of Rs), which is essentil for ctivtion of the mitogen-ctivted protein pthwy Rs Rf MEK ERK. The upregultion of this signlling cscde hs een proven to tke prt in mplifiction of mitogenic stimuli nd promotion of cellulr prolifertion of glioms (Lo 2010). Such oservtions provide rtionle for ttempting to disrupt this pthwy s good cndidte in nticncer strtegy. Recently, numer of specific inhiitors hve een developed. Among these, sorfeni seems to hve pivotl role. It hs een demonstrted in vrious tumour cell lines tht sorfeni induced mitochondril dmge mnifested y cytochrome c relese into the cytosol, cspse 9 nd 3 ctivtion, nd in consequence, poptosis medited through n intrinsic pthwy (Hung et l. 2010; Rhmni et l. 2007;Yuetl.2006). On the other hnd, other experiments showed tht poptosis fter sorfeni tretment ws correlted with n externl pthwy with cspse 8 ctivtion (Prk et l. 2010). There re lso some reports indicting tht sorfeni lso induced utophgy (Sieglin et l. 2010). Considering such multiple effects of sorfeni on induction of progrmmed cell deth nd different mechnisms ctivted within cells, we decided to evlute the type of cell deth tht will e predominnt in humn gliolstom multiforme (T98G) nd nplstic strocytom (MOGGCCM) cells. Our experiments showed tht in nplstic strocytom, the drug induced minly poptosis, ut t level only slightly higher thn tht oserved in non-treted, control cells. The process ws initited through mitochondril pthwy. In the cse of gliolstom multiforme cells, sorfeni ppered to e very effective utophgy inducer. At the moleculr level, this ws correlted with the incresed conversion of LC3I form of LC3 into LC3II the first mmmlin protein indentified tht specificlly ssocites with utophgosome memrnes. Therefore, the clevge of LC3I into LC3II is the hllmrk of utophgy (Key et l. 2000). Incresed eclin 1 expression, criticl component involved in utophgosome formtion in the erly stge of utophgy, ws lso oserved. It hs lso een shown tht ctivtion of cspse 3 inhiits utophgosome formtion nd my lock eclin 1 ctivity or even cleve the protein (Luo nd Ruinsztein 2010; Pirtoli et l. 2009; Wng 2008; Wirwn et l. 2010). In the T98G cells, the ctivity of cspse 3 ws not significnt, in contrst to the nplstic strocytom cells, where the ctivity of the enzyme ws incresed. The very interesting morphology of utophgic T98G cells should e emphsized. Deth vesicles were concentrted t the edges of cells ut not dispersed throughout the cytoplsm. The uthenticity of the peripherl vcuoles s utophgic ones ws confirmed y loclistion of eclin 1 within them. The cuse of such n unusul distriution is unknown. Nturl ioctive compounds my ct in synergy with drugs used in nticncer therpy (Brgnhol et l. 2006; Jkuowicz-Gil et l. 2010, 2011, 2013; Rmos 2007; Russo 2007; Schültke et l. 2005). In the present pioneer study, the use of the comintion of sorfeni with quercetin resulted in n effective cell type-specific induction of poptosis nd utophgy. After the tretment with oth the drugs, poptosis ws most frequent in the nplstic strocytom cells. The percentge of ded cells ws higher thn tht oserved fter the single sorfeni pplictions. At the moleculr level, poptosis ws medited y n intrinsic pthwy. In the cse of T98G cells treted with sorfeni nd quercetin, the level of ded cells ws comprle with tht oserved fter the seprte sorfeni tretment; nd simultneous incution with oth the drugs induced oth poptosis nd utophgy. Keeping in mind tht quercetin induces minly poptosis while sorfeni minly utophgy, it seems tht these drugs initited different pthwys in the gliolstom multiforme cell line. As descried erlier, the lnce etween cspse 3 nd eclin 1 my hve criticl role in such diversity. In our study, sorfeni s well s quercetin pplied lone or in comintion decresed the level of Rs nd Rf expression in the MOGGCCM nd T98G cells, therey upregulting the Rs Rf MEK ERK pthwy. The level of inhiition ws similr in oth the cell lines, irrespective of the drug precedence nd concentrtion. This indictes tht decresed signl trnsmission through the Rs Rf MEK ERK cscde increses the sensitivity of gliom cells to deth upon sorfeni nd quercetin tretment nd stimultes poptosis nd utophgy induction with the sme effect. Het shock proteins re moleculr chperones whose ctivity protects cells ginst deth. They re engged in preventing cytochrome c relese, mitochondril memrne permeiliztion, mturtion of procspse 9 nd cspse 3 ctivtion. Depletion of Hsp27 nd Hsp72 severely decreses degrdtion of misfolded proteins (Stetler et l. 2010; Turturicietl.2011). In cncer cells, where the level of het shock proteins is normlly high, these moleculr chperones ct s negtive prognostic mrkers. It ws lso oserved tht the expression of the proteins rose with the grde of the tumour nd tht they might prticipte in oncogenesis. Therefore, inhiition of het shock protein expression hs ecome novel strtegy for cncer therpy (Grrido et l. 2006; Grner et l. 2007; Jkuowicz-Gil et l. 2013; Sreedhr nd Csermley 2007). In our

13 76 Neurotox Res (2014) 26:64 77 experiments, oth sorfeni nd quercetin prtilly inhiited Hsp27 nd Hsp72 expression in the T98G nd MOGGCCM cells. Additionl lock of their expression y specific sirna showed very good predisposition of the comintion of the drugs to induce poptosis in trnsfected cells. This type of progrmmed cell deth ws detected in out 90 % of the cell popultion, where most cells were in the erly stge of the process. As reveled y the moleculr nlysis, poptosis in the trnsfected cells ws medited y n intrinsic signl. Blocking of Hsp27 nd Hsp72 expression in the T98G nd MOGGCCM cells did not increse sensitivity to utophgy induction upon sorfeni nd quercetin tretment. It is known tht therpeuticlly incresed utophgy could represent n lterntive wy to destroy cncer cells. This would e eneficil in the light of recent investigtions, s glioms nturlly resist poptosis. On the other hnd, mny studies demonstrte tht utophgy represents protective mechnism helping cncer cells to get rid of toxic prticles nd, in consequence, to increse their survivl. In the light of these oservtions, redirecting cncer cells to enter the poptotic pthwy would e eneficil (Grci-Arencii et l. 2010; Lefrnc nd Kiss 2006; Lin et l. 2012). Eliminting Hsp expression y sirna significntly incresed the sensitivity of the T98G cell line to induction of poptosis ut not utophgy. This my e explined y the fct tht Hsps re very effective cspse 3 inhiitors. Elimintion of these moleculr chperones from cells does not result in locking cspse 3 ctivity, which in turn my decrese the level of eclin 1 nd lock utophgy in consequence. In summry, we demonstrted for the first time tht sorfeni pplied in comintion with quercetin is very potent progrmmed cell inducer in gliom multiforme T98G nd nplstic strocytom MOGGCCM cells ut the type of deth, poptosis or utophgy, is cell type nd drug precedence specific. Our results indicte tht locking of Hsp27 nd Hsp72 expression mkes T98G nd MOG- GCCM cells extremely vulnerle to poptosis induction upon sorfeni nd quercetin tretment nd tht progrmmed cell deth is initited y n internl signl. They lso confirm tht moleculr chperones re responsile for gliom cell resistnce to progrmmed cell deth nd tht the nturl ioctive compound cts in synergy with nticncer drugs. We demonstrted tht sorfeni dministered with quercetin seems to e potent nd promising comintion tht might e useful in gliom therpy. Conflict of interest Authors clim no conflict of interest. Open Access This rticle is distriuted under the terms of the Cretive Commons Attriution License which permits ny use, distriution, nd reproduction in ny medium, provided the originl uthor(s) nd the source re credited. References Blsurmnin K, Schroit AJ (2003) Aminophospholipid symmetry: mtter of life nd deth. Annu Rev Physiol 65: Brdford MM (1976) A rpid nd sensitive method for quntittion of microgrm quntities of protein utilizing the principle of proteindye inding. Anl Biochem 72: Brgnhol E, Zmin LL, Cnedo AD, Horn F, Tmjusuku AS, Wink MR, Slego C, Bttstini AM (2006) Antiprolifertive effect of quercetin in the humn U138MG gliom cell line. Anticncer Drugs 17: Grci-Arencii M, Hochfeld WE, Toh PPC, Ruinsztein DC (2010) Autophgy, gurdin ginst neurodegenertion. Semin Cell Dev Biol 21: Grrido C, Brunet M, Didelot C, Zermti Y, Schmitt E, Kroemer G (2006) Het shock proteins 27 nd Hsp 70: nti-poptotic proteins with tumorigenic properties. Cell Cycle 5: Ghoril IM, Witzig TE, Adjei A (2005) Trgeting poptosis pthwys in cncer therpy. CA Cncer J Clin 5: Grner MW, Cumming RI, Binger DD (2007) The het shock response nd chperone/het shock proteins in rin tumors: surfce expression, relese nd possile immune consequences. J Neurosci 27: Hhn O, Stdler W (2006) Sorfeni. Curr Opin Oncol 18: Hosokw N, Hiryoshi K, Kudo H, Tkechi H, Aoike A, Kwi K, Ngt K (1992) Inhiition of the ctivtion of het shock fctor in vivo nd in vitro y flvonoids. Mol Cell Biol 12: Hung R, Chen XQ, Hung Y, Chen N, Zeng H (2010) The multikinse inhiitor sorfeni induces cspse-dependent poptosis in PC-3 prostte cncer cells. Asin J Androl 12: Jkuowicz-Gil J, Lngner E, Wertel I, Piersik T, Rzeski W (2010) Temozolomide, quercetin nd cell deth in the MOGGCCM strocytom cell line. Chem Biol Interct 188: Jkuowicz-Gil J, Lngner E, Rzeski W (2011) Kinetic studies of the effects of Temodl nd quercetin on strocytom cells. Phrmcol Rep 63: Jkuowicz-Gil J, Lngner E, Bdziul D, Wertel I, Rzeski W (2013) ) Apoptosis induction in humn gliolstom multiforme T98G cells upon temozolomide nd quercetin tretment. Tumor Biol 34: Jkuowicz-Gil J, Lngner E, Bdziul D, Wertel I, Rzeski W (2013) Silencing of Hsp27 nd Hsp72 in gliom cells s tool for progrmmed cell deth induction upon temozolomide nd quercetin tretment. Toxicol App Phrmcol. doi: / s Jnkowsk A, Skonieczn D, Rommerts FFC, Wrchoł JB (1997) Investigtions on poptosis in Leydig cells cultured in vitro. Foli Histochem Cytoiol 35:97 98 Key Y, Mizushim N, Ueno T, Ymmoto A, Kirisko T, Nod T, Kominmi E, Ohsumi Y, Yoshimori T (2000) LC3 mmmlin homologue of yest Apg8p is loclized in utophgosome memrnes fter processing. EMBO J 19: Kim EJ, Choi CH, Prk JK, Kng SK, Kim YK (2008) Underlying mechnism of quercetin induced cell deth in humn gliom cells. Neurochem Res 33: Kleihues P, Louis DN, Scheithuer BW, Rorke LB, Reifenerger G, Burger PC, Cnenee WK (2002) The WHO clssifiction of tumors of the nervous system. J Neuropthol Exp Neurol 61: Lemmli UK (1970) Clevge of structurl proteins during the ssemly of the hed of cteriophge T4. Nture 227: Lefrnc F, Kiss R (2006) Autophgy, the Trojn horse to comt gliolstoms. Neurosurg Focus 20:E7

14 Neurotox Res (2014) 26: Lin CJ, Lee CC, Shih YL, Lin CH, Wng SH, Chen TH, Shih CM (2012) Inhiition of mitochondri nd endoplsmic reticulum stress-medited utophgy ugments temozolomide-induced poptosis in gliom cells. PLoS ONE 7:e38706 Lo HW (2010) Trgeting Rs RAF ERK nd its interctive pthwys s novel therpy for mlignnt glioms. Curr Cncer Drug Trgets 10: Luo S, Ruinsztein DC (2010) Apoptosis locks eclin 1-dependent utophgosome synthesis n effect reduced y Bcl-xL. Cell Deth Differ 17: Nors LB, Supko JG, Rosenfeld M, Chmerlin M, Phuphnich S, Btchelor T, Desideri S, Ye X, Wright J, Gujr S, Grossmn SA (2011) Phse I tril of sorfeni in ptients with recurrent or progressive mlignnt gliom. Neur Oncol 13: Ohgki H, Kleihues P (2005) Popultion-sed studies on incidence, survivl rtes nd genetic ltertions in strocytic nd oligodendroglil glioms. J Neuropthol Exp Neurol 64: Omuro AM, Fivre S, Rymond E (2007) Lessons lerned in the development of trgeted therpy for mlignnt glioms. Mol Cncer Ther 6: Prk MA, Mitchell C, Zhng G, Ycou A, Allegood J, Häussinger D, Reinehr R, Lrner A, Spiegel S, Fisher PB, Voelkel-Johnson C, Ogretmen B, Grnt S, Dent P (2010) Vorinostt nd sorfeni increse CD95 ctivtion in gstrointestinl tumor cells through C(2?) de novo cermide-pp2a-rective oxygen speciesdependent signling pthwy. Cncer Res 70: Pirtoli L, Cevenini G, Tini P, Vnnini M, Oliveri G, Mrsisli S, Mourmorus V, Ruino G, Mircco C (2009) The prognostic role of eclin 1 protein expression in high-grde glioms. Autophgy 5: Rhmni M, Dvis EM, Crtree TR, Hii JR, Nguyen TK, Dent P, Grnt S (2007) The kinse inhiitor sorfeni induces cell deth through process involving induction of endoplsmic reticulum stress. Mol Cell Biol 27: Rmos S (2007) Effects of dietry flvonoids on poptotic pthwys relted to cncer chemoprevention. J Nutr Biochem 18: Russo GL (2007) Ins nd outs of dietry phytochemicls in cncer chemoprevention. Biochem Phrmcol 33: Schültke E, Kmencic H, Zho M, Tin GF, Bker AJ, Grieel RW, Juurlink BHJ (2005) Neuroprotection following fluid percussion rin trum: pilot study using quercetin. J Neurotrum 22: Sieglin MD, Rskett CM, Gilert CA, Ross AH, Altieri DC (2010) Sorfeni exerts nti-gliom ctivity in vitro nd in vivo. Neurosci Lett 478: Sreedhr AS, Csermley P (2007) Het shock proteins in the regultion of poptosis: new strtegies in tumor therpy. A comprehensive review. Phrmcol Ther 101: Stetler RA, Gn Y, Zhng W, Liou AK, Go Y, Co G, Chen J (2010) Het shock proteins: cellulr nd moleculr mechnisms in the CNS. Prog Neuroiol 92: Tkeuchi H, Kondo Y, Fujiwr K, Knzw T, Aoki H, Mills GB, Kondo S (2005) Synergistic ugmenttion of rpmycin-induced utophgy in mlignnt gliom cells y phosphtidylinositol 3-kinse/protein kinse B inhiitors. Cncer Res 65: Turturici G, Sconzo G, Gerci F (2011) Hsp70 nd its moleculr role in nervous system diseses. Biochem Res Int 2011: Wng J (2008) Beclin 1 ridges utophgy, poptosis nd differentition. Autophgy 4: Wilhelm SM, Crter C, Tng L, Wilkie D, McNol A, Rong H, Chen C, Zhng X, Vincent P, McHugh M, Co Y, Shujth J, Gwlk S, Eveleigh D, Rowley B, Liu L, Adnne L, Lynch M, Auclir L, Tylor I, Gedrich R, Voznesensky A, Riedl B, Post LE, Bollg G, Tril PA (2004) BAY exhiits rod spectrum orl ntitumor ctivity nd trgets the RAF/MEK/ERK pthwy nd receptor tyrosine kinses involved in tumor progression nd ngiogenesis. Cncer Res 64: Wirwn EVW, Vnde Wlle L, Kersse K, Cornelis S, Clerhout S, Vnovererghe I, Roelndt R, De Rycke R, Verspurten J, Declerq W, Agostinis P, Vnden Berghe T, Lippens S, Vndeneele P (2010) Cspse-medited clevge of eclin 1 inctivtes eclin1-induced utophgy nd enhnces poptosis y promoting the relese of propoptotic fctors from mitochondri. Cell Deth Dis 1:e18 Yng F, Brown C, Buettner R, Hedvt M, Strr R, Scuto A, Schroeder A, Jensen MC, Jove R (2010) Sorfeni induces growth rrest nd poptosis of humn gliolstom cells vi dephosphoryltion of STAT3. Mol Cncer Ther 9: Yu C, Fridy BB, Li JP, Yng L, Srkri J, Ky NE, Crter CA, Roerts LR, Kufmnn SH, Adjei AA (2006) Cytotoxic synergy etween the multikinse inhiitor sorfeni nd the protesome inhiitor ortezomo in vitro: induction of poptosis through Akt nd c-jun NH2-terminl kinse pthwy. Mol Cncer Ther 5: