Article Upregulation of DR5 and Downregulation of by IITZ-01, Lysosomotropic Autophagy Inhibitor, Potentiates TRAIL-Mediated in Renal Cells via Ubiquitin-Proteasome Pathway

Sk Abrar Shahriyar 1, Seung Un Seo 1, Kyoung-jin Min 2, Peter Kubatka 3,4 , Do Sik Min 5, Jong-Soo Chang 6, Dong Eun Kim 7, Seon Min Woo 1,* and Taeg Kyu Kwon 1,*

1 Department of Immunology, School of Medicine, Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu 42601, Korea; [email protected] (S.A.S.); [email protected] (S.U.S.) 2 New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, 80 Chembok-ro, Dong-gu, Daegu 41061, Korea; [email protected] 3 Department of Medical Biology, Jessenius Faculty of Medicine, Comenius University in Bratislava, 03601 Martin, Slovak; [email protected] 4 Department of Experimental Carcinogenesis, Division of Oncology, Biomedical Center Martin, Jessenius Faculty of Medicine, Comenius University in Bratislava, 03601 Martin, Slovak 5 College of Pharmacy, Yonsei University, 85 Songdogwahak-ro, Yeonsu-gu, Incheon 21983, Korea; [email protected] 6 Department of Life Science and Chemistry, College of Natural Science Daejin University, Pochon-shi, Kyeinggido 11150, Korea; [email protected] 7 Department of Otolaryngology, School of Medicine, Keimyung University, 1095 Dalgubeoldaero, Dalseo-Gu, Daegu 42601, Korea; [email protected] * Correspondence: [email protected] (S.M.W.); [email protected] (T.K.K.); Tel.: +82-53-258-7358 (T.K.K.)  Received: 14 July 2020; Accepted: 20 August 2020; Published: 21 August 2020 

Abstract: -related apoptosis-inducing (TRAIL) selectively is able to increase apoptosis in cancer cells as agent with minimum toxicity to noncancerous cells. However, all cancer cells are not sensitive to TRAIL-induced apoptosis. In this study, we showed the sub-lethal concentrations of a lysosomotropic autophagy inhibitor, IITZ-01, sensitizes cancer cells (renal, lung, and breast carcinoma) to TRAIL-induced apoptosis through DR5 upregulation and survivin downregulation through ubiquitin-proteasome pathway. Knockdown of DR5 or overexpression of survivin inhibited combined treatment with IITZ-01 and TRAIL-induced apoptosis. IITZ-01 downregulated protein expression of Cbl, ubiquitin E3 ligase, and decreased expression level of Cbl markedly led to increase DR5 protein expression and TRAIL sensitivity. Moreover, IITZ-01 decreased expression level of survivin protein via downregulation of deubiquitinase ubiquitin-specific protease 9X (USP9X) expression. Taken together, these results provide the first evidence that IITZ-01 enhances TRAIL-mediated apoptosis through DR5 stabilization by downregulation of Cbl and USP9X-dependent survivin ubiquitination and degradation in renal carcinoma cells.

Keywords: IITZ-01; TRAIL; DR5; survivin; USP9X

1. Introduction Lysosomes are acidic organelles within cells that degrade and reuse macromolecules through endocytosis, autophagy and phagocytosis, and are involved in regulation of cellular homeostasis [1,2]. Lysosomotropic agents can selectively diffuse, accumulate into lysosomes, and consequently induce pH

Cancers 2020, 12, 2363; doi:10.3390/cancers12092363 www.mdpi.com/journal/cancers Cancers 2020, 12, 2363 2 of 15 alteration, decrease of enzyme activity and inhibition of calcium signaling in lysosome [3]. In addition, several lysosomotropic drugs break down phagocytosis, endocytosis and autophagy by disrupting membrane fusion between intracellular organelles [4–6]. Chloroquine (CQ) and hydroxychloroquine (HCQ), well-known lysosomotropic drugs, increase lysosomal membrane permeabilization by neutralizing intracellular lysosomal pH, resulting in the induction of apoptotic cell death in cancer [7,8]. Moreover, two lysosomotropic agents can potentiate anti-cancer effect to various chemotherapeutic drugs and overcome resistance to anti-cancer drugs or irradiation in many cancer cells [9–13]. Therefore, lysosomotropic agents are capable of increasing therapeutic efficacy of tumors, but the underlying molecular mechanism is not clear. IITZ-01, as benzimidazole analogs of morpholino s-triazine, has been kwon to contain lysosomotrpopic properties and anti-cancer activity [14,15]. IITZ-01 accumulates autophagosome by inhibiting autophagic flux through damaged lysosome. In addition, IITZ-01 decreases mitochondria membrane potential through downregulation of Bcl-2 and IAP family proteins followed by -dependent apoptosis in breast cancer cells [16]. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) targets malignant cells by binding to the death receptors (DRs), followed by the increase of caspase-dependent apoptosis except normal cells [17,18]. Although TRAIL is considered to be a strong therapeutic agent of malignant cells, many malignant cells have been demonstrated to be resistant to TRAIL. Therefore, many researchers have reported that combined treatment with numerous sensitizing agents is able to overcome TRAIL resistance [19,20]. In our present study, we found the effect of IITZ-01 as a novel TRAIL sensitizer. IITZ-01 induced Cbl-mediated DR5 stabilization and USP9X-dependent survivin degradation, resulted in the enhancement to TRAIL sensitivity in cancer.

2. Results

2.1. IITZ-01 Increases TRAIL Sensitivity in Renal Carcinoma Cells IITZ-01 exerts an anticancer effect in breast, colon and prostate cancer cells [15,16]. We examined whether IITZ-01 could augment TRAIL-induced apoptosis in human renal carcinoma cells. As shown in Figure1a, individual IITZ-01 (0.5, 1 µM) and TRAIL (30, 50 ng/mL) had no effect on apoptosis in human renal carcinoma (Caki-1 and ACHN) cells, but combined treatment of IITZ-01 and TRAIL remarkably increased the sub-G1 population and cleavage of PARP. Furthermore, IITZ-10 plus TRAIL treatment increased Annexin V/7-Aminoactinomycin D (7-AAD) double positive cells (Figure1b). We observed typical apoptotic morphologies, including blebbing, apoptotic bodies, exemplary chromatin damage in the nuclei and DNA fragmentation in Caki-1 cells treated with IITZ-01 and TRAIL (Figure1c). We investigated whether activation plays a significant role in cell death by combined treatment with IITZ-01 plus TRAIL. Combined treatment increased the caspase-3 activity (Figure1d). Furthermore, when we used z-VAD-fmk (z-VAD), a pan-caspase inhibitor, pretreatment of z-VAD completely abolished apoptosis and cleavage of caspase-3 by IITZ-01 plus TRAIL (Figure1e). These results suggest that IITZ-01 enhances TRAIL-induced apoptosis in human renal carcinoma cells. Cancers 2020, 12, 2363 3 of 15 Cancers 2020, 12, x 3 of 15

FigureFigure 1. 1. IITZ-01IITZ-01 sensitizes sensitizes renal renal carcinoma carcinoma cells cells to TNF to-related TNF-related apoptosis apoptosis-inducing-inducing ligand (TRAIL ligand- (TRAIL-inducedinduced apoptosis. apoptosis. (a) Caki (a)- Caki-11 and ACHN and ACHN cells cellswere wereincubated incubated with withindicated indicated concentrations concentrations with withIITZ IITZ-01-01 and and/or/or TRAIL TRAIL for for 18 18 h. h. The The apoptotic apoptotic population population (sub (sub-G1)-G1) and and protein levels were analyzedanalyzed by flowby flow cytometry cytometry and and Western Western blotting, blotting, respectively; respectively (; b(,bc,)c) Caki-1 Caki-1 cells cells were were incubatedincubated withwith 11 µμMM IITZ-01IITZ- and01 and/or/or 50 50 ng /ng/mLmL TRAIL TRAIL for for 18 18 h. h. Cell Cell death death was was detected detected using using Annexin Annexin VV/7/7-AAD-AAD stainingstaining (b).(b The). The cell cell morphology morphology was was examined examined using using interference interference light light microscopy. microscopy. Nuclei Nuclei condensationcondensation andand DNA fragmentation were detected using 4′,6′-diamidino-2-phenylindole (DAPI) staining and DNA DNA fragmentation were detected using 40,60-diamidino-2-phenylindole (DAPI) staining and DNA fragmentationfragmentation detection detection , kit respectively, respectively (c );(c); (d ()d Caki-1) Caki-1 and and ACHN ACHN cells cells were were incubatedincubated withwith 11 µμMM IITZ-01IITZ- and01 and/or/or 50 50 ng /ng/mLmL TRAIL TRAIL for for 18 18 h. h. DEVDase DEVDase (caspase-3) (caspase-3) activity activity was was examined examined usingusing kitkit asas a describeddescribed in ain Material a Material and and Methods; Methods; (e )(e Caki-1) Caki-1 and and ACHN ACHN cells cells were were incubated incubated withwith 11 µμMM IITZ-01IITZ-01 plusplus 50 ng50 /ng/mLmL TRAIL TRAIL in in the the presence presence or or absence absence of of 20 20 μMµM z-VAD-fmk z-VAD-fmk (z-VAD). (z-VAD). The The values values inin graphgraph (a,c(–a,ce)– represente) represent the the mean meanSD ± SD of of three three independent independent experiments. experiments. * *p p< < 0.050.05 comparedcompared to the control. ± # p#< p0.05 < 0.0 compared5 compared to to IITZ-01 IITZ-01 plus plus TRAIL. TRAIL.

2.2.2.2. Eff ectEffect of IITZ-01of IITZ-01 on on Expression Expression Levels Levels of of Apoptosis-Related Apoptosis-Related Proteins Proteins ToTo elucidate elucidate the molecular the molecular mechanisms mechanisms leading leading to apoptosis to apoptosis in IITZ-01 in IITZ treated-01 treated Caki-1 and Caki ACHN-1 and cells,ACHN we investigated cells, we investigated the changes the of apoptosis-related changes of apoptosis proteins-related expression. proteins IITZ-01expression significantly. IITZ-01 diminishedsignificantly survivin diminishe expressiond survivin and increasedexpression DR5 and expression,increased DR5 while expression other apoptosis-related, while other apoptosis proteins- (Mcl-1,related XIAP, proteins Bcl-2, (Mcl Bcl-xL,-1, XIAP, Bim, cIAP1, Bcl-2, Bcl cIAP2,-xL, Bim, DR4, cIAP1, c-FLIP cIAP2, and Bax) DR4, were c-FLIP not a andffected Bax) by were IITZ-01 not treatmentaffected (Figure by IITZ2).-01 These treatment results (Fig indicateure 2). that These IITZ-01 results induces indicate survivin that downregulationIITZ-01 induces and survivin DR5 upregulation,downregulation resulting and inDR5 the upregulation, increase of TRAIL-mediated resulting in the apoptosisincrease of in TRAIL caspase-dependent-mediated apoptosis manner. in caspase-dependent manner.

Cancers 2020, 12, 2363 4 of 15 Cancers 2020, 12, x 4 of 15

Figure 2. IITZ-01 induces upregulation of DR5 and downregulation of survivin. Caki-1 and ACHN Figure 2. IITZ-01 induces upregulation of DR5 and downregulation of survivin. Caki-1 and ACHN µ cellscells were were incubated incubated with with 0.25–1 0.25–1 μMM IITZ-01 IITZ-01 for for 18 18 h. h. The The protein protein expression expression levels levels of of apoptosis apoptosis related proteinsrelated proteins and actin and were actin determined were determined by Western by Western blotting. blotting. 2.3. Upregulation of DR5 by IITZ-01 Is Associated with TRAIL-Induced Apoptosis 2.3. Upregulation of DR5 by IITZ-01 Is Associated with TRAIL-Induced Apoptosis DR5DR5 playsplays aa majormajor role in in TRAIL TRAIL-mediated-mediated apopto apoptosissis [21,22] [21,,22 and], and other other lysosomotropic lysosomotropic agents agents induceinduce DR5 DR5 upregulationupregulation resulting resulting in in the the enhancement enhancement of ofTRAIL TRAIL sensitivity sensitivity [23].[ 23Expectably]. Expectably,, DR5 DR5 proteinprotein level level waswas increasedincreased by by IITZ IITZ-01-01 after after 3 3h h(Fig (Figureure 3a)3a).. ToTo explore explore the theIITZ IITZ-01-mediated-01-mediated DR5 DR5 upregulationupregulation at at the the transcriptional transcriptional levels, levels, we we checked checked DR5 DR5 mRNA mRNA level level and andpromoter promoter activity. activity. However, IITZ-01However, did IITZ not-01 alter did DR5 not mRNAalter DR5 level mRNA and promoter level and promoter activity (Figure activity3 b,c).(Figure Next, 3b,c we). Next investigated, we theinvestigated impact of IITZ-01the impact on DR5 of IITZ protein-01 on stability DR5 protein using the stability cycloheximide using the (CHX). cycloheximide Combined (CHX treatment). withCombined CHX and treatment IITZ-01 with more CHX maintained and IITZ- DR501 more protein maintaine level comparedd DR5 protein to CHX level alonecompared (Figure to 3CHXd). Next, wealone investigated (Figure 3d). the Next importance, we investigated of the DR5 the upregulationimportance of inthe IITZ-01 DR5 upregulation plus TRAIL-induced in IITZ-01 apoptosis,plus Caki-1TRAIL and-induced ACHN apoptosis cells were, Caki transiently-1 and ACHN transfected cells were with transiently DR5 siRNA. transfected Knockdown with of DR5 DR5 siRNA. significantly reducedKnockdown apoptosis of DR5 by significantly the combined reduced treatment apoptosis of IITZ-01 by the combined and TRAIL treatment (Figure of3e). IITZ Therefore,-01 and TRAIL these data (Figure 3e). Therefore, these data indicate that upregulation of DR5 expression is involved in IITZ-01 indicate that upregulation of DR5 expression is involved in IITZ-01 plus TRAIL-induced apoptosis. plus TRAIL-induced apoptosis.

Cancers 2020, 12, 2363 5 of 15 Cancers 2020, 12, x 5 of 15

Figure 3. DR5DR5 stabilization stabilization contributes contributes to to the the sensitization sensitization effect effect of of IITZ IITZ-01-01 on TRAIL-inducedTRAIL-induced apoptosis in CakiCaki-1-1 cells cells.. ( (aa,b,b)) Caki Caki-1-1 cells cells were were incubated incubated with with 1 1 μMµM IITZIITZ-01-01 for indicated time periodsperiods.. The The prote proteinin and and mRNA mRNA levels levels were were determined determined by by Western Western blotting blotting ( (aa)) and and RT RT-PCR-PCR (upper (upper panel)/qPCRpanel)/qPCR (low(low pane),pane), respectivelyrespectively ( b(b););(c) (c)Caki-1 Caki-1 cells cells were were transiently transiently transfected transfected with with DR5(-605) DR5(-605) or orDR5(SacI) DR5(SacI) promoter promoter and and incubated incubated with with1 µM 1 IITZ-01 μM IITZ for- 1801 h.for The 18 cells h. The were cells lysed were and lysed the luciferase and the luciferaseactivity was activity measured was asmeasured a described as ain described a Material in and a MaterialMethods; and (d) Caki-1 Methods; cells (d were) Caki incubated-1 cells were with incubated1 µM IITZ-01 with for 181 μM h, washed IITZ-01 with for PBS, 18 and h, washed then treated with with PBS, 20 andµg/mL then cycloheximide treated with (CHX) 20 μg/mL and/or cyclohex1 µM IITZ-01imide for (CHX) the indicated and/or 1 time μM periods.IITZ-01 Thefor the band indica intensityted time of the periods. DR5 protein The band was measuredintensity of using the DR5ImageJ; protein (e) Caki-1 was measured and ACHN using cells Image were J; transiently (e) Caki-1 transfectedand ACHN with cells control were transiently (Cont) siRNA transfected or DR5 withsiRNA, control and then (Cont) incubated siRNAwith or DR5 1 µM siRNA IITZ-01, and and then 50 ng incubated/mL TRAIL with for 181 μMh. The IITZ apoptotic-01 and population50 ng/mL TRA(sub-G1)IL for and 18 protein h. The levels apoptot wereic analyzed population by flow (sub- cytometryG1) and protein (e) and Western levels were blot ( analyzedd,e). The values by flow in cytometrygraph (b,c ,(e)e) representand Western the meanblot (d,eSD). The of three values independent in graph (b,c,e experiments.) represent * p the< 0.05 mean compared ± SD of tothree the ± independentIITZ-01 plus TRAILexperiments. in control * p < siRNA. 0.05 compared to the IITZ-01 plus TRAIL in control siRNA.

2.4. D Downregulationownregulation of of Cbl Cbl Plays Plays a Critical Role in DR5 Upregulation and TRAIL Sensitivity by IITZ-01IITZ-01 Previous studies studies reported reported that that E3 ligase E3 ligaseCbl contributes Cbl contributes to DR5 ubiquitination to DR5 ubiquitination and degradation and [24,25]degradation. We also[24, 25 reported]. We also that reported CQ-mediated that CQ-mediated Cbl downregulation Cbl downregulation induce decrease induce decrease of DR5 protein of DR5 expressionprotein expression level [23] level. Following [23]. Following these, we these, checked we checked the role theof Cbl role in of DR5 Cbl expression in DR5 expression in IITZ-01 in treated IITZ-01 cellstreated. IITZ cells.-01 IITZ-01downregulated downregulated Cbl protein Cbl proteinlevel in level a time in- adependent time-dependent manner manner,, but not but mRNA not mRNA level (Figurelevel (Figure 4a). We4a). further We further examined examined whether whether IITZ-01 IITZ-01 can regulate can regulate Cbl stabilization. Cbl stabilization. As shown As shown in Figure in 4Figureb, IITZ4b,-01 IITZ-01 significantly significantly inhibited inhibited Cbl Cblstability stability compared compared with with CHX CHX alone. alone. To To further further confirm confirm the involvementinvolvement ofof Cbl Cbl on DR5 on upregulation DR5 upregulation by IITZ-01, by we IITZ transiently-01, we overexpressed transiently ov Cbl.erexpressed Overexpression Cbl. Overexpressionof Cbl markedly diminished of Cbl markedly IITZ-01 induceddiminished DR5 IITZ upregulation-01 induced (Figure DR54c). upregulation Downregulation (Figure of Cbl 4 byc). DownregulationsiRNA induced TRAIL-mediatedof Cbl by siRNA apoptosis,induced TRAIL cleavage-mediated of PARP apoptosis, and DR5 cleavage upregulation of PARP in Caki-1 and DR and5 upregulationACHN cells (Figurein Caki4-d).1 and These ACHN data cells demonstrate (Figure 4 thatd). These Cbl is data important demonstrate role in that stability Cbl is of important DR5 and rolesensitization in stability of of TRAIL DR5 and by IITZ-01. sensitization of TRAIL by IITZ-01.

Cancers 2020, 12, 2363 6 of 15 Cancers 2020, 12, x 6 of 15

Figure 4. Figure 4. DDownregulationownregulation of of Cbl is involved in IITZ-01-mediatedIITZ-01-mediated DR5 upregulation and TRAIL sensitization. (a) Caki-1 cells were incubated with 0.25–1 µM IITZ-01 for 18 h. The protein and sensitization. (a) Caki-1 cells were incubated with 0.25–1 μM IITZ-01 for 18 h. The protein and mRNA mRNA levels were determined by Western blotting and RT-PCR, respectively; (b) Caki-1 cells were levels were determined by Western blotting and RT-PCR, respectively; (b) Caki-1 cells were incubated incubated with 20 µg/mL CHX and/or 1 µM IITZ-01 for the indicated time periods. The band intensity with 20 μg/mL CHX and/or 1 μM IITZ-01 for the indicated time periods. The band intensity of the of the Cbl protein was measured using Image J; (c) Caki-1 cells were transfected with pCMV- Cbl protein was measured using Image J; (c) Caki-1 cells were transfected with pCMV-myc (Vector) (Vector) or pCMV-myc/Cbl and treated with 1 µM IITZ-01 for 18 h; (d) Caki-1 and ACHN cells were or pCMV-myc/Cbl and treated with 1 μM IITZ-01 for 18 h; (d) Caki-1 and ACHN cells were transiently transiently transfected with Cont siRNA or Cbl siRNA, and then incubated with 50 ng/mL TRAIL transfected with Cont siRNA or Cbl siRNA, and then incubated with 50 ng/mL TRAIL for 18 h. The for 18 h. The apoptotic population (sub-G1) and protein levels were analyzed by flow cytometry (d) apoptotic population (sub-G1) and protein levels were analyzed by flow cytometry (d) and Western and Western blotting (b–d), respectively. The values in graph (d) represent the mean SD of three blotting (b–d), respectively. The values in graph (d) represent the mean ± SD of three± independent independent experiments. * p < 0.05 compared to the TRAIL in control siRNA. experiments. * p < 0.05 compared to the TRAIL in control siRNA. 2.5. Survivin Degradation Is Important for IITZ-01 Plus TRAIL-Induced Apoptosis 2.5. Survivin Degradation Is Important for IITZ-01 Plus TRAIL-Induced Apoptosis We next investigated whether IITZ-01 modulates survivin expression at the transcriptional level.We Survivin next investigated mRNA expression whether IITZ was-01 not modulates altered bysurvivin IITZ-01 expression treatment at the (Figure transcriptional5a). Therefore, level. weSurvivin investigated mRNA whether expression IITZ-01 was reduces not alteredsurvivin by expression IITZ-01 at treatment the post-translational (Figure 5a). level. Therefore, Combined we treatmentinvestigated with whether IITZ-01 IITZ and- CHX01 reduces more quicklysurvivin decreased expression survivin at the proteinpost-translational expression level rather. Combined than CHX alonetreatment (Figure with5b). IITZ In- addition,01 and CHX MG132, more a quickly proteasome decreased inhibitor, survivin reversed protein IITZ-01-induced expression rather survivin than downregulationCHX alone (Figure (Figure 5b).5 c). In Toaddition, investigate MG132, the functional a proteasome role inhibitor, of survivin re inversed combined IITZ-01 treatment-induced IITZ-01survivin plus downregulation TRAIL-mediated (Figure apoptosis, 5c). To we investigate used survivin-overexpressed the functional role of Caki-1 survivin and in ACHN combined cells. Ectopictreatment expression IITZ-01 of plus survivin TRAIL prevented-mediated sub-G1 apoptosis, population we used and PARP survivin cleavage-overexpressed by combined Caki treatment-1 and (FigureACHN5 cells.d). Therefore, Ectopic expression these data suggestedof survivin that prevented IITZ-01 reducessub-G1 survivinpopulation expression and PARP via proteasomecleavage by activitycombined at the treatment posttranslational (Figure 5d). level, Therefore, and downregulation these data ofsuggested survivin thatcontributes IITZ-01 to reduces IITZ-01-induced survivin TRAILexpression sensitization. via proteasome activity at the posttranslational level, and downregulation of survivin contributes to IITZ-01-induced TRAIL sensitization.

Cancers 2020, 12, 2363 7 of 15 Cancers 2020, 12, x 7 of 15

FigureFigure 5. 5.Survivin Survivin downregulationdownregulation contributes contributes to to the the sensitization sensitization e effectffect of of IITZ-01 IITZ-01 on on TRAIL-induced TRAIL-induced apoptosisapoptosis in in Caki-1 Caki-1 cells. cells. ( (aa)) Caki-1 Caki-1 cellscells werewere incubatedincubated withwith 11µ μMM IITZ-01IITZ-01 forfor indicatedindicated timetime periods.periods. TheThe protein protein and and mRNA mRNA levels levels were were determined determined by Western by Western blotting blotting or RT-PCR, or RT respectively;-PCR, respectively; (b) Caki-1 (b) cellsCaki were-1 cells incubated were incubated with 20 µ withg/mL 20 CHX μg/mL and /CHXor 1 µ and/orM IITZ-01 1 μM for IITZ the- indicated01 for the timeindicated periods. time The periods. band intensityThe band of the intensity survivin of protein the survivin was measured protein using was Image measured J; (c) Caki-1 using cells Image were J; pretreated (c) Caki-1 with cells 0.5 wereµM MG132,pretreated and with then 0.5 μM incubated MG132, with and 1 µ thenM IITZ-01 incubated for 18with h; (1d )μM Vector-transfected IITZ-01 for 18 h; cells(d) Vector (Caki-1-transfected/Vec and ACHNcells / (CakiVec) and-1/Vec survivin-overexpressing and ACHN/Vec) and cells (Caki-1 survivin/Survivin-overexpre andssing ACHN cells/Survivin) (Caki- were1/Survivin incubated and with 1 µM IITZ-01 and/or 50 ng/mL TRAIL for 18 h. The values in graph (b,d) represent the mean SD ACHN/Survivin) were incubated with 1 μM IITZ-01 and/or 50 ng/mL TRAIL for 18 h. The values± in ofgraph three ( independentb,d) represent experiments. the mean ± *SDp < of0.05 three compared independent to the experiments. IITZ-01 plus TRAIL* p < 0.05 in Vectorcompared cells. to the 2.6. USP9X-DependentIITZ-01 plus TRAIL Survivin in Vector Degradation cells. Is Important for IITZ-01 Plus TRAIL-Induced Apoptosis 2.6. AmongUSP9X-Dependent deubiquitinases, Survivin ubiquitin Degradation specific Is Important peptidase for 9X IITZ (USP9X)-01 Plus and TRAIL STAM-binding-Induced Apoptosis protein-like 1 (STAMBPL1) are involved in stabilization of survivin [26,27]. Therefore, we examined protein Among deubiquitinases, ubiquitin specific peptidase 9X (USP9X) and STAM-binding protein- expression levels of USP9X and STAMBPL1 by IITZ-01. As shown in Figure6a, IITZ-01 decreased like 1 (STAMBPL1) are involved in stabilization of survivin [26,27]. Therefore, we examined protein USP9X protein expression, but not STAMBPL1. Moreover, USP9X mRNA expression was not changed expression levels of USP9X and STAMBPL1 by IITZ-01. As shown in Figure 6a, IITZ-01 decreased by IITZ-01 (Figure6b). Therefore, we examined post-translational regulation of IITZ-01-mediated USP9X protein expression, but not STAMBPL1. Moreover, USP9X mRNA expression was not UPS9X downregulation and found CHX plus IITZ-01 more degraded USP9X expression (Figure6c). changed by IITZ-01 (Figure 6b). Therefore, we examined post-translational regulation of IITZ-01- To examine whether USP9X physically interacts with survivin, we performed an immunoprecipitation mediated UPS9X downregulation and found CHX plus IITZ-01 more degraded USP9X expression assay by using Caki-1 cells. Immunoprecipitation assay revealed that USP9X reciprocally binds to (Figure 6c). To examine whether USP9X physically interacts with survivin, we performed an survivin, but not control IgG (Figure6d). To verify the functional role of USP9X in degradation immunoprecipitation assay by using Caki-1 cells. Immunoprecipitation assay revealed that USP9X of survivin by IITZ-01, we used constructs with wild-type (WT) and catalytically inactive mutant reciprocally binds to survivin, but not control IgG (Figure 6d). To verify the functional role of USP9X (C1566S) of USP9X. USP9X WT markedly impeded survivin degradation by IITZ-01 in Caki-1/Vector in degradation of survivin by IITZ-01, we used constructs with wild-type (WT) and catalytically cells, whereas USP9X/C1566S still revealed IITZ-01-inhibited survivin expression (Figure6e). Next, inactive mutant (C1566S) of USP9X. USP9X WT markedly impeded survivin degradation by IITZ-01 we examined whether USP9X regulates survivin deubiquitination in cells. Transfection of cells with in Caki-1/Vector cells, whereas USP9X/C1566S still revealed IITZ-01-inhibited survivin expression HA-Ub expressing vector induced survivin ubiquitination (Figure6f). However, ubiquitination of (Figure 6e). Next, we examined whether USP9X regulates survivin deubiquitination in cells. survivin was significantly decreased in USP9X WT-overexpressed cells. In contrast, catalytically Transfection of cells with HA-Ub expressing vector induced survivin ubiquitination (Figure 6f). inactive mutant USP9X increased survivin ubiquitination (Figure6f). Taken together, our results However, ubiquitination of survivin was significantly decreased in USP9X WT-overexpressed cells. suggest that IITZ-01 can inhibit survivin ubiquitination via downregulation of USP9X, resulted in In contrast, catalytically inactive mutant USP9X increased survivin ubiquitination (Figure 6f). Taken degradation of survivin protein expression. together, our results suggest that IITZ-01 can inhibit survivin ubiquitination via downregulation of USP9X, resulted in degradation of survivin protein expression.

Cancers 2020, 12, 2363 8 of 15 Cancers 2020, 12, x 8 of 15

FigureFigure 6.6. DegradationDegradation ofof survivinsurvivin isis criticalcritical toto inductioninduction ofof IITZ-01IITZ-01 plusplus TRAIL-inducedTRAIL-induced apoptosisapoptosis throughthrough downregulation of of USP9X. USP9X. (a (,ab,b) )Caki Caki-1-1 cells cells were were incubated incubated with with 0.25 0.25–1-1 μM µIITZM IITZ-01-01 for 18 for h. 18USP9X h. USP9XmRNA mRNAlevels were levels determined were determined by RT-PCR; by RT-PCR;(c) Caki- (1c )cells Caki-1 were cells incu werebated incubatedwith 20 μg/mL with 20CHXµg /mLand/or CHX 1 μM and /IITZor 1-µ01M for IITZ-01 the indicated for the indicated time periods. time periods.The band The intensity band intensity of the USP9X of the USP9Xprotein proteinwas measured was measured using using Image Image J; (d)J; The (d) The interaction interaction with with survivin survivin and and USP9X USP9X was was indicated by by immunoprecipitationimmunoprecipitation (IP)(IP) assay;assay; ((ee)) Caki-1Caki-1 cellscells werewere transientlytransiently transfectedtransfected with pDESTS1 (Vector), pDESTS1-USP9XpDESTS1-USP9X (USP9X(USP9X WT)WT) oror pDESTS1-USP9XpDESTS1-USP9X/C1566S/C1566S (USP9X(USP9X C1566S),C1566S), andand thenthen incubatedincubated withwith 11 µμMM IITZ-01IITZ-01 forfor 1818 h;h; ((ff)) ToTo analyze analyze thethe ubiquitination ubiquitination ofof endogenousendogenous survivin,survivin, Caki-1Caki-1 cellscells werewere transfectedtransfected with with HA-ubiquitin HA-ubiquitin (HA-Ub), (HA-Ub), USP9X USP9X WT WT and and USP9X USP9X C1566S C1566S and treated and treated with 0.5 withµM MG132.0.5 μM CellsMG132. were Cells lysed were in 1% lysed sodium in 1% dodecyl sodium sulfate dodecyl (SDS) sulfate buffer to(SDS) disrupt buffer interacting to disrupt proteins interacting and cell proteins lysates wereand cell then lysates diluted were to 0.1% then SDS, diluted followed to 0.1% by SDS, the immunoprecipitation followed by the immunoprecipitation using an anti-survivin. using Survivin an anti- ubiquitinationsurvivin. Survivin was ubiquitination detected by Western was detected blotting by using Western an horseradish blotting using peroxidase an horseradish (HRP)-conjugated peroxidase anti-Ub(HRP)-conjugated antibody. The anti protein-Ub antibody. levels wereThe protein analyzed levels by Western were analyzed blotting by (a ,Westernc–f). blotting (a,c–f). 2.7. IITZ-01 Plus TRAIL Treatment Effects on Apoptosis in Various Cancerous but Not in Normal Cells 2.7. IITZ-01 Plus TRAIL Treatment Effects on Apoptosis in Various Cancerous but Not in Normal Cells We further investigate whether IITZ-01 plus TRAIL trigger apoptosis in other carcinoma cells and We further investigate whether IITZ-01 plus TRAIL trigger apoptosis in other carcinoma cells normal cells. As shown in Figure7a, combined treatment with IITZ-01 and TRAIL augmented sub-G1 and normal cells. As shown in Figure 7a, combined treatment with IITZ-01 and TRAIL augmented population and PARP cleavage in other renal cancer cells (A498), human lung cancer cells (A549) and sub-G1 population and PARP cleavage in other renal cancer cells (A498), human lung cancer cells breast cancer cells (MCF7). Moreover, IITZ-01 upregulated DR5 and downregulated Cbl, USP9X and (A549) and breast cancer cells (MCF7). Moreover, IITZ-01 upregulated DR5 and downregulated Cbl, survivin in all examined cancer cells (Figure7b). However, IITZ-01 plus TRAIL treatment had no USP9X and survivin in all examined cancer cells (Figure 7b). However, IITZ-01 plus TRAIL treatment effect on morphological apoptotic bodies and sub-G1 populations in normal human mesangial cells had no effect on morphological apoptotic bodies and sub-G1 populations in normal human (MC) and normal human skin fibroblast (HSF) cells (Figure7c). In addition, we did not distinguish mesangial cells (MC) and normal human skin fibroblast (HSF) cells (Figure 7c). In addition, we did survivin protein level in normal cells (Figure7d). We also analyzed survivin and DR5 expression in not distinguish survivin protein level in normal cells (Figure 7d). We also analyzed survivin and DR5 renal clear cell carcinoma patient using TCGA databases through University of California Santa Cruz expression in renal clear cell carcinoma patient using TCGA databases through University of (UCSC) Xena Public Data Hub (xena.ucsc.edu) [28]. Survivin and DR5 is highly expressed in tumor California Santa Cruz (UCSC) Xena Public Data Hub (xena.ucsc.edu) [28]. Survivin and DR5 is highly (Figure S1a). Moreover, high expression of survivin and DR5 is shorter the survival rate and showed expressed in tumor (Figure S1a). Moreover, high expression of survivin and DR5 is shorter the poor prognosis (Figure S1b). Therefore, these data indicate that IITZ-01 sensitizes to TRAIL-induced survival rate and showed poor prognosis (Figure S1b). Therefore, these data indicate that IITZ-01 cell death in cancer cells. sensitizes to TRAIL-induced cell death in cancer cells.

Cancers 2020, 12, 2363 9 of 15 Cancers 2020, 12, x 9 of 15

Figure 7. IITZIITZ-01-01 pl plusus TRAIL TRAIL induces induces apoptosis apoptosis on onother other carcinoma carcinoma cells cells,, but butnot notnormal normal cells. cells. (a) Renal(a) Renal carcinoma carcinoma (A498), (A498), lung lung carcinoma carcinoma (A549) (A549) and and breast breast carcinoma carcinoma (MCF7) (MCF7) were were incuba incubatedted with 1 μM1 µM IITZ-01IITZ-01 andand/or/or 5050 ng/mL ng/mL TRAIL for 1818 h; h; (b)) A498, A549 and MCF7 cells were incubated with 0.250.25–1–1 μµM IITZ-01IITZ-01 for 18 h; 18 h; (c) Caki-1,Caki-1, human renal normal mesangial cells (MC) and human skin fibroblastfibroblast (HSF) cells were incubated with 1 μM 1 µM IITZ-01IITZ-01 andand/or/or 5050 ng/mL ng/mL TRAILTRAIL for 1818 h. h. The cell morphology was exa examinedmined using interference light microscopy; (d) MC and HSF cells cells were were incubated incubated with 0.25–10.25–1 µμM IITZ-01IITZ-01 for 1818 h. h. (positive control (p.c); Caki-1Caki-1 cell lysate). The apoptotic population (sub(sub-G1)-G1) and protein levels were analyzed by by flow flow cytometry cytometry ( (aa,,cc)) and and Wes Westerntern blotting blotting ( (aa,,bb,,dd),), respectivelyrespectively.. The values in graph (a,c) represent thethe meanmean ± SD of three independent experiments. * p ± < 0.00.055 compared to the control control.. 3. Discussion 3. Discussion In this study, we demonstrated that lysosomotropic autophagy inhibitor, IITZ-01, enhanced In this study, we demonstrated that lysosomotropic autophagy inhibitor, IITZ-01, enhanced TRAIL-mediated apoptosis in cancer cells, but not in normal cells. We found that stabilization TRAIL-mediated apoptosis in cancer cells, but not in normal cells. We found that stabilization of DR5 of DR5 and degradation of survivin by IITZ-01 play a critical role in TRAIL-mediated apoptosis. and degradation of survivin by IITZ-01 play a critical role in TRAIL-mediated apoptosis. Downregulation of Cbl E3 ligase is associated with DR5 upregulation in IITZ-01 treated cells. In addition, Downregulation of Cbl E3 ligase is associated with DR5 upregulation in IITZ-01 treated cells. In IITZ-01 decreased survivin stability via downregulation of USP9X expression. Therefore, we suggested addition, IITZ-01 decreased survivin stability via downregulation of USP9X expression. Therefore, that IITZ-01 could enhance TRAIL-induced apoptosis via modulation of DR5 and survivin expression we suggested that IITZ-01 could enhance TRAIL-induced apoptosis via modulation of DR5 and (Figure8). survivin expression (Figure 8). IITZ-01, a novel potent lysosomotropic autophagy inhibitor, induced vacuolated appearance of cells due to deacidify lysosomes. IITZ-01 showed more than 10-fold potent autophagy inhibition compare with chloroquine [16]. As shown in Figure1b, IITZ-01 also induced vacuolation in renal carcinoma Caki-1 cells. Previous study reported about the mechanism of IITZ-01 decreases IAP family protein involving cIAP1, XIAP and survivin in triple-negative breast cancer cells [16]. However, in our study, survivin was downregulated by IITZ-01, whereas expression of other IAP family protein (cIAP1, cIAP2 and XIAP) was not changed (Figure2). This contradiction is believed to be due to the di fference between the cell line used and the concentration of the IITZ-01. DRs expression is critical for TRAIL-mediated apoptosis [29]. IITZ-01 increased DR5 protein level not DR4 (Figure2). We reported the anti-cancer e ffect of lysosomotropic compounds (CQ, Monensin, and Nigericin) in renal carcinoma Caki-1 cells [23]. Other lysosomotropic compounds also increased DR5 expression. CQ (30 µM) induced both mRNA and protein of DR5 expression, while monensin, and nigericin only increased stability of DR5 protein. Interestingly, all drugs decreased Cbl expression.

Cancers 2020, 12, 2363 10 of 15

Although Cbl induces proteasome-dependent degradation via ubiquitination of target proteins, Cbl also could modulate lysosomal degradation of target proteins in a ubiquitin-dependent or -independent manner. For examples, Cbl mediates lysosomal degradation of K63-linked polyubiquitinating gp130 in interleukin 6-treated cells, and mono-ubiquitinating Notch1 in skeletal myoblast [30]. Cbl regulates lysosomal degradation of cystic fibrosis transmembrane conductance regulator in an ubiquitin-independent manner [31]. Furthermore, Cbl negatively regulates -mediated signaling activation via lysosome-dependent degradation. After ligand engagement, internalized T cell receptor (TCR) is degraded by Cbl in lysosomes [32], and ligand binding EphB1 receptor is also Cbl-dependently degraded in lysosome [33]. In addition, Cbl negatively modulates activation of protein tyrosine kinase through the lysosomal pathway [26,27]. Although proteasome inhibitors upregulate DR5 expression [34], lysosome is also important for modulation of DR5 proteins expression [35]. In our study, since IITZ-01 induced DR5 expression in a Cbl-dependent manner (Figure4c) and IITZ-01 is a Cancerslysosomotropic 2020, 12, x agent, it is a possibility that IITZ-01 inhibits lysosomal degradation of DR5. 10 of 15

FigureFigure 8. 8. TheThe diagram diagram indicating indicating the the mechanism mechanism of of IITZ IITZ-01-induced-01-induced TRAIL TRAIL sensitization. sensitization.

IITZSurvivin-01, a containsnovel potent only lysosomotropic a single BIR domainautophagy and inhibitor, is one of induced the inhibitor vacuolated of apoptosis appearance (IAP) of cellsfamily due proteins. to deacidify Upregulation lysosomes. of IITZ survivin-01 showed protein more inhibit than apoptosis 10-fold both potent in autophagy a caspase-dependent inhibition compareand -independent with chloroquine way by interacting[16]. As shown with XIAPin Figure and mitochondrial1b, IITZ-01 also apoptosis-inducing induced vacuolation factor in (AIF),renal carcinomarespectively Caki [36-,371 cells.]. Therefore, Previous downregulation study reported of about survivin the protein mechanism expression of IITZ is- targeted01 decrease to induces IAP familycancer cell protein death. involving Here, we cIAP1,identified XIAP that andIITZ-01 survivin induced in survivin triple-negative expression breast via protein cancer stabilization cells [16]. However,(Figure5a,b). in our Degradation study, survivin of survivin was downregulated is mainly mediated by IITZ by-01, proteasome whereas expression via ubiquitination of other [IAP38]. familyE3 ligase protein XIAP (cIAP1, regulates cIAP2 the degradationand XIAP) w ofas survivin not changed by ubiquitin-proteasome (Figure 2). This contradiction pathway [ 39is believed]. However, to beIITZ-01 due to did the not difference affect on between expression the levelscell line of used XIAP and (Figure the 2concentration). Deubiquitinating of the IITZ enzymes-01. (DUBs) can preventDRs protein expression degradation is critical by for removing TRAIL- ubiquitinmediated fromapoptosis target [29]. substrate IITZ- [0140 ].increased Previously, DR5Chen protein et al. levelreported not thatDR4 USP9X (Figure is 2). involved We repor inted deubiquitination the anti-cancer and effect degradation of lysosomotropic of survivin compounds by inhibition (CQ, of Monensin,long noncoding and Nigericin) RNA LNC473 in renal [41 carcinoma]. Furthermore, Caki-1 cells we reported[23]. Other that lysosomotropic STAM-binding compounds protein-like also 1 increased(STAMBPL1) DR5 directly expression. interacts CQ (30 and μM) deubiquitinates induced both survivin mRNA expression and protein [42 of]. DR5 However, expression, IITZ-01 while only monendecreasedsin, USP9Xand nigericin expression only and increased ectopic stability expression of DR5 of USP9X protein. prevented Interestingly, downregulation all drugs ofdecreased survivin Cblin IITZ-01 expression. treated Although cells (Figure Cbl 6 inducesa,c). However, proteasome it need-dependent to further degradation studies to identify via ubiquitination the molecular of targetmechanism proteins, of IITZ-01-induced Cbl also coulddownregulation modulate lysosomal of USP9X. degradation Moreover, of we tar foundget proteins IITZ-01 in downregulated a ubiquitin- dependentUSP9X through or -independent post-translation manner. level (Figure For examples,6b,c). Cbl mediates lysosomal degradation of K63- linked polyubiquitinating gp130 in interleukin 6-treated cells, and mono-ubiquitinating Notch1 in skeletal myoblast [30]. Cbl regulates lysosomal degradation of cystic fibrosis transmembrane conductance regulator in an ubiquitin-independent manner [31]. Furthermore, Cbl negatively regulates receptor-mediated signaling activation via lysosome-dependent degradation. After ligand engagement, internalized T cell receptor (TCR) is degraded by Cbl in lysosomes [32], and ligand binding EphB1 receptor is also Cbl-dependently degraded in lysosome [33]. In addition, Cbl negatively modulates activation of protein tyrosine kinase through the lysosomal pathway [26,27]. Although proteasome inhibitors upregulate DR5 expression [34], lysosome is also important for modulation of DR5 proteins expression [35]. In our study, since IITZ-01 induced DR5 expression in a Cbl-dependent manner (Figure 4c) and IITZ-01 is a lysosomotropic agent, it is a possibility that IITZ- 01 inhibits lysosomal degradation of DR5. Survivin contains only a single BIR domain and is one of the inhibitor of apoptosis (IAP) family proteins. Upregulation of survivin protein inhibit apoptosis both in a caspase-dependent and - independent way by interacting with XIAP and mitochondrial apoptosis-inducing factor (AIF), respectively [36,37]. Therefore, downregulation of survivin protein expression is targeted to induce cancer cell death. Here, we identified that IITZ-01 induced survivin expression via protein stabilization (Figure 5a,b). Degradation of survivin is mainly mediated by proteasome via

Cancers 2020, 12, 2363 11 of 15

Taken all together, we suggest that ubiquitin-proteasome pathway-mediated DR5 upregulation and survivin downregulation have a critical role in lysosomotropic autophagy inhibitor (IITZ-01)-mediated sensitization of cancer cells to TRAIL-induced apoptosis.

4. Materials and Methods

4.1. Cell Cultures and Materials Human renal carcinoma (Caki-1, ACHN and A498), human lung cancer (A549) and human breast cancer (MCF7) were procured from American Type Culture Collection (Manassas, VA, USA). Lonza (Basel, Switzerland) provided human mesangial cells, and Korea Cell Line Bank (Seoul, Korea) provided normal human skin fibroblasts cells. These cells were cultured in appropriate medium containing 10% fetal bovine serum, 1% penicillin/streptomycin and 100 µg/mL gentamicin at 37 ◦C in a humidified atmosphere with 5% CO2. IITZ-01 was purchased from Selleckchem (Houston, TX, USA). Human recombinant TRAIL and zVAD-fmk were provided by R&D system (Minneapolis, MN, USA). MG132 and lactacystin were supplied from Calbiochem (San Diego, CA, USA) and Enzo Life Sciences (Ann Arbor, MI, USA), respectively. Cycloheximide was provided from Sigma Chemical Co. (St. Louis, MO, USA). The primary antibodies were obtained as follows: anti-PARP, anti-cleaved caspase-3, anti-Bcl-xL, anti-DR5 from Cell Signaling Technology (Beverly, MA, USA). Anti-Bim, anti-Bax and anti-XIAP from BD Biosciences (San Jose, CA, USA). Anti-Mcl-1, anti-Bcl-2, anti-cIAP2, anti-Cbl from Santa Cruz Biotechnology (St. Louis, MO, USA). Anti-survivin from R&D system (Minneapolis, MN, USA). Anti-cIAP1 and anti-DR4 from Abcam (Cambridge, MA, USA), Anti-USP9X from Abnova (Taipei City, Taiwan). Anti-caspase-3 and anti-c-FLIP from Enzo Life Sciences (San Diego, CA, USA). Anti-actin from Sigma Chemical Co. (St. Louis, MO, USA). pCMV-Myc-Cbl plasmid was a gift from Dr. S. J. Kim (CHA University, Korea), and USP9X wild-type and USP9X/C1566S plasmids were a gift from Dr. D. S. Lim (KAIST, Korea).

4.2. Flow Cytometry Analysis

To analyze apoptosis, cells were harvested and fixed with 95% ethanol at least 1 h at 4 ◦C. Furthermore, cells were incubated in 1.12% sodium citrate buffer containing RNase at 37 ◦C for 30 min, added to 50 µg/mL propidium iodide, and analyzed using BD Accuri™ C6 flow cytometer (BD Biosciences, San Jose, CA, USA) [43].

4.3. Western Blotting Cells have been lysed in RIPA lysis buffer (20 mM HEPES and 0.5% Triton X-100, pH 7.6) and supernatant fraction were collected. Proteins were separated by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) and transferred to the nitrocellulose membranes (GE Healthcare Life Science, Pittsburgh, PO, USA). Incubated with specific antibody and bands were detected using Immobilon Western Chemiluminescent horseradish peroxidase (HRP) Substrate (EMD Millipore, Darmstadt, Germany).

4.4. Annexin V and 7-AAD Staining FITC-conjugated Annexin V and 7-aminoactinomycin D (7-AAD) (BD Pharmingen, San Jose, CA, USA) were used to estimate cell death mode. Cells were washed in cold PBS and resuspended in Annexin V-binding buffer. We added Annexin V-FITC and 7-AAD into the suspended cells, and then incubated for 15 min at room temperature in the dark. The cell death population was detected by flu BD Accuri™ C6 flow cytometer (BD Biosciences, San Jose, CA, USA).

4.5. DAPI, DNA Fragmentation Assay and Caspase Activity Assay

To investigate the nuclei condensation, cellular nuclei cells were stained with 300 nM 40, 60-diamidino-2-phenylindole solution (Roche, Mannheim, Germany), and we viewed fluorescence Cancers 2020, 12, 2363 12 of 15 images using fluorescence microscopy (Carl Zeiss, Jena, Germany) [44]. To analyze DNA fragmentation, we used death detection ELISA plus kit (Boehringer Mannheim, Indianapolis, IN, USA) according to the manufacturer’s recommendations. To measure DEVDase activity, cells were treated with IITZ-01 and/or TRAIL, harvested, and incubated with reaction buffer containing acetyl-Asp-Glu-Val-Asp p-nitroanilide (Ac-DEVD-pNA) substrate, as previously mentioned [45].

4.6. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Quantitative PCR (qPCR) To isolate the total RNA, we used TriZol reagent (Life Technologies, Gaithersburg, MD, USA), and obtained cDNA using M-MLV reverse transcriptase (Gibco-BRL, Gaithersburg, MD, USA). For PCR, we used Blend Taq DNA polymerase (Toyobo, Osaka, Japan) with primers targeting DR5, Cbl, survivin and actin as mentioned in our previous studies [46,47]. For qPCR we utilize SYBR Fast qPCR Mix (Takara Bio Inc., Shiga, Japan) and reactions were performed on Thermal Cycler Dice® Real Time System III (Takara Bio Inc., Shiga, Japan). The following primers were used for the amplification of DR5 and actin as described as our previous study [23]. We used actin as a reference Gene to calculate the threshold cycle number (Ct) of DR5 gene and reported the delta-delta Ct values of the genes.

4.7. Luciferase Activity Assay The DR5 (-605) or DR5 (SacI) promoter-constructs transfected into the cells using Lipofectamine™2000 (Invitrogen, Carlsbad, CA, USA). Furthermore, cells were collected and harvested in lysis buffer (25 mM Tris-phosphate pH 7.8, 2 mM EDTA, 1% Triton X-100, and 10% glycerol). The supernatants were used to measure the luciferase activity according to the manufacturer’s instructions (Promega, Madison, WI, USA).

4.8. Transfection For developing stable cell lines, Caki-1 cells were transfected with the control plasmid pcDNA 3.1(+) vector or pcDNA 3.1(+)/survivin-flag plasmids using Lipofectamine™2000 (Invitrogen, Carlsbad, CA, USA). After 24 h, cells were picked by 700 µg/mL G418 (Invitrogen, Carlsbad, CA, USA). For knockdown of gene, Caki-1 cells were transfected with the control siRNA (Bioneer, Daejeon, Korea), DR5 siRNA (Invitrogen, Carlsbad, CA, USA) or Cbl siRNA (Santa Cruz Biotechnology, St. Louis, MO, USA) using Lipofectamine® RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA). Furthermore, protein expressions were checked by western blotting.

4.9. Immunoprecipitation Assay Cells were collected, washed with PBS, lysed with RIPA lysis buffer containing 10 mM nethylmaleimide (NEM) (EMD Millipore, Darmstadt, Germany) and 1 mM PMSF, and then sonicated for protein extraction in ice. After sonication, cell lysates were centrifuged at 13,000 g for 15 min at × 4 ◦C. The supernatants were incubated with 1 µg of anti-USP9X or anti-survivin antibody overnight at 4 ◦C, and then attached to 20 µL of Protein G agarose bead using the rotator at 4 ◦C for 2 h. Cell lysates were washed with RIPA lysis buffer containing 10 mM NEM and 1 mM PMSF, and boiled in 2 sample × buffer for 10 min. Protein–protein interactions were checked by Western blotting.

4.10. Ubiquitination Assay The assay was performed as described in our previous study [45]. Cells were co-transfected with HA-tagged ubiquitin (HA-Ub), USP9X/wild type and UPS9X/C1566S plasmid, and treated with MG132 for 12 h. Immunoprecipitation was performed using the anti-survivin, and ubiquitination of endogenous survivin was checked using HRP-conjugated anti-Ub under denaturing conditions. Cancers 2020, 12, 2363 13 of 15

4.11. Statistical Analysis The data were analyzed using a one-way ANOVA and post-hoc comparisons (Student-Newman-Keuls) using the Statistical Package for Social Sciences 22.0 software (SPSS Inc.; Chicago, IL, USA).

5. Conclusions Our study suggests that IITZ-01, a lysosomotropic agent, sensitizes TRAIL-induced apoptosis via DR5 upregulation and survivin downregulation in cancer cells, but not normal cells. In mechanisms, IITZ-01 induces DR5 stabilization by downregulation of Cbl and USP9X-dependent survivin ubiquitination and degradation.

Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6694/12/9/2363/s1, Figure S1: Analysis of expression and prognostic significance of survivin (BIRC5), DR5 (TNFRSF10B), USP9X and Cbl in renal clear cell carcinoma. Results were generated using the UCSC Xena browser based on data in TCGA. Figure S2. Uncropped western blots for Figure1. Figure S3 Uncropped western blots for Figure2. Figure S4. Uncropped western blots for Figure3. Figure S5. Uncropped western blots for Figure4. Figure S6. Uncropped western blots for Figure5. Figure S7. Uncropped western blots for Figure6. Figure S8. Uncropped western blots for Figure7. Author Contributions: Conceptualization, T.K.K., K.-j.M. and S.M.W.; investigation, S.A.S. and S.U.S.; data curation, P.K.,D.S.M., J.-S.C. and D.E.K.; writing—original draft preparation, T.K.K., S.M.W. and S.A.S.; supervision, T.K.K.; funding acquisition, T.K.K. All authors have read and agreed to the published version of the manuscript. Funding: This work was supported by an NRF grant funded by the Korea Government (MSIP) (2014R1A5A2010008, NRF-2018R1D1A3B07049596 and NRF-2019R1A2C2005921). Conflicts of Interest: The authors declare that they have no conflict of interest.

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