(PDF) Increased lanosterol turnover: a metabolic buRDen for daunorubicin-resistant leukemia cells ORIGINAL PAPERIncreased lanosterol turnover: a metabolic burdenfor daunorubicin-resistant leukemia cellsClaudia Sta¨ubert1,2,5•Rosanna Krakowsky5•Hasanuzzaman Bhuiyan3•Barbara Witek1•Anna Lindahl4•Oliver Broom1•Anders Nordstro¨m1,2,4Received: 30 November 2015 / Accepted: 10 December 2015 / Published online: 23 December 2015ÓThe Author(s) 2015. This article is published with open access at Springerlink.comAbstract The cholesterol metabolism is essential forcancer cell proliferation. We found the expression of genesinvolved in the cholesterol biosynthesis pathway up-regu-lated in the daunorubicin-resistant leukemia cell line CEM/R2, which is a daughter cell line to the leukemia cell lineCCRF-CEM (CEM). Cellular2H2O labelling, mass spec-trometry, and isotopomer analysis revealed an increase inlanosterol synthesis which was not accompanied by anincrease in cholesterol flux or pool size in CEM/R2 cells.Exogenous addition of lanosterol had a negative effect onCEM/R2 and a positive effect on sensitive CEM cell via-bility. Treatment of CEM and CEM/R2 cells with choles-terol biosynthesis inhibitors acting on the enzymessqualene epoxidase and lanosterol synthase, both alsoinvolved in the 24,25-epoxycholesterol shunt pathway,revealed a connection of this pathway to lanosterol turn-over. Our data highlight that an increased lanosterol fluxposes a metabolic weakness of resistant cells that poten-tially could be therapeutically exploited.Keywords Leukemia Drug resistance Cholesterolbiosynthesis LC–MS Stable isotope labelling massspectrometry CancerAbbreviationsALL Acute lymphoblastic leukemiaCoA Coenzyme ADNR DaunorubicinFBS Fetal bovine serumLC–MS Liquid chromatography–mass spectrometryP-gp P-glycoproteinIntroductionCholesterol is an essential component of mammalian cellmembranes and serves as a precursor for bile acids andvarious endocrine steroid hormones. The biosynthesis,cellular absorption, and efflux of cholesterol are tightlyregulated to maintain homeostatic levels required for nor-mal cell proliferation. A link between cholesterol andcancer was proposed over a century ago, with the discoverythat tumor cells had accumulated cholesterol [1]. Sincethen, many studies have provided evidence for a linkbetween carcinogenesis/tumor progression and cholesterolbiosynthesis and efflux [2]. Elevated activity of hydrox-ymethylglutaryl-coenzyme A reductase (HMGCR), the firstenzyme of the mevalonate pathway, has been shown in arange of different tumors including hepatocellular carci-noma [3], leukemia [4] and lymphoma [5]. Moreover,Electronic supplementary material The online version of thisarticle (doi:10.1007/s12032-015-0717-5) contains supplementarymaterial, which is available to authorized users. Anders Nordstro¨manders.nordstrom@umu.se1Department of Molecular Biology, Umea˚University,90187 Umea˚, Sweden2Department of Forest Genetics and Plant Physiology,Swedish Metabolomics Centre, Swedish University ofAgricultural Sciences, Umea˚, Sweden3Doping Laboratory, Department of Clinical Pharmacology,Karolinska University Hospital, Stockholm, Sweden4Department of Oncology-Pathology, Science for LifeLaboratory, Karolinska Institutet, Stockholm, Sweden5Institute of Biochemistry, Faculty of Medicine, University ofLeipzig, Leipzig, Germany123Med Oncol (2016) 33:6DOI 10.1007/s12032-015-0717-5Content courtesy of Springer Nature, terms of use apply. Rights reserved. inhibition of HMGCR, the initial and rate-limiting step ofcholesterol biosynthesis, with statins inhibits tumor growthin mouse xenograft models [6–8]. Epidemiological datafurther support a role for statins in reducing the risk ofdeveloping pancreatic cancer [9] and with an increasedprogression-free survival in inflammatory breast cancer[10].Cancer is a clonal disease whereby therapeutic inter-vention poses a selective pressure resulting in cancer cellsescaping therapy. Surviving cells are characterized by drugresistance and are often associated with disease relapse[11]. Several reports support a function of cholesterol inestablishing and maintaining increased drug tolerance incancer cells. Cholesterol has been found to be increased by50 % in isolated plasma membranes of vinblastine-resistantversus sensitive acute lymphoblastic leukemia cells (ALL)[12]. The observation that drug-resistant myeloid leukemiacell lines are more sensitive to statins than their sensitiveparental lines further substantiates a role for cholesterol inchemoresistance [13]. Moreover, in vitro treatment of acutemyeloid leukemia (AML) cells with chemo- or radiother-apy causes increased intracellular cholesterol levelsaccompanied by an increased drug tolerance, whereasinhibition of cholesterol biosynthesis with statins couldrestore drug sensitivity [14]. Further, it has been shown thatrat and human hepatocellular carcinoma cells displayincreased mitochondrial cholesterol levels and HMGCR orsqualene synthase (FDFT1) inhibition sensitizes those cellsto mitochondria-directed chemotherapy [15]. Similarly, ina doxorubicin-resistant bladder cancer cell line, simulta-neous administration of statin with doxorubicin revertedthe resistant phenotype [16].We have recently shown that resistance to daunorubicin(DNR) in an ALL cell line is associated with a rewiredmetabolism [17]. RNA Sequencing revealed the cholesterolbiosynthetic pathway as the top canonical pathway up-regulated in the resistant cells [17].In the present study, we validate these previous findingsusing quantitative real-time PCR (RT-qPCR) and measurerelative quantity and synthesis rates of cholesterol itselfand lanosterol, the first committed intermediate in choles-terol biosynthesis, by application of2H2O labelling andmass spectrometry isotopomer analysis. We found that thetranscriptional up-regulation of the cholesterol biosynthesispathway does not translate into an increased cholesterolsynthesis rate or quantity in the resistant cells, but rather anincreased flux through the lanosterol pool. With this reportwe shift the focus from the importance of solely cholesterolfor cancer progression and drug sensitivity to the upstreambiosynthetic intermediate lanosterol. Our data reveal apreviously unrecognized metabolic cost of cancer drugresistance and point toward a potential novel regulatoryrole of lanosterol in maintaining cholesterol homeostasis,which may be particularly critical for drug-resistant leu-kemia cancer cells.Materials and methodsCell lines and growth conditionsCCRF-CEM [CCRF CEM] (ATCCÒCCL-119TM) (CEM)leukemia cells were acquired through LGC Standards(Teddington, UK) from the American Type Culture Col-lection and maintained following the recommendationsfrom ATCC. Detailed description of the generation of theDNR-resistant CEM/R2 is described in [17].Proliferation assays: ATPliteTM(Perkin Elmer)Cells were seeded in black plates with a final density of15,000 cells/well. Simultaneously, the respective treatmentwas started. ATPliteTMwas performed following themanufacturer’s instructions. Cells were incubated for 48 hwith or without cholesterol biosynthesis inhibitors, namely,atorvastatin (100 lM), terbinafine (25 lM), ketoconazole(20 lM), triparanol (2 lM), CI976 (25 lM), all purchasedfrom Sigma-Aldrich (MO, USA), and hymeglusin(10 lM), YM-53601 (10 lM) and BIBB-515 (25 lM) allordered from Santa Cruz Biotechnology (TX, USA) in theabsence (vehicle control DMSO or MeOH) or presence ofDNR (CEM, 1 nM and CEM/R2, 0.5 lM) in RPMI 1640(HyClone, Fisher scientific) supplemented with 10 % FBS.Lanosterol, cholesterol, and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (PC), all obtained from Sigma-Aldrich(MO, USA), were dissolved in chloroform/methanol (1:1).Cholesterol or lanosterol was mixed in equimolar propor-tion with PC and dried by vacuum in a speed vacuumconcentrator. The lanosterol/PC, cholesterol/PC mixture, orPC alone was re-suspended in serum-free RPMI 1640 onthe day of the experiment and used within the day ofpreparation.To analyze the effect of lanosterol and cholesterol, cellswere incubated in serum-free RPMI 1640 medium(HyClone, Fisher scientific) for 48 h in the absence orpresence of DNR (CEM, 100 nM and CEM/R2, 1 lM).The negative/vehicle control always contained respectiveamounts of DMSO, MeOH, or PC.RNA isolation, reverse transcription,and quantitative real-time PCROne million cells of each CEM and CEM/R2 cells wereseeded in a 6-w plate and cultured for 24 h in RPMI sup-plemented with 10 % FBS without or with 50 lM ator-vastatin, 12.5 lM terbinafine, 12.5 lM BIBB515, or6Page 2 of 10 Med Oncol (2016) 33:6123Content courtesy of Springer Nature, terms of use apply. Rights reserved. 10 lM ketoconazole before harvested for RNA prepara-tion. Total RNA isolation was performed using RNeasyÒMini Kit (Qiagen, Germany) following manufacturer’sinstructions. RNA (1 lg) was treated with DnaseI (NEB)prior reverse transcription using iScriptTMcDNA SynthesisKit (Bio-Rad) following the manufacturer’s instructions.The qPCR was set up using iTaqTMUniversal SYBRÒGreen Supermix (Bio-Rad), and real-time PCR and datacollection were performed on Bio-RadÒiQTM5 Real-TimePCR Detection System. Primer design procedure anddetailed description of each step can be found in [18]. AllqPCR primer pairs are stated in Table S1. Expression ofgene-encoding proteins involved in cholesterol biosynthe-sis was normalized to the reference genes RPL13A, RPS18,ACTB, and GAPDH.Liquid chromatography–mass spectrometry (LC–MS) measurementsFor experiments with2H2O, 1 9106cells were culturedfor 4 h (without or with 50 lM atorvastatin, 12.5 lMterbinafine, 12.5 lM BIBB515 or 10 lM ketoconazole) or24 h (untreated comparison of lanosterol and cholesterolturnover in CEM versus CEM/R2), in RPMI 1640 med-ium supplemented with 10 % FBS which was eitherdiluted with sterile H2Oor2H2O (99 %) to a final con-centration of 30 %2H2O (Cambridge Isotope Laboratories(MA, USA)).The cells were centrifuged, washed at least once withPBS, transferred to an 1.5-mL Eppendorf tube and lysed/extracted using 200 lL 50:50 chloroform/methanol towhich a small lab spoon of 0.2 lm i.d. glass beads wasadded (Retsch). Tubes were placed in a Retsch BeadmillMM 400 and shaken at 30 Hz for 2 min. Eppendorf tubeswere transferred to a centrifuge kept at 4 °C and spun at14,000 rpm for 10 min after which the supernatant wastransferred to LC–MS glass vials, dried down in a speedvacuum concentrator, and stored at -20 °C until analysis.Samples were dissolved in 20 lL chloroform out of which2–4 lL were injected into the Agilent 1290 LC systemconnected to either a 6540 or 6550 Agilent Q-TOF massspectrometer (CA, USA) and an atmospheric pressureionization (APCI) source was used. Data were collectedbetween m/z 70 and 1700 in positive ion mode only. Thefollowing APCI settings were used: gas temperature200 °C, vaporizer 350 °C, gas flow 11 l/min, nebulizerpressure 40 psig, Vcap 3500, corona 4, fragmentor 100,Skimmer1 45, and OctapoleRFPeak 750. All samples wereseparated using reverse phase only, Kinetex C18,100 mm 92.1 mm, 2.6 lM 100 A˚, Phenomenex (CA,USA). For elution, solvents reversed phase (A) H2O, 0.1 %formic acid (B) 75:25 methanol/isopropanol, 0.1 % formicacid were used. All solvents were of HPLC grade. Lineargradients were used for all separations and were devised asfollows for reversed phase separation (0.5 mL/min) min 0:5 %B, min 8: 95 %B, min 10: 95 %B, min 10.2: 5 %B,min 12: 5 %B. Raw data were processed and analyzedusing MassHunter Qual, Agilent (CA, USA). Identificationof metabolites in all experiments was carried out usingsynthetic standards obtained from Sigma-Aldrich (MO,USA) and Inventia Pty. Ltd (NSW, Australia) comparingaccurate mass, retention time, and in some cases MS/MSspectra.ResultsThe cholesterol biosynthetic pathway is up-regulated in DNR-resistant CEM/R2 cellsLevels of gene expression for all cholesterol biosyntheticgenes in CEM and CEM/R2 were monitored using RT-qPCR (Fig. 1a, b). Interestingly, mRNA levels of HMGCRare significantly lower in CEM/R2 cells (Fig. 1b), whereasmRNA levels of all other enzymes involved in cholesterolbiosynthesis except five are significantly up-regulated(Fig. 1b). The highest fold change in mRNA expressionwas obtained for CYP51A1 (cytochrome P450, family 51,subfamily A, polypeptide 1) and ABCA1 with approxi-mately fivefold higher mRNA levels in CEM/R2 whencompared to CEM cells followed by squalene epoxidase(SQLE) with approximately threefold higher mRNA levelsin CEM/R2 cells (Fig. 1b).Increased flux through the lanosterolbut not the cholesterol pool in resistant CEM/R2cellsNext, we measured relative quantity of lanosterol, the firstcholesterol biosynthesis intermediate committed solely tothe cholesterol pathway, and cholesterol using liquidchromatography–mass spectrometry (LC–MS). Surpris-ingly, we found that in spite of a transcriptionally up-reg-ulated cholesterol pathway in CEM/R2 cells, the relativeconcentration of lanosterol and cholesterol was lower inCEM/R2 cells (Fig. 2a). This observation led us to analyzewhether the lower relative quantity of lanosterol andcholesterol can be explained by lower synthesis rate inCEM/R2 cells. Thus, we set out to measure de novo syn-thesis of cholesterol itself and lanosterol using2H2Olabelling and LC–MS [19]. By growing cells in mediadiluted with2H2O, the stable isotope2H will be incorpo-rated throughout the cellular metabolism which can befollowed in the individual metabolites by mass spectrom-etry and isotopomer analysis, illustrated in Fig. 2b. Virtu-ally, no de novo formation of cholesterol could be observedMed Oncol (2016) 33:6 Page 3 of 10 6123Content courtesy of Springer Nature, terms of use apply. Rights reserved. (Fig. 2b, c). Lanosterol on the other hand which displayeda lower relative concentration in CEM/R2 cells comparedto CEM (Fig. 2a) exhibited at the same time a higher rel-ative2H incorporation in CEM/R2 cells, suggesting ahigher flux through the lanosterol pool in the resistant cells(Fig. 2c).Exogenous addition of lanosterol is beneficialfor drug-sensitive CEM but disadvantageousfor resistant CEM/R2 cellsWith no apparent transfer of the2H label from lanosterol tocholesterol (Fig. 2b), it is reasonable to assume that inFig. 1 Cholesterol biosynthesis pathway is altered in CEM/R2 cells.aOutlined cholesterol synthesis pathway in which all genes withincreased mRNA expression are highlighted in green and HMGCR asits mRNA expression level is lower in CEM/R2 cells is highlighted inred. Major points of inhibition by cholesterol biosynthesis inhibitorsare highlighted in red. bmRNA expression levels of all genesinvolved in the cholesterol biosynthetic pathway is shown as log2 foldover relative mRNA expression for each gene in CEM cells.Significance was assessed using a two-tailed unpaired ttest. Dataare shown as mean ±SEM of six independent experiments.*PB0.05; **PB0.01; ***PB0.0016Page 4 of 10 Med Oncol (2016) 33:6123Content courtesy of Springer Nature, terms of use apply. Rights reserved. CEM/R2 cells the increased lanosterol production reflectsthat lanosterol, rather than just being an intermediate inthe cholesterol biosynthesis either is exported out of thecells or fills another function. Thus, to probe whetherincreased lanosterol flux is essential to maintain resistanceor rather a metabolic consequence of resistance, weinvestigated the influence of exogenous addition oflanosterol and cholesterol on CEM and CEM/R2 cellviability (Fig. 2d). Lanosterol addition was beneficial forCEM but disadvantageous for CEM/R2 cell viability(Fig. 2d). Cholesterol had no apparent effect on CEMcells and was slightly disadvantageous for CEM/R2 cells(Fig. 2d). Next, we evaluated the pro-survival effect oflanosterol and cholesterol on DNR sensitivity of CEMand CEM/R2 cells (Fig. 2e, f). Presence of both lanosteroland cholesterol decreased the sensitivity of CEM cells toDNR (Fig. 2e), thus supporting a pro-survival effect oflanosterol for cancer cells. However, in CEM/R2 cells,exogenous lanosterol addition did not trigger such aneffect and cholesterol addition decreased sensitivity toDNR only slightly (Fig. 2f). We conclude therefore thatthe increased lanosterol flux represents a metabolic costrather than a survival advantage for the resistant CEM/R2cells.Fig. 2 Resistant leukemia cells CEM/R2 exhibit an increased fluxthrough the lanosterol but not the cholesterol pool and are negativelyaffected by exogenous lanosterol addition. aRelative concentration oflanosterol and cholesterol in CEM versus CEM/R2 cells as deter-mined by LC–MS. bMass spectra of lanosterol (left) and cholesterol(right) after cell growth for 24 h using regular media (top) and mediawith addition of 30 %2H2O(bottom). cData showing de novosynthesis of lanosterol and cholesterol measured on cells grown in30 %2H2O. a–cData of a single experiment carried out in fivereplicates are shown as minimum to maximum with line at mean.dViability of CEM and CEM/R2 cells (n=4) that were grown for48 h in serum-free RPMI 1640 in presence of 50 lM 1,2-dimyristoyl-sn-glycero-3-phosphocholine (PC), lanosterol/PC mixture (each25 lM), or cholesterol/PC mixture (each 25 lM). Viability of CEM(e) and CEM/R2 (f) cells that were incubated for 48 h in absence orpresence of DNR (CEM, 100 nM and CEM/R2, 1 lM) and 50 pM PC,lanosterol/PC mixture (each 25 lM), or cholesterol/PC mixture (each25 lM). d–fData are shown as mean ±SEM of four independentexperiments carried out in triplicate, Pvalues were determined usingan ordinary one-way ANOVA with Dunnett’s multiple comparisonstest.#B0.1; *PB0.05; **PB0.0110Med Oncol (2016) 33:6 Page 5 of 10 6123Content courtesy of Springer Nature, terms of use apply. Rights reserved. Differential sensitivity of CEM versus CEM/R2 cellstoward cholesterol biosynthesis inhibitorsTo gain further insights into the differences of the choles-terol biosynthetic pathway between sensitive and resistantleukemia cells, with special focus on both rate-limiting stepsand steps producing or consuming lanosterol, we evaluatedthe potential of different cholesterol biosynthesis inhibitorsas both cytostatic agents (Fig. 3a) and positive modulatorsof drug sensitivity (Figure S1) in CEM and CEM/R2 cells.If cholesterol lowering per se has an anticancereffect, the transcriptional up-regulation of the cholesterolbiosynthesis pathway in CEM/R2 cells points toward anincreased relevance of this pathway in resistant cells.Therefore, one would expect that all inhibitors of choles-terol biosynthesis, administered alone, would have astronger effect on CEM/R2 cell viability. This did not turnout to be the case. However, terbinafine, an antifungalcompound inhibiting SQLE, triparanol, an inhibitor of24-dehydrocholesterol reductase (DHCR24), both shownto suppress tumor growth [20–23], as well as CI976, apotent and selective Acyl-CoA/cholesterol acyltransferase(SOAT1) inhibitor [24,25] did affect CEM/R2 more thanthe sensitive CEM cells (Fig. 2a).Fig. 3 Effect of cholesterol biosynthesis inhibitors on cell viabilityand lanosterol as well as cholesterol relative synthesis rate of CEMand CEM/R2 cells. aCEM and CEM/R2 cells were incubated for48 h with 10 lM hymeglusin, 100 lM atorvastatin, 10 lM YM-53601, 25 lM terbinafine, 25 lM BIBB-515,10 lM ketoconazole,2lM triparanol, or 25 lM CI976 in RPMI 1640 supplemented with10 % FBS in comparison with vehicle control DMSO or MeOH.Hymeglusin, atorvastatin, and ketoconazole had a stronger effect onCEM cells, whereas terbinafine, triparanol, and CI976 were moreeffective on CEM/R2 cells, and no difference in sensitivity betweenCEM and CEM/R2 cells was observed for YM-53601 and BIBB-515.Data are shown as mean ±SEM of three independent experimentscarried out in triplicates. Relative synthesis rate of lanosterol (b) andcholesterol (c) in CEM and CEM/R2 cells as determined by LC–MSafter cells were cultured for 4 h in presence or absence of 50 lMatorvastatin or 12.5 lM terbinafine, 12.5 lM BIBB515 or 10 lMketoconazole, in RPMI 1640 supplemented with 10 % FBS and 30 %2H2O. Data of a single experiment carried out in four replicates areshown as minimum to maximum with line at mean. a–cSignificancewas assessed using a two-tailed unpaired ttest#B0.1; *PB0.05;**PB0.01; ***PB0.0016Page 6 of 10 Med Oncol (2016) 33:6123Content courtesy of Springer Nature, terms of use apply. Rights reserved. Lanosterol de novo synthesis rate correlateswith sensitivity toward cholesterol biosynthesisinhibitorsIf the increased lanosterol flux is a metabolic burden forthe resistant cells, while being associated with increasedviability in sensitive cells, one could argue that cholesterolsynthesis inhibitors inducing a relatively larger reductionin lanosterol flux in resistant compared to sensitive cellsshould result in lower viability of sensitive CEM to CEM/R2 cells or vice versa. To test this hypothesis, we analyzedthe effect on de novo synthesis of lanosterol and choles-terol when exposed to atorvastatin acting at HMGCR, thefirst rate-limiting enzyme, terbinafine, acting at the pro-posed second rate-limiting step SQLE, BIBB-515, whichinhibits lanosterol synthase (LSS), as well as ketoconazole,an inhibitor of the CYP51A1 [26,27] directly downstreamof lanosterol (Fig. 3b, c). All four inhibitors reduced the denovo synthesis rate of lanosterol and cholesterol (except ofatorvastatin in CEM cells) significantly in both cell lineswhen compared to control (Fig. 3b, c). Next, we comparedthe relative degree of reduction of lanosterol de novosynthesis for sensitive versus resistant cells. We observedthat atorvastatin reduced the de novo synthesis through thelanosterol pool more in resistant cells, whereas the oppo-site was true for terbinafine and BIBB515 (Fig. 3b). Nodifference in lanosterol biosynthesis rate comparing CEMand CEM/R2 cells was observed when cells were treatedwith ketoconazole (Fig. 3b). The comparison of the via-bility data obtained for atorvastatin and terbinafine(Fig. 3a) with the lanosterol flux information gained byLC–MS (Fig. 3b) reveals a connection between sensitivityto those inhibitors and their effect on lanosterol de novosynthesis rate.Inhibition of the 24,25-epoxycholesterol shuntpathway suggests its differential regulationin resistant cellsThe resistant cells show an increased flux of lanosterolwhich is disadvantageous for them. It is reasonable toassume that these cells have regulatory mechanisms that aredifferent when compared to sensitive cells. These mecha-nisms can either be the cause of or are required to managethe observed increased lanosterol flux. One such regulatorymechanism of cholesterol biosynthesis is the 24,25-epoxy-cholesterol (24,25-EC) shunt pathway (Figs. 1a, 4a). 24,25-EC has been shown to cause a decreased cholesterol syn-thesis rate by inhibition of HMGCR [28] and DHCR24 [29]and thus prevents accumulation of newly synthesizedcholesterol which could cause ER stress and cell toxicity[30,31]. Since the inhibitors of this shunt pathway, namelyterbinafine inhibiting SQLE and BIBB515 inhibiting LSS,had both reduced de novo synthesis of lanosterol more inCEM/R2 cells, we evaluated their effect on mRNA level ofgenes involved in the cholesterol biosynthetic pathway, incomparison with the effects induced by atorvastatin andketoconazole treatment (Fig. 4b, Figure S2).Both BIBB515 and terbinafine but not atorvastatin andketoconazole induce differential expression of HMGCR,SQLE, and DHCR24 when comparing sensitive andresistant CEM cells (Fig. 4b). Several other genes that areinvolved in the main cholesterol biosynthesis pathwaydisplay the same pattern (Figure S2). The inhibition of the24,25-EC shunt pathway, but not the main cholesterolbiosynthesis pathway, produces an up-regulation of rate-limiting steps of the cholesterol biosynthetic pathwayspecific to resistant cells (Fig. 4b). We are therefore sug-gesting that either the shunt pathway is critical for resistantcells to regulate an increased lanosterol flux or a differ-entially regulated shunt pathway is the cause for theincreased lanosterol flux.DiscussionAn altered cholesterol metabolism is critical for rapidlyproliferating cancer cells and plays a role in developmentof resistance. Based on our findings, we suggest thatlanosterol, the first intermediate committed solely towardcholesterol biosynthesis, is a valuable marker to detectalterations of the cellular cholesterol homeostasis. Weshow that the increased lanosterol flux in a DNR-resistantdaughter cell line of the T-ALL leukemia cell line CEMrepresents a metabolic cost that can potentially have ther-apeutic implications. In a broader sense, our results high-light that phenotyping cancers with respect to cholesterolmetabolism can be useful for therapy guidance.We used RT-qPCR and compared mRNA levels of allproteins involved in the cholesterol biosynthesis pathway(Fig. 1a) between DNR-sensitive CEM cells and theresistant daughter cell line CEM/R2. The almost uniformlyincreased expression in the resistant cells (Fig. 1b) was notmatched by increased levels of cholesterol or lanosterol(Fig. 2a). Using2H2O labelling of cultured cells, we coulddemonstrate an increased biosynthetic flux of lanosterol(Fig. 2b, c) with no concomitant accumulation of lanos-terol or cholesterol (Fig. 2a). It is therefore reasonable toassume that in CEM/R2 cells the increased lanosterolproduction reflects that lanosterol, rather than just being anintermediate in the cholesterol biosynthesis, is exported outof the cell [32–34] or fills another function. Membraneassociated lanosterol will alter plasma membrane organi-zation relative to cholesterol [35], which potentially couldimpact drug tolerance [12]. Further, lanosterol has beenproposed to act as a survival factor for dopaminergicMed Oncol (2016) 33:6 Page 7 of 10 6123Content courtesy of Springer Nature, terms of use apply. Rights reserved. neurons, potentially via a mitochondrial decouplingmechanism [36]. We observed that exogenously appliedlanosterol negatively affected viability of resistant cell buthad a positive effect on cell viability of DNR-sensitiveCEM cells. Moreover, since lanosterol has been describedas a survival factor [36], we tested the effect of lanosterolon DNR sensitivity of both CEM and CEM/R2 by co-ad-ministration of DNR and lanosterol, which revealed thatlanosterol presence decreased DNR sensitivity of CEMcells but had no such effect on CEM/R2 cells. We concludefrom the results from all those experiments that theincreased lanosterol flux is a stressor for the resistant cellsand thus a negative consequence of the resistance. Con-sequently, it is reasonable to hypothesize that resistant cellshave different regulatory mechanisms of the cholesterolbiosynthetic pathway to cope with the increased lanosterolflux. Inhibition of different steps of the cholesterol pathwayrevealed differential effects on viability when comparingsensitive and resistant cells (Fig. 3a). Further, using2H2Olabelling mass spectrometry, we show that inhibitors thatreduce the lanosterol synthesis rate more in the resistantcells affected their viability less upon treatment with therespective inhibitors (Fig. 3b, c).Lanosterol itself has been shown to be involved inposttranslational regulation of the cholesterol biosynthesispathway through induction of proteasomal degradation ofHMGCR [37] and SQLE [38]. The enzyme SQLE, which isinhibited by terbinafine, is further transcriptionally [39,40]and posttranslationally regulated by cholesterol [41,42]and participates, together with LSS, which can be inhibitedby BIBB515, in a shunt of the mevalonate pathway thatproduces 24,25-EC (Figs. 1a, 4a). By measuring geneexpression of all genes in the cholesterol biosynthesis whenapplying one of the inhibitors, atorvastatin, terbinafine,BIBB515, or ketoconazole, a pattern emerged. Inhibitors ofSQLE or LSS, both of which are enzymes involved in themain pathway and in the 24,25-EC shunt pathway, resultedin a reciprocal expression pattern for many genes whencomparing CEM versus CEM/R2. In contrast, atorvastatinand ketoconazole that do not affect the 24,25-EC shuntpathway exert the same effects on mRNA levels ofcholesterol biosynthesis genes in both sensitive and resis-tant cells (Figs. 4b, S2).In conclusion, our data provide a novel connectionbetween drug resistance and increased lanosterol flux andalso links the 24,25-EC shunt pathway with resistance. WeFig. 4 Differentially regulated 24,25-EC shunt pathway in CEM/R2cells potentially cause or effect of increased lanosterol flux.aHMGCR, the first rate-limiting step of cholesterol biosynthesis isnegatively regulated by lanosterol, 24,25-dihydrolanosterol and24,25-EC [50]. SQLE is regulated by lanosterol and cholesterol[41–45] and participates, together with LSS, in a shunt of themevalonate pathway that produces 24,25-EC. 24,25-EC itself inhibitsHMGCR [46] and DHCR24 [47]. bBIBB515 and terbinafine, actingat LSS and SQLE, respectively, induce differential expression ofHMGCR, SQLE, and DHCR between CEM and CEM/R2. Cells werecultured for 24 h in presence or absence of 50 lM atorvastatin,12.5 lM terbinafine, 12.5 lM BIBB515, or 10 lM ketoconazole inRPMI 1640 supplemented with 10 % FBS. mRNA expression levelsare shown as log2 relative mRNA expression fold over vehicle-treatedCEM or CEM/R2 cells, respectively. Data are shown as mean ±SEMof four independent experiments, Pvalues were determined using anunpaired ttest. *PB0.05; **PB0.01; ***PB0.0016Page 8 of 10 Med Oncol (2016) 33:6123Content courtesy of Springer Nature, terms of use apply. Rights reserved. believe that there is a high potential for exploitation of thisknowledge in personalized therapy guidance.Acknowledgments This research was supported by grants from theSwedish Foundation for Strategic Research (to A. N.), the SwedishResearch Council 2007-5143 (to A. N.), the Erling-Persson FamilyFoundation (to Umea˚University), and Jane and Dan Olsson Foun-dation (to A. N.).Compliance with ethical standardsConflict of interest None.Open Access This article is distributed under the terms of theCreative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,distribution, and reproduction in any medium, provided you giveappropriate credit to the original author(s) and the source, provide alink to the Creative Commons license, and indicate if changes weremade.References1. White CP. On the occurrence of crystals in tumours. J PatholBacteriol. 1909;13(1):3–10. doi:10.1002/path.1700130103.2. 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If you would like to use or distribute our Springer Nature journal content to a wider audience or on a regular basis or in any other manner notexpressly permitted by these Terms, please contact Springer Nature at onlineservice@springernature.com Content uploaded by Anders NordströmAuthor contentAll content in this area was uploaded by Anders Nordström on Jan 04, 2016 Content may be subject to copyright.Supplementary resources (2)Supplementary MaterialDataJanuary 2016Claudia Stäubert · Rosanna Krakowsky · Hasanuzzaman Bhuiyan · Barbara Witek · Anders NordströmSupplementary material 1DataDecember 2015Claudia Stäubert · Rosanna Krakowsky · Hasanuzzaman Bhuiyan · Barbara Witek · Anders NordströmCitations (9)References (47)... To date, reports on bioactive compounds against tea pathogenic fungi are limited. Previously, we identified two new macrolactins [10,11] from Bacillus subtilis B5, a bacterium isolated from the 3000 m deep sea sediment of the Pacific Ocean that exhibit antifungal activity against P. theae and C. gloeosporioides. In the present study, Fusarium solani H915, a fungal strain that originated from the mangrove sediment of the Zhangjiangkou Mangrove National Nature Reserve, was found to possess antifungal activity. ...... The EtOAc extract (15 g) was fractionated over a column packed with silica gel (300 g, Yantai Chemical Industry Research Institute, Yantai, China), eluted with petroleum ether-ethyl acetate (v/v) (20:1; 10:1; 5:1; 2:1; 1:1; each 1.0 L) and chloroform-methyl alcohol (v/v) (50:1; 20:1; 10:1; 5;1; 2:1; 0:1; each 1.0 L), to afford 10 fractions (Fr. [1][2][3][4][5][6][7][8][9][10]. Further separation was conducted on the fractions with antifungal activity (Fr. ...... The antifungal activity against tea pathogenic fungi P. theae and C. gloeosporioides was evaluated according to a previously described method [10,11]. Petri plates were used in the test. ...New Bis-Alkenoic Acid Derivatives from a Marine-Derived Fungus Fusarium solani H915ArticleFull-text availableDec 2018MAR DRUGSShun-Zhi LiuXia YanXi-Xiang Tang Ying-Kun QiuFusarium solani H915 is a fungus derived from mangrove sediments. From its ethyl acetate extract, a new alkenoic acid, fusaridioic acid A (1), three new bis-alkenoic acid esters, namely, fusariumester A1 (2), A2 (3) and B (4), together with three known compounds (5–7), were isolated. The structures of the new compounds were comprehensively characterized by high resolution electrospray ionization-mass spectrometry (HR-ESI-MS), 1D and 2D nuclear magnetic resonance (NMR). Additionally, the antifungal activities against tea pathogenic fungi Pestalotiopsis theae and Colletotrichum gloeosporioides were studied. The new compound, 4, containing a β-lactone ring, exhibited moderate inhibitory activity against P. theae, with an MIC of 50 μg/disc. Hymeglusin (6), a typical β-lactone antibiotic and a terpenoid alkaloid, equisetin (7), exhibited potent inhibitory activities against both fungal species. The isolated compounds were evaluated for their effects on zebrafish embryo development. Equisetin clearly imparted toxic effect on zebrafish even at low concentrations. However, none of the alkenoic acid derivatives exhibited significant toxicity to zebrafish eggs, embryos, or larvae. Thus, the β-lactone containing alkenoic acid derivatives from F. solani H915 are low in toxicity and are potent antifungal agents against tea pathogenic fungi.ViewShow abstract... LSS activity increases in the daunorubicin-resistant leukemia cell line (CEM/R2). [74] CYP51A1 CYP51A1 is significantly upregulated in the drug-tolerant (DT) human lung cancer cell lines. The CYP51A1 inhibitor, ketoconazole, shows the synergy in apoptosis induction with tyrosine kinase inhibitors of epidermal growth factor receptor. ...Enzymes in the Cholesterol Synthesis Pathway: Interactomics in the Cancer ContextArticleFull-text availableJul 2021 P. V. Ershov Leonid A. Kaluzhskiy Alexis IvanovA global protein interactome ensures the maintenance of regulatory, signaling and structural processes in cells, but at the same time, aberrations in the repertoire of protein-protein interactions usually cause a disease onset. Many metabolic enzymes catalyze multistage transformation of cholesterol precursors in the cholesterol biosynthesis pathway. Cancer-associated deregulation of these enzymes through various molecular mechanisms results in pathological cholesterol accumulation (its precursors) which can be disease risk factors. This work is aimed at systematization and bioinformatic analysis of the available interactomics data on seventeen enzymes in the cholesterol pathway, encoded by HMGCR, MVK, PMVK, MVD, FDPS, FDFT1, SQLE, LSS, DHCR24, CYP51A1, TM7SF2, MSMO1, NSDHL, HSD17B7, EBP, SC5D, DHCR7 genes. The spectrum of 165 unique and 21 common protein partners that physically interact with target enzymes was selected from several interatomic resources. Among them there were 47 modifying proteins from different protein kinases/phosphatases and ubiquitin-protein ligases/deubiquitinases families. A literature search, enrichment and gene co-expression analysis showed that about a quarter of the identified protein partners was associated with cancer hallmarks and over-represented in cancer pathways. Our results allow to update the current fundamental view on protein-protein interactions and regulatory aspects of the cholesterol synthesis enzymes and annotate of their sub-interactomes in term of possible involvement in cancers that will contribute to prioritization of protein targets for future drug development.ViewShow abstract... While deregulated cellular metabolism appears to be an emerging hallmark of cancer drug resistance [12][13][14][15], deregulated energetics involving alterations in cellular metabolism to support neoplastic proliferation is a fundamental hallmark of cancer [16]. Central to this metabolic deregulation is Warburg s discovery of aerobic glycolysis, where glucose is converted to lactate even when oxygen supply is sufficient to support oxidative phosphorylation. ...GLUL Ablation Can Confer Drug Resistance to Cancer Cells via a Malate-Aspartate Shuttle-Mediated MechanismArticleFull-text availableDec 2019 Magesh Muthu Ranjeet Kumar Azharuddin Sajid Syed Khaja Anders NordströmGlutamate-ammonia ligase (GLUL) is important for acid-base homeostasis, ammonia detoxification, cell signaling, and proliferation. Here, we reported that GLUL ablation conferred resistance to several anticancer drugs in specific cancer cell lines while leaving other cell lines non-resistant to the same drugs. To understand the biochemical mechanics supporting this drug resistance, we compared drug-resistant GLUL knockout (KO) A549 non-small-cell lung carcinoma (NSCLC) cells with non-resistant GLUL KO H1299 NSCLC cells and found that the resistant A549 cells, to a larger extent, depended on exogenous glucose for proliferation. As GLUL activity is linked to the tricarboxylic acid (TCA) cycle via reversed glutaminolysis, we probed carbon flux through both glycolysis and TCA pathways by means of 13C5 glutamine, 13C5 glutamate, and 13C6 glucose tracing. We observed increased labeling of malate and aspartate in A549 GLUL KO cells, whereas the non-resistant GLUL KO H1299 cells displayed decreased 13C-labeling. The malate and aspartate shuttle supported cellular NADH production and was associated with cellular metabolic fitness. Inhibition of the malate-aspartate shuttle with aminooxyacetic acid significantly impacted upon cell viability with an IC50 of 11.5 μM in resistant GLUL KO A549 cells compared to 28 μM in control A549 cells, linking resistance to the malate-aspartate shuttle. Additionally, rescuing GLUL expression in A549 KO cells increased drug sensitivity. We proposed a novel metabolic mechanism in cancer drug resistance where the increased capacity of the malate-aspartate shuttle increased metabolic fitness, thereby facilitating cancer cells to escape drug pressure.ViewShow abstract... Besides, diverse intermediary metabolites of mevalonate via are active molecules that regulate different reproductive processes. One of them is the lanosterol that has been related to inflammation (Araldi et al., 2017) and cell proliferation (Stäubert et al., 2016). Lanosterol 14demethylase (CYP51A1), the enzyme that catalyzes lanosterol to cholesterol conversion, is present in the ovary (Wang et al., 2006(Wang et al., , 2009Nakamura et al., 2015) and uterus (Song et al., 2008). ...Inferring lanosterol functions in the female rabbit reproductive tract based on the immunolocalization of lanosterol 14-demethylase and farnesoid beta-receptorArticleNov 2019ACTA HISTOCHEMMaribel Méndez-TepepaDafne Zepeda-Pérez Leticia Nicolás-Toledo Estela CuevasFemale reproductive organs have de novo synthesis of cholesterol. Some sterol molecules, intermediaries in the cholesterol synthesis, have important paracrine/autocrine actions. Lanosterol binds to the farnesoid beta-receptor (FXRβ), a molecule widely expressed in the ovaries, suggesting that it may play a role in reproduction. Up to date, we know little about lanosterol functions across female reproductive organs. We described immunolocalized lanosterol 14-demethylase (LDM or CYP51A1), responsible for catalyzing the conversion of lanosterol in cholesterol, and FXRβ in the ovary, oviduct, uterus, and vagina of virgin and pregnant rabbits. In virgin rats, we found CYP51A1 and FXRβ immunoreactivity was found in all ovarian follicles, epithelial cells, stroma, and Graafian follicles. Also, the epithelium and stroma, as well as the smooth muscle of the oviduct, vagina, and uterus showed CYP51A1 and FXRβ immunoreactivity. In pregnant dams, we observed the presence of CYP51A1 and FXRβ immunoreactivity in the corpora lutea, giant uterine cells, and trophoblastic cells. The presence of CYP51A1 and FXRβ support that lanosterol participates in diverse reproductive processes, including follicular maturation, transport of gametes and zygote, implantation of blastocyst, lubrication, and contraction of the vagina, secretion of female prostate, and control of delivery mediated by pelvic muscles contraction.ViewShow abstract... Identifying differences further away from central aspects such as aerobic glycolysis and glutamine addiction may provide better therapeutic windows. The homeostatic mechanisms complicate discovery as quantitative differences may not be apparent and, to address this, global flux probing methods are being developed [42,43,[150][151][152]. From a basic understanding of cancer cell nutrient requirements, it could be worth revisiting classical experiments [34,36] investigating cellular needs with more powerful metabolomics and flux techniques available today. ...Current Status and Future Prospects of Clinically Exploiting Cancer-specific Metabolism—Why Is Tumor Metabolism Not More Extensively Translated into Clinical Targets and Biomarkers?ArticleFull-text availableMar 2019INT J MOL SCI Magesh Muthu Anders NordströmTumor cells exhibit a specialized metabolism supporting their superior ability for rapid proliferation, migration, and apoptotic evasion. It is reasonable to assume that the specific metabolic needs of the tumor cells can offer an array of therapeutic windows as pharmacological disturbance may derail the biochemical mechanisms necessary for maintaining the tumor characteristics, while being less important for normally proliferating cells. In addition, the specialized metabolism may leave a unique metabolic signature which could be used clinically for diagnostic or prognostic purposes. Quantitative global metabolic profiling (metabolomics) has evolved over the last two decades. However, despite the technology’s present ability to measure 1000s of endogenous metabolites in various clinical or biological specimens, there are essentially no examples of metabolomics investigations being translated into actual utility in the cancer clinic. This review investigates the current efforts of using metabolomics as a tool for translation of tumor metabolism into the clinic and further seeks to outline paths for increasing the momentum of using tumor metabolism as a biomarker and drug target opportunity.ViewShow abstract... Concerning therapy resistance, enhanced cholesterol pathway activity has been reported in cancer cell models resistant to e.g. platinum drugs or doxorubicin [78][79][80][81][82][83]. Accordingly, also in our experiments dtxA sensitivity could be significantly restored by blockade of the mevalonate pathway by both statins and zoledronic acid, while cholesterol supplementation of destruxinsensitive parental cells enhanced cell viability. ...Altered membrane rigidity via enhanced endogenous cholesterol synthesis drives cancer cell resistance to destruxinsArticleFull-text availableMay 2018 Daniela HeilosClemens RöhrlChristine Pirker Walter BergerDestruxins, secondary metabolites of entomopathogenic fungi, exert a wide variety of interesting characteristics ranging from antiviral to anticancer effects. Although their mode of action was evaluated previously, the molecular mechanisms of resistance development are unknown. Hence, we have established destruxin-resistant sublines of HCT116 colon carcinoma cells by selection with the most prevalent derivatives, destruxin (dtx)A, dtxB and dtxE. Various cell biological and molecular techniques were applied to elucidate the regulatory mechanisms underlying these acquired and highly stable destruxin resistance phenotypes. Interestingly, well-known chemoresistance-mediating ABC efflux transporters were not the major players. Instead, in dtxA- and dtxB-resistant cells a hyper-activated mevalonate pathway was uncovered resulting in increased de-novo cholesterol synthesis rates and elevated levels of lanosterol, cholesterol as well as several oxysterol metabolites. Accordingly, inhibition of the mevalonate pathway at two different steps, using either statins or zoledronic acid, significantly reduced acquired but also intrinsic destruxin resistance. Vice versa, cholesterol supplementation protected destruxin-sensitive cells against their cytotoxic activity. Additionally, an increased cell membrane adhesiveness of dtxA-resistant as compared to parental cells was detected by atomic force microscopy. This was paralleled by a dramatically reduced ionophoric capacity of dtxA in resistant cells when cultured in absence but not in presence of statins. Summarizing, our results suggest a reduced ionophoric activity of destruxins due to cholesterol-mediated plasma membrane re-organization as molecular mechanism underlying acquired destruxin resistance in human colon cancer cells. Whether this mechanism might be valid also in other cell types and organisms exposed to destruxins e.g. as bioinsecticides needs to be evaluated.ViewShow abstract... Finally, a recent study suggests the involvement of SE in leukemia. In particular, approximately threefold-higher SQLE transcript levels were observed in the daunorubicin-resistant leukemia CEM/R2 cell line [92]. ...Squalene epoxidase as a promising metabolic target in cancer treatmentArticleFull-text availableMar 2018CANCER LETT Gabriella Cirmena Paola Franceschelli Edoardo Isnaldi Zoppoli GabrieleOncogenic alteration of the cholesterol synthesis pathway is a recognized mechanism of metabolic adaptation. In the present review, we focus on squalene epoxidase (SE), one of the two rate-limiting enzymes in cholesterol synthesis, retracing its history since its discovery as an antimycotic target to its description as an emerging metabolic oncogene by amplification with clinical relevance in cancer. We review the published literature assessing the association between SE over-expression and poor prognosis in this disease. We assess the works demonstrating how SE promotes tumor cell proliferation and migration, and displaying evidence of cancer cell demise in presence of human SE inhibitors in in vitro and in vivo models. Taken together, robust scientific evidence has by now accumulated pointing out SE as a promising novel therapeutic target in cancer treatment.ViewShow abstractA preliminary investigation into the unsaponifiable fraction of donkey milk: Sterols of animal origin, phytosterols, and tocopherolsArticleNov 2020J Dairy Sci Mina Martini Iolanda Altomonte Rosario Licitra Federica SalariWe investigated the main sterols, phytosterols, and the α- and γ-tocopherol content in donkey milk during the first 2 mo of lactation. Cholesterol was the main sterol in milk (mean ± standard deviation = 0.97 ± 0.443 g/100 g of fat). Lanosterol was the main minor sterol of animal origin, followed by desmosterol (0.003 ± 0.001 and 0.001 ± 0.001 g/100 g of fat, respectively). Of the phytosterols, β-sitosterol was the main sterol of vegetal origin in donkey milk (0.005 ± 0.002 g/100 g of fat), but lower levels of campesterol, brassicasterol, and stigmasterol were also recorded. Mean levels of α- and γ-tocopherol were 0.01 ± 0.007 and 0.003 ± 0.001 g/100 g of fat, respectively. We observed no significant changes in sterol or tocopherol content during the first 2 mo of lactation. The presence of lanosterol in donkey milk is of particular interest, because lanosterol is a potential drug and has important physiological effects. The presence of phytosterols, which are considered nutraceutical molecules, enhances the nutritional quality of donkey milk fat for consumers.ViewShow abstractSeparation and purification of lanosterol, dihydrolanosterol and cholesterol from lanolin by high‐performance counter‐current chromatography dual‐mode elution methodArticleApr 2019J SEP SCIHairun PeiXiaotong MaYan Pan Jimin ZhengLanosterol is a potential drug for cataracts treatment which can reverse the aggregation of intracrystalline proteins. The low concentration in lanolin calls for high‐performance separation methods. In this study, a counter‐current chromatography (CCC) dual‐mode elution method was developed for the first time to separate and purify lanosterol from hexane extract of lanolin after saponification, in which the column was firstly eluted with the lower phase as mobile phase in head‐to‐tail mode, followed by the upper phase in the tail‐to‐head mode. High‐purity of lanosterol, dihydrolanosterol and cholesterol can be obtained simultaneously. A solvent system composed of n‐heptane / acetonitrile / ethyl acetate (5/5/1, v/v/v) was selected and optimized via partition coefficient (K) determination. 111 mg lanosterol, 84 mg dihydrolanosterol and 183 mg cholesterol with high purity of 99.77 %, 95.71 % and 91.43 % respectively analyzed by HPLC were obtained within 80 min from 700 mg crude extract from 1.78 g lanolin. The method was also used to improve the purity of commercial lanosterol product from 66.97% to above 99%. Counter‐Current Chromatography could serve as a potential and powerful technique for commercial production of highly pure lanosterol.This article is protected by copyright. All rights reservedViewShow abstractHydroxycarboxylic acid receptors are essential for breast cancer cells to control their lipid/fatty acid metabolismArticleFull-text availableMar 2015 Claudia Stäubert Oliver Broom Anders NordströmCancer cells exhibit characteristic changes in their metabolism with efforts being made to address them therapeutically. However, targeting metabolic enzymes as such is a major challenge due to their essentiality for normal proliferating cells. The most successful pharmaceutical targets are G protein-coupled receptors (GPCRs), with more than 40% of all currently available drugs acting through them.We show that, a family of metabolite-sensing GPCRs, the Hydroxycarboxylic acid receptor family (HCAs), is crucial for breast cancer cells to control their metabolism and proliferation.We found HCA1 and HCA3 mRNA expression were significantly increased in breast cancer patient samples and detectable in primary human breast cancer patient cells. Furthermore, siRNA mediated knock-down of HCA3 induced considerable breast cancer cell death as did knock-down of HCA1, although to a lesser extent. Liquid Chromatography Mass Spectrometry based analyses of breast cancer cell medium revealed a role for HCA3 in controlling intracellular lipid/fatty acid metabolism. The presence of etomoxir or perhexiline, both inhibitors of fatty acid β-oxidation rescues breast cancer cells with knocked-down HCA3 from cell death.Our data encourages the development of drugs acting on cancer-specific metabolite-sensing GPCRs as novel anti-proliferative agents for cancer therapy.ViewShow abstractRewired Metabolism in Drug-resistant Leukemia Cells a metabolic switch hallmarked by reduced dependence on exogenous glutamineArticleFull-text availableFeb 2015J BIOL CHEM Claudia Stäubert Hasanuzzaman BhuiyanAnna Lindahl Anders NordströmCancer cells which escape induction therapy are a major cause of relapse. Understanding metabolic alterations associated with drug resistance opens up unexplored opportunities for the development of new therapeutic strategies. Here, we applied a broad spectrum of technologies including RNA sequencing, global untargeted metabolomics and stable isotope labelling mass spectrometry to identify metabolic changes in P-glycoprotein overexpressing T-cell acute lymphoblastic leukemia (ALL) cells, which escaped a therapeutically relevant daunorubicin treatment. We show that compared to sensitive ALL cells, resistant leukemia cells possess a fundamentally rewired central metabolism characterized by reduced dependence on glutamine despite a lack of expression of glutamate-ammonia ligase (GLUL), a higher demand for glucose and an altered rate of fatty acid β-oxidation, accompanied by a decreased pantothenic acid uptake capacity. We experimentally validate our findings by selectively targeting components of this metabolic switch, using approved drugs and starvation approaches followed by cell viability analyses in both the ALL cells and in an acute myeloid leukemia (AML) sensitive/resistant cell line pair. We demonstrate how comparative metabolomics and RNA expression profiling of drug-sensitive and -resistant cells expose targetable metabolic changes and potential resistance markers. Our results show that drug resistance is associated with significant metabolic costs in cancer cells, which could be exploited using new therapeutic strategies.Copyright © 2015, The American Society for Biochemistry and Molecular Biology.ViewShow abstractEstablishment and characterization of a bladder cancer cell line with enhanced doxorubicin resistance by mevalonate pathway activationArticleFull-text availableJan 2015TUMOR BIOL Annemarie GreifeJ. Tukova Christine Steinhoff Jirí HatinaResistance to chemotherapy is a major problem in the treatment of urothelial bladder cancer. Several mechanisms have been identified in resistance to doxorubicin by analysis of resistant urothelial carcinoma (UC) cell lines, prominently activation of drug efflux pumps and diminished apoptosis. We have derived a new doxorubicin-resistant cell line from BFTC-905 UC cells, designated BFTC-905-DOXO-II. A doxorubicin-responsive green fluorescent protein (GFP) reporter assay indicated that resistance in BFTC-905-DOXO-II was not due to increased drug efflux pump activity, whereas caspase-3/7 activation was indeed diminished. Gene expression microarray analysis revealed changes in proapoptotic and antiapoptotic genes, but additionally induction of the mevalonate (cholesterol) biosynthetic pathway. Treatment with simvastatin restored sensitivity of BFTC-905-DOXO-II to doxorubicin to that of the parental cell line. Induction of the mevalonate pathway has been reported as a mechanism of chemoresistance in other cancers; this is the first observation in bladder cancer. Combinations of statins with doxorubicin-containing chemotherapy regimens may provide a therapeutic advantage in such cases.ViewShow abstractTracking the clonal origin of lethal prostate cancerArticleFull-text availableOct 2013J Clin InvestigMichael C. HaffnerTimothy Mosbruger David EsopiSrinivasan YegnasubramanianRecent controversies surrounding prostate cancer overtreatment emphasize the critical need to delineate the molecular features associated with progression to lethal metastatic disease. Here, we have used whole-genome sequencing and molecular pathological analyses to characterize the lethal cell clone in a patient who died of prostate cancer. We tracked the evolution of the lethal cell clone from the primary cancer to metastases through samples collected during disease progression and at the time of death. Surprisingly, these analyses revealed that the lethal clone arose from a small, relatively low-grade cancer focus in the primary tumor, and not from the bulk, higher-grade primary cancer or from a lymph node metastasis resected at prostatectomy. Despite being limited to one case, these findings highlight the potential importance of developing and implementing molecular prognostic and predictive markers, such as alterations of tumor suppressor proteins PTEN or p53, to augment current pathological evaluation and delineate clonal heterogeneity. Furthermore, this case illustrates the potential need in precision medicine to longitudinally sample metastatic lesions to capture the evolving constellation of alterations during progression. Similar comprehensive studies of additional prostate cancer cases are warranted to understand the extent to which these issues may challenge prostate cancer clinical management.ViewShow abstractThe role of cholesterol metabolism and cholesterol transport in carcinogenesis: A review of scientific findings, relevant to future cancer therapeuticsArticleFull-text availableSep 2013 Pedro Miguel Cruz Huanbiao MoWalter J. McConathy Andras LackoWhile the unique metabolic activities of malignant tissues as potential targets for cancer therapeutics has been the subject of several recent reviews, the role of cholesterol metabolism in this context is yet to be fully explored. Cholesterol is an essential component of mammalian cell membranes as well as a precursor of bile acids and steroid hormones. The hypothesis that cancer cells need excess cholesterol and intermediates of the cholesterol biosynthesis pathway to maintain a high level of proliferation is well accepted, however the mechanisms by which malignant cells and tissues reprogram cholesterol synthesis, uptake and efflux are yet to be fully elucidated as potential therapeutic targets. High and low density plasma lipoproteins are the likely major suppliers of cholesterol to cancer cells and tumors, potentially via receptor mediated mechanisms. This review is primarily focused on the role(s) of lipoproteins in carcinogenesis, and their future roles as drug delivery vehicles for targeted cancer chemotherapy.ViewShow abstractStatin use in primary inflammatory breast cancer: A cohort studyArticleFull-text availableJul 2013Br J Canc Takae BrewerH MasudaDD Liu Naoto T UenoBackground:Some studies have suggested that statins, which have cholesterol-lowering and anti-inflammatory properties, may have antitumor effects. Effects of statins on inflammatory breast cancer (IBC) have never been studied.Methods:We reviewed 723 patients diagnosed with primary IBC in 1995–2011 and treated at The University of Texas MD Anderson Cancer Center. Statin users were defined as being on statins at the initial evaluation. Based on Ahern et al s statin classification (JNCI, 2011), clinical outcomes were compared by statin use and type (weakly lipophilic to hydrophilic (H-statin) vs lipophilic statins (L-statin)). We used the Kaplan–Meier method to estimate the median progression-free survival (PFS), overall survival (OS) and disease-specific survival (DSS), and a Cox proportional hazards regression model to test the statistical significance of potential prognostic factors.Results:In the multivariable Cox model, H-statins were associated with significantly improved PFS compared with no statin (hazard ratio=0.49; 95% confidence interval=0.28–0.84; P 0.01); OS and DSS P-values were 0.80 and 0.85, respectively. For L-statins vs no statin, P-values for PFS, DSS, and OS were 0.81, 0.4, and 0.74, respectively.Conclusion:H-statins were associated with significantly improved PFS. A prospective randomised study evaluating the survival benefits of statins in primary IBC is warranted.ViewShow abstractMultilevel regulation of low-density lipoprotein receptor and 3-hydroxy- 3-methylglutaryl coenzyme A reductase gene expression in normal and leukemic cellsArticleOct 1994BLOOD Sigurd Vitols Svante Norgren Gunnar JuliussonH LuthmanAltered cholesterol homeostasis has been noted in malignant cells, which led us to explore the regulation of cholesterol metabolism in normal and leukemic cells. The mean low-density lipoprotein (LDL) receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities were fivefold and threefold higher in mononuclear blood cells from 33 patients with leukemia, compared with cells from 23 healthy subjects, whereas elevations in RNA levels were twofold and 40% only. The activities of the two proteins correlated in normal cells (r = .46), whereas an inverse correlation was found in leukemic cells (r = -.40). Relatively weak correlations were found between LDL receptor RNA levels and receptor activity in normal (r = .48) and leukemic cells (r = .49), and HMG-CoA reductase RNA levels correlated (r = .53) with reductase activity in leukemic cells only. The ratios of protein activities to RNA levels in cells were constant during consecutive blood samplings and similar in leukemic blood and bone marrow cells from the same individual. During cholesterol deprivation, protein activities increased more than RNA levels, and leukemic cells with high LDL receptor activity showed a partial resistance to the suppressing effect of sterols on LDL receptor gene expression. The results demonstrate that LDL receptor RNA levels alone can not explain variation in receptor activity, suggesting post-RNA regulation of LDL receptor expression, similar to what has been described for HMG-CoA reductase. Taken together, the present results suggest multilevel regulation of both proteins and demonstrate that each cell clone, normal or malignant, has a unique ratio of protein activity to RNA level. Leukemic cells, in contrast to normal cells, can meet increased cholesterol requirements by either elevated LDL receptor activity or increased cholesterol synthesis, which is of potential interest for diagnosis and specific treatment of leukemia.ViewShow abstractOn the occurrence of crystals in tumoursArticleJ Pathol BacteriolR.M. WhiteViewCombination of Lipitor and Celebrex Inhibits Prostate Cancer VCaP Cells In Vitro and In VivoArticleJul 2014ANTICANCER RESHuarong HuangXiao-Xing CuiShaohua Chen Xi ZhengBackground/aim: Lipitor is a cholesterol-lowering drug and Celebrex is a Cyclooxygenase-2 inhibitor. We investigated the effects of Lipitor and Celebrex on human prostate cancer VCaP cells cultured in vitro and grown as orthotopic xenograft tumors in SCID mice.Materials and methods: Apoptosis was measured by morphological assessment and caspase-3 assay. Nuclear factor-kappa B (NF-κB) activation was determined by luciferase reporter assay. B-cell lymphoma-2 (Bcl2) was measured by western blotting and immunohistochemistry. Orthotopic prostate tumors were monitored by the IVIS imaging system.Results: the combination of Lipitor and Celebrex had stronger effects on the growth and apoptosis of VCaP cells than did either drug alone. The combination more potently inhibited activation of NFκB and expression of Bcl2 than either drug alone. The growth of orthotopic VCaP prostate tumors was strongly inhibited by treatment with the drug combination.Conclusion: Administration of Lipitor and Celebrex in combination may be an effective strategy for inhibiting the growth of prostate cancer.ViewShow abstractThe E3 Ubiquitin Ligase MARCH6 Degrades Squalene Monooxygenase and Affects 3-Hydroxy-3-Methyl-Glutaryl Coenzyme A Reductase and the Cholesterol Synthesis PathwayArticleJan 2014Mol Cell Biol Noam Zelcer Laura Sharpe Anke LoreggerAndrew J BrownThe mevalonate pathway is used by cells to produce sterol and nonsterol metabolites and is subject to tight metabolic regulation.We recently reported that squalene monooxygenase (SM), an enzyme controlling a rate-limiting step in cholesterol biosynthesis,is subject to cholesterol-dependent proteasomal degradation. However, the E3-ubiquitin (E3) ligase mediating this effect wasnot established. Using a candidate approach, we identify the E3 ligase membrane-associated RING finger 6 (MARCH6, also knownas TEB4) as the ligase controlling degradation of SM. We find that MARCH6 and SM physically interact, and consistent withMARCH6 acting as an E3 ligase, its overexpression reduces SM abundance in a RING-dependent manner. Reciprocally, knockdownof MARCH6 increases the level of SM protein and prevents its cholesterol-regulated degradation. Additionally, this increasescell-associated SM activity but is unexpectedly accompanied by increased flux upstream of SM. Prompted by this observation,we found that knockdown of MARCH6 also controls the level of 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGCR) in hepatocytesand model cell lines. In conclusion, MARCH6 controls abundance of both SM and HMGCR, establishing it as a major regulatorof flux through the cholesterol synthesis pathway.ViewShow abstractShow moreAdvertisementRecommendationsDiscover moreProjectCancer Metabolomics Magesh Muthu Anders Nordström Jenny Liao Persson[...]Jonathan D. GilthorpeView projectArticleAn assay to measure adriamycin binding in osteosarcoma cellsSeptember 1994 · Journal of Orthopaedic Research Mark C GebhardtKatsuyuki Kusuzaki Henry MankinD S SpringfieldAdjuvant chemotherapy is currently employed in the treatment of patients with osteosarcoma, but the drug regimens, although effective in improving disease-free survival, are unsuccessful in 20-40% of patients and very toxic. It would be useful to know whether tumor cells are sensitive to a given drug prior to its use. To this end, we developed a method of assessing Adriamycin (doxorubicin) ... [Show full abstract] binding to tumor nuclei as a possible means of detecting sensitivity to the drug. Adriamycin-sensitive murine osteosarcoma cells were used to develop the assay. The in vitro conditions (drug concentration, duration of incubation, and temperature) were optimized with use of the murine osteosarcoma cells in culture. After the cells had been incubated with Adriamycin, cell viability was determined and Adriamycin fluorescence intensity was measured with a cytofluorometer. The optimal parameters for Adriamycin binding were found to be a 30-minute incubation in a 10 micrograms/ml concentration of Adriamycin at 37 degrees C; the frequency of cells that emitted Adriamycin fluorescence from the nucleus compared with the total number of living cells reached 100% under these conditions. In a murine leukemia cell line with known sensitivity to Adriamycin, the cells emitted red fluorescence from the nucleus and cytoplasm, whereas in a resistant line the cells emitted Adriamycin fluorescence from only the cytoplasm. We demonstrated that it is possible to differentiate nuclear from cytoplasmic concentration of Adriamycin in a tumor cell with use of a fluorescent microscope and that resistant cell lines can be distinguished from sensitive cell lines by this method.(ABSTRACT TRUNCATED AT 250 WORDS)Read moreArticleEffect of high glucose on cholesterol efflux in renal tubular cell and intervention of anthocyaninsJanuary 2016C.-Y. DuY.-H. ShiY. Zhu[...]H.-J. DuanAim: To investigate the effects of high glucose on cholesterol metabolism in renal tubular cells and the intervention of the anthocyanins. Methods HK-2 cells were grown in the DMEM medium supplemented with 10% FBS and were divided into 5 groups; normal glucose group, high glucose group, mannitol group, C3G group and Cy group. Effect of anthocyanins on cell viability was detected with MTT, and ... [Show full abstract] cholesterol accumulation was detected with Amplex Red Cholesterol Assay kit and Filipin staining. Expression of ABCA1 was detected with RT-qPCR and Western Blot. Results: In compared with control groups, HG significantly promoted cholesterol mass inside the cell and decreased the cholesterol concentration in the me dium after treatment for 24 h or 48 h. The levels of mRNA and protein of ABCA1 were detected with RT-qPCR and Western blot, and both were decreased in the presence of HG. Whereas treatment with C3G and Cy markedly attenuated HG-induced cholesterol mass inside the cell by up-regulating the expression of ABCA1. Conclusions: High glucose can reduce the expressions of the ABCA1, and then decrease cholesterol efflux and increase the cholesterol accumulation in HK-2 cells. Anthocyanins can decrease cholesterol accumulation by up-regulating the expression of ABCA1.Read moreArticleFull-text availableProteasomal Degradation of Mcl-1 by Maritoclax Induces Apoptosis and Enhances the Efficacy of ABT-73...November 2013 · PLoS ONE Manoj Pandey Krishne Gowda Kenichiro Doi[...]Shantu AminMetastatic melanoma remains one of the most invasive and highly drug resistant cancers. The over expression of anti-apoptotic protein Mcl-1 has been associated with inferior survival, poor prognosis and chemoresistance of malignant melanoma. A BH3 mimetic, ABT-737, has demonstrated efficacy in several forms of cancers. However, the efficacy of ABT-737 depends on Mcl-1. Because the over expression ... [Show full abstract] of Mcl-1 is frequently observed in melanoma, specifically targeting of Mcl-1 may overcome the resistance of ABT-737. In this study, we investigated the effects of Maritoclax, a novel Mcl-1-selective inhibitor, alone and in combination with ABT-737, on the survival of human melanoma cells.For cell viability assessment we performed MTT assay. Apoptosis was determined using western blot and flow cytometric analysis.The treatment of Maritoclax reduced the cell viability of melanoma cells with an IC50 of between 2.2-5.0 µM. Further, treatment of melanoma cells with Maritoclax showed significant decrease in Mcl-1 expression. We found that Maritoclax was able to induce apoptosis in melanoma cells in a caspase-dependent manner. Moreover, Maritoclax induced Mcl-1 degradation via the proteasome system, which was associated with its pro-apoptotic activity. We also found that Maritoclax treatment increased mitochondrial translocation of Bim and Bmf. Importantly, Maritoclax markedly enhanced the efficacy of ABT-737 against melanoma cells in both two- and three-dimensional spheroids.Taken together, these results suggest that targeting of Mcl-1 by Maritoclax may represent a new therapeutic strategy for melanoma treatment that warrants further investigation as a single therapy or in combination with other agents such as Bcl-2 inhibitors.View full-textArticleFull-text availableDeregulation of Mitochondrial ATPsyn-?? in Acute Myeloid Leukemia Cells and with Increased Drug Resi...December 2013 · PLoS ONEXiang Xiao Jingke Yang Ruijuan Li[...]Guangsen ZhangThe mechanisms underlying the development of multidrug resistance in acute myeloid leukemia are not fully understood. Here we analyzed the expressions of mitochondrial ATPsyn-β in adriamycin-resistant cell line HL-60/ADM and its parental cell line HL-60. Meanwhile we compared the differences of mitochondrial ATPsyn-β expression and ATP synthase activity in 110 acute myeloid leukemia (AML, non-M3) ... [Show full abstract] patients between relapsed/refractory and those in remission. Our results showed that down-regulation of ATPsyn-β expression by siRNA in HL-60 cells increased cell viability and apoptotic resistance to adriamycin, while up-regulation of mitochondrial ATPsyn-β in HL-60/ADM cells enhanced cell sensitivity to adriamycin and promoted apoptosis. Mitochondrial ATPsyn-β expression and ATP synthase activity in relapsed/refractory acute myeloid leukemia patients were downregulated. This downregulated ATPsyn-β expression exhibited a positive correlation with the response to adriamycin of primary cells. A lower expression of ATPsyn-β in newly diagnosed or relapsed/refractory patients was associated with a shorter first remission duration or overall survival. Our findings show mitochondrial ATPsyn-β plays an important role in the mechanism of multidrug resistance in AML thus may present both a new marker for prognosis assessment and a new target for reversing drug resistance.View full-textArticleFull-text availableAssociation of Sphingosine‐1‐phosphate (S1P)/S1P Receptor‐1 Pathway with Cell Proliferation and Surv...June 2015 · Journal of Veterinary Internal MedicineA.M. RodriguezAshley J. Graef Dana N Levine[...] Jong Hyuk KimSphingosine-1-phosphate (S1P) is a key biolipid signaling molecule that regulates cell growth and survival, but it has not been studied in tumors from dogs.S1P/S1P1 signaling will contribute to the progression of hemangiosarcoma (HSA).Thirteen spontaneous HSA tissues, 9 HSA cell lines, 8 nonmalignant tissues, including 6 splenic hematomas and 2 livers with vacuolar degeneration, and 1 ... [Show full abstract] endothelial cell line derived from a dog with splenic hematoma were used.This was a retrospective case series and in vitro study. Samples were obtained as part of medically necessary diagnostic procedures. Microarray, qRT-PCR, immunohistochemistry, and immunoblotting were performed to examine S1P1 expression. S1P concentrations were measured by high-performance liquid chromatography/mass spectrometry. S1P signaling was evaluated by intracellular Ca(2+) mobilization; proliferation and survival were evaluated using the MTS assay and Annexin V staining.Canine HSA cells expressed higher levels of S1P1 mRNA than nonmalignant endothelial cells. S1P1 protein was present in HSA tissues and cell lines. HSA cells appeared to produce low levels of S1P, but they selectively consumed S1P from the culture media. Exogenous S1P induced an increase in intracellular calcium as well as increased proliferation and viability of HSA cells. Prolonged treatment with FTY720, an inhibitor of S1P1 , decreased S1P1 protein expression and induced apoptosis of HSA cells.S1P/S1P1 signaling pathway functions to maintain HSA cell viability and proliferation. The data suggest that S1P1 or the S1P pathway in general could be targets for therapeutic intervention for dogs with HSA.Copyright © 2015 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.View full-textDiscover the world s researchJoin ResearchGate to find the people and research you need to help your work.Join for free ResearchGate iOS AppGet it from the App Store now.InstallKeep up with your stats and moreAccess scientific knowledge from anywhere orDiscover by subject areaRecruit researchersJoin for freeLoginEmail Tip: Most researchers use their institutional email address as their ResearchGate loginPasswordForgot password? 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