ﬁthrin-m Prevents Cisplatin-Induced Chemobrain by ... · Piﬁthrin-m Prevents Cisplatin-Induced Chemobrain by Preserving Neuronal Mitochondrial Function Gabriel S. Chiu, Magdalena

Therapeutics, Targets, And Chemical Biology

Pifithrin-m Prevents Cisplatin-InducedChemobrain by Preserving NeuronalMitochondrial FunctionGabriel S. Chiu, Magdalena A. Maj, Sahar Rizvi, Robert Dantzer, Elisabeth G. Vichaya,Geoffroy Laumet, Annemieke Kavelaars, and Cobi J. Heijnen

Abstract

Cognitive impairment, termed chemobrain, is a common neu-rotoxicity associated with chemotherapy treatment, affecting anestimated 78% of patients. Prompted by the hypothesis thatneuronal mitochondrial dysfunction underlies chemotherapy-induced cognitive impairment (CICI), we explored the efficacy ofadministering the small-molecule pifithrin (PFT)-m, an inhibitor ofmitochondrial p53accumulation, inpreventingCICI.MaleC57BL/6J mice injected with cisplatin � PFT-m for two 5-day cycles wereassessed for cognitive functionusingnovelobject/place recognitionand alternation in a Y-maze. Cisplatin impairedperformance in thenovel object/place recognition and Y-maze tests. PFT-m treatmentprevented CICI and associated cisplatin-induced changes in coher-ency ofmyelin basic protein fibers in the cingular cortex and loss of

doublecortinþ cells in the subventricular zone and hippocampaldentate gyrus. Mechanistically, cisplatin decreased spare respiratorcapacity of brain synaptosomes and caused abnormal mitochon-drial morphology, which was counteracted by PFT-m administra-tion.Notably, increasedmitochondrial p53did not lead to cerebralcaspase-3 activation or cytochrome-c release. Furthermore, PFT-madministration did not impair the anticancer efficacy of cisplatinand radiotherapy in tumor-bearing mice. Our results supportedthe hypothesis that neuronal mitochondrial dysfunction inducedby mitochondrial p53 accumulation is an underlying cause ofCICI and that PFT-m may offer a tractable therapeutic strategy tolimit this common side-effect of many types of chemotherapy.Cancer Res; 77(3); 742–52. �2016 AACR.

IntroductionAdvances in the efficacy of cancer treatment have led to a sharp

increase in the number of cancer survivors, which has reachednearly 15 million in the United States alone (1, 2). However, inmany cases cancer treatment is associated with severe neurotoxicside effects, including fatigue, peripheral pain (3), paresthesia,and cognitive deficits, that can persist long after completion oftreatment (4). These neurotoxicities not only disrupt social,educational, and occupational functioning, but also decreasesurvival by interfering with adherence to medication and healthbehavior.

In humans, chemotherapy-induced cognitive impairment(CICI), so-called "chemobrain," is characterized by subtle tomoderate cognitive deficits, including a decrease in processingspeed, memory, executive functioning, and attention, as assessedby neuropsychological tests (4–6). CICI has been noted in 78%ofcross-sectional and 69% of prospective longitudinal studies per-formed between 1995 and 2012 in patients treated for breastcancer (7–9). One in two adults is projected to be diagnosed withcancer in his or her lifetime, implying that CICI represents agrowing public health concern.

Advanced neuroimaging techniques have revealed that che-motherapy causes structural alterations in white and graymatter in patients with cancer, along with an increased levelof activation of the fronto-parietal attentional network (5).However, little is known about the mechanism underlyingCICI, and no preventive or curative interventions have beenapproved by the FDA.

Platinum-based chemotherapeutic agents like cisplatin arewide-ly used to treat solid tumors (10). Traditionally, these agents havebeen thought to act on tumor cells by preferential binding to theguanine residues in DNA to induce crosslinking (11). The body'ssubsequent inability to repair damaged DNA leads to an arrest inmitosis, inhibition of cell proliferation, and tumor cell death.

Preclinical studies have shown that cisplatin crosses the blood–brain barrier and impairs adult neurogenesis in the brain (12).Werecently showed that cisplatin-induced cognitive impairment inmice was associated with increased coherency of white-matterstructures and sharp decrease in the number of neuronal dendriticspines and arborizations (2).

In rodent models, peripheral neuropathy induced by cisplatinor other chemotherapeutic agents such as taxanes is associatedwith structural damage tomitochondria in the dorsal root gangliaand peripheral nerves (13). In general, damaged mitochondriacan give rise to increased oxidative stress, inflammation, andproapoptotic signaling (14). We recently showed that co-admin-istration of the small-molecule pifithrin (PFT)-m, an inhibitor ofmitochondrial p53 accumulation, protects against taxane-induced and cisplatin-induced peripheral neuropathy (3). Inaddition, we have demonstrated that PFT-m is neuroprotective ina model of neonatal hypoxic–ischemic brain damage by prevent-ing neuronal apoptosis (15).

Laboratory of Neuroimmunology, Department of Symptom Research, TheUniversity of Texas MD Anderson Cancer Center, Houston, Texas.

Corresponding Author: Cobi J. Heijnen, The University of Texas MD AndersonCancer Center, 1515 Holcombe Boulevard, Unit 384, Houston, TX 77030. Phone:713-563-0162; Fax: 713-745-3475; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-16-1817

�2016 American Association for Cancer Research.

CancerResearch

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In this study, we investigated whether cerebral neuronal mito-chondrial damage is the underlying mechanism of cisplatin-induced cognitive impairment. Moreover, we tested whetherPFT-m is capable of preventing the development of cisplatin-induced chemobrain and precluding structural alterations in thebrainwith respect to damage towhitematter andneurogenic areasin the brain.

Materials and MethodsAnimals

C57BL/6Jmalemice were used in this study.Mice were bred in-house and were allowed water and food ad libitum. Housing waskept constant at 22 � 2�C, and a 12/12 hours reverse dark–lightcycle (dark 1,000–2,200hours)was employed. Video recording ofanimal behavior was performed under red light using a SonyHandycam DCR-SR100.

All experiments were conducted at The University of Texas MDAnderson Cancer Center in Houston, Texas. Animal usage wascarried out in accordance with Institutional Animal Care and UseCommittee-approved protocols.

InjectionsCisplatin (2.3 mg/kg; Fresenius Kabi USA) or PBS was admin-

istered intraperitoneally daily for 5 days, followed by a 5-day restwith no injection and then another 5-day injection cycle. Toinvestigate the effects of PFT-m on cognitive function, PFT-m(8 mg/kg; Sigma-Aldrich/Millipore Sigma) or vehicle (5%dimethyl sulfoxide in saline) was administered intraperitoneally1 hour before cisplatin injection.

Novel object/place recognition and Y-maze testsTo assess cognitive function, we subjected mice to novel

object/place recognition (NOPR) and Y-maze tests 7 days afterthe final cisplatin injection. All behavioral assays were per-formed in a blinded setup. NOPR was performed as we havepreviously described (2). In brief, mice were transferred to atesting arena (46.99 cm � 25.4 cm) containing two identicalobjects placed against one side of the arena for 5 minutes(training phase) and then returned to their home cages for30 minutes. Mice were transferred back to the arena, nowcontaining one familiar object placed at the same location asin training, and one novel object placed on the opposite end ofthe arena (testing phase). Investigative behavior toward eitherobject during the 5-minute testing period was evaluated usingEthoVision XT 10.1 video tracking software (Noldus Informa-tion Technology Inc.). Discrimination index was determined bythe equation (TNovel � TFamiliar)/(TNovel þ TFamiliar).

For the Y-maze test, spontaneous alternations were performedas previously described (16). In brief, mice were placed in asymmetrical three-arm, gray plastic Y-maze (35 cm length� 5 cmwidth� 15.5 cm height per arm, with an arm angle of 120�) withexternal spatial room cues. Mice were randomly placed in one ofthe arms. Movement was recorded for 5 minutes, and mouseexploration was evaluated. Perfect alternations were defined asexploration of all three arms sequentially before reentering apreviously visited arm. All four paws must have been within thearm to be counted as an entrance. Results are represented as theratio of the number of perfect alternations to the total number ofpossible alternations.

LocomotionSpontaneous locomotor activity was measured as described

previously (16–20). In brief, mice were video recorded for 5minutes in their home cage. Distance travelled was quantifiedusing EthoVisionXT10.1 (Noldus Information Technology, Inc.).

Body weightBody weights were measured daily at the time of injection. The

percentage of change from baseline (day 0) was calculated.

Tumor response to chemoradiationWe used a heterotopic syngeneic murine model of human

papillomavirus (HPV)–related head and neck cancer to assesswhether PFT-m might potentially interfere with the tumor'sresponse to chemoradiation (21–23). Male C57BL/6J mice wereinjected in the hind leg with 1 � 106 cells derived from C57BL/6oropharyngeal epithelial cells that were transfected with onco-genes E6/7 ofHPV 16 and hRAS (21, 22). Mice were injected withPFT-m (8 mg/kg) or vehicle 24 hours before a curative regimen ofcisplatin (5.28 mg/kg intraperitoneally; Calbiochem, EMD Milli-pore) and local irradiation (8 Gy provided by a small-animalcesium137 irradiator that collimates parallel opposed radiationbeams to a 3-cm field) starting 12 days after tumor implantationand repeated weekly for 3 weeks (22). Tumor volume was deter-mined using Vernier calipers from three mutually orthogonaltumor diameters, where volume ¼ (p/6)(d1�d2�d3).

Quantitative real-time polymerase chain reactionChemotherapy is thought to induce a low-grade inflammation

in patients with cancer that may contribute to neurotoxic sideeffects, including cognitive dysfunction (24–26).We analyzed theeffect of cisplatin on the expression of certain prototypic proin-flammatory cytokines and glial activation markers in the hippo-campus and frontal cortex at the mRNA level. RNA was isolatedas described previously (16, 18, 20). In brief, mice were perfusedpostmortem with ice-cold PBS, and the hippocampus andprefrontal cortex were dissected from whole brain. RNA wasreverse transcribed using the High-Capacity cDNA ReverseTranscription Kit (Applied Biosystems/Thermo Fisher Scientif-ic). The Prime-Time qPCR Assays used were IL1b (NM_008361(1), IL6 (NM_031168(1), TNFa (NM_013693(1), CD11b[Itgam; NM_008401(2)], glial fibrillary acidic protein (GFAP;NM_010277(1), and glyceraldehyde 3-phosphate dehydroge-nase [GAPDH; NM_008084(1); Integrated DNA Technologies].qRT-PCR was performed on a CFX384 Real-Time System (Bio-Rad Laboratories) using TaqMan Universal PCR Master Mix(Applied Biosystems).

ImmunohistochemistryThe integrity of the neurogenic stem cell niche is crucial for

adequate cognitive function (27, 28);wehave found that cisplatinreduces white-matter integrity as analyzed at the level of thecoherency of myelin basic protein (MBP) fibers in the cingulatecortex (2). We therefore performed fixation and immunostainingof neuronal precursors in the subventricular zone (SVZ) anddentate gyrus (DG), as previously described (3). In brief, micewere perfused with 4% paraformaldehyde (PFA). Brains wereremoved and fixed in 4% PFA for 48 hours, then paraffin embed-ded and sectioned at 8 mm for the SVZ and cingulum and 10 mmfor the DG. Sectioned were stained for doublecortin (DCX, 1:50;

PFT-m Protects against Cisplatin-Induced Chemobrain

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Abcam) andMBP (1:100; Abcam). Images were taken at 40� and63� objectives and quantified for positive staining using ImageJ.Cell counting was performed by two experienced and indepen-dent researchers blinded to treatment.

Synaptosome isolationTo study whether cisplatin treatment resulted in altered

mitochondrial morphology at the time of the behavioral anal-yses, we prepared synaptosomes of the brains of mice at 7 to 9days after the last dose of cisplatin. Synaptosomes were isolatedaccording to Kamat and collegues (29). Briefly, after perfusionwith PBS, brains were dissected and homogenized (10% w/v)into a 0.32 mol/L sucrose 4-(2-hydroxyethyl)-1-piperazi-neethanesulfonic acid (HEPES) buffer (145 mmol/L NaCl,5 mmol/L KCl, 2 mmol/L CaCl2, 1 mmol/L MgCl2, 5 mmol/Lglucose, 5 mmol/L HEPES, pH 7.4) using a glass Douncehomogenizer with approximately 10 up-and-down strokes. Thebrain lysate was centrifuged at 4�C for 10 minutes at 1,000 � g.The supernatant was extracted and diluted 1:1 with 1.3 mol/Lsucrose HEPES buffer (final concentration of 0.8 mol/Lsucrose) and centrifuged at 4�C for 30 minutes at 20,000 �g. The pellet containing isolated synaptosomes was resus-pended in base media (Seahorse Biosciences/Agilent Technol-ogies) supplemented with 11 mmol/L glucose, 2 mmol/Lglutamine, and 1 mmol/L pyruvate for oxygen-consumptionanalysis or 2% glutaraldehyde plus 2% PFA in PBS for trans-mission electron microscopy.

Transmission electron microscopySimilar to methods we have previously described (3), synapto-

somes were isolated and fixed in a solution containing 2%glutaraldehyde plus 2% PFA in PBS for at least 24 hours. Sampleswere then fixed with a solution containing 3% glutaraldehydeplus 2% PFA in 0.1 mol/L cacodylate buffer, pH 7.3, then washedin 0.1 mol/L sodium cacodylate buffer and treated with 0.1%Millipore-filtered cacodylate-buffered tannic acid, post-fixed with1%buffered osmium tetroxide for 30minutes, and stained en blocwith 1% Millipore-filtered uranyl acetate. The samples weredehydrated in increasing concentrations of ethanol, infiltrated,and embedded in LX-112 medium. The samples were polymer-ized in a 60�C oven for approximately 2 days. Ultrathin sectionswere cut in a Leica Ultracut microtome (Leica Microsystems Inc.),stained with uranyl acetate and lead citrate in a Leica EM Stainer,and examined in a JEM 1010 transmission electron microscope(JEOL USA, Inc.) at an accelerating voltage of 80 kV. Digitalimages were obtained using AMT Imaging System (AdvancedMicroscopy Techniques Corp.). Quantifications were performedby two independent researchers using ImageJ.

Oxygen consumption rateTo determine whether the mitochondrial morphologic abnor-

malities induced by cisplatin treatment and the protective effectsof PFT-m were associated with changes in mitochondrial bioen-ergetics, we analyzed synaptosomal oxygen consumption rates(OCR) using the Seahorse XFe 24 analyzer. Synaptosomes wereisolated and plated at 50 mg of total protein on a Seahorse XFe 24microplate (Seahorse Biosciences) precoatedwithGelTrex (1hourin 37�C; Life Technologies/Thermo Fisher Scientific). The platewas centrifuged at 600� g for 30minutes and then allowed to restat 37�C for 30 minutes. The assay cartridge was loaded with

4 mmol/L oligomycin in Port A, 6 mmol/L carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone (FCCP) in Port B, and 2mmol/L of rotenone and 2 mmol/L of antimycin A in Port C. Thedecrease in OCR in response to addition of the complex-IIIinhibitor oligomycin represents oxygen consumption related toATP production, whereas the uncoupler FCCP induces maximalOCR. The addition of rotenone/antimycin A inhibits all mito-chondrial respiration, leaving only non-mitochondrial oxygenconsumption. All values were normalized to mitochondrial-dependent respiration.

Mitochondrial isolationTo investigate the mechanism underlying the protective effect

of PFT-m, we examined the effect of cisplatin on mitochondrialp53 and on HSP70, a secondary target of PFT-m in proliferatingtumor cells (30, 31). Because it is generally accepted that increasesin mitochondrial p53 induce apoptosis through mitochondrialouter membrane permeabilization, leading to activation of cas-pase-3 and subsequent apoptosis (32), we also examined theeffect of cisplatin treatment on brain cytosolic cytochrome-c andactivation of caspase-3.

Brains were pulverized using liquid nitrogen-cooled mortarand pestle. Approximately 100 mg of brain sample was homog-enized in 700 mL of ice-cold buffer containing 70mmol/L sucrose,210 mmol/L mannitol, 5 mmol/L HEPES, 1 mmol/L ethylene-diaminetetraacetic acid (EDTA), and protease and phosphataseinhibitors. Homogenates were incubated on ice for 30 minutes,followed by centrifugation at 800 � g for 10 minutes at 4�C.Collected supernatant 1 was centrifuged at 10,000 � g for 10minutes at 4�C toobtain cytoplasmic fraction (supernatant 2) anda mitochondrial pellet.

Mitochondrial protein was obtained by resuspending mito-chondrial pellets in ice-cold buffer containing 50 mmol/L Tris,pH 8.0, 1 mmol/L EDTA, 150 mmol/L NaCl, and proteaseand phosphatase inhibitors, followed by sonication. After incu-bation on ice for 30 minutes, homogenates were centrifugedat 13,000 � g for 15 minutes at 4�C, and the supernatant wasused as mitochondrial protein. Protein concentration was mea-sured using Bradford assay, according to the manufacturer'sinstructions.

Western blot analysisProteins (35 mg total protein per sample) were separated on

10% SDS-PAGE gel and transferred onto polyvinylidene difluor-ide membranes (Millipore). Membranes were blocked with 5%nonfat dry milk in 0.1% Tween-PBS (TBST) for 1 hour at roomtemperature and incubated overnight at 4�C with primary anti-bodies against p53 (1:500; Cell Signaling Technology, Inc.),HSP70 (1:500; Santa Cruz Biotechnology, Inc.), cytochrome-coxidoreductase (COX-IV; 1:1,000, Invitrogen/Thermo Fisher Sci-entific), cleaved caspase-3 (1:1,000, Cell Signaling Technology,Inc.), cytochrome-c (1:1,000, BD Biosciences), Histone H1(1:800, Abcam), and horseradish peroxidase (HRP)–conjugatedmouse anti–b-actin (1:5,000, Sigma-Aldrich) in 5% BSA/TBST.Membranes were washed in TBST and then incubated with HRP-conjugated secondary antibodies, either mouse immunoglobulin(Ig)G or rabbit IgG (Jackson ImmunoResearch, West Grove, PA;1:5,000 in 5% nonfat dry milk/TBST). HRP-conjugated mouseb-actin (Sigma-Aldrich) was incubated for 1 hour at room tem-perature, washed, and developed. Specific bands were detectedwith Amersham ECL Western Blotting Detection Reagent (GE

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Healthcare Bio-Sciences) and captured in the LAS system usingImage Quant software for quantification of bands.

Statistical analysisAll data are presented as mean � SEM. Data were analyzed

using GraphPad Prism 6 (GraphPad). One-way or two-wayANOVA was used with or without repeated measure to test forstatistical significance where applicable. Post hoc pair-wise,multiple comparisons were performed using the Tukey testwhen needed. Differences were considered statistically significantat P < 0.05.

ResultsEffect of PFT-m on cisplatin-induced cognitive impairment

Cisplatin (2.3mg/kg)orPBSwas administered intraperitoneallydaily for 5 days, followed by a 5-day rest period and another 5-daycycle of cisplatin. To investigate the effects of PFT-m on cognitivefunction, PFT-m (8 mg/kg) or vehicle was administered intraper-itoneally 1 hour before cisplatin. The data presented in Fig. 1A andC demonstrate that performance in the NOPR and Y-maze testswas impaired in cisplatin-treated mice, whereas coadministrationof PFT-m preventedCICI completely. No differences were observedin the total time the animals interacted with objects in the NOPR,indicating that cisplatin had no effect on interest in the objects oron locomotor activity (Fig. 1B). Consistently, locomotor activity inthe home cage was not affected by cisplatin (Fig. 1D). PFT-mtreatment did not prevent cisplatin-induced loss of body weight(Fig. 1E). Cisplatin (in the presence or absence of PFT-m) did notinduce depressive-like behavior in comparison with PBS-treatedanimals, as analyzed by sucrose preference (saline: 88.1 � 1.4%;cisplatin: 90.9 � 1.3%) and forced-swim tests (saline: 55.2 � 8.4seconds; cisplatin: 50.7 � 11.9 seconds).

Effect of cisplatin and PFT-m on neuronal precursors in thebrain

The results in Fig. 2 show that our regimen of cisplatin treat-ment led to a decrease in the number of DCXþ neural progenitorsin the two major neurogenic niches in the brain. After 2 cycles ofcisplatin, the number of DCXþ neuroblasts in the DG of thehippocampus decreased by 70% (Fig. 2A–E). The number ofDCXþ cells in the SVZ decreased by 30% (Fig. 2F–I). Preventivetreatment with PFT-m completely prevented the loss of DCXþ

neuroblasts in both the DG and SVZ (Fig. 2D and I).

Effect of PFT-m on cisplatin-induced changes in white-matterintegrity

As shown in Fig. 3A–E, the two 5-day cycles of cisplatin used inthe current study increased white-matter coherency, suggesting aloss of arborization and lateral fibers. The latter assumption issupported by the skeletonizedMBPþ structures shown in Fig. 3A–D0. Coadministration of PFT-m prevented the cisplatin-inducedincrease inwhite-matter coherency in the cingulate cortex (Fig. 3Dand D0), implying that PFT-m protects against structural white-matter damage induced by cisplatin.

Effect of cisplatin on the inflammatory response in the brainIn our analysis of the effect of cisplatin on the expression of

IL1b, IL6, and TNFa, GFAP, and CD11b, we did not detect anysignificant increase in cytokinemRNA after cisplatin treatment, asmeasured 24 hours after the first injection or after the behavioral

testing 1 week after the last dose of cisplatin (Table 1). Takentogether, these data indicate that CICI in our mouse model is notassociated with typical (neuro)inflammation.

Early effects of cisplatin and PFT-m on p53 translocation tomitochondria and caspase-3 activation

Figure 4A shows that the protein level of p53 in isolated brainmitochondrial fractions was increased at 4 and 24 hours after asingle dose of cisplatin, as determined by Western blotting.Coadministration of PFT-m completely prevented this cisplatin-induced increase in brain mitochondrial p53 (Fig. 4B). Cisplatintreatment did not lead to an increase in brain cytosolic cytochrome-cor to activation of caspase-3 (Fig. 4C). The results in Fig. 4D

Figure 1.

Effect of PFT-mon cisplatin-induced cognitive impairment. Animalswere treatedwith cisplatin and PFT-m for two 5-day cycles. A, NOPR was performed 7 daysafter the last injection, and discrimination index was calculated. B, Totalinvestigation time between both objects in the NOPR was recorded. Results areexpressed as means� SEM; n¼ 8. *, P < 0.05. C, Y-maze was performed 7 daysafter the last injection, and percentage of perfect alternation was calculated.Results are expressed as means � SEM; n ¼ 4. *, P < 0.05. D, Spontaneouslocomotor activity (total distance traveled) was measured. Results areexpressed as means � SEM; n ¼ 8. E, The percentage of baseline body weightwas recorded. Results are expressed as means� SEM; n¼ 12; � , P < 0.05 saline/PBS versus saline/cisplatin; $, P < 0.05 PFT-m/PBS versus PFT-m/cisplatin.






confirm the purity of the subcellular fractions and show that therewas no effect of cisplatin on cytosolic/nuclear p53. We did notdetect changes in brain HSP70 after cisplatin treatment (Fig. 4E).

Effects of cisplatin and PFT-m on mitochondrial morphologyand mitochondrial function

The results in Fig. 5A–F demonstrate that synaptosomal mito-chondria of brains of cisplatin-treated animals were swollenand had abnormal cristae when compared with synaptosomalmitochondria from PBS-treated control mice. PFT-m coadminis-tration prevented this long-lasting abnormal morphology of themitochondria induced by two cycles of cisplatin (Fig. 5D–F).

Basal respiration of the synaptosomes was not affected bycisplatin (Fig. 5G), and there was no effect of cisplatin on theoxygen consumption related to ATP production (Fig. 5H). How-ever, the maximal respiration as determined after addition of themitochondrial uncoupler FCCP was significantly lower in synap-tosomal fractions of cisplatin-treated mice versus PBS-treated

control mice (Fig. 5I). Consequently, the difference betweenmaximal and basal respiration, also known as spare respiratorycapacity, was decreased in the synaptosomes from cisplatin-trea-ted mice (Fig. 5J). Coadministration of PFT-m prevented thecisplatin-induced impairment in maximal and spare respiratorycapacity (Fig. 5G–J). No differences in proton leak betweencisplatin-treated and PBS-treated mice were observed (saline:0.28 � 0.01 pmol O2/mg of protein; cisplatin 0.33 � 0.11 pmolO2/mg of protein). We did not detect cisplatin-induced changesin lipid peroxidation as quantified using a TBARS assay (saline:4.32� 0.24 nmol/L/mg total protein; cisplatin: 4.39� 0.38 nmol/L/mg total protein).

Effect of PFT-m on the sensitivity of the tumor tochemoradiation

We used a heterotopic syngeneic murinemodel of HPV-relatedhead and neck cancer to assess whether PFT-m might potentiallyinterfere with the tumor's response to chemoradiation (cisplatinþ

Figure 2.

Effect of cisplatin and PFT-m on neuronal precursors cells in the brain. Animals were treated with cisplatin and PFT-m for two 5-day cycles. A and F, PBS-treatedanimals. B and G, Cisplatin-treated animals. C and H, PFT-m–treated animals. D and I, PFT-m/cisplatin-treated animals. DCXþ neuronal precursors wereobserved in the DG of the hippocampus (A–D) and SVZ (F–I). A0–D0, �40 magnification of the DG tip. DCXþ cells were counted in the DG (E) and the SVZ (J).� , P < 0.05.

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radiation,CRT).CRT inhibited tumorgrowth as expected (Fig. 6A).The response toCRTwasnot adversely affectedbyPFT-m treatment.Furthermore, the addition of PFT-m to the cancer treatment did notsignificantly influence body-weight loss (Fig. 6B).

DiscussionWe show for the first time that the inhibitor of mitochondrial

p53 accumulation, PFT-m, prevented cisplatin-induced cognitiveimpairment or "chemobrain" in mice, as assessed in the NOPRand Y-maze tests (Fig. 1A andC). The impaired cognitive functionobserved in cisplatin-treated mice was associated with impaired

cerebral neurogenesis (Fig. 2) and structural abnormalities incerebral white matter that were prevented by PFT-m (Fig. 3).Mechanistically, we show that cisplatin increased brain mito-chondrial p53 levels (Fig. 4) and induced abnormal mitochon-drial morphology and impaired the spare respiratory capacityof synaptosomal mitochondria, as shown by Seahorse analysis(Fig. 5). Notably, PFT-m prevented not only the functional andstructural abnormalities induced by cisplatin, but also the cis-platin-induced increase in mitochondrial p53 and the abnormal-ities in brain mitochondrial function and morphology.

Although (neuro)inflammation has often been proposed as amechanism for chemotherapy-induced neurotoxicities, we did

Figure 3.

Effect of cisplatin and PFT-m on white-matter coherency in the brain. Animals were treated with cisplatin and PFT-m for two 5-day cycles. A, PBS-treated animals.B, Cisplatin-treated animals. C, PFT-m–treated animals. D, PFT-m/cisplatin–treated animals. A–D, MBP staining was performed at the cingulum of thebrain. A0–D0, Skeletonized display of the MBP staining. E, Coherency index was calculated using ImageJ with the OrientationJ plugin. Skeletonized structureswere created using ImageJ by converting the sample image to a binary 8-bit image and then processed with the Skeletonize (2D/3D) plugin. � , P < 0.05.

Table 1. Effect of cisplatin on the inflammatory response in the brain

Region Gene Saline Cisplatin

24 Hours post-treatmentHippocampus IL1b 1.00 (0.10) 0.75 (0.05)a

IL6 1.00 (0.12) 1.15 (0.11)TNFa 1.00 (0.13) 0.80 (0.08)GFAP 1.00 (0.06) 0.87 (0.03)a

CD11b 1.00 (0.05) 0.86 (0.04)a

Prefrontal cortex IL1b 1.00 (0.16) 0.97 (0.09)IL6 1.00 (0.06) 0.91 (0.07)TNFa 1.00 (0.37) 0.73 (0.11)GFAP 1.00 (0.06) 0.81 (0.01)a

CD11b 1.00 (0.05) 0.80 (0.17)7 Days post-treatmentHippocampus IL1b 1.00 (0.22) 1.06 (0.25)

IL6 1.00 (0.30) 1.44 (0.28)TNFa 1.00 (0.07) 0.76 (0.05)a

GFAP 1.00 (0.10) 1.03 (0.14)CD11b 1.00 (0.15) 1.03 (0.14)

Prefrontal cortex IL1b 1.00 (0.08) 0.88 (0.13)IL6 1.00 (0.13) 1.15 (0.06)TNFa 1.00 (0.18) 1.07 (0.30)GFAP 1.00 (0.03) 1.20 (0.24)CD11b 1.00 (0.04) 1.02 (0.10)

NOTE: Results are expressed as fold change means (SEM). n ¼ 5–9.aP < 0.05.






not find evidence for increased cytokine production in the brainsof cisplatin-treatedmice 24 hours or 1 week after the last injectionof cisplatin (Table 1). In line with these results, we also did notdetect microglial or astrocyte activation, as analyzed by Iba-1 andGFAP fluorescence, or a change in the morphological appearanceof microglia and astrocytes. Therefore, we suggest that neuroin-flammation does not play a major role in CICI. We cannot

exclude, however, thepossibility of limited region-specific inflam-mation and/or short-lasting inflammation at other time points.

Interestingly, our current data point toward a key role fordamage to brain mitochondria as the mechanism underlyingCICI. It has been shown that cisplatin-induced peripheral neu-ropathy is associated with changes in mitochondrial morphologyin peripheral sensory neurons (3, 13). However, to the best of our

Figure 4.

Acute effects of cisplatin on p53 translocation to themitochondria and caspase-3 activation.A andB,Mitochondrial p53 wasmeasured 4 and 24 hours after cisplatininjection (A) and 4 hours after cisplatin and PFT-m injections (B). COX-IV was used as a protein loading control. Relative expression of p53 was calculated 4and 24 hours after cisplatin treatment. C, The cytosolic fractions were analyzed for the apoptotic markers cytochrome-c and cleaved caspase-3 4, 24, and 72 hoursafter a single dose of cisplatin. "Con" designates positive controls: brain mitochondria fraction for cytochrome-c (top) and whole-cell lysate of H2O2-treatedN2A cells for active caspase-3 (middle).b-Actinwas used as a protein loading control for cytosolic proteins.D,Cisplatin increasesmitochondrial p53without inducingp53 in cytosol or nucleus. Subcellular fractions collected at 4 hours after cisplatin injection were analyzed for p53, Cox-IV (mitochondria), b-actin (cytosol andmitochondria), and Histone H1 (nuclear fraction). E, Cytosolic HSP70. b-actin was used as a protein loading control. Results are expressed as means � SEM;n ¼ 6; � , P < 0.05.

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knowledge, we are the first to show that systemic administrationof cisplatin induces structural changes in synaptosomal mito-chondria (Fig. 5A–F). This abnormal morphology is associatedwith a decrease in mitochondrial bioenergetic function, asreflected by a lower spare respiratory capacity in cerebralsynaptosomes of cisplatin-treated mice (Fig. 5G–J). Spare respi-ratory capacity is viewed as an index of mitochondrial healthand is a measure of how well a cell can produce energy understressful energy-demanding conditions (13, 33). Notably, thereis evidence that decreased spare respiratory capacity is associ-ated with cognitive impairment in rodent models of Alzheimerdisease (34).

In support of our hypothesis that mitochondrial dysfunction isa central mechanism of CICI, we show here that treatment withPFT-m, which prevents mitochondrial p53 translocation, pre-vented the reduction in synaptosomal mitochondrial spare respi-ratory capacity as well as the behavioral deficits, implying a causalrelation between mitochondrial protection and CICI. It remainsto be determined whether this reduction in spare respiratorycapacity in response to cisplatin reflects a general deficit in allmitochondria in thebrainor if it is specific formitochondria in thesynapses. The relatively high mitochondrial density in nerveterminals might render this cellular compartment most sensitiveto cellular stress (33). A decrease in mitochondrial function and

Figure 5.

Effects of cisplatin and PFT-m on mitochondrial morphology. Synaptosomes were isolated from the brains of animals treated with PBS (A), cisplatin (B), PFT-m (C),or PFT-m plus cisplatin (D) for two 5-day cycles. E and F, Mitochondrial length (E) and percentage of atypical mitochondria (F) were quantified. Results areexpressed as means � SEM; n ¼ 8. Oxygen consumption rates were analyzed in isolated synaptosomes using the Seahorse XFe 24 Analyzer. G–J, Basal (G),ATP production-related (H), maximum (I), and spare respiratory capacity (J) were calculated. Results are expressed as means � SEM; n ¼ 8. *, P < 0.05.






spare respiratory capacity has also been associated with decreasedcognitive ability in Alzheimer's disease (35) and aging (36, 37). Inthis respect, it is of interest that advanced neuroimaging hasshown that in cancer patients, the severity of cognitiveimpairment is analogous to multiple years of brain aging (38).

Strom and colleagues (39) originally identified PFT-m as acompound that inhibits accumulationof p53 at themitochondriawithout inhibiting p53 transcriptional activity. These authorsshowed that PFT-m inhibits radiation-induced p53-dependentthymocyte apoptosis in vitro and in vivo. PFT-m was proposed toprotect against apoptosis by reducing the binding of p53 to bothBcl-xL and Bcl-2, resulting in preservation of mitochondrialintegrity (39).

We previously investigated the protective effects of PFT-m in amodel of neonatal hypoxic–ischemic (HI) brain damage. PFT-mwas shown topreventHI-induced loss ofwhite and graymatter andto preserve behavioral features like motoric and cognitive function(15).Mechanistically,HIwas shown to increase early neuronal p53mitochondrial accumulation, reactive oxygen species, and cytosolicaccumulation of pro-apoptotic proteins, as measured by increasedcytosolic cytochrome-c and activated caspase-3 levels (32). Here,we show that administration of cisplatin rapidly increased mito-chondrial levels of p53, which was prevented by co-administrationof PFT-m (Fig. 4A and B). However, the increase in mitochondrialp53 as a result of cisplatin treatment did not initiate a generalapoptotic cascade, because neither cytosolic cytochrome-c noractivated caspase-3 were increased either at 4 or 24 hours aftercisplatin treatment (Fig. 4C). The absence of apoptosis may havebeen caused by the fact that cisplatin does not induce early nuclearfactor-kB activation and subsequent cytokine synthesis, aswas seenearly after HI brain damage at the moment of mitochondrial p53accumulation (32). However, we did observe structural changesand functional mitochondrial impairment after cisplatin treat-ment, all of which were prevented by PFT-m (Fig. 5). Cisplatintreatment did not lead to increases in cytosolic p53. Moreover, asalready shown by Strom and colleagues (39), PFT-m did notinterfere with cytosolic p53 levels.

Therefore, we propose that the cisplatin-induced increase inmitochondrial p53 reduces synaptosomal mitochondrial integ-rity, possibly by decreasing mitochondrial membrane potential(40), and thereby decreasing the availability of cellular energyunder demanding conditions, which may result in oxidativecellular damage in neuronal circuitries engaged in cognition.

At the level of brain structure, we observed cisplatin-inducedabnormalities in white-matter structure in the cingulum that werealso prevented by PFT-m (Fig. 3). Moreover, we recently showedthat cisplatin reduces arborization and spine density of pyramidalneurons in the cingulum (2). Van Schependom and colleagues(41) described that neural disconnections, especially of white-matter tracts connecting both hemispheres seen in cognitivelyimpaired versus cognitively preserved patients with multiplesclerosis, were responsible for the cognitive impairment observedin these patients. Diffusion tensor neuroimaging of the brains oflung cancer patients indicates that cisplatin treatment results in anincreased fractional anisotropy in areas of white matter in thecingulate cortex. Kesler and colleagues (38, 42) recently publishedadvanced neuroimaging data of patients treated with the anthra-cycline doxorubicin and concluded that lower memory perfor-mance was associated with decreased functional and structuralconnectivity. In line with these findings, we demonstrate here inmice that cisplatin treatment resulted in increased overall coher-ency of white-matter fibers in the cingulum, a feature underlyingthe changes in fractional anisotropy in patients, which maycontribute to the observed cognitive impairment. We do not haveevidence for an overall decrease in intensity or area of MBPstaining, indicating that the major fibers are not lost. Therefore,we conclude that cisplatin treatment leads to a loss of MBPpositive branches rather than an overall loss of MBPþ tissue. Ina previous study, we showed that cisplatin treatment reducesdendritic branching, as assessed at the individual neuron levelusing Golgi staining to visualize dendrites (2). It is likely that thereduction in the complexity of the organization of MBP-stainingin the same area is a consequence of the loss of dendritic com-plexity in response to cisplatin. If so, it is likely that the primary

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Figure 6.

Effects of PFT-m on the tumor's response to cancer therapy. Mice were implanted with a syngeneic murine model of HPV-positive head and neck cancer and weretreated once weekly for 3 weeks with a regimen of cisplatin þ local radiation (CRT). This regimen of CRT effectively delayed tumor growth, and this effectwas not adversely affected by concurrent treatment with PFT-m (A). A repeated-measures ANOVA revealed a significant effect of time, P < 0.01, with no significanteffect of group or group-by-time interaction. As anticipated, CRT resulted in mild weight loss that was not significantly exacerbated by PFT-m treatment(B). A repeated-measures ANOVA showed a significant effect of time, P < 0.001, with no significant effect of group or group-by-time interaction. Results areexpressed as means � SEM; n ¼ 6. Arrows, day of CRT treatment; gray bars, PFT-m treatment.

Chiu et al.





damage is caused by increased p53 binding to neuronal mito-chondria, leading to mitochondrial insufficiency, dendritic dam-age, and subsequent loss ofMBP-positive small fibers.However, itis possible that there is a direct effect of cisplatin onmitochondriain oligodendrocytes that could contribute to abnormal patterns ofMBP staining in cisplatin-treated mice. For example, it has beenshown that cisplatin reduces survival of oligodendrocytes in vitroand in vivo (43). Therefore, we cannot exclude that in vivo PFT-mprevents the mitochondrial accumulation of p53 in oligoden-drocytes as well.

Although we do not have evidence for global activation ofapoptotic pathways after cisplatin treatment, we cannot excludethat a limited number of cells, such as proliferating cells in thestem cell compartment, undergo apoptotic cell death.

Adult neurogenesis is required for integration of circuitryduring learning andmemory, as reviewed by Zhao and colleagues(27), and the sharp decrease in DCXþ cells in the neurogenicniches induced by cisplatin may well contribute to the observedcognitive deficits. Interestingly, the decrease in the number ofDCXþ neuroblasts as a result of cisplatin treatment was alsocompletely prevented by PFT-m (Fig. 2). Preliminary data indicatethat the number of uncommittedNestinþGFAPþ stem cells in theSVZ did not change under influence of cisplatin, which mightindicate that cisplatin selectively affects migrating proliferatingneuroblasts (DCXþ) and/or that the differentiation of uncom-mitted stem cells to neuroblasts is arrested possibly in favor ofglial cells. In this respect, the study of Wang and colleagues (44),which showed that oxidative damage to mitochondrial DNA inneural stem cells inhibits neural stem cell differentiation andrepresents a primary signal for elevated astrogliosis, is of interest.Because cisplatin has been shown to damage mtDNA in neurons(45), the sharp decrease in DCXþ cells may well be a reflection ofthe relatively high sensitivity of neural stem cells for oxidativedamage tomtDNA, in comparisonwith adult neurons. These datamay also imply that the differentiation of the neural progenitors isinhibited in favor of glial differentiation.

On thebasis of our current data,wepropose thatmitochondrialprotectants may represent an attractive, efficacious treatment forchemotherapy-induced neurotoxicities. It remains to be deter-mined whether the observed reduction in neuronal precursorsand the white matter abnormalities are a direct consequence ofmitochondrial abnormalities in the synaptosomes or are due toeffects of cisplatin on the neuronal stem cells and oligodendro-cytes themselves. We recently showed that PFT-m also preventstaxane-induced and cisplatin-induced peripheral neuropathy (3).Emerging data suggest that PFT-m acts as an anticancer agent (30,46–48). Consistently, we show here in vivo that PFT-m does notinterfere with the antitumor efficacy of a cisplatin-based chemor-adiation regimen in a heterotopic murine model of HPV-relatedhead and neck cancer. Mechanistically, it has been shown thatPFT-m promotes tumor-cell death in vitro and in vivo via anautophagy-promoting mechanism involving interaction with the

stress proteinHSP70 and lysosome-associatedmembrane protein2 in rapidly proliferating tumor cells (30, 31, 46, 48,49). Theseeffects of PFT-m on tumor cells are independent of p53.

Taken together, our results indicate that systemic administrationof a mitochondrial protectant such as PFT-m may not only becapable of protecting against peripheral and central neurotoxicities,but alsowill act as an adjuvant therapy topromote tumor cell deathwithout interfering with the efficacy of the cancer therapy. Wepropose that prevention of mitochondrial p53 accumulation dueto chemotherapy is an important therapeutic target to preventcancer treatment-induced peripheral and central neurotoxicities.The addition of PFT-m to cisplatin may well represent a promisingnovel therapeutic approach to help fight tumor growth and thedevelopment of neurotoxicities in patients with cancer.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: G.S. Chiu, M.A. Maj, R. Dantzer, A. Kavelaars,C.J. HeijnenDevelopment of methodology: G.S. Chiu, M.A. MajAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.):G.S. Chiu, M.A. Maj, S. Rizvi, E.G. Vichaya, G. LaumetAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):G.S.Chiu,M.A.Maj, S. Rizvi, E.G. Vichaya,G. Laumet,A. Kavelaars, C.J. HeijnenWriting, review, and/or revision of the manuscript: G.S. Chiu, R. Dantzer,E.G. Vichaya, A. Kavelaars, C.J. HeijnenAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): G.S. ChiuStudy supervision: G.S. Chiu, A. Kavelaars, C.J. Heijnen

AcknowledgmentsThe authors are greatly indebted to Dr. Kathy Mason and Jessica M. Molk-

entine for performing the CRT on mice with the heterotopic HPV-tumor. Wethank Dr. John Lee (Sanford Research) for providing the MEER cell line for thetumor model. Processing for electron microscopy was performed by KennethDunner, Jr. at theHigh-Resolution ElectronMicroscopy Facility atMDAndersonCancer Center. We thank Jason Lu and Siddiqua Noor for quantification ofDCXþ staining and quantification of mitochondrial morphology. The authorsacknowledge the editorial assistance of Jeanie F. Woodruff, BS, ELS.

Grant SupportThis research was funded by grants from the NIH (R21 CA183736 to C.J.

Heijnen and R. Dantzer, R01 CA193522 to R. Dantzer, and MD AndersonCancer Center Support Grant P30 CA016672, which supports the institution'sHigh-Resolution ElectronMicroscopy Facility) and fromTheUniversity of TexasMDAnderson Cancer Center (the Institutional Research Grant Program and theKnowledge Gap Chemobrain Project of the Moon Shots Program).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received July 6, 2016; revised October 6, 2016; accepted November 4, 2016;published OnlineFirst November 22, 2016.

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2017;77:742-752. Published OnlineFirst November 22, 2016.Cancer Res Gabriel S. Chiu, Magdalena A. Maj, Sahar Rizvi, et al. Neuronal Mitochondrial Function

Prevents Cisplatin-Induced Chemobrain by PreservingµPifithrin-

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ﬁthrin-m Prevents Cisplatin-Induced Chemobrain by ... · Piﬁthrin-m Prevents Cisplatin-Induced Chemobrain by Preserving Neuronal Mitochondrial Function Gabriel S. Chiu, Magdalena