MEDROXYPROGESTERONE ACETATE INDUCES C6 GLIOMA CHEMOSENSITIZATION VIA ANTIDEPRESSANT-LIKE LYSOSOMAL PHOSPHOLIPIDOSIS/MYELINOSIS IN VITRO

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MEDROXYPROGESTERONE ACETATE INDUCES C6 GLIOMACHEMOSENSITIZATION VIA ANTIDEPRESSANT-LIKE LYSOSOMALPHOSPHOLIPIDOSIS/MYELINOSIS IN VITROMeric A. Altinoz a; Gunduz Gedikoglu b; Aydin Sav c; Emin Ozcan d; Kursat Ozdilli a; Ayhan Bilir e; Rolando F.Del Maestro f

a Institute of Health, Halic University, Istanbul, Turkey b Pediatric Oncology, Halic University, Istanbul, Turkey c

Neuropathology, Marmara University, Istanbul, Turkey d 3rd Neurology Clinic, Bakirkoy State and EducationHospital and Research Hospital for Psychiatric Diseases, Istanbul, Turkey e Department of Histology andEmbryology, Istanbul Medical Faculty of Istanbul University, Istanbul, Turkey f Brain Tumor ResearchInstitute, Montreal Neurological Institute, Montreal, Quebec, Canada

Online Publication Date: 01 October 2007

To cite this Article Altinoz, Meric A., Gedikoglu, Gunduz, Sav, Aydin, Ozcan, Emin, Ozdilli, Kursat, Bilir, Ayhan and Maestro, RolandoF. Del(2007)'MEDROXYPROGESTERONE ACETATE INDUCES C6 GLIOMA CHEMOSENSITIZATION VIA ANTIDEPRESSANT-LIKE LYSOSOMAL PHOSPHOLIPIDOSIS/MYELINOSIS IN VITRO',International Journal of Neuroscience,117:10,1465 — 1480

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Intern. J. Neuroscience, 117:1465–1480, 2007Copyright C© 2007 Informa Healthcare USA, Inc.ISSN: 0020-7454 / 1543-5245 onlineDOI: 10.1080/00207450701540062

MEDROXYPROGESTERONE ACETATE INDUCESC6 GLIOMA CHEMOSENSITIZATION VIAANTIDEPRESSANT-LIKE LYSOSOMALPHOSPHOLIPIDOSIS/MYELINOSIS IN VITRO

MERIC A. ALTINOZ

Institute of Health, Halic UniversityIstanbul, Turkey

GUNDUZ GEDIKOGLU

Pediatric OncologyHalic UniversityIstanbul, Turkey

AYDIN SAV

Neuropathology, Marmara UniversityIstanbul, Turkey

EMIN OZCAN

3rd Neurology ClinicBakirkoy State and Education Hospital andResearch Hospital for Psychiatric DiseasesIstanbul, Turkey

KURSAT OZDILLI

Institute of HealthHalic UniversityIstanbul, Turkey

Received 4 April 2007.Address correspondence to Meric A. Altinoz, M.D., Halic University, Institute of Health,

Mecidiyekoy, Istanbul, Turkey. E-mail: [email protected]

1465

Downloaded By: [Canadian Research Knowledge Network] At: 19:52 16 March 2009

1466 M. A. ALTINOZ ET AL.

AYHAN BILIR

Department of Histology and EmbryologyIstanbul Medical Faculty of Istanbul UniversityIstanbul, Turkey

ROLANDO F. DEL MAESTRO

Brain Tumor Research InstituteMontreal Neurological InstituteMontreal, Quebec, Canada

The authors have previously shown that medroxyprogesterone acetate (MPA)inhibits growth and increases drug sensitivity in C6 glioma with myeloidbodies. Myeloid bodies can occur in cells either due to robust toxicity withmitochondrial membrane disruption or due to milder events such as seenin lysosomal-phospholipidosis. Exact patterns of myelinosis accompanying toMPA chemo-sensitization is important, because uncoupling of nuclear versusmitochondrial toxicity of anti-neoplastics by MPA would lead to safer employmentof glioma chemotherapy with reduced neurotoxicity. By monitoring and comparingcell kinetics with fine structural features of cell death, the authors estimatedsubcellular effects accompanying growth-inhibitory drug actions in C6 glioma.The analysis revealed that MPA induced mainly lysosomal phospholipidosis, whileinhibiting clonogenicity alone and augmenting procarbazine efficacy. It inducedapoptosis in combination with cisplatin. It reduced mitochondrial-damage-basedearly cytotoxicity of methotrexate, yet it did not hinder its anti-clonogenicefficacy. Progesterone analogues—similar to antidepressants—inhibit cholesterolesterification, and this efficacy relates with their P-glycoprotein inhibition. Reducingesterification and plasma-membrane localization of cholesterol may lead MPAinduction of lysosomal phospholipidosis, growth indolency, and drug sensitizationin glioma.

Keywords chemotherapy, glioma, medroxyprogesterone

INTRODUCTION

Despite extensive investigations and treatment strategies, prognosis of malig-nant glial tumor remains poor, and cytotoxic chemotherapy often leads severeneurotoxicity (O’Sobe et al.,). Thus, novel brain tumor treatment strategiesshould be developed, which would selectively inhibit growth or induce celldeath in glial tumors, while saving healthy neural parencyhma. To achieve thisaim, selective ultrastructural/biochemical changes leading to clonogenic and


MPA INDUCES C6 GLIOMA CHEMOSENSITIZATION 1467

apoptotic cell death of tumoral cells should be investigated in further detail.This study aimed to compare in vitro ultrastructural changes in C6 gliomacells accompanying to the long-term clonogenic cell death against to thoseaccompanying to early necrapoptotic signs. Therefore, methotrexate, cisplatin,and procarbazin were selected as cytotoxic drugs, and medroxyprogesteroneacetate as a non-toxic growth inhibitory agent, which is used in breast cancertherapy and that could also induce drug sensitization (Zibera et al., 1995).

MATERIALS AND METHODS

Cell Culture

C6 cells established from ATCC (American Type Culture Collection) weremaintained using RPMI-1640 (Biological Industries, Israel) with 15% heatinactivated fetal calf serum (FCS), 0.2 mM glutamine, 50 µg/ml neomycin, and100 µg/ml streptomycin. The flasks were kept in an incubator with humidifiedatmosphere of 5% CO2 at 37◦C.

Drugs, Plating Efficacy, and Treatment Schedule

Single 1 µg/ml dose of MPA (2.6 µM) was used. Doses of cisplatin,(Cisplatyl R©, Rhone Poulenc) methotrexate (Roger Bellon) and procarbazin(Natulan, Roche R©) were 0.1 (0.33 µM), 0.3 (0.66 µM), and 50 µg/ml (0.19mM), respectively. Cells prepared in 5 ml of RPMI-1640 (Biological Industries,Haemec, Israel) were plated into each well of a six-well plate in 1 × 105/mlconcentration with 100% vitality. All drugs were given in equal volumes of100 µl into corresponding plates 24 h after plating. Continuous incubationwas applied and the drugs were kept for 96 h; thereafter cells were harvestedwith trypsin-EDTA solution C (Biological Industries, Israel) and vital cellnumbers were determined in a hemocytometer using trypan blue exclusionmethod.

Soft Agar Colony Assay

Stock agar solution (with 0.3% concentration) was prepared at 45◦C. Fourvolumes of RPMI-1640 were then rapidly mixed with one volume of stockagar solution and 1 ml of this solution was put into each well of a six-well plateas an under layer. Plates were then incubated at 4◦C for 15 min. For each well103, 100% vital cells were suspended in 1 ml of RPMI-1640 medium.



They were then mixed with 2 ml of the mixture of 4 volumes of RPMIplus 1 volume of stock agar solution. One milliliter of this suspension wasthen put onto each underlayer of the 6-well plates, and drugs were added ina final volume of 100 µl to the corresponding groups. The 6-well plates werethen kept in an incubator in a humidified atmosphere of 4.5% CO2 and air mixfor one week. Colonies containing more than 50 cells were counted under aninverted microscope. Colony forming inhibition was calculated according thefollowing formula: Colony forming inhibition% = 100 × (1- colony numberof drug treated group/colony number of control).

3H-Thymidine Labelling Index

Half an hour before the end of each 24-h period of monitoring of the plating,cells were incubated with 1 µCi/ml 3H-thymidine (Amersham, England,sp. act. 185 GBq/mmol, 5Ci/mmol). Following 30 min of incubation, cellswere trypsinized and smear slides were prepared. Then slides were fixedwith Carnoy’s fixative (ethanol/glacial acetic acid at a ratio of 3:1). Inboundradioactive materials were washed twice with 2% perchloric acid at 4◦C for30 min. To determine the thymidine labelling index, cover slips were coatedwith gel emulsion film (Ilford K2, England). After 3 days of exposure at 4◦C,autoradiograms were washed with D-19 developer and slides were evaluatedafter Giemsa staining. With 100 × 12.5 magnification 3 × 103 cells wereevaluated in 100 different areas, cells with at least 5 grain in their nuclei wereconsidered to be labelled. Background grain number was less than one per area.

Transmission Electron Microscopy Analysis

Cells were collected by centrifugation. Pellets were fixed with 2% glutaralde-hyde in 0.1 M sodium cacodylate for 1 h at 4◦C. Samples were then washedtwice for 10 min with 0.1 M sodium cacodylate with a pH value of 7.4. Theywere then fixed with 2% osmium tetroxide in 0.1 M sodium cacodylate for 1 h atroom temperature. Then, samples were washed 3 times for 5 min each time withthe same buffer. Cells were exposed to 1% uranyl acetate for 1 h and washedagain 3 times for 10 min again each time with the same buffer. Samples weredehydrated via a graded ethanol series. After embedding in Epon 812, sampleswere sectioned to a thickness of 700–800 Angstrom on a Richert Ultramicrotom(OMS, Austria). Sections were post-stained with 5% uranyl acetate for 30 minfollowed by Reynold’s lead citrate incubation. Samples were then examinedwith a Jeol-100 C transmission electron microscope. For quantification of



the general percentage of the presence of myelin like multilamellar bodies,mitochondrial whorls, apoptotic or necrotic figures, 30 different areas with5000 × magnification were analyzed. For determining the intensity of thesebodies per cell, higher magnifications (> 2,0000 ×) were applied.

Statistics

Plating efficacy and thymidine labeling indices were compared using two-tailedStudent’s t-test, colony growth values were compared via Wilcoxon matched-pairs signed-ranked test.

RESULTS

Qualitative and Quantitative Changes in Cellular Ultrastructure

As seen in Figure 1a and b, C6 glioma cells bear oval nuclei (4 to 7 µm) anda cytoplasm rich in secret vacuoles. Filopodia were notified. As seen in Figure1c, treatment with MPA resulted in autophagosomes containing ring-forminginclusion particles. Multilamellar myeloid bodies (0.7 to 0.9 µm) surroundedby electron loose halo were noteworthy. As seen in Figure 2a, b, and c, treatmentwith methotrexate resulted in most dramatic ultrastructural changes; most cellswere necrotic and the remaining few were apoptotic. Condensing chromatinformed large or small clumps protruding the nuclear membrane. Vacuoles(0.9–1.4 µm) with whorled or dispersed content were abundant. In highermagnification, proximity of these whorls to the sequestered mitochondria werenotified. Their presence has not always coincided with latest stages of chromatincondensation.

During MTX-induced cell death, two different processes are occuringsimultaneously in nuclear demise of apoptotic cells. One is from outside toinside that tries to divide the nucleus into two compartments, another forceoriginates from within the nucleus to sweep away chromatin material towardthe nuclear membrane. Thus, the almost chromatin-free nucleus seen in Figure3c is presumably reflecting one step later stage of the process seen in cellnucleus of Figure 3d. Another feature is the presence of fibrillar remnants ofthe nucleolus until the late stages of chromatin condensation. Vacuoles withwhorled figures were also seen in cisplatin treated cells, albeit rare and less percell. As seen in Figure 4a, procarbazin treatment induced single deep nuclearindentations, autophagosome formation and a few multilammellar bodies. Withcombined treatment of MTX and MPA, cell cytoplasm showed a morphologyresembling lipid vacuolization or swelling of endoplasmic reticulum cisternae.



Figure 1. (a and b) Control: C6 glioma cells bear oval nuclei (n) with around 4 to 7 µm diameter,and a cytoplasm rich from secret vacuoles, with electron loose (s′—0.8 µm) or dense content(s′ ′—0.6 µm). Gap junction like connections between adjacent cells (double arrowheads), andfilopodia among single dispersed ones (f) were notified. (c and d) MPA: Multilamellar myeloidbodies (0.7 to 0.9 µm, asterisk in c), and autophagosomes (m—0.7–1.33 µm) containing myeloidspherical inclusion particles, which are surrounded with intense osmiophilic sheet (asterisk in d),giving a ringlike appearance.

Percentage Inhibiton of Colony Growth (%ICG)—Figure 5a

Colony growth inhibition was 61.33%, 57.33%, 45.33%, and 66.67% for MPA,procarbazin, cisplatin and methotrexate groups, respectively. When MPA wascombined with drugs, 68.33%, 56.33%, and 62.67% inhibition were found for



Figure 2. (a, b, c) MTX: Dramatic ultrastructural changes were seen, parallel to drastic cellkinetic perturbations. Most cells were either necrotic or showed signs of several hallmarks ofapoptosis. Condensing chromatin formed large or small (thick or thin double arrow heads in 2a)clumps protruding nuclear membrane. Vacuoles (v, 0.9–1.4 µm) with whorled or dispersed contentwere abundant. In addition to chromatin condensation, nuclear indentation occured in some cellssimultaneously (four oblique arrows indicating nuclear membrane invaginations—in 2a). In somecell nuclei, fibrous remnants of nucleolus (nu—in 2b) were observed, when chromatin condensationhas almost finished, with protruded nuclear membrane (surrounding multiple asterisks). Some cellshad chromatin figures resembling to a apoptotic process but with disrupted outer membrane (asteriskin 2c), secondary necrosis.



Figure 3. MTX: Whorled figures (w, 0.9–1.2 µm) in greater magnification. Note their proximityto sequestered mitochondria. (b) MPA + MTX: Note the segregation of inner and outer nuclearmembranes, and nuclear lamina capping over nuclear pores (oblique arrows). Inside the nucleus,fibrillar (f) nucleolar remnants are still discernible despite the profound margination of coarsechromatin. Cytoplasm bears abundant vacuoles (v), and inclusions reminiscent of Rosenthal fibers(rf). (3c and d) MPA + MTX: Protrusion of nuclear membrane with condensed chromatin (doublearrowheads) and invaginations of nuclear membrane (arrows) were seen simultaneously. Electronloose patches (asterisks) were seen in nucleus (n). Fibrillar remnants of the nucleolus (nu in 3c,and asterisk in 3d) were present until late stages of chromatin condensation. Structures resemblingto lipid droplets, or enwidenings of endoplasmic reticulum cisternae (lv) were notified, which canbe described as paraptosis.

its combinations with procarbazin, cisplatin, and methotrexate, respectively.Among these values enhancement of procarbazin and cisplatin induced colonygrowth inhibitions with MPA occured with a statistical significance (p < .05for both values), but MPA induced depletion of methotrexate’s colony growthinhibitory potential lacked significance.

Percentage Inhibiton of Plating efficacy (%IPE)—Figure 5a

Ninety-six-h values of inhibition on plating efficacy were 37.00%, 9.00%,25.70%, and 94.70% for MPA, procarbazin, cisplatin, and methotrexategroups, respectively. When MPA was combined with drugs, 24.70%, 47.40%,



Figure 4. (a, b) MPA: Note the extensive myelinosis in a cell with completely healthy nucleus(n). Dispersed in a cytoplasm enriched in ribosomal stacks (ri), lysosomes with double membranesbegin to step-wise (1-2-3, 0.95 µm) maturate to form more condensed nascent autophagic vacuoles(av, 0.8 µm) with electron-loose halo or form multilamellated myelin bodies (ml, 1′–2′, 1 and0.8 µm). Mitochondria (mi) were not entrapped and bearing intact morphology.



and 97.40% inhibition were found for its combinations with procarbazin,cisplatin, and methotrexate, respectively. Among these values enhancement ofcisplatin and procarbazin induced plating inhibitions with MPA occured witha high statistical significance (p < .01 for both values). MPA induced increasein methotrexate-induced plating inhibition was near to significance limits(p = .042).

Percentage Inhibition of Thymidine Labelling Indices or S-Phase(%ISP)—Figure 5a

Ninety-six-h values of inhibition on thymidine labelling index were 72.75%,35.62%, 73.95%, and 100.00% for MPA, procarbazin, cisplatin, and methotrex-ate groups respectively. When MPA was combined with drugs, 48.92%,78.67%, and 86.70% inhibition were found for combinations with procarbazin,cisplatin, and MTX, respectively. Among these, enhancement of cisplatin-, andprocarbazin-induced TLI inhibitions with MPA occured with high statisticalsignificance (p < .00001 and p < .001, respectively); also the MPA decrease ofmethotrexate induced TLI inhibition had a high statistical significance (p < .01).

DISCUSSION

Apoptosis and Clonogenic Stem Cell Death as Two Ways toInduce Glioma Regression

Considering the neurotoxicity and the limited efficacy of chemotherapyfor malignant glial tumors, new strategies are needed to improve theiroutcome. Astrocytoma patients, bearing specimens with more apoptosis thanproliferation could have long survivals among high-grade cases (Rhodes, 1998).Thus, one plausible approach would be inducing apoptosis. Another optionwould be blocking stem cell characteristics of tumor cells as described by agarhuman tumor cloning studies (Hamburger & Salmon, 1977), which also bearefficacy to predict clinical glial tumor drug resistance and sensitivity (Kimmelet al., 1987). There is a correspondence between the in vitro chemosensitivity tohuman glioma cells and the rat C6 glioma cells, suggesting that the C6 gliomarepresents a suitable model of human glioma for drug sensitization studies(Hand et al., 1988).

Low-Dose Cisplatin Efficacy to Suppress Glioma Growth and toInduce Drug Sensitization

Due to the existence of regional chemotherapy for glioma therapy, the authorsstudied the effects of low doses of cytotoxic drugs in their sustained presence.



Figure 5. (a) Percentage inhibition of colony growth (%ICG), plating efficacy (%IPE) and of S-phase (%ISP) in C6 glioma cells treated with medroxyprogesterone acetate (MPA), cisplatin (Cisp),methotrexate (MTX), and procarbazin (Proc) alone or in combination. For statistical comparisons,please refer to results section. (b) Lysosomal phospholipidosis (LysoPhos), apoptosis (Apop),mitochondrial whorls (MitoWhor), and necrosis (Necros) rates in C6 glioma following treatmentdescribed earlier. For statistical comparisons, please refer to Results section.

Benefits of some therapeutic regimes may be masked with the toxicity ofunnecessarily high doses. Researchers apply 25 µM cisplatin to induce DNAstrand breaks and cytotoxicity in human glioma cells (Ali-Osman et al., 1993).Injection of even 40 nM of cisplatin into rat cerebrospinal fluid can induce



structure changes in medulla spinalis (Olivi et al., 1993). As low as 0.2 µg/ml(0.66 µM) of cisplatin could repress S-phase after 18 h in human gliomacells (Honda & Tabuchi, 1980). In the present study, half of this dosage wasable to repress C6 glioma colony growth by 45%, which occurred with lackof early cytotoxicity (only 25% depletion of plating). Methionine depletionis seen during consecutive lower doses of cisplatin treatment (Araki et al.,2000). The significant growth-inhibitory and chemo-sensitizing potentials ofthe low-dose cisplatin in our study may have occured via methione depletion,since cisplatin inhibits methionine uptake of C6 glioma (Mineura et al., 1999);and the depletion of exogenous methionine lead to a drug-insensitive gliomaxenograft to decrease in growth by 80% with carmustine treatment (Poirson-Bichat et al., 2000). MPA synergism with cisplatin occured via both apoptosisand clonogenic cell death, which is concordant to progesterone accelerationof cisplatin-induced clonogenic death of breast cancer cells via inducing highmobility group domain (HMG) proteins to shield DNA adducts from nucleotideexcision repair (He et al., 2000). C6 glioma is a p53 positive cell line, and p53drives apoptosis, if the DNA damage is irrepairable (Asai et al., 1994).

Clonogenic, but not Apoptotic Chemosensitization towardProcarbazin by MPA

The therapeutic dose for procarbazin is 215 mg/kg in humans (Ehling &Neuhauser, 1979); if it would have been absorbed for 100%, its plasmaconcentrations would reach 3 mg/ml. In the present study, 50 µg/ml ofprocarbazine synergized with MPA on colony inhibition with increase ofmyeloid figures. Procarbazine is a DNA methylating triazene, and repair ofits DNA damage does not involve mechanisms similar to cisplatin damage,which would be expected to upregulate p53. Thus, it is plausible that MPAsensitization toward procarbazin occured only via clonogenic cell death, whichis probably an accelerated senescence of stem cells existing in the C6 gliomapopulation (Kondo et al., 2004).

MPA Reduces Early and Robust S-phase Fraction Depletion andMitochondrial Toxicity of Methotrexate, without Sacrificing itsAnti-Clonogenic Efficacy

Intrathecal MTX induced neurotoxicity is seen in up to 40% of patients, andit is dose related (Nelson & Frank, 1981). In patients without developingneurotoxicity following intrathecal application, MTX peak concentration is



0.2 µM (Bleyer & Dedrick, 1977). A closer dosage less than 1 µM (target dosein leptomeningeal carcinomatosis), 0.66 µM was here very effective. Duringmethotrexate-induced cell death, whorled vacuoles appeared in proximity tothe sequestered mitochondria. Such whorled myelin figures form as remnantsof mitochondria after sudden insults, which destroy mitochondrial outermembrane (Maeda, 1982; Orvoine et al., 1978).

Mitochondria are involved in cell demise through the opening ofmitochondrial permeability transition channels, via executing necrapoptosis(Lemasters, 1999). The term “necrapoptosis” indicates pathways leading toboth apoptosis and necrosis, in which if ATP levels fall profoundly, necrosisensues; if ATP levels are partially maintained, apoptosis follows mitochondrialdestruction (Lemasters, 1999). MTX at doses inhibiting cell growth withoutcell kill alters cellular oxygen consumption; and after 24 h, there is a closerelationship between cellular oxygen uptake and MTX-cytotoxicity (Koumauoet al., 1981). In tumors transport of reduced equivalents from cytoplasmicNADH into the respiratory chain is via the malate-aspartate shuttle, and itis selectively inhibited by MTX leading a decrease in cellular energetic gain(Bastos et al., 1990). This would explain both high necrosis and apoptosis seenin MTX-treated C6 cells. MPA reduced early mitochondrial toxicity of MTX,yet it did not hinder MTX-anticlonogenic potential. MPA may have shifted theanti-tumor mechanism of action of MTX from a mixed nucleus–mitochondriatoxicity to a dominant nuclear effect, because main anti-tumor mechanismof MTX is via blocking folate availability for nucleotide biosynthesis.Progesterone is capable of protecting cardiac mitochondria against alkalineenvironment–induced swelling (Jung & Brierley, 1981), and MPA is shownto reverse liver mitochondrial damage and swelling following CCl4-inducedcirrhosis (Arranto, 1983). Uncoupling clonogenic death-inducing activitiy ofanti-neoplastics from their mitochondrial toxicity may mean obviated neuraland cardiac toxicity with MPA, which the authors are currently investigating inanimal models.

MPA-Induced Myelinosis is a Lysosomal PhospholipidosisReminiscent of Anti-Depressant Action

MPA-induced multilamellar bodies or “myeloid figures” were differing fromthe MTX-induced changes because cell membrane or nucleus did not revealany signs of apoptosis or necrosis. These were much rather resembling tomyeloid body formations during antidepressant induced lipidosis, reflectinglysosomal accumulation of phospholipids (Xia et al., 2000). There is no



study until now questioning the nature and extent of progesterone andantidepressant-induced membrane lipid perturbations in glial tumors. Studiesshowing that antidepressants also induce apoptosis in C6 glioma cells (Spanovaet al., 1997), may indicate the importance of research in this area. Boththe antidepressant imipramin and progesterone (Lange et al., 1997) inhibitre-esterification of cholesterol in the endoplasmic reticulum. P-glycoprotein,which is involved in glioma chemo-resistance, also implicates in mediatingcholesterol esterification; and vice versa progestin-induced blockage ofcholesterol esterification inhibits P-glycoprotein activity (Debry et al., 1997).

Among glial tumors more drug sensitive types such as oligodendrogliomaand pleomorphic xantho-astrocytoma contain eosinophilic hyaline droplets andround granular bodies at light microscopy, which show lamellated myelinfigures (not mitochondrial myelin whorls) at electron microscopic analysis(Hitotsumatsu et al., 1994; Iwaki et al., 1987). These figures are absent inglioblastomas and they reduce during anaplastic recurrence of pleomorphicxanthoastrocytoma, and they are accepted as signs of more indolent and lessaggressive tumor growth (Hitotsumatsu et al., 1994; Iwaki et al., 1987). Itis herein proposed that reduced cholesterol esterification capability amongthese tumors renders cholesterol entrapped in endosomal-lysosomal pathwayand hinders its accumulation in plasma membrane. Thus, helping membranesynthesis for cell division, enhancing invasion, and reducing permeability tochemotherapeutics via plasma membrane cholesterol would be blocked leadingto more indolent and more chemo-sensitive characteristics in these tumors.

CONCLUSIONS ON THE LIKELY SIGNIFICANCEOF THE FINDINGS FOR THE CLINICAL APPLICATION

MPA-induced lysososomal phospholipidosis in gliomas may switch the tumorphenotype to an indolent and more drug-responsive stage as discussed forthe aforementioned tumors with inherent high content of myelin figures; andmay also lead to uncoupling of neuro-toxicity versus therapeutic efficacy ofchemotherapy.

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MEDROXYPROGESTERONE ACETATE INDUCES C6 GLIOMA CHEMOSENSITIZATION VIA ANTIDEPRESSANT-LIKE LYSOSOMAL PHOSPHOLIPIDOSIS/MYELINOSIS IN VITRO

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