ANTICANCER RESEARCHInternational Journal of Cancer Research and Treatment

ISSN: 0250-7005

Reprinted fromANTICANCER RESEARCH 34: 4153-4160 (2014)

Comparative Analysis of Hsp10 and Hsp90 Expression inHealthy Mucosa and Adenocarcinoma of the Large Bowel

FRANCESCA RAPPA1,2*, CARMELO SCIUME3*, MARGHERITA LO BELLO1, CELESTE CARUSO BAVISOTTO1,ANTONELLA MARINO GAMMAZZA1,2, ROSARIO BARONE1,2, CLAUDIA CAMPANELLA1,2,

SABRINA DAVID1, FRANCESCO CARINI1, FEDERICA ZARCONE3, STEFANO RIZZUTO3, ADRIANA LENA3,GIOVANNI TOMASELLO2,3, MARIA LAURA UZZO4, GIOVANNI FRANCESCO SPATOLA4,

GIUSEPPE BONAVENTURA4, ANGELO LEONE4, ALDO GERBINO4, FRANCESCO CAPPELLO1,2, FABIO BUCCHIERI1,2, GIOVANNI ZUMMO1 and FELICIA FARINA1

1Department of Experimental Biomedicine and Clinical Neuroscience, Section of Human Anatomy, University of Palermo, Palermo, Italy;

2Euro-Mediterranean Institute of Science and Technology (IEMEST), Palermo, Italy; 3Department of Surgical and Oncological Sciences, University of Palermo, Palermo, Italy;

4Department of Experimental Biomedicine and Clinical Neuroscience, Section of Histology and Embryology, University of Palermo, Palermo, Italy

P.A. ABRAHAMSSON, Malmö, SwedenB. B. AGGARWAL, Houston, TX, USAT. AKIMOTO, Kashiwa, Chiba, JapanA. ARGIRIS, San Antonio, TX, USAJ. P. ARMAND, Toulouse, FranceV. I. AVRAMIS, Los Angeles, CA, USAR. C. BAST, Houston, TX, USAG. BAUER, Freiburg, GermanyE. E. BAULIEU, Le Kremlin-Bicetre, FranceY. BECKER, Jerusalem, IsraelE. J. BENZ, Jr., Boston, MA, USAJ. BERGH, Stockholm, SwedenD. D. BIGNER, Durham, NC, USAA. BÖCKING, Düsseldorf, GermanyG. BONADONNA, Milan, ItalyF. T. BOSMAN, Lausanne, SwitzerlandG. BROICH, Monza, ItalyJ. M. BROWN, Stanford, CA, USAØ. S. BRULAND, Oslo, NorwayM. M. BURGER, Basel, SwitzerlandM. CARBONE, Honolulu, HI, USA C. CARLBERG, Kuopio, FinlandJ. CARLSSON, Uppsala, SwedenA. F. CHAMBERS, London, ON, CanadaP. CHANDRA, Frankfurt am Main, GermanyL. CHENG, Indianapolis, IN, USAJ.-G. CHUNG, Taichung, Taiwan, ROCE. DE CLERCQ, Leuven, BelgiumW. DE LOECKER, Leuven, BelgiumW. DEN OTTER, Amsterdam, The NetherlandsE. P. DIAMANDIS, Toronto, ON, Canada G. TH. DIAMANDOPOULOS, Boston, MA, USAD. W. FELSHER, Stanford, CA, USAJ. A. FERNANDEZ-POL, Chesterfield, MO, USAI. J. FIDLER, Houston, TX, USAA. P. FIELDS, Jacksonville, FL, USAB. FUCHS, Zurich, SwitzerlandG. GABBIANI, Geneva, SwitzerlandR. GANAPATHI, Charlotte, NC, USAA. F. GAZDAR, Dallas, TX, USAJ. H. GESCHWIND, Baltimore, MD, USAA. GIORDANO, Philadelphia, PA, USAG. GITSCH, Freiburg, GermanyR. H. GOLDFARB, Saranac Lake, NY, USAS. HAMMARSTRÖM, Umea° , SwedenI. HELLSTRÖM, Seattle, WA, USAL. HELSON, Quakertown, PA, USAR. M. HOFFMAN, San Diego, CA, USAK.-S. JEONG, Daegu, South KoreaS. C. JHANWAR, New York, NY, USAJ. V. JOHANNESSEN, Oslo, NorwayB. KAINA, Mainz, GermanyP. -L. KELLOKUMPU-LEHTINEN, Tampere,

FinlandB. K. KEPPLER, Vienna, AustriaD. G. KIEBACK, Riesa (Dresden), GermanyR. KLAPDOR, Hamburg, GermanyU. R. KLEEBERG, Hamburg, GermanyP. KLEIHUES, Zürich, SwitzerlandE. KLEIN, Stockholm, SwedenS. D. KOTTARIDIS, Athens, Greece

G. R. F. KRUEGER, Köln, GermanyD. W. KUFE, Boston, MA, USAPat M. KUMAR, Manchester, UKShant KUMAR, Manchester, UKM. KUROKI, Fukuoka, JapanO. D. LAERUM, Bergen, NorwayF. J. LEJEUNE, Lausanne, SwitzerlandL. F. LIU, Piscataway, NJ, USAD. M. LOPEZ, Miami, FL, USAE. LUNDGREN, Umea° , SwedenH. T. LYNCH, Omaha, NE, USAY. MAEHARA, Fukuoka, JapanJ. MAHER, London, UKJ. MARESCAUX, Strasbourg, FranceJ. MARK, Skövde, SwedenS. MITRA, Houston, TX, USAM. MUELLER, Heidelberg, GermanyF. M. MUGGIA, New York, NY, USAM. J. MURPHY, Jr., Dayton, OH, USAM. NAMIKI, Kanazawa, Ishikawa, JapanR. NARAYANAN, Boca Raton, FL, USAK. NILSSON, Uppsala, SwedenS. PATHAK, Houston, TX, USAJ.L. PERSSON, Malmö, SwedenS. PESTKA, Piscataway, NJ, USAG. J. PILKINGTON, Portsmouth, UK C. D. PLATSOUCAS, Norfolk, VA, USAF. PODO, Rome, ItalyA. POLLIACK, Jerusalem, IsraelG. REBEL, Strasbourg, FranceM. RIGAUD, Limoges, FranceU. RINGBORG, Stockholm, SwedenM. ROSELLI, Rome, ItalyA. SCHAUER, Göttingen, GermanyM. SCHNEIDER, Wuppertal, GermanyA. SETH, Toronto, ON, Canada G. V. SHERBET, Newcastle-upon-Tyne, UKG.-I. SOMA, Tokushima, JapanG. S. STEIN, Burlington, VT, USAT. STIGBRAND, Umea° , SwedenT. M. THEOPHANIDES, Athens, GreeceB. TOTH, Omaha, NE, USAP. M. UELAND, Bergen, NorwayH. VAN VLIERBERGHE, Ghent, BelgiumR. G. VILE, Rochester, MN, USAM. WELLER, Zurich, SwitzerlandB. WESTERMARK, Uppsala, SwedenY. YEN, Duarte, CA, USAM.R.I. YOUNG, Charleston, SC, USAB. ZUMOFF, New York, NY, USA

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ISSN (print): 0250-7005ISSN (online): 1791-7530

Abstract. Background: Heat shock proteins (Hsps) assist otherproteins in their folding and drive the degradation of defectiveproteins. During evolution, these proteins have also acquiredother roles. Hsp10 is involved in immunomodulation and tumorprogression. Hsp90 stabilizes a range of “client” proteinsinvolved in cell signaling. The present study evaluated theexpression levels of Hsp10 and Hsp90 in normal mucosa andadenocarcinoma samples of human large bowel. Materials andMethods: Samples of normal mucosa and adenocarcinoma werecollected and Reverse transcriptase-polymerase chain reactionRT-PCR, western blotting (WB) analyses, as well asimmunohistochemistry were performed to evaluate the expressionlevels of Hsp10 and Hsp90. Results: RT-PCR showed a highergene expression of Hsp10 and Hsp90 in adenocarcinoma samplescompared to healthy mucosa. WB results confirmed thesefindings. Immunohistochemistry revealed higher levels of Hsp10in adenocarcinoma in both the epithelium and the laminapropria, while Hsp90 expression was higher in the adeno-carcinoma samples only in the lamina propria. Conclusion:Hsp10 and Hsp90 may be involved in large bowel carcinogenesis.

Colorectal cancer is a malignancy with significant social andhealth care-related effects. The carcinogenetic model of thelarge bowel is a multi-step process in which the transformationof normal mucosa into invasive tumor entails the accumulationof different genetic alterations (1). Many studies havesuggested the involvement of heat shock proteins (Hsps) inlarge bowel carcinogenesis (2, 3). Hsps are among the mostevolutionarily conserved proteins. They are an important classof proteins that have different functions essential for cell lifeand survival. Their classic role is to assist other proteins infolding and re-folding and, in the case of defective orirreversibly misfolded proteins, to drive their degradation (4).For this reason, some Hsps are also known as molecularchaperones. During evolution, this class of proteins has alsoacquired “extra-chaperoning” roles, such as participating inimmune system regulation (5), cell senescence (5), celldifferentiation, programmed cell death and carcinogenesis.These molecules have also been implicated in the pathogenesisof a number of chronic inflammatory and autoimmunediseases, like inflammatory bowel disease (IBD), in whichHsps have been identified as potential biomarkers fordiagnostics, prognostics and etiopathogenetic factors, ortherapeutic targets (6, 7). Moreover, Hsps have other importantfunctions such as their involvement in various metabolicmechanisms of neoplastic cells such as tissue invasion,induction of angiogenesis and metastasis. Hsp10 is a 10-kDa,highly conserved, mitochondrion-resident protein that co-chaperones with another mitochondrial heat shock protein,Hsp60, for protein folding and the assembly and disassemblyof protein complexes (8). Moreover, Hsp10 plays otherimportant roles in a variety of mechanisms such us

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*These Authors contributed equally to this study.

Correspondence to: Dr. Francesca Rappa, Department of ExperimentalBiomedicine and Clinical Neuroscience, Section of Human Anatomy,University of Palermo, Palermo, Italy. Tel: +39 0916553519, Fax: +390916553580, e-mail: francyrappa@hotmail.com

Key Words: Hsp10, Hsp90, large bowel adenocarcinoma, RT-PCR,immunohistochemistry.

ANTICANCER RESEARCH 34: 4153-4160 (2014)

Comparative Analysis of Hsp10 and Hsp90 Expression inHealthy Mucosa and Adenocarcinoma of the Large Bowel

FRANCESCA RAPPA1,2*, CARMELO SCIUME3*, MARGHERITA LO BELLO1, CELESTE CARUSO BAVISOTTO1,ANTONELLA MARINO GAMMAZZA1,2, ROSARIO BARONE1,2, CLAUDIA CAMPANELLA1,2,

SABRINA DAVID1, FRANCESCO CARINI1, FEDERICA ZARCONE3, STEFANO RIZZUTO3, ADRIANA LENA3,GIOVANNI TOMASELLO2,3, MARIA LAURA UZZO4, GIOVANNI FRANCESCO SPATOLA4,

GIUSEPPE BONAVENTURA4, ANGELO LEONE4, ALDO GERBINO4, FRANCESCO CAPPELLO1,2, FABIO BUCCHIERI1,2, GIOVANNI ZUMMO1 and FELICIA FARINA1

1Department of Experimental Biomedicine and Clinical Neuroscience, Section of Human Anatomy, University of Palermo, Palermo, Italy;

2Euro-Mediterranean Institute of Science and Technology (IEMEST), Palermo, Italy; 3Department of Surgical and Oncological Sciences, University of Palermo, Palermo, Italy;

4Department of Experimental Biomedicine and Clinical Neuroscience, Section of Histology and Embryology, University of Palermo, Palermo, Italy

0250-7005/2014 $2.00+.40

immunomodulation and cell proliferation and differentiation(9, 10). The latter are linked to its localization either in thecytosol (11) or the extracellular space. Hsp90 is a molecularchaperone that performs its functions inside cells by formingcytosolic chaperoning machines (4). This chaperone isinvolved in signal transduction and other key pathways criticalfor malignancy in several cancers (12, 13), including largebowel carcinoma (14). Hsp90, that also includes two isoforms,Hsp90α and Hsp90β, is an ubiquitous protein highlyexpressed (accounting for up to 1-2% of total cellular proteincontent) in the cytosol, both in normal and stress conditions(15). Hsp90 can accumulate in cancer cells and is implicatedin the carcinogenesis process for many reasons. This moleculefavors tumorigenicity and promotes cancer cell growth andsurvival (16, 17) by inhibiting programmed cell death andsenescence (18). Hsp90 client proteins involved in suchactivities include, for example, mutated p53, BRAF andErbB2 (19). Hsp90 also influences tumor neoangiogenesisbecause it stabilizes proteins important for the metabolism ofendothelial cells such as vascular endothelial growth factorand nitric oxide synthase (20, 21).

In the present study, we evaluated by immuno-morphological experiments, western blotting (WB) and RT-PCR analyses the levels and expression of Hsp10 and Hsp90in a series of large bowel mucosa samples obtained fromhealthy controls and patients with adenocarcinomas withmoderate grade of differentiation.

Materials and Methods

Sample collection. A total of 40 large bowel mucosa biopsies fromhealthy controls (n=20) and adenocarcinomas with moderate gradeof differentiation (n=20) were obtained from the DICHIRONSDepartment of the University of Palermo, Italy. The samplescollected for WB and PCR analyses were kept frozen at –80˚C untiluse, while the samples for immunomorphological analysis werefixed in formalin and embedded in paraffin.

RT-PCR analysis. Total RNA extraction from all tissue samples wasperformed using TRI REAGENT® (Catalog Number T9424, Sigma-Aldrich, Saint Lous, MO, USA) according to the manufacturer’sinstructions. cDNA was synthesized using ImProm–II ReverseTranscriptase (Catalog Number A3800, Promega Corporation,Madison, WI, USA) and amplified using GoTaq® Flexi DNAPolymerase (Catalog Number M8291, Promega Corporation,Madison, WI, USA). PCR amplification was performed by addingspecific primers for human Hsp10, Hsp90α and Hsp90β : for humanHsp10 cDNA, the forward primer sequence was 5’CTCCCAGAATATGGAGGCACC-3’ and the reverse primer sequence was 5’-TGGAATGGGCAGCATCATGT-3’; for human Hsp90α cDNA, theforward primer sequence was 5’-GTCTAGTTGACCGTTCCGCA-3’and the reverse primer sequence was 5’-GAGGAGGCACCCTCAAGTTC-3’; for human Hsp90β cDNA, the forwardprimer sequence was 5’-CTCTCTCGAGTCA CTCCGGC-3’ and thereverse primer sequence was 5’-GTACGTTCCTGAGG GTTGGG-3’). The PCR product was visualized on 1.5% agarose gel with the

Syber stain (SYBER SafeTM DNA gel stain, Invitrogen, Carlsbad,CA, USA). Experiments were performed in triplicate. Quantitativemeasurements of bands were performed using the NIH Image J 1.40analysis program (National Institutes of Health, Bethesda, MD, USA).

Western blotting analysis. 50 mg of tissue samples from normalmucosa and adenocarcinoma were incubated on ice in a Radio-Immunoprecipitation Assay (RIPA) lysis buffer (0.3M NaCl, 0.1%SDS, 25mm HEPES pH 7.5, 1.5 mm MgCl2, 0.2 mm EDTA, 1%Triton X-100, 0.5mm DTT, 0.5% sodium deoxycholate) containingproteases and phosphatase inhibitors (0.1 mg/ml phenylmethylsulfonylfluoride, 20 mg/ml aprotinin, 20 mg/ml leupeptin, 10 mg/ml NaF, 1mM DTT, 1 mM sodium orthovanadate, 20 mM β-glycerol phosphate)for 1 h. Subsequently, the samples were centrifuged at 13,000 rpm for10 min at 4˚C and supernatants were isolated. Protein concentrationwas measured by the Bradford method. Proteins (40 mg) wereseparated on a 12% SDS-PAGE gel, and then electrophoreticallytransferred to a nitrocellulose membrane (Nitrocell Membrane, Bio-Rad Laboratories Inc. Karlsruhe, Germany). After blocking, themembranes were incubated overnight with primary antibodies (rabbitanti-Hsp10 polyclonal antibody, sc-20958 and mouse anti-Hsp90, F-8clone,sc-13119, Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA)diluted 1:1000 in Tris Buffered Saline with Tween 20 (T-TBS)containing 1% dry milk. Blots were washed in T-TBS and incubatedfor 1 h with Horseradish Peroxidase (HRP) conjugated secondaryantibodies (ECLTM anti-rabbit IgG HRP-conjugated whole antibody;ECLTM anti-mouse IgG HRP-conjugated whole antibody, AmershamBiosciences) diluted 1:5000 in T-TBS containing 1% dry milk. β-actin(mouse anti-β-actin monoclonal antibody, C4 clone, sc-47778, SantaCruz Biotechnology, Inc.) was used as a loading control. The finaldetection procedure was carried out using the ECL Western BlottingDetection Reagent (RPMN2232, Amersham Biosciences, Uppsala,Sweden) according to the manufacturer’s instructions. Experimentswere performed in triplicate.

Densitometric analysis of blots was performed using the ImageJ1.41 software (National Institutes of Health, USA, http://rsb.info.nih.gov/ij).

Immunohistochemical analysis. Samples of normal mucosa andadenocarcinoma were fixed in formalin and embedded in paraffin.From the paraffin blocks, sections with a thickness of 4-5 mm wereobtained using a cutting microtome. These sections were dewaxed inxylene for 10 min and rehydrated by sequential immersion in adescending scale of alcohols and transitioned in water for 5 min.Subsequently, the sections were immersed for 8 min in sodiumcitrate buffer (pH 6) at 95˚C for antigen retrieval and, subsequently,immersed for 8 min in acetone at –20˚C to prevent the detachmentof the sections from the slide. All subsequent reactions wereconducted at room temperature. The reactions were performed by astreptavidin–biotin complex method using Histostain®-Plus 3rd GenIHC Detection Kit (Invitrogen Corporation, Cat no. 85-8943). Theprimary antibodies used were anti-human Hsp10 antigen (Gene Texpolyclonal, dilution 1:300) and anti-human Hsp90 antigen (SantaCruz Biotechnology, clone F-8, dilution 1:200). Non-immune serawere substituted for negative controls. NCI-H292 cells were usedas positive control. Nuclear counterstaining was carried out usinghematoxylin (Hematoxylin aqueosus formula, N. Cat. S2020,DAKO). Finally, the slides were prepared for observation withcoverslips with an aqueous mounting solution. The observation ofthe sections was performed with an optical microscope (Leica DM

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5000 B, Heidelberg, Germany.) connected to a digital camera (LeicaDC 300F). Each tissue section was analysed on two separateoccasions by two independent observers (FC and FR).

Statistical analyses. Statistical analyses were carried out using theGraphPad Prism 4.0 package (GraphPad Inc., San Diego, CA,USA). Standard statistical analyses were employed to calculate themeans and the standard deviations (SD). One-way ANOVA (andnonparametric) analysis of variance was used to assess significantdifferences within the data. Differences between the means wereconsidered significant when p<0.001 as indicated in the figures.

Results

RT-PCR analysis. RT-PCR experiments carried-out onsamples of normal mucosa and adenocarcinoma showed thatmRNA expression of Hsp10, Hsp90α and Hsp90β werehigher in adenocarcinoma samples than in normal mucosa(Figure 1). These data suggest that there is an increased geneexpression of these molecules in colon adenocarcinoma.

Western blotting analysis. WB experiments showed thatHsp10 and Hsp90 levels increase significantly from normalmucosa to adenocarcinoma (Figure 2). The data obtainedwith the WB experiments are in agreement with the RT-PCRresults, and suggest that there is an increased synthesis ofthese protein molecules in colon adenocarcinoma.

Immunohistochemical analysis. The immunohistochemicalevaluation, carried-out on all samples, showed that thelocalization of cellular immunopositivity of Hsp10 and Hsp90was cytoplasmic, and sometimes granular. The levels of Hspswere investigated in the epithelial and lamina propria cells.The percentage of positive cells was calculated in 10 randomhigh power fields (HPF) at a magnification of 400× andexpressed as means (Figure 3). Hsp10 levels, both in theepithelium and lamina propria, were higher inadenocarcinoma samples compared to normal mucosa. Incontrast, Hsp90 levels resulted higher only in the laminapropria cells of the adenocarcinoma samples, compared tonormal mucosa, while no differences in expression wereobserved between the epithelial cells of the normal mucosaand adenocarcinoma samples (Figure 4). Data obtained fromimmunohistochemistry were plotted using the MicrosoftExcel software (Microsoft Italia, Milan, Italy). Standardstatistical analyses were employed to calculate the means ofpositivity percentage and the standard deviations (SD).

Discussion

In the present work, we studied the gene expression of Hsp10and Hsp90 (Hsp90α and Hsp90β isoforms) by RT-PCRanalysis in samples of normal large bowel mucosa andadenocarcinoma of large bowel. The data obtained showed a

higher gene expression of Hsp10 and Hsp90 inadenocarcinoma samples than in healthy mucosa. Moreover,the Western blotting analysis confirmed greaterconcentrations of Hsp10 and Hsp90 proteins in theadenocarcinoma samples compared to healthy mucosa. Theresults obtained from comparative evaluation of the proteinlevels and gene expression of Hsp10 and Hsp90 confirmedthat these proteins seem to be implicated in thecarcinogenesis of large bowel. However, the RT-PCR and WBtechniques have the limitation of not differentiating the levelsof proteins and RNA between the different tissue structures,i.e., the epithelial and lamina propria components. Therefore,we performed further immunohistochemical experiments toevaluate the immunopositivity in the epithelial and in thelamina propria cells. In the currently available literature,several reports support the idea that Hsps are involved in thepathogenesis and the progression of different humanneoplasms for many reasons. Possibly the most importantreason is that the Hsps have acquired, probably duringcellular evolution, various functions within the cell such as

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Figure 1. RT-PCR analysis: 1.5% agarose gels of the gene HSP10,Hsp90 isoform α and Hsp90 isoform β from Homo sapiens amplified byRT-PCR. Histogram of quantitative measurements of bands of mRNAexpression levels for Hsp10, Hsp90α and Hsp90β genes.

participating in immune system regulation (22-24), cellproliferation and differentiation (25, 26) and carcinogenesis(27, 28). Higher Hsp10 levels have been found in differentmalignancies (29) such as large bowel cancer (11, 30), exo-cervical cancer (30), prostate cancer (31), mantle celllymphoma (32) and serous ovarian cancer (33), while lowerexpression levels of this protein have been detected inbronchial cancer (29, 34). In our experiments, Hsp10, which

in normal cells is generally localized in the mitochondrialmatrix, accumulated in the cytosol of cancerous cells and thelamina propria of adenocarcinoma, while Hsp90 showed ahigher immunopositivity in the lamina propria of the tumor.This finding, in agreement with other data present in literature(11, 30, 35) is of importance because it suggests an activetrafficking of Hsp10 between the inner and outer regions ofthe tumor cell. Moreover, the expression of Hsp10 and Hsp90

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Figure 2. Western blotting Analysis: Representative immunoblots for Hsp10 and Hsp90 (on the left) and densitometric quantification of Hsp10 andHsp90 protein levels (on the right) in normal mucosa (NM) and adenocarcinoma (AC). β-actin was used as a loading control.

Figure 3. Histogram showing immunohistochemical results: The percentage of positive cells was calculated in 10 random high power fields (HPF)of epithelial cells (EC) and lamina propria cells (LPC) of normal mucosa (NM) and adenocarcinoma (AC) samples of large bowel at a magnificationof ×400.

in the lamina propria cells suggests a possible role of theseproteins in tumor progression and immunomodulation. Manystudies have suggested that Hsp10 could be involved in theimmunomodulation mechanism that occurs in tumors (36,37). For example, in a study performed in patients withovarian cancer, the authors suggest that Hsp10 plays animmunosuppressive role being considered as a critical factorin the suppression of T-cell activation acting as a means toescape immune surveillance in cancer (38). Both Hsp10 andHsp90, like other Hsps, protect cancer cells against apoptosis(17, 39). In particular, Hsp10 seems to be a molecule thatinhibits the proapoptotic activity by interacting with the Rafsignaling cascade and shifting the balance towards cellsurvival (40, 41). The anti-apoptotic action of Hsp90 involvesthe inhibition of cytochrome c-mediated activation of pro-caspase-9 (42) and its interaction with proteins able togenerate signals in response to growth factor stimulation (43,44). Other studies have shown that tumor cells overexpressingHsps have an increased tendency to invade surrounding

tissues and to spread to distant organs (45, 46). In particular,the higher expression of Hsp90 in the lamina propria cells inlarge bowel adenocarcinoma samples may suggest aninvolvement of this protein in the mechanisms of tumorprogression and angiogenesis. In fact, some previous reportsdemonstrate that Hsp90 stabilizes vascular endothelial growthfactor and nitric oxide synthase in endothelial cells (45) andmay induce neovascularization (46). We have carried-out astudy based on gene and protein expression analyses andimmunomorphological experiments, obtaining results that arein accordance with various previously published studies.Further investigations are certainly needed to betterunderstand the molecular mechanisms that underlie theinvolvement of Hsp10 and Hsp90 in large bowelcarcinogenesis. Once the role of these molecules in largebowel carcinogenesis has been established, this type of cancermay be considered a “chaperonopathy by mistake” (47) andHsps could become a key target in the search for newtherapeutic strategies for cancer (48, 49).

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Figure 4. Representative images of immunohistochemical results: Representative images of immunohistochemical experiments performed for Hsp10and Hsp90 in large bowel mucosa samples of Hsp10 immunostaining: A) Normal mucosa. B) Adenocarcinoma with moderate grade of differentiation.Hsp90 immunostaining: C) Normal mucosa. D) Adenocarcinoma with moderate grade of differentiation. Magnification ×200; scale bar 100 μm.Histograms show statistical results for the evaluation of immunopositivity for Hsp10 and Hsp90 in epithelial cells (upper histogram) and in laminapropria cells (lower histogram) in normal mucosa (NM) and adenocarcinoma (AC) samples of human large bowel.

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7 Tomasello G, Sciumé C, Rappa F, Rodolico V, Zerilli M,Martorana A, Cicero G, De Luca R, Damiani P, Accardo FM,Romeo M, Farina F, Bonaventura G, Modica G, Zummo G,Conway de Macario E, Macario AJ and Cappello F: Hsp10, Hsp70,and Hsp90 immunohistochemical levels change in ulcerative colitisafter therapy. Eur J Histochem 55(4): e38, 2011.

8 Jia H, Halilou AI, Hu L, Cai W, Liu J and Huang B: Heat shockprotein 10 (Hsp10) in immune-related diseases: one coin, twosides Int J Biochem Mol Biol 2(1): 47-57, 2011.

9 David S, Bucchieri F, Corrao S, Czarnecka AM, Campanella C,Farina F, Peri G, Tomasello G, Sciumè C, Modica G, La RoccaG, Anzalone R, Giuffrè M, Conway De Macario E, Macario AJ,Cappello F and Zummo G: Hsp10: anatomic distribution,functions, and involvement in human disease. Front Biosci (EliteEd) 5: 768-778, 2013.

10 Cappello F, Tripodo C, Farina F, Franco V and Zummo G: HSP10selective preference for myeloid and megakaryocytic precursors innormal human bone marrow. Eur J Histochem 48(3): 261-265, 2004.

11 Cappello F, David S, Rappa F, Bucchieri F, Marasà L, BartolottaTE, Farina F and Zummo G: The expression of HSP60 andHSP10 in large bowel carcinomas with lymphnode metastase.BMC Cancer 5: 139, 2005.

12 Cappello F, David S, Ardizzone N, Rappa F, Marasà L,Bucchieri F and Zummo G: Expression of heat-shock proteinsHsp10, Hsp27, Hsp60, Hsp70 and Hsp90 in urothelial carcinomaof urinary bladder. J Cancer Mol 2: 73-77, 2006.

13 Lebret T, Watson RW, Molinié V, O’Neill A, Gabriel C,Fitzpatrick JM and Botto H: Heat-shock proteins HSP27,HSP60, HSP70, and HSP90: Expression in bladder carcinoma.Cancer 98: 970-977, 2003.

14 Chen WS, Chen CC, Chen LL, Lee CC and Huang TS: Secretedheat shock protein 90α (HSP90α) induces nuclear factor-κB-mediated TCF12 protein expression to down-regulate E-cadherinand to enhance colorectal cancer cell migration and invasion. JBiol Chem 288(13): 9001-9010, 2013.

15 Di Felice V, Cappello F, Montalbano A, Ardizzone NM, DeLuca A, Macaluso F, Amelio D, Cerra MC and Zummo G:HSP90 and eNOS partially co-localize and change cellularlocalization in relation to different ECM components in 2D and3D cultures of adult rat cardiomyocytes. Biol Cell 99(12): 689-699, 2007.

16 Neckers L: Chaperoning oncogenes: Hsp90 as a target ofgeldanamycin. Handb Exp Pharmacol 172: 259-277, 2006.

17 Joly AL, Wettstein G, Mignot G, Ghiringhelli F and Garrido C:Dual role of heat shock proteins as regulators of apoptosis andinnate immunity. J Innate Immun 2(3): 238-47, 2010.

18 Beere HM: Stressed to death: regulation of apoptotic signalingpathways by the heat shock proteins. Sci STKE 93: re1, 2001.

19 Neckers L: Hsp90 inibhitors as a novel cancer chemotherapeuticagents. Trends Mol Med 8: S55-61, 2002.

20 Sun J and Liao JK: Induction of angiogenesis by heat-shockprotein 90 mediated by protein kinase Akt and endothelial nitricoxide syntethase. Aterioscler Thromb Vasc Biol 24: 2238-2244,2004.

21 Pfosser A, Thalgott M, Büttner K, Brouet A, Feron O,Boekstegers P and Kupatt C: Liposomal Hsp90 cDNA inducesneovascularization via nitric oxide in chronic ischemia.Cardiovasc Res 65: 728-736, 2005.

22 Pockley AG, Muthana M and Calderwood SK: The dualimmunoregulatory roles of stress proteins. Trends Biochem Sci33: 71-79, 2008.

23 Agnello D, Scanziani E, Di Giancamillo M, Leoni F, Modena D,Mascagni P, Introna M, Grezzi P and Villa P: Preventiveadministration of Mycobacterium tubercolosis 10-kDa heatshock protein (hsp10) suppresses adjuvant arthritis in Lewis rats.Int Immunopharmacol International 2(4): 463-474, 2002.

24 Vanags D, Williams B, Johnson B, Hall S, Nash P, Taylor A,Weiss J and Feeney D: Therapeutic efficacy and safety ofchaperonin 10 in patients with rheumatoid arthritis: a double-blind randomised trial. Lancet 368(9538): 855-863, 2006.

25 Walsh D, Grantham J, Zhu XO, Wei Lin J, van Oosterum M,Taylor R and Edwards M: The role of heat shock proteins inmammalian differentiation and development. Environ Med 43:79-87, 1999.

26 Voellmy R: Transduction of the stress signal and mechanisms oftranscriptional regulation of heat shock/stress protein expressionin higher eukaryotes. Crit Rev Eukaryot Gene Expr 4: 357-401,1994.

27 Czarnecka AM, Campanella C, Zummo G and Cappello F:Mitochondrial chaperones in cancer: From molecular biology toclinical diagnostics. Cancer Biol Ther 5: 714-720, 2006.

28 Calderwood SK, Khaleque MA, Sawyer DB and Ciocca DR:Heat shock proteins in cancer: chaperones of tumorigenesis.Trends Biochem Sci 31(3): 164-172, 2006.

29 Cappello F, Czarnecka AM, La Rocca G, Di Stefano A, ZummoG and Macario AJ: Hsp60 and Hspl0 as antitumor molecularagents. Cancer Biol Ther 6(4): 487-489, 2007.

30 Cappello F, Bellafiore M, David S, Anzalone R and Zummo G:Ten kilodalton heat shock protein (HSP10) is overexpressedduring carcinogenesis of large bowel and uterine exocervix.Cancer Lett 196(1): 35-41, 2003.

31 Cappello F, Rappa F, David S, Anzalone R and Zummo G:Immunohistochemical evaluation of PCNA, p53, HSP60, HSP10and MUC-2 presence and expression in prostate carcinogenesis.Anticancer Res 23(2B): 1325-1331, 2003.

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32 Ghobrial IM, McCormick DJ, Kaufmann SH, Leontovich AA,Loegering DA, Dai NT, Krajnik KL, Stenson MJ, Melhem MF,Novak AJ, Ansell SM and Witzig TE: Proteomic analysis ofmantle-cell lymphoma by protein microarray. Blood 105(9):3722–3730, 2005.

33 Têtu B, Popa I, Bairati I, L’Esperance S, Bachvarova M, PlanteM, Harel F and Bachvarov D: Immunohistochemical analysis ofpossible chemoresistance markers identified by micro-arrays onserous ovarian carcinomas. Mod Pathol 21(8): 1002-1010, 2008.

34 Cappello F, Di Stefano A, David S, Rappa F, Anzalone R, LaRocca G, D’Anna SE, Magno F, Donner CF, Balbi B andZummo G: Hsp60 and Hsp10 down-regulation predicts bronchialepithelial carcinogenesis in smokers with chronic obstructivepulmonary disease. Cancer 107(10): 2417-2424, 2006.

35 He Y, Shang X, Sun J, Zhang L, Zhao W, Tian Y, Cheng H andZhou R: Gonadal apoptosis during sex reversal of the rice fieldeel: implications for an evolutionarily conserved role of themolecular chaperone heat shock protein 10. J Exp Zool B MolDev Evol 314(4): 257-266, 2010.

36 Czarnecka AM, Campanella C, Zummo G and Cappello F: Heatshock protein 10 and signal transduction: a "capsula eburnea" ofcarcinogenesis? Cell Stress Chaperones 11(4): 287-294, 2006.

37 Corrao S, Campanella C, Anzalone R, Farina F, Zummo G,Conway de Macario E, Macario AJ, Cappello F and La RoccaG: Human Hsp10 and Early Pregnancy Factor (EPF) and theirrelationship and involvement in cancer and immunity: currentknowledge and perspectives. Life Sci 86(5-6): 145-152, 2010.

38 Akyol S, Gercel-Taylor C, Reynolds LC and Taylor DD: HSP-10in ovarian cancer: expression and suppression of T-cell signaling.Gynecol Oncol 101(3): 481-486, 2006.

39 Rappa F, Farina F, Zummo G, David S, Campanella C, Carini F,Tomasello G, Damiani P, Cappello F, DE Macario EC and MacarioAJ: HSP-molecular chaperones in cancer biogenesis and tumortherapy: an overview. Anticancer Res 32(12): 5139-5150, 2012.

40 Lin KM, Hollander JM, Kao VY, Lin B, Macpherson L andDillmann WH: Myocyte protection by 10 kD heat shock protein(Hsp10) involves the mobile loop and attenuation of the RasGTP-ase pathway. FASEB J 18(9): 1004-1006, 2004.

41 Lin KM, Lin B, Lian IY, Mestril R, Scheffler IE and DillmannWH: Combined and individual mitochondrial HSP60 and HSP10expression in cardiac myocytes protects mitochondrial functionand prevents apoptotic cell deaths induced by simulatedischemia-reoxygenation. Circulation 103(13): 1787-1792, 2001.

42 Pandey P, Saleh A, Nakazawa A, Kumar S, Srinivasula SM,Kumar V, Weichselbaum R, Nalin C, Alnemri ES, Kufe D andKharbanda S: Negative regulation of cytochrome c-mediatedoligomerization of Apaf-1 and activation of procaspase-9 by heatshock protein 90. EMBO J 19: 4310-4322, 2000.

43 Garrido C, Gurbuxani S, Ravagnan L and Kroemer G: Heatshock proteins: endogenous modulators of apoptotic cell death.Biochem Biophys Res Commun 286: 433-442, 2001.

44 Westerheide SD, Kawahara TL, Orton K and Morimoto RI:Triptolide, an inhibitor of the human heat shock response thatenhances stress-induced cell death. J Biol Chem 281: 9616-9622,2006.

45 Sun J and Liao JK: Induction of angiogenesis by heat shockprotein 90 mediated by protein kinase Akt and endothelial nitricoxide synthase. Arterioscler Thromb Vasc Biol 24(12): 2238-2244, 2004.

46 Pfosser A, Thalgott M, Büttner K, Brouet A, Feron O,Boekstegers P and Kupatt C: Liposomal Hsp90 cDNA inducesneovascularization via nitric oxide in chronic ischemia.Cardiovasc Res 65(3): 728-736, 2005.

47 Macario AJL and Conway de Macario E: Chaperonopathies bydefect, excess, or mistake. Ann NY Acad Sci 1113: 178-191,2007.

48 Macario AJL and Conway de Macario E: Chaperonopathies andchaperonotherapy. FEBS let 581: 3681-3688, 2007.

49 Rappa F, Cappello F, Halatsch ME, Scheuerle A and Kast RE:Aldehyde dehydrogenase and HSP90 co-localize in humanglioblastoma biopsy cells. Biochimie 95(4): 782-786, 2013.

Received April 16, 2014Revised June 5, 2014

Accepted June 6, 2014

Rappa et al: Hsp10 and Hsp90 in Large Bowel Adenocarcinoma

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