Hyaluronan: Towards novel anti-cancer therapeuticsif-pan.krakow.pl/pjp/pdf/2013/5_1056.pdf ·...

Review

Hyaluronan: Towards novel anti-cancer therapeutics

Micha³ S. Karbownik1, Jerzy Z. Nowak2,3

1Department of Pharmacology, Medical University of Lodz, ¯eligowskiego 7/9, PL 90-752 £ódŸ, Poland

2Institute of Pharmacology, Polish Academy of Sciences, Scientific Board, Smêtna 12, PL 31-343 Kraków, Poland

3Medical Center MEDYCEUSZ, Bazarowa 9, PL 90-053 £ódŸ, Poland

Correspondence: Micha³ S. Karbownik, e-mail: [email protected];Jerzy Z. Nowak, e-mail: [email protected]

Abstract:

The understanding of the role of hyaluronan in physiology and various pathological conditions has changed since the complex natureof its synthesis, degradation and interactions with diverse binding proteins was revealed. Initially perceived only as an inert compo-nent of connective tissue, it is now known to be involved in multiple signaling pathways, including those involved in cancer patho-genesis and progression.Hyaluronan presents a mixture of various length polymer molecules from finely fragmented oligosaccharides, polymersintermediate in size, to huge aggregates of high molecular weight hyaluronan. While large molecules promote tissue integrity andquiescence, the generation of breakdown products enhances signaling transduction, contributing to the pro-oncogenic behavior ofcancer cells. Low molecular weight hyaluronan has well-established angiogenic properties, while the smallest hyaluronan oligomersmay counteract tumor development. These equivocal properties make the role of hyaluronan in cancer biology very complex.This review surveys recent data on hyaluronan biosynthesis, metabolism, and interactions with its binding proteins called hyaladherins(CD44, RHAMM), providing the molecular background underlying its differentiated biological activity. In particular, the article criticallypresents current ideas on actual role of hyaluronan in cancer. The paper additionally maps a path towards promising novel anti-cancertherapeutics which target hyaluronan metabolic enzymes and hyaladherins, and constitute hyaluronan-based drug delivery systems.

Key words:

hyaluronan, hyaluronan metabolism, hyaluronan oligomers, CD44, RHAMM, cancer, anticancer drugs, drug delivery systems

Abbreviations: CD44 – cluster of differentiation 44, HA –hyaluronan, HAS – hyaluronan synthase, HMWH – high mo-lecular weight hyaluronan, HYAL – hyaluronidase, LMWH –low molecular weight hyaluronan, RHAMM – receptor forhyaluronan-mediated motility

Historical perspective

The dawn of the “hyaluronan era” began in 1841,when German anatomist Friedrich G.J. Henle, cred-

ited, among others, with the discovery of the loop ofHenle in the kidney, described the amorphous mate-rial between cells and called it “ground substance”[46]. Nowadays, “ground substance” is known to bepredominantly composed of hyaluronan. As a matterof fact, “ground substance” is a mistranslation of Ger-man “Grundsubstanz”, which would be more accu-rately translated as “fundamental”, “primordial” or“basic” rather than “ground”.

In 1928, Francisco Duran-Reynals reported that ex-tracts of normal rabbit testicles injected subcutane-

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ously on the backs of shaved rabbits greatly facilitatedthe spread of concomitantly administered vaccine vi-rus [27]. Duran-Reynals called the extract “spreadingfactor”. Later, a similar increase in spread was pro-duced by adding testicular extract to diphtheria toxinor to a suspension of Staphylococcus aureus and in-jecting in the same manner as described above. Accel-erated intradermal spread did not depend on the typeof substance, because the same result has been ob-served even with carbon particles. The phenomenonis neither mediated by vascular nor nervous response,since increased diffusion was seen to occur even infragments of excised skin. These observations en-abled the conclusion to be drawn that “spreading fac-tor” contains an enzyme which splits Henle’s “groundsubstance”.

In 1934, Karl Meyer and John Palmer isolateda polysaccharide from the vitreous body of the bovineeye. For convenience, they constructed a name “hya-luronic acid”, connecting substitute name for the vit-reous – “hyaloid” with the name of a component ofthat polysaccharide – “uronic acid” [83]. In the fewfollowing years, hyaluronan was isolated from manysources such as umbilical cord, rooster comb, andstreptococci [68]. Step by step, it became evident thatHenle’s “ground substance”, renamed later to “acidmucopolysaccharides”, is predominantly composed ofhyaluronan, while Duran-Reynals’s “spreading fac-tor” is a “mucolysine” or literally hyaluronidase,which dissolves the extracellular matrix by breakingdown the hyaluronan polymer. The chemical structureof “hyaluronic acid” was essentially solved in 1954,and the name “hyaluronan”, corresponding to thechemical nature of the molecule under physiologicalconditions, was proposed in 1986 [95].

General profile of hyaluronan

Hyaluronic acid is a linear high-molecular-weightbiopolymer composed of alternating units of D-glucuronic acid (GlcA) and N-acetyl-D-glucosamine(Glc-NAc), connected to each other with b-1,3- andb-1,4-glycosidic bonds (Fig. 1). The polymer is nearlyperfect in its chemical repeats except for occasionaldeacetylated glucosamine residues [119]. The car-boxyl group of D-glucuronic acid is dissociated atphysiological pH values, resulting in the formulation

of a negatively charged polymer that combines withthe most prevalent extracellular cation, Na+ to formsodium hyaluronate. Hence, it would be advisable toabandon the name “hyaluronic acid”, suggesting thatthe molecule is not ionized, in favor of “hyaluronan”.However, the abbreviation “HA”, standing for “hyalu-ronic acid”, has been widely accepted.

Hyaluronan belongs to the family of glycosamino-glycans (GAGs), but it differs from the other mem-bers. Hyaluronan is a non-sulfated polymer consistingof a variably-sized but larger molecule of up to 25,000disaccharide units, giving a molecular mass of up to10 MDa. Within the structure of the extracellular ma-trix (ECM), all the GAGs, apart from hyaluronan, arecovalently linked with a protein core, creating struc-tures known as proteoglycans. Hyaluronan constitutesa frame for coordinately attached proteoglycans or theother GAGs, creating huge aggregates (Fig. 2) [95].

Hyaluronan, in low concentrations, occurs ubiqui-tously in human body. It has been estimated that anadult human organism contains about 12–15 g of hya-

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Hyaluronan and cancerMicha³ S. Karbownik and Jerzy Z. Nowak

Fig. 1. Structural formula of the disaccharide unit of hyaluronic acid –HA (n – the number of mers in a polymer molecule)

Fig. 2. Hyaluronan aggregates in extracellular matrix (adapted from[55])

luronan. Over half of the total body hyaluronan oc-curs in the skin. Moreover, it is found in connectivetissue, synovial fluid, vitreous body of the eye and in-tervertebral discs. Hyaluronan is also found in highconcentrations during embryogenesis: in fetal tissues,amniotic fluid, as a major constituent of fetal struc-tures. Interestingly, hyaluronan is abundant in malig-nancies [95, 117, 119].

Hyaluronan has remarkable physical properties.Due to negative charge, it creates numerous coordi-nate bonds with water molecules. One gram of so-dium hyaluronate can hold up to 6 liters of water. Ata concentration as low as 0.1%, lower than in moistur-izing eye drops, the HA chains are entangled, result-ing in an extremely high viscosity which is shear de-pendent. These properties contribute to maintain ap-propriate tissue hydration and tension. The presenceof hyaluronan in synovial fluid within joint cavitiesprovides necessary lubrication and serves as a shockabsorber. The remarkable rheological properties ofhyaluronan make it crucial in decreasing the frictionbetween sliding surfaces in joints and tendon sheaths.Hyaluronan also plays a role of free radical scavenger,protecting the skin from the negative impact of ultra-violet rays in sunlight [22].

The production of hyaluronan used in clinical medi-cine was initiated by Endre A. Balazs, who extractedthe first highly purified HA from rooster combs, theanimal tissue with by far the highest HA content, andumbilical cords. Hyaluronan has an identical structurein all living organisms, making it possible to obtainwell-tolerated molecules from various biologicalsources. However, animal tissues are not perfect mate-rials, because HA isolated in that way may containtraces of contaminating ingredients evoking allergic re-actions. Nowadays, hyaluronan is mainly produced via

recombinant streptococcal fermentation [68].

Biomedical applications of hyaluronan

Hyaluronan has found a number of applications inmedicine. The first product containing hyaluronan(Hyalgan) was registered in the 1960s by the Italiancompany Fidia and since then it has been used topi-cally for the treatment of burns and skin ulcers. Someof the medical uses of hyaluronan are reviewed below[54, 58].

• Ophthalmology. Because of the viscoelastic proper-ties of hyaluronan, it is used in a number of key oph-thalmologic surgeries. It protects delicate eye tissues,provides space and replaces vitreous fluid lost duringsurgical manipulation such as cataract surgery, lensimplantation, corneal transplantation. In addition,hyaluronic acid in an aqueous solution represents oneof the most common type of lubricant eye drops (arti-ficial tears) that can be used to alleviate dry eye syn-drome, a very common condition characterized bydryness, burning and a sandy-gritty eye irritation.• Rheumatology. A progressive degradation of poly-meric carbohydrates in synovial fluid (mainly hyalu-ronan) can be observed in the course of rheumatoidarthritis, while degradation of cartilage and bone ac-companies osteoarthritis. Patients suffering fromthese diseases benefit from viscosupplementationwith hyaluronan intra-articular injections. Admin-istration of HA preparations has been reported to im-prove symptoms and decrease the use of potentiallyharmful nonsteroidal anti-inflammatory drugs(NSAIDs) in patients with osteoarthritis. It has beensuggested that the beneficial effect of HA treatmentmay be also attributed to its anti-inflammatory, anal-gesic and antioxidant features. Moreover, someorally-administered dietary supplements contain hya-luronic acid. Although producers describe these prod-ucts as “joint lubricants” or “skin enhancers”, theirclinical efficacy has not been proven and the negligi-ble oral bioavailability of HA [65] makes it doubtful.• Otolaryngology. Viscoaugmentation of the vocalcords, the repair of injured or scarred vocal cords, andtreatment of glottal insufficiency are additional usesof hyaluronan. In hearing disorder therapy, films ofhyaluronan derivatives are used in ear and sinus sur-gery, promoting wound healing, facilitating re-epithelization, and preventing adhesion between lay-ers of mucousal tissues.• Gynecology. Hyaluronan preparations available asvaginal suppositories or creams are intended to en-hance healing processes in such contexts as followingsurgery, in mucosal atrophy, after chemo- or radio-therapy and childbirth.• Dermatology and plastic surgery. Preparations ofhyaluronic acid fillers composed of slightly cross-linked hyaluronan are widely used for filling facialwrinkles and depressed scars. Such gels are more ef-fective in maintaining cosmetic correction thancollagen-based products. Hyaluronan preparations ap-plied as topical gels are helpful in the process of heal-


ing fresh or chronic skin wounds and venous leg ul-cers. Due to its antioxidant properties, HA serves alsoas an anti-inflammatory component in wound dress-ing materials.• Oncology. Oncology? How is it possible, that theviscoelastic biopolimer, with the ability to bind watermolecules, decreasing the friction between slidingsurfaces or filling intraoperative tissue loss can con-tribute to cancer treatment? The answer is hidden be-hind the complexity of hyaluronan metabolism andinteractions with its binding proteins, called hyalad-herins. Although hyaluronan has been perceived fordecades as being only an inert component of connec-tive tissue, functioning as one of the building materi-als of the body, it is in fact, a “dynamic” molecule.The evolution of the attitude towards hyaluronan hasbeen referred to by one of the most respected authori-ties on hyaluronan, Bryan P. Toole, as “from extracel-lular glue to pericellular cue” [130]. This review pro-vides a molecular and pathophysiological backgroundto understanding hyaluronan-based anti-cancer thera-peutics.

Hyaluronan metabolism and cancer

Biosynthesis...

Contrary to all glycosaminoglycans, which are pro-duced in the Golgi apparatus, hyaluronan is synthe-sized at the inner side of plasma membranes to formhuge aggregates of molecular mass up to 1 MDa. Af-terwards, it is extruded through pore-like structures tothe cell surface [107]. Hyaluronan is produced by cellmembrane-bound proteins – glycosyltransferases –called hyaluronan synthases (HASs). The enzymesmediate the chemical reaction of transglycosylation ofD-glucuronic acid and N-acetylglucosamine nucleo-tide precursors elongating the HA molecule accordingto the scheme:

GlcA-UDP + GlcNAc-b-1,4-HA ®

UDP + GlcA-b-1,3-GlcNAc-b-1,4-HA

UDP – uridine-5’-diphosphate; HA – chain of hyalu-ronic acid.

The discovery of three types of HASs has enableda better understanding of hyaluronan biosynthesis.HAS-1 is encoded by the gene has1 linked on

19q13.3 human chromosome and is responsible forthe synthesis of high-molecular-weight hyaluronan.The gene has2 is localized at chromosome 8q24.12,HAS-2 is responsible for generation of HA in re-sponse to shock, inflammation and tissue repair; has3,localized on chromosome 16q22.1, produces mole-cules of masses up to 100 kDa [33, 50, 55].

...and its role in cancer

The level of expression of has genes is regulated dur-ing physiological and pathological processes by vari-ous growth factors, cytokines and cellular stress. Themalignant transformation of cells influences extensiveHA production. Only has2 expression is elevated inv-Ha-ras transformed cells which show little malig-nancy, whereas both has1 and has2 expression isincreased in highly malignant cells transformed withv-src. Clinicopathological studies have confirmed thatoverexpression of has1, has2 and/or intronic genesplicing is seen in highly metastatic carcinomas andcorrelates with poor prognosis in some human cancers[50, 98]. On the other hand, suppression of overex-pressed has2 results in decreased tumor cell migra-tion, decreased tumor cell growth, increased cisplatinsensitivity in oral cancer cells [137] and decreased in-vasive capability of breast cancer cells [6]. HAS-3 ac-tivity also has a similar role; it mediates tumor growthin colon cancer cell lines. Inhibition of the HAS-3isoenzyme activity decreases in vivo colon cancergrowth [64, 127]. Interestingly, studies of nonmalig-nant cells overexpressing different HASs reveal thatthe high levels of HA induced by HAS-3 were, con-trary to malignant cells, inversely correlated with cellmotility [12]. Thus, it can be generalized and con-cluded that hyaluronan, the product of HASs activity,may positively correlate with the malignant pheno-type of cancer cells.

The more hyaluronan, the more of a “pro-cancerenvironment” it becomes. It resembles a “lesson inembryology”. Studies in embryogenesis show thathyaluronan plays a crucial role in physiological devel-opment processes and in tissue modeling. HA is espe-cially abundant when undifferentiated cells are prolif-erating rapidly and move from their stem niche to thesite of organ development. The hyaluronan environ-ment inhibits differentiation and promotes prolifera-tion. In order for the cell to differentiate, it has to re-move its HA coat [117, 119]. Cancer cells seem tolearn “the lesson in embryology” trying to copy nor-



mal, undifferentiated cells in embryogenesis. It maybe the abundance of hyaluronan or failure to shed theHA coat that promotes malignant cell proliferationand development of cancer [119].

On the other hand, it has been reported that somemalignancies show diminished HA content. Poorly-differentiated squamous cell carcinomas have a lowHA concentration, which correlates with increasedlymph node metastases. The presence of reduced lev-els of HA and its main hyaladherin, CD44, has beenassociated with poor prognosis in stage I cutaneousmelanoma, although it has been shown earlier thathyaluronan enhances growth and metastatic capacityof melanoma cells in vitro [117].

These scientific reports demonstrate that hyaluro-nan expression may be involved in different stages ofmalignant tumor progression. HASs expression maycorrelate with cancer cell malignancy. The up-regu-lation of these enzymes, which is observed in somecancers, may contribute to poor prognosis. HASsseem to be also valuable potential targets for anti-cancer drugs and attempts to create such drugs are be-ing made.

Degradation…

Hyaluronan is catabolized in two main processes: en-zymatic and non-enzymatic. In the enzymatic process,hyaluronoglucosaminidases are involved. These en-zymes are more commonly known by the more con-venient name hyaluronidases (HYALs). The enzymescatalyze the reaction of hydrolysis of b-1,4-glycosidicbonds (Fig. 1). Six genes have been identified whichencode hyaluronidases in the human genome: hyal1,hyal2 and hyal3 linked on 3p21.3 human chromo-some and hyal4, ph-20 (or spam1) and pseudogenephyal1 (that lost its protein-coding ability) localizedat chromosome 7q31.3 [122]. Hyaluronidases pre-dominantly degrade hyaluronan, however, they have

also a limited ability to degrade chondroitin and chon-droitin sulfates [120].

HYAL-1 is the main blood plasma and tissue hya-luronidase, responsible for degrading high molecularweight hyaluronan (HMWH) to small tetrasaccha-rides [18]. HYAL-2 is a tissue isoenzyme anchored tothe cell membrane. It decomposes HMWH to inter-mediate in length molecules (low molecular weighthyaluronan – LMWH) of ~ 20 kDa (i.e., containingabout 50 repeating disaccharide units) [66]. HYAL-3and HYAL-4 are widely expressed, but their functionsremain unknown. PH-20 isoenzyme, firstly discov-ered by Duran-Reynals in extracts of rabbit testiclesand called “spreading factor”, is essential in the fer-tilization process. Its function is to digest ECM in thecorona radiata surrounding the oocyte and to allowsperm to penetrate inside the oocyte [76].

Hyaluronan catabolism is a two-stage process:HMWH aggregates in the extracellular matrix, boundto specific hyaladherin CD44, undergo enzymatic de-pletion by HYAL-2 isoenzyme to intermediate hyalu-ronan fragments – LMWH. These fragments becomecompletely degraded after endocytosis by lysosomalHYAL-1 (the isoenzyme which is active in acidic pH)or may be transported via the lymphatic system andthe blood to the liver and kidney [117] (Fig. 3).

Reactive oxygen species – ROS (and/or reactive ni-trogen species – RNS) may also contribute to hyaluro-nan catabolism through non-enzymatic breakdown.Hyaluronan is sliced nonselectively to fragments ofvarious lengths. Free radicals not only directly de-grade HA, but also may increase hyaluronidaseHYAL-2 expression, which was demonstrated in pri-mary airway epithelial cells [90]. There is no doubtthat the free-radical process plays an important role inhyaluronan metabolism, however, the extent of thatmechanism is still uncertain when compared to over-all catabolism. In fact, any inflammatory process, par-ticularly a chronic one, contributes to free radical pro-


Fig. 3. Hyaluronan degradation process

duction. As an illustration, the average molecularmass of hyaluronan in the synovial fluid of healthypeople ranges from 6 to 7 MDa, whereas in rheuma-toid arthritis, it falls to 3-5 MDa. It seems likely thatthis molecular mass reduction is due to the activity ofreactive oxygen species [95].

As mentioned earlier, hyaluronan is a “dynamic”molecule. It undergoes a permanent process of bio-synthesis followed by degradation. Actually, the rateof hyaluronan turnover is extraordinarily high, but itvaries and depends on location and physiology. Thehalf-life of blood plasma hyaluronan is about 2–5 min(however, a half-life of intravenously infused hyalu-ronan solution is dose-dependent and varies from 2 to14 h [44]), in synovial fluid it is about 12 h, in carti-lage – 1–3 weeks, and in the vitreous body of the eye– up to 70 days. About one third of body hyaluronanis replaced every day [95].

…and its role in cancer

The well-defined positive impact of hyaluronan syn-thases activity on the growth, migration and malig-nancy of most types of cancer cell as well as the poorprognosis of cancer itself, suggests that hyaluroni-dases may play a role as tumor suppressors. A body ofresearch confirms this hypothesis. Firstly, both chro-mosomal loci of genes encoding hyaluronidases occurat the sites of putative tumor suppressor genes [77].Gene hyal1 was previously named LuCa1 (LungCan-

cer1), because it has been reported that loss of hetero-zygosity or homozygous deletion occurs in most lungcancers [144], and squamous cell carcinomas of thehead and neck [32]. Secondly, hyal1 overexpressionresults in the opposite outcome: the suppression of tu-morigenicity in an experimental model for colon car-cinoma [51]. Thirdly, endometrial cancer tissue dem-onstrates lower HYAL-1 and HYAL-2 mRNA levelscompared to normal endometrial tissue [96]. Finally,Shuster et al. [114] report that the tumor volumes ofhuman breast carcinoma xenografts implanted into se-vere combined immunodeficiency mice (i.e., SCIDmice) noticeably decreased by up to 50% in only4 days after administration with intravenous superhigh concentrations of hyaluronidase.

In spite of this evidence, a considerable body of con-trary data exists, which suggests that hyaluronidasesexpression and activity is elevated in many other can-cers. Clinical studies indicate that both hyal1 and hyal2

are overexpressed in samples of colorectal cancer, but

especially in advanced stages of the disease [8]. In vi-

tro studies on breast cancer demonstrate that knock-down of hyal1 expression in cells results in decreasedcell growth, adhesion, invasion and angiogenesis,while forced overexpression of the isoenzyme pro-moted cell malignancy. Moreover, in a nude mousemodel, forced hyal1 expression induced breast cancercell xenograft tumor growth and angiogenesis [125].The hyal1-expressing bladder and prostate tumors had4–9 times more microvessel density and larger capillar-ies than tumors lacking hyal1 expression, resemblingbenign neoplasms [69]. A similar correlation betweenHYALs and cancerous growth has been observed inother tumors of the genitourinary tract [116], the headand neck [31], as well as the brain [24].

The rise in hyaluronidase level is so significant insome malignancies that it may serve as a diagnosticmarker of the disease. The detection of urinary hyalu-ronidase RNA and its enzyme activity is a promisingnoninvasive test with high sensitivities and specificitiesfor detection of bladder cancer [28], whereas the meas-urement of hyal-1 gene expression may be a potentialbiomarker of some subtypes of epithelial ovarian can-cer [142]. De Sá et al. [23] report that referring to typesof hyaluronidases is not accurate enough, becauseHYAL isoforms have been described as various prod-ucts of alternative splicing responsible for differencesin enzyme activity. They revealed HYAL-3-v1 protein,which is enzymatically inactive, to be characteristic oflow-grade prostate cancer, according to the Gleasonscore, and a marker of better prognosis in adenocarci-noma and squamous cell carcinoma of the lung. Thistest can be used to select patients for watchful waitingprotocols. HYAL-3-v2 and HYAL-1-v3 were expressedpreferentially by prostate tumors that had not recurred.HYAL-1-wild-type was associated with a poorer prog-nosis of lung cancer mentioned above.

The contrary data presented above suggest that hyalu-ronidases may function as both tumor suppressors andtumor promoters. A step towards resolving this paradoxhas been taken recently. It was shown that whileHYAL-1 acts as a tumor promoter at the naturally oc-curring levels expressed by tumor tissues, HYAL levelsexceeding 100 mU/106 cells, not naturally expressedby tumor cells, significantly reduces tumor incidenceand growth due to induction of apoptosis [69, 131].Therefore, the function of HYAL as a tumor promoteror a suppressor is a concentration-dependent phenome-non, but in tumor tissues, the tumor cell-derived HYALacts mainly as a tumor promoter [73].



Some studies indicate that both hyaluronan synthe-sizing and degrading enzymes are elevated in malig-nancies, and may “cooperate” in cancerous growth.Enegd et al. [29] reveal that HAS overexpressionalone is not enough to promote in vivo astrocytomagrowth. Increased synthesis of hyaluronan by cancercells promotes tumor cell growth only if the cells ex-press hyaluronidases as well. Such specific crosstalkbetween HASs and HYALs has been confirmed bySimpson in human prostate tumor cell line in mice:the most significant tumorigenic potential was real-ized by artificial coexpression of both hyal1 and has2

genes [115]. In another study by Bharadwaj et al. [7],has2-expressing prostate tumor cell line exhibited lit-tle tumorigenicity and no metastatic potential. In con-trast, co-expression of has2 and hyal1 genes in concertshowed a greater than sixfold increase in tumorigene-sis, significant motility and spontaneous metastasisin vivo. Based on the findings cited above, it can beproposed that, at least in some types of cancers, it isnot the increased amount of hyaluronan, but rather itsturnover, that influences its tumor-promoting ability[50]. Rapid hyaluronan turnover may be regarded asthe constant presence of hyaluronan mixture in themyriad of molecular sizes: from finely fragmentedoligosaccharides, intermediate LMWH to huge aggre-gates of HMWH.

Hyaluronan fragments

Hyaluronan as a polymer molecule exhibits a varietyof chain lengths. High molecular weight hyaluronan(HMWH) attaining about 0.1 – 10 MDa (hundreds tothousands mers), low molecular weight hyaluronan(LMWH) of about tens kDa (about 50 mers) andoligosaccharides (oHA) of up to a few kDa (up toa dozen of mers) have distinctly different biologicalfunctions. HMWH is mainly involved in a variety offunctions based on rheological properties as tissue hy-dration, shock absorption, decreasing frictions andspace-filling functions. HMWH has been also re-ported to possess anti-inflammatory, anti-angiogenicand immunosuppressive potential. It reduces signal-ing cascades in inflammation model of mouse chon-drocytes [13] and inhibits phagocytosis in monocytes,macrophages, and granulocytes. The rate of inhibitionis proportional to HA molecular size. The mechanismof inhibiting angiogenesis is poorly known, but itseems to be due to phosphorylation of range of pro-

teins. The immunosuppressive effect derives in partfrom the ability of high-molecular-size HA to coatcell surfaces thus preventing ligand access to surfacereceptors [61, 121].

In physiological conditions, hyaluronan has a highaverage molecular mass in excess of 1 MDa. However,following e.g., tissue injury, hyaluronan fragments oflower molecular mass accumulate [52]. While high-molecular-size HA, in general, promotes tissue integrityand quiescence, the generation of breakdown productsenhances signaling transduction and is usually perceivedby cells as a “danger signal” [13]. Hyaluronan of lowermolecular size mediates wound healing processes, chon-drogenesis, infection, and cancer biology. LMWH andHA oligosaccharides smaller than 36 kDa (100 dissacha-rade units) induce proteolytic cleavage of CD44 – one ofthe major hyaladherins on the surface of cancer cells. Itpromotes tumor cell migration, and subsequently tumorprogression. Inhibition of the interaction by anti-CD44neutralizing monoclonal antibody results in completeabrogation of these cellular events [123]. IncreasedCD44 cleavage has been documented in gliomas, breast,colon, and ovarian cancers, and in non-small cell carci-nomas of the lung [97]. A recent study also revealed dis-tinct role of high- and low-molecular weight HA. It wasdocumented that LMWH increases adhesion capacity offibrosarcoma cells, which plays a critical role in the pro-cess of metastatic tumor dissemination, whereasHMWH has an opposite effect [60]. Another very recentstudy confirms the pro-metastatic role of hyaluronan oli-gosaccharides (6.5 kDa) [62].

The hyaluronan oligosaccharides and LMWH havewell-established angiogenic properties. Tumor vascu-larization is observed along HA-rich areas associatedwith the generation of HA fragments. Highly invasivebladder cancers, for instance, are able to generate angio-genic HA fragments of 10–15 disaccharide units that canstimulate endothelial cell proliferation, adhesion andcapillary formation [124]. The effect of intensified in vi-

tro endothelial cell proliferation, tube formation and/orincreased mRNA of vascular endothelial growth factor(VEGF) and in vivo revascularization elicited by HA oli-gosaccharides and LMWH of 4–25 mers (but not largerHA molecules) have been reported in several studies[19, 35, 48, 78, 89, 103, 118, 139]. Moreover, smallangiogenic HA fragments may serve as prognosticmarkers. oHA are found in the urine of high-grade blad-der cancer patients [71], saliva of patients with high-stage head and neck squamous cell carcinoma [31] andprostate cancer tissue [72].


The pathways through which HA fragments modulateangiogenesis are relatively well understood. Activationof Raf-1, extracellularly regulated kinases 1/2 (ERK1/2)and early response genes including c-fos and c-jun areinvolved in endothelial cell migration and prolifera-tion [118]. Generally, the induction of angiogenesisby oHA seems to be the mechanism for cancer cell in-vasiveness, and serves another example of how ma-lignancies can commandeer normal physiologicalfunctions, attributed originally to wound healing pro-cess, for their own purposes [121].

Interestingly, some authors report that, the smallestoHA fragments such as exhaustively digested HA ma-terial, especially tetrasaccharides, had no angiogenicpotential, as opposed to longer chain oHA [19, 78].Still, a number of findings suggest that the smallestoHA may counteract tumor development. Oligomersof around 7 disaccharide units, inhibit anchorage-independent growth of several tumor cell types andinduce apoptosis in vitro [37]. Toole et al. [132] re-ported that small oHA (3-9 disaccharide units), butnot large polymers, even kill many types of cancercells by triggering apoptosis while leaving normalcells unaffected. Zeng et al. [145] reveal that injectedhyaluronan oligomers can markedly inhibit in vivo

melanoma growth. Ween et al. [140], artificially pro-moted motility, invasion, and metastatic behavior ofsome ovarian cell lines, by adding versican and/or ex-ogenous hyaluronan; they observed that small HAoligomers of 6–10 disaccharides were able to reversecancer cell adhesion, motility and invasion both in thepresence and absence of exogenous HA. They con-clude that oHA are promising inhibitors of ovariancancer dissemination. Urakawa et al. [134] deter-mined the effective size of HA oligosaccharides re-quired to inhibit cell growth in a highly-invasivebreast cancer cell line. They found that HA decasac-charides significantly inhibited cell growth, motility,and invasion, whereas tetrasaccharides did not.Moreover, decasaccharides suppressed the expansionof osteolytic lesions in a mouse bone metastasismodel of breast cancer. Interestingly, both oHA, butnot HMWH, have has-expression inhibiting potential.The fact that oHA inhibit endogenous hyaluronan pro-duction was also reported previously [87].

Hyaluronan oligosaccharides represent a very in-teresting group of molecules that may exhibit equivo-cal properties. They can be considered as factors ei-ther inducing tumor cells adhesion, angiogenesis andmetastatic potential or inhibiting cell growth andevoking apoptosis. The precise role of hyaluronan

oligomers in a specific tumor may depend on the sizeof the molecule, its concentration, nature of the cancerand surrounding cells. It is notable that most of thementioned experiments have been performed usingisolated cells or tissues, or have investigated onlya single biological process. In order to describe thefully integrated picture of how hyaluronan oligomersinfluence cancer, there is a need to identify furthermodels for basic and pre-clinical studies. The researchshould eventually aim for investigating parametersused in clinical oncology, e.g., disease free survival,progression free survival, time to progression, oroverall survival.

The reason why larger and smaller hyaluronanmolecules have opposite functions is determined notonly by the factors already reported above, i.e., sup-pression of HA production and CD44 cleavage [131].Pharmacodynamic analysis revealed HA-CD44 inter-action to have a non-homogenous quality. HMWHbinding at the cell surface is a complex interplay ofmultivalent binding events. Small oligosaccharidesbetween 3 and 9 disaccharide units, because of theirshortness, exhibit only monovalent binding to CD44,displacing any hyaluronan polymer from membrane-bound receptors (receptor antagonists). At approxi-mately 10 to 19 disaccharide units, a progressive in-crease in avidity of about 3× was seen, suggesting thatdivalent binding was occurring [67, 132]. Thus, HAmolecule length subsequently affects intracellular sig-naling pathways and biological effects. The overallhypothesis for pro- and anti-cancer activity of variouslength hyaluronan molecules is presented in Figure 4.

Hyaluronan-binding proteins

It was in 1979, when hyaluronan was, for the firsttime, demonstrated to bind specifically and with highaffinity to a variety of cells [133]. Now it is evidentthat the actual pharmacological effects of hyaluronanmolecules of various sizes described above are medi-ated through interactions with certain proteins calledhyaladherins. The group of hyaluronan-binding pro-teins comprise cell surface receptors (CD44,RHAMM, Toll-like receptors, LYVE-1 and HARE)and extracellular matrix or blood plasma proteins(link protein, aggrecan, brevican, versican, TSG-6 –tumor necrosis factor-a-stimulated gene 6 and SHAP



– serum-derived hyaluronan-associated protein) [95].The structures and functions of the main hyaladher-ins, CD44 and RHAMM, are briefly highlightedbelow.

CD44

CD44 protein (cluster of differentiation 44) is a mainhyaluronan-binding receptor. It is expressed on themajority of cells. CD44 is a transmembrane proteinencoded by a single gene. However, it exhibits con-siderable structural diversity, because of extensive al-ternate splicing: theoretically there can be more than800 isoforms, but not all are expressed. The variantwithout any alternatively spliced exons is called stan-dard CD44 or CD44s, while the others are known asvariant CD44 or CD44v. The diversity of CD44 maybe further increased by undergoing reversible post-translational glycosylation. The short cytoplasmic tailof CD44 binds to ankyrin and ezrin-radixin-moesinproteins, providing a link to the cytoskeleton, as wellas to merlin, which abrogates this binding. It isthought that CD44 may exist in a variety of states: in-active and unable to bind HA, inducible (can bind HAon contact with inducers) or constitutively active.CD44 variants exhibit especially high affinity to HA[55, 86]. The schematic structure of CD44 is pre-sented in Figure 5.

As already described, various length hyaluronanmolecules exhibit complex pro- or anti-cancer activi-ties. Most of these activities are mediated throughCD44 receptor [86, 93, 131, 139]. While standard

CD44 is ubiquitous, variants of CD44 are expressedmostly by tumor cells. Pathological conditions pro-mote alternate splicing and post-translational modifi-cations to produce diversified CD44 molecules withenhanced HA binding, which leads to increased tumo-rigenicity [86]. Thus, CD44v (especially often CD44-v6, v4-v7 and v6-v9) is observed to be up-regulatedon the surface of cancer cells, involved in cancer in-vasion, correlated with malignant properties and/oruseful for early molecular diagnosis of many cancersincluding breast [3], gastric [21], gynecologic [47],


Fig. 5. Schematic structure of CD44 (adapted from [55])

Fig. 4. The overall hypothesis for pro- and anti-cancer activity of various length hyaluronan molecules

head and neck [138], lymphoma [91], osteosarcoma[75], prostate [25], colorectal [59] and lung [94] can-cer.

CD44 seems to be essential in the onset of malig-nant transformation, as it has been identified at thesurface of cancer stem cells: a small population ofcancer cells responsible for maintaining the tumorand, possibly, the formation of new tumors at meta-static areas. This finding suggests that CD44 may bea potential diagnostic target for early cancer detection,contributing to early initiation of anti-cancer therapyand its better outcomes [26, 102].

The interaction between various hyaluronan frag-ments and CD44 receptor (HA-CD44), contributing tocancer cell adhesion, migration, invasion and growth,triggers multiple intracellular signaling pathways. Theexact nature of the interaction is given in more detailearlier [55] and is presented in Figure 6. In short,ankyrin, which is attached to cytoplasmic tail ofCD44, promotes the cytoskeleton activation essentialto cell adhesion and migration [146] (branch A). Acti-vation of RhoA [11] and Rac1 [9], small GTPase pro-teins, results in myosin light chain phosphorylation,microtubule organization and cell migration. RhoAalso mediates the lowering of extracellular pH, activa-tion of ECM-degrading enzymes and, in consequence,cancer cell invasion [10] (branch B). HA-CD44 acti-vates receptor tyrosine kinases contributing to manyaspects of malignant cell behavior [36, 88, 100]

(branch C). CD44 may associate with the proteolyticform of the matrix metalloproteinase-9 (MMP-9),which promotes collagen degradation and tumor cellinvasion [143] (branch D). This considerably simpli-fied scheme does not elucidate the whole complexityof HA-CD44 interaction, but indicates only a fewpossible signaling pathways. The aim of the scheme isto clarify the overall concept of HA-CD44 interac-tion, highlight its importance and lay the grounds forunderstanding the target for potential anti-cancertherapeutics.

The involvement of the CD44 molecule in cancerprogression is not simple as described above. The cor-relation between CD44 and tumorigenicity is not ab-solute and discordant results have been reported.Firstly, the role of standard CD44 may be the oppositeto that of CD44v: the down-regulation of CD44s incolon cancer [17], neuroendocrine carcinomas [63],and prostate cancer [34] is postulated to result inmetastatic and/or aggressive cancer behavior. Sec-ondly, in a mouse model of spontaneously metastasiz-ing breast cancer, the loss of CD44 (both standard andvariant forms) promoted metastasis to the lung [74].Thirdly, CD44 acts as metastasis suppressor gene ofprostate cancer [56]. Altogether, although most of theevidence suggests a positive correlation betweenCD44 and cancer, attempts to make a monosemousevaluation of CD44 have sparked debate over its ex-act role in tumor progression. Various factors affect



Fig. 6. Impact of HA-CD44 interactionon intracellular signaling and cancercell growth (adapted from [55] withchanges)

the complexity of the role of CD44, which need to betaken into consideration in order to gain a clear pic-ture.

RHAMM

RHAMM protein (receptor for hyaluronan-mediatedmotility), recently designated as CD168, is encoded bythe RHAMM (HMMR) gene. It was originally discov-ered in 1982 as a soluble molecule that altered cell mi-gration behavior, when bound to hyaluronan. As withCD44, it may be expressed in multiple splice variants.The HA-binding region of RHAMM is highly con-served and bears no structural resemblance to the linkmodule in CD44. RHAMM is poorly expressed inmost normal human tissues. In the mouse model, it hasbeen shown that RHAMM expression is essential toneither embryonic development nor adult homeostasis.RHAMM expression increases following tissue injuryand its only known physiological function is to pro-mote wound repair. Thus, RHAMM deletion results inslow healing of skin wounds [81, 129].

RHAMM expression is increased during the neoplas-tic progression of a variety of human tumors [126, 128],e.g., renal cell carcinoma [14], prostate cancer [42], oralsquamous cell carcinomas [113], acute myeloid leuke-mia [40] and multiple myeloma [82], and seems to beessential for some cancer cell functions [60]. RHAMMalternate splice variants dominate over standard forms ofa protein in cancers [82, 113]. High RHAMM expres-sion predicts shorter survival rates of patients [113] andis associated with metastasis [14, 42].

Interestingly, two populations of RHAMM areknown: intra- and extracellular. RHAMM normally islocalized intracellularly and is only released by certainpoorly-defined stimuli. The intracellular RHAMM pro-tein is co-localized with microtubules, both in the inter-phase and in dividing cells, representing a member ofthe family of microtubule-associated proteins (MAPs).High levels of RHAMM correlate with genomic insta-bility in multiple myeloma [80], and the molecularmechanisms of the phenomenon have been describedto some extent [112, 113, 126, 128]. RHAMM has beenproposed to be a novel breast cancer susceptibility genewith the potential to promote genomic instability [126].Two case-control studies indicated that variations inthe RHAMM locus were linked to an elevated risk ofbreast cancer [108]. It has been revealed that RHAMMis mechanistically associated with breast cancer type 1

susceptibility protein (BRCA1). BRCA1 loss-of-function combined with RHAMM overexpression maycontribute to chromosomal instability during cancerprogression [79, 126].

The presence of intracellular hyaladherin RHAMMraises an interesting possibility that hyaluronan maybe located not only extracellularly. In 1976, rat brainnuclei were isolated and found to be rich in glyco-saminoglycans such as hyaluronan. The fact was sur-prising, but did not receive widespread attention.Nowadays, both the intracellular and nuclear forms ofhyaluronan are evident. Intracellular HA is involvedin cell signaling, whereas nuclear HA may promotechromatin condensation and facilitate mitosis, sinceelevated synthesis of hyaluronan occurs during theG2/M mitotic stages. In the mitotic spindle, hyaluro-nan has been shown to colocalize with tubulin andwith RHAMM [30]. Perinuclear hyaluronan may ini-tiate the synthesis and organization of so-called cablehyaluronan structures. The hyaluronan cables may beinvolved in monocyte adhesion during the process ofinflammation [45]. Despite the relatively long historyof intracellularly located hyaluronan and RHAMM,there is still very little data about their precise role.

The mechanism of RHAMM transport out of thecell remains unclear, the gene does not encode a tradi-tional leader sequence to permit secretion via the tradi-tional Golgi/endoplasmic reticulum export pathway,but may involve transport channels or proteins, flip-pase activity, or exocytosis [81]. Extracellularly,RHAMM is a non-integral cell surface protein associ-ated with CD44 that, upon binding to HA, activatespro-migration and invasion functions. An effect ofCD44 on tumor cell motility may depend in part on itsability to partner with RHAMM [43]. This hypothesismay reconcile the discrepancies in CD44 function intumor progression described above. ExtracellularRHAMM may enhance the localization of CD44 onthe cell surface, intensifying subsequent CD44-depen-dent signaling through mitogen-activated protein ki-nases (MAPK/ERK1,2 pathway) [126, 129]. On theother hand, CD44 is one of the components that maybe necessary for the induction of RHAMM expression.Co-expression of any of the CD44v variants withRHAMM identifies a subgroup of diffuse large B-celllymphoma patients with a very poor prognosis [91].HA-promoted endothelial cell tube formation in angio-genesis is mediated through CD44-RHAMM interplay[101]. In conclusion, a strong link between CD44-RHAMM implies their crucial involvement in concert


in cancer growth. Both CD44 and RHAMM appearnot only vitally important molecules in HA-dependenttumorigenicity, but also promising targets for abrogat-ing tumor growth by hyaluronan-based therapeutics.

Towards hyaluronan-based therapeutics

The research on hyaluronan involvement in cancerpathogenesis laid the foundations for development ofnovel anti-cancer therapeutic strategies. HA-inducedcancer cell growth and metastatic potential may be at-tenuated by targeting HA biochemical pathways.Three different therapeutic approaches may be indi-cated: (1) targeting hyaluronan metabolism, (2) tar-geting hyaladherins, and (3) HA-based drug deliverysystems.

1. Targeting hyaluronan metabolism

As already reported, hyaluronan abundance due tosynthase overactivity may contribute to tumorgrowth. Therefore, inhibition of the enzymes mayserve as an interesting anti-cancer approach. 4-Me-thylumbelliferone (MU) is a drug known also underthe name hymecromone as a choleretic and antispas-modic used in gastroenterology in the treatment ofmotor disorders of the bile ducts. MU has been widelyinvestigated as a hyaluronan synthase inhibitor andproposed as a promising candidate for cancer treat-ment. It has been shown to inhibit growth and motilityor induce apoptosis of several cancer cell lines [2,104, 135]. In vivo it reduces tumor microvessel den-sity [70] and development of distant metastasis [2].MU has been also reported in a mouse model to sensi-tize human pancreatic cancer cells to the cytotoxicdrug gemcitabine [92]. Convenient oral administra-tion and low toxicity profile are additional advantagesof MU [70]. No clinical trials have been yet con-ducted to determine its anti-cancer activity.

Hyaluronan-degrading enzymes, hyaluronidases,have been shown to possess both cancer-promotingand suppressing properties. Consequently, two oppo-site approaches have been proposed. Sulfated oligo-saccharides of hyaluronan (sHA) have been known toinhibit enzymatic activity of hyaluronidases for over60 years, however, just very recently, it was shown

that sHA blocks the proliferation, motility, and inva-sion of some prostate cancer cell lines and inducesapoptosis. In xenograft models, sulfated hyaluronanoligomers are highly effective in inhibiting tumorgrowth [5]. Phlorotannins from the brown algae Eis-

enia bicyclis and Ecklonia kurome and polysaccharidefrom hot water extract of the sporophyll of Undaria

pinnatifida also show inhibitory activity against hya-luronidases. The extracts have been proposed as use-ful medicinal food with potential anti-cancer activity[39].

On the other hand, hyaluronidase itself may con-tribute to a better outcome of anti-cancer treatment. Ithas been demonstrated in several clinical trials thatthe addition of bovine HYAL to chemotherapeuticprotocols results in longer survival and/or lower re-currence [4, 57, 105]. The mechanism of synergisticeffect is explained by reduction of intratumoral pres-sure or by increasing tumor-specific accumulation ofchemotherapy. Despite such promising results, furtherinvestigations have been abandoned due to allergic re-actions and induction of inflammation or pain in thejoints because of enhanced HYAL activity in normaltissues. Nowadays, it seems that hyaluronidases “de-serve a second look” [141]. A resumption of interestin “second-generation” PEGylated recombinant hu-man HYAL has been observed [53].

2. Targeting hyaladherins

Targeting hyaladherins presents a very promising ap-proach against HA-induced tumorigenesis. HA-CD44interaction, which deserves particular attention, initi-ates signal transduction pathways leading to cancercell growth, adhesion, migration, invasion and metas-tasis. Therefore, some ways of antagonizing HA-CD44 interaction have been proposed [1, 86, 99]. (1)Anti-CD44 antibodies themselves may induce apop-tosis of cancer cells [109]. Antibodies against highlyexpressed variants are also designated to selectivelydeliver a cytotoxic drug to cancer cells. Anti-CD44v6conjugated with a cytotoxic drug mertansine has beenused in early phase clinical trials. The patients withbreast or head and neck tumors experienced stabilizeddisease [106]. However, the trial was later aborted dueto limited success. (2) Another example of interrupt-ing HA-CD44 interaction involves small hyaluronanoligosaccharides (3-9 disaccharide units) [132, 134].oHA inhibit downstream cell survival and prolifera-tion pathways, stimulate apoptosis and expression of



cancer suppressor PTEN. In vivo oHA inhibit thegrowth of several tumors implanted as xenografts[37]. oHA also sensitize cancer cells to some chemo-therapeutic drugs inhibiting expression of MDR1 andother ABC transporters [20, 84]. (3) Small interferingRNA (siRNA) and, even more advantageous, shorthairpin RNA (shRNA) targeting CD44v6 in coloncancer also interrupt HA-CD44 interaction. Targetingwith shRNA inhibits distant tumor growth in mice.Due to its selectivity, a number of normal cells ex-pressing CD44 remain unaffected [85, 86].

Anti-cancer vaccination with CD44 and RHAMMantigens seems to represent a very promising ap-proach. Vaccination with cDNA of particular CD44vinduced an immune resistance to progression of mam-mary tumor in mice model, and decreased tumor massand metastatic potential [136]. Vaccination withRHAMM-R3 peptide in patients with hematologicmalignancies induced immunological responses andshowed positive preliminary clinical effects [41].

3. HA-based drug delivery systems

The concept of polymer-drug conjugates was firstproposed by Helmut Ringsdorf in 1975. Conjugatesof hyaluronan and anti-cancer drugs have been intro-duced to the therapy on the basis of CD44 ability tointernalize hyaluronan and drugs bound to it. HA-carrying drugs alone or encapsulated drugs in lipo-somes present a model of therapy selectively targetedto cells highly expressing CD44, i.e., cancer cells.Moreover, HA particles have advantages over otherdrug delivery systems due to biocompatibility, biode-gradability and non-immunogenicity. HA-drug conju-gates may also improve the inadequate water solubil-ity of some anti-cancer agents [16, 33, 86, 106].

A prodrug targeting cancer cells containing pacli-taxel was developed using hyaluronan as drug carrier.The bioconjugate showed selective toxicity againstthe human cancer cell lines (breast, colon, and ovar-ian) that are known to overexpress HA receptors,while no toxicity was observed against a mouse fibro-blast cell line at the same concentrations. Hyaluro-nan-paclitaxel bioconjugate, HYTAD1-p20, showssignificant advantage over conventional paclitaxel interms of in vitro activity against bladder carcinomacells, in vivo safety profile and pharmacokinetics[111]. Currently, a delivery system labelled ON-COFIDTM-P, is undergoing phase II clinical studies insix European countries for the treatment of refractory

bladder cancer [16]. Another hyaluronan-irinotecanconjugate was tested in phase II clinical trial in pa-tients with metastatic colorectal cancer. HA-irino-tecan demonstrated improved efficacy compared toirinotecan alone. The study is currently recruiting par-ticipants for phase III trials [38]. Despite all the clini-cal successes, HA-drug conjugates have their owndisadvantages: no simple chemical conjugation methodexists and the process of hyaladherin-mediated uptakeof the conjugate may be significantly reduced by che-motherapeutic drug attachment. Self-assembled PE-Gylated HA-nanoparticles has been invented to over-come these difficulties [15].

Concluding remarks

Hyaluronan is no longer perceived as only an inertcomponent of connective tissue, functioning as one ofthe building materials of the body. It is a “dynamic”molecule with a rapid metabolism resulting in thepresence of molecules of various sizes: finely frag-mented oligosaccharides, intermediate LMWH tohuge aggregates of HMWH. Hyaluronan is involvedin intracellular signaling mediated by CD44 andRHAMM receptors that leads to cancer cell growth,adhesion, migration, invasion and metastasis. Thus,therapeutics targeting HA-mediated signaling are be-lieved to be promising anti-cancer drugs.

A number of successful scientific outcomes maystoke enthusiasm regarding the future of hyaluronan-based anti-cancer therapeutics. However, a number ofkey barriers are still to be overcome. Only a fewtherapeutic approaches described above have been yettested clinically. The concomitant use of hyaluroni-dase with chemotherapeutic drugs has shown positiveclinical results, but the incidence of adverse effects re-sulting from HYAL administration has constrainedfurther investigations for several years. Up to now,targeted therapy with HA-drug bioconjugates appearsthe only clinically successful approach that might beapproved for the medical practice in the nearest futureif technological difficulties are resolved.

“Cancer is a disease of the organism and not onlythe result of abnormal cell growth” [110]. Thus, thetransfer of in vitro experiments or even in vivo studiescarried on animal models into clinical practice mayproduce other difficulties. Numerous agents showing


promising activity in preclinical models have turnedout to be almost useless in clinical settings. Recentdisappointments have evoked reasonable skepticismabout models for the testing of anti-cancer drugs.Moreover, human malignancies are very diverse, evenwithin particular histological classifications. This di-versity is quickly displayed when exposed to anti-cancer agents. Therefore, genetically engineered and“humanized” mouse models or the stem cell modelshould be harnessed for the development of futurecancer treatments. Follow-up investigations based onmore accurate cancer models and clinical trials willreveal the actual role of hyaluronan-based medica-tions in oncological practice.

Acknowledgment:

The paper has been supported by the Medical University of Lodzwith the grant for young scientists No. 502-03/1-023-01/502-14-159.

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Received: December 31, 2012; in the revised form: May 7, 2013;accepted: May 16, 2013.


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