Hyaluronan oligosaccharides sensitize lymphoma resistant cell lines to vincristine by modulating P-glycoprotein activity and PI3K/Akt pathway Rosal  ıa I. Cordo Russo * , Mariana G. Garc  ıa, Laura Alaniz, Guillermo Blanco, Elida Alvarez and Silvia E. Hajos * Department of Immunology, School of Pharmacy and Biochemistry, University of Buenos Aires (UBA), IDEHU-CONICET, Buenos Aires, 1113, Argentina Multidrug resistance (MDR) is one of the main reasons for failure of cancer therapy. It may be mediated by overexpression of ATP- dependent efflux pumps or by alterations in survival or apoptotic pathways. Fragments generated by enzymatic degradation of hya- luronan (oHA) were able to modulate growth and cell survival and sensitize MDR breast cancer cells to cytotoxic drugs. In this work the relationship between oHA and MDR in lymphoid malig- nancies was analyzed using murine lymphoma cell lines resistant to doxorubicin (LBR-D160) or vincristine (LBR-V160) and a sen- sitive line (LBR-). After oHA treatment, higher apoptosis levels were observed in the resistant cell lines than in the sensitive one. Besides, oHA sensitized LBR-D160 and LBR-V160 to vincristine showing increased apoptosis induction when used in combination with vincristine. Native hyaluronan failed to increase apoptosis levels. As different survival factors could be modulated by hyalur- onan, we investigated the PI3K/Akt pathway through PIP3 pro- duction and phosphorylated Akt (p-Akt) and survivin expression was also evaluated. Our results showed that oHA decreased p-Akt in the 3 cell lines while anti-CD44 treatment abolished this effect. Besides, survivin was downregulated only in LBR-V160 by oHA. When Pgp function was evaluated, we observed that oHA were able to inhibit Pgp efflux in murine and human resistant cell lines in a CD44-dependent way. In summary, we report for the first time that oHA per se modulate MDR in lymphoma cells by decreasing p-Akt as well as Pgp activity, thus suggesting that oHA could be useful in combination with classical chemotherapy in MDR hematological malignancies. ' 2007 Wiley-Liss, Inc. Key words: multidrug resistance; P-glycoprotein; hyaluronan oligosaccharides; hematological malignancies; PI3K/Akt The molecular mechanisms by which anticancer drugs fail to kill cancer cells have been widely studied; however, they are not com- pletely understood yet. Multidrug resistance (MDR), one of the major obstacles to effective chemotherapy, may be mediated by overexpression of ATP-dependent efflux pumps such as phospho- glycoprotein (Pgp), reduced drug uptake, activation of detoxifying systems, or alterations in survival or apoptotic pathways. 1 Human Pgp (MDR1) and its rodent homologues mdr-1 and mdr-3 are con- served proteins that translocate antitumor agents across the plasma membrane of resistant cancer cells, reducing intracellular drug con- centrations to sublethal levels. 2 Pgp-mediated drug efflux can be modulated by MDR inhibitors, which block transport in a competi- tive or noncompetitive way. Although many Pgp inhibitors (including verapamil, cyclosporin A and PSC833) are able to sensi- tize drug-resistant cell lines to antitumor agents in vitro, they have not always been useful in clinical trails since they appear to be either toxic or ineffective in vivo. Nowadays research is focused on the ‘‘fourth generation’’ inhibitors among old drugs, such as disul- firam or herbal components, which have been shown to modulate Pgp in vitro and could be used in safety doses in vivo. 3 The balance between apoptotic and survival signals determines the susceptibility of cells to apoptosis. Since many chemothera- peutic drugs exert their cytotoxic effects by inducing apoptosis, alterations in cell survival and apoptotic signaling pathways appear to be important in MDR. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a central role in oncogenesis by regulat- ing several biological processes such as cell survival and prolifera- tion, apoptosis, cell growth, angiogenesis, tumor invasion and me- tastasis. PI3K activates Akt by phosphorylation. Once activated, phosphorylated Akt (p-Akt) phosphorylates multiple proteins implicated in cellular processes leading to induction of cell sur- vival and inhibition of apoptosis. Akt overexpression is a frequent molecular alteration in human malignancies closely associated with chemoresistance, and it therefore constitutes a critical target for cancer intervention. 4,5 Survivin, one of the members of the inhibitor of apoptosis pro- tein (IAP) family is a protein that suppresses apoptosis by inhibi- tion of caspases. This protein is normally expressed in embryonic and fetal organs but not in adult differentiated tissues. However, several reports have demonstrated its expression in human tumors and enhanced survivin levels have been correlated with tumor pro- gression. 6 Besides, it has been recently shown that Akt may regu- late survivin expression in neuroblastoma. 7 Hyaluronan (HA) is a large, linear glycosaminoglycan with a mo- lecular weight ranging from 10 5 to 10 7 Da. It is the major compo- nent of the mammalian extracellular matrix (ECM) where it has a structural function influencing hydration and physical properties of tissues. 8 Besides, upon interaction with CD44 or RHAMM cell sur- face receptors, HA is able to modulate cell-signaling pathways. 9 Increased levels of HA have been found in the ECM within tumors and have been related with tumor progression and metastasis. 10–13 However, HA oligosaccharides (oHA)—fragments generated by en- zymatic degradation of hyaluronan—can stimulate dissimilar tumor behavior. 14 It has been described that perturbation of hyaluronan- cell interactions by treatment with oHA suppresses PI3K/Akt cell survival signaling pathway in breast carcinoma cell lines, induces apoptosis and reduces tumor growth in vivo. 15,16 Moreover, oHA sensitize multidrug resistant breast tumor cells to a variety of che- motherapeutic agents while HA enhances MDR. 17 Recent investiga- tions have shown that oHA reduce constitutive MDR-1 expression in MDR breast tumor cells; in contrast, HA induces MDR-1 expres- sion in drug sensitive cells. 18 Although many studies have been per- formed in carcinoma models, little is known about the role of oHA and native HA on MDR in hematological malignancies. The aim of this work was to analyze the relationship between oHA and MDR in murine lymphoma cell lines resistant to doxoru- bicin (LBR-D160) or to vincristine (LBR-V160) obtained previ- ously in our laboratory. 19 The ability of oHA to induce apoptosis and to sensitize MDR cells to antineoplasic drugs as well as the Grant sponsor: CONICET. Grant sponsor: University of Buenos Aires. The first two authors contributed equally to this paper. *Correspondence to: Department of Immunology, School of Pharmacy and Biochemistry, University of Buenos Aires (UBA), IDEHU-CONICET, Buenos Aires, 1113, Argentina. Fax: 154-11-4964-0024. E-mail: rcordo@ffyb.uba.ar and E-mail: shajos@ffyb.uba.ar Received 2 February 2007; Accepted after revision 8 August 2007 DOI 10.1002/ijc.23122 Published online 5 November 2007 in Wiley InterScience (www.interscience. wiley.com). Abbreviations: CsA, cyclosporine A; Da, dalton; DNR, daunorubicin; DOX, doxorubicin; HA, hyaluronan; Hase, hyaluronidase; K562-Vinc, vin- cristine resistant human leukemic cell line; K562-WT, wild type human my- eloid leukemic cell line; LBR-, sensitive cell line; LBR-D160, doxorubicin resistant cell line; LBR-V160, vincristine resistant cell line; mAb, monoclo- nal antibody; MDR, multidrug resistance; oHA, hyaluronan oligosaccha- rides; p-Akt, phosphorylated Akt; PI3K, Phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol trisphosphate; Pgp, P-glycoprotein; RT-PCR, reverse transcription-polymerase chain reaction; SD, standard deviation; TBS, tris-buffered saline; TCA, trichloroacetic acid; VCR, vincristine. Int. J. Cancer: 122, 1012–1018 (2008) ' 2007 Wiley-Liss, Inc. Publication of the International Union Against Cancer