Hyaluronan oligosaccharides modulate multidrug resistance in leukemic cell lines

ALANIZ L; CORDO RUSSO R; GARCIA MG; BLANCO G; ALVAREZ E; HAJOS SE

Charleston, Carolina del Sur, USA

Congreso; 7º Conferencia Internacional de Ácido Hialurónico; 2007

Multidrug resistance (MDR) is the main reason for failure of cancer therapy. Molecular mechanisms by which anticancer drugs fail to kill cancer cells have been widely studied, however, they are not completely understood yet. MDR can be mediated by several mechanisms: overexpression of ATP-dependent efflux pumps such as P-glycoprotein (Pgp), reduced drug uptake, activation of detoxifying systems, or alterations in survival or apoptotic pathways leading to  failure to undergo apoptosis (1). Several studies indicate that fragments generated by degradation of hyaluronan, hyaluronan oligosaccharides (oHA), are able to modulate growth and cell survival (2) and sensitize MDR breast cancer cells to cytotoxic drugs (3). The aim of this work was to analyze the relationship between oHA and MDR in lymphoid malignancies.  In this study we used murine lymphoma cell lines selected for resistance to vincristine (LBR-V160) and doxorubicin (LBR-D160) and a sensitive line (LBR-) obtained previously (4). The effect of oHA, per se and in combination with doxorubicin (DOX) or vincristine (VCR),  was evaluated on apoptosis induction by Annexin V-staining method and acridine orange-ethidium bromide staining. We observed higher apoptosis levels in LBR-D160 (13.5%±3.1%; 18.9%±4.5%) and in LBR-V160 (13.5%±3.4%; 20.4%±3.5%) compared to LBR- (4.5%±2.2%; 7.4%±3.4%) with 150 and 300 mg/ml oHA respectively (P<0.001). Such doses of oHA used in combination with VCR increased apoptosis in LBR-D160 (38.6%±2.7%; 33.9%±4.4%) and in LBR-V160 (30.9%±1.9%; 26.6%±1.5%) while no effect was observed with DOX. When similar experiments were done with native hyaluronan, no significant apoptosis levels were obtained. As different survival factors could be modulated by hyaluronan, we investigated PI3K/Akt pathway through phosphorylated Akt (p-Akt) and survivin expression by western blot. Our results showed that treatment with oHA decreased p-Akt in the three cell lines while survivin expression was only down regulated in the resistant cell lines. After oHA treatment, Pgp function was evaluated by flow cytometry through daunorubicin accumulation and its expression by RT-PCR. Our results showed that oHA were able to inhibit Pgp efflux without modyfing mRNA levels in both resistant cell lines. We conclude that oHA modulate MDR by decreasing p-Akt and survivin expression as well as Pgp activity suggesting that hyaluronan oligosaccharides could be useful in combination with classical chemotherapy in MDR lymphomas.