RESUMEN
Fragments of the extracellular matrix component hyaluronan (HA) promote tissue inflammation, fibrosis and tumor progression. HA fragments act through HA receptors including CD44, LYVE1, TLR2, 4 and the receptor for hyaluronan mediated motility (RHAMM/HMMR). RHAMM is a multifunctional protein with both intracellular and extracellular roles in cell motility and proliferation. Extracellular RHAMM binds directly to HA fragments while intracellular RHAMM binds directly to ERK1 and tubulin. Both HA and regions of tubulin (s-tubulin) are anionic and bind to basic amino acid-rich regions in partner proteins, such as in HA and tubulin binding regions of RHAMM. We used this as a rationale for developing bioinformatics and SPR (surface plasmon resonance) based screening to identify high affinity anionic RHAMM peptide ligands. A library of 12-mer peptides was prepared based on the carboxyl terminal tail sequence of s-tubulin isoforms and assayed for their ability to bind to the HA/tubulin binding region of recombinant RHAMM using SPR. This approach resulted in the isolation of three 12-mer peptides with nanomolar affinity for RHAMM. These peptides bound selectively to RHAMM but not to CD44 or TLR2,4 and blocked RHAMM:HA interactions. Furthermore, fluorescein-peptide uptake by PC3MLN4 prostate cancer cells was blocked by RHAMM mAb but not by CD44 mAb. These peptides also reduced the ability of prostate cancer cells to degrade collagen type I. The selectivity of these novel HA peptide mimics for RHAMM suggest their potential for development as HA mimetic imaging and therapeutic agents for HA-promoted disease.
Asunto(s)
Materiales Biomiméticos/química , Materiales Biomiméticos/metabolismo , Movimiento Celular/fisiología , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Secuencia de Aminoácidos , Materiales Biomiméticos/farmacología , Neoplasias de la Mama/metabolismo , Carbocianinas , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Femenino , Colorantes Fluorescentes , Humanos , Receptores de Hialuranos/efectos de los fármacos , Ligandos , Masculino , Datos de Secuencia Molecular , Invasividad Neoplásica/prevención & control , Biblioteca de Péptidos , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/metabolismo , Resonancia por Plasmón de Superficie , Tubulina (Proteína)/farmacologíaRESUMEN
The effect of oral hyaluronan (HA) on bone loss in ovariectomized (OVX) 3-month-old rats was measured using serum markers of bone turnover and bone mineral density. OVX rats were administered 1 mg/kg HA (OVX + HA) or phosphate-buffered saline (PBS) (OVX + PBS) by oral gavage (5 days/week for 54 days). Additional controls included sham ovariectomy with PBS gavage (Sham + PBS) and no treatment. Oral administration of HA resulted in approximately 50% (p < 0.05) increases in serum HA. Gel filtration analyses showed this was high molecular weight HA (300-500 kDa). Osteopenia was mild due to the young age of the animals. Thus, ovariectomy resulted in a 30% increase in serum collagen N-terminal telopeptides (p < 0.001), a 20% increase in serum nitrate/nitrite levels (p = 0.05), and a 5-6% decrease in femur bone mineral density/content (p < 0.05). HA gavage blunted the development of osteopenia in this model as determined by preventing the 30% increase in serum collagen N-terminal telopeptide levels (p < 0.001) and by reducing bone mineral content loss from 6 to 4%. These results show that oral supplements of HA (gavage solution, 0.12% solution) significantly reduce bone turnover associated with mild osteopenia in rats.