muramidase and Osteosarcoma

Current therapy of osteosarcoma (OS), the most common primary bone malignancy, is based on a combination of surgery and chemotherapy. Multidrug resistance mediated by P-glycoprotein (P-gp) overexpression has been previously associated with treatment failure and progression of OS, although other mechanisms may also play a role. We considered the typical acidic extracellular pH (pHe) of sarcomas, and found that doxorubicin (DXR) cytotoxicity is reduced in P-gp negative OS cells cultured at pHe 6.5 compared to standard 7.4. Short-time (24-48 hours) exposure to low pHe significantly increased the number and acidity of lysosomes, and the combination of DXR with omeprazole, a proton pump inhibitor targeting lysosomal acidity, significantly enhanced DXR cytotoxicity. In OS xenografts, the combination treatment of DXR and omeprazole significantly reduced tumor volume and body weight loss. The impaired toxicity of DXR at low pHe was not associated with increased autophagy or lysosomal acidification, but rather, as shown by SNARF staining, with a reversal of the pH gradient at the plasma membrane (ΔpHcm), eventually leading to a reduced DXR intracellular accumulation. Finally, the reversal of ΔpHcm in OS cells promoted resistance not only to DXR, but also to cisplatin and methotrexate, and, to a lesser extent, to vincristine. Altogether, our findings show that, in OS cells, short-term acidosis induces resistance to different chemotherapeutic drugs by a reversal of ΔpHcm, suggesting that buffer therapies or regimens including proton pump inhibitors in combination to low concentrations of conventional anticancer agents may offer novel solutions to overcome drug resistance.

Topics: Animals; Antineoplastic Agents; Apoptosis; ATP Binding Cassette Transporter, Subfamily B, Member 1; Bone Neoplasms; Cell Membrane; Cell Proliferation; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Humans; Hydrogen-Ion Concentration; Male; Mice; Mice, Inbred NOD; Mice, SCID; Muramidase; Osteosarcoma; Tumor Cells, Cultured; Tumor Microenvironment; Xenograft Model Antitumor Assays

Chitosan has shown promise as a coating for dental/craniofacial and orthopaedic implants. However, the effects of degree of deacetylation (DDA) of chitosan on coating bond strength, degradation, and biological performance is not known. The aim of this project was to evaluate bonding, degradation, and bone cell growth on titanium coated with chitosans of different DDA and from different manufacturers. Three different chitosans, 80.6%, 81.7%, and 92.3% DDA were covalently bonded to titanium coupons via silane-glutaraldehyde molecules. Bond strengths were evaluated in mechanical tensile tests, and degradation, over 5 weeks, was conducted in cell culture medium with and without 100 microg/mL lysozyme. Cytocompatibility was evaluated for 10 days using UMR 106 osteoblastic cells. Results showed that mean chitosan coating bond strengths ranged from 2.2-3.8 MPa, and that there was minimal affect of DDA on coating bond strengths. The coatings exhibited little dissolution over 5 weeks in medium with or without lysozyme. However, the molecular weight (MW) of the chitosan coatings remaining on the titanium samples after 5 weeks decreased by 69-85% with the higher DDA chitosan coatings exhibiting less percent change in MW than the lower DDA materials. The growth of the UMR 106 osteoblast cells on the 81.7% DDA chitosan coating was lower on days 3 and 5, as compared with the other two coatings, but by day 10, there were no differences in growth among three coatings or to the uncoated titanium controls. Differences in growth were attributed to differences in manufacturer source material, though all coatings were judged to be osteocompatible in vitro.

Topics: Bone and Bones; Cell Culture Techniques; Chitosan; Coated Materials, Biocompatible; Humans; Implants, Experimental; Materials Testing; Muramidase; Orthopedics; Osteosarcoma; Polymers; Stress, Mechanical; Surface Properties; Tensile Strength; Titanium

A series of experiments was conducted to examine the effect of glucan on the reticuloendothelial system (RES) and on the development of 90Sr-induced osteosarcomas and malignant lymphomas in CBA/S mice. Glucan demonstrated a strong RES-stimulating effect, as evidenced by a dose-related increase in lysozyme levels in the plasma and an enlargement of the liver and spleen. Weekly injections of glucan between 150 and 250 days after exposure to 90Sr suppressed the actuarial appearance of the fibroblastic type of osteosarcomas and stimulated the emergence of malignant lymphomas. Glucan itself had no tumorigenic effect in mice not exposed to 90Sr.

Topics: Animals; Bone Neoplasms; Drug Administration Schedule; Female; Glucans; Lymphoma; Macrophages; Male; Mice; Mice, Inbred CBA; Mononuclear Phagocyte System; Muramidase; Neoplasms, Radiation-Induced; Organ Size; Osteosarcoma; Spleen; Strontium Radioisotopes

An organ-specific alkaline phosphatase (AIP) inhibitor, L-homoarginine at 44.5 mM concentration inhibited 3H-thymidine uptake by mouse C3H/He osteosarcoma (OS) cells, while L-arginine, L-phenylalanine and L-glycine had little effect on the uptake. This inhibitory effect by L-homoarginine persisted even after the cells were washed free of the amino acid with fresh media. L-homoarginine did not affect 3H-thymidine uptake by mouse myeloma MOPC 104E cells. In the long-term culture, 22.3 mM L-homoarginine inhibited proliferation of OS cells. L-Arginine at the same concentration inhibited the proliferation to a lesser extent. On the other hand, L-phenylalanine and L-glycine did not affect in vitro proliferation of OS cells. When similar numbers of viable OS cells were inoculated s. c. after culturing with 44.5 mM L-homoarginine or L-arginine for 24 hr, the tumor growth in mice injected with L-homoarginine (but not L-arginine) treated cells was delayed markedly. Electron microscopic studies indicated that the inhibitory effect on OS cell proliferation was associated with a marked increase in lysosomal granules and a decrease in virus-like structures. Similarly, a biochemical assay of acid phosphatase (AcP) of the cell homogenates demonstrated two-fold increase of the activity in L-homoarginine treated cells when compared to the controls and L-arginine treated cells. Thus, L-homoarginine inhibits proliferation and AIP activity of mouse OS cells and appears to promote cell differentiation as evidenced by the increased synthesis of cytoplasmic granules and acid phosphatase activity.

Topics: Alkaline Phosphatase; Amino Acids; Animals; Arginine; Cells, Cultured; Female; Homoarginine; Mice; Mice, Inbred C3H; Muramidase; Osteosarcoma; Sarcoma, Experimental; Stereoisomerism