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HDAC Inhibitor-Loaded Bone Cement for Advanced Local Treatment of Osteosarcoma and Chondrosarcoma

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HDAC Inhibitor-Loaded Bone Cement for Advanced Local Treatment of Osteosarcoma and Chondrosarcoma ANTICANCER RESEARCH 34: 6459-6466 (2014) HDAC Inhibitor-loaded Bone Cement for Advanced Local Treatment of Osteosarcoma and Chondrosarcoma MARCUS TONAK2,4*, MARC BECKER1,5*, CLAUDINE GRAF3, LUKAS ECKHARD4, MATTHIAS THEOBALD3, POL-MARIA ROMMENS4, THOMAS C. WEHLER3,5* and DIRK PROSCHEK4* Interdisciplinary Sarcoma Working Groups of 1First Medical Department, 3Third Medical Department, and 4Department of Orthopaedics and Traumatology, University Medical Center Mainz, Mainz, Germany; 2Department for Orthopaedic and Trauma Surgery, University Hospital Mannheim, Mannheim, Germany 5Department of Internal Medicine V – Pulmonology, Allergology and Respiratory Critical Care Medicine, Saarland University, Homburg, Germany Abstract. The treatment of osteosarcoma, especially wide control group. SaOs-2 and SW1353 cells with medium from resection, is challenging. An additional local drug therapy native cement clots without drug therapy presented a cell after resection using anti-neoplastic bone cement activity of 100% in all groups and during all measurements. (Polymethylmethacrylate (PMMA)) could help improve the Human mesenchymal stem cells were not significantly outcome of therapy. In this study, we evaluated the effects of affected during therapy with VPA and low concentrations of PMMA loaded with valproic acid (VPA) and suberoylanilide SAHA. In contrast, cell activity of human mesenchymal stem hydroxamic acid (SAHA) on the cell activity of a SaOs-2 cell cells was significantly reduced under therapy with higher culture, as well as the elution rate of the drugs out of the concentrations of SAHA, with an approximately linear bone cement. Materials and Methods: In our experiments, decrease between days 0-3 and a rapidly decreasing activity we used the SaOs-2 osteosarcoma and the SW1353 between days 4-7. Conclusion: A local cytotoxic therapy in chondrosarcoma cell line. Bone cement clots (5 g) were the treatment of osteosarcoma and chondrosarcoma might prepared and loaded with different drug concentrations of improve the rate of metastasis and survival of patients. Our VPA (25 mg and 50 mg) and SAHA (1 mg, 2.5 mg and 5 mg). results present an encouraging approach to loading PMMA Two control groups were established, one with a native with anti-neoplastic drugs. cement clot, the other with human mesenchymal stem cells, in order to evaluate toxicity on non tumor-cells. Cell activity Osteosarcoma and chondrosarcoma are very aggressive was measured using an Alamar Blue assay on days 1, 2, 3, 4 neoplasms and the most common types of bone cancer. Most and 7. The cement clots were additionally examined in a often (more than 50% of all cases) osteosarcoma affects the material testing unit for biomechanical and structural distal femur and proximal tibia. The overall survival has changes. Results: Tumor cells showed a significant and improved dramatically during the last centuries. Comparing complete reduction of activity under therapy with VPA and the 1960s, with a survival rate of about 15%, actually SAHA. Drug release of VPA was extensive between days 0 approximately 70% of all patients with osteosarcoma survive and 3 and decreased progressively to day 7. Cumulative drug nowadays (2). The metastatic disease is the most challenging concentration in the medium continuously increased. difficulty in treatment. The presence of metastasis at Biomechanical testing of the cement clots showed no diagnosis reduces the survival rate to 30% (1, 2) differences in stability and architecture compared to the Chondrosarcoma often affects the lower extremity and the pelvic bone and has a peak of incidence in the second half of life. The survival rate for patients with high-grade chondrosarcoma is below 50%, with a high risk *These Authors contributed equally to this article. (approximately 70%) of pulmonary metastasis. Traditional radiotherapy and chemotherapy is not very effective (1, 2). Correspondence to: Claudine Graf, MD, Ph.D., III Medical Biopsy as well as therapy should be carried out in Department, University Medical Center, Langenbeckstraße 1, specialized oncological centers. Surgery with a wide 55131 Mainz, Germany. Tel: +49 6131177302, Fax: +49 6131176689, e-mail: [email protected] resection of the tumor is still the most important step in therapy. Wide resection reduces local and systemic Key Words: Bone cement, PMMA, valproic acid, SAHA, HDAC metastasis significantly (2, 3). Surgery is combined with a inhibitor, local therapy. multi-agent chemotherapy. Several agents, such as cisplatin, 0250-7005/2014 $2.00+.40 6459 ANTICANCER RESEARCH 34: 6459-6466 (2014) doxorubicin, methotrexate and ifosfamide, are well-examined cells. The interaction between bone cement and tumor cells, and are active against osteosarcoma (4). However, as well as the drug elution rate and the biomechanical postoperative treatment with cytotoxic agents is rarely changes of the PMMA, were evaluated. Two different HDAC curative and is accompanied by severe side-effects such as inhibitors were examined. Local cytotoxic effects were tested bone marrow depression, cardiotoxicity or nephrotoxicity. in tumor cells, as well as in human mesenchymal stem cells Targeted therapy is a new part of chemotherapy. Tumor (hMSCs), to evaluate the effects on non-tumoral cells. cells often achieve their resistance towards apoptosis by epigenetic deactivation of apoptosis-regulating genes. In this Materials and Methods context histone deacetylase (HDAC) inhibitors is a promising group of medications. Treatment with these inhibitors causes Bone marrow-derived hMSCs, SaOs-2 cells and SW1353 cells. chromatin condensation and down-regulation of gene hMSC cultures were generated from bone marrow. After hip expression which is necessary for carcinogenesis and tumor replacement, pieces of stromal bone marrow were excised from the growth (4-6). Several in vitro and in vivo studies have femoral head with a medical forceps. The femoral head was demonstrated a tumor response to HDAC inhibitor (4-6). considered to be excess material after surgical treatment and would otherwise have been discarded. Informed consent was obtained from HDAC inhibitors have actually undergone clinical the donating patient. assessment (7). Therefore, two HDAC inhibitors, valproic The pieces of stromal bone marrow were washed several times acid (VPA) and suberoylanilide hydroxamic acid (SAHA), with phosphate buffered saline (PBS) by mixing vigorously until the were used in this study. supernatant remained clear. The PBS from the washing steps was Bone cement is a commonly used material in surgery. It centrifuged and the supernatants were discarded. The cells were can be applied for direct stabilization of bone (e.g. cultivated in 6-well plates with an initial density of 1×106/ml in pathological fractures, bone defects/voids), or as support for Dulbecco’s Modified Eagle’s Medium (DMEM)/F12-GlutaMAX™ (Invitrogen Life Technologies, Darmstadt, Germany) supplemented osteosynthesis or endoprosthesis. The cement is a compound with 10% fetal calf serum (FCS). The medium was changed every consisting of 90% polymethylmethacrylate (PMMA) and 2-3 days and non-adherent cells were discarded. 10% of contrast media crystals. The structure of the bone The osteosarcoma cell line SaOs-2 and the chondrosarcoma cell cement is made out of two substances, small particles of pre- line SW1353 were purchased from Cell Line Service (Eppelheim, polymerized PMMA being supplied as a white powder, and Germany). The cells were cultured in DMEM/F12-GlutaMAX™ a liquid monomer of MMA. Both substances are mixed and supplemented with 10% FCS. glued together in a polymerization reaction. Microscopically PMMA, VPA and SAHA. A standard bone cement without antibiotic this reaction leads to formation of a net-like structure in load was used in this trial (VertebroX; Somatex Inc., Berlin-Teltow, which the PMMA particles are embedded. The polymer net Germany). The cement procedure started with the mixing of the also enables the bone cement to be loaded with other PMMA and the drugs in different concentrations. A standard substances such as antibiotics (8, 9). vacuum-assisted mixing device was used (SOMIX; Somatex A possible extension in the treatment of sarcoma would Medical Technologies). A standard cement clot of 5 g each with the be to add local drug therapy to the surgical therapy. A benefit shape of a tablet was produced (Figure 1). The drug concentration might be the possibility of achieving a high local drug used was chosen to be high in order to achieve a high local concentration. The amount of PMMA-loaded chemotherapy concentration, reducing the number of free tumor cells, as followed the usually applied concentration for systemic therapy and well as micrometastases. Local control of the tumor as well was chosen in this experiment to be between 25- and 50-times as the risk of metastasis might be improved. For local drug higher than that used in systemic therapy. This approach is known delivery, certain material properties are necessary. The from the loading of PMMA with antibiotics (10, 15). The 5 g chemotherapy agent should elute a sufficient local drug cement clots with different drug concentrations were embedded in concentration constantly over a period of several days. 10 ml of DMEM/F12-GlutaMAX™ each. On day 1, medium from Additionally, the stability of the bone cement should not be the cement clots was removed and stored at −20˚C until administration to the hMSC, SW1353 and SaOs-2 cells. After affected by the added drug. removal, the cement clots were immediately embedded again with A reasonable approach would be to use bone cement as a 10 ml cell culture medium due to local tissue effects being taken carrier for a local chemotherapy. PMMA is already into account. This procedure was repeated on days 2, 3, 4 and 7. commonly loaded with antibiotics and there is much clinical Drug concentration eluted into the medium was measured each day and experimental experience in the therapy of bone infection for VPA. Drug concentration of SAHA could not be measured due (10-13). Research in local therapy of sarcoma using drug- to there being no measurement procedure. loaded PMMA is in early stages (14). No clinical data are Cell activity assay.
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