Effects of a combined therapy of bortezomib and ionizing radiation on chondrosarcoma three-dimensional spheroid cultures.

Chondrosarcomas represent a heterogeneous group of primary bone cancers that are characterized by hyaline cartilaginous neoplastic tissue and are predominantly resistant to radiation and chemotherapy. However, adjuvant radiotherapy is often recommended in inoperable cases or after incomplete resections. To improve the efficiency of treatment, the present study tested a combination therapy with ionizing radiation (IR) and the proteasome inhibitor bortezomib. Using a three-dimensional (3D) spheroid model, 0-20 Gy of IR was applied to chondrosarcoma cells and healthy human chondrocytes. Following combined treatment with IR and bortezomib, the cell cycle distribution, apoptotic induction, the survivin pathway, autophagy and DNA damage were evaluated. Both cell types exhibited a slight decrease in viability following increasing doses of IR; the chondrosarcoma cells demonstrated a significant dose-dependent increase in the expression levels of the DNA damage marker histone H2AX phosphorylation at serine 139 (γH2AX). The combination treatment with bortezomib significantly decreased the cell viability after 48 h compared with that in irradiated cells. High-dose IR induced a G2/M phase arrest, which was accompanied by a decrease in the number of cells at the G1 and S phase. Co-treatment with bortezomib changed the distribution of the cell cycle phases. The mRNA expression levels of the proapoptotic genes Bcl-2-associated X protein (Bax) and Bak were significantly increased by bortezomib treatment and combination therapy with IR. In addition, the combination therapy resulted in a synergistic decrease of the expression levels of survivin and its corresponding downstream pathway molecules, including heat shock protein 90, X-linked inhibitor of apoptosis protein, smad 2 and smad 3. Comparative analyses of γH2AX at 1 and 24 h post-IR revealed efficient DNA repair in human chondrosarcoma cells. Therefore, additional bortezomib treatment may only temporarily improve the radiation sensitivity of chondrosarcoma cells. However, the inhibition of the survivin pathway by the combined treatment with IR and bortezomib, observed in the present study, revealed a novel aspect in the tumor biology of chondrosarcoma 3D spheroid cultures and may represent a potential target for therapy.

Surface Modifications of Titanium Aluminium Vanadium Improve Biocompatibility and Osteogenic Differentiation Potential.

Osteogenic cells are strongly influenced in their behaviour by the surface properties of orthopaedic implant materials. Mesenchymal stem and progenitor cells (MSPCs) migrate to the bone-implant interface, adhere to the material surface, proliferate and subsequently differentiate into osteoblasts, which are responsible for the formation of the bone matrix. Five surface topographies on titanium aluminium vanadium (TiAl6V4) were engineered to investigate biocompatibility and adhesion potential of human osteoblasts and the changes in osteogenic differentiation of MSPCs. Elemental analysis of TiAl6V4 discs coated with titanium nitride (TiN), silver (Ag), roughened surface, and pure titanium (cpTi) surface was analysed using energy-dispersive X-ray spectroscopy and scanning electron microscopy. In vitro cell viability, cytotoxicity, adhesion behaviour, and osteogenic differentiation potential were measured via CellTiter-Glo, CytoTox, ELISA, Luminex® technology, and RT-PCR respectively. The Ag coating reduced the growth of osteoblasts, whereas the viability of MSPCs increased significantly. The roughened and the cpTi surface improved the viability of all cell types. The additive coatings of the TiAl6V4 alloy improved the adhesion of osteoblasts and MSPCs. With regard to the osteogenic differentiation potential, an enhanced effect has been demonstrated, especially in the case of roughened and cpTi coatings.

Cobalt Chromium Molybdenum Surface Modifications Alter the Osteogenic Differentiation Potential of Human Mesenchymal Stem Cells.

Surface roughness on orthopedic implant materials has been shown to be highly influential on the behavior of osteogenic cells. Mesenchymal stem and progenitor cells (MSPCs) migrate to the interface, adhere, proliferate, and differentiate into osteoblasts, which subsequently form bone matrix. Modifications of the implant surfaces should accelerate this process and improve biocompatibility. In this study, five surface topographies on cobalt chromium molybdenum (CoCrMo) were engineered to examine the influence on MSPCs. Scanning electron microscopy revealed significant differences in the morphology of untreated CoCrMo discs in comparison with CoCrMo with a titanium nitride (TiN) coating, polished and porous coated CoCrMo surfaces, and CoCrMo with a pure titanium (cpTi) coating. Elemental analysis was performed using energy-dispersive X-ray spectroscopy (EDX). Human primary MSPCs were expanded from tissue samples of spongiosa bone and characterized according to the criteria of the International Society for Cellular Therapy. The characteristic phenotype of MSPC was confirmed by flow cytometry and multilineage differentiation. Alcaline phosphatase and osteopontin expression increased significantly in all groups about 5-fold and 10-fold, respectively, in comparison to the undifferentiated controls. The porous coated surface showed a reduced expression of osteogenic markers. Due to the osteogenic differentiation, the expression of integrin α5ß1, which is particularly important for cell-material contact, increased 4-7-fold. In the dynamic process of bone biology, MSPCs cultured and differentiated on cpTi, showed significant upregulation of IL6 and leptin.

Periplocin mediates TRAIL-induced apoptosis and cell cycle arrest in human myxofibrosarcoma cells via the ERK/p38/JNK pathway.

BACKGROUND: Periploca sepium is traditionally used in Chinese medicine to treat particularly rheumatic disorders and as a tonic. Periplocin was found as the most cytotoxic compound of its root bark and induced death receptor mediated apoptosis in liposarcoma cells. Sarcomas are a rare type of cancer with only a few treatment options. The five-year survival rate of advanced tumors is low. PURPOSE: In this study, we investigated the effects of periplocin in two myxofibrosarcoma (MFS)cell lines, MUG-Myx2a and MUG-Myx2b, which are subclones of the same tumor and reflect the tumor´s heterogeneity, and in T60 primary myxofibrosarcoma cells. METHODS: The xCELLigence system and the CellTiter 96® AQueous assay were used for studying cell viability. FACS and Western blot experiments were used to investigate the effects of periplocin on apoptosis induction, cell cycle distribution, and the expression of cleaved PARP, caspase 3, p53, phospho-histone γH2AX, ERK/phospho ERK, p38/phospho p38, and, finally, JNK/phospho JNK. Additionally, the expression of the apoptotic markers Bim, NOXA, Bak, Bcl-2, Bcl-xl, and the death receptors IGFR, FADD, TRADD, TNFR1A, TRAIL-R1, and TRAIL-R2 were evaluated using reversed real-time PCR. RESULTS: Periplocin decreased dose-dependently the viability of all MFS cell lines and was more effective than the standard chemotherapeutic doxorubicin. It arrested the cells in the G2/M phase and led to caspase activation. Moreover, periplocin increased the mRNA expression of NOXA, Bak, Bcl-2, and death receptors such as TRAIL-R1 and TRAIL-R2 and the protein expression of ERK/phospho ERK, p38/phospho p38, and JNK/phospho JNK. In all cases, differences in the effects in the different subclones were observed. CONCLUSION: Periplocin showed promising effects in MFS cells. The higher effectiveness compared to doxorubicin is an important aspect for further research with regard as a treatment option. The different effects of periplocin in the two subclones showed the great importance of intratumoral heterogeneity in MFS therapy.

CoCrMo surface modifications affect biocompatibility, adhesion, and inflammation in human osteoblasts.

In this study, different surface modifications were performed on a Cobalt-Chrome-Molybdenum (CoCrMo) alloy and the effects on cell viability and cytotoxicity as well as the adhesion potential of human osteoblasts (hFOB) and their inflammation reaction were investigated in vitro. CoCrMo discs were coated with TiN, with polished and porous coated surfaces, or with pure titanum (cpTi) surfaces and examined by Scanning Electron Microscopy to evaluate surface modifications. In vitro cell viability, adhesion behaviour, and expression of inflammation markers of hFOB human osteoblasts were measured via CellTiter-Glo, CytoTox, ELISA, and RT-PCR respectively. All results were compared to CoCrMo without surface modifications. The biocompatibility data showed high compatibility for the TiN hard coatings. Likewise, the porous surface coating increased cell viability significantly, compared to an untreated CoCrMo alloy. None of the investigated materials influenced cytotoxicity. Different surface modifications did not influence expression of fibronectin, although TiN, porous surface coatings and polished surfaces showed highly significant reductions in integrin subunit expression. In addition to the regulation of adhesion potential these three surfaces stimulated an anti-inflammatory response by osteocytes. Improved biocompatibility and adhesion properties may contribute to better osteointegration of prosthetics.