Targeted sensitization of tumor cells for radiation through monocarboxylate transporters 1 and 4 inhibition in vitro.

OBJECTIVES: Monocarboxylate transporters (MCT) 1, 2 and 4 play an important role in tumor metabolism. The amount of lactate transported by MCT's highly correlates with overall survival. Furthermore, glycolysis and hypoxia are possible causes for radiation resistance. MATERIALS AND METHODS: An oral squamous cell carcinoma cell line (CAL27, ATCC) was analyzed in an in vitro cell assay. After incubation with two different inhibitors for MCT1 (AR-C122982/SR-13800 and AR-C155858/SR-13801, Tocris) or for MCT4 (simvastatin, Sigma-Aldrich and 2-cyano-3-(4-hydroxyphenyl)-2-propenoic acid (CHC), Tocris), cells were irradiated with six gray with a Gammacell 2000 (Nuklear Data). For analysis, cell counting assay, wound healing assay, MTT assay and clonogenic assay were applied. RESULTS: Cell counting assay showed significant lower results for simvastatin, CHC and for the highest concentrations of AR-C122982 and AR-C155858 (p < 0.03). Additionally, cell counts decreased significantly with irradiation after 72 hours (p < 0.05) only for AR-C122982, CHC and simvastatin. The clonogenic assay confirmed these results with substantially reduced growth when incubated with CHC, simvastatin and AR-C155858 (p < 0.002). Furthermore, MCT1 and 4 inhibition led to highly reduced migration (p < 0.05). There again, comparing the wound healing assay of irradiated to non-irradiated tests showed contrary results (controls: p < 0.001; AR-C155858: p > 0.05; AR-C122982: p > 0.32; CHC: p > 0.1; simvastatin p > 0.1). The MTT assay presented significant effects with MCT1 and 4 inhibition (simvastatin/AR-C122982/CHC: p < 0.007). Irradiated cells showed significantly lower expression after only 48 h compared to non-irradiated cells (simvastatin/AR-C122982/CHC: p < 0.02). CONCLUSIONS: Inhibition of MCT, especially MCT4 may represent a possible tool to overcome radiation resistance in tumor cell lines. CLINICAL RELEVANCE: MCT Inhibitors may be used as a possible therapeutic approach to sensitize OSCC to radiation.

Mechanically-induced GDF15 Secretion by Periodontal Ligament Fibroblasts Regulates Osteogenic Transcription.

The alveolar bone provides structural support against compressive and tensile forces generated during mastication as well as during orthodontic treatment. To avoid abnormal alveolar bone resorption and tooth loss, a balanced bone turnover by bone-degrading osteoclasts and bone-generating osteoblasts is of great relevance. Unlike its contradictory role in regulating osteoclast and osteoblast cell differentiation, the TGF-ß/BMP-family member GDF15 is well known for its important functions in the regulation of cell metabolism, as well as cell fate and survival in response to cellular stress. Here, we provide first evidence for a potential role of GDF15 in translating mechanical stimuli into cellular changes in immature osteoblasts. We detected enhanced levels of GDF15 in vivo in periodontal ligament cells after the simulation of tooth movement in rat model system as well as in vitro in mechanically stressed human periodontal ligament fibroblasts. Moreover, mechanical stimulation enhanced GDF15 secretion by periodontal ligament cells and the stimulation of human primary osteoblast with GDF15 in vitro resulted in an increased transcription of osteogenic marker genes like RUNX2, osteocalcin (OCN) and alkaline phosphatase (ALP). Together, the present data emphasize for the first time a potential function of GDF15 in regulating differentiation programs of immature osteoblasts according to mechanical stimulation.

Comparative metabolic analysis in head and neck cancer and the normal gingiva.

OBJECTIVES: Chronic accumulation of lactate in malignant tumor tissue is associated with increased malignancy and radioresistance. For this study, biopsies of primary head and neck squamous cell carcinoma (HNSCC) and of the normal gingiva of the same patient were compared via metabolic profiling to the healthy gingiva from cancer-free patients. MATERIALS AND METHODS: Cryobiopsies of 140 HNSCC patients were used to determine ATP, lactate, and glucose concentrations of the tumor and normal gingiva via induced metabolic bioluminescence imaging (imBI). Additionally, these metabolites were quantified in a collective of 79 healthy (non-tumor-bearing) patients. Furthermore, tumor samples were analyzed via immunofluorescence imaging and quantitative real-time PCR for the expression of lactate and glucose transporters. RESULTS: There were significant differences in ATP concentrations detectable between the tumor, normal gingiva of tumor patients, and gingiva from healthy patients. Lactate concentrations were significantly increased in tumor tissue compared to the normal gingiva of tumor patients as well as the gingiva from healthy patients. Concerning glucose, there was a significant decrease in glucose concentrations detectable in the tumor biopsies compared to the normal gingiva of tumor patients. On the other hand, tumor samples from patients revealed significantly elevated relative expression levels of monocarboxylate transporters (MCT-1 and MCT-4), as well as glucose transporters (GLUT-1 and GLUT-3) compared to the corresponding normal gingiva of each patient. CONCLUSIONS: We could demonstrate that the lactate concentration in HNSCC correlates with primary tumor (T) stage. CLINICAL RELEVANCE: The aim of this study was to identify metabolic parameters to improve early cancer diagnosis, allow predictions on the degree of malignancy, and contribute to a personalized tumor therapy.

Effect of compressive loading and incubation with clodronate on the RANKL/OPG system of human osteoblasts.

OBJECTIVES: In vivo studies have shown that bisphosphonates result in slow rates of orthodontic tooth movement. This study investigated whether clodronate modifies the impact of mechanical loading on the RANKL/OPG system of human osteoblasts. METHODS: Osteoblasts were cultured in vitro with 0.5 or 5.0 µM clodronate for 48 h and/or subjected to 3 h of compressive loading at 34.9 g/cm(2). Cell viability was determined by MTT assay. Real-time polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and immunocytochemical staining were used to analyze the cells for their production of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL) at the transcriptional and protein levels. RESULTS: Compressive loading did not affect osteoblast viability in a significant way. Clodronate (5.0 µM) mildly reduced the viability of both compressed and uncompressed cells. Compressive loading induced a 4.2-fold increase in RANKL gene expression, while clodronate led to a concentration-dependent inhibition of this effect (1.8-fold increase at 5.0 µM). OPG gene expression was decreased by compressive loading both in the presence of 0.5 µM clodronate and in the absence of clodronate, and OPG protein synthesis in the compressed cells was significantly decreased in the presence of clodronate. Immunocytochemical staining revealed an increase of RANKL protein synthesis in compressed cells, while clodronate and cell compression reduced this increase. CONCLUSION: This study demonstrates that clodronate decreases the compression-induced RANKL/OPG ratio expressed by human osteoblasts. Reported in vivo findings of reduced osteoclast numbers on the compression side of orthodontic tooth movement under the action of clodronate-and the associated slow rate of tooth movement-might be attributable not only to a direct impact on osteoclasts but also to changes in osteoblast-osteoclast interaction resulting from the presence of clodronate.

ΔNp63 expression in four carcinoma cell lines and the effect on radioresistance--a siRNA knockdown model.

OBJECTIVES: This study investigated the expression of ΔNp63α in carcinoma cell lines of the upper aerodigestive tract and their potential influence on radioresistance using a small interfering RNA (siRNA) knockdown approach. MATERIALS AND METHODS: Four carcinoma cell lines were investigated for the expression of the ΔNp63 isoform by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) (0, 24, 48 h) with and without single dose irradiation of 6 Gy. Furthermore, all cell lines were transfected with siRNA against the ΔNp63α isoform over 24 h. Knockdown effectiveness was controlled by qRT-PCR and Western blot. Apoptotic events were evaluated by terminal transferase dUTP nick end labeling (TUNEL) assay and cross-checked by a test for cell viability (WST-1, Roche) over 48 h. RESULTS: All cell lines presented varying expression of the ΔNp63α isoform with and without irradiation. A sufficient knockdown rate was established by siRNA transfection. Knockdown of the ΔNp63 isoform showed an effect on radiation sensitivity proven by an increase of apoptotic events detectable by immunofluorescence (TUNEL assay) and likewise a significant reduction of formazan production (WST-1 test) in three cell lines. CONCLUSIONS: We found overexpression of ΔNp63α with and without irradiation in three cell lines, and the knockdown of ΔNp63α led to increased apoptotic events and fewer viable cells. Thus, the overexpression of ΔNp63α might protect carcinoma cells against irradiation effects. CLINICAL RELEVANCE: The present work supports the hypothesis that protein 63 might serve as a negative predictor for irradiation response and survival in a clinical setting and may be a target for future therapeutic strategies.

BMP-2 and bFGF release and in vitro effect on human osteoblasts after adsorption to bone grafts and biomaterials.

OBJECTIVES: Combination of scaffolds and growth factors is a promising option for several clinical problems in bone biomaterials. Simplified growth factor loading by adsorption from aqueous solution is one important option for this technology. We evaluated the adsorption followed by PBS rinsing, release and biological effect of transient loading with basic fibroblast growth factor (bFGF) and bone morphogenic protein 2 (BMP-2) on fresh frozen bone, processed bone matrix, collagen, and a ceramic material with immunofluorescence, enzyme-linked immunosorbent assay (ELISA), and qRT-PCR. MATERIALS AND METHODS: The study consisted of three in vitro experiments (immunofluorescence, ELISA, and qRT-PCR) in human osteoblasts (HOB). The first evaluated the adsorption of the growth factors bFGF and BMP-2 to the biomaterials, analyzed by immunofluorescence assays. The second experiment used ELISA to analyze the release of the growth factors from the matrix. The biological effect of the growth factors on HOB was then studied with qRT-PCR experiments as the third step. RESULTS: Strongest sustained release peaks in ELISA were observed in bFGF loading on processed bone matrix (steam-resistant mineralized bone matrix, SMBM) with up to 553 pg/ml medium. BMP-2 loading was less effective in ELISA peak release experiments with up to 257 pg/ml medium in processed bone matrix (SMBM). bFGF showed also higher release peaks in collagen material (192 pg/ml) compared with BMP-2 (101 pg/ml). Cumulative release values 0-72 h were estimated. The expression of runX2, osteocalcin, and alkaline phosphatase as markers for osteoblast activity was correlating. CONCLUSION: The results showed sustained release of BMP-2 and bFGF after transient loading on bone biomaterials with a stronger effect in biological scaffolds. This is interesting for therapeutic growth factor loading as well as insights in natural growth factor matrix deposition during bone healing.