Sequential activation of the AKT pathway in human osteoblasts treated with Oscarvit: a bioactive product with positive effect both on skeletal pain and mineralization in osteoblasts.

BACKGROUND: Oscarvit (OSC) is an in-house preparation consisting of calcium, magnesium, phosphorus, strontium, Vitamin D, and eggshell membrane hydrolysate containing naturally occurring glycosaminoglycans and sulfated glycoproteins. OSC has been used both in an open-label human study and in vitro in osteoblasts. METHODS: Fifteen patients divided into three groups received oral OSC 0.6 g three times daily for 20 days. The main outcome measures were regional skeletal pain over the treatment period. For the in vitro experiments eight primary human osteoblasts cultures were established from trabecular bone, six of them from the femoral head, and two from the tibia. Cells were cultured for five to 20 days in the presence of 20 µg/ml OSC. Immunocytochemistry and RT-PCR were used to detect molecular alterations involved in the mineralization process. Calcium concentration was measured by means of a colorimetric assay and cell viability was analyzed using the LDH cytotoxicity assay. To investigate whether the osteoblasts response to OSC is associated with signaling processes the ERK1/2 and AKT signal transduction pathways were analyzed. RESULTS: Open label human study: OSC, 0.6 g three times daily, resulted in a significant positive effect on pain alleviation of 42% after 5 days (p < 0.001), 57% after 10 days and 68% after 20 days (p < 0.0001; for both time points), with no side-effects being reported. In vitro analysis: In osteoblasts, growing in OSC-supplemented media significant overexpression of bone γ-carboxylglutamic acid-containing protein, secreted phosphoprotein-1, integrin binding sialoprotein, and dentin matrix phosphoprotein genes could be detected when compared to control osteoblasts grown in the absence of OSC. Moreover, OSC-treated osteoblasts produced over the study period vast extracellular calcium deposits without any loss of cellular integrity or signs of cellular toxicity. In addition OSC promotes osteoblast differentiation and activates the AKT signaling pathway. CONCLUSION: This open label study provides preliminary evidence of the efficacy of OSC. Despite the limitations (small heterogeneous patient group) the findings can be viewed as a necessary investigation that supports further clinical trials with a double-blind controlled design. Experiments at cellular and molecular level provide supplementary information about OSC that increases mineralization in osteoblasts and activation of the AKT pathway. TRIAL REGISTRATION: DRKS00013233 , 06th November 2017, retrospectively registered.

Lysyl oxidase like-4 monoclonal antibody demonstrates therapeutic effect against head and neck squamous cell carcinoma cells and xenografts.

A new member of the lysyl oxidase (LOX) family, lysyl oxidase-like 4 (LOXL4), is overexpressed in head and neck squamous cell carcinoma (HNSCC) compared to normal squamous epithelium. A monoclonal antibody (mAb) derived from fusion of Balb/c mouse splenocytes immunized with LOXL4 specific peptide was used to evaluate its therapeutic efficacy in 15 HNSCC cell lines associated with LOXL4 overexpression. For xenograft experiments 41 severe combined immunodeficient (SCID) mice were used to analyze LOXL4-mAb mediated tumor regression. Cell viability was analyzed using cytotoxicity-, and clonogenic-assays. Significant suppression of tumor cell growth was observed in 12 out of 15 (80%) tumor cell lines after 48 hr exposure to the mAb (LD50 of 15 µg/ml to 45 µg/ml). The effect induced by the antibody could be blocked by pre-incubation of the antibody with the peptide used for immunization of the mice and antibody generation, indicating that the effect of the antibody is specific. In mice inoculated with HNSCC cells, i.v. injections of the LOXL4-mAb resulted within 70 days in extensive tumor destruction in all treated animals whereas no tumor regression occurred in control animals. In mice pre-immunized i.v. with LOXL4-mAb and subsequently injected with HNSCC cells, tumor development was considerably delayed in contrast to non LOXL4-mAb pre-immunized animals. These results demonstrate that the LOXL4-mAb has potent antitumor activity and suggest its suitability as a therapeutic immune agent applicable to HNSCC exhibiting tumor specific upregulation of LOXL4.

Neutrophil uptake of nitrogen-bisphosphonates leads to the suppression of human peripheral blood γδ T cells.

Nitrogen-bisphosphonates (n-BP), such as zoledronate, are the main class of drugs used for the prevention of osteoporotic fractures and the management of cancer-associated bone disease. However, long-term or high-dose use has been associated with certain adverse drug effects, such as osteonecrosis of the jaw and the loss of peripheral of blood Vγ9Vδ2 T cells, which appear to be linked to drug-induced immune dysfunction. In this report we show that neutrophils present in human peripheral blood readily take up zoledronate, and this phenomenon is associated with the potent immune suppression of human peripheral blood Vγ9Vδ2 T cells. Furthermore, we found this zoledronate-mediated inhibition by neutrophils could be overcome to fully reconstitute Vγ9Vδ2 T cell proliferation by concomitantly targeting neutrophil-derived hydrogen peroxide, serine proteases, and arginase I activity. These findings will enable the development of targeted strategies to mitigate some of the adverse effects of n-BP treatment on immune homeostasis and to improve the success of immunotherapy trials based on harnessing the anticancer potential of peripheral blood γδ T cells in the context of n-BP treatment.

Head and neck cancer cells and xenografts are very sensitive to palytoxin: decrease of c-jun n-terminale kinase-3 expression enhances palytoxin toxicity.

OBJECTIVES: Palytoxin (PTX), a marine toxin isolated from the Cnidaria (zooanthid) Palythoa caribaeorum is one of the most potent non-protein substances known. It is a very complex molecule that presents both lipophilic and hydrophilic areas. The effect of PTX was investigated in a series of experiments conducted in head and neck squamous cell carcinoma (HNSCC) cell lines and xenografts. MATERIALS AND METHODS: Cell viability, and gene expression of the sodium/potassium-transporting ATPase subumit alpha1 (ATP1AL1) and GAPDH were analyzed in HNSCC cells and normal epithelial cells after treatment with PTX using cytotoxicity-, clonogenic-, and enzyme inhibitor assays as well as RT-PCR and Northern Blotting. For xenograft experiments severe combined immunodeficient (SCID) mice were used to analyze tumor regression. The data were statistically analyzed using One-Way Annova (SPSS vs20). RESULTS: Significant toxic effects were observed in tumor cells treated with PTX (LD50 of 1.5 to 3.5 ng/ml) in contrast to normal cells. In tumor cells PTX affected both the release of LDH and the expression of the sodium/potassium-transporting ATPase subunit alpha1 gene suggesting loss of cellular integrity, primarily of the plasma membrane. Furthermore, strong repression of the c-Jun N-terminal kinase 3 (JNK3) mRNA expression was found in carcinoma cells which correlated with enhanced toxicity of PTX suggesting an essential role of the mitogen activated protein kinase (MAPK)/JNK signalling cascades pathway in the mechanisms of HNSCC cell resistance to PTX. In mice inoculated with carcinoma cells, injections of PTX into the xenografted tumors resulted within 24 days in extensive tumor destruction in 75% of the treated animals (LD50 of 68 ng/kg to 83 ng/kg) while no tumor regression occurred in control animals. CONCLUSIONS: These results clearly provide evidence that PTX possesses preferential toxicity for head and neck carcinoma cells and therefore it is worth further studying its impact which may extend our knowledge of the biology of head and neck cancer.

Surface contamination of dental implants assessed by gene expression analysis in a whole-blood in vitro assay: a preliminary study.

AIM: We aimed at evaluating pyrogen contamination of dental implants made of titanium and zirconia by using gene expression analysis in a whole-blood in vitro assay. MATERIAL AND METHODS: Titanium and zirconia implants (five each) were incubated in human whole blood. Samples were assayed for gene expression levels of toll-like receptor 4 (TLR4), TLR9, interleukin (IL)-1ß, nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-kB), tumour necrosis factor (TNF)-α, and Fas-associated protein with death domain (FADD) as indicators of surface contamination resulting in lipopolysaccharides (LPS)-stimulated TLR- or TNF-mediated immune responses. Gene expression was assayed using real-time quantitative polymerase chain reaction (RT-qPCR). Non-stimulated blood from the same donor served as a negative control, and blood stimulated with LPS served as a positive control. After dry-heat treatment with dry heat, all implants were re-analysed as described above. RESULTS: Both implant systems contained surface contaminants evoking a pro-inflammatory response similar to that induced by LPS. After dry-heat treatment, gene expression was significantly decreased to levels similar to those of negative control samples. CONCLUSIONS: The results demonstrated LPS-like surface-bound contaminants in both tested implant systems. Depyrogenation with dry heat seems to be an effective means of reducing such contamination in dental implants.

Smoking-Induced SLPI Expression Hinders HPV Infections Also in Squamous Cell Carcinomas of the Vulva.

In HNSCC, protein- and mRNA-expression of the antileukoproteinase SLPI are significantly inverse correlated with HPV-infection suggesting that elevated expression of SLPI protects against HPV-infections. Moreover, SLPI-expression is up-regulated in HNSCC-patients reporting a smoking habit. Here, we investigate the described correlation in other HPV-driven cancers, namely vulvar squamous cell carcinoma (VSCC). FFPE samples of 99 VSCC were analyzed by PCR for HPV-DNA-expression and by RT-qPCR for SLPI-mRNA-expression. Of 99 VSCC 10 (10.1%) are HPV-positive; 9 were HPV16; 1 HPV18; all were E6/E7 mRNA-positive. 33 of the 99 patients (33.3%) reported a smoking habit; 7 (21.1%) of these were HPV-positive. Of 66 (66.7%) non-smokers 3 (4.5%) were HPV-positive. SLPI-expression was 4.0-fold lower in HPV-positive than HPV-negative patients. Smoking resulted in 2.3-fold higher SLPI expression. The data presented here indicate that SLPI plays a pivotal role in HPV-infection not only in HNSCC but also in VSCC and possibly also in other HPV-driven cancers. This however, needs to be analyzed in future studies. Furthermore these data lead to the hypothesis that the smoking induced SLPI-increase is systemic rather than local, as assumed based on the HNSCC data.

SLPI and AnxA2 expression in neoplasm-free palatine tonsils is associated with smoking habit of individuals.

In order to confirm the inverse correlation between secretory leucocyte protease inhibitor (SLPI) expression, and human papillomavirus (HPV) infection previously observed in head and neck squamous-cell carcinoma, the present study retrospectively investigated the association between SLPI and Annexin A2 (AnxA2) expression, and HPV status in non-neoplastic chronic tonsillitis (n=118), and tonsillar hyperplasia (n=96) tissue. We hypothesised that smoking induces the upregulation of SLPI, resulting in reduced binding of HPV to AnxA2, a known modulator of HPV entry into the cell. SLPI and cyclin-dependent kinase inhibitor 2A (p16INK4A) protein expression was measured using immunohistochemistry in 214 specimens; SLPI and AnxA2 gene expression was measured using reverse transcription-quantitative polymerase chain reaction in 213 cases; and DNA was isolated from all the specimens to determine HPV status. The association between the results of the aforementioned analyses and the smoking habits of patients was analysed. The samples were HPV-negative. p16INK4A expression demonstrated moderate and strong staining in 38, and 0 cases, respectively. SLPI expression presented negative, weak and moderate signals in 163, 45, and 6 cases, respectively. A positive correlation was identified between smoking and SLPI (P=0.0001). Gene expression analysis (n=213) revealed that smoking (n=48) resulted in a significant increase in SLPI and AnxA2 expression. A significant positive correlation between AnxA2 and SLPI, indicating a surplus of AnxA2 in relation to SLPI, was exclusively identified in non-smokers. The data demonstrated that smoking results in increased SLPI and AnxA2 expression also in non-neoplastic tonsillar tissue. The observed surplus of AnxA2 in relation to SLPI identified exclusively in the tonsillar tissue of non-smokers indicates a higher possibility of a successful HPV infection of the tonsillar tissue of non-smokers, given the properties of AnxA2 to function as an infection modulator.

The antileukoprotease secretory leukocyte protease inhibitor (SLPI) and its role in the prevention of HPV-infections in head and neck squamous cell carcinoma.

Recently, we demonstrated a significant inverse correlation between HPV-infection and SLPI-expression suggesting that SLPI protects against HPV-infection of HNSCC. Here we analyzed in a single lab setting 307 formalin-fixed paraffin-embedded HNSCC cases (tonsillar n = 135; non-tonsillar: n = 172) from eight health care centers. Samples were analyzed for SLPI gene- and protein-expression. Annexin A2, its heterotetramer A2t, putatively facilitating HPV- and SLPI-cell entry, was measured to study the correlation between SLPI and annexin A2. Data were correlated with tobacco consumption and HPV-status. Overall, HPV-DNA prevalence was 23.5% (72/307); attributed to: 43.7% (59/135) tonsillar and 7.6% (13/172) non-tonsillar cases. Smoking resulted in 6.44-fold increased and HPV-infection in 3.46-fold decreased SLPI-gene expression in all HNSCC with similar significant results obtained in tonsillar and non-tonsillar SCC separately. Correlating annexin A2- and SLPI-gene expression showed a significant surplus of annexin A2 in HPV-positive tumors (4.21× more annexin A2) and 6.72× more annexin A2 than SLPI in nonsmokers in all HNSCCs and similar significant results for both tumor entities separately. The surplus of annexin A2 in non-smokers and HPV-positive patients supports our hypothesis that decreased SLPI levels facilitate HPV-infection i.e., increased SLPI-expression may protect against HPV-infection of tonsillar and non-tonsillar SCC.

Human papillomavirus infection in head and neck cancer: the role of the secretory leukocyte protease inhibitor.

We previously showed that secretory leukocyte protease inhibitor (SLPI) gene and protein expression is significantly lower in metastatic versus non-metastatic head and neck squamous cell carcinoma (HNSCC). However, we did not assess the human papillomavirus (HPV) status of these cases. Since SLPI plays a role in HIV and herpes simplex virus (HSV) infections, we hypothesized that SLPI may be involved in HPV-infected HNSCC. In HNSCC tissue (n=54), HPV DNA was determined and correlated with SLPI expression. Additionally, to investigate a possible role of smoking on SLPI expression in clinically normal mucosa, 19 patients treated for non­malignant diseases (non-HNSCC) were analyzed for SLPI expression and correlated with smoking habits. In HNSCC patients, SLPI expression showed a significant inverse correlation with HPV status. In patients with moderate/strong SLPI expression (n=19), 10.5% were HPV-positive. By contrast, patients with absent/weak SLPI expression (n=35), 45.7% were HPV-positive. Low SLPI expression was correlated with metastasis (P=0.003) independent of HPV status. HPV-positivity was clearly associated with lymph node status (81.3% N1-3 cases). In smoking non-HNSCC patients (n=7), 42.9% showed absent/weak and 57.1% moderate/strong SLPI staining. In non-smoking non-HNSCC patients (n=10) 83.3% showed absent/weak and 16.7% moderate/strong SLPI expression. For the first time, a correlation between SLPI downregulation and HPV infection was demonstrated, suggesting that high levels of SLPI, possibly induced by environmental factors such as tobacco smoking, correlate with protective effects against HPV infection. SLPI may be a potential biomarker identifying head and neck cancer patients not at risk of developing metastases (SLPI-positive), and those at risk to be infected by HPV (SLPI-negative) and likely to develop metastases.