Influence of type 2 diabetes on immunohistochemical detection of TRAF6, cFos and NFATC1 in the gingiva in cases of chronic periodontitis.

Type 2 diabetes (T2D) and chronic periodontitis (CP) are common diseases worldwide. Although T2D increases the severity of CP and alveolar bone loss, the mechanism of this is not well understood. We investigated using immunohistochemistry the expression of three osteoclast proteins, TRAF6, cFos and NFATc1, in gingival tissues. Gingival tissues were obtained from three groups: HC group, healthy controls; CP group, patients with CP; T2D + CP group, patients with both T2D and CP. Strong immunostaining for TRAF6, cFos and NFATc1 was observed in the gingival epithelium as well as in inflammatory cells in the CP and T2D + CP groups. Immunostaining was most intense in the T2D + CP group. We found strong up-regulation of TRAF6, cFos and NFATC1 in gingiva tissue of subjects with both T2D and CP, which corroborates our hypothesis that T2D potentiates osteoclastogenesis in CP.

Inhibitory Effects of Cathepsin K Inhibitor (ODN-MK-0822) on the Paracrine Pro-Osteoclast Factors of Breast Cancer Cells.

BACKGROUND: Breast cancer (BC) produces bone resorptive cytokines and growth factors that accelerate the development of osteoclasts (OCs), leading to osteolytic bone metastases. In the Long-term Odanacatib Fracture Trial (LOFT), the skeletal-metastasized breast cancer subjects who received odanacatib (ODN) had a delayed tumour progression and skeletal tumour burden as a result of anti-resorptive effects through inhibition of cathepsin K (CTSK). In this study, we explored the effect of ODN, a CTSK inhibitor, on the paracrine pro-osteoclast activity of breast cancer cells. METHODS: An immunohistochemistry study was performed to demonstrate CTSK and PTHrP expression in the samples of primary breast carcinoma. Expression of CTSK mRNA and protein was confirmed by the reverse transcription PCR and western blotting analysis in two human breast cancer cell lines, MDA-MB-231 and MCF-7 BC cell lines. Cells were incubated with sub-lethal amounts of ODN, and their conditioned supernatants were assessed for their capacity to differentiate PBMCs of healthy donors into osteoclast and its interference on bone-resorbing activities. We also measured the mRNA levels of major pro-osteoclast (pro-OC) factors in ODN-treated breast cancer cells and their secreted levels by semi-quantitative reverse transcription PCR and protein expression by immunoblotting. RESULTS: Different staining intensity was observed in samples containing PTHrP and CTSK in various histological grades of breast carcinoma. A significant positive relationship was found between CTSK expression and histological grade of BC and presence or absence of distant metastasis. The present study results also indicate that ODN has no effects on OCs number, however, ODN decreases the mRNA expression of secreted pro-OC factors such as PTHrP, CXCR-4, and TNF-α. Immunoblot indicates that ODN treatment decreased the protein expression of CTSK, IL-6, and IL-1ß, and thus lowered protein levels paralleled the defective phosphorylation of NF-κB. Moreover, there was a significant reduction in the level of growth factors such as IGF-1, PDGF, and TGFß expression at transcriptional level after ODN treatment as compared to control. CONCLUSION: ODN has shown to prevent osteolytic metastasis by interacting with the NF-κB pathway, inhibiting bone resorptive cytokines and growth factors. This effect can also be taken into account the delayed development of metastatic bone disease found in the long-term odanacatib fracture trial (LOFT) study.

Analysis of short-term variation and long-term drift during reagent kit lot change in an NABL accredited clinical biochemistry laboratory.

BACKGROUND: Kit lot change in clinical biochemistry labs leads to variations in patient results. This study planned to identify variations during 60 reagent lot changes in our laboratory during the period from June 2018 to May 2019. METHODS: A statistical analysis was performed to identify the difference between patient samples results variations and QC results. The long term drift was analyzed using a regression test. RESULTS: There was a significant difference between the patient and QC results in 16.7% of reagent lot changes. Moreover, the extent of variation in QC results was 3.3%. No long-term drift was seen in three analytes which were studied using regression analysis. CONCLUSIONS: Our results showed that, during reagent kit lot change, along with QC material, the patient samples should also be run in order to identify the variation. However, this practice is presently ignored by most of the laboratories. There was no accumulated effect in our laboratory due to reagent kit lot change.

Inhibitory effect of cathepsin K inhibitor (ODN-MK-0822) on invasion, migration and adhesion of human breast cancer cells in vitro.

Approximately 90% of patients with advanced breast cancer develop bone metastases; an event that results in severe decrease of quality of life and a drastic deterioration in prognosis. Therefore, to increase the survival of breast cancer patients, the development of new therapeutic strategies to impair metastatic process and skeletal complications is critical. Previous studies on the role of cathepsin K (CTSK) in metastatic spreading led to several strategies for inhibition of this molecule such as MIV-711 (Medivir), balicatib and odanacatib (ODN) which were on trial in the past. The present study intended to assess the anti-metastatic efficacy of ODN in breast cancer cells. Human breast cancer cell lines MDA-MB-231 were treated with different concentrations of ODN and performed invasion, adhesion and migration assays and, RT-PCR and western blot to evaluate the effect of ODN on the metastatic potential of breast cancer cells. ODN markedly decreased wound healing cell migration, invasion and adhesion at a dose dependent manner. ODN inhibits cell invasion by decreasing the matrix metalloproteinase (MMP-9) with the upregulation of TIMP-1 expression. ODN effectively inhibited the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal Kinase (JNK), and blocked the expression of ß-integrins and FAK proteins. ODN also significantly inhibited PI3K downstream targets Rac1, Cdc42, paxillin and Src which are critical for cell adhesion, migration and cytoskeletal reorganization. ODN exerts anti-metastatic action through inhibition of signaling pathway for MMP-9, PI3K and MAPK. This indicates potential therapeutic effects of ODN in the treatment of metastatic breast cancer.

Obesity and Cathepsin K: A Complex Pathophysiological Relationship in Breast Cancer Metastases.

BACKGROUND: Breast cancer appears in a strong inclination to metastasize in bone tissue. Several strategies are discussed in combating bone metastasis in breast cancer. However, therapy is only palliative and does not provide any improvement in survival to the majority of patients with advanced cancer. Obese and overweight women with breast cancer are three times more likely to develop metastatic disease compared to normal-weight women with the same treatment regimen. Overweight greatly intensify adipocytes formation in the bone marrow affecting bone metabolism by decreasing osteoblast differentiation and bone formation. Cathepsin K (CTSK), a cysteine protease, effectively degrades several components of the extracellular matrix and has the ability to differentiate adipocytes from bone marrow lineage. Therefore, the purpose of this review is to emphasize the underlying mechanism of CTSK and obesity role in breast cancer metastasis. METHODS: Systematic review was performed using PubMed, EMBASE. The evidence of obesity and CTSK in breast cancer skeletal metastasis were analyzed, summarized and compared. RESULTS: The present investigation argues for a specific association of CTSK with breast cancer skeletal metastasis by promoting adipocyte differentiation. The potential tumor-supporting roles of adipocytes are well documented, and in fact, suppressing adipocyte could be a new therapeutic option in the battle against lethal metastatic breast cancers. CONCLUSION: This review emphasizes CTSK through its multifaceted role in differentiating adipocytes, inflammation, and extracellular degradation, may be a critical factor in an obesity-cancer connection. Thus, integration of CTSK targeting strategies into established traditional therapies seems to hold substantial promise.

Platelet-rich fibrin/biphasic calcium phosphate impairs osteoclast differentiation and promotes apoptosis by the intrinsic mitochondrial pathway in chronic periodontitis.

BACKGROUND: The study explored the effect of platelet-rich fibrin/biphasic calcium phosphate (PRF/BCP) on differentiation and survival of osteoclasts obtained from peripheral blood of CP patients. METHODS: Peripheral blood mononuclear cells (PBMCs) from 25 patients with chronic periodontitis (CP) and 25 healthy individuals were assayed for cluster of differentiation14+ (CD14+ ) expression and monocytes were induced to differentiate into osteoclasts for 21 days in-vitro in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL). We assessed the number of osteoclasts by tartrate-acid resistant acid phosphatase (TRAP)-positivity. The mechanism of apoptosis was studied with reference to expression of Bcl-2, Bax, Bcl-xL, nuclear factor kappa-light chain enhancer of activated B cells (NF-κB), caspase 3/9 and DNA fragmentation. RESULTS: We observed a relative increase in the proportion of circulating osteoclasts in test group than control group (healthy individuals). In addition, osteoclast precursors in untreated cells (CP) were more osteoclastogenic as compared to cells treated with PRF/BCP and hence, there was a significant increase in the number of osteoclasts in CP. In PRF/BCP treated cells, we found a direct inhibition of transcription factor NF-κB with an increased caspase 3/9 levels and caspase 3 activity. Additionally, the protein expression and transcriptional profile of Bax was upregulated and Bcl-2 and Bcl-xL levels were down-regulated on treatment with PRF/BCP. CONCLUSION: Our results revealed that the PRF/BCP displayed an inhibitory role in osteoclasts formation and its molecular mechanism of action was related to the apoptosis induction through intrinsic mitochondrial pathway.