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Background: Breast cancer (BC) produces bone resorptive cytokines and growth factors which 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: 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. 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 TNFα. Immuno blot indicates that ODN treatment decreased the protein expression of CTSK, IL-6, and IL-1β, 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 TGFβ 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 take into account the delayed development of metastatic bone disease found in the long-term odanacatib fracture trial (LOFT) study.

Naringin is a citrus flavonoid recently studied for anti-inflammatory activity in numerous cancer cells. In this study, the anti-inflammatory properties of naringin along with 5-fluorouracil in human skin cancer cell lines A375 was analyzed. A375 cells were treated with naringin, 5-fluorouracil alone, and combination. MTT assay and cell viability assays was demonstrated to detect the inhibitory effects of naringin or 5-fluorouracil on cell proliferation. mRNA expression of TNFα, IL-6, IL-1β, and NFκB were determined using quantitative RT-PCR. The effect of naringin and 5-fluorouracil combination significantly inhibited the growth and proliferation of the A375 cells in a concentration dependent manner with the IC50 values of naringin (24.75 μM) 5-fluorouracil (2.5 μM). The combination of naringin+5-fluorouracil on A375 cell lines at a concentration of half IC50 values (12µM+1 μM). Naringin and 5-fluorouracil combination also decreased the level of TNFα, IL-6, IL-1β, and NFκB mRNA in the A375 cell line. Naringin and 5-fluorouracil exerted anti-inflammatory effect through the suppression of NF-kB, IL-1β, TNFα, IL-6 in A375 cells. Taken together, our results suggested that treating A375 with naringin and 5-fluorouracil combination may have future applications in treating skin cancers through its anti-inflammatory effect.

RAW264.7, a murine macrophage cell line, an osteoclast model used for the differentiation of Osteoclast by inducing RANKL. Carvacrol, a monoterpenoid phenol that possess various medicinal property, was used for the treatment of osteoclast cells in this study. We investigate the effect of carvacrol in osteoclast cells induced by RANKL in RAW264.7 cells. RAW264.7 cells, cultured along with 40ng/ml of RANKL, on day 5, the cells were treated with TRAP staining to assess the formation of osteoclast cells, the presence of multinucleated TRAP positive cells were visualised using an inverted light microscope. The osteoclast cells were treated with varying concentrations of carvacrol (0-200µm) respectively for 48 hours. By MTT assay, it was found that there was no cytotoxic effect induced by carvacrol in RAW264.7 cells, whereas in RANKL induced osteoclast cells, there was a significant change in a dose dependent manner for 48 hours. By western blot and agarose gel electrophoresis, the levels of osteoclastogenic marker genes such as TRAP and cathepsin k were assessed and the developed osteoclast cells were treated with 150µm of carvacrol and found a significant change on treatment with carvacrol when compared with control. The present study reveals the anti-osteoclastogenic effect of carvacrol on osteoclast cells induced by RANKL in RAW264.7 cells.

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 β-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.

Background: Hesperetin is a natural compound known for its cholesterol-lowering effect and a wide range of pharmacological activities. Objectives: Investigating the potential anticancer activities of Hesperetin in malignant hematolymphoid cell lines HuT78 and MJ, derived from patients with Cutaneous T-Cell Lymphomas (CTCL). Methods: The cytotoxic effect of Hesperetin on two different CTCL cell lines, HuT78 and MJ, was assessed by MTS-based colorimetric assay. Apoptosis, cell cycle, ROS (Reactive Oxygen Species) and molecular analysis were performed using flow-cytometry and immunoblotting. Results: Hesperetin-treated CTCL cells were arrested at the sub-G1 phase of cell cycle with the concomitant decrease in the expression of the cell cycle regulator protein cyclin B. In addition, the study found that the cellular treatment with Hesperetin caused an induction of apoptosis, which was independent of ROS generation. Hesperetin caused a significant decrease in the expression level of anti-apoptotic protein Bcl-xL and an increase in cleaved caspase-3 and PARP proteins in CTCL cells. Furthermore, Hesperetin treatment in CTCL cells down-regulated the expression of Notch1 and phosphorylation of STAT3 (Tyr705) and inhibited NFκBp65. Conclusion: This study highlights the anticancer properties of Hesperetin. Which induces apoptosis in CTCL cells via STAT3/Notch1/NFκB mediated signaling pathway, suggesting that further development of this novel class of flavonoid may contribute to new drug discovery for certain hematolymphoid malignancies.

In this study, we report that the integrin LFA-1 cross-linking with its ligand ICAM-1 in human PBMCs or CD4+ T cells promotes Th1 polarization by upregulating IFN-γ secretion and T-bet expression. LFA-1 stimulation in PBMCs, CD4+ T cells, or the T cell line HuT78 activates the Notch pathway by nuclear translocation of cleaved Notch1 intracellular domain (NICD) and upregulation of target molecules Hey1 and Hes1. Blocking LFA-1 by a neutralizing Ab or specific inhibition of Notch1 by a γ-secretase inhibitor substantially inhibits LFA-1/ICAM-1–mediated activation of Notch signaling. We further demonstrate that the Notch pathway activation is dependent on LFA-1/ICAM-1–induced inactivation of glycogen synthase kinase 3β (GSK3β), which is mediated via Akt and ERK. Furthermore, in silico analysis in combination with coimmunoprecipitation assays show an interaction between NICD and GSK3β. Thus, there exists a molecular cross-talk between LFA-1 and Notch1 through the Akt/ERK–GSK3β signaling axis that ultimately enhances T cell differentiation toward Th1. Although clinical use of LFA-1 antagonists is limited by toxicity related to immunosuppression, these findings support the concept that Notch inhibitors could be attractive for prevention or treatment of Th1-related immunologic disorders and have implications at the level of local inflammatory responses.