Boeravinone B, a natural rotenoid, inhibits osteoclast differentiation through modulating NF-κB, MAPK and PI3K/Akt signaling pathways.

Osteoporosis is a major public health concern, which requires novel therapeutic strategies to prevent or mitigate bone loss. Natural compounds have attracted attention as potential therapeutic agents due to their safety and efficacy. In this study, we investigated the regulatory activities of boeravinone B (BOB), a natural rotenoid isolated from the medicinal plant Boerhavia diffusa, on the differentiation of osteoclasts and mesenchymal stem cells (MSCs), the two main cell components responsible for bone remodeling. We found that BOB inhibited osteoclast differentiation and function, as determined by TRAP staining and pit formation assay, with no significant cytotoxicity. Furthermore, our results showing that BOB ameliorates ovariectomyinduced bone loss demonstrated that BOB is also effective in vivo. BOB exerted its inhibitory effects on osteoclastogenesis by downregulating the RANKL/RANK signaling pathways, including NF-κB, MAPK, and PI3K/Akt, resulting in the suppression of osteoclast-specific gene expression. Further experiments revealed that, at least phenomenologically, BOB promotes osteoblast differentiation of bone marrow-derived MSCs but inhibits their differentiation into adipocytes. In conclusion, our study demonstrates that BOB inhibits osteoclastogenesis and promotes osteoblastogenesis in vitro by regulating various signaling pathways. These findings suggest that BOB has potential value as a novel therapeutic agent for the prevention and treatment of osteoporosis. [BMB Reports 2023; 56(10): 545-550].

Oxysterol 25-hydroxycholesterol as a metabolic pathophysiological factors of osteoarthritis induces apoptosis in primary rat chondrocytes.

The aim of the present study was to investigate the pathophysiological etiology of osteoarthritis that is mediated by the apoptosis of chondrocytes exposed to 25-hydroxycholesterol (25-HC), an oxysterol synthesized by the expression of cholesterol-25-hydroxylase (CH25H) under inflammatory conditions. Interleukin-1ß induced the apoptosis of chondrocytes in a dose- dependent manner. Furthermore, the production of 25-HC increased in the chondrocytes treated with interleukin-1ß through the expression of CH25H. 25-HC decreased the viability of chondrocytes. Chondrocytes with condensed nucleus and apoptotic populations increased by 25-HC. Moreover, the activity and expression of caspase-3 were increased by the death ligand-mediated extrinsic and mitochondria-dependent intrinsic apoptotic pathways in the chondrocytes treated with 25-HC. Finally, 25-HC induced not only caspase-dependent apoptosis, but also induced proteoglycan loss in articular cartilage ex vivo cultured rat knee joints. These data indicate that 25-HC may act as a metabolic pathophysiological factor in osteoarthritis that is mediated by progressive chondrocyte death in the articular cartilage with inflammatory condition.

Formononetin induces apoptotic cell death through the suppression of mitogen­activated protein kinase and nuclear factor­κB phosphorylation in FaDu human head and neck squamous cell carcinoma cells.

Formononetin, a phytoestrogen extracted from various herbal plants, has been investigated as an anticancer agent against diverse types of cancer. The aim of the present study was to investigate the induction of apoptotic cell death by formononetin in the FaDu pharyngeal squamous cell carcinoma cell line. Formononetin significantly increased FaDu cell death, with an estimated IC50 value of 50 µM; however, it did not affect the viability of normal L929 mouse fibroblasts used as normal control at 5­25 µM. Typical characteristics of apoptosis, such as morphological alterations, chromatin condensation, DNA fragmentation and the size of the apoptotic cell population, were increased in FaDu cells treated with formononetin for 24 h. Furthermore, formononetin­induced FaDu cell death involved the death receptor­mediated extrinsic and the mitochondria­dependent intrinsic apoptotic pathways by activating the caspase cascade. The chemotherapeutic effects of formononetin were mediated by the suppression of mitogen­activated protein kinases, including extracellular signal­regulated kinase 1/2 and p38, and nuclear factor­κB phosphorylation in FaDu cells. Finally, the oral administration of formononetin decelerated tumor growth through the expression of cleaved caspase­3 in a FaDu cell xenograft animal model. Taken together, these findings indicate that formononetin holds promise as a chemotherapeutic agent and may be of value in the treatment of human head and neck squamous cell carcinoma.

Anticatabolic Effects of Morin through the Counteraction of Interleukin-1ß-Induced Inflammation in Rat Primary Chondrocytes.

Morin, a flavonoid isolated from various medicinal herbal plants, has an anti-inflammatory effect. This study aimed to elucidate the anticatabolic effects and cellular mechanism of morin against interleukin-1ß (IL-1ß) in rat primary chondrocytes. Morin at 10-100 µM did not affect the viability of rat primary chondrocytes. Treatment with morin for 21 days ameliorated the IL-1ß-induced decrease in extracellular matrix. Furthermore, treatment with morin attenuated IL-1ß-induced proteoglycan loss in the articular cartilage through suppression of catabolic factors, such as matrix metalloproteinases, inflammatory mediators, and pro-inflammatory cytokines. These data indicated that morin exerted anticatabolic effects that can prevent and reduce progressive degeneration of the articular cartilage, and thus may be a potential candidate treatment for osteoarthritis.

Reversine induces caspase-dependent apoptosis of human osteosarcoma cells through extrinsic and intrinsic apoptotic signaling pathways.

BACKGROUND: The 2-(4-morpholinoanilino)-6-cyclohexylaminopurine (reversine) acts as a chemopreventive agent and induces apoptotic cell death in various cancer cells. However, the anticancer effects of reversine on osteosarcoma cells are not clearly established. OBJECTIVE: The purpose of this study was to investigate the effect of reversine on cell proliferation and induction of apoptosis in human osteosarcoma cells. METHODS: Cell viability assay, histological analysis, DAPI staining, caspase activation analysis, flow cytometric analysis and immunoblotting were carried out in MG-63 osteosarcoma cells. RESULTS: Reversine inhibited the growth of cells in a dose-dependent manner and induced nuclear condensation and fragmentation. Reversine-treated cells showed caspase-3/7 activation and increased apoptosis versus control cells. FasL, a death ligand associated with extrinsic apoptotic signaling pathways, was significantly up-regulated by reversine treatment. Moreover, the caspase-8, a part of the extrinsic apoptotic pathway, was activated by reversine treatments. Expressions of anti-apoptotic factors such as Bcl-2 and Bcl-xL, components of the mitochondria dependent intrinsic apoptosis pathway, significantly decreased following reversine treatment. The expressions of pro-apoptotic factors such as BAX, BAD and caspase-9 increased by reversine treatments. In addition, reversine activated caspase-3 and Poly (ADP-ribose) polymerase (PARP) to induce cell death. The Z-VAD-fmk significantly inhibited cell death through the suppression of caspase-3 expression in MG-63 cells treated with reversine. CONCLUSION: These results suggest that the reversine may inhibit cell proliferation and induce apoptotic cell death in MG-63 osteosarcoma cells through both the mitochondria-mediated intrinsic pathway and the death receptor-mediated extrinsic pathway, and may have potential properties for the discovery of anti-cancer agents.

Formononetin Antagonizes the Interleukin-1ß-Induced Catabolic Effects Through Suppressing Inflammation in Primary Rat Chondrocytes.

In the present study, we demonstrated the anti-catabolic effects of formononetin, a phytoestrogen derived from herbal plants, against interleukin-1ß (IL-1ß)-induced severe catabolic effects in primary rat chondrocytes and articular cartilage. Formononetin did not affect the viability of primary rat chondrocytes in both short- (24 h) and long-term (21 days) treatment periods. Furthermore, formononetin effectively antagonized the IL-1ß-induced catabolic effects including the decrease in proteoglycan content, suppression of pericellular matrix formation, and loss of proteoglycan through the decreased expression of cartilage-degrading enzymes like matrix metalloproteinase (MMP)-13, MMP-1, and MMP-3 in primary rat chondrocytes. Moreover, catabolic oxidative stress mediators like nitric oxide, inducible nitric oxide synthase, cyclooxygenase-2, and prostaglandin E2 were significantly downregulated by formononetin in primary rat chondrocytes treated with IL-1ß. Sequentially, the upregulation of pro-inflammatory cytokines (like IL-1α, IL-1ß, IL-6, and tumor necrosis factor α), chemokines (like fractalkine, monocyte chemoattractant protein-1, and macrophage inflammatory protein-3α), and vascular endothelial growth factor were significantly downregulated by formononetin in primary rat chondrocytes treated with IL-1ß. These data suggest that formononetin may suppress IL-1ß-induced severe catabolic effects and osteoarthritic condition. Furthermore, formononetin may be a promising candidate for the treatment and prevention of osteoarthritis.

Counting-based cell-free DNA screening test fails to identify triploidy-A case report.

Although noninvasive prenatal testing (NIPT) is a good test with high sensitivity and specificity for trisomy 21, 18, and 13, it remains a screening test and cannot be used for diagnostic purposes. It is important to consider the outcomes of this test and interpret the results and offer consultation accordingly.

Biochanin-A induces apoptosis and suppresses migration in FaDu human pharynx squamous carcinoma cells.

The aim of the present study was to investigate biochanin-A-induced anticancer effects and their cellular signaling pathway in FaDu pharyngeal squamous carcinoma cells. Biochanin-A induced cell death through increased cytotoxicity of FaDu cells in a dose- and time-dependent manner. The number of cells with nucleus condensation and the apoptotic population were increased in the FaDu cells stimulated with biochanin-A for 24 h. Furthermore, extrinsic apoptotic factors such as FasL and their downstream target caspase-8 were increased and activated in the FaDu cells treated with biochanin-A in a dose-dependent manner. Moreover, biochanin-A decreased the expression of intrinsic anti-apoptotic factors such as Bcl-2 and Bcl-xL, and increased the level and activation of intrinsic apoptotic factors such as Bad and caspase-9. Finally, biochanin-A induced the activation of caspase-3 and Poly(ADP ribose) polymerase (PARP) in FaDu cells. Our results suggest that biochanin-A-induced apoptosis was mediated by death receptor mediated-extrinsic and mitochondria-dependent intrinsic apoptotic signaling pathways. Biochanin-A also inhibited wound healing migration and proliferation of FaDu cells via the downregulation and inactivation of matrix metalloproteinase-2 and -9 that are mediated by the suppression of p38, mitogen activated protein kinase (MAPK), NF-κB and Akt cellular signaling pathways. Therefore, these data suggest that the biochanin-A may act as a potential chemotherapeutic compound to treat head and neck cancer.

Licochalcone-E induces caspase-dependent death of human pharyngeal squamous carcinoma cells through the extrinsic and intrinsic apoptotic signaling pathways.

The aim of the present study was to investigate licochalcone-E (Lico-E)-induced apoptosis and the associated apoptotic signaling pathway in FaDu cells, a human pharyngeal squamous carcinoma cell line. Treatment with Lico-E exhibited significant cytotoxicity on FaDu cells in a concentration-dependent manner. The IC50 value of Lico-E in FaDu cells was ~50 µM. Treatment with Lico-E increased the number of dead FaDu cells. Furthermore, chromatin condensation, which is associated with apoptotic cell death, was observed in FaDu cells treated with Lico-E for 24 h. By contrast, Lico-E did not produce cytotoxicity or increase the number of dead cells when applied to human normal oral keratinocytes (hNOKs). Furthermore, chromatin condensation was not observed in hNOKs treated with Lico-E. Treatment with Lico-E increased the expression of Fas ligand and the cleaved form of caspase-8 in FaDu cells. Furthermore, treatment with Lico-E increased the expression of pro-apoptotic factors, including apoptosis regulator BAX, Bcl-2-associated agonist of cell death, apoptotic protease-activating factor 1, caspase-9 and tumor suppressor p53, while decreasing the expression of anti-apoptotic factors, including apoptosis regulator Bcl-2 and Bcl-2-like protein 1 in FaDu cells. The expression of cleaved caspases-3 and poly (ADP-ribose) polymerase was significantly upregulated following treatment with Lico-E in FaDu cells, while Lico-E-induced apoptotic FaDu cell death was partially suppressed by treatment with Z-VAD-FMK, a pan caspase inhibitor. Therefore, Lico-E-induced oral cancer (OC) cell-specific apoptosis is mediated by the death receptor-dependent extrinsic and mitochondrial-dependent intrinsic apoptotic signaling pathways. In conclusion, these data suggested that Lico-E exhibits potential chemopreventive effects and warrants further developed as a chemotherapeutic agent against OC.

Synthesis and Characterization of ß-Tricalcium Phosphate Derived From Haliotis sp. Shells.

PURPOSE: To develop a methodology for the synthesis of ß-tricalcium phosphate (ß-TCP, Ca3(PO4)2) from the shell of Haliotis sp. (abalone shell) and to verify its characterization and biocompatibility. MATERIALS AND METHODS: Calcium oxide (CaO) was synthesized from abalone shell by sintering and was suspended in distilled water to prepare calcium hydroxide (Ca(OH)2). For the synthesis of calcium carbonate (CaCO3), carbon dioxide was used to infuse Ca(OH)2 at pH 7.4. CaCO3 was reacted with phosphoric acid at pH 6.0 to obtain dicalcium phosphate (CaHPO4). Subsequently, ß-TCP was synthesized by a chemical reaction between CaHPO4 and CaO at 950°C to 1100°C for 3 hours. Fourier transform infrared spectroscopy (FT-IR) and x-ray diffraction (XRD) was performed to verify the physiochemical characteristics of the composite synthesized from abalone shell. RESULTS: FT-IR and XRD results showed that ß-TCP was successfully synthesized from abalone shell. The synthesized ß-TCP did not affect cell viability of either normal human oral keratinocytes or osteoblastic MG-63 cells. These data indicate that ß-TCP synthesized from abalone shell is biologically safe. CONCLUSIONS: ß-TCP (Ca3(PO4)2) synthesized from abalone shell can be used as a potential source of bone grafting material.

Coumestrol Counteracts Interleukin-1ß-Induced Catabolic Effects by Suppressing Inflammation in Primary Rat Chondrocytes.

In the present study, we investigated the anti-catabolic effects of coumestrol, a phytoestrogen derived from herbal plants, against interleukin-1ß-induced cartilage degeneration in primary rat chondrocytes and articular cartilage. Coumestrol did not affect the viability of human normal oral keratinocytes and primary rat chondrocytes treated for 24 h and 21 days, respectively. Although coumestrol did not significantly increase the proteoglycan contents in long-term culture, it abolished the interleukin-1ß-induced loss of proteoglycans in primary rat chondrocytes and knee articular cartilage. Furthermore, coumestrol suppressed the expression of matrix-degrading enzymes such as matrix metalloproteinase-13, -3, and -1 in primary rat chondrocytes stimulated with interleukin-1ß. Moreover, the expression of catabolic factors such as nitric oxide synthase, cyclooxygenase-2, prostaglandin E2, and inflammatory cytokines in interleukin-1ß-stimulated primary rat chondrocytes was suppressed by coumestrol. In summary, these results indicate that coumestrol counteracts the catabolic effects induced by interleukin-1ß through the suppression of inflammation. Therefore, based on its biological activity and safety profile, coumestrol could be used as a potential anti-catabolic biomaterial for osteoarthritis.

Biochanin-A antagonizes the interleukin-1ß-induced catabolic inflammation through the modulation of NFκB cellular signaling in primary rat chondrocytes.

Biochanin-A, a phytoestrogen derived from herbal plants, protected from the IL-1ß-induced loss of proteoglycans through the suppression of matrix degrading enzymes such as matrix metalloproteinase (MMP)-13, MMP-3, MMP-1, and ADAMTS-5 in primary rat chondrocytes and the knee articular cartilage. It also suppressed the expression of IL-1ß-induced catabolic factors such as nitric oxide synthase 2, cyclooxygenase-2, prostaglandin E2, and inflammatory cytokines. Furthermore, biochanin-A suppressed the IL-1ß-induced phosphorylation of NFκB, and inhibited its nuclear translocation in primary rat chondrocytes. These results indicate that biochanin-A antagonizes the IL-1ß-induced catabolic effects through its anti-inflammatory activity that involves the modulation of NFκB signaling.

Biological Effects of the Herbal Plant-Derived Phytoestrogen Bavachin in Primary Rat Chondrocytes.

The aim of this study was to examine the anabolic and anticatabolic functions of bavachin in primary rat chondrocytes. With bavachin treatment, chondrocytes survived for 21 d without cell proliferation, and the proteoglycan content and extracellular matrix increased. Short-term monolayer culture of chondrocytes showed that gene induction of both aggrecan and collagen type II, major extracellular matrix components, was significantly upregulated by bavachin. The expression and activities of cartilage-degrading enzymes such as matrix metalloproteinases and a disintegrin and metalloproteinase with thrombospondin motifs were inhibited significantly by bavachin, while tissue inhibitors of metalloprotease were significantly upregulated. Bavachin inhibits the expression of inducible nitric oxide synthase, a representative catabolic factor, and downregulated the expression of nitric oxide, cyclooxygenase-2, and prostaglandin E2 in a dose-dependent manner in chondrocytes. Our results suggest that the bavachin has anabolic and potent anticatabolic biological effects on chondrocytes, which may have considerable promise in treating articular cartilage degeneration in the future.

Licochalcone-A induces intrinsic and extrinsic apoptosis via ERK1/2 and p38 phosphorylation-mediated TRAIL expression in head and neck squamous carcinoma FaDu cells.

We investigated Licochalcone-A (Lico-A)-induced apoptosis and the pathway underlying its activity in a pharyngeal squamous carcinoma FaDu cell line. Lico-A purified from root of Glycyrrhiza inflata had cytotoxic effects, significantly increasing cell death in FaDu cells. Using a cell viability assay, we determined that the IC50 value of Lico-A in FaDu cells was approximately 100 µM. Chromatin condensation was observed in FaDu cells treated with Lico-A for 24 h. Consistent with this finding, the number of apoptotic cells increased in a time-dependent manner when FaDu cells were treated with Lico-A. TRAIL was significantly up-regulated in Lico-A-treated FaDu cells in a dose-dependent manner. Apoptotic factors such as caspases and PARP were subsequently activated in a caspase-dependent manner. In addition, levels of pro-apoptotic factors increased significantly in response to Lico-A treatment, while levels of anti-apoptotic factors decreased. Lico-A-induced TRAIL expression was mediated in part by a MAPK signaling pathway involving ERK1/2 and p38. In xenograft mouse model, Lico-A treatment effectively suppressed the growth of FaDu cell xenografts by activating caspase-3, without affecting the body weight of mice. Taken together, these data suggest that Lico-A has potential chemopreventive effects and should therefore be developed as a chemotherapeutic agent for pharyngeal squamous carcinoma.

Berberine induces FasL-related apoptosis through p38 activation in KB human oral cancer cells.

In the present study, we examined the anticancer properties of berberine in KB oral cancer cells with a specific focus on its cellular mechanism. Berberine did not affect the cell viability of the primary human normal oral keratinocytes that were used as a control. However, the viability of KB cells was found to decrease significantly in the presence of berberine in a dose-dependent manner. Furthermore, in KB cells, berberine induced the fragmentation of genomic DNA, changes in cell morphology, and nuclear condensation. In addition, caspase-3 and -7 activation, and an increase in apoptosis were observed. Berberine was also found to upregulate significantly the expression of the death receptor ligand, FasL. In turn, this upregulation triggered the activation of pro-apoptotic factors such as caspase-8, -9 and -3 and poly(ADP-ribose) polymerase (PARP). Furthermore, pro-apoptotic factors such as Bax, Bad and Apaf-1 were also significantly upregulated by berberine. Anti-apoptotic factors such as Bcl-2 and Bcl-xL were downregulated. Z-VAD-FMK, a cell-permeable pan-caspase inhibitor, suppressed the activation of caspase-3 and PARP. These results clearly indicate that berberine-induced cell death of KB oral cancer cells was mediated by both extrinsic death receptor-dependent and intrinsic mitochondrial-dependent apoptotic signaling pathways. In addition, berberine-induced upregulation of FasL was shown to be mediated by the p38 MAPK signaling pathway. We also found that berberine-induced migration suppression was mediated by downregulation of MMP-2 and MMP-9 through phosphorylation of p38 MAPK. In summary, berberine has the potential to be used as a chemotherapeutic agent, with limited side-effects, for the management of oral cancer.