GPR183 Regulates 7α,25-Dihydroxycholesterol-Induced Oxiapoptophagy in L929 Mouse Fibroblast Cell.

7α,25-dihydroxycholesterol (7α,25-DHC) is an oxysterol synthesized from 25-hydroxycholesterol by cytochrome P450 family 7 subfamily B member 1 (CYP7B1) and is a monooxygenase (oxysterol-7α-hydroxylase) expressed under inflammatory conditions in various cell types. In this study, we verified that 7α,25-DHC-induced oxiapoptophagy is mediated by apoptosis, oxidative stress, and autophagy in L929 mouse fibroblasts. MTT assays and live/dead cell staining revealed that cytotoxicity was increased by 7α,25-DHC in L929 cells. Consequentially, cells with condensed chromatin and altered morphology were enhanced in L929 cells incubated with 7α,25-DHC for 48 h. Furthermore, apoptotic population was increased by 7α,25-DHC exposure through the cascade activation of caspase-9, caspase-3, and poly (ADP-ribose) polymerase in the intrinsic pathway of apoptosis in these cells. 7α,25-DHC upregulated reactive oxygen species (ROS) in L929 cells. Expression of autophagy biomarkers, including beclin-1 and LC3, was significantly increased by 7α,25-DHC treatment in L929 cells. 7α,25-DHC inhibits the phosphorylation of Akt associated with autophagy and increases p53 expression in L929 cells. In addition, inhibition of G-protein-coupled receptor 183 (GPR183), a receptor of 7α,25-DHC, using GPR183 specific antagonist NIBR189 suppressed 7α,25-DHC-induced apoptosis, ROS production, and autophagy in L929 cells. Collectively, GPR183 regulates 7α,25-DHC-induced oxiapoptophagy in L929 cells.

25-Hydroxycholesterol-Induced Oxiapoptophagy in L929 Mouse Fibroblast Cell Line.

25-hydroxycholesterol (25-HC) is an oxysterol synthesized from cholesterol by cholesterol-25-hydroxylase during cholesterol metabolism. The aim of this study was to verify whether 25-HC induces oxiapoptophagy in fibroblasts. 25-HC not only decreased the survival of L929 cells, but also increased the number of cells with condensed chromatin and altered morphology. Fluorescence-activated cell sorting results showed that there was a dose-dependent increase in the apoptotic populations of L929 cells upon treatment with 25-HC. 25-HC-induced apoptotic cell death was mediated by the death receptor-dependent extrinsic and mitochondria-dependent intrinsic apoptosis pathway, through the cascade activation of caspases including caspase-8, -9, and -3 in L929 cells. There was an increase in the levels of reactive oxygen species and inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide, and prostaglandin E2 in L929 cells treated with 25-HC. Moreover, 25-HC caused an increase in the expression of beclin-1 and microtubule-associated protein 1A/1B-light chain 3, an autophagy biomarker, in L929 cells. There was a significant decrease in the phosphorylation of protein kinase B (Akt) in L929 cells treated with 25-HC. Taken together, 25-HC induced oxiapoptophagy through the modulation of Akt and p53 cellular signaling pathways in L929 cells.

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.

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.

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.

Demethoxycurcumin induces apoptosis via inhibition of NF-κB pathway in FaDu human head and neck squamous cell carcinoma.

Background: Demethoxycurcumin (DMC) is a curcumin analog with antitumor properties. However, its effects have not been investigated in human head and neck squamous cell carcinoma (HNSCC). The aim of the present study was to verify the antitumor effect and cellular signaling pathways of DMC in FaDu HNSCC cells. Methods: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cell Live/Dead staining, hematoxylin and eosin staining, DAPI staining, FACS, western blotting, caspase-3 activity assay, and nuclear translocation were performed to verify apoptosis and the cellular signaling pathway of DMC in FaDu cells. Results: DMC increased FaDu cell death, with cells presenting altered morphology and condensed nuclei. DMC increased significantly the apoptotic population of FaDu cells. Sequentially, DMC increased the expression of cleaved caspase-3 and PARP through the up-regulation of pro-apoptotic factors such as FasL, cleaved caspase-8, Bax, Bad, and cleaved caspase-9 and the suppression of anti-apoptotic factors including Bcl-xL and Bcl-2 in FaDu cells. Furthermore, DMC not only suppressed the phosphorylation of NF-κB, but also inhibited the translocation of NF-κB from cytosol to nucleus of FaDu cells. Conclusions: Present study demonstrates that DMC-induced cell death is mediated caspase-dependently by death receptor-mediated extrinsic and mitochondria-dependent intrinsic apoptosis through the inhibition of NF-κB translocation from the cytosol to the nucleus of FaDu cells. DMC is a curcuminoid with antitumor properties that modulates the NF-κB cellular signaling pathway in FaDu cells. Taken together, this study suggests that DMC has a considerable chemotherapeutic potential for HNSCC.

25-Hydroxycholesterol Induces Death Receptor-mediated Extrinsic and Mitochondria-dependent Intrinsic Apoptosis in Head and Neck Squamous Cell Carcinoma Cells.

BACKGROUND/AIM: Oxysterol plays important physiological roles in diverse biological processes including apoptosis. However, the mechanisms underlying oxysterol-induced apoptosis remain unknown. 25-hydroxycholesterol (25-HC) is an oxysterol synthesized by cholesterol 25-hydroxylase from cholesterol during sterol metabolism. The aim of present study was to investigate 25-HC-induced apoptosis and associated signalling pathways in FaDu cells, which is originated form human head and neck squamous cell carcinoma cells. MATERIALS AND METHODS: 25-HC-induced apoptosis was investigated by cell cytotoxicity assay using MTT, cell viability assay using cell LIVE/DEAD cell viability assay, haematoxylin & eosin staining, nuclear staining, fluorescence-activated cell sorting, western blotting using specific antibodies associated with extrinsic and intrinsic apoptosis pathways, and caspase-3/-7 activity assay in FaDu cells. RESULTS: 25-HC dose-dependently decreased the viability of FaDu cells and up-regulated apoptotic events, such as alteration in morphology, and nuclear condensation. Flow cytometric analysis showed an increase in apoptotic population upon 25-HC treatment, suggesting that 25-HC induces apoptosis in FaDu cells. Moreover, 25-HC-induced apoptosis in FaDu cells was dependent on the activation of caspases by Fas antigen ligand-triggered death receptor-mediated extrinsic pathway and mitochondria-dependent intrinsic pathway via mitogen activated protein kinases. CONCLUSION: Cholesterol-derived oxysterol, 25-HC has potential anti-cancer function in FaDu cells and may have potential properties for the discovery of anti-cancer agents.

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.

A Comparative Study with Biphasic Calcium Phosphate to Deproteinized Bovine Bone in Maxillary Sinus Augmentation: A Prospective Randomized and Controlled Clinical Trial.

PURPOSE: The purpose of this study was to evaluate a new graft material, biphasic calcium phosphate, composed of 60% hydroxyapatite and 40% ß-Tricalcium phosphate and deproteinized bovine bone mineral, which is established as a predictable graft material for maxillary sinus augmentation. MATERIALS AND METHODS: Maxillary sinus augmentation was performed with different bone materials. Bone biopsies were performed on tissue harvested from the future implant bed using a trephine bur at 6 months after maxillary sinus augmentation. Resonance frequency analysis was performed immediately and at 6 months after the implant placement. Microcomputed tomography and histomorphometric analysis were performed in all patients. RESULTS: Fifty-six patients (60 sinuses) were included in the study. At 6 months postoperative, 31 biopsies were performed on tissues harvested from the calcium phosphate, and 29 biopsies on tissues from the bovine bone grafts. There was no implant failure during the 21-month mean follow-up period. The overall implant stability quotient values were higher than 60, and gradually increased for 6 months. Higher new bone volume fraction and new bone surface density were observed in the calcium phosphate group compared with the bovine bone group. In contrast, residual bone graft volume in the bovine bone group was higher than that in the calcium phosphate group. Nevertheless, there was no significant difference between groups in the microcomputed tomography and histomorphometric parameters. CONCLUSION: Within the study's limitations, both graft materials demonstrated similar biocompatibility and osteoconductivity in the maxillary sinus augmentation.

Phenformin Induces Caspase-dependent Apoptosis of FaDu Head and Neck Squamous Cell Carcinoma Cells.

BACKGROUND/AIM: The present study aimed to investigate the apoptotic effects of phenformin, a therapeutic agent for diabetes, on head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: Cytotoxicity was measured by the MTT and live/dead cell assay. Phenformin-induced apoptotic FaDu cell death and its associated cellular signaling pathways were investigated by hematoxylin and eosin staining, 4',6-diamidino-2-phenylindole staining, caspase-3 activity assay, fluorescence-activated cell sorting analysis, and western blotting. RESULTS: Phenformin promoted death of and apoptotic processes in FaDu cells, including morphological alterations and nuclear condensation. Furthermore, treatment with phenformin increased caspase-3 activity and apoptotic populations via the caspase cascade through cleavage of capspase-8, -9, and -3 and poly(ADP-ribose) polymerase in FaDu cells. Moreover, phosphorylation levels of mitogen-activated protein kinases, nuclear factor-κB, and AKT were down-regulated in FaDu cells by phenformin. CONCLUSION: Phenformin induced death of FaDu cells via caspase-dependent extrinsic and intrinsic apoptosis pathways and is a promising novel therapeutic agent for HNSCC.

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.

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.

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.

Berberine-induced anticancer activities in FaDu head and neck squamous cell carcinoma cells.

In the present study, we investigated berberine­induced apoptosis and the signaling pathways underlying its activity in FaDu head and neck squamous cell carcinoma cells. Berberine did not affect the viability of primary human normal oral keratinocytes. In contrast, the cytotoxicity of berberine was significantly increased in FaDu cells stimulated with berberine for 24 h. Furthermore, berberine increased nuclear condensation and apoptosis rates in FaDu cells than those in untreated control cells. Berberine also induced the upregulation of apoptotic ligands, such as FasL and TNF-related apoptosis-inducing ligand, and triggered the activation of caspase-8, -7 and -3, and poly(ADP ribose) polymerase, characteristic of death receptor-dependent extrinsic apoptosis. Moreover, berberine activated the mitochondria­dependent apoptotic signaling pathway by upregulating pro-apoptotic factors, such as Bax, Bad, Apaf-1, and the active form of caspase-9, and downregulating anti-apoptotic factors, such as Bcl-2 and Bcl-xL. In addition, berberine increased the expression of the tumor suppressor p53 in FaDu cells. The pan-caspase inhibitor Z-VAD-fmk suppressed the activation of caspase-3 and prevented cytotoxicity in FaDu cells treated with berberine. Interestingly, berberine suppressed cell migration through downregulation of vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, and MMP-9. Moreover, the phosphorylation of extracellular signal-regulated kinase (ERK1/2) and p38, components of the mitogen-activated protein kinase pathway that are associated with the expression of MMP and VEGF, was suppressed in FaDu cells treated with berberine for 24 h. Therefore, these data suggested that berberine exerted anticancer effects in FaDu cells through induction of apoptosis and suppression of migration. Berberine may have potential applications as a chemotherapeutic agent for the management of head and neck 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.