Autophagy inhibition by cudraxanthone D regulates epithelial–mesenchymal transition in SCC25 cells

Cudraxanthone D (CD) is a natural xanthone compound derived from the root barks of Cudrania tricuspidata . However, the biological functions of CD in human metabolism have been rarely reported until now. Autophagy is the self-degradation process related to cancer cell metastasis. Here, we elucidated the effects of CD on human oral squamous cell carcinoma (OSCC) cells’ metastatic ability. We confirmed that CD effectively decreased the proliferation and viability of SCC25 human OSCC cells in time- and dose-dependent manners. Also, the metastasis phenotype of the SCC25 cell (migration, invasion, and epithelial–mesenchymal transition [EMT]) was inhibited by CD. To further investigate the mechanism by which CD inhibited the metastatic capacity, we detected the relationship between EMT and autophagy in the SCC25 cells. The results revealed that CD inhibited the metastasis of the SCC25 cells by attenuating autophagy. Thus, our findings produced a potential novel agent for the treatment of human OSCC metastasis.

Autophagy inhibition by cudraxanthone D regulates epithelial–mesenchymal transition in SCC25 cells

Cudraxanthone D (CD) is a natural xanthone compound derived from the root barks of Cudrania tricuspidata . However, the biological functions of CD in human metabolism have been rarely reported until now. Autophagy is the self-degradation process related to cancer cell metastasis. Here, we elucidated the effects of CD on human oral squamous cell carcinoma (OSCC) cells’ metastatic ability. We confirmed that CD effectively decreased the proliferation and viability of SCC25 human OSCC cells in time- and dose-dependent manners. Also, the metastasis phenotype of the SCC25 cell (migration, invasion, and epithelial–mesenchymal transition [EMT]) was inhibited by CD. To further investigate the mechanism by which CD inhibited the metastatic capacity, we detected the relationship between EMT and autophagy in the SCC25 cells. The results revealed that CD inhibited the metastasis of the SCC25 cells by attenuating autophagy. Thus, our findings produced a potential novel agent for the treatment of human OSCC metastasis.

Anticancer effects of D-pinitol in human oral squamous carcinoma cells

D-pinitol is an analog of 3-methoxy-D-chiro-inositol found in beans and plants. D-pinitol has anti-inflammatory, antidiabetic, and anticancer effects. Additionally, D-pinitol induces apoptosis and inhibits metastasis in breast and prostate cancers. However, to date, no study has investigated the anticancer effects of D-pinitol in oral cancer. Therefore, in this study, whether the anticancer effects of D-pinitol induce apoptosis, inhibit the epithelialto-mesenchymal transition (EMT), and arrest cell cycle was investigated in squamous epithelial cells. D-pinitol decreased the survival and cell proliferation rates of CAL-27 and Ca9-22 oral squamous carcinoma cells in a concentration- and time-dependent manner. Evidence of apoptosis, including nuclear condensation, poly (ADP-ribose) polymerase, and caspase-3 fragmentation, was also observed. D-pinitol inhibited the migration and invasion of both cell lines. In terms of EMT-related proteins, E-cadherin was increased, whereas N-cadherin, Snail, and Slug weredecreased. D-pinitol also decreased the expression of cyclin D1, a protein involved in the cell cycle, but increased the expression of p21, a cyclin-dependent kinase inhibitor. Hence, D-pinitol induces apoptosis and cell cycle arrest in CAL-27 and Ca9-22 cells, demonstrating an anticancer effect by decreasing the EMT.

Anticancer effects of D-pinitol in human oral squamous carcinoma cells

D-pinitol is an analog of 3-methoxy-D-chiro-inositol found in beans and plants. D-pinitol has anti-inflammatory, antidiabetic, and anticancer effects. Additionally, D-pinitol induces apoptosis and inhibits metastasis in breast and prostate cancers. However, to date, no study has investigated the anticancer effects of D-pinitol in oral cancer. Therefore, in this study, whether the anticancer effects of D-pinitol induce apoptosis, inhibit the epithelialto-mesenchymal transition (EMT), and arrest cell cycle was investigated in squamous epithelial cells. D-pinitol decreased the survival and cell proliferation rates of CAL-27 and Ca9-22 oral squamous carcinoma cells in a concentration- and time-dependent manner. Evidence of apoptosis, including nuclear condensation, poly (ADP-ribose) polymerase, and caspase-3 fragmentation, was also observed. D-pinitol inhibited the migration and invasion of both cell lines. In terms of EMT-related proteins, E-cadherin was increased, whereas N-cadherin, Snail, and Slug weredecreased. D-pinitol also decreased the expression of cyclin D1, a protein involved in the cell cycle, but increased the expression of p21, a cyclin-dependent kinase inhibitor. Hence, D-pinitol induces apoptosis and cell cycle arrest in CAL-27 and Ca9-22 cells, demonstrating an anticancer effect by decreasing the EMT.

Piperlongumine suppressed osteoclastogenesis in RAW264.7 macrophages

Piperlongumine (PL) is a natural product found in long pepper (Piper longum). The pharmacological effects of PL are well known, and it has been used for pain, hepatoprotection, and asthma in Oriental medicine. No studies have examined the effects of PL on bone tissue or bone-related diseases, including osteoporosis. The current study investigated for the first time the inhibitory effects of PL on osteoclast differentiation, bone resorption, and osteoclastogenesis-related factors in RAW264.7 macrophages stimulated by the receptor activator for nuclear factor-κB ligand (RANKL). Cytotoxicity was examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and osteoclast differentiation and bone resorption were confirmed by tartrate-resistant acid phosphatase (TRAP) staining and pit formation analysis. Osteoclast differentiation factors were confirmed by western blotting. PL exhibited toxicity in RAW264.7 macrophages, inhibiting osteoclast formation and bone resorption, in addition to inhibiting the expression of osteoclastogenesis-related factors, such as tumor necrosis factor receptor-associated factor 6 (TRAF6), c-Fos, and NFATc1, in RANKL-stimulated RAW264.7 macrophages. These findings suggest that PL is suitable for the treatment of osteoporosis, and it serves as a potential therapeutic agent for various bone diseases.

Combined effect of recombinant human bone morphogenetic protein-2 and low level laser irradiation on bisphosphonate-treated osteoblasts

OBJECTIVES: The purpose of this study was to evaluate the synergic effect of recombinant human bone morphogenetic protein-2 (rhBMP-2) and low-level laser therapy (LLLT) on bisphosphonate-treated osteoblasts. MATERIALS AND METHODS: Human fetal osteoblast cells (hFOB 1.19) were cultured with 100 µM alendronate. Low-level Ga-Al-As laser alone or with 100 ng/mL rhBMP-2 was then applied. Cell viability was measured with MTT assay. The expression levels of receptor activator of nuclear factor kappa-B ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG) were analyzed for osteoblastic activity inducing osteoclastic activity. Collagen type and transforming growth factor beta-1 were also evaluated for bone matrix formation. RESULTS: The results showed that rhBMP-2 and LLLT had a synergic effect on alendronate-treated osteoblasts for enhancing osteoblastic activity and bone matrix formation. Between rhBMP-2 and LLLT, rhBMP-2 exhibited a greater effect, but did not show a significant difference. CONCLUSION: rhBMP-2 and LLLT have synergic effects on bisphosphonate-treated osteoblasts through enhancement of osteoblastic activity and bone formation activity.

Combined Treatment with Low-Level Laser and rhBMP-2 Promotes Differentiation and Mineralization of Osteoblastic Cells under Hypoxic Stress

BACKGROUND: The aim of this study was to evaluate the combined effect of low-level laser treatment (LLLT) and recombinant human bone morphological protein-2 (rhBMP-2) applied to hypoxic-cultured MC3T3-E1 osteoblastic cells and to determine possible signaling pathways underlying differentiation and mineralization of osteoblasts under hypoxia. METHODS: MC3T3-E1 cells were cultured under 1% oxygen tension for 72 h. Cell cultures were divided into four groups: normoxia control, low-level laser (LLL) alone, rhBMP-2 combined with LLLT, and rhBMP-2 under hypoxia. Laser irradiation was applied at 0, 24, and 48 h. Cells were treated with rhBMP-2 at 50 ng/mL. Alkaline phosphatase activity was measured at 3, 7, and 14 days to evaluate osteoblastic differentiation. Cell mineralization was determined with Alizarin red S staining at 7 and 14 days. Western blot assays were performed to evaluate whether p38/protein kinase D (PKD) signaling was involved. RESULTS: The results indicate that LLLT and rhBMP-2 synergistically increased alkaline phosphatase (ALP) activity and mineralization. Western blot analyses showed that expression of type I collagen, runt-related transcription factor 2 (RUNX2), and Osterix (Osx), increased and expression of hypoxia-inducible factor 1-alpha (HIF-1α), decreased more in the LLLT and rhBMP-2 combined group than in the rhBMP-2 or LLL alone groups. Moreover, LLLT and rhBMP-2 stimulated p38 phosphorylation and rhBMP-2 and LLLT increased Prkd1 phosphorylation. CONCLUSION: Combined treatment with rhBMP-2 and LLL induced differentiation and mineralization of hypoxiccultured MC3T3-E1 osteoblasts by activating p38/PKD signaling in vitro.

Luteolin Induces Apoptosis via Mitochondrial Pathway and Inhibits Invasion and Migration of Oral Squamous Cell Carcinoma by Suppressing Epithelial-Mesenchymal Transition Induced Transcription Factors

Oral squamous cell carcinoma (OSCC) is the most common type of oral malignancy. Numerous therapies have been proposed for its cure. Research is continually being conducted to develop new forms of treatment as current therapies are associated with numerous side-effects. Luteolin, a common dietary flavonoid, has been demonstrated to possess strong anti-cancer activity against various human cancer cell lines. Nevertheless, research into luteolin-based anticancer activity against oral cancer remains scarce. Thus, the objective of this study was to assess the effect of luteolin as an anti-cancer agent. After treatment with luteolin, Ca9-22 and CAL-27 oral cancer cells showed condensed nuclei and enhanced apoptotic rate with evidence of mitochondria-mediated apoptosis. Epithelialmesenchymal transition (EMT) is closely related to tumor migration and invasion. Luteolin suppressed cancer cell invasion and migration in the current study. Elevated expression of E-cadherin, an adherens junction protein, was evident in both cell lines after luteolin treatment. Luteolin also significantly inhibited transcription factors (i.e., N-cadherin, Slug, Snail, Twist, and ZEB-1) that regulated expression of tumor suppressors such as E-cadherin based on Western blot analysis and quantitative PCR. Thus, luteolin could induce mitochondrial apoptosis and inhibit cancer cell invasion and migration by suppressing EMT-induced transcription factors.

Low-level laser therapy affects osseointegration in titanium implants: resonance frequency, removal torque, and histomorphometric analysis in rabbits

OBJECTIVES: The purpose of this study was to investigate the effects of low-level laser therapy (LLLT) with a diode gallium-aluminum-arsenide (Ga-Al-As) low-level laser device on the healing and attachment of titanium implants in bone. MATERIALS AND METHODS: Thirteen New Zealand white male rabbits weighing 3.0+/-0.5 kg were used for this study. Dental titanium implants (3.75 mm in diameter and 8.5 mm in length, US II RBM plus fixture; Osstem, Seoul, Korea) were implanted into both femurs of each rabbit. The rabbits were randomly divided into a LLLT group and a control group. The LLLT was initiated immediately after surgery and then repeated daily for 7 consecutive days in the LLLT group. Six weeks and 12 weeks after implantation, we evaluated and compared the osseointegration of the LLLT group and control group, using histomorphometric analysis, removal torque testing, and resonance frequency analysis (RFA). The results were statistically significant when the level of probability was 0.05 or less based on a non-parametric Mann-Whitney U-test. RESULTS: The implant survival rate was about 96%. Histologically and histomorphometrically, we observed that the titanium implants were more strongly attached in LLLT group than in control group. However, there was no significant difference between the LLLT group and control group in removal torque or RFA. CONCLUSION: Histologically, LLLT might promote cell-level osseointegration of titanium implants, but there was no statistically significant effects.

Anticancer Properties of Icariside II in Human Oral Squamous Cell Carcinoma Cells

OSCC is currently the most common malignancy of the head and neck, affecting tens of thousands of patients per year worldwide. Natural flavonoids from plants are potential sources for novel anti-cancer drugs. Icariin is the active ingredient of flavonol glycoside, which is derived from the medical plant Herba Epimedii. A metabolite of icariin, icariside II exhibits a variety of pharmacological actions, including anti-rheumatic, anti-depressant, cardiovascular protective, and immunomodulatory functions. However, the exact mechanism causing the apoptosis-inducing effect of icariside II in OSCC is still not fully understood. In the present study, we assessed the anti-cancer effect of icariside II in OSCC cell lines by measuring its effect on cell viability, cell proliferation, and mitochondria membrane potential (MMP). Icariside II treatment of OSCC cells resulted in a dose- and time-dependent decrease in cell viability. Hoechst staining indicated apoptosis in icariside II-treated HSC cells. Icariside II inhibited cell proliferation and induced apoptosis in HSC cells, with significant increases in all present parameters in HSC-4 cells. The results clearly suggested that icariside II induced apoptosis via activation of intrinsic pathways and caspase cascades in HSC-4 cell lines. The collective findings of the study suggested that Icariside II is a potential treatment for OSCC; in addition, the data could provide a basis for the development of a novel anti-cancer strategy.

Effect of low-level laser therapy on bisphosphonate-treated osteoblasts / 대한악안면성형재건외과학회지

BACKGROUND: This study investigates the effect of alendronate-treated osteoblasts, as well as the effect of low-level laser therapy (LLLT) on the alendronate-treated osteoblasts. Bisphosphonate decreases the osteoblastic activity. Various treatment modalities are used to enhance the bisphosphonate-treated osteoblasts; however, there were no cell culture studies conducted using a low-level laser. METHODS: Human fetal osteoblastic (hFOB 1.19) cells were treated with 50 μM alendronate. Then, they were irradiated with a 1.2 J/cm² low-level Ga-Al-As laser (λ = 808 ± 3 nm, 80 mW, and 80 mA; spot size, 1 cm²; NDLux, Seoul, Korea). The cell survivability was measured with the MTT assay. The three cytokines of osteoblasts, receptor activator of nuclear factor κB ligand (RANKL), osteoprotegerin (OPG), and macrophage colony-stimulating factor (M-CSF) were analyzed. RESULTS: In the cells treated with alendronate at concentrations of 50 μM and higher, cell survivability significantly decreased after 48 h (p < 0.05). After the applications of low-level laser on alendronate-treated cells, cell survivability significantly increased at 72 h (p < 0.05). The expressions of OPG, RANKL, and M-CSF have decreased via the alendronate. The RANKL and M-CSF expressions have increased, but the OPG was not significantly affected by the LLLT. CONCLUSIONS: The LLLT does not affect the OPG expression in the hFOB cell line, but it may increase the RANKL and M-CSF expressions, thereby resulting in positive effects on osteoclastogenesis and bone remodeling.

Synergistic Effects of Chios Gum Mastic Extract and Low Level Laser Therapy on Osteoblast Differentiation

In the present study, we evaluated the effect of CGM on osteogenic differentiation of cultured osteoblasts, and determined whether combination treatment with LLLT had synergistic effects on osteogenic differentiation. The results indicated that CGM promoted proliferation, differentiation, and mineralization of osteoblasts at the threshold concentration of 10 µg/ml; whereas, CGM showed cytotoxic properties at concentrations above 100 µg/ml. ALP activity and mineralization were increased at concentrations above 10 µg/ml. CGM in concentrations up to 10 µg/ml also increased the expression of osteoblast-activated factors including type I collagen, BMP-2, RUNX2, and Osterix. The CGM (50 µg/ml) and LLLT (80 mW for 15 sec) combination treatment group showed the highest proliferation levels, ALP activity, and mineralization ratios. The combination treatment also increased the levels of phosphorylated forms of p38, ATF2, PKD, ERK, and JNK. In addition, the osteoblast differentiation factors including type I collagen, BMP-2, RUNX2, and Osterix protein levels were clearly increased in the combination treatment group. These results suggested that the combination treatment of CGM and LLLT has synergistic effects on the differentiation and mineralization of osteoblastic cells.

Mechanism Underlying Shikonin-induced Apoptosis and Cell Cycle Arrest on SCC25 Human Tongue Squamous Cell Carcinoma Cell Line

Shikonin, a major ingredient in the traditional Chinese herb Lithospermumerythrorhizon, exhibits multiple biological functions including antimicrobial, anti-inflammatory, and antitumor effects. It has recently been reported that shikonin displays antitumor properties in many cancers. This study was aimed to investigate whether shikonin could inhibit oral squamous carcinoma cell (OSCC) growth via mechanisms of apoptosis and cell cycle arrest. The effects of shikonin on the viability and growth of OSCC cell line, SCC25 cells were assessed by MTT assay and clonogenic assays, respectively. Hoechst staining and DNA electrophoresis indicated that the shikonin-treated SCC25 cells were undergoing apoptosis. Western blotting, immunocytochemistry, confocal microscopy, flow cytometry, MMP activity, and proteasome activity also supported the finding that shikonin induces apoptosis. Shikonin treatment of SCC25 cells resulted in a time- and dose-dependent decrease in cell viability, inhibition of cell growth, and increase in apoptotic cell death. The treated SCC25 cells showed several lines of apoptotic manifestation as follows: nuclear condensation; DNA fragmentation; reduced MMP and proteasome activity; decrease in DNA contents; release of cytochrome c into cytosol; translocation of AIF and DFF40 (CAD) onto the nuclei; a significant shift in Bax/Bcl-2 ratio; and activation of caspase-9, -7, -6, and -3, as well as PARP, lamin A/C, and DFF45 (ICAD). Shikonin treatment also resulted in down-regulation of the G1 cell cycle-related proteins and up-regulation of p27(KIP1). Taken together, our present findings demonstrate that shikonin strongly inhibits cell proliferation by modulating the expression of the G1 cell cycle-related proteins, and that it induces apoptosis via the proteasome, mitochondria, and caspase cascades in SCC25 cells.

Autophagy Inhibition Promotes Quercetin Induced Apoptosis in MG-63 Human Osteosarcoma cells

Quercetin is a natural flavonoid phytochemical that is extracted from various plants. Having an advantages due to its varied biological properties, such as anti-inflammatory, anti-viral, anti-oxidant, and anti-cancer effects, quercetin is used to treat many diseases. Recently, it has been reported that autophagy inhibition may play a key role in anti-cancer therapy. Therefore, in this study, we investigated the molecular mechanisms and anti-cancer effects of quercetin in human osteosarcoma cells via autophagy inhibition. We ascertained that quercetin inhibited cell proliferation and induced cell death, these process is demonstrated that apoptosis via the mitochondrial pathway and the caspase cascade. Quercetin also induced autophagy which was inhibited by 3-MA, autophagy inhibitor and the blockade of autophagy promoted the quercetin-induced apoptosis, confirming that autophagy is a pro-survival process. Thus, these findings demonstrate that quercetin is an effective anti-cancer agent, and the combination of quercetin and an autophagy inhibitor should enhance the effect of anti-cancer therapy.

Combined effect of bisphosphonate and recombinant human bone morphogenetic protein 2 on bone healing of rat calvarial defects / 대한악안면성형재건외과학회지

PURPOSE: This study aimed to investigate new bone formation using recombinant human bone morphogenetic protein 2 (rhBMP-2) and locally applied bisphosphonate in rat calvarial defects. METHODS: Thirty-six rats were studied. Two circular 5 mm diameter bony defect were formed in the calvaria using a trephine bur. The bony defect were grafted with Bio-Oss(R) only (group 1, n = 9), Bio-Oss(R) wetted with rhBMP-2 (group 2, n = 9), Bio-Oss(R) wetted with rhBMP-2 and 1 mM alendronate (group 3, n = 9) and Bio-Oss(R) wetted with rhBMP-2 and 10 mM alendronate (group 4, n = 9). In each group, three animals were euthanized at 2, 4 and 8 weeks after surgery, respectively. The specimens were then analyzed by histology, histomorphometry and immunohistochemistry analysis. RESULTS: There were significant decrease of bone formation area (p < 0.05) between group 4 and group 2, 3. Group 3 showed increase of new bone formation compared to group 2. In immunohistochemistry, collagen type I and osteoprotegerin (OPG) didn't show any difference. However, receptor activator of nuclear factor kappaB ligand (RANKL) decreased with time dependent except group 4. CONCLUSION: Low concentration bisphosphonate and rhBMP-2 have synergic effect on bone regeneration and this is result from the decreased activity of RANKL of osteoblast.

The Inhibition of Oxidative Stress by Chios Gum Mastic is Associated with Autophagy

Chios Gum Mastic (CGM) is a natural resin extracted from the leaves of Pistacia lentiscus, a plant endemic to the Greek island of Chios. It has been used by traditional healers, and it has antibacterial, antifungal properties, and therapeutic benefits for the skin. The CGM reduces the formation of dental plaque and bacterial growth in oral saliva, and recent studies have demonstrated the role of antioxidant activity of CGM. Although CGM has been widely investigated, its protective effect against oxidative-damage to keratinocytes, as well as the relationship between CGM and autophagy, has not been investigated. The aim of this study was to assess the protective effect of CGM against H2O2-induced oxidative stress and to evaluate the autophagic features induced by CGM in human keratinocytes. The pretreatment with CGM significantly reduced apoptosis in H2O2-exposed HaCaT cells. It promoted the degradation of caspase-3, caspase-8, and caspase-9; and it induced the formation of the processed PARP. The treatment with CGM caused an increase in vesicle formation compared to control group. The level of p62 was reduced and the conversion of LC3-I to LC3-II was increased in CGM treated HaCaT cells. Also, the treatment with CGM increased cleavage of ATG5-ATG12 complex. In summary, CGM helps the cells to survive under stressful conditions by preventing apoptosis and enhancing autophagy. Besides, the present investigation provides evidence to support the antioxidant potential of CGM in vitro and opens up a new horizon for future experiments.

Protective Effect of Propofol against Hypoxia-reoxygenation Injury in HaCaT Human Keratinocytes

The aim of this study was to determine the beneficial effect of propofol on human keratinocytes that have undergone hypoxia reoxygenation (H/R) injury and to investigate whether autophagy is associated with the protective mechanism. Thus, we evaluated how propofol influences the intracellular autophagy and apoptosis during the H/R process in the HaCaT cells. The cultured human keratinocyte cells were exposed to 24 h of hypoxia (5% CO2, 1% O2, 94% N2) followed by 12 h of reoxygenation (5% CO2, 21% O2, 74% N2). The experiment was divided into 4 groups: (1) Control=Normoxia ; (2) H/R=Hypoxia Reoxygenation ; (3) PPC+H/R=Propofol Preconditioning+Hypoxia Reoxygenation; (4) 3-MA+PPC+H/R=3-MA-Methyladenine+Propofol Preconditioning+Hypoxia Reoxygenation. In addition, Western blot analysis was performed to identify the expression of apoptotic pathway parameters, including Bcl-2, Bax, and caspase 3 involved in mitochondrial-dependent pathway. Autophagy was determined by fluorescence microscopy, MDC staining, AO staining, and western blot. The H/R produced dramatic injuries in keratinocyte cells. In our study, the viability of Propofol in H/R induced HaCaT cells was first studied by MTT assay. The treatment with 25, 50, and 100 microM Propofol in H/R induced HaCaT cells enhanced cell viability in a dose-dependent manner and 100 microM was the most effective dose. The Atg5, Becline-1, LC3-II, and p62 were elevated in PPC group cells, but H/R-induced group showed significant reduction in HaCaT cells. The Atg5 were increased when autophagy was induced by Propofol, and they were decreased when autophagy was suppressed by 3-MA. These data provided evidence that propofol preconditioning induced autophagy and reduced apoptotic cell death in an H/R model of HaCaT cells, which was in agreement with autophagy playing a very important role in cell protection.

Effect of recombinant human bone morphogenetic protein-2 on bisphosphonate-treated osteoblasts

OBJECTIVES: Bisphosphonate-related osteonecrosis of the jaw (BRONJ) is a side effect of bisphophonate therapy that has been reported in recent years. Osteoclastic inactivity by bisphosphonate is the known cause of BRONJ. Bone morphogenetic protein-2 (BMP-2) plays an important role in the development of bone. Recombinant human BMP-2 (rhBMP-2) is potentially useful as an activation factor for bone repair. We hypothesized that rhBMP-2 would enhance the osteoclast-osteoblast interaction related to bone remodeling. MATERIALS AND METHODS: Human fetal osteoblast cells (hFOB 1.19) were treated with 100 microM alendronate, and 100 ng/mL rhBMP-2 was added. Cells were incubated for a further 48 hours, and cell viability was measured using an MTT assay. Expression of the three cytokines from osteoblasts, receptor activator of nuclear factor-kappaB ligand (RANKL), osteoprotegerin (OPG), and macrophage colony-stimulating factor (M-CSF), were analyzed by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. RESULTS: Cell viability was decreased to 82.75%+/-1.00% by alendronate and then increased to 110.43%+/-1.35% after treatment with rhBMP-2 (P<0.05, respectively). OPG, RANKL, and M-CSF expression were all decreased by alendronate treatment. RANKL and M-CSF expression were increased, but OPG was not significantly affected by rhBMP-2. CONCLUSION: rhBMP2 does not affect OPG gene expression in hFOB, but it may increase RANKL and M-CSF gene expression.

Apoptotic Effect of Co-treatment with Curcumin and Cisplatin on SCC25 Human Tongue Squamous Cell Carcinoma Cell Line

Curcumin is a widely used flavoring agent in food, and it has been reported to inhibit cell growth, to induce apoptosis, and to have antitumor activity in many cancers. Cisplatin is one of the most potent known anticancer agents and shows significant clinical activity against a variety of solid tumors. This study was undertaken to investigate the synergistic apoptotic effects of co-treatment with curcumin and cisplatin on human tongue SCC25 cells. To investigate whether the co-treatment efficiently reduced the viability of the SCC25 cells compared with the two treatments separately, an MTT assay was conducted. The induction and the augmentation of apoptosis were confirmed by DNA electrophoresis, Hoechst staining, and an analysis of DNA hypoploidy. Western blot, MMP and immunofluorescence tests were also performed to evaluate the expression levels and the translocation of apoptosis-related proteins following the co-treatment. In this study, following the co-treatment with curcumin and cisplatin, the SCC25 cells showed several forms of apoptotic manifestation, such as nuclear condensation, DNA fragmentation, reduction of MMP, increased levels of Bax, decreased levels of Bcl-2, and decreased DNA content. In addition, they showed a release of cytochrome c into the cytosol, translocation of AIF and DFF40 (CAD) to the nuclei, and activation of caspase-7, caspase-3, PARP, and DFF45 (ICAD). In contrast, separate treatments of 5 microM of curcumin or 4 microg/ml of cisplatin, for 24 hours, did not induce apoptosis. Therefore, our data suggest that combination therapy with curcumin and cisplatin could be considered as a novel therapeutic strategy for human oral squamous cell carcinoma.

NaF-induced Autophagy on SCC25 Human Tongue Squamous Cell Carcinoma Cells

Fluoride has been accepted as an important material for oral health and is widely used to prevent dental caries in dentistry. However, its safety is still questioned by some. Autophagy has been implicated in cancer cell survival and death, and may play an important role in oral cancer. This study was undertaken to examine whether sodium fluoride (NaF) modulates autophagy in SCC25 human tongue squamous cell carcinoma cells. NaF demonstrated anticancer activity via autophagic and apoptotic cell death. Autophagic vacuoles were detectable using observed to form by monodansylcadaverine (MDC) and acridine orange (AO). Analysis of NaF-treated SCC25 cells for the presence of biochemical markers revealed direct effects on the conversion of LC-3II, degradation of p62/SQSTM1, cleavage formation of ATG5 and Beclin-1, and caspase activation. NaF-induced cell death was suppressed by the autophagy inhibitor 3-methyladenine (3-MA). NaF-induced autophagy was confirmed as a pro-death signal in SCC25 cells. These results implicate NaF as a novel anticancer compound for oral cancer therapy.