Isolation of Pro-Osteogenic Compounds from Euptelea polyandra That Reciprocally Regulate Osteoblast and Osteoclast Differentiation.

Plants contain a large number of small-molecule compounds that are useful for targeting human health and in drug discovery. Healthy bone metabolism depends on the balance between bone-forming osteoblast activity and bone-resorbing osteoclast activity. In an ongoing study searching for 22 plant extracts effective against osteoporosis, we found that the crude extract of Euptelea polyandra Sieb. et Zucc (E. polyandra) had osteogenic bioactivity. In this study, we isolated two compounds, isoquercitrin (1) and astragalin (2), responsible for osteogenic bioactivity in osteoblastic MC3T3-E1 cells from the leaf of E. polyandra using column chromatography and the spectroscopic technique. This is the first report to isolate astragalin from E. polyandra. Compounds (1) and (2) promoted osteoblast differentiation by increasing alkaline phosphatase (ALP) activity and alizarin red S stain-positive calcium deposition, while simultaneously suppressing tartrate-resistant acid phosphatase (TRAP)-positive osteoclast differentiation in RAW264.7 cells at non-cytotoxic concentrations. Isoquercitrin (1) and astragalin (2) increased the expression of osteoblastic differentiation genes, Osterix, ALP, and Osteoprotegerin in the MC3T3-E1 cells, while suppressing osteoclast differentiation genes, TRAP, Cathepsin K, and MMP 9 in the RAW264.7 cells. These compounds may be ideal targets for the treatment of osteoporosis due to their dual function of promoting bone formation and inhibiting bone resorption.

Potentiation of Anticancer Activity of G2/M Blockers by Mild Hyperthermia.

BACKGROUND/AIM: Hyperthermia (HT), combined with chemotherapy, has been used to treat various types of cancer. This study aimed to investigate the HT-sensitivity of malignant and non-malignant cells, and then evaluate the combination effect of docetaxel (DTX) and a newly synthesized chromone derivative (compound A) with HT. MATERIALS AND METHODS: The number of viable cells was determined using the MTT method. Cell cycle distribution was analyzed using a cell sorter, and DNA fragmentation pattern was detected using agarose gel electrophoresis. RESULTS: Among 12 cultured cells, oral squamous cell carcinoma (OSCC), especially Ca9-22 cells, and myelogenous leukemia cells showed higher sensitivity to HT than lung carcinoma and glioblastoma cell lines, while normal oral cells were the most resistant. Cytotoxicity of DTX on Ca9-22 cells was maximum at 41-42°C and 45~60 min exposure to HT. DXT, compound A, and HT induced G2/M arrest of Ca-22 cells. Mild HT enhanced the DTX- and compound A-induced subG1 arrest, in a synergistic fashion. CONCLUSION: The combination G2/M blockers and mild-HT can potentially be used for the treatment of OSCC.

Lemur tail kinase 1 (LMTK1) regulates the endosomal localization of ß-secretase BACE1.

Lemur tail kinase 1 (LMTK1), previously called apoptosis-associated tyrosine kinase (AATYK), is an endosomal Ser/Thr kinase. We recently reported that LMTK1 regulates axon outgrowth, dendrite arborization and spine formation via Rab11-mediated vesicle transport. Rab11, a small GTPase regulating recycling endosome trafficking, is shown to be associated with late-onset Alzheimer's disease (LOAD). In fact, genome-wide association studies identified many proteins regulating vesicle transport as risk factors for LOAD. Furthermore, LMTK1 has been reported to be a risk factor for frontotemporal dementia. Then, we hypothesized that LMTK1 contributes to AD development through vesicle transport and examined the effect of LMTK1 on the cellular localization of AD-related proteins, amyloid precursor protein (APP) and ß-site APP cleaving enzyme 1 (BACE1). The ß-cleavage of APP by BACE1 is the initial and rate-limiting step in Aß generation. We found that LMTK1 accumulated BACE1, but not APP, to the perinuclear endosomal compartment, whereas the kinase-negative(kn) mutant of LMTK1A did not. The ß-C-terminal fragment was prone to increase under overexpression of LMTK1A kn. Moreover, the expression level of LMTK1A was reduced in AD brains. These results suggest the possibility that LMTK1 is involved in AD development through the regulation of the proper endosomal localization of BACE1.

Purification and Biological Function of Caldecrin.

Blood calcium homeostasis is critical for biological function. Caldecrin, or chymotrypsin-like elastase, was originally identified in the pancreas as a serum calcium-decreasing factor. The serum calcium-decreasing activity of caldecrin requires the trypsin-mediated activation of the protein. Protease activity-deficient mature caldecrin can also reduce serum calcium concentration, indicating that structural processing is necessary for serum calcium-decreasing activity. Caldecrin suppresses the differentiation of bone-resorbing osteoclasts from bone marrow macrophages (BMMs) by inhibiting receptor activator of NF-κB ligand (RANKL)-induced nuclear factor of activated T-cell cytoplasmic 1 expression via the Syk-PLCγ-Ca2+ oscillation-calcineurin signaling pathway. It also suppresses mature osteoclastic bone resorption by RANKL-stimulated TRAF6-c-Src-Syk-calcium entry and actin ring formation. Caldecrin inhibits lipopolysaccharide (LPS)-induced osteoclast formation in RANKL-primed BMMs by inducing the NF-κB negative regulator A20. In addition, caldecrin suppresses LPS-mediated M1 macrophage polarization through the immunoreceptor triggering receptor expressed on myeloid cells (TREM) 2, suggesting that caldecrin may function as an anti-osteoclastogenic and anti-inflammatory factor via TREM2. The ectopic intramuscular expression of caldecrin cDNA prevents bone resorption in ovariectomized mice, and the administration of caldecrin protein also prevents skeletal muscle destruction in dystrophic mice. In vivo and in vitro studies have indicated that caldecrin is a unique multifunctional protease and a possible therapeutic target for skeletal and inflammatory diseases.

Hyperactive and impulsive behaviors of LMTK1 knockout mice.

Lemur tail kinase 1 (LMTK1), previously called Apoptosis-Associated Tyrosine Kinase (AATYK), remains an uncharacterized Ser/Thr protein kinase that is predominantly expressed in the brain. It is recently reported that LMTK1A, an isoform of LMTK1, binds to recycling endosomes through its palmitoylation and regulates endosomal trafficking by suppressing the activity of Rab11 small GTPase. In neurons, knockdown or knockout of LMTK1 results in longer axons, greater branching of dendrites and increased number of spines, suggesting that LMTK1 plays a role in neuronal circuit formation. However, its in vivo function remained to be investigated. Here, we examined the brain structures and behaviors of LMTK1 knockout (KO) mice. LMTK1 was expressed in most neurons throughout the brain. The overall brain structure appeared to be normal in LMTK1 KO mice, but the numbers of synapses were increased. LMTK1 KO mice had a slight impairment in memory formation and exhibited distinct psychiatric behaviors such as hyperactivity, impulsiveness and high motor coordination without social interaction deficits. Some of these abnormal behaviors represent core features of attention deficit hyperactive disorder (ADHD), suggesting the possible involvement of LMTK1 in the pathogenesis of ADHD.

LMTK1, a Novel Modulator of Endosomal Trafficking in Neurons.

Neurons extend long processes known as axons and dendrites, through which they communicate with each other. The neuronal circuits formed by the axons and dendrites are the structural basis of higher brain functions. The formation and maintenance of these processes are essential for physiological brain activities. Membrane components, both lipids, and proteins, that are required for process formation are supplied by vesicle transport. Intracellular membrane trafficking is regulated by a family of Rab small GTPases. A group of Rabs regulating endosomal trafficking has been studied mainly in nonpolarized culture cell lines, and little is known about their regulation in polarized neurons with long processes. As shown in our recent study, lemur tail (former tyrosine) kinase 1 (LMTK1), an as yet uncharacterized Ser/Thr kinase associated with Rab11-positive recycling endosomes, modulates the formation of axons, dendrites, and spines in cultured primary neurons. LMTK1 knockdown or knockout (KO) or the expression of a kinase-negative mutant stimulates the transport of endosomal vesicles in neurons, leading to the overgrowth of axons, dendrites, and spines. More recently, we found that LMTK1 regulates TBC1D9B Rab11 GAP and proposed the Cdk5/p35-LMTK1-TBC1D9B-Rab11 pathway as a signaling cascade that regulates endosomal trafficking. Here, we summarize the biochemical, cell biological, and physiological properties of LMTK1.

Augmentation of Neurotoxicity of Anticancer Drugs by X-Ray Irradiation.

BACKGROUND: In order to investigate the combination effect of anticancer drugs and X-ray irradiation on neurotoxic side-effects (neurotoxicity), a method that provides homogeneously X-ray-irradiated cells was newly established. MATERIALS AND METHODS: PC12 cell suspension was irradiated by X-ray (0.5 Gy) in serum-supplemented medium, immediately inoculated into 96-microwell plates and incubated overnight. The medium was replaced with fresh serum-depleted medium containing 50 ng/ml nerve growth factor to induce differentiation toward nerve-like cells with characteristic neurites according to the overlay method without changing the medium. The differentiated cells were treated by anticancer drugs as well as antioxidants, oxaliplatin or bortezomib, and the viable cell number was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. RESULTS: Antioxidants and anticancer drugs were cytotoxic to differentiating PC12 cells. Combination of anticancer drugs and X-ray irradiation slightly reduced cell viability. CONCLUSION: The present 'population irradiation method' may be useful for the investigation of the combination effect of X-ray irradiation and any pharmaceutical drug.

Isoform-dependent subcellular localization of LMTK1A and LMTK1B and their roles in axon outgrowth and spine formation.

Lemur kinase 1 (LMTK1) is a membrane-bound Ser/Thr kinase that is expressed in neurons. There are two splicing variants of LMTK1 with different membrane binding modes, viz., cytosolic LMTK1A that binds to membranes through palmitoylation at the N-terminal cysteines and LMTK1B, an integral membrane protein with transmembrane sequences. We recently reported that LMTK1A regulates axon outgrowth and spine formation in neurons. However, data about LMTK1B are scarce. We analysed the expression and cellular localization of LMTK1B along with its role in axon and spine formation. We found that both LMTK1B and LMTK1A were expressed equally in the cerebral cortex and cerebellum of the mouse brain. Similar to LMTK1A, the wild type of LMTK1B was localized to Rab11-positive pericentrosomal compartment. The kinase negative (kn) mutant of LMTK1B was found to be associated with an increase in the tubular form of endoplasmic reticulum (ER), which was not the case with LMTK1A kn. Furthermore, unlike LMTK1A kn, LMTK1B kn did not stimulate the axon outgrowth and spine formation. These results suggest that while LMTK1A and LMTK1B share a common function in recycling endosomal trafficking at the pericentrosomal compartment, LMTK1B has an additional unique function in vesicle transport in the ER region.

Caldecrin inhibits lipopolysaccharide-induced pro-inflammatory cytokines and M1 macrophage polarization through the immunoreceptor triggering receptor expressed in myeloid cells-2.

Caldecrin was previously isolated as a serum calcium-decreasing factor from the pancreas and is known to suppress receptor activator of nuclear factor-κB ligand (RANKL)-induced calcium oscillation pathways in osteoclasts. Here, we explored the effects of caldecrin on lipopolysaccharide (LPS)-Toll-like receptor-4 (TLR-4) signaling pathways in macrophages. Caldecrin inhibited the LPS-induced gene expression of pro-inflammatory cytokines and M1 macrophage polarization in mouse bone marrow macrophages and the RAW264.7 mouse macrophage cell line. Next, we focused on triggering receptor expressed in myeloid cells-2 (TREM-2) as a co-receptor common to RANKL receptor and TLR-4, and established Trem2-KO RAW264.7 cells, in which Trem2 gene was deleted using the CRISPR/Cas9 system. Caldecrin-mediated alterations in pro-inflammatory cytokine expression and M1 macrophage polarization were not observed in Trem2-KO RAW264.7 cells. These results suggest that caldecrin is not only an inhibitor of osteoclast activation but also a negative regulator of LPS-induced inflammatory responses, functioning via TREM-2.

Further Quantitative Structure-Cytotoxicity Relationship Analysis of 3-Styrylchromones.

BACKGROUND/AIM: Very few studies are available about the biological activity of 3-styrylchromones. Our previous study demonstrated the importance of methoxy group at 6-position of the chromone ring and hydroxyl group at 4'-position of phenyl group in styryl moiety. As a sequel of this study, we synthesized fourteen compounds that include eight 3-styrylchromones where methoxy group was introduced at 7-position of chromone rings, and then evaluated their tumor-specificity. MATERIALS AND METHODS: Tumor-specificity (TS) was calculated by relative cytotoxicity against human oral squamous cell carcinoma cell lines versus human normal oral cells. Apoptosis induction and growth arrest were monitored by cell-cycle analysis. Quantitative structure-activity relationship analysis of TS was performed with 3,167 chemical descriptors. RESULTS AND DISCUSSION: Two compounds, 7-methoxy-3-[(1E)-2-phenylethenyl]-4H-1-benzopyran-4-one [7] and 3-[(1E)-2-(4-hydroxyphenyl)ethenyl]-7-methoxy-4H-1-benzopyran-4-one [14] showed higher tumor-specificity than doxorubicin and 5-FU, suggesting the importance of methoxy group in 7-position of the chromone ring. These compounds induced the apoptosis and mitotic arrest in HSC-2 cells. The tumor-specificity of 3-styrylchromone derivatives were most correlated with descriptors for molecule shape and electronic charge. The present study suggested that modification by introducing methoxy group at 7-position, instead at 6-position, further increased the tumor-specificity of 3-styrylchromone.

Quantitative Structure-Cytotoxicity Relationship of 2-Arylazolylchromones and 2-Triazolylchromones.

BACKGROUND/AIM: 4H-1-Benzopyran-4-one (chromone), present in various flavonoids as a backbone structure, has been used for the synthesis of anticancer drugs. The study aimed at investigating the cytotoxicity of eight 2-arylazolylchromones and twelve 2-triazolylchromones against four human oral squamous cell carcinoma (OSCC) cell lines and three human normal mesenchymal oral cells, and then performed a quantitative structure-activity relationship (QSAR) analysis. MATERIALS AND METHODS: Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. The distribution of cells to various phases of cell cycle was determined by cell cycle analysis. A total of 3,218 physicochemical, structural and quantum chemical features were calculated for QSAR analysis from the most stabilized structure optimized using CORINA. RESULTS: 2-[4-(4-fluorophenyl)-1H-imidazol-1-yl]-4H-1-benzopyran-4-one [6] had the highest tumor-specificity (TS), comparable with that of 5-flurouracil (5-FU) and doxorubicin, inducing cytostatic growth inhibition, accumulation of G2+M phase cells with no cells in the G1 phase. All eight 2-triazolylchromones showed much lower tumor-specificity, confirming our previous finding. Tumor-specificity was also correlated with 3D shape, topological shape, size, ionization potential, and the presence of more than two aromatic rings in the molecule and imidazole ring in the nitrogen-containing heterocyclic ring. CONCLUSION: [6] can be a lead compound for designing anticancer drugs.

Quantitative Structure-Cytotoxicity Relationship of 2-Styrylchromones.

BACKGROUND/AIM: Studies of biological activity of 2-styrylchromone derivatives focusing on antioxidant, anti-inflammatory, antiviral and antitumor activity are limited. In this study, eighteen synthetic 2-styrylchromone derivatives were investigated for their cytotoxicity against human malignant and non-malignant cells, and then subjected to quantitative structure-activity relationship (QSAR) analysis. MATERIALS AND METHODS: Tumor-specificity was calculated by the ratio of mean 50% cytotoxic concentration (CC50) against four normal oral cells to that against oral squamous cell carcinoma cell lines. Induction of apoptosis and growth arrest were evaluated by cell-cycle analysis. For QSAR analysis, 3,117 types of physicochemical, structural, and quantum chemical features were calculated from the most stabilized structure of 2-styrylchromone derivatives. RESULTS: Two 2-styrylchromone derivatives in which a methoxy group was introduced at the 4-position of the benzene ring showed tumor-specificity equivalent to or higher than doxorubicin in TS value. These compounds accumulated the subG1 and G2/M phase cells, suggesting the induction of apoptosis. Their tumor-specificity can be explained mainly by molecular shape and electronic state. CONCLUSION: These findings suggest the applicability of 2-styrylchromone to develop safe and effective anticancer agents as seed compounds.

The LMTK1-TBC1D9B-Rab11A Cascade Regulates Dendritic Spine Formation via Endosome Trafficking.

Dendritic spines are postsynaptic protrusions at excitatory synapses that are critical for proper neuronal synaptic transmission. While lipid and protein membrane components are necessary for spine formation, it is largely unknown how they are recruited to developing spines. Endosomal trafficking is one mechanism that may influence this development. We recently reported that Lemur kinase 1A (LMTK1A), a membrane-bound Ser/Thr kinase, regulates trafficking of endosomes in neurons. LMTK1 has been shown to be a p35 Cdk5 activator-binding protein and a substrate for Cdk5-p35; however, its neuronal function has not been sufficiently studied. Here, we investigate the role of LMTK1 in spine formation. Depletion of LMTK1 increases spine formation, maturation, and density in primary cultured neurons and in mouse brain of either sex. Additionally, expression of kinase-negative LMTK1 stimulates spine formation in primary neurons and in vivo LMTK1 controls spine formation through Rab11, a regulator of recycling endosome trafficking. We identify TBC1D9B, a Rab11A GTPase-activating protein (Rab11A GAP), as a LMTK1 binding protein, and find that TBC1D9B mediates LMTK1 activity on Rab11A. TBC1D9B inactivates Rab11A under the control of LMTK1A. Further, by analyzing the effect of decreased TBC1D9B expression in primary neurons, we demonstrate that TBC1D9B indeed regulates spine formation. This is the first demonstration of the biological function of TBC1D9B. Together, with the regulation of LMTK1 by Cdk5-p35, we propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel signaling mechanism regulating endosomal transport for synapse formation and function.SIGNIFICANCE STATEMENT Dendritic spines are postsynaptic specializations essential for synaptic transmission. However, it is not known how critical membrane components are recruited to spines for their formation. Endosomal trafficking is one such mechanism that may mediate this process. Here we investigate regulators of endosomal trafficking and their contribution to spine formation. We identify two novel factors, LMTK1 and TBC1D9B, which regulate spine formation upstream of Rab11A, a small GTPase. LMTK1 is a membrane bound Ser/Thr kinase regulated by Cdk5-p35, and TBC1D9B is a recently identified Rab11 GAP. LMTK1 controls the GAP activity of TBC1D9B on Rab11A, and TBC1D9B mediates the LMTK1 activity on Rab11A. We propose the Cdk5-LMTK1-TBC1D9B-Rab11A cascade as a novel mechanism controlling spine formation and function.

Quantitative Structure-Cytotoxicity Relationship of Azulene Amide Derivatives.

BACKGROUND/AIM: Very few studies of anticancer activity of azulene amides led us to investigate the cytotoxicity of 21 N-alkylazulene-1-carboxamides introduced either with 3-methyl [1-7], 7-isopropyl-3-methyl [8-14] or 2-methoxy group [15-21] Materials and Methods: Tumor-specificity (TS) was calculated by the ratio of mean 50% cytotoxic concentration (CC50) against three normal human oral mesenchymal cells to that against four human oral squamous cell carcinoma (OSCC) cell lines. Potency-selectivity expression (PSE) was calculated by dividing TS value by CC50 value against OSCC cell lines. Apoptosis-inducing activity was evaluated by caspase-3 activation and appearance of subG1 cell population. RESULTS: [8-14] showed higher TS and PSE values, than [1-7] and [15-21] The most active compound [8-14] induced apoptosis in C9-22 OSCC cells at 4-times higher CC50 Quantitative structure-activity relationship analysis of [1-14] demonstrated that their tumor-specificity was correlated with chemical descriptors that explain the molecular shape and hydrophobicity. CONCLUSION: 7-Isopropyl-3-methyl-N-propylazulene-1-carboxamide [8] can be a potential candidate of lead compound for manufacturing new anticancer drug.

Screening for natural medicines effective for the treatment of osteoporosis.

Bone-forming osteoblasts are differentiated from mesenchymal stem cells and dysregulation of this differentiation can lead to osteoporosis. Meanwhile, bone-resorbing osteoclasts are both differentiated and multinucleated from hematopoietic precursor cells of monocyte and/or macrophage lineage. Bone resorption inhibitors such as bisphosphonates and estrogen are used to treat osteoporosis. However, the adverse effects of the long-term use of these medicines are of concern, and so the development of new therapies to ameliorate osteoporosis is desirable. Therefore, in the present study, we screened 22 plant extracts and found that nine methanolic extracts of plants promote the differentiation of MC3T3-E1 cells to osteoblasts. These nine extracts were then evaluated for their inhibitory activity on osteoclast differentiation in RAW264.7 mouse macrophage cells. Of the nine extracts, Daucus carota, Vitis spp., Sasa veitchii, Euptelea polyandra, and Sesamum indicum exhibited pro-osteoblastic and anti-osteoclastic activity with low cytotoxicity, suggesting their potential effectiveness against osteoporosis.

Quantitative Structure-Cytotoxicity Relationship of Pyrano[4,3-b]chromones.

BACKGROUND/AIM: 4H-1-Benzopyran-4-one (chromone) provides a backbone structure for the chemical synthesis of potent anticancer drugs. Since studies of the biological activity of pyrano[4,3-b]chromones are limited, we investigated a total of 20 pyrano[4,3-b]chromones (10 sets of diastereomers) for their cytotoxicity against four human oral squamous cell carcinoma (OSCC) cell lines and human normal oral cells, and then carried out a quantitative structure-activity relationship (QSAR) analysis. MATERIALS AND METHODS: Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Tumor-specificity (TS) was evaluated by the ratio of mean 50% cytotoxic concentration (CC50) against normal oral cells to that against human OSCC cell lines. Potency-selectivity expression (PSE) value was calculated by dividing the TS value by the CC50 against tumor cells. Apoptosis induction was evaluated by morphological observation, western blot analysis and cell-cycle analysis. For QSAR analysis, a total of 3,072 physicochemical, structural and quantum chemical features were calculated from the most stabilized structure optimized using CORINA. RESULTS: 8-Chloro-4,4a-dihydro-3-methoxy-3-methyl-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (16) and 3-ethoxy-4,4a-dihydro-8-methoxy-3H,10H-pyrano[4,3-b][1]benzopyran-10-one (17) had the highest TS, higher than that of 5-flurouracil and melphalan, without induction of apoptosis. Compound 16 induced cytostatic growth inhibition and much lower cytotoxicity against human normal oral keratinocytes compared to doxorubicin. TS of 20 pyrano[4,3-b]chromones was correlated with 3D structure, polarity, ionic potential and electric state. CONCLUSION: Chemical modification of 16 may be a potential choice for designing a new type of anticancer drug.

Quantitative Structure-Cytotoxicity Relationship of 3-(N-Cyclicamino)chromone Derivatives.

BACKGROUND/AIM: 4H-1-Benzopyran-4-ones (chromones) provide a backbone structure for the chemical synthesis of potent anticancer drugs. In contrast to 2-(N-cyclicamino)chromones, the biological activity of 3-(N-cyclicamino)chromones has not been reported. In this study, cytotoxicity of 15 3-(N-cyclicamino)chromone derivatives was investigated and subjected to quantitative structure-activity relationship (QSAR) analysis. MATERIALS AND METHODS: Cytotoxicity against four human oral squamous cell carcinoma cell lines and three oral normal mesenchymal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Tumor-specificity (TS) was evaluated as the ratio of mean 50% cytotoxic concentration (CC50) against normal oral cells to that against human oral squamous cell carcinoma cell lines. Potency-selectivity expression (PSE) value was calculated by dividing the TS value by the CC50 against tumor cells. Apoptosis induction was evaluated by morphological observation, western blot analysis and cell-cycle analysis. For QSAR analysis, a total of 3,096 physicochemical, structural and quantum chemical features were calculated from the most stabilized structure optimized using CORINA. RESULTS: 3-(4-phenyl-1-piperazinyl)-4H-1-benzopyran-4-one (3a) had the highest tumor specificity, comparable with that of melphalan, without induction of apoptosis. Compound 3a caused cytostatic growth inhibition and had much lower cytotoxicity against human oral keratinocytes compared to doxorubicin. TS of the 15 3-(N-cyclicamino)chromones was correlated with 3D structure and lipophilicity. CONCLUSION: Chemical modification of 3a may be a potential choice for designing a new type of anticancer drug.

Quantitative Structure-Cytotoxicity Relationship of 2-(N-cyclicamino)chromone Derivatives.

BACKGROUND/AIM: 4H-1-Benzopyran-4-ones (chromones) have provided backbone structure for the chemical synthesis of potent anticancer drugs. In this study, the cytotoxicity of fifteen 2-(N-cyclicamino)chromone derivatives was investigated and subjected to quantitative structure-activity relationship (QSAR) analysis. MATERIALS AND METHODS: Cytotoxicity against four human oral squamous cell carcinoma cell lines and three oral normal mesenchymal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Tumor specificity (TS) was evaluated by ratio of mean 50% cytotoxic concentration (CC50) against normal oral cells to that against human oral squamous cell carcinoma cell lines. Potency-selectivity expression (PSE) value was calculated by dividing the TS value by CC50 against tumor cells. Apoptosis induction was evaluated by morphological observation, western blot analysis and cell-cycle analysis. For QSAR analysis, a total of 3,089 physicochemicals, structural and quantum chemical features were calculated from the most stabilized structure optimized using Corina. RESULTS: 7-Methoxy-2-(4-morpholinyl)-4H-1-benzopyran-4-one (5c) showed highest tumor-specificity, comparable with that of doxorubicin, without inducing apoptosis. Tumor-specificity of fifteen 2-(N-cyclicamino) chromones was correlated with molecular shape, especially 3D-structure. CONCLUSION: Chemical modification of 5c may be a potential choice for designing a new type of anticancer drugs.

Search of Neuroprotective Polyphenols Using the "Overlay" Isolation Method.

Previous studies of the neuroprotective activity of polyphenols have used ununiform culture systems, making it difficult to compare their neuroprotective potency. We have established a new and simple method for preparing differentiated PC12 cells by removing the toxic coating step. Cells were induced to differentiate with the nerve growth factor (NGF) in a serum-free medium, without a medium change, but with a one-time overlay supplementation of NGF. The optimal inoculation density of the cells was 6⁻12 × 10³ cells/cm², and the presence of serum inhibited the differentiation. Neuroprotective activity could be quantified by the specific index (SI) value, that is, the ratio of the 50% cytotoxic concentration to the 50% effective concentration. Alkaline extract from the leaves of Sasa senanensis Rehder (SE), having had hormetic growth stimulation, showed the highest SI value, followed by epigallocatechin gallate. The SI value of curcumin and resveratrol was much lower. This simple overly method, that can prepare massive differentiated neuronal cells, may be applicable for the study of the differentiation-associated changes in intracellular metabolites, and the interaction between neuronal cells and physiological factors.

Dental Application of Natural Products.

This review article summarizes the recent progress in dental applications of natural products. Catechin gel showed selective antimicrobial activity, whereas the alkaline extract of various plant species rich in lignin carbohydrate complex (LCC) showed much higher antiviral activity than lower molecular weight polyphenols. Mouthwash with the alkaline extract of a plant classified as OTC effectively reduced halitosis. Unexpectedly, many polyphenolic compounds purified from the natural kingdom showed much lower tumor-specificity against human oral squamous cell lines as compared with antitumor agents, although they showed apoptosis-inducing activity. The alkaline extract of bamboo leaf, which exerted various common biological activities with LCC, showed osteogenic activity by stimulating differentiation toward osteoblasts while inhibiting differentiation toward osteoclasts. LCC enhanced the dectin-2 mRNA expression in macrophages, whereas glucan showed anti-osteoblastic action via dectin-1. These data suggest that natural products exert their biological activity by interacting with these molecules.