Terbium promotes adhesion and osteogenic differentiation of mesenchymal stem cells via activation of the Smad-dependent TGF-ß/BMP signaling pathway.

With its special physical and chemical properties, terbium has been widely used, which has inevitably increased the chance of human exposure to terbium-based compounds. It was reported that terbium mainly deposited in bone after introduction into the human body. Although some studies revealed the effects of terbium on bone cell lines, there have been few reports about the potential effect of terbium on adhesion and differentiation of mesenchymal stem cells (MSCs). In this study, we investigated the effects of terbium on the adhesion and osteogenic and adipogenic differentiation of MSCs and the associated molecular mechanisms. Our data reveal that terbium promoted the osteogenic differentiation in a time-dependent manner and conversely inhibited the adipogenic differentiation of MSCs. Meanwhile, the cell-cell or cell-matrix interaction was enhanced by activating adherent-related key factors, which were evaluated by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Real-time RT-PCR and Western blot analysis were also performed to further detect osteogenic and adipogenic biomarkers of MSCs. The regulation of terbium on differentiation of MSCs led to the interaction between the transforming growth factor ß/bone morphogenetic protein and peroxisome-proliferator-activated receptor γ (PPARγ) signaling pathways, resulting in upregulation of the osteogenic master transcription factors, such as Runt-related transcription factor 2, bone morphogenetic protein 2, collagen I, alkaline phosphatase, and osteocalcin, and downregulation of the adipogenic master transcription factors, such as PPARγ2. The results provide novel evidence to elucidate the mechanisms of bone metabolism by terbium and may be helpful for more rational application of terbium-based compounds in the future.

TGF-ß/BMP signaling pathway is involved in cerium-promoted osteogenic differentiation of mesenchymal stem cells.

The extensive applications of cerium (Ce) increased the chance of human exposure to Ce and its compounds. It was reported that Ce was mainly deposited in the bone after administration. However, the potential effect and mechanism of Ce on bone metabolism are not well understood. In this study, we investigated the cellular effects of Ce on the differentiation of mesenchymal stem cells (MSCs) and the associated molecular mechanisms. The results indicated that Ce promoted the osteogenic differentiation and inhibited the adipogenic differentiation of MSCs at cell level. Genes involved in transforming growth factor-ß/bone morphogenetic proteins (TGF-ß/BMP) signaling pathway were significantly changed when the MSCs were exposed to 0.0001 µM Ce by RT(2) Profiler™ PCR Array analysis. The expression of genes and proteins related to pathways, osteogenic, and adipogenic biomarkers of MSCs upon interaction with Ce was further confirmed by quantitative real-time reverse transcriptase polymerase chain reaction (Q-PCR) and Western blot analysis. The results suggest that Ce exerts the effects by interacting with bone morphogenetic protein receptor (BMPR) and activates TGF-ß/BMP signaling pathway, leads to the up-regulation of the osteogenic master transcription factor, runt-related transcription factor 2 (Runx 2), and the down-regulation of the adipocytic master transcription factor, peroxisome proliferator-activated receptor gamma 2 (PPARγ2). Runx2, which subsequently up-regulates osteoblast (OB) marker genes collagen I (Col I) and BMP2 at early stages, alkaline phosphatase (ALP), and osteocalcin (OCN) at later stages of differentiation, thus driving MSCs to differentiate into OBs. The results provide novel evidence to elucidate the mechanisms of bone metabolism by Ce.

Differentially expressed genes from the glioblastoma cell line SHG-44 treated with all-trans retinoic acid in vitro.

Morphology, immunocytochemistry, growth curve assay, and flow cytometry were used to investigate the effects of all-trans retinoic acid (RA) on cell proliferation, cell cycle progression and differentiation of the astrocytoma cell line SHG-44 from glioblastoma multiforme (World Health Organization grade IV). The differentially expressed genes from RA-treated and normal SHG-44 were identified by cDNA microarray after the cell line SHG-44 was treated with 10muM RA for 3 days. Validation of some differentially expressed genes was performed by Northern Blot analysis. The expression of glial fibrillary acidic protein (GFAP) was markedly increased in RA-treated SHG-44 cells. Other changes included a short shuttle shape, small nucleus, decreased karyoplasm proportion, the formation of increased thin cytoplasmic processes, reduced cell growth and a 15% increase in G0/G1 phase cell populations. In addition, 42 known genes were identified with altered expression in our cDNA microarray. There was stable down-regulation of MDM2 and UGB as well as overexpression of SOD2, CSTB, and G3BP when RA-treated SHG-44 was compared with normal SHG-44. RA simultaneously suppressed the proliferation of SHG-44 cells significantly as well as induced differentiation and altered gene expression.

Acrylamide-induced molecular mutation spectra at HPRT locus in human promyelocytic leukaemia HL-60 and NB4 cell lines.

Acrylamide (AA) is a compound widely used in many industries around the world. The recent finding that it is formed naturally in foods by heating raises human health concerns. AA is a proven carcinogen in animals and a probable carcinogen in humans, while its mutagenicity detected using in vitro mammalian gene mutation assays is still inconsistent in different cell systems. In the present study, we investigated the mutagenicity of AA in human promyelocytic leukaemia cells, HL-60 and NB4 cells, by examining the mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene locus. In a 6-h treatment without the exogenous activation, AA exerted a weak mutagenic effect at the highest concentration used in the study (700 mg/l) in HL-60 cells (P < 0.01) as well as in NB4 cells (P < 0.05). Molecular analysis of AA-induced mutants revealed a different mutation spectrum, when compared to that of spontaneous mutants. The most frequent spontaneous mutations were point mutations, whereas AA-induced mutations were mainly single exon deletions besides point mutations, and an increase in the proportion of partial deletion was associated with the increase of AA treatment. There was no obvious difference in the mutation spectra observed between the HL-60 and NB4 cell lines. These results showed that AA has a weak mutagenic effect at HPRT gene locus in human promyelocytic leukaemia HL-60 and NB4 cell lines and those molecular mutation spectra (single exon deletions and point mutations) may be related to some specific and precise mechanism.

Differential expression of genes associated with cell proliferation and apoptosis induced by okadaic acid during the transformation process of BALB/c 3T3 cells.

Okadaic acid (OA) is a tumor promoter in two-stage carcinogenesis experiments. Nevertheless, the effects of OA on cell transformation, cell proliferation and apoptosis vary widely, and the molecular events underlying these effects of OA are not well understood. In the present study, we examined the promoting activity and the associated effects on cell growth and apoptosis mediated by OA in BALB/c 3T3 cells, and evaluated alterations of gene transcriptional expression by microarray analysis. The promoting activity of OA was estimated by a two-stage transformation assay, in which cells were treated first with a low dose of the initiator N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and then with OA for 14 days. It showed that OA, at concentrations of 7.8-31.3 ng/ml, enhanced the transformation of MNNG-treated cells. In the promotion phase, cells exposed to OA (7.8 ng/ml) grew slowly for the first 2 days and subsequently died. As determined by Hoechst 33342 fluorescent dye and Annexin-V/PI dual-colored flow cytometry, OA induced morphologically apoptotic cells and increased the percentage of early apoptotic cells. The gene expression profile induced by OA at five time points in the promotion phase was determined by use of a specific mouse toxicological microarray containing 1796 clones, and a total of 177 differentially expressed genes were identified. By gene ontology analysis, 31 of these were determined to be functionally involved with cell growth and/or maintenance. In this group, numerous genes associated with the cell proliferation and cell cycle progression were down-regulated at early and/or middle time points. Among these was a subset of genes associated with apoptosis, in which Bnip3, Cycs, Casp3 and Bag1 genes are involved in the mitochondrial pathway of apoptosis. Ier3, Mdm2 and Bnip3 genes may be p53 targets. Furthermore, real-time PCR confirmed the expression changes of five genes selected at random from the differentially expressed genes. We conclude that OA induces cell growth inhibition and apoptosis in the two-stage, MNNG-initiated transformation of BALB/c 3T3 cells. The results of gene expression profile analysis imply that multiple molecular pathways are involved in OA-induced proliferation inhibition and apoptosis. Mitochondrial and p53-associated apoptotic pathways also may contribute to OA-induced apoptosis.

Effect of zinc ion on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and the adipocytic trans-differentiation of mouse primary osteoblasts.

A series of experimental methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test, alkaline phosphatase (ALP) activity measurement and Oil Red O stain and measurement were employed to assess the effect of zinc ion on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells (MSCs) and the adipogenic trans-differentiation of mouse primary osteoblasts. The results showed that except for individual concentrations of zinc ion there was no effect on the proliferation of MSCs and osteoblasts. Zinc ion inhibited the osteogenic differentiation of MSCs at all the concentrations tested. It also inhibited adipogenic differentiation at all concentrations tested except 10(-9)mol/L. Both of the inhibition effects were attenuated with time increasing. Zinc ion depressed adipocytic trans-differentiation of osteoblasts at concentrations of 10(-11) and 10(-10)mol/L, but the effect could be reversed to promote or even be removed when concentration was increased. It suggests that the influence of zinc ion on osteogenic, adipogenic differentiation of MSCs and adipocytic trans-differentiation of osteoblasts depends on zinc ion concentrations and incubation time. The protective effects of zinc ion on bone may be mediated by modulating differentiation of MSCs away from the adipocytes and inhibiting adipocytic trans-differentiation of osteoblasts. This may in turn promote osteoblast formation and reduce secretion of cytokines which may inhibit osteoclast formation and activation. These findings may be valuable for better understanding the mechanism of the effect of zinc ion on bone.

[Correlation between ITS genotype and geographical distribution of Pogostemon cablin].

To investigate the correlation between genotype and distribution of Pogostemon cablin by sequencing ITS1 and ITS2 genes, and provide molecular information for its germplasm evaluation, ITS1 and ITS2 genes of Pogostemon cablin from different localities were identified by PCR direct sequencing. The sequences of ITS1 and ITS2 genes were 424 bp and 380 bp in length, respectively. And nineteen base substitutions were observed in ITS1 gene, and five in ITS2 gene. The results showed a good correlation between genotype and distribution of Pogostemon cablin, and ITS gene sequencing could provide useful molecular information for germplasm evaluation of the plant species verification.

[Comparison of antioxidative and antitumor activities of six flavonoids from Epimedium koreanum].

OBJECTIVE: To study the antioxidative and antitumor activities of flavonoids isolated from Epimedium koreanum. METHOD: The compounds were separated by column chromatography with silica gel and Sephadex LH-20, and identified by spectral a- nalysis (ESI-MS, 1H-NMR and 13C-NMR) respectively. DPPH radical scavenging assay and MTT assay were used to observe the antioxidative and antitumor abilities. RESULT: Six compounds were isolated from the the ethyl acetate extract of the aerial part. Their structures were identified as icariin (I), luteolin (II), baohuoside II (III), hyperoside (IV), epimedokoreanin B (V) and baohuoside I (VI). The results indicated that at concentrations of 3. 125-200 micromol x L(-1), compound I, III and VI had no ability to scavenge the DPPH radical, but the scavenging ability of compounds II, IV and V were stronger than that of Vit C in dose-dependant manner. Compounds I, II, V and VI could inhibit the proliferation of MCF-7 and HepG2 in dose-dependant manner, but compounds III and IV had no effect on the proliferation. CONCLUSION: The antitumor activity of E. koreanum may be partially related to the antioxidantive activity of flavonoids.

[Study on chemical constituents from rhizomes of Actaea asiatica].

OBJECTIVE: To investigate the chemical constituents of the rhizomes of Actaea asiatica in order to obtain a more comprehensive understanding of its effective components. METHOD: Compounds were separated by silica gel chromatography, RP-C18 chromatography and semi-preparative high performance liquid chromatography, and their structures were established by spectral analysis and chemical evidence. RESULT: Six compounds were isolated from the ethyl acetate extract. Their structures were identified as 25-O-acetylcimigenol (1), 12beta-hydroxycimigenol (2), 23-epi-26-deoxyactein (3), 27-deoxyacetylacteol (4), 26-deoxycimicifugenin (5) and beta-sitosterol (6). CONCLUSION: All these compounds mentioned above were isolated from the plant for the first time.

Anti-hepatoma activity and mechanism of ursolic acid and its derivatives isolated from Aralia decaisneana.

AIM: To investigate the anti-tumor activity of ursolic acid (UA) and its derivatives isolated from Aralia decaisneana on hepatocellular carcinoma both in vitro and in vivo. METHODS: In vivo cytotoxicity was first screened by 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay. Morphological observation, DNA ladder, flow cytometry analysis, Western blot and real time PCR were employed to elucidate the cytotoxic mechanism of UA. Implanted mouse hepatoma H22 was used to evaluate the growth inhibitory effect of UA in vivo. RESULTS: UA could significantly inhibit the proliferation of HepG2 and its drug-resistance strain, R-HepG2 cells, but had no inhibitory effect on primarily cultured normal mouse hepatocytes whereas all the six derivatives of UA could not inhibit the growth of all tested cell lines. Further study on mechanism demonstrated that apoptosis and G0/G1 arrest were involved in the cytotoxicity and cleavage of poly-(ADP-ribose)-polymerase (PARP). Downregulation of cyclooxygenase-2 (COX-2) protein and upregulation of heat shock protein (HSP) 105 mRNA correlated to the apoptosis of HepG2 cells treated with UA. In addition, UA also could inhibit the growth of H22 hepatoma in vivo. CONCLUSION: UA is a promising anti-tumor agent, but further work needs to be done to improve its solubility.

[Progress in anti-osteoporosis activity of traditional Chinese medicine and medicinal plants].

The anti-osteoporosis activity and mechanism of traditional Chinese medicine and medicinal plants were discussed. It is hoped that we can provide some reference for future drug development and introduction of traditional Chinese medicine to world.

[Cytotoxicity and mechanism of 23-O-acetylcimigenol-3-O-beta-D-xylopyranoside on HepG2 cells].

OBJECTIVE: To elucidate the cytotoxicity and mechanism of 23-O-acetylcimigenol-3-O-beta-D-xylopyranoside isolated from C. dahurica on HepG2 cells and to find the leading compound for new drug development. METHOD: MTT, AO/EB staining observation, flow cytometry and western blot methods were used to study the cytotoxicity, morphological changes, cell cycle distribution and protein expression profile of 23-O-acetylcimigenol-3-O-beta-D-xylopyranoside on HepG2 cells. RESULT: 23-O-acetylcimigenol-3-O-beta-D-xylopyranoside could inhibit the proliferation of HepG2 cells with IC50 at 16 micromol x L(-1), and could also induce apoptosis and G2-M cell cycle arrest. Further study demonstrated that the compound could cleavage PARP, regulate protein expression of bcl-2 family and decrease the expression of cdc 2 and cyclin B. CONCLUSION: 23-O-acetylcimigenol-3-O-beta-D-xylopyranoside exerts its cytotoxicity on HepG2 cells via apoptosis and G2-M arrest. In addition, caspases family activation, regulation of protein expression of bcl-2 family and down regulation of cdc 2 and cyclin B were involved in apoptosis and G2-M arrest induced by it.

[Review of bioactivities of natural cycloartane triterpenoids].

Cycloartane triterpenoids, which exist widely in nature, are mainly distributed in Astragalus (Leguminosae) species, Trib. Cimicifuga (Ranunculaceae) and Thalictrium (Ranunculaceae) species and possess various bioactivities. Along with the development of isolation techniques of phytochemistry, more and more this kind of compounds are isolated and identified. However, bioactivity researches on the compounds are relatively lagged behind. Most researches are still in screening level, deficient in mechanism elucidation, short of action proven in vivo and SAR analysis. The author summarized the bioactivity of this kind of compounds from all aspects: anti-tumor, anti-virus, antibacterial, anti-inflammation, immune-regulatory, cardiovascular system, hepatic protection and so forth. This will be benefit for the further research and development of the compounds.

Differential gene expression profiles in paclitaxel-induced cell cycle arrest and apoptosis in human breast cancer MCF-7 cells.

AIM: To elucidate the molecular mechanism of cell cycle arrest and apoptosis of MCF-7 cells induced by paclitaxel. METHODS: Flow cytometry was used to determine the cell cycle changes of MCF-7 cells upon paclitaxel treatment. Gene expression profiles of MCF-7 cells induced by paclitaxel were obtained by using cDNA microarrays containing 9984 genes and expressed sequence tags (ESTs). RESULTS: Cell cycle analysis showed that 77.8% of cells arrested at G2/M phase and 1.3% of cells underwent apoptosis upon 100 nmol x L(-1) paclitaxel treatment for 24 hours; cDNA microarray results revealed that 27 and 77 genes were differentially expressed upon 12.5 nmol x L(-1) (IC50) and 100 nmol x L(-1) paclitaxel treatment, respectively. CONCLUSION: Paclitaxel stabilized microtubules and caused G2/M cell cycle arrest and apoptotic cell death in a concentration-dependent manner, which is associated with the regulation of selected genes related to microtubule assembly and cytoskeleton, cell cycle regulation, and DNA repair and apoptosis.

[Gene expression profiles in early stage of BALB/c 3T3 cells' transformation promoted with 12-O-tetradecanoylphorbol-13-acetate].

OBJECTIVE: To elucidate the potential molecular mechanism responsible for the early time of tumor promotion, gene expression profile was studied in the transformed BALB/c 3T3 cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). METHODS: The two-stage cell transformation model was established by using the initiator of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and promoter of TPA. Cell proliferation was measured by trypan blue staining and cell cycle analysis was carried out by flow cytometry assay. A cDNA microarray representing 1 152 genes was used to investigate the gene expression profiles of BALB/c 3T3 cells exposed to TPA at 4 h and 24 h respectively. RESULTS: TPA could effectively inhibit cell proliferation and induce the G1 and S cell cycle arrested in the early time. Moreover 19 genes were found differentially expressed at least twofold in the TPA treated cells as compared with the control cells, 9 of them were upregulated and 10 downregulated. Most of the differentially expressed genes were involved in cell proliferation, differentiation or apoptosis, and related to ras or p53 signal transduction pathway. CONCLUSION: TPA could influence the transcriptional expression of some genes related to cell cycle modulation and ultimately result in the cell growth arrest.

Reversal of multidrug resistance in cancer cells by pyranocoumarins isolated from Radix Peucedani.

The pyranocoumarins, (+/-)-3'-angeloyl-4'-acetoxy-cis-khellactone, were isolated from Radix Peucedani, the dry root of Peucedanum praeruptorum Dunn, through bioassay-guided fractionation. The chemical structure of pyranocoumarins was determined by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. X-ray crystallography showed that there are eight molecules (i.e. two each of four conformers) in each unit cell with their optical activities equally cancelled out. The four conformers are 3'(R)-angeloyl-4'(R)-acetoxy-khellactone in two conformational forms, and 3'(S)-angeloyl-4'(S)-acetoxy-khellactone in two conformational forms. Pyranocoumarins caused apoptotic cell death with IC50 of 41.9+/-2.8 and 17.3+/-8.2 microM for drug-sensitive KB-3-1 and multidrug resistant (MDR) KB-V1, respectively. The two- to threefold sensitivity difference between the two cell lines is interesting considering that the same ratio for doxorubicin is 50-300. Strong synergistic interactions were demonstrated when pyranocoumarins were combined with common anti-tumor drugs including doxorubicin, paclitaxel, puromycin or vincristine in MDR KB-V1 cell line, but not in drug-sensitive KB-3-1 cells. Pyranocoumarins increased doxorubicin accumulation in KB-V1 cells by about 25% after 6 h of incubation. Pyranocoumarins treatment for 24 h down-regulated the expression of P-glycoprotein in KB-V1 cells at both protein and mRNA levels. Pyranocoumarins also transiently reduced the cellular ATP contents in KB-V1 cells in a dose-dependent manner. Our results suggest that pyranocoumarins could be a potential MDR reversing agent.

[Lab-on-a-chip (microfluidics) technology].

Over the last decade, the lab-on-a-chip concept has received considerable attention. This technology promises significant advantages in terms of speed, cost, sample/reagent consumption, contamination, efficiency and automation. In this review, we focus on lab-on-a-chip with fluid flow along micro-scale channels (microfluidics) and its application on life science and biotechnology.

Molecular mechanism of granulocytic differentiation of human promyelocytic leukemia HL-60 cells induced by all-trans retinoic acid.

AIM: To elucidate the molecular mechanism of granulocytic differentiation of human promyelocytic leukemia HL-60 cells induced by all-trans-retinoic acid (ATRA). METHODS: Flow cytometry was used to determine the cell cycle changes of HL-60 cells upon ATRA treatment. Gene expression profiles of HL-60 cells induced by 1 mumol.L-1 ATRA were obtained by using cDNA microarrays containing 9,984 genes and expressed sequence tags (ESTs). RESULTS: Cell cycle analysis showed that 48%-73% of cells were arrested at G1/G0 phase upon ATRA treatment; cDNA microarray results demonstrated that the expression of genes encoding adhesion molecules, tissue remodeling proteins, transporters and ribosomal proteins were up-regulated in ATRA treated of HL-60 cells. Several genes involved in oxidase activation pathway were also differentially expressed. CONCLUSION: ATRA treatment induced growth arrest and differentiation in HL-60 cells, which is associated with regulation of the oxidase activation pathway and the expression of tissue remodeling proteins.

Genotyping and species identification of Fritillaria by DNA chips.

AIM: To investigate the genetic polymorphism of several species of Fritillaria and to develop a DNA chip for the genotyping and identification of the origin of various species of Fritillaria at molecular level. METHODS: Genomic DNA from bulbs of several Fritillaria species was extracted and the polymorphisms of the D2 and D3 regions inside the 26S rDNA gene were identified by direct sequencing. Oligonucleotide probes specific for these polymorphisms were designed and printed on the poly-lysine coated slides to prepare the DNA chip. PCR products from the Fritillaria species were labeled with fluorescence by incorporation of dye-labeled dideoxyribonucleotides and hybridized to the immobilized probes on the chip. RESULTS: The polymorphisms were used as markers for discrimination among various species. Specific oligonucleotide probes were designed and immobilized on a DNA chip. Differentiation of the various Fritillaria species was accomplished based on hybridization of fluorescent labeled PCR products with the DNA chip. CONCLUSION: The results demonstrated the reliability of using DNA chips to identify different species of Fritillaria, and the DNA chip technology can provide a rapid, high throughput tool for genotyping and quality assurance of the plant species verification.