Amygdalin Influences Bladder Cancer Cell Adhesion and Invasion In Vitro

The cyanogenic diglucoside amygdalin, derived from Rosaceae kernels, is employed by many patients as an alternative anti-cancer treatment. However, whether amygdalin indeed acts as an anti-tumor agent is not clear. Metastasis blocking properties of amygdalin on bladder cancer cell lines was, therefore, investigated. Amygdalin (10 mg/ml) was applied to UMUC-3, TCCSUP or RT112 bladder cancer cells for 24 h or for 2 weeks. Tumor cell adhesion to vascular endothelium or to immobilized collagen as well as tumor cell migration was examined. Effects of drug treatment on integrin α and β subtypes, on integrin-linked kinase (ILK) and total and activated focal adhesion kinase (FAK) were also determined. Integrin knock-down was carried out to evaluate integrin influence on migration and adhesion. A 24 h or 2 week amygdalin application distinctly reduced tumor cell adhesion and migration of UMUC-3 and RT112 cells. TCCSUP adhesion was also reduced, but migration was elevated under amygdalin. Integrin subtype expression was significantly and specifically altered by amygdalin depending on the cell line. ILK was moderately, and activated FAK strongly, lost in all tumor cell lines in the presence of amygdalin. Knock down of β1 integrin caused a significant decrease in both adhesion and migration of UMUC-3 cells, but a significant increase in TCCSUP adhesion. Knock down of β4 integrin caused a significant decrease in migration of RT112 cells. Since the different actions of amygdalin on the different cell lines was mirrored by β1 or β4 knock down, it is postulated that amygdalin influences adhesion and migratory properties of bladder cancer cells by modulating β1 or β4 integrin expression. The amygdalin induced increase in TCCSUP migratory behavior indicates that any anti-tumor benefits from amygdalin (seen with the other two cell lines) may depend upon the cancer cell type.     

Red Blood Cells Preconditioned with Hemin Are Less Permissive to Plasmodium Invasion In Vivo and In Vitro

Malaria is a parasitic disease that causes severe hemolytic anemia in Plasmodium-infected hosts, which results in the release and accumulation of oxidized heme (hemin). Although hemin impairs the establishment of Plasmodium immunity in vitro and in vivo, mice preconditioned with hemin develop lower parasitemia when challenged with Plasmodium chabaudi adami blood stage parasites. In order to understand the mechanism accounting for this resistance as well as the impact of hemin on eryptosis and plasma levels of scavenging hemopexin, red blood cells were labeled with biotin prior to hemin treatment and P. c. adami infection. This strategy allowed discriminating hemin-treated from de novo generated red blood cells and to follow the infection within these two populations of cells. Fluorescence microscopy analysis of biotinylated-red blood cells revealed increased P. c. adami red blood cells selectivity and a decreased permissibility of hemin-conditioned red blood cells for parasite invasion. These effects were also apparent in in vitro P. falciparum cultures using hemin-preconditioned human red blood cells. Interestingly, hemin did not alter the turnover of red blood cells nor their replenishment during in vivo infection. Our results assign a function for hemin as a protective agent against high parasitemia, and suggest that the hemolytic nature of blood stage human malaria may be beneficial for the infected host.     

Response-Predictive Gene Expression Profiling of Glioma Progenitor Cells In Vitro

Background

High-grade gliomas are amongst the most deadly human tumors. Treatment results are disappointing. Still, in several trials around 20% of patients respond to therapy. To date, diagnostic strategies to identify patients that will profit from a specific therapy do not exist.

Methods

In this study, we used serum-free short-term treated in vitro cell cultures to predict treatment response in vitro. This approach allowed us (a) to enrich specimens for brain tumor initiating cells and (b) to confront cells with a therapeutic agent before expression profiling.

Results

As a proof of principle we analyzed gene expression in 18 short-term serum-free cultures of high-grade gliomas enhanced for brain tumor initiating cells (BTIC) before and after in vitro treatment with the tyrosine kinase inhibitor Sunitinib. Profiles from treated progenitor cells allowed to predict therapy-induced impairment of proliferation in vitro.

Conclusion

For the tyrosine kinase inhibitor Sunitinib used in this dataset, the approach revealed additional predictive information in comparison to the evaluation of classical signaling analysis.     

Lipo-Chitin Oligosaccharides,Plant Symbiosis Signalling Molecules That Modulate Mammalian Angiogenesis In Vitro

Lipochitin oligosaccharides (LCOs) are signaling molecules required by ecologically and agronomically important bacteria and fungi to establish symbioses with diverse land plants. In plants, oligo-chitins and LCOs can differentially interact with different lysin motif (LysM) receptors and affect innate immunity responses or symbiosis-related pathways. In animals, oligo-chitins also induce innate immunity and other physiological responses but LCO recognition has not been demonstrated. Here LCO and LCO-like compounds are shown to be biologically active in mammals in a structure dependent way through the modulation of angiogenesis, a tightly-regulated process involving the induction and growth of new blood vessels from existing vessels. The testing of 24 LCO, LCO-like or oligo-chitin compounds resulted in structure-dependent effects on angiogenesis in vitro leading to promotion, or inhibition or nil effects. Like plants, the mammalian LCO biological activity depended upon the presence and type of terminal substitutions. Un-substituted oligo-chitins of similar chain lengths were unable to modulate angiogenesis indicating that mammalian cells, like plant cells, can distinguish between LCOs and un-substituted oligo-chitins. The cellular mode-of-action of the biologically active LCOs in mammals was determined. The stimulation or inhibition of endothelial cell adhesion to vitronectin or fibronectin correlated with their pro- or anti-angiogenic activity. Importantly, novel and more easily synthesised LCO-like disaccharide molecules were also biologically active and de-acetylated chitobiose was shown to be the primary structural basis of recognition. Given this, simpler chitin disaccharides derivatives based on the structure of biologically active LCOs were synthesised and purified and these showed biological activity in mammalian cells. Since important chronic disease states are linked to either insufficient or excessive angiogenesis, LCO and LCO-like molecules may have the potential to be a new, carbohydrate-based class of therapeutics for modulating angiogenesis.     

Graptopetalum Paraguayense Ameliorates Chemical-Induced Rat Hepatic Fibrosis In Vivo and Inactivates Stellate Cells and Kupffer Cells In Vitro

Background

Graptopetalum paraguayense (GP) is a folk herbal medicine with hepatoprotective effects that is used in Taiwan. The aim of this study was to evaluate the hepatoprotective and antifibrotic effects of GP on experimental hepatic fibrosis in both dimethylnitrosamine (DMN)- and carbon tetrachloride (CCl₄)-induced liver injury rats.

Methods

Hepatic fibrosis-induced rats were fed with the methanolic extract of GP (MGP) by oral administration every day. Immunohistochemistry, biochemical assays, and Western blot analysis were performed. The effects of MGP on the expression of fibrotic markers and cytokines in the primary cultured hepatic stellate cells (HSCs) and Kupffer cells, respectively, were evaluated.

Results

Oral administration of MGP significantly alleviated DMN- or CCl₄-induced liver inflammation and fibrosis. High levels of alanine transaminase, aspartate transaminase, bilirubin, prothrombin activity and mortality rates also decreased in rats treated with MGP. There were significantly decreased hydroxyproline levels in therapeutic rats compared with those of the liver-damaged rats. Collagen I and alpha smooth muscle actin (α-SMA) expression were all reduced by incubation with MGP in primary cultured rat HSCs. Furthermore, MGP induced apoptotic cell death in activated HSCs. MGP also suppressed lipopolysaccharide-stimulated rat Kupffer cell activation by decreasing nitric oxide, tumor necrosis factor-α and interleukin-6 production, and increasing interleukin-10 expression.

Conclusions

The results show that the administration of MGP attenuated toxin-induced hepatic damage and fibrosis in vivo and inhibited HSC and Kupffer cell activation in vitro, suggesting that MGP might be a promising complementary or alternative therapeutic agent for liver inflammation and fibrosis.     

Neurotrophin-Induced Migration and Neuronal Differentiation of Multipotent Astrocytic Stem Cells In Vitro

Hypoxic ischemic encephalopathy (HIE) affects 2–3 per 1000 full-term neonates. Up to 75% of newborns with severe HIE die or have severe neurological handicaps. Stem cell therapy offers the potential to replace HIE-damaged cells and enhances the autoregeneration process. Our laboratory implanted Multipotent Astrocytic Stem Cells (MASCs) into a neonatal rat model of hypoxia-ischemia (HI) and demonstrated that MASCs move to areas of injury in the cortex and hippocampus. However, only a small proportion of the implanted MASCs differentiated into neurons. MASCs injected into control pups did not move into the cortex or differentiate into neurons. We do not know the mechanism by which the MASCs moved from the site of injection to the injured cortex. We found neurotrophins present after the hypoxic-ischemic milieu and hypothesized that neurotrophins could enhance the migration and differentiation of MASCs. Using a Boyden chamber device, we demonstrated that neurotrophins potentiate the in vitro migration of stem cells. NGF, GDNF, BDNF and NT-3 increased stem cell migration when compared to a chemokinesis control. Also, MASCs had increased differentiation toward neuronal phenotypes when these neurotrophins were added to MASC culture tissue. Due to this finding, we believed neurotrophins could guide migration and differentiation of stem cell transplants after brain injury.     

Potential Therapeutic Effect of Natural Killer Cells on Doxorubicin-Resistant Breast Cancer Cells In Vitro

Objective

The aim of this study was to explore the therapeutic effect of natural killer (NK) cells on human doxorubicin-sensitive and resistant breast adenocarcinoma.

Methods

Human doxorubicin-sensitive and resistant breast cancer cell lines (MCF-7 and MCF-7/ADR) were tagged with renilla luciferase (Rluc) (MCF-7/RC and MCF-7/ADR/RC). NK cells were tagged with enhanced firefly luciferase (effluc) using a recombinant retrovirus transfection (NKF). Expression of Rluc, effluc, and NK cell surface markers CD16, CD56 as well as death receptors, DR4 and DR5, were assessed by using flow cytometry. In vitro cytotoxic effect of NK to MCF-7 and MCF-7/ADR was measured and in vivo bioluminescence imaging was also performed to visualize MCF-7/RC, MCF-7/ADR, and NKF in an animal model.

Results

NK92-MI, MCF-7, and MCF-7/ADR cells were successfully labeled with Rluc or effluc. Both the target breast cancer cells (with Rluc) and therapeutic NK cells (with effluc) were noninvasively visualized in nude mice. Doxorubicin-resistant breast cancer cells (MCF-7/ADR) presented a higher expression of DR5 and were more sensitive to NK cells compared with doxorubicin-sensitive breast cancer cells (MCF-7).

Conclusion

The results of present study suggest that NK cell therapy has a therapeutic effect on doxorubicin-sensitive and resistant breast cancer cells.     

The Campylobacter jejuni Cj0268c Protein Is Required for Adhesion and Invasion In Vitro

Adherence of Campylobacter jejuni to its particular host cells is mediated by several pathogen proteins. We screened a transposon-based mutant library of C. jejuni in order to identify clones with an invasion deficient phenotype towards Caco2 cells and detected a mutant with the transposon insertion in gene cj0268c. In vitro characterization of a generated non-random mutant, the mutant complemented with an intact copy of cj0268c and parental strain NCTC 11168 confirmed the relevance of Cj0268c in the invasion process, in particular regarding adherence to host cells. Whereas Cj0268c does not impact autoagglutination or motility of C. jejuni, heterologous expression in E. coli strain DH5α enhanced the potential of the complemented E. coli strain to adhere to Caco2 cells significantly and, thus, indicates that Cj0268c does not need to interact with other C. jejuni proteins to develop its adherence-mediating phenotype. Flow cytometric measurements of E. coli expressing Cj0268c indicate a localization of the protein in the periplasmic space with no access of its C-terminus to the bacterial surface. Since a respective knockout mutant possesses clearly reduced resistance to Triton X-100 treatment, Cj0268c contributes to the stability of the bacterial cell wall. Finally, we could show that the presence of cj0268c seems to be ubiquitous in isolates of C. jejuni and does not correlate with specific clonal groups regarding pathogenicity or pathogen metabolism.     

Natural Mineral Particles Are Cytotoxic to Rainbow Trout Gill Epithelial Cells In Vitro

Worldwide increases in fluvial fine sediment are a threat to aquatic animal health. Fluvial fine sediment is always a mixture of particles whose mineralogical composition differs depending on the sediment source and catchment area geology. Nonetheless, whether particle impact in aquatic organisms differs between mineral species remains to be investigated. This study applied an in vitro approach to evaluate cytotoxicity and uptake of four common fluvial mineral particles (quartz, feldspar, mica, and kaolin; concentrations: 10, 50, 250 mg L⁻¹) in the rainbow trout epithelial gill cell line RTgill-W1. Cells were exposed for 24, 48, 72, and 96 h. Cytotoxicity assays for cell membrane integrity (propidium iodide assay), oxidative stress (H₂DCF-DA assay), and metabolic activity (MTT assay) were applied. These assays were complemented with cell counts and transmission electron microscopy. Regardless of mineral species, particles ≤2 µm in diameter were taken up by the cells, suggesting that particles of all mineral species came into contact and interacted with the cells. Not all particles, however, caused strong cytotoxicity: Among all assays the tectosilicates quartz and feldspar caused sporadic maximum changes of 0.8–1.2-fold compared to controls. In contrast, cytotoxicity of the clay particles was distinctly stronger and even differed between the two particle types: mica induced concentration-dependent increases in free radicals, with consistent 1.6–1.8-fold-changes at the 250 mg L⁻¹ concentration, and a dilated endoplasmic reticulum. Kaolin caused concentration-dependent increases in cell membrane damage, with consistent 1.3–1.6-fold increases at the 250 mg L⁻¹ concentration. All effects occurred in the presence or absence of 10% fetal bovine serum. Cell numbers per se were marginally affected. Results indicate that (i.) natural mineral particles can be cytotoxic to gill epithelial cells, (ii.) their cytotoxic potential differs between mineral species, with clay particles being more cytotoxic, and (iii.) some clays might induce effects comparable to engineered nanoparticles.     

Oral Community Interactions of Filifactor alocis In Vitro

Filifactor alocis is a gram positive anaerobe that is emerging as an important periodontal pathogen. In the oral cavity F. alocis colonizes polymicrobial biofilm communities; however, little is known regarding the nature of the interactions between F. alocis and other oral biofilm bacteria. Here we investigate the community interactions of two strains of F. alocis with Streptococcus gordonii, Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans, organisms with differing pathogenic potential in the oral cavity. In an in vitro community development model, S. gordonii was antagonistic to the accumulation of F. alocis into a dual species community. In contrast, F. nucleatum and the type strain of F. alocis formed a synergistic partnership. Accumulation of a low passage isolate of F. alocis was also enhanced by F. nucleatum. In three species communities of S. gordonii, F. nucleatum and F. alocis, the antagonistic effects of S. gordonii superseded the synergistic effects of F. nucleatum toward F. alocis. The interaction between A. actinomycetemcomitans and F. alocis was strain specific and A. actinomycetemcomitans could either stimulate F. alocis accumulation or have no effect depending on the strain. P. gingivalis and F. alocis formed heterotypic communities with the amount of P. gingivalis greater than in the absence of F. alocis. However, while P. gingivalis benefited from the relationship, levels of F. alocis in the dual species community were lower compared to F. alocis alone. The inhibitory effect of P. gingivalis toward F. alocis was dependent, at least partially, on the presence of the Mfa1 fimbrial subunit. In addition, AI-2 production by P. gingivalis helped maintain levels of F. alocis. Collectively, these results show that the pattern of F. alocis colonization will be dictated by the spatial composition of microbial microenvironments, and that the organism may preferentially accumulate at sites rich in F. nucleatum.     

Effect of Acrylamide on Oocyte Nuclear Maturation and Cumulus Cells Apoptosis in Mouse In Vitro

Acrylamide (ACR) is a chemical compound with severe neurotoxicity, genotoxicity, carcinogenicity and reproductive toxicity. Recent studies showed that ACR impairs the function of reproductive organs, e.g., epididymis and testes. In vitro maturation of mouse oocyte is a sensitive assay to identify potential chemical hazard to female fertility. The aim of this study was to evaluate the adverse effects of ACR on the nuclear maturation and cumulus cells apoptosis of mouse oocytes in vitro. Cumulus–oocyte complexes were incubated in a maturation medium containing 0, 5, 10 and 20 μM of ACR. Chromosome alignment and spindle morphology of oocytes was determined by immunofluorescence and confocal microscopy. Our results showed that oocytes exposed to different doses of ACR in vitro were associated with a significant decrease of oocyte maturation, significant increase of chromosome misalignment rate, occurrence of abnormal spindle configurations, and the inhibition of oocyte parthenogenetic activation. Furthermore, apoptosis of cumulus cells was determined by TUNEL and CASPASE-3 assay. Results showed that apoptosis in cumulus cells was enhanced and the expression of CASPASE-3 was increased after cumulus–oocyte complexes were exposed to ACR. Therefore, ACR may affect the nuclear maturation of oocytes via the apoptosis of cumulus cells in vitro.     

Antitumor Effects and Mechanism of Novel Emodin Rhamnoside Derivatives against Human Cancer Cells In Vitro

A series of novel anthracene L-rhamnopyranosides compounds were designed and synthesized and their anti-proliferative activities on cancer cell lines were investigated. We found that one derivative S-8 (EM-d-Rha) strongly inhibited cell proliferation of a panel of different human cancer cell lines including A549, HepG2, OVCAR-3, HeLa and K562 and SGC-790 cell lines, and displayed IC50 values in low micro-molar ranges, which are ten folds more effective than emodin. In addition, we found EM-d-Rha (3-(2”,3”-Di-O-acetyl-α-L-rhamnopyranosyl-(1→4)-2’,3’-di-O-acetyl-α-L-rhamnopyranosyl)-emodin) substantially induced cellular apoptosis of HepG2 and OVCAR-3 cells in the early growth stage. Furthermore, EM-d-Rha led to the decrease of mitochondrial transmembrane potential, and up-regulated the express of cells apoptosis factors in a concentration- and time-dependent manner. The results indicated the EM-d-Rha may inhibit the growth and proliferation of HepG2 cells through the pathway of apoptosis induction, and the possible molecular mechanism may due to the activation of intrinsic apoptotic signal pathway.     

Effects of p21 Gene Down-Regulation through RNAi on Antler Stem Cells In Vitro

Cell cycle is an integral part of cell proliferation, and consists mainly of four phases, G1, S, G2 and M. The p21 protein, a cyclin dependent kinase inhibitor, plays a key role in regulating cell cyclevia G1 phase control. Cells capable of epimorphic regeneration have G2/M accumulation as their distinctive feature, whilst the majority of somatic cells rest at G1 phase. To investigate the role played byp21 in antler regeneration, we studied the cell cycle distribution of antler stem cells (ASCs), via down-regulation of p21 in vitro using RNAi. The results showed that ASCs had high levels of p21 mRNA expression and rested at G1 phase, which was comparable to the control somatic cells. Down-regulation of p21 did not result in ASC cell cycle re-distribution toward G2/M accumulation, but DNA damage and apoptosis of the ASCs significantly increased and the process of cell aging was slowed. These findings suggest that the ASCs may have evolved to use an alternative, p21-independent cell cycle regulation mechanism. Also a unique p21-dependent inhibitory effect may control DNA damage as a protective mechanism to ensure the fast proliferating ASCs do not become dysplastic/cancerous. Understanding of the mechanism underlying the role played by p21 in the ASCs could give insight into a mammalian system where epimorphic regeneration is initiated whilst the genome stability is effectively maintained.     

Cell Migration Is Regulated by AGE-RAGE Interaction in Human Oral Cancer Cells In Vitro

Advanced glycation end products (AGEs) are produced in an irreversible non-enzymatic reaction of carbohydrates and proteins. Patients with diabetes mellitus (DM) are known to have elevated AGE levels, which is viewed as a risk factor of diabetes-related complications. In a clinical setting, it has been shown that patients with oral cancer in conjunction with DM have a higher likelihood of cancer metastasis and lower cancer survival rates. AGE-RAGE (a receptor of AGEs) is also correlated with metastasis and angiogenesis. Recent studies have suggested that the malignancy of cancer may be enhanced by glyceraldehyde-derived AGEs; however, the underlying mechanism remains unclear. This study examined the apparently close correlation between AGE-RAGE and the malignancy of SAS oral cancer cell line. In this study, AGEs increased ERK phosphorylation, enhanced cell migration, and promoted the expression of RAGE, MMP2, and MMP9. Using PD98059, RAGE antibody, and RAGE RNAi to block RAGE pathway resulted in the inhibition of ERK phosphorylation. Cell migration, MMP2 and MMP9 expression were also reduced by this treatment. Our findings demonstrate the importance of AGE-RAGE with regard to the malignancy of oral cancer, and help to explain the poor prognosis of DM subjects with oral cancer.     

Protein Distribution during Human Erythroblast Enucleation In Vitro

Enucleation is the step in erythroid terminal differentiation when the nucleus is expelled from developing erythroblasts creating reticulocytes and free nuclei surrounded by plasma membrane. We have studied protein sorting during human erythroblast enucleation using fluorescence activated cell sorting (FACS) to obtain pure populations of reticulocytes and nuclei produced by in vitro culture. Nano LC mass spectrometry was first used to determine the protein distribution profile obtained from the purified reticulocyte and extruded nuclei populations. In general cytoskeletal proteins and erythroid membrane proteins were preferentially restricted to the reticulocyte alongside key endocytic machinery and cytosolic proteins. The bulk of nuclear and ER proteins were lost with the nucleus. In contrast to the localization reported in mice, several key erythroid membrane proteins were detected in the membrane surrounding extruded nuclei, including band 3 and GPC. This distribution of key erythroid membrane and cytoskeletal proteins was confirmed using western blotting. Protein partitioning during enucleation was investigated by confocal microscopy with partitioning of cytoskeletal and membrane proteins to the reticulocyte observed to occur at a late stage of this process when the nucleus is under greatest constriction and almost completely extruded. Importantly, band 3 and CD44 were shown not to restrict specifically to the reticulocyte plasma membrane. This highlights enucleation as a stage at which excess erythroid membrane proteins are discarded in human erythroblast differentiation. Given the striking restriction of cytoskeleton proteins and the fact that membrane proteins located in macromolecular membrane complexes (e.g. GPA, Rh and RhAG) are segregated to the reticulocyte, we propose that the membrane proteins lost with the nucleus represent an excess mobile population of either individual proteins or protein complexes.