Amygdalin blocks bladder cancer cell growth in vitro by diminishing cyclin A and cdk2.

Amygdalin, a natural compound, has been used by many cancer patients as an alternative approach to treat their illness. However, whether or not this substance truly exerts an anti-tumor effect has never been settled. An in vitro study was initiated to investigate the influence of amygdalin (1.25-10 mg/ml) on the growth of a panel of bladder cancer cell lines (UMUC-3, RT112 and TCCSUP). Tumor growth, proliferation, clonal growth and cell cycle progression were investigated. The cell cycle regulating proteins cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin D1, p19, p27 as well as the mammalian target of rapamycin (mTOR) related signals phosphoAkt, phosphoRaptor and phosphoRictor were examined. Amygdalin dose-dependently reduced growth and proliferation in all three bladder cancer cell lines, reflected in a significant delay in cell cycle progression and G0/G1 arrest. Molecular evaluation revealed diminished phosphoAkt, phosphoRictor and loss of Cdk and cyclin components. Since the most outstanding effects of amygdalin were observed on the cdk2-cyclin A axis, siRNA knock down studies were carried out, revealing a positive correlation between cdk2/cyclin A expression level and tumor growth. Amygdalin, therefore, may block tumor growth by down-modulating cdk2 and cyclin A. In vivo investigation must follow to assess amygdalin's practical value as an anti-tumor drug

Familial cleft tongue caused by a unique translation initiation codon variant in TP63

Variants in transcription factor p63 have been linked to several autosomal dominantly inherited malformation syndromes. These disorders show overlapping phenotypic characteristics with various combinations of the following features: ectodermal dysplasia, split-hand/foot malformation/syndactyly, lacrimal duct obstruction, hypoplastic breasts and/or nipples, ankyloblepharon filiforme adnatum, hypospadias and cleft lip/palate. We describe a family with six individuals presenting with a striking novel phenotype characterized by a furrowed or cleft tongue, a narrow face, reddish hair, freckles and various foot deformities. Whole-exome sequencing (WES) identified a novel heterozygous variant, c.3G>T, in TP63 affecting the translation initiation codon (p.1Met?). Sanger sequencing confirmed dominant inheritance of this unique variant in all six affected family members. In summary, our findings indicate that heterozygous variants in TP63 affecting the first translation initiation codon result in a novel phenotype dominated by a cleft tongue, expanding the complex genotypic and phenotypic spectrum of TP63-associated disorders

Loss-of-function variants in DNM1 cause a specific form of developmental and epileptic encephalopathy only in biallelic state

Background Developmental and epileptic encephalopathies (DEEs) represent a group of severe neurological disorders characterised by an onset of refractory seizures during infancy or early childhood accompanied by psychomotor developmental delay or regression. DEEs are genetically heterogeneous with, to date, more than 80 different genetic subtypes including DEE31 caused by heterozygous missense variants in DNM1. Methods We performed a detailed clinical characterisation of two unrelated patients with DEE and used whole-exome sequencing to identify causative variants in these individuals. The identified variants were tested for cosegregation in the respective families. Results We excluded pathogenic variants in known, DEE-associated genes. We identified homozygous nonsense variants, c.97C>T; p.(Gln33*) in family 1 and c.850C>T; p.(Gln284*) in family 2, in the DNM1 gene, indicating that biallelic, loss-of-function pathogenic variants in DNM1 cause DEE. Conclusion Our finding that homozygous, loss-of-function variants in DNM1 cause DEE expands the spectrum of pathogenic variants in DNM1. All parents who were heterozygous carriers of the identified loss-of-function variants were healthy and did not show any clinical symptoms, indicating that the type of mutation in DNM1 determines the pattern of inheritance

FACS analysis of integrin α and β subtype expression on UMUC-3 cells.

<p>The left panel depicts integrin expression as histogram plots with a dotted line indicating background fluorescence and a solid line indicating specific fluorescence in untreated cells. The right panel shows integrin subtype expression after 24 h and 2 weeks amygdalin exposure, compared to controls set at 100%. n.c.  =  not calculated. * indicates significant difference to controls.</p

Knock-down of β1 (UMUC-3, TCCSUP) or β4 (RT112) integrin alters chemotaxis.

<p>Tumor cells were transfected with integrin β1 or β4 siRNA or scrambled siRNA (siRNA control). Controls remained untreated (control). Values are shown as migrated cells per 0.25 mm². One representative of six experiments is shown. * indicates significant difference to the untreated control. Insert taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110244#pone-0110244-g008" target="_blank">figure 8</a>.</p

Up: Growth of UMUC-3, TCCSUP and RT112 bladder cancer cells treated with different concentrations of amygdalin after 24 h, 48 h and 72 h.

<p>Controls remained untreated. Down: Tumor cell growth after 2 weeks treatment with 10 mg/ml amygdalin. Each experiment was done in triplicate and repeated 5 times. Data from one representative experiment is shown. *indicates significant difference to controls (p = 0.0022).</p

Effect of amygdalin on bladder cancer cell migration.

<p>Tumor cells treated with amygdalin for 24 h or for 2 weeks were seeded in the upper chamber with a chemo-attractant in the lower well. Cells were allowed to move for 20 h, either in amygdalin-free medium (amygdalin-A) or in amygdalin-containing medium (amygdalin-B). Cells migrating to the lower membrane surface were counted. Controls were set to 100%. One representative of six experiments is shown. *  =  significant difference to controls. #  =  significant difference between amygdalin-A and amygdalin-B.</p

Influence of β1 (UMUC-3, TCCSUP) or β4 (RT112) knock-down on bladder cancer cell adhesion to collagen.

<p>Tumor cells were transfected with integrin β1 or β4 siRNA. Non- treated cells (control) and cells treated with scrambled siRNA (siRNA control) served as controls. Efficacy of receptor knockdown was evaluated by western blotting (lower right). One representative of six experiments is shown. *indicates significant difference to the controls.</p

Adhesion of UMUC-3, TCCSUP and RT112 bladder cancer cells to immobilized collagen.

<p>Tumor cells were treated with 10 mg/ml amygdalin for either 24 h or for 2 weeks. Cells not treated with amygdalin served as the controls. 0.5×10⁶ cells/well were added to immobilized collagen for 60 min. Mean number of adherent tumor cells from five fields was calculated. One representative of six experiments is shown. *indicates significant difference to controls.</p