[Abstract unavailable

Crocin, the main pigment of Crocus sativus L., has been shown to have antiproliferative effects on cancer cells, but the involved mechanisms are only poor understood. This study focused on probable effect of crocin on the immortality of hepatic cancer cells. Cytotoxicity of crocin (IC 50 3 mg/ml) in hepatocarcinoma HepG2 cells was determined after 48 h by neutral red uptake assay and MTT test. Immortality was investigated through quantification of relative telomerase activity with a quantitative real-time PCR-based telomerase repeat amplification protocol (qTRAP). Telomerase activity in 0.5 μg protein extract of HepG2 cells treated with 3 mg/ml crocin was reduced to about 51% as compared to untreated control cells. Two mechanisms of inhibition, i.e. interaction of crocin with telomeric quadruplex sequences and down regulation of hTERT expression, were examined using FRET analysis to measure melting temperature of a synthetic telomeric oligonucleotide in the presence of crocin and quantitative real-time RT-PCR, respectively. No significant changes were observed in the T m telomeric oligonucleotides, while the relative expression level of the catalytic subunit of telomerase (hTERT) gene showed a 60% decrease as compared to untreated control cells. In conclusion, telomerase activity of HepG2 cells decreases after treatment with crocin, which is probably caused by down-regulation of the expression of the catalytic subunit of the enzyme.

Cancer cells are often immortal through up-regulation of the hTERT gene, which encodes the catalytic subunit of a special reverse transcriptase to overcome end-replication problem of chromosomes. This study demonstrates that papaverine, an isoquinoline alkaloid from the Papaveraceae, can overcome telomerase dependent immortality of HepG-2 cells that was used as a model of hepatocarcinoma. Although this alkaloid does not directly interact with telomeric sequences, papaverine inhibits telomerase through down-regulation of hTERT, which was analysed using thermal FRET and qRT-PCR, respectively. The IC 50 values for the reduction of both telomerase activity and hTERT expression was 60 µM, while IC 50 for cytotoxicity was 120 µM. Repeated treatments of the cells with very low non-toxic concentrations of papaverine resulted in growth arrest and strong reduction of population doublings after 40 days. This treatment induced senescent morphology in HepG-2 cells, which was evaluated by beta-galactosidase staining. Altogether, papaverine can be regarded as a promising model compound for drug design targeting cancer development.

Chelidonine reduces telomerase activity through down-regulation of hTERT expression. Senescence induction might not be directly caused by reducing telomerase activity as it occurs after a few population doublings.

BackgroundNatural bioproducts are invaluable resources in drug discovery. Isoquinoline alkaloids of Chelidonium majus constitute a structurally diverse family of natural products that are of great interest, one of them being their selectivity for human telomeric G-quadruplex structure and telomerase inhibition. MethodsThe study focuses on the mechanism of telomerase inhibition by stabilization of telomeric Gquadruplex structures by berberine, chelerythrine, chelidonine, sanguinarine and papaverine. Telomerase activity and mRNA levels of hTERT were estimated using quantitative telomere repeat amplification protocol and qPCR, in MCF-7 cells treated with different groups of alkaloids. The selectivity of the main isoquinoline alkaloids of Chelidonium majus towards telomeric Gquadruplex forming sequences were explored using a sensitive modified thermal FRET-melting measurement in the presence of the complementary oligonucleotide CT22. We assessed and monitored G-quadruplex topologies using circular dichroism (CD) methods, and compared spectra to previously well-characterized motifs, either alone or in the presence of the alkaloids, Molecular modeling was performed to rationalize ligand binding to the G-quadruplex structure. ResultsThe results highlight strong inhibitory effects of chelerythrine, sanguinarine and berberine on telomerase activity, most likely through substrate sequestration. These isoquinoline alkaloids interacted strongly with telomeric sequence G-quadruplex. In comparison, chelidonine and papaverine had no significant interaction with the telomeric quadruplex, while they strongly inhibited telomerase at transcription level of hTERT. Altogether, all of the studied alkaloids showed various levels and mechanisms of telomerase inhibition. ConclusionsWe report on a comparative study of anti-telomerase activity of the isoquinoline alkaloids of Chelidonium majus. Chelerythrine was most effective in inhibiting telomerase activity by substrate sequesteration through G-quadruplex stabilization. General SignificanceUnderstanding structural and molecular mechanisms of anti-cancer agents can help in developing new and more potent drugs with fewer side effects. Isoquinolines are the most biologically active agents from Chelidonium majus, which have shown to be telomeric G-quadruplex stabilizers and potent telomerase inhibitors.

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AIM:To identify the altered gene expression patterns in squamous cell carcinoma of esophagus (ESCC) in relation to adjacent normal esophageal epithelium. METHODS:Total RNA was extracted using SV total RNA isolation kit from snap frozen tissues of ESCC samples and normal esophageal epithelium far from the tumor. Radiolabeled cDNA were synthesized from equal quantities of total RNAs of tumor and normal tissues using combinations of 24 arbitrary 13-mer primers and three different anchoring oligo-dT primers and separated on sequencing gels. cDNA with considerable different amounts of signals in tumor and normal tissue were reamplified and cloned. Using southern blot, the clones of each band were controlled for false positive results caused by probable heterogeneity of cDNA population with the same size. Clones that confirmed differential expression by slot blot selected for sequencing and northern analysis. Corresponding full-length gene sequences was predicted using human genome project data, related transcripts were translated and used for various protein/motif searches to speculate their probable functions.

The Persian racerunner Eremias persica Blanford, 1875 is confined to the Iranian plateau, and forms one of the most widespread but rarely studied species of the family Lacertidae. With many local populations inhabiting a variety of habitats, and exhibiting considerable morphological, genetic, and ecological variations, it represents a species complex. We analysed sequences of mitochondrial cytochrome b and 12S ribosomal RNA (rRNA) genes derived from 13 geographically distant populations belonging to the E. persica complex. Using our knowledge of palaeogeographical events, a molecular clock was calibrated to assess the major events in fragmentation, radiation, and intraspecific variation. The sequence data strongly support a basal separation of the highland populations of western Iran from those of the open steppes and deserts, occurring in the east. The subsequent radiation, fragmentation, and evolution of these major assemblages have led to several discernable geographical lineages across the wide area of the Iranian plateau. The results indicate a middle-Miocene origin for the clade as a whole. The first split, isolating the western and eastern clades, appears to have occurred 11-10 Mya. Further fragmentations and divergence within the major clades began about 8 Mya, with an evolutionary rate of 1.6% sequence divergence per million years among the lineages in the genes studied (combined data set). Molecular and morphological data strongly support a taxonomic revision of this species complex. At least four of the discovered clades should be raised to species, and two to subspecies, rank.