Selectivity of major isoquinoline alkaloids from Chelidonium majus towards telomeric G-quadruplex: A study using a transition-FRET (t-FRET) assay

Background Natural 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. Methods The study focuses on the mechanism of telomerase inhibition by stabilization of telomeric G-quadruplex structures by berberine, chelerythrine, chelidonine, sanguinarine and papaverine. Telomerase activity and mRNA levels of hTERT were estimated using quantitative telomere repeat amplification protocol (q-TRAP) and qPCR, in MCF-7 cells treated with different groups of alkaloids. The selectivity of the main isoquinoline alkaloids of Chelidonium majus towards telomeric G-quadruplex 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. Results The 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. Conclusions We 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 significance Understanding 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

ORIGINAL ARTICLES Transcriptional down regulation of hTERT and senescence

induction in HepG2 cells by chelidonin

Dose-Dependent Cytotoxic Effects of Boldine in HepG-2 Cells—Telomerase Inhibition and Apoptosis Induction

Plant metabolites are valuable sources of novel therapeutic compounds. In an anti-telomerase screening study of plant secondary metabolites, the aporphine alkaloid boldine (1,10-dimethoxy-2,9-dihydroxyaporphine) exhibited a dose and time dependent cytotoxicity against hepatocarcinoma HepG-2 cells. Here we focus on the modes and mechanisms of the growth-limiting effects of this compound. Telomerase activity and expression level of some related genes were estimated by real-time PCR. Modes of cell death also were examined by microscopic inspection, staining methods and by evaluating the expression level of some critically relevant genes. The growth inhibition was correlated with down-regulation of the catalytic subunit of telomerase (hTERT) gene (p < 0.01) and the corresponding reduction of telomerase activity in sub-cytotoxic concentrations of boldine (p < 0.002). However, various modes of cell death were stimulated, depending on the concentration of boldine. Very low concentrations of boldine over a few passages resulted in an accumulation of senescent cells so that HepG-2 cells lost their immortality. Moreover, boldine induced apoptosis concomitantly with increasing the expression of bax/bcl2 (p < 0.02) and p21 (p < 0.01) genes. Boldine might thus be an interesting candidate as a potential natural compound that suppresses telomerase activity in non-toxic concentrations

Transcriptional down regulation of hTERT and senescence induction in HepG2 cells by chelidonine

AIM: To investigate the potential effects of chelidonine, the main alkaloid of Chelidonium majus, on telomerase activity and its regulation in HepG2 cells

Antiproliferative Effect of the Isoquinoline Alkaloid Papaverine in Hepatocarcinoma HepG-2 Cells — Inhibition of Telomerase and Induction of Senescence

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 IC50 values for the reduction of both telomerase activity and hTERT expression was 60 µM, while IC50 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

Dose-Dependent Cytotoxic Effects of Boldine in HepG-2 Cells—Telomerase Inhibition and Apoptosis Induction

Plant metabolites are valuable sources of novel therapeutic compounds. In an anti-telomerase screening study of plant secondary metabolites, the aporphine alkaloid boldine (1,10-dimethoxy-2,9-dihydroxyaporphine) exhibited a dose and time dependent cytotoxicity against hepatocarcinoma HepG-2 cells. Here we focus on the modes and mechanisms of the growth-limiting effects of this compound. Telomerase activity and expression level of some related genes were estimated by real-time PCR. Modes of cell death also were examined by microscopic inspection, staining methods and by evaluating the expression level of some critically relevant genes. The growth inhibition was correlated with down-regulation of the catalytic subunit of telomerase (hTERT) gene (p < 0.01) and the corresponding reduction of telomerase activity in sub-cytotoxic concentrations of boldine (p < 0.002). However, various modes of cell death were stimulated, depending on the concentration of boldine. Very low concentrations of boldine over a few passages resulted in an accumulation of senescent cells so that HepG-2 cells lost their immortality. Moreover, boldine induced apoptosis concomitantly with increasing the expression of bax/bcl2 (p < 0.02) and p21 (p < 0.01) genes. Boldine might thus be an interesting candidate as a potential natural compound that suppresses telomerase activity in non-toxic concentrations

Antiproliferative Effect of the Isoquinoline Alkaloid Papaverine in Hepatocarcinoma HepG-2 Cells — Inhibition of Telomerase and Induction of Senescence

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 IC50 values for the reduction of both telomerase activity and hTERT expression was 60 µM, while IC50 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

Telomere shortening in breast cancer cells (MCF7) under treatment with low doses of the benzylisoquinoline alkaloid chelidonine.

Telomeres, the specialized dynamic structures at chromosome ends, regularly shrink with every replication. Thus, they function as an internal molecular clock counting down the number of cell divisions. However, most cancer cells escape this limitation by activating telomerase, which can maintain telomere length. Previous studies showed that the benzylisoquinoline alkaloid chelidonine stimulates multiple modes of cell death and strongly down-regulates telomerase. It is still unknown if down-regulation of telomerase by chelidonine boosts substantial telomere shortening. The breast cancer cell line MCF7 was sequentially treated with very low concentrations of chelidonine over several cell passages. Telomere length and telomerase activity were measured by a monochrome multiplex quantitative PCR and a q-TRAP assay, respectively. Changes in population size and doubling time correlated well with telomerase inhibition and telomere shortening. MCF7 cell growth was arrested completely after three sequential treatments with 0.1 μM chelidonine, each ending after 48 h, while telomere length was reduced to almost 10% of the untreated control. However, treatment with 0.01 μM chelidonine did not have any apparent consequence. In addition to dose and time dependent telomerase inhibition, chelidonine changed the splicing pattern of hTERT towards non-enzyme coding isoforms of the transcript. In conclusion, telomere length and telomere stability are strongly affected by chelidonine in addition to microtubule formation