Limits...
Identification of a novel topoisomerase inhibitor effective in cells overexpressing drug efflux transporters.

Fayad W, Fryknäs M, Brnjic S, Olofsson MH, Larsson R, Linder S - PLoS ONE (2009)

Bottom Line: Thaspine was found to induce conformational activation of the pro-apoptotic proteins Bak and Bax, mitochondrial cytochrome c release and mitochondrial membrane permeabilization in HCT116 cells.Inhibition of both topoisomerase I and II was observed using in vitro assays, and thaspine was found to have a reduced cytotoxic effect on a cell line with a mutated topoisomerase II enzyme.The alkaloid thaspine from the cortex of Croton lechleri is a dual topoisomerase inhibitor effective in cells overexpressing drug efflux transporters and induces wide-spread apoptosis in multicellular spheroids.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology and Pathology, Karolinska Institute and Hospital, Stockholm, Sweden.

ABSTRACT

Background: Natural product structures have high chemical diversity and are attractive as lead structures for discovery of new drugs. One of the disease areas where natural products are most frequently used as therapeutics is oncology.

Method and findings: A library of natural products (NCI Natural Product set) was screened for compounds that induce apoptosis of HCT116 colon carcinoma cells using an assay that measures an endogenous caspase-cleavage product. One of the apoptosis-inducing compounds identified in the screen was thaspine (taspine), an alkaloid from the South American tree Croton lechleri. The cortex of this tree is used for medicinal purposes by tribes in the Amazonas basin. Thaspine was found to induce conformational activation of the pro-apoptotic proteins Bak and Bax, mitochondrial cytochrome c release and mitochondrial membrane permeabilization in HCT116 cells. Analysis of the gene expression signature of thaspine-treated cells suggested that thaspine is a topoisomerase inhibitor. Inhibition of both topoisomerase I and II was observed using in vitro assays, and thaspine was found to have a reduced cytotoxic effect on a cell line with a mutated topoisomerase II enzyme. Interestingly, in contrast to the topoisomerase II inhibitors doxorubicin, etoposide and mitoxantrone, thaspine was cytotoxic to cell lines overexpressing the PgP or MRP drug efflux transporters. We finally show that thaspine induces wide-spread apoptosis in colon carcinoma multicellular spheroids and that apoptosis is induced in two xenograft mouse models in vivo.

Conclusions: The alkaloid thaspine from the cortex of Croton lechleri is a dual topoisomerase inhibitor effective in cells overexpressing drug efflux transporters and induces wide-spread apoptosis in multicellular spheroids.

Show MeSH

Related in: MedlinePlus

Thaspine is a topoisomerase inhibitor.(A) Connectivity Map (CMAP) results after treatment with thaspine. The bar view is constructed from 6100 horizontal lines, each representing a single treatment and ordered according to their corresponding enrichment to the query signatures generated after thaspine treatment. Black horizontal lines in the barview represent the individual instances for ellipticine and camptothecin when the thaspine expression signature was used as query signature. Score according to the CMAP database; (B) inhibition of topoisomerase I activity: 1: plasmid +5U topoisomerase I; 2: plasmid; 3: plasmid +5U topoisomerase I+DMSO (1 µm); 4. Marker for nicked plasmid; 5: plasmid +5U topoisomerase I +50 µM thaspine; 6: plasmid +5U topoisomerase I +25 µM thaspine; 7: plasmid +5U topoisomerase I +10 µM thaspine; 8: plasmid + DMSO (1 µm). Topoisomerase I primarily induced nicked plasmid DNA, note the inhibition by thaspine. (C) inhibition of topoisomerase II activity: 1: plasmid +15U topoisomerase II; 2: plasmid; 3: plasmid +15U topoisomerase II + DMSO (1 µm); 4. Marker for nicked plasmid; 5: plasmid +15U topoisomerase II +50 µM thaspine; 6: plasmid +15U topoisomerase II +25 µM thaspine; 7: plasmid +15U topoisomerase II +10 µM thaspine; 8: plasmid + DMSO (1 µm). Topoisomerase II primarily induced nicked plasmid DNA, note the inhibition by thaspine.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2749935&req=5

pone-0007238-g004: Thaspine is a topoisomerase inhibitor.(A) Connectivity Map (CMAP) results after treatment with thaspine. The bar view is constructed from 6100 horizontal lines, each representing a single treatment and ordered according to their corresponding enrichment to the query signatures generated after thaspine treatment. Black horizontal lines in the barview represent the individual instances for ellipticine and camptothecin when the thaspine expression signature was used as query signature. Score according to the CMAP database; (B) inhibition of topoisomerase I activity: 1: plasmid +5U topoisomerase I; 2: plasmid; 3: plasmid +5U topoisomerase I+DMSO (1 µm); 4. Marker for nicked plasmid; 5: plasmid +5U topoisomerase I +50 µM thaspine; 6: plasmid +5U topoisomerase I +25 µM thaspine; 7: plasmid +5U topoisomerase I +10 µM thaspine; 8: plasmid + DMSO (1 µm). Topoisomerase I primarily induced nicked plasmid DNA, note the inhibition by thaspine. (C) inhibition of topoisomerase II activity: 1: plasmid +15U topoisomerase II; 2: plasmid; 3: plasmid +15U topoisomerase II + DMSO (1 µm); 4. Marker for nicked plasmid; 5: plasmid +15U topoisomerase II +50 µM thaspine; 6: plasmid +15U topoisomerase II +25 µM thaspine; 7: plasmid +15U topoisomerase II +10 µM thaspine; 8: plasmid + DMSO (1 µm). Topoisomerase II primarily induced nicked plasmid DNA, note the inhibition by thaspine.

Mentions: The molecular target of thaspine is not known. To generate hypotheses regarding the mechanism of action, we used the Connectivity Map (CMAP) [18], which is a large compendium of gene expression signatures from drug-treated cell lines. We analyzed the drug-induced gene expression changes compared to vehicle control in the breast cancer cell line MCF-7, since all of the 1,309 compounds in CMAP have been tested on this cell line. The gene expression response to thaspine was similar to that of ellipticine, a known topoisomerase II inhibitor, and similar to the response to the topoisomerase I inhibitor camptothecin (Figure 4A).


Identification of a novel topoisomerase inhibitor effective in cells overexpressing drug efflux transporters.

Fayad W, Fryknäs M, Brnjic S, Olofsson MH, Larsson R, Linder S - PLoS ONE (2009)

Thaspine is a topoisomerase inhibitor.(A) Connectivity Map (CMAP) results after treatment with thaspine. The bar view is constructed from 6100 horizontal lines, each representing a single treatment and ordered according to their corresponding enrichment to the query signatures generated after thaspine treatment. Black horizontal lines in the barview represent the individual instances for ellipticine and camptothecin when the thaspine expression signature was used as query signature. Score according to the CMAP database; (B) inhibition of topoisomerase I activity: 1: plasmid +5U topoisomerase I; 2: plasmid; 3: plasmid +5U topoisomerase I+DMSO (1 µm); 4. Marker for nicked plasmid; 5: plasmid +5U topoisomerase I +50 µM thaspine; 6: plasmid +5U topoisomerase I +25 µM thaspine; 7: plasmid +5U topoisomerase I +10 µM thaspine; 8: plasmid + DMSO (1 µm). Topoisomerase I primarily induced nicked plasmid DNA, note the inhibition by thaspine. (C) inhibition of topoisomerase II activity: 1: plasmid +15U topoisomerase II; 2: plasmid; 3: plasmid +15U topoisomerase II + DMSO (1 µm); 4. Marker for nicked plasmid; 5: plasmid +15U topoisomerase II +50 µM thaspine; 6: plasmid +15U topoisomerase II +25 µM thaspine; 7: plasmid +15U topoisomerase II +10 µM thaspine; 8: plasmid + DMSO (1 µm). Topoisomerase II primarily induced nicked plasmid DNA, note the inhibition by thaspine.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2749935&req=5

pone-0007238-g004: Thaspine is a topoisomerase inhibitor.(A) Connectivity Map (CMAP) results after treatment with thaspine. The bar view is constructed from 6100 horizontal lines, each representing a single treatment and ordered according to their corresponding enrichment to the query signatures generated after thaspine treatment. Black horizontal lines in the barview represent the individual instances for ellipticine and camptothecin when the thaspine expression signature was used as query signature. Score according to the CMAP database; (B) inhibition of topoisomerase I activity: 1: plasmid +5U topoisomerase I; 2: plasmid; 3: plasmid +5U topoisomerase I+DMSO (1 µm); 4. Marker for nicked plasmid; 5: plasmid +5U topoisomerase I +50 µM thaspine; 6: plasmid +5U topoisomerase I +25 µM thaspine; 7: plasmid +5U topoisomerase I +10 µM thaspine; 8: plasmid + DMSO (1 µm). Topoisomerase I primarily induced nicked plasmid DNA, note the inhibition by thaspine. (C) inhibition of topoisomerase II activity: 1: plasmid +15U topoisomerase II; 2: plasmid; 3: plasmid +15U topoisomerase II + DMSO (1 µm); 4. Marker for nicked plasmid; 5: plasmid +15U topoisomerase II +50 µM thaspine; 6: plasmid +15U topoisomerase II +25 µM thaspine; 7: plasmid +15U topoisomerase II +10 µM thaspine; 8: plasmid + DMSO (1 µm). Topoisomerase II primarily induced nicked plasmid DNA, note the inhibition by thaspine.
Mentions: The molecular target of thaspine is not known. To generate hypotheses regarding the mechanism of action, we used the Connectivity Map (CMAP) [18], which is a large compendium of gene expression signatures from drug-treated cell lines. We analyzed the drug-induced gene expression changes compared to vehicle control in the breast cancer cell line MCF-7, since all of the 1,309 compounds in CMAP have been tested on this cell line. The gene expression response to thaspine was similar to that of ellipticine, a known topoisomerase II inhibitor, and similar to the response to the topoisomerase I inhibitor camptothecin (Figure 4A).

Bottom Line: Thaspine was found to induce conformational activation of the pro-apoptotic proteins Bak and Bax, mitochondrial cytochrome c release and mitochondrial membrane permeabilization in HCT116 cells.Inhibition of both topoisomerase I and II was observed using in vitro assays, and thaspine was found to have a reduced cytotoxic effect on a cell line with a mutated topoisomerase II enzyme.The alkaloid thaspine from the cortex of Croton lechleri is a dual topoisomerase inhibitor effective in cells overexpressing drug efflux transporters and induces wide-spread apoptosis in multicellular spheroids.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology and Pathology, Karolinska Institute and Hospital, Stockholm, Sweden.

ABSTRACT

Background: Natural product structures have high chemical diversity and are attractive as lead structures for discovery of new drugs. One of the disease areas where natural products are most frequently used as therapeutics is oncology.

Method and findings: A library of natural products (NCI Natural Product set) was screened for compounds that induce apoptosis of HCT116 colon carcinoma cells using an assay that measures an endogenous caspase-cleavage product. One of the apoptosis-inducing compounds identified in the screen was thaspine (taspine), an alkaloid from the South American tree Croton lechleri. The cortex of this tree is used for medicinal purposes by tribes in the Amazonas basin. Thaspine was found to induce conformational activation of the pro-apoptotic proteins Bak and Bax, mitochondrial cytochrome c release and mitochondrial membrane permeabilization in HCT116 cells. Analysis of the gene expression signature of thaspine-treated cells suggested that thaspine is a topoisomerase inhibitor. Inhibition of both topoisomerase I and II was observed using in vitro assays, and thaspine was found to have a reduced cytotoxic effect on a cell line with a mutated topoisomerase II enzyme. Interestingly, in contrast to the topoisomerase II inhibitors doxorubicin, etoposide and mitoxantrone, thaspine was cytotoxic to cell lines overexpressing the PgP or MRP drug efflux transporters. We finally show that thaspine induces wide-spread apoptosis in colon carcinoma multicellular spheroids and that apoptosis is induced in two xenograft mouse models in vivo.

Conclusions: The alkaloid thaspine from the cortex of Croton lechleri is a dual topoisomerase inhibitor effective in cells overexpressing drug efflux transporters and induces wide-spread apoptosis in multicellular spheroids.

Show MeSH
Related in: MedlinePlus