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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.

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Induction of apoptosis by thaspine.(A) chemical structure of thaspine (NSC76022); (B) induction of caspase-cleaved CK18 by thaspine, cisplatin, doxorubicin and mechlorethamine in HCT116 colon carcinoma cells. Treatment was for 24 hours with the indicated concentrations of compounds. Cells were lysed and CK18-Asp396 was determined using the M30 CytoDeath ELISA. Results are shown with S.D. from triplicate determinations. Similar results (including the biphasic response to doxorubicin) were observed in independent experiments.
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pone-0007238-g001: Induction of apoptosis by thaspine.(A) chemical structure of thaspine (NSC76022); (B) induction of caspase-cleaved CK18 by thaspine, cisplatin, doxorubicin and mechlorethamine in HCT116 colon carcinoma cells. Treatment was for 24 hours with the indicated concentrations of compounds. Cells were lysed and CK18-Asp396 was determined using the M30 CytoDeath ELISA. Results are shown with S.D. from triplicate determinations. Similar results (including the biphasic response to doxorubicin) were observed in independent experiments.

Mentions: We used HCT116 colon carcinoma cells as target cells to screen for apoptosis-inducing agents present in NCI Natural Product Set (www.dtp.nci.nih.gov). Apoptosis was determined using a modification of the M30-Apoptosense® method [9] which specifically measures caspase-cleaved cytokeratin 18 formed in apoptotic cells. Activity in this assay is inhibited by the pan-caspase inhibitor zVAD-fmk [9]. The M30-Apoptosense® method is a useful screening tool since it measures the accumulation of the apoptotic product in cell cultures, leading to an integrative determination of apoptosis to the point of harvesting the cells. Using a compound concentration of 25 µM and an exposure time of 24 hours, 20 compounds were identified as inducing apoptosis above a preselected threshold value (Table 1). Molecular targets have been reported on 14 of these 20 compounds (Table 1). The alkaloid thaspine (taspine; NSC76022) was one of the remaining 6 compounds with unknown mechanism of action (Figure 1A). Thaspine is of interest since it is an alkaloid from Dragon's blood, a latex prepared from the cortex of the tree Croton lechleri and used by tribes in the Amazonas basin for medicinal purposes. Thaspine induced strong caspase-cleavage of cytokeratin-18 in HCT116 cells at a concentration of ∼10 µM (Fig. 1B). This concentration requirement is similar to that of other cancer therapeutic drugs such as cisplatin (∼20 µM), doxorubicin (∼3 µM) and mechlorethamine (∼20 µM) for induction of caspase activity of this cell line (Fig. 1B). Thaspine was also found to induce activation of caspase-3 at 10 and 16 hours (see below).


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)

Induction of apoptosis by thaspine.(A) chemical structure of thaspine (NSC76022); (B) induction of caspase-cleaved CK18 by thaspine, cisplatin, doxorubicin and mechlorethamine in HCT116 colon carcinoma cells. Treatment was for 24 hours with the indicated concentrations of compounds. Cells were lysed and CK18-Asp396 was determined using the M30 CytoDeath ELISA. Results are shown with S.D. from triplicate determinations. Similar results (including the biphasic response to doxorubicin) were observed in independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0007238-g001: Induction of apoptosis by thaspine.(A) chemical structure of thaspine (NSC76022); (B) induction of caspase-cleaved CK18 by thaspine, cisplatin, doxorubicin and mechlorethamine in HCT116 colon carcinoma cells. Treatment was for 24 hours with the indicated concentrations of compounds. Cells were lysed and CK18-Asp396 was determined using the M30 CytoDeath ELISA. Results are shown with S.D. from triplicate determinations. Similar results (including the biphasic response to doxorubicin) were observed in independent experiments.
Mentions: We used HCT116 colon carcinoma cells as target cells to screen for apoptosis-inducing agents present in NCI Natural Product Set (www.dtp.nci.nih.gov). Apoptosis was determined using a modification of the M30-Apoptosense® method [9] which specifically measures caspase-cleaved cytokeratin 18 formed in apoptotic cells. Activity in this assay is inhibited by the pan-caspase inhibitor zVAD-fmk [9]. The M30-Apoptosense® method is a useful screening tool since it measures the accumulation of the apoptotic product in cell cultures, leading to an integrative determination of apoptosis to the point of harvesting the cells. Using a compound concentration of 25 µM and an exposure time of 24 hours, 20 compounds were identified as inducing apoptosis above a preselected threshold value (Table 1). Molecular targets have been reported on 14 of these 20 compounds (Table 1). The alkaloid thaspine (taspine; NSC76022) was one of the remaining 6 compounds with unknown mechanism of action (Figure 1A). Thaspine is of interest since it is an alkaloid from Dragon's blood, a latex prepared from the cortex of the tree Croton lechleri and used by tribes in the Amazonas basin for medicinal purposes. Thaspine induced strong caspase-cleavage of cytokeratin-18 in HCT116 cells at a concentration of ∼10 µM (Fig. 1B). This concentration requirement is similar to that of other cancer therapeutic drugs such as cisplatin (∼20 µM), doxorubicin (∼3 µM) and mechlorethamine (∼20 µM) for induction of caspase activity of this cell line (Fig. 1B). Thaspine was also found to induce activation of caspase-3 at 10 and 16 hours (see below).

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