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, 4 (10), e7238

Identification of a Novel Topoisomerase Inhibitor Effective in Cells Overexpressing Drug Efflux Transporters


Identification of a Novel Topoisomerase Inhibitor Effective in Cells Overexpressing Drug Efflux Transporters

Walid Fayad et al. PLoS One.


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.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. 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.
Figure 2
Figure 2. In vivo induction of tumor apoptosis by thaspine.
(A) Induction of apoptosis in HCT116 tumors in vivo. SCID mice were injected with 10 mg/kg thaspine. Mice were sacrificed after 48 hours and tumors stained for active caspase-3 (upper panel: thaspine treated mice; lower panel: PBS injected mice). (B, C) SCID mice carrying HCT116 tumors (B) or FaDu head-neck carcinoma tumors (C) were injected with 10 mg/kg of thaspine or with PBS and the levels of human caspase-cleaved CK18 (CK18-Asp396) were determined in mouse plasma 48 hours after treatment using ELISA. The antibodies used to detect CK18-Asp396 do not bind mouse CK18. Four mice in each group; bars represent S.D. (D) SCID mice carrying FaDu tumors were treated with 10 mg/kg of thaspine (day 1, arrow) and tumor volume was calculated; black circles: untreated mice, red squares: treated mice (4 mice in each group). Error bars are presented as unidirectional for figure clarity. Animals were sacrificed when tumors were approximately 1 cm3 according to local animal welfare regulations. Statistical significance was calculated using Student's t-test.
Figure 3
Figure 3. Induction of the mitochondrial apoptosis pathway by thaspine.
(A) loss of mitochondrial membrane potential in thaspine-treated HCT116 cells. Control and thaspine-treated cells (10 µM, 18 hours) were stained with tetramethyl-rhodamine ethyl ester (TMRE) and fluorescence was measured by flow cytometry; (B) Release of cytochrome c to the cytosol of thaspine-treated cells. Cells were treated with 10 µM thaspine for 7 or 16 hours. Cytochrome c was quantified in mitochondrial and cytosolic fractions by Western blotting. (C) thaspine induces the active conformation of Bak and Bax. Cells were treated with 10 µM thaspine, fixed and stained with conformation-specific antibodies to Bak and Bax. Note induction of the active conformation of both molecules by thaspine (red arrows: immunofluorescent signal in untreated cells; green arrows: immunofluorescent signal in drug-treated cells). (D) Inhibition of thaspine-induced apoptosis by siRNA to Bik and Bid. Cells were transfected with siRNAs in triplicate 96 well plates and treated with thaspine or solvent (see Material and Methods). After 24 hours the levels of caspase-cleaved CK18 were determined using the M30 CytoDeath ELISA.
Figure 4
Figure 4. 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.
Figure 5
Figure 5. Thaspine induces wide-spread activation of caspase-3 in spheroids.
HCT116 spheroids with homogeneous diameters were formed using the hanging drop technique as described . Five days after formation spheroids were compact and contained proliferating cells only in the surface layers (Ki67 staining, top left). Spheroids were treated with drugs for the times indicated, fixed, sectioned and stained for active caspase-3. Thaspine was used at 20 µM, doxorubicin at 20 µM, and cisplatin at 40 µM.

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