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

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.

SUBMITTER: Fayad W

PROVIDER: S-EPMC2749935 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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

Fayad Walid W Fryknäs Mårten M Brnjic Slavica S Olofsson Maria Hägg MH Larsson Rolf R Linder Stig S

PloS one 20091002 10

<h4>Background</h4>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.<h4>Method and findings</h4>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-ind ...[more]

PMID: 19798419

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Project description:ATP-binding cassette (ABC) drug efflux transporters could contribute to low intracellular concentrations of antiretroviral drugs in HIV-1 cell reservoirs and sanctuary sites. Furthermore, the functional expression of these transporters could be induced in activated T-cells. Therefore, we investigated the expression of ABC drug efflux transporters in human T-cells exposed to an HIV pseudotype virus (pHIVNL4-3), and further examined the potential involvement of the mammalian target of rapamycin (mTOR) signaling pathway in regulating their expression following exposure to pHIVNL4-3. Additionally, we investigated the contribution of the drug efflux transporters to the inflammatory response following pHIVNL4-3-induced T-cell activation. Human peripheral blood mononuclear cells (PBMCs) were exposed to HIV-1 envelope glycoprotein gp120IIIB, pHIVNL4-3 and/or mTOR inhibitors. The expression of ABC transporters, T-cell activation marker CD69, mTOR and pHIVNL4-3 was assessed in CD4+ T-cells by Flow cytometry. mRNA and protein levels of proinflammatory cytokines (IL6, TNFα and INFγ) were examined in PBMCs by qPCR and ELISA analyses, respectively, following exposure to pHIVNL4-3 with or without inhibitors of mTOR or ABC transporters. The expression of ABC transporters (P-glycoprotein, breast cancer resistance protein and multi-drug resistance associated protein-1) was significantly increased in CD4+ T-cells exposed to pHIVNL4-3. Treatment with mTOR inhibitors attenuated pHIVNL4-3-induced transporter expression, as well as mRNA and protein levels of IL6, TNFα and INFγ. Additionally, inhibition of P-gp or MRP1 activity resulted in lower concentrations of proinflammatory cytokines in supernatants of PBMC exposed to pHIVNL4-3. Herein we present novel data demonstrating that upregulation of ABC drug efflux transporters could involve the mTOR signaling pathway in CD4+ T-cells exposed to an HIV pseudotype. These transporters could limit antiretroviral drug penetration in HIV target T-cells. Furthermore, ABC transporters could potentially contribute to HIV-associated proinflammatory cytokine secretion.

Project description:Although the prognostic value of the ATP-binding cassette, subfamily C (ABCC) transporters in childhood neuroblastoma is usually attributed to their role in cytotoxic drug efflux, certain observations have suggested that these multidrug transporters might contribute to the malignant phenotype independent of cytotoxic drug efflux.A v-myc myelocytomatosis viral related oncogene, neuroblastoma derived (MYCN)-driven transgenic mouse neuroblastoma model was crossed with an Abcc1-deficient mouse strain (658 hMYCN(1/-), 205 hMYCN(+/1) mice) or, alternatively, treated with the ABCC1 inhibitor, Reversan (n = 20). ABCC genes were suppressed using short interfering RNA or overexpressed by stable transfection in neuroblastoma cell lines BE(2)-C, SH-EP, and SH-SY5Y, which were then assessed for wound closure ability, clonogenic capacity, morphological differentiation, and cell growth. Real-time quantitative polymerase chain reaction was used to examine the clinical significance of ABCC family gene expression in a large prospectively accrued cohort of patients (n = 209) with primary neuroblastomas. Kaplan-Meier survival analysis and Cox regression were used to test for associations with event-free and overall survival. Except where noted, all statistical tests were two-sided.Inhibition of ABCC1 statistically significantly inhibited neuroblastoma development in hMYCN transgenic mice (mean age for palpable tumor: treated mice, 47.2 days; control mice, 41.9 days; hazard ratio [HR] = 9.3, 95% confidence interval [CI] = 2.65 to 32; P < .001). Suppression of ABCC1 in vitro inhibited wound closure (P < .001) and clonogenicity (P = .006); suppression of ABCC4 enhanced morphological differentiation (P < .001) and inhibited cell growth (P < .001). Analysis of 209 neuroblastoma patient tumors revealed that, in contrast with ABCC1 and ABCC4, low rather than high ABCC3 expression was associated with reduced event-free survival (HR of recurrence or death = 2.4, 95% CI = 1.4 to 4.2; P = .001), with 23 of 53 patients with low ABCC3 expression experiencing recurrence or death compared with 31 of 155 patients with high ABCC3. Moreover, overexpression of ABCC3 in vitro inhibited neuroblastoma cell migration (P < .001) and clonogenicity (P = .03). The combined expression of ABCC1, ABCC3, and ABCC4 was associated with patients having an adverse event, such that of the 12 patients with the "poor prognosis" expression pattern, 10 experienced recurrence or death (HR of recurrence or death = 12.3, 95% CI = 6 to 27; P < .001).ABCC transporters can affect neuroblastoma biology independently of their role in chemotherapeutic drug efflux, enhancing their potential as targets for therapeutic intervention.

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

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

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