Differential induction of Leishmania donovani bi-subunit topoisomerase I–DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I

Emergence of the bi-subunit topoisomerase I in the kinetoplastid family (Trypanosoma and Leishmania) has brought a new twist in topoisomerase research related to evolution, functional conservation and preferential sensitivities to the specific inhibitors of type IB topoisomerase family. In the present study, we describe that naturally occurring flavones baicalein, luteolin and quercetin are potent inhibitors of the recombinant Leishmania donovani topoisomerase I. These compounds bind to the free enzyme and also intercalate into the DNA at a very high concentration (300 µM) without binding to the minor grove. Here, we show that inhibition of topoisomerase I by these flavones is due to stabilization of topoisomerase I–DNA cleavage complexes, which subsequently inhibit the religation step. Their ability to stabilize the covalent topoisomerase I–DNA complex in vitro and in living cells is similar to that of the known topoisomerase I inhibitor camptothecin (CPT). However, in contrast to CPT, baicalein and luteolin failed to inhibit the religation step when the drugs were added to pre-formed enzyme substrate binary complex. This differential mechanism to induce the stabilization of cleavable complex with topoisomerase I and DNA by these selected flavones and CPT led us to investigate the effect of baicalein and luteolin on CPT-resistant mutant enzyme LdTOP1Δ39LS lacking 1–39 amino acids of the large subunit [B. B. Das, N. Sen, S. B. Dasgupta, A. Ganguly and H. K. Majumder (2005) J. Biol. Chem. 280, 16335–16344]. Baicalein and luteolin stabilize duplex oligonucleotide cleavage with LdTOP1Δ39LS. This observation was further supported by the stabilization of in vivo cleavable complex by baicalein and luteolin with highly CPT-resistant L.donovani strain. Taken together, our data suggest that the interacting amino acid residues of topoisomerase I may be partially overlapping or different for flavones and CPT. This study illuminates new properties of the flavones and provide additional insights into the ligand binding properties of L.donovani topoisomerase I

The Somatic Genomic Landscape of Glioblastoma

We describe the landscape of somatic genomic alterations based on multi-dimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer

Cytotoxic activity and apoptosis-inducing potential of di-spiropyrrolidino and di-spiropyrrolizidino oxindole andrographolide derivatives.

Anticancer role of andrographolide is well documented. To find novel potent derivatives with improved cytotoxicity than andrographolide on cancer cells, two series of di-spiropyrrolidino- and di-spiropyrrolizidino oxindole andrographolide derivatives prepared by cyclo-addition of azomethine ylide along with sarcosine or proline (viz. sarcosine and proline series respectively) and substitution of different functional groups (-CH3, -OCH3 and halogens) were examined for their cytotoxic effect on a panel of six human cancer cell lines (colorectal carcinoma HCT116 cells, pancreatic carcinoma MiaPaCa-2 cells, hepatocarcinoma HepG2 cells, cervical carcinoma HeLa cells, lung carcinoma A549 and melanoma A375 cells). Except halogen substituted derivatives of proline series (viz. CY2, CY14 and CY15 for Br, Cl and I substitution respectively), none of the other derivatives showed improved cytotoxicity than andrographolide in the cancer cell lines examined. Order of cytotoxicity of the potent compounds is CY2>CY14>CY15>andrographolide. Higher toxicity was observed in HCT116, MiaPaCa-2 and HepG2 cells. CY2, induced death of HCT116 (GI50 10.5), MiaPaCa-2 (GI50 11.2) and HepG2 (GI50 16.6) cells were associated with cell rounding, nuclear fragmentation and increased percentage of apoptotic cells, cell cycle arrest at G1 phase, ROS generation, and involvement of mitochondrial pathway. Upregulation of Bax, Bad, p53, caspases-3,-9 and cleaved PARP; downregulation of Bcl-2, cytosolic NF-κB p65, PI3K and p-Akt; translocation of P53/P21, NF-κB p65 were seen in CY2 treated HCT116 cells. Thus, three halogenated di-spiropyrrolizidino oxindole derivatives of andrographolide are found to be more cytotoxic than andrographolide in some cancer cells. The most potent derivative, CY2 induced death of the cancer cells involves ROS dependent mitochondrial pathway like andrographolide

Analysis of drug induced covalent topoisomerase I–DNA complex formation in promastigotes by KCl-SDS precipitation assay

<p><b>Copyright information:</b></p><p>Taken from "Differential induction of bi-subunit topoisomerase I–DNA cleavage complex by selected flavones and camptothecin: activity of flavones against camptothecin-resistant topoisomerase I"</p><p>Nucleic Acids Research 2006;34(4):1121-1132.</p><p>Published online 18 Feb 2006</p><p>PMCID:PMC1373691.</p><p>© The Author 2006. Published by Oxford University Press. All rights reserved</p> Exponentially growing promastigotes (5 × 10 cells/ml) were labeled with [H]thymidine at 22°C for 24 h and then treated with different concentrations of drugs as indicated. Parts of the labeled cells were treated with DHBA (150 µM) for 10 min before the addition of different concentration of baicalein as indicated. SDS-K precipitable complex were measured as described in Materials and Methods. Experiments were performed three times and representative data from one set of experiments are expressed as means ± SD. Variations among different set of experiments wer

Analysis of apoptosis by flow cytometry in HCT116 cells.

<p>Cells were treated with 20 µM of CY2 (left panel) and andrographolide (right panel) for 24 h and 36 h. Binding of annexin-V/FITC to phosphatidyl serine was determined by flow cytometry. Percentages of apoptotic cells determined by the number of annexin V(+)/propidium iodide cells are shown in the scattered plot.</p