DNA Topo Research Articles

Antitumor activities on HL-60 human leukemia cell line, molecular docking, and quantum-chemical calculations of some sulfonamide-benzoxazoles

We previously synthesized some novel benzoxazole derivatives-containing sulfonamide. In this study, the compounds were investigated for their antitumor activities against the HL-60 human leukemia cells, using the MTT assay. Moreover, quantum chemical calculations using the DFT methods were applied for understanding the difference in antitumor activity. Additionally, molecular docking into active site of the DNA Topo II enzyme was performed on 3QX3. PDB file in order to find out possible mechanism of antitumor effect.According to all obtained results showed that compounds 1b, 1c, and 1d could be potential drug candidates as new antitumor agents, and are promising for cancer therapy.

Abstract 4792: The effects of KPT-330, a selective inhibitor of nuclear export, on nuclear topoisomerase II alpha levels and etoposide activity in human myeloid leukemia HL-60 cells

Abstract DNA topoisomerase IIá (topo II) is a known cargo protein for exportin 1 (XPO1, CRM1) (Seminars Cancer Biol. 27: 62-73, 2014) for nuclear export. Here we utilized KPT-330, a selective inhibitor of nuclear export, to evaluate its cytotoxicity, its effects on the nuclear content of topo II in human myeloid leukemia HL60 cells and on the subsequent activity of the topo II targeting agent etoposide. Since KPT-330 is a potential michael acceptor and is known to interact with the CRM1 active site Cys528 to inhibit its activity, we first analyzed interaction of KPT-330 with glutathione (GSH)-containing cysteine. In an in vitro binding study KPT-330 was weakly bound to GSH with a Kd = 130+/-24 μM. In contrast, the natural product compound parthenolide, also a michael acceptor, bound to GSH with 30-fold greater affinity. Depletion of cellular GSH with buthionine sulfoximine (BSO) did not significantly alter the 72 hr growth inhibitory effects of KPT-330 yielding I50-values of 109 +/- 19 and 80 +/- 44 nM (p = 0.133) in the absence and presence of BSO. In contrast, GSH depletion significantly enhanced the activity of parthenolide yielding I50-values of 2.59 +/- 0.16 and 1.35 +/- 0.15 μM (p<0.001) in the absence or presence of BSO. KPT-330 (50 μM) did not alter cellular GSH levels in HL60 cells. Using 3’-(p-hydroxyphenyl) fluorescein, KPT-330 was antioxidant in GSH replete and depleted HL60 cells. Together results indicate that conjugation with GSH does not play a significant role in KPT-330 activity. Less than additive cytotoxicity (trypan blue) and apoptosis (Hoescht) were observed using a single fixed ratio of KPT-330 and etoposide incubated either simultaneously or after overnight pre-incubation with KPT-330. Similarly, using the Chou and Talalay technique, KPT-330/etoposide combinations were less than additive in exponentially growing HL60 cells. The mechanism(s) for this apparent antagonism using this combination are under investigation. Using fluorescently labeled topo II antibody, nuclear content of exponentially growing HL60 cells was 85.9 +/- 2.3% of total in the absence of KPT-330 and 86.5+/- 1.8% after an 16 hr incubation with 100 nM KPT-330. In plateau phase cells there was a statistically significant decrease in nuclear topoisomerase IIá to 74.1+/-1.8% compared to exponentially growing cells (p<0.05). In these plateau phase cells, a similar 16 hr incubation with 100 nM KPT-330 resulted in a significant increase in nuclear topoisomerase IIá to 82.6+/-2.5% of total compared to controls (p<0.05). Results are in accord with the known CRM1-mediated shuttling of topo II from the nucleus only in cells approaching or in plateau phase (Exp. Cell Res. 313: 627-637, 2007). Studies are underway to evaluate etoposide-mediated DNA damage and topo II-covalent complexes in HL60 cells in various growth phases after KPT-330 treatment as a correlate with nuclear topo II residency. Citation Format: Erica Godley, Ragu Kanagasabai, Soumendrakrishna Karmahapatra, Jack C. Yalowich. The effects of KPT-330, a selective inhibitor of nuclear export, on nuclear topoisomerase II alpha levels and etoposide activity in human myeloid leukemia HL-60 cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4792.

Dual inhibition of Staphylococcus aureus DNA gyrase and topoisomerase IV activity by phenylalanine-derived (Z)-5-arylmethylidene rhodanines

Methicillin resistant Staphylococcus aureus (MRSA) is a major drug resistant bacteria that persists in both community and clinical settings due to growing resistance to current drug regimens. Thus, there is a continued need for novel compounds that are active against this organism. Previously, we reported that various rhodanine derivatives inhibited the supercoiling activity of DNA gyrase. In this study, we determined the effect of new phenylalanine-derived (Z)-5-arylmethylidene rhodanines (which are efficacious against MRSA) on the activity of the two type II bacterial topoisomerases, DNA gyrase and topoisomerase IV (Topo IV). Compounds 1 and 9 showed the greatest efficacy against DNA gyrase with a minimal inhibitory concentration (MIC) of 5μM while compounds 2 and 3 were the most efficacious against Topo IV with MIC values of 0.75μM and 0.5μM, respectively. Induced fit docking, using the crystallographic structures of the target enzymes, indicated that these rhodanine derivatives bind to the ATPase domain of gyrB and ParE subunits on DNA gyrase and Topo IV, respectively. These new compounds were efficacious against both DNA gyrase and Topo IV. The increased efficacy of these new rhodanine compounds, as compared to other rhodanine derivatives, results from their dual inhibition of DNA gyrase and Topo IV, thereby making them good candidates for further drug design and development.

Analysis of gene expression during aging of CGNs in culture: implication of SLIT2 and NPY in senescence.

Senescence is the major key factor that leads to the loss of neurons throughout aging. Cellular senescence is not the consequence of single cause, but there are multiple aspects which may induce senescence in a cell. Various causes such as gene expression, molecular interactions and protein processing and chromatin organization are described as causal factor for senescence. It is well known that the damage to the nuclear or mitochondrial DNA contributes to the aging either directly by inducing the apoptosis/cellular senescence or indirectly by altering cellular functions. The significant nuclear DNA damage with the age is directly associated with the continuous declining in DNA repair. The continuous decline in expression of topoisomerase 2 beta (Topo IIβ) in cultured cerebellar granule neurons over time indicated the decline in the repair of damage DNA. DNA Topo IIβ is an enzyme that is crucial for solving topological problems of DNA and thus has an important role in DNA repair. The enzyme is predominantly present in non-proliferating cells such as neurons. In this paper, we have studied the genes which were differentially expressed over time in cultured cerebellar granule neurons (CGNs) and identified potential genes associated with the senescence. Our results showed that the two genes neuropeptide Y (Npy) and Slit homolog 2 (Drosophila) (Slit2) gradually increase during aging, and upon suppression of these two genes, there was gradual increase in cell viability along with restoration of the expression of Topo IIβ and potential repair proteins.

New compound with DNA Topo I inhibitory activity purified from Penicillium oxalicum HSY05

Strain HSY05 was isolated from sea sediment collected from the South China Sea and was later identified as Penicillium oxalicum by 16S rDNA sequence analysis. Various chromatographic processes led to the isolation and purification of two metabolites from the fermentation culture of HSY05, including one new compound, 2,2′,4,4′-tetrahyoxy-8′-methyl-6-methoxy-acyl-ethyl-diphenylmethanone (1), and a known compound secalonic acid D (SAD, 2), as characterised by UV, IR, 1D, 2D-NMR and MS data. The inhibitory activities against topoisomerase I of these two compounds were evaluated. The result showed that in addition to the known topo I inhibitor SAD (2), compound 1 also exhibited a moderate inhibitory effect.

β-Elemene Inhibits Cell Proliferation by Regulating the Expression and Activity of Topoisomerases I and IIα in Human Hepatocarcinoma HepG-2 Cells.

Objective. To investigate the effects of β-Elemene (β-ELE) on the proliferation, apoptosis, and topoisomerase I (TOPO I) and topoisomerase IIα (TOPO IIα) expression and activity of human hepatocarcinoma HepG-2 cells. Methods. After treatment with β-ELE, morphological alterations of HepG-2 cells were observed under an inverted microscope. Cell proliferation was assessed using an MTT assay, cell cycles were analyzed using flow cytometry, and apoptosis was detected by Annexin V/PI staining. The expression of TOPO I and TOPO IIα was analyzed by Western blot techniques, and their activity was measured using the TOPO I-mediated, supercoiled pBR322 DNA relaxation and TOPO IIα-mediated Kinetoplast DNA (kDNA) decatenation assays, respectively. Supercoiled pBR322 and kDNA were also used to determine the direct effect of β-ELE on DNA breaks. Results. β-ELE significantly inhibited HepG-2 cell proliferation in a dose- and time-dependent manner. β-ELE also induced tumor cell arrest at S phase, induced cell apoptosis, and downregulated the protein expression of TOPO I and TOPO IIα in a dose-dependent manner. β-ELE also inhibited TOPO I- and TOPO IIα-mediated DNA relaxation but did not directly induce DNA breakage at any concentration. Conclusion. β-ELE could inhibit the proliferation of HepG-2 cells and interfere with the expression and activity of TOPO I and TOPO IIα.

Abstract 826: Myeloperoxidase (MPO) dependency for DNA damage, genotoxicity and cytotoxicity induced by etoposide (VP-16): Implications for therapy-induced second malignancies

Abstract Use of the phenolic anticancer agent etoposide (VP-16), which targets DNA topoisomerase II (topo II), can lead to an increased incidence of acute myeloid leukemia linked to rearrangements in the MLL gene. Previous work (Mol. Pharm. 79: 479-87, 2011) demonstrated that MPO, found in myeloid progenitor cells, oxidized VP-16 to its phenoxyl radical form and led to enhanced DNA topo II-mediated strand cleavage through redox cycling resulting in MLL translocations. In the present study, we utilized MPO shRNA in myeloid leukemia HL-60 cells to further examine MPO dependency for VP-16-, VP-16 catechol (VP-OH)-, and VP-16 ortho-quinone (VP-oQ)-induced: 1) DNA strand breaks; 2) oxidative DNA damage; 3) caspase 3 activation; 4) cytotoxicity. Using Comet assays, VP-16 induced a concentration-dependent increase in DNA strand breaks which was attenuated in MPO knockdown cells, as well as in cells incubated with succinylacetone (SA), a heme synthesis inhibitor which depletes cells of active MPO. Similar MPO dependency was demonstrated using VP-OH, and parthenolide (PTL), a sesquiterpene lactone, whose activity is known to be dependent on MPO. In contrast, DNA damage induced by VP-oQ, a fully oxidized VP-16 metabolite, and by the topo I inhibitor camptothecin (CPT), was independent of MPO. VP-16 and VP-OH also induced DNA abasic sites in MPO-replete HL-60 cells. In MPO depleted cells, VP-16 diminished oxidative DNA damage to levels below those seen in controls. Paradoxically, caspase 3 activation induced by VP-16 and VP-OH was decreased in MPO-replete compared to MPO-depleted cells. By comparison, PTL-induced caspase 3 activation was MPO dependent and CPT-induced caspase 3 activation was MPO independent. Cytotoxicity (trypan blue exclusion) induced by VP-16 and VP-OH was inversely related to, while that of PTL corresponded to, MPO levels and activity. VP-oQ, CPT, and podophyllotoxin (PDT) were equally cytotoxic regardless of MPO levels or activity. Clonogenic assays revealed similar paradoxes where VP-16 cytotoxicity was diminished in MPO-replete compared to MPO knockdown cells. In contrast, the effects of VP-oQ, CPT, and PDT on colony formation were not influenced by expression of MPO. Mass spectrometric analysis demonstrated MPO-dependent production of VP-oQ GSH adducts. The production of these inactive adducts were reduced in MPO depleted cells accounting, in part, for the unexpected increase in VP-16 cytotoxicity in MPO knockdown cells. Together, results strongly suggest that MPO-catalyzed oxidation of VP-16 to redox active species leads to enhanced genotoxic events linked to the known leukemogenic action of this anticancer agent. Future studies will be directed toward use of nutritional antioxidants to diminish formation of genotoxic radical species of VP-16 in MPO-containing myeloid precursors as a strategy to prevent drug-induced second malignancies. Citation Format: Ragu Kanagasabai, Jason Goodspeed, Soumendra Krishna Karmahapatra, Alex Klausing, Anna Skwarska, Michael Darby, Yuan Zhao, Jiang Wang, Mitchell A. Phelps, Jack C. Yalowich. Myeloperoxidase (MPO) dependency for DNA damage, genotoxicity and cytotoxicity induced by etoposide (VP-16): Implications for therapy-induced second malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 826. doi:10.1158/1538-7445.AM2014-826

Dioscin, a natural steroid saponin, induces apoptosis and DNA damage through reactive oxygen species: A potential new drug for treatment of glioblastoma multiforme

Dioscin, a natural product obtained from medicinal plants shows lipid-lowering, anti-cancer and hepatoprotective effects. However, the effect of it on glioblastoma is unclear. In this study, dioscin significantly inhibited proliferation of C6 glioma cells and caused reactive oxygen species (ROS) generation and Ca²⁺ release. ROS accumulation affected levels of malondialdehyde, nitric oxide, glutathione disulfide and glutathione, and caused cell apoptosis. In addition, ROS generation caused mitochondrial damage including structural changes, increased mitochondrial permeability transition and decreased mitochondria membrane potential, which led to the release of cytochrome C, nuclear translation of programmed cell death-5 and increased activities of caspase-3,9. Simultaneously, dioscin down-regulated protein expression of Bcl-2, Bcl-xl, up-regulated expression of Bak, Bax, Bid and cleaved poly (ADP-ribose) polymerase. Also, oxygen stress induced S-phase arrest of cancer cells by way of regulating expression of DNA Topo I, p53, CDK2 and Cyclin A and caused DNA damage. In a rat allograft model, dioscin significantly inhibited tumor size and extended the life cycle of the rats. In conclusion, dioscin shows noteworthy anti-cancer activity on glioblastoma cells by promoting ROS accumulation, inducing DNA damage and activating mitochondrial signal pathways. Ultimately, we believe dioscin has promise as a new therapy for the treatment of glioblastoma.

Induction of DNA damage and ATF3 by retigeric acid B, a novel topoisomerase II inhibitor, promotes apoptosis in prostate cancer cells

Retigeric acid B (RB) has been reported to exhibit its anti-tumor activity in vitro and in vivo. Here, we found that RB significantly inhibited activity of topoisomerase IIα (Topo IIα), leading to remarkable DNA damage in prostate cancer (PCa) cells as evidenced by a strong induction of γH2AX and DNA fragmentation. Activation of ATM and ATR sequentially led to induction of phospho-Chk1/2 and phospho-Cdc25 in response to RB. Blockade of ATM/ATR signaling resulted in the attenuation of RB-induced γH2AX, and partially rescued RB-mediated cell death. RB treatment also resulted in inactivation of DNA repair proteins such as phospho-BRCA1, impairment of HR, and NHEJ repair as indicated by DNA end-joining assays. Meanwhile, a stress-responsive gene activating transcription factor 3 (ATF3) was noted for its predominant expression in response to RB-induced DNA damage. Knockdown of ATF3 inhibited the RB-induced expression changes of cell cycle- and apoptosis-related genes such as DR5, DDIT4, CDC25A, GADD45A, and partially blocked RB-mediated inhibition on cell proliferation and induction of apoptosis, suggesting the crucial involvement of ATF3 in this event. Microarray data displayed that RB caused changes of genes required for damaged-DNA binding and repair, as well as ATF3 and its target genes. Our data firstly demonstrated that RB was a novel DNA Topo II inhibitor and triggered cell death by inducing DNA damage and stress-response, suggesting a promising anticancer agent.

Synthesis and Insilico Assessment of Novel Benzothiazole Series against Human DNA Topo Isomerase I

Human topoisomerase I (Top1) plays a pivotal role in cell replication and transcription, and therefore is an important anti-cancer target. Thiazolidinone and Azetidinone derivatives of 6-substituted 2-aminobenzothiazoles were synthesized and tested for their topoisomerase inhibitory activity and cytotoxicity by computational methods. Among the synthesized thiazolidinone and azetidinone analogs, compound 5avi and 6cvi exhibited potent topo I inhibitory activity and strong cytotoxicity. Interestingly, consistency was observed between the cytotoxicities and topo I activities in these thiazolidinone and azetidinone analogs, suggesting that the target of compound 5avi and 6cvi is limited to topo I. Molecular docking studies were performed with the Molecular Operating Environment (London DG Scoring Function) program to afford the ideal interaction mode of the compound into the binding site of the DNA–topo I complex in order to clarify the topo I activity of 5avi and 6cvi..

TCH-1030 targeting on topoisomerase I induces S-phase arrest, DNA fragmentation, and cell death of breast cancer cells

Camptothecin (CPT) and its derivatives are powerful anticancer agents, but these compounds are chemically unstable due to their α-hydroxy lactone six-membered E-ring structure, which is essential for trapping topoisomerase I (topo I)-DNA cleavage complexes. Moreover, the reversibility of trapping the topo I-DNA cleavage complex and the tight binding of CPTs to human serum albumin limit the levels of available active drug. CPT analogs are the only clinically available drugs that target topo I. Owing to the clinical importance of CPT analogs, the development of new anticancer agents which inhibit topo I is urgently needed. In the present study, we report the synthesis, biologic evaluation, and molecular mechanism of a series of substituted indeno[1,2-c]quinoline derivatives against the growth of several human cancer cell lines. We found that 9-methoxy-6-(piperazin-1-yl)-11H-indeno[1,2-c]quinoline-11-one O-3-(dimethylamino)propyl oxime (TCH-1030) intercalated into DNA and preferentially inhibited DNA topo I relaxation. Flow cytometric analysis and BrdU incorporation assays indicate that TCH-1030 alters cell cycle progression, induces S-phase arrest, and causes DNA polyploidy (>4N) that is distinct from the typical G2-M arrest reported with known topoisomerase toxins. Our data indicate that TCH-1030 induces caspase 3 activation, PARP cleavage, γ-H2AX phosphorylation, and, consequently, DNA fragmentation and apoptosis. We also demonstrated that treatment with TCH-1030 significantly inhibits tumor growth in a BT483-xenograft nude mouse model. Taken together, we conclude that the primary mechanism of action of TCH-1030-induced cell cycle retardation and apoptosis-mediated DNA damage involves DNA binding and intercalation as well as topo I inhibition.

In Vitro and in Vivo Anticancer Activity of Copper Bis(thiosemicarbazone) Complexes

Neutral and cationic copper bis(thiosemicarbazone) complexes bearing methyl, phenyl, and hydrogen, on the diketo-backbone of the ligand have been synthesized. All of them were characterized by spectroscopic methods and in three cases by X-ray crystallography. In vitro cytotoxicity studies revealed that they are cytotoxic unlike the corresponding zinc complexes. Copper complexes Cu(GTSC) and Cu(GTSCHCl) derived from glyoxal-bis(4-methyl-4-phenyl-3-thiosemicarbazone) (GTSCH(2)) are the most cytotoxic complexes against various human cancer cell lines, with a potency similar to that of the anticancer drug adriamycin and up to 1000 fold higher than that of the corresponding zinc complex. Tritiated thymidine incorporation assay revealed that Cu(GTSC) and Cu(GTSCHCl) inhibit DNA synthesis substantially. Cell cycle analyses showed that Cu(GTSC) and Cu(GTSCHCl) induce apoptosis in HCT116 cells. The Cu(GTSCHCl) complex caused distinct DNA cleavage and Topo IIα inhibition unlike that for Cu(GTSC). In vivo administration of Cu(GTSC) significantly inhibits tumor growth in HCT116 xenografts in nude mice.

Detection of the Epstein-Barr Virus and DNA-Topoisomerase II-αin Recurrent and Nonrecurrent Giant Cell Lesion of the Jawbones

The aims of this study were to determine whether the expression of Topo II-α correlates with presence of EBV in giant cell lesion of the jawbones and whether it is predictive of clinical biologic behavior of these lesions. Paraffin-embedded tissues from 8 recurrent and 7 nonrecurrent cases of bony GCLs and 9 peripheral giant cell lesions (PGCLs) as a control group were assessed for the expression of EBV and Topo II-α using immunohistochemistry. The results showed positive staining for Topo II-α in mononuclear stromal cells (MSCs) and multinucleated giant cells (MGCs). Student t-test showed that mean Topo II-α labelling index (LI) in recurrent cases was significantly higher than that in non-recurrent cases (P = 0.0001). Moreover, Spearman's correlation coefficients method showed a significant correlation between DNA Topo II-α LI and both of gender and site in these lesions. Moderate EBV expression in relation to the highest Topo II-α LI was observed in two cases of GCT. It was concluded that high Topo II-α LIs could be identified as reliable predicators for the clinical behavior of GCLs. Moreover, EBV has no etiological role in the benign CGCLs in contrast to its role in the pathogenesis of GCTs.

Guanidine-reactive agent phenylglyoxal induces DNA damage and cancer cell death

BackgroundDNA-damaging compounds (e.g., alkylating agents, cytotoxic antibiotics and DNA topoisomerase poisons) are the most widely used anticancer drugs. The inability of tumor cells to properly repair some types of DNA damage may explain why specific DNA-damaging drugs can selectively kill tumor cells. Phenylglyoxal is a dicarbonyl compound known to react with guanidine groups such as that of the DNA base guanine, therefore suggesting that phenylglyoxal could induce DNA damage and have anticancer activity. MethodsCellular DNA damage was measured by the alkaline comet assay and the γH2AX focus assay. Formation of topoisomerase I- and topoisomerase II-DNA complexes was assessed by the TARDIS assay, an immunofluorescence technique that employs specific antibodies to DNA topo I or topo II to detect the protein covalently bound to the DNA in individual cells. Cell growth inhibition and cytotoxicity were determined by XTT, MTT and clonogenic assays. Apoptosis was assessed by the Annexin V flow cytometry assay. ResultsPhenylglyoxal induced cellular DNA damage and formation of high levels of topoisomerase I- and topoisomerase II-DNA complexes in cells. These topoisomerase-DNA complexes were abolished by catalase pretreatment and correlated well with the induction of apoptosis. Phenylglyoxal-induced cell death was partially prevented by catalase pretreatment and was higher in lung cancer cells (A549) than in normal lung fibroblasts (MRC5). Mammalian cell lines defective in nucleotide excision repair (NER), homologous recombination (HR) and non-homologous end joining (NHEJ) were more sensitive to phenylglyoxal than parental cells; this suggests that phenylglyoxal may induce bulky distortions in the shape of the DNA double helix (which are repaired by the NER pathway) and DNA double-strand breaks (which are repaired by HR and NHEJ). ConclusionsThis report shows that phenylglyoxal is a new DNA-damaging agent with anticancer activity, and suggests that tumor cells with defects in NER, HR and NHEJ may be hypersensitive to the cytotoxic activity of phenylglyoxal.

Inhibitory effect of riccardin D on growth of human non-small cell lung cancer: In vitro and in vivo studies

Riccardin D is a macrocyclic bisbibenzyl compound extracted from liverwort plant Dumortiera hirsuta. Our previous study showed that riccardin D induced apoptosis of human leukemia cells by targeting DNA topoisomerase II (topo II). Riccardin D has been considered as a novel DNA topo II inhibitor and potential chemotherapeutic agent for treatment of cancers. In this study, we evaluated the inhibitory effects of riccardin D on growth of human non-small cell lung cancer (NSCLC) both in vitro and in vivo. Riccardin D effectively inhibited the proliferation of NSCLC cells as estimated by the MTT assay. Further examination showed that the ability of invasion and migration of NSCLC cells was suppressed on exposure to riccardin D as estimated by the assays of scratch and transwell chamber. The anticancer activity of riccardin D was verified in mice bearing human NSCLC H460 xenografts. Riccardin D injection produced a 44.5% inhibition of cancer growth without apparent signs of toxicity to animals. Further, riccardin D induced apoptosis of NSCLC cells as evidenced by the increases of cells with externalization of phosphatidylserine and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive in H460 xenografts. The analysis of apoptotic proteins showed that riccardin D activated the caspases cascade signaling pathway as demonstrated by the increases of cleaved caspase-3 and cleaved PARP in NSCLC cells in vitro and in H460 xenografts in mice. The pBR322 DNA relaxation assay indicated that riccardin D inhibited the activity of DNA topo II in H460 and A549 cells, suggesting the mechanism of riccardin D in induction of NSCLC apoptosis. In addition, we studied the activity and expression of matrix metalloproteinases (MMPs) in NSCLC cells. The activities of MMP-2 and MMP-9 in supernatants of NSCLC cells were suppressed on exposure to riccardin D as estimated by gelatin zymography assay. The inhibitory effects of riccardin D on expressions of MMP-2 and MMP-9 were verified in H460 xenografts in mice and the decreases of vascular endothelial growth factor (VEGF) and Erk1/2 might associate with the inhibition of MMPs and NSCLC growth. Together, our results suggest that riccardin D has a high inhibitory effect on human NSCLC growth through induction of apoptosis.

Inhibition of angiogenesis involves in anticancer activity of riccardin D, a macrocyclic bisbibenzyl, in human lung carcinoma

Riccardin D is a novel macrocyclic bisbibenzyl compound extracted from Chinese liverwort plant Dumortiera hirsuta. Our previous studies showed that riccardin D is a DNA topo II inhibitor and has therapeutic potential for treatment of cancers. In this combined in vitro and in vivo study, we examined the inhibitory effects of riccardin D on tumor angiogenesis and the subsequent effect of anticancer activity was evaluated. Incubation with riccardin D weakly inhibited the proliferation of human umbilical vascular endothelial cells (HUVEC) as estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( MTT) assay. The scratch wound experiment showed that riccardin D effectively decreased the motility and migration of HUVEC cells. Riccardin D inhibited the formation of capillary tube as demonstrated by decrease of branch points formed by HUVEC cells on 3-D Matrigel. We examined the levels of angiogenic factors including vascular endothelial growth factor (VEGF), VEGF receptor 2, epidermal growth factor receptor (EGF receptor), and matrix metalloproteinase (MMPs) in HUVEC cells. The expressions of VEGF, phospho-VEGF receptor 2, EGF receptor and MMP-2 were significantly reduced by riccardin D as estimated by Western blot assay and real-time quantitative PCR analysis. The decrease of VEGF was also detected in riccardin D-treated human lung cancer H460 cells. The anticancer activity of riccardin D was then evaluated in a mouse model in which riccardin D delayed the growth of H460 xenografts without obvious toxicity to animals after three weeks injection. To evaluate the role of antiangiogenesis of riccardin D in mice, CD34 immunohistochemical staining was employed to analyze the mean vascular density in H460 xenograft tissues. The number of blood vessels was significantly decreased after riccardin D treatment. These results suggest that riccardin D display the inhibitory effect on growth of human lung carcinoma cells and that the inhibition of angiogenesis may involve in anticancer activity of riccardin D.

Design and synthesis of 4-amino-2-phenylquinazolines as novel topoisomerase I inhibitors with molecular modeling

4-Amino-2-phenylquinazolines 7 were designed as bioisosteres of 3-arylisoquinolinamines 6 that were energy minimized to provide stable conformers. Interestingly, the 2-phenyl ring of 4-amino-2-phenylquinazolines was parallel to the quinazoline ring and improved their DNA intercalation ability in the DNA–topo I complex. Among the synthesized 4-amino group-substituted analogs, 4-cyclohexylamino-2-phenylquinazoline 7h exhibited potent topo I inhibitory activity and strong cytotoxicity. Interestingly, consistency was observed between the cytotoxicities and topo I activities in these quinazoline analogs, suggesting that the target of 4-amino-2-phenylquinazolines is limited to topo I. Molecular docking studies were performed with the Surflex–Dock program to afford the ideal interaction mode of the compound into the binding site of the DNA–topo I complex in order to clarify the topo I activity of 7h.

Selective silencing of DNA topoisomerase IIβ in human mesenchymal stem cells by siRNAs (small interfering RNAs)

hMSCs (human mesenchymal stem cells) express two isoforms of DNA topo II (topoisomerase II). Although both isoforms have the same catalytic activity, they are specialized for different functions in the cell: while topo IIα is essential for chromosome segregation in mitotic cells, topo IIβ is involved in more specific cellular functions. A number of inhibitors are available that inhibit the catalytic activity of both topo II isoforms. However, in order to investigate the isoform-specific inhibition of these two enzymes, it is necessary to use other techniques such as siRNA (small interfering RNA) interference to selectively silence either one of the isoforms individually. Depending on the lipid charge densities and protein varieties of the cell membrane, previous studies have demonstrated that transfection efficiencies of siRNAs to hMSCs are very low. In the study reported here, we demonstrate the use of Lipofectamine RNAiMAX as an efficient transfection reagent to introduce siRNAs into human mesenchymal stem cells with significantly great efficiency to silence topo IIβ selectively. A high level of transfection efficiency (80%) was achieved by using unlabelled topo IIβ-specific siRNA oligos. Specifically, it was confirmed repeatedly that green labelled siRNAs interfere with the transfection of siRNAs. The reagent induced minimal cytotoxicity (3.5–4.5%), and cell viability of the transfected hMSCs decreased 20–30% compared with untreated cells, depending on the concentration of the reagent.

Secalonic acid D as a novel DNA topoisomerase I inhibitor from marine lichen-derived fungus Gliocladium sp. T31

Context: DNA topoisomerase I (topo I) is an essential enzyme which regulates the conformational changes in DNA topology by cleaving and rejoining DNA strands during normal cell growth. The inhibitors of topo I represent a major class of anticancer drugs. In our projects to isolate new anticancer agents from marine-derived fungi, secalonic acid D (SAD) with inhibitory activity on topo I was isolated from the fermentation broth of marine lichen-derived fungus Gliocladium sp. T31, which was collected from marine sediments in South Pole.Objective: The inhibitory activity of SAD on topo I was investigated for the first time.Materials and methods: The inhibitory effect of SAD on topo I was determined via in vitro supercoil relaxation assays and electrophoretic mobility shift assay (EMSA) using plasmid substrate, pBR322.Results: SAD displays a considerable inhibition on topo I in a dose-dependent manner with the minimum inhibitory concentration (MIC) of 0.4 µM. Unlike the prototypic DNA topo I poison camptothecin (CPT), SAD inhibits the binding of topo I to DNA but does not induce the formation of topo I-DNA covalent complexes.Discussion and conclusion: SAD is an excellent topo I inhibitor and thus a significantly potential anticancer candidate.

Alteration of fibroblast phenotype by asbestos-induced autoantibodies

Pulmonary fibrosis is a relentlessly progressive disease for which the etiology can be idiopathic or associated with environmental or occupational exposures. There is not a clear explanation for the chronic and progressive nature of the disease, leaving treatment and prevention options limited. However, there is increasing evidence of an autoimmune component, since fibrotic diseases are often accompanied by production of autoantibodies. Because exposure to silicates such as silica and asbestos can lead to both autoantibodies and pulmonary/pleural fibrosis, these exposures provide an excellent tool for examining the relationship between these outcomes. This study explored the possibility that autoantibodies induced by asbestos exposure in mice would affect fibroblast phenotype. L929 fibroblasts and primary lung fibroblasts were treated with serum IgG from asbestos- or saline-treated mice, and tested for binding using cell-based ELISA, and for phenotypic changes using immunofluorescence, laser scanning cytometry and Sirius Red collagen assay. Autoantibodies in the serum of C57Bl/6 mice exposed to asbestos (but not sera from untreated mice) bound to mouse fibroblasts. The autoantibodies induced differentiation to a myofibroblast phenotype, as demonstrated by increased expression of smooth muscle α-actin (SMA), which was lost when the serum was cleared of IgG. Cells treated with purified IgG of exposed mice produced excess collagen. Using ELISA, we tested serum antibody binding to DNA topoisomerase (Topo) I, vimentin, TGFβ-R, and PDGF-Rα. Antibodies to DNA Topo I and to PDGF-Rα were detected, both of which have been shown by others to be able to affect fibroblast phenotype. The anti-fibroblast antibodies (AFA) also induced STAT-1 activation, implicating the PDGF-R pathway as part of the response to AFA binding. These data support the hypothesis that asbestos induces AFA that modify fibroblast phenotype, and suggest a mechanism whereby autoantibodies may mediate some of the fibrotic manifestations of asbestos exposure.