2,3,5,4'-Tetrahydroxystilbene-2-O-ß-glucoside Isolated from Polygoni Multiflori Ameliorates the Development of Periodontitis.

Periodontitis, a chronic infection by periodontopathic bacteria, induces uncontrolled inflammation, which leads to periodontal tissue destruction. 2,3,5,4'-Tetrahydroxystilbene-2-O-beta-glucoside (THSG), a polyphenol extracted from Polygoni Multiflori, reportedly has anti-inflammatory properties. In this study, we investigated the mechanisms of THSG on the Porphyromonas gingivalis-induced inflammatory responses in human gingival fibroblasts and animal modeling of ligature-induced periodontitis. Human gingival fibroblast cells were treated with lipopolysaccharide (LPS) extracted from P. gingivalis in the presence of resveratrol or THSG to analyze the expression of TNF-α, IL-1ß, and IL-6 genes. Increased AMP-activated protein kinase (AMPK) activation and SirT1 expression were induced by THSG. Treatment of THSG decreased the expression of LPS-induced inflammatory cytokines, enhanced AMPK activation, and increased the expression of SirT1. In addition, it suppressed the activation of NF-κB when cells were stimulated with P. gingivalis LPS. The anti-inflammatory effect of THSG and P. Multiflori crude extracts was reproduced in ligature-induced periodontitis animal modeling. In conclusion, THSG inhibited the inflammatory responses of P. gingivalis-stimulated human gingival fibroblasts and ameliorated ligature-induced periodontitis in animal model.

Topoisomerase IIß deficiency enhances camptothecin-induced apoptosis.

Camptothecin (CPT), a topoisomerase (Top) I-targeting drug that stabilizes Top1-DNA covalent adducts, can induce S-phase-specific cytotoxicity due to the arrest of progressing replication forks. However, CPT-induced non-S-phase cytotoxicity is less well characterized. In this study, we have identified topoisomerase IIß (Top2ß) as a specific determinant for CPT sensitivity, but not for many other cytotoxic agents, in non-S-phase cells. First, quiescent mouse embryonic fibroblasts (MEFs) lacking Top2ß were shown to be hypersensitive to CPT with prominent induction of apoptosis. Second, ICRF-187, a Top2 catalytic inhibitor known to deplete Top2ß, specifically sensitized MEFs to CPT. To explore the molecular basis for CPT hypersensitivity in Top2ß-deficient cells, we found that upon CPT exposure, the RNA polymerase II large subunit (RNAP LS) became progressively depleted, followed by recovery to nearly the original level in wild-type MEFs, whereas RNAP LS remained depleted without recovery in Top2ß-deficient cells. Concomitant with the reduction of the RNAP LS level, the p53 protein level was greatly induced. Interestingly, RNAP LS depletion has been well documented to lead to p53-dependent apoptosis. Altogether, our findings support a model in which Top2ß deficiency promotes CPT-induced apoptosis in quiescent non-S-phase cells, possibly due to RNAP LS depletion and p53 accumulation.

Mechanisms of ceramide-induced COX-2-dependent apoptosis in human ovarian cancer OVCAR-3 cells partially overlapped with resveratrol.

Ceramide is a member of the sphingolipid family of bioactive molecules demonstrated to have profound, diverse biological activities. Ceramide is a potential chemotherapeutic agent via the induction of apoptosis. Exposure to ceramide activates extracellular-signal-regulated kinases (ERK)1/2- and p38 kinase-dependent apoptosis in human ovarian cancer OVCAR-3 cells, concomitant with an increase in the expression of COX-2 and p53 phosphorylation. Blockade of cyclooxygenase-2 (COX-2) activity by siRNA or NS398 correspondingly inhibited ceramide-induced p53 Ser-15 phosphorylation and apoptosis; thus COX-2 appears at the apex of the p38 kinase-mediated signaling cascade induced by ceramide. Induction of apoptosis by ceramide or resveratrol was inhibited by the endocytosis inhibitor, cytochalasin D (CytD); however, cells exposed to resveratrol showed greater sensitivity than ceramide-treated cells. Ceramide-treated cells underwent a dose-dependent reduction in trans-membrane potential. Although both ceramide and resveratrol induced the expressions of caspase-3 and -7, the effect of inducible COX-2 was different in caspase-7 expression induced by ceramide compared to resveratrol. In summary, resveratrol and ceramide converge on an endocytosis-requiring, ERK1/2-dependent signal transduction pathway and induction of COX-expression as an essential molecular antecedent for subsequent p53-dependent apoptosis. In addition, expressions of caspase-3 and -7 are observed. However, a p38 kinase-dependent signal transduction pathway and change in mitochondrial potential are also involved in ceramide-induced apoptosis.

Macrocyclic biphenyl tetraoxazoles: synthesis, evaluation as G-quadruplex stabilizers and cytotoxic activity.

A series of macrocyclic biphenyl tetraoxazoles was synthesized. The latter stages of the synthetic approach allowed for the addition of varied N-protected α-amino acids, which were subsequently deprotected and condensed to provide the desired macrocycles. Improved yields could be realized in the macrocyclization step of their synthesis relative to other macrocyclic G-quadruplex stabilizers. These 24-membered macrocycles were evaluated for their ability to stabilize G-quadruplex DNA and for their relative cytotoxicity against human tumor cells. These biphenyl tetraoxazoles were not strong ligands for G-quadruplex DNA relative to other macrocyclic polyoxazoles. This reduced stabilizing potential did correlate with their comparatively lower cytotoxic activity as observed in the human tumor cell lines, RPMI 8402 and KB3-1. These studies provide useful insights into the conformational requirements for the development of selective and more potent G-quadruplex ligands.

Dietary isothiocyanate-induced apoptosis via thiol modification of DNA topoisomerase IIα.

Studies in animal models have indicated that dietary isothiocyanates (ITCs) exhibit cancer preventive activities through carcinogen detoxification-dependent and -independent mechanisms. The carcinogen detoxification-independent mechanism of cancer prevention by ITCs has been attributed at least in part to their ability to induce apoptosis of transformed (initiated) cells (e.g. through suppression of IκB kinase and nuclear factor κB as well as other proposed mechanisms). In the current studies we show that ITC-induced apoptosis of oncogene-transformed cells involves thiol modification of DNA topoisomerase II (Top2) based on the following observations. 1) siRNA-mediated knockdown of Top2α in both SV40-transformed MEFs and Ras-transformed human mammary epithelial MCF-10A cells resulted in reduced ITC sensitivity. 2) ITCs, like some anticancer drugs and cancer-preventive dietary components, were shown to induce reversible Top2α cleavage complexes in vitro. 3) ITC-induced Top2α cleavage complexes were abolished by co-incubation with excess glutathione. In addition, proteomic analysis revealed that several cysteine residues on human Top2α were covalently modified by benzyl-ITC, suggesting that ITC-induced Top2α cleavage complexes may involve cysteine modification. Interestingly, consistent with the thiol modification mechanism for Top2α cleavage complex induction, the thiol-reactive selenocysteine, but not the non-thiol-reactive selenomethionine, was shown to induce Top2α cleavage complexes. In the aggregate, our results suggest that thiol modification of Top2α may contribute to apoptosis induction in transformed cells by ITCs.

Proteasome-dependent processing of topoisomerase I-DNA adducts into DNA double strand breaks at arrested replication forks.

Reversible topoisomerase I (Top1)-DNA cleavage complexes are the key DNA lesion induced by anticancer camptothecins (CPTs) (e.g. topotecan and irinotecan) as well as structurally perturbed DNAs (e.g. oxidatively damaged, UV-irradiated, or alkylated DNA). It has been proposed that Top1 cleavage complexes arrest advancing replication forks, triggering the formation of DNA double strand breaks (DSBs) because of replication fork runoff at the Top1 cleavage complex sites on the leading strand. In this study, we show that the formation of replication-dependent DSBs requires the ubiquitin-proteasome pathway in CPT-treated cells. First, the proteasome inhibitor MG-132 specifically inhibited CPT-induced but not ionizing radiation- or hydroxyurea-induced DSBs as revealed by both the neutral comet assay and measurements of the specific DNA damage signals (e.g. gamma-H2AX, phosphorylated ataxia telangiectasia mutated (Ser-1981), and phosphorylated Chk2 (Ser-33/35)) that are characteristic for DSBs. Knocking down the 20 S proteasome maturation protein also supported the requirement of the proteasome activity for CPT-induced DSBs. Second, CPT-induced DSB signals were shown to require ubiquitin, ubiquitin-activating enzyme (E1), a CUL-3-based ubiquitin ligase (E3), and the formation of Lys-48-linked polyubiquitin chains on Top1. Third, immunocytochemical studies revealed that the CPT-induced formation of gamma-H2AX foci occurred at the replication forks and was attenuated by co-treatment with the proteasome inhibitor MG-132. In the aggregate, these results support a replication fork collision model in which Top1 cleavage complexes at the arrested replication forks are degraded by proteasome prior to replication fork runoff on the leading strand to generate DSBs.

A G-quadruplex stabilizer induces M-phase cell cycle arrest.

G-quadruplex stabilizers such as telomestatin and HXDV bind with exquisite specificity to G-quadruplexes, but not to triplex, duplex, or single-stranded DNAs. Studies have suggested that the antiproliferative and possibly anti-tumor activities of these compounds are linked to their inhibitory effect on telomerase and/or telomere function. In the current studies, we show that HXDV, a synthetic analog of telomestatin, exhibits antiproliferative activity against both telomerase-positive and -negative cells and induces robust apoptosis within 16 h of treatment, suggesting a mode of action independent of telomerase. HXDV was also shown to inhibit cell cycle progression causing M-phase cell cycle arrest, as evidenced by accumulation of cells with 4 n DNA content, increased mitotic index, separated centrosomes, elevated histone H3 phosphorylation at Ser-10 (an M-phase marker), and defective chromosome alignment and spindle fiber assembly (revealed by time-lapse microscopy). The M-phase arrest caused by HXDV paralleled with reduction in the expression level of the major M-phase checkpoint regulator Aurora A. All these cellular effects appear to depend on the G-quadruplex binding activity of HXDV as its non-G-quadruplex binding analog, TXTLeu, is completely devoid of all these effects. In the aggregate, our results suggest that HXDV, which exhibits anti-proliferative and apoptotic activities, is also a novel M-phase blocker, with a mode of action dependent on its G-quadruplex binding activity.

Genistein induces topoisomerase IIbeta- and proteasome-mediated DNA sequence rearrangements: Implications in infant leukemia.

Genistein is a bioflavonoid enriched in soy products. However, high levels of maternal soy consumption have been linked to the development of infant leukemia ALL and AML. The majority of infant leukemia is linked to mixed lineage leukemia gene (MLL) translocations. Previous studies have implicated topoisomerase II (Top2) in genistein-induced infant leukemia. In order to understand the roles of the two Top2 isozymes in and the molecular mechanism for genistein-induced infant leukemia, we carried out studies in vitro using purified recombinant human Top2 isozymes, as well as studies in cultured mouse myeloid progenitor cells (32Dc13) and Top2beta knockout mouse embryonic fibroblasts (MEFs). First, we showed that genistein efficiently induced both Top2alpha and Top2beta cleavage complexes in the purified system as well as in cultured mouse cells. Second, genistein induced proteasomal degradation of Top2beta in 32Dc13 cells. Third, the genistein-induced DNA double-strand break (DSB) signal, gamma-H2AX, was dependent on the Top2beta isozyme and proteasome activity. Fourth, the requirement for Top2beta and proteasome activity was mirrored in genistein-induced DNA sequence rearrangements, as monitored by a DNA integration assay. Together, our results suggest a model in which genistein-induced Top2beta cleavage complexes are processed by proteasome, leading to the exposure of otherwise Top2beta-concealed DSBs and subsequent chromosome rearrangements, and implicate a major role of Top2beta and proteasome in genistein-induced infant leukemia.

Macrocyclic hexaoxazoles: Influence of aminoalkyl substituents on RNA and DNA G-quadruplex stabilization and cytotoxicity.

A series of 24-membered macrocyclic hexaoxazoles containing one or two aminoalkyl substituents was synthesized and evaluated for cytotoxicity and for their ability to selectively stabilize G-quadruplex DNA and RNA. The most cytotoxic analog 4a, with IC(50) values of 25 and 130 nM using KB3-1 and RPMI 8402 cells, is efficacious in vivo in athymic nude mice with a human tumor xenograft from the breast cancer cell line MDA-MB-435.

Roles of DNA topoisomerase II isozymes in chemotherapy and secondary malignancies.

Drugs that target DNA topoisomerase II (Top2), including etoposide (VP-16), doxorubicin, and mitoxantrone, are among the most effective anticancer drugs in clinical use. However, Top2-based chemotherapy has been associated with higher incidences of secondary malignancies, notably the development of acute myeloid leukemia in VP-16-treated patients. This association is suggestive of a link between carcinogenesis and Top2-mediated DNA damage. We show here that VP-16-induced carcinogenesis involves mainly the beta rather than the alpha isozyme of Top2. In a mouse skin carcinogenesis model, the incidence of VP-16-induced melanomas in the skin of 7,12-dimethylbenz[a]anthracene-treated mice is found to be significantly higher in TOP2beta(+) than in skin-specific top2beta-knockout mice. Furthermore, VP-16-induced DNA sequence rearrangements and double-strand breaks (DSBs) are found to be Top2beta-dependent and preventable by cotreatment with a proteasome inhibitor, suggesting the importance of proteasomal degradation of the Top2beta-DNA cleavage complexes in VP-16-induced DNA sequence rearrangements. VP-16 cytotoxicity in transformed cells expressing both Top2 isozymes is, however, found to be primarily Top2alpha-dependent. These results point to the importance of developing Top2alpha-specific anticancer drugs for effective chemotherapy without the development of treatment-related secondary malignancies.

Role of topoisomerase IIbeta in the expression of developmentally regulated genes.

Mice lacking topoisomerase IIbeta (TopIIbeta) are known to exhibit a perinatal death phenotype. In the current study, transcription profiles of the brains of wild-type and top2beta knockout mouse embryos were generated. Surprisingly, only a small number (1 to 4%) of genes were affected in top2beta knockout embryos. However, the expression of nearly 30% of developmentally regulated genes was either up- or down-regulated. By contrast, the expression of genes encoding general cell growth functions and early differentiation markers was not affected, suggesting that TopIIbeta is not required for early differentiation programming but is specifically required for the expression of developmentally regulated genes at later stages of differentiation. Consistent with this notion, immunohistochemical analysis of brain sections showed that TopIIbeta and histone deacetylase 2, a known TopIIbeta-interacting protein, were preferentially expressed in neurons which are in their later stages of differentiation. Chromatin immunoprecipitation analysis of the developing brains revealed TopIIbeta binding to the 5' region of a number of TopIIbeta-sensitive genes. Further studies of a TopIIbeta-sensitive gene, Kcnd2, revealed the presence of TopIIbeta in the transcription unit with major binding near the promoter region. Together, these results support a role of TopIIbeta in activation/repression of developmentally regulated genes at late stages of neuronal differentiation.

12-Substituted 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridines as novel topoisomerase I-targeting antitumor agents.

2,3-Dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine and a few of its 12-substituted analogs are active as TOP1-targeting agents. Studies were performed to further evaluate the potential of this series of non-camptothecin TOP1-targeting agents. The influence of a hydroxymethyl, formyl, N,N-dimethylaminomethyl, 2-(N,N-dimethylamino)ethyl, 3-(N,N-dimethylamino)propyl), and 4-(N,N-dimethylamino)butyl substituent at the 12-position on TOP1-targeting activity and tumor cell growth was evaluated. In addition, the relative pharmacologic activities of the 12-carboxamide analog, as well as its N-methyl and N,N-dimethyl derivatives were assessed.

Topoisomerase IIbeta mediated DNA double-strand breaks: implications in doxorubicin cardiotoxicity and prevention by dexrazoxane.

Doxorubicin is among the most effective and widely used anticancer drugs in the clinic. However, cardiotoxicity is one of the life-threatening side effects of doxorubicin-based therapy. Dexrazoxane (Zinecard, also known as ICRF-187) has been used in the clinic as a cardioprotectant against doxorubicin cardiotoxicity. The molecular basis for doxorubicin cardiotoxicity and the cardioprotective effect of dexrazoxane, however, is not fully understood. In the present study, we showed that dexrazoxane specifically abolished the DNA damage signal gamma-H2AX induced by doxorubicin, but not camptothecin or hydrogen peroxide, in H9C2 cardiomyocytes. Doxorubicin-induced DNA damage was also specifically abolished by the proteasome inhibitors bortezomib and MG132 and much reduced in top2beta(-/-) mouse embryonic fibroblasts (MEF) compared with TOP2beta(+/+) MEFs, suggesting the involvement of proteasome and DNA topoisomerase IIbeta (Top2beta). Furthermore, in addition to antagonizing Top2 cleavage complex formation, dexrazoxane also induced rapid degradation of Top2beta, which paralleled the reduction of doxorubicin-induced DNA damage. Together, our results suggest that dexrazoxane antagonizes doxorubicin-induced DNA damage through its interference with Top2beta, which could implicate Top2beta in doxorubicin cardiotoxicity. The specific involvement of proteasome and Top2beta in doxorubicin-induced DNA damage is consistent with a model in which proteasomal processing of doxorubicin-induced Top2beta-DNA covalent complexes exposes the Top2beta-concealed DNA double-strand breaks.

ISG15 as a novel tumor biomarker for drug sensitivity.

Tumor cells are known to exhibit highly varied sensitivity to camptothecins (CPT; e.g., irinotecan and topotecan). However, the factors that determine CPT sensitivity/resistance are largely unknown. Recent studies have shown that the ubiquitin-like protein, IFN-stimulated gene 15 (ISG15), which is highly elevated in many human cancers and tumor cell lines, antagonizes the ubiquitin/proteasome pathway. In the present study, we show that ISG15 is a determinant for CPT sensitivity/resistance possibly through its effect on proteasome-mediated repair of topoisomerase I (TOP1)-DNA covalent complexes. First, short hairpin RNA-mediated knockdown of either ISG15 or UbcH8 (major E2 for ISG15) in breast cancer ZR-75-1 cells decreased CPT sensitivity, suggesting that ISG15 overexpression in tumors could be a factor affecting intrinsic CPT sensitivity in tumor cells. Second, the level of ISG15 was found to be significantly reduced in several tumor cells selected for resistance to CPT, suggesting that altered ISG15 regulation could be a significant determinant for acquired CPT resistance. Parallel to reduced CPT sensitivity, short hairpin RNA-mediated knockdown of either ISG15 or UbcH8 in ZR-75-1 cells resulted in increased proteasomal degradation of CPT-induced TOP1-DNA covalent complexes. Taken together, these results suggest that ISG15, which interferes with proteasome-mediated repair of TOP1-DNA covalent complexes, is a potential tumor biomarker for CPT sensitivity.

Thyroid hormone-induced expression of inflammatory cytokines interfere with resveratrol-induced anti-proliferation of oral cancer cells.

Thyroid hormone, L-thyroxine (T4), induces inflammatory genes expressions and promotes cancer growth. It also induces expression of the checkpoint programmed death-ligand 1 (PD-L1), which plays a vital role in cancer progression. On the other hand, resveratrol inhibits inflammatory genes expressions. Moreover, resveratrol increases nuclear inducible cyclooxygenase (COX)-2 accumulation, complexes with p53, and induces p53-dependent anti-proliferation. In this study, we investigated the effect of T4 on resveratrol-induced anti-proliferation in oral cancer. T4 increased the expression and cytoplasmic accumulation of PD-L1. Increased expressions of pro-inflammatory genes, interleukin (IL)-1ß and transforming growth factor (TGF)-ß1, were shown to stimulate PD-L1 expression. T4 stimulated pro-inflammatory and proliferative genes expressions, and oral cancer cells proliferation. In contrast, resveratrol inhibited those genes and activated anti-proliferative genes. T4 retained resveratrol-induced COX-2 in cytoplasm and prevented COX-2 nuclear accumulation when resveratrol treated cancer cells. A specific signal transducer and activator of transcription 3 (STAT3) inhibitor, S31-201, blocked T4-induced inhibition and restored resveratrol-induced nuclear COX-2 accumulation. By inhibiting the T4-activated STAT3 signal transduction axis with S31-201, resveratrol was able to sequentially reestablish COX-2/p53-dependent gene expressions and anti-proliferation. These findings provide a novel understanding of the inhibitory effects of T4 on resveratrol-induced anticancer properties via the sequential expression of PD-L1 and inflammatory genes.

Ring-closing metathesis for the synthesis of a highly G-quadruplex selective macrocyclic hexaoxazole having enhanced cytotoxic potency.

The synthesis of a 24-membered macrocyclic hexaoxazole via ring-closing metathesis is described. The target compound selectively stabilizes G-quadruplex DNA with no detectable stabilization of duplex DNA. An MTT cytotoxicity assay indicated that this unsaturated macrocyclic hexaoxazole exhibits significant cytotoxicity toward P388, RPMI 8402, and KB3-1 cell lines with IC50 values of 45, 25, and 38 nM, respectively.

Facile formation of hydrophilic derivatives of 5H-8,9-dimethoxy-5-[2-(N,N-dimethylamino)ethyl]-2,3-methylenedioxydibenzo[c,h] [1,6]naphthyridin-6-one (ARC-111) and its 12-aza analog via quaternary ammonium intermediates.

Several new TOP1-targeting agents were prepared using as intermediates the N,N,N-trimethyl quaternary ammonium salts of either ARC-111 or its 12-aza analog (ARC-31), 3 and 4, respectively. Direct displacement of the quaternary ammonium group with water, imidazole, alkylethylenediamines, or polyhydroxylated alkylamines provides a convenient means for furthering the structure-activity relationships associated with these non-camptothecin TOP1-targeting agents.

11-Substituted 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine derivatives as novel topoisomerase I-targeting agents.

Several 11-substituted benzo[i]phenanthridine derivatives were synthesized, and their TOP1-targeting activity and cytotoxicity were assessed. Comparative data indicate that TOP1-targeting was often the primary molecular target associated with their cytotoxicity. Several 11-aminoalkyl derivatives, 11-aminocarboxy derivatives as well as the 11-[(2-dimethylamino)ethyl]carboxamide of 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine were synthesized and did exhibit considerable cytotoxicity with IC(50) values ranging from 20 to 120 nM in the human lymphoblast tumor cell line RPMI8402.

Syntheses and biological evaluation of topoisomerase I-targeting agents related to 11-[2-(N,N-dimethylamino)ethyl]-2,3-dimethoxy-8,9-methylenedioxy-11H-isoquino[4,3-c]cinnolin-12-one (ARC-31).

Several 11-ethyl-2,3-dimethoxy-8,9-methylenedioxy-11H-isoquino[4,3-c]cinnolin-12-ones with varied functionality on the ethyl substituent have exhibited potent topoisomerase I (TOP1) targeting activity and antitumor activity. The influence of various polar substituents at the 2-position of the 11-ethyl substituent, including N-methylamine, N-isopropylamine, hydroxyl, and hydroxylamino groups, on TOP1-targeting activity and cytotoxicity was assessed. The N-methylamine and N-isopropylamine derivatives were also evaluated as antitumor agents in athymic nude mice with MDA-MB-435 human tumor xenografts. Both compounds were active as antitumor agents upon either parenteral or oral administration.

Synthesis of N-substituted 5-[2-(N-alkylamino)ethyl]dibenzo[c,h][1,6]naphthyridines as novel topoisomerase I-targeting antitumor agents.

Several N-alkyl and N,N-dialkyl 5H-8,9-dimethoxy-5-(2-aminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-ones have been identified as topoisomerase I-targeting agents with potent antitumor activity. In the present study, the impact on biological activity of substitution of a trifluoromethyl, cyano, aminocarbonyl, or ethynyl group on a N-methyl substituent of N,N-dimethyl-, N-methyl-N-ethyl-, and N-methyl-N-isopropyl 5H-8,9-dimethoxy-5-(2-aminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-ones was assessed.