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In mammalian cells, extracellular vesicles (EVs) derived from the endosomal system carry many different kinds of bioactive molecule to deliver to recipient cells in a paracrine or endocrine manner. EVs can mediate local and systemic intercellular communications, including reeducating stromal cells, remodeling the architecture of the tumor microenvironment, modulating cancer metabolism and metastases, or even conferring drug resistance. Because the molecular and functional characteristics of prostate cancer (PCa) evolve over time, the bioactive molecule profiles/signatures of tumor-derived EVs (TDEs) reflect the real-time status of cancer cells. TDEs appear to be valuable diagnostic and prognostic biomarkers as well as potential therapeutic vehicles, suggesting their essential role in precision medicine of disease management. We summarized critical aspects of TDEs in PCa and discussed their potential clinical applications.

A subpopulation of cancer stem cells (CSCs) plays a critical role of cancer progression, recurrence, and therapeutic resistance. Many studies have indicated that castration-resistant prostate cancer (CRPC) is associated with stem cell phenotypes, which could further promote neuroendocrine transdifferentiation. Although only a small subset of genetically pre-programmed cells in each organ has stem cell capability, CSCs appear to be inducible among a heterogeneous cancer cell population. However, the inductive mechanism(s) leading to the emergence of these CSCs are not fully understood in CRPC. Tumor cells actively produce, release, and utilize exosomes to promote cancer development and metastasis, cancer immune evasion as well as chemotherapeutic resistance; the impact of tumor-derived exosomes (TDE) and its cargo on prostate cancer (PCa) development is still unclear. In this study, we demonstrate that the presence of Cav-1 in TDE acts as a potent driver to induce CSC phenotypes and epithelial–mesenchymal transition in PCa undergoing neuroendocrine differentiation through NFκB signaling pathway. Furthermore, Cav-1 in mCRPC-derived exosomes is capable of inducing radio- and chemo-resistance in recipient cells. Collectively, these data support Cav-1 as a critical driver for mCRPC progression.

Background DNA topoisomerase and telomerase enzymes are popular targets of several anti-tumor drugs. Smooth proceeding of telomeric recombination requires Topoisomerase II (Top2), which is involved in telomere-telomere recombination through functioning in relaxation of positive supercoils among the cells adopting telomerase-independent Alternative lengthening of telomere (ALT) pathway. Most of the inhibitors reported so far have been designed to targetsolely telomerase-positive cells, which can potentially lead to therapeutic failure because tumor cells treated with telomerase inhibitors can activate the ALT pathway for telomere maintenance. Knowing that ALT cells are more sensitive against a Top2 inhibitor, ICRF-93 agent, compared to telomerase-positive cells, we analyzed two selected ellipticine derivatives that we recently reported as TopII-targeting compounds, to assess their effects on the formation of DNA breaks and suppression of ALT pathway. Methods Cell viability, Comet, C-Circle assays, dot blot, immunofluorescence staining, and telomere fluorescence in situ hybridization (FISH) staining were used for determining the effect of the compounds on ALT status of tumor cells. Results and conclusions Treatment of ALT cells with ellipticine derivatives resulted in the formation of DNA breaks and suppression of ALT-associated phenotypes in vitro. Our results will contribute to the development of therapeutic strategies combining telomerase and ALT pathway inhibitors., Turkish Scientific and Technical Research AssemblyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [115Z349], This research was supported by Grant 115Z349 (Z.T. and S.Z.) from the Turkish Scientific and Technical Research Assembly. the cell lines used in this study were provided by Drs. Shu-Chun Teng and Tsai-Kun Li (College of Medicine, National Taiwan University, Taipei, Taiwan).

A series of thiochromeno [2,3-c]quinolin-12-one derivatives with various substitutions were synthesized and evaluated as topoisomerase (Topo) inhibitors. Six (8, 10, 12, 14, 19, and 26) of 23 compounds showed strong inhibitory activities against Topo-mediated DNA relaxation and proliferation of five human cell lines including breast (MDA-MB-231, MDA-MB-468 and MCF7), colorectal (HCT116) and non-small cell lung (H1299) cancers. Among these, compounds 14 and 26 exhibited full inhibitory activities against Topo I at 3 μM and Topo IIα at 1 μM. Cancer cells treated with 26 accumulated DNA damage and were arrested at the G2/M phase. With time, cells proceeded to apoptosis, as revealed by increased amounts of cells with fragmented DNA and cleavage of caspase-8 and -9. In contrast, normal breast epithelial cells showed low sensitivity to 26. Taken together, our study identifies 26 as a potent Topo dual-inhibitor with low toxicity to normal cells, and elucidates that the terminal amino group of N-2-aminoethylamino or N-3-aminopropylamino at the 6th position and 8,10-di-halogen substituents on thiochromeno [2,3-c]quinolin-12-one are critical for the Topo-inhibiting and cancer-killing activities.

Background Tumor microenvironments (TMEs) activate various axes/pathways, predominantly inflammatory and hypoxic responses, impact tumorigenesis, metastasis and therapeutic resistance significantly. Although molecular pathways of individual TME are extensively studied, evidence showing interaction and crosstalk between hypoxia and inflammation remain unclear. Thus, we examined whether interferon (IFN) could modulate both inflammatory and hypoxic responses under normoxia and its relation with cancer development. Methods IFN was used to induce inflammation response and HIF-1α expression in various cancer cell lines. Corresponding signaling pathways were then analyzed by a combination of pharmacological inhibitors, immunoblotting, GST-Raf pull-down assays, dominant-negative and short-hairpin RNA-mediated knockdown approaches. Specifically, roles of functional HIF-1α in the IFN-induced epithelial-mesenchymal transition (EMT) and other tumorigenic propensities were examined by knockdown, pharmacological inhibition, luciferase reporter, clonogenic, anchorage-independent growth, wound-healing, vasculogenic mimicry, invasion and sphere-formation assays as well as cellular morphology observation. Results We showed for the first time that IFN induced functional HIF-1α expression in a time- and dose- dependent manner in various cancer cell lines under both hypoxic and normoxic conditions, and then leading to an activated HIF-1α pathway in an IFN-mediated pro-inflammatory TME. IFN regulates anti-apoptosis activity, cellular metastasis, EMT and vasculogenic mimicry by a novel mechanism through mainly the activation of PI3K/AKT/mTOR axis. Subsequently, pharmacological and genetic modulations of HIF-1α, JAK, PI3K/AKT/mTOR or p38 pathways efficiently abrogate above IFN-induced tumorigenic propensities. Moreover, HIF-1α is required for the IFN-induced invasiveness, tumorigenesis and vasculogenic mimicry. Further supports for the HIF-1α-dependent tumorigenesis were obtained from results of xenograft mouse model and sphere-formation assay. Conclusions Our mechanistic study showed an induction of HIF-1α and EMT ability in an IFN-mediated inflammatory TME and thus demonstrating a novel interaction between inflammatory and hypoxic TMEs. Moreover, targeting HIF-1α may be a potential target for inhibiting tumor tumorigenesis and EMT by decreasing cancer cells wound healing and anchorage-independent colony growth. Our results also lead to rationale guidance for developing new therapeutic strategies to prevent relapse via targeting TME-providing IFN signaling and HIF-1α programming. Electronic supplementary material The online version of this article (10.1186/s13046-018-0730-6) contains supplementary material, which is available to authorized users.

Cordyceps militaris is a traditional Chinese medicine frequently used for tonic and therapeutic purposes. Reports from our laboratory and others have demonstrated that extracts of the cultivated fruiting bodies of C. militaris (CM) exhibit a potent cytotoxic effect against many cancer cell lines, especially human leukemia cells. Here, we further investigated the underlying mechanism through which CM is cytotoxic to cancer cells. The CM-mediated induction of PARP cleavage and its related DNA damage signal (γH2AX) was diminished by caspase inhibitor I. In contrast, a ROS scavenger failed to prevent CM-mediated leukemia cell death. Moreover, two signaling molecules, AKT and p38 MAPK, were activated during the course of apoptosis induction. Employing MTT analysis, we found that a p38 MAPK inhibitor but not an AKT inhibitor could rescue cells from CM-mediated cell death, as well as inhibit the cleavage of PARP, formation of apoptotic bodies and up-regulation of the γH2AX signal. These results suggest that CM-mediated leukemia cell death occurs through the activation of the p38 MAPK pathway, indicating its potential therapeutic effects against human leukemia.

Human type II topoisomerase (Top2) isoforms, hTop2α and hTop2β, are targeted by some of the most successful anticancer drugs. These drugs induce Top2-mediated DNA cleavage to trigger cell-death pathways. The potency of these drugs correlates positively with their efficacy in stabilizing the enzyme-mediated DNA breaks. Structural analysis of hTop2α and hTop2β revealed the presence of methionine residues in the drug-binding pocket, we therefore tested whether a tighter Top2-drug association may be accomplished by introducing a methionine-reactive Pt2+ into a drug to further stabilize the DNA break. Herein, we synthesized an organoplatinum compound, etoplatin-N2β, by replacing the methionine-juxtaposing group of the drug etoposide with a cis-dichlorodiammineplatinum(II) moiety. Compared to etoposide, etoplatin-N2β more potently inhibits both human Top2s. While the DNA breaks arrested by etoposide can be rejoined, those captured by etoplatin-N2β are practically irreversible. Crystallographic analyses of hTop2β complexed with DNA and etoplatin-N2β demonstrate coordinate bond formation between Pt2+ and a flanking methionine. Notably, this stable coordinate tether can be loosened by disrupting the structural integrity of drug-binding pocket, suggesting that Pt2+ coordination chemistry may allow for the development of potent inhibitors with protein conformation-dependent reversibility. This approach may be exploited to achieve isoform-specific targeting of human Top2s.

Purpose: Human DNA topoisomerase III alpha (hTOP3α) is involved in DNA repair surveillance and cell-cycle checkpoints possibly through formatting complex with tumor suppressors. However, its role in cancer development remained unsolved. Experimental Design: Coimmunoprecipitation, sucrose gradient, chromatin immunoprecipitation (ChIP), real time PCR, and immunoblotting analyses were performed to determine interactions of hTOP3α with p53. Paired cell lines with different hTOP3α levels were generated via ectopic expression and short hairpin RNA (shRNA)-mediated knockdown approaches. Cellular tumorigenic properties were analyzed using cell counting, colony formation, senescence, soft agar assays, and mouse xenograft models. Results: The hTOP3α isozyme binds to p53 and cofractionizes with p53 in gradients differing from fractions containing hTOP3α and BLM. Knockdown of hTOP3α expression (sh-hTOP3α) caused a higher anchorage-independent growth of nontumorigenic RHEK-1 cells. Similarly, sh-hTOP3α and ectopic expression of hTOP3α in cancer cell lines caused increased and reduced tumorigenic abilities, respectively. Genetic and mutation experiments revealed that functional hTOP3α, p53, and p21 are required for this tumor-suppressive activity. Mechanism-wise, ChIP data revealed that hTOP3α binds to the p53 and p21 promoters and positively regulates their expression. Two proteins affect promoter recruitments of each other and collaborate in p21 expression. Moreover, sh-hTOP3α and sh-p53 in AGS cells caused a similar reduction in senescence and hTOP3α mRNA levels were lower in gastric and renal tumor samples. Conclusion: We concluded that hTOP3α interacts with p53, regulates p53 and p21 expression, and contributes to the p53-mediated tumor suppression. Clin Cancer Res; 20(6); 1489–501. ©2014 AACR.

Both cancer-suppressing and cancer-promoting properties of reactive nitrogen and oxygen species (RNOS) have been suggested to play a role in tumor pathology, particularly those activities associated with chronic inflammation. Here, we address the impact of nitric oxide (NO) on the induction of DNA damage and genome instability with a specific focus on the involvement of topoisomerase II (TOP2). We also investigate the contribution of NO to the formation of skin melanoma in mice.Similar to the TOP2-targeting drug, etoposide (VP-16), the NO-donor, S-nitrosoglutathione (GSNO), induces skin melanomas formation in 7,12-dimethyl- benz[a]anthracene (DMBA)-initiated mice. To explore the mechanism(s) underlying this NO-induced tumorigenesis, we use a co-culture model system to demonstrate that inflamed macrophages with inducible NO synthase (iNOS) expression cause γ-H2AX activation, p53 phosphorylation, and chromosome DNA breaks in the target cells. Inhibitor experiments revealed that NO and TOP2 isozymes are responsible for the above described cellular phenotypes. Notably, NO, unlike VP-16, preferentially induces the formation of TOP2β cleavable complexes (TOP2βcc) in cells. Moreover, GSNO induced TOP2-dependent DNA sequence rearrangements and cytotoxicity. Furthermore, the incidences of GSNO- and VP-16-induced skin melanomas were also observed to be lower in the skin-specific top2β-knockout mice. Our results suggest that TOP2 isozymes contribute to NO-induced mutagenesis and subsequent cancer development during chronic inflammation.We provide the first experimental evidence for the functional role of TOP2 in NO-caused DNA damage, mutagenesis, and carcinogenesis. Notably, these studies contribute to our molecular understanding of the cancer-promoting actions of RNOS during chronic inflammation.

Pancreatic cancer is an aggressive malignancy, which generally responds poorly to chemotherapy. In this study, trichodermin, an endophytic fungal metabolite from Nalanthamala psidii, was identified as a potent and selective antitumor agent in human pancreatic cancer. Trichodermin exhibited antiproliferative effects against pancreatic cancer cells, especially p53-mutated cells (MIA PaCa-2 and BxPC-3) rather than normal pancreatic epithelial cells. We found that trichodermin induced caspase-dependent and mitochondrial intrinsic apoptosis. Trichodermin also increased apoptosis through mitotic arrest by activating Cdc2/cyclin B1 complex activity. Moreover, trichodermin promoted the activation of c-Jun N-terminal kinase (JNK), and inhibition of JNK by its inhibitor, shRNA, or siRNA significantly reversed trichodermin-mediated caspase-dependent apoptosis. Trichodermin triggered DNA damage stress to activate p53 function for executing apoptosis in p53-mutated cells. Importantly, we demonstrated that trichodermin with efficacy similar to gemcitabine, profoundly suppressed tumor growth through inducing intratumoral DNA damage and JNK activation in orthotopic pancreatic cancer model. Based on these findings, trichodermin is a potential therapeutic agent worthy of further development into a clinical trial candidate for treating cancer, especially the mutant p53 pancreatic cancer.

Combination therapy, which can optimize killing activity to cancers and minimize drug resistance, is a mainstream therapy against hormone-refractory prostate cancers (HRPCs). Rottlerin, a natural polyphenolic component, synergistically increased PC-3 (a HRPC cell line) apoptosis induced by camptothecin (a topoisomerase I inhibitor). Using siRNA technique to knockdown protein kinase C-δ (PKCδ), the data showed that rottlerin-mediated synergistic effect was PKCδ-independent, although rottlerin has been used as a PKCδ inhibitor. Rottlerin potentiated camptothecin-induced DNA fragmentation at S phase and ATM phosphorylation at Ser1981. The effect was correlated to apoptosis (r2 = 0.9). To detect upstream signals, the data showed that camptothecin acted on and stabilized topoisomerase I-DNA complex, leading to the formation of camptothecin-trapped cleavage complexes (TOP1cc). The effect was potentiated by rottlerin. To determine DNA repair capability, the time-related γH2A.X formation was examined after camptothecin removal. Consequently, rottlerin significantly inhibited camptothecin removal-mediated decline of γH2A.X formation at S phase, indicating the impairment of DNA repair activity in the presence of rottlerin. The combinatory treatment of camptothecin and rottlerin induced conformational change and activation of Bax and formation of truncated Bad, suggesting the contribution of mitochondria stress to apoptosis. In summary, the data suggest that rottlerin-mediated camptothecin sensitization is through the augmented stabilization of TOP1cc, leading to an increase of DNA damage stress and, possibly, an impairment of DNA repair capability. Subsequently, mitochondria-involved apoptosis is triggered through Bax activation and truncated Bad formation. The novel discovery may provide an anticancer approach of combinatory use between rottlerin and camptothecin for the treatment of HRPCs.

Purpose: Solid tumors encounter a growth-limiting hypoxic microenvironment as they develop. Hypoxia-inducible factors (HIF) play important roles in hypoxia-associated tumor development and therapeutic resistance. Targeting the HIF pathway (especially HIF-1α) represents a promising cancer treatment strategy. Here, we report a novel class of HIF-1α inhibitors and the possible molecular basis of inhibition. Experimental Design: We analyzed the inhibitory effects of clinically used topoisomerase II (TOP2)–targeting drugs on HIF-1α expression with a primary focus on mitoxantrone. The potential role of TOP2 in mitoxantrone-inhibited HIF-1α expression was studied using pharmacologic inhibition, a knockdown approach, and TOP2 mutant cells. Moreover, involvement of mitoxantrone in proteasome-mediated degradation, transcription, and translation of HIF-1α was examined. Results: The TOP2-targeting mitoxantrone, but neither doxorubicin nor etoposide (VP-16), strongly inhibited HIF-1α expression under hypoxic conditions in a dose- and time-dependent manner. Surprisingly, the mitoxantrone-mediated inhibition of HIF-1α expression was largely independent of two TOP2 isozymes, proteasomal degradation, and transcription. Furthermore, mitoxantrone inhibited HIF-1α expression and function in a similar fashion as cycloheximide, suggesting that mitoxantrone might inhibit HIF-1α via a blockage at its translation step. In vitro translation experiments using HIF-1α mRNA further confirmed inhibition of HIF-1α translation by mitoxantrone. Interestingly, levels of the polysome-bound HIF-1α and VEGF-A mRNA were elevated and decreased after mitoxantrone treatment, respectively. Conclusions: We have identified the TOP2-targeting compound, mitoxantrone, as an HIF-1α inhibitor possibly through a translation inhibition mechanism, suggesting the possibility of an additional anticancer activity for mitoxantrone. Clin Cancer Res; 17(15); 5026–37. ©2011 AACR.

Type II topoisomerases (TOP2s) resolve the topological problems of DNA by transiently cleaving both strands of a DNA duplex to form a cleavage complex through which another DNA segment can be transported. Several widely prescribed anticancer drugs increase the population of TOP2 cleavage complex, which leads to TOP2-mediated chromosome DNA breakage and death of cancer cells. We present the crystal structure of a large fragment of human TOP2β complexed to DNA and to the anticancer drug etoposide to reveal structural details of drug-induced stabilization of a cleavage complex. The interplay between the protein, the DNA, and the drug explains the structure-activity relations of etoposide derivatives and the molecular basis of drug-resistant mutations. The analysis of protein-drug interactions provides information applicable for developing an isoform-specific TOP2-targeting strategy.

A novel series of topoisomerase I (Top I) inhibitors were designed on the basis of camptothecin using scaffold modification strategy. Thirty-one new compounds were synthesized and evaluated for anticell proliferation activity. The most potent compound 26 presented a significant inhibitory effect on Top I, leading to Top I-mediated cleavage and influences on Top I expression at the cellular level. Moreover, 26 was proved to induce cell death via apoptosis and accelerated DNA strand breaks without significant alteration in cell cycle populations. All of the experimental results herein indicated that 26 could interact with DNA-Top I complex and induce cancer cell apoptosis to produce antitumor effects. The in vivo evaluation of 26 on the growth of HT-29 tumor xenografts in nude mice suggested its therapeutic potential for further development.

Hepatocellular carcinoma is a very common malignancy and is chemoresistant to currently available chemotherapeutic agents. Endoplasmic reticulum (ER) stress-induced apoptotic pathway is suggested to be less affected by the resistance mechanisms, becoming a potential target of chemotherapeutic strategy. The anticancer effects and expression of GADD153, a transcription factor induced by ER stress, were examined in hepatocellular carcinoma Hep3B cells. The correlation between these two parameters was constructed under flavonoid stimulation with a correlation coefficient (r) of 0.8. The data also showed that genistein (isoflavone) was the most effective one. Genistein induced the activation of several ER stress-relevant regulators, including m-calpain, GADD153, GRP78 and caspase-12. Furthermore, genistein-induced effect was inhibited in cells transfected with antisense GADD153 cDNA, indicating a functional role of GADD153. Notably, genistein induced the activation of caspase-2, whereas did not cause the DNA damage. It also triggered the production of ROS. The antioxidant trolox significantly reduced ROS accumulation, but did not modify genistein-induced apoptotic cell death. The long-term exposure (48 h) of cells to genistein caused Mcl-1 down-regulation and Bad cleavage; furthermore, cyclosporin A (an inhibitor of mitochondrial permeability transition pore) almost completely abolished genistein-induced loss of mitochondrial membrane potential, and induced a 30% reverse of apoptosis caused by long-term treatment (48 h) of genistein, suggesting the involvement of mitochondrial stress in the late phase of genistein-induced effect. Taken together, it is suggested that genistein induces the anticancer effect through a mechanism initiated by ER stress and facilitated by mitochondrial insult in Hep3B cells.

Cyclopentenone prostaglandins (PGs) such as PGA1, PGA2 and delta12-PGJ2 have been shown to suppress tumor cell growth and to induce apoptosis in prostate cancer cells. Bromovulone III, which is isolated from the soft coral Clavularia viridis, is a cyclopentenone prostanoid. In this study, the anti-tumor activity as well as action mechanism of bromovulone III was identified in prostate cancer cells. Bromovulone III displayed anti-tumor activity of 30 to 100 times more effective than PGA1, PGA2 and delta12-PGJ2 in PC-3 cells. Several targets of caspases and Bcl-2 family of proteins were detected and the data demonstrated that bromovulone III induced the activation of caspase-8, -9 and -3, and Bid cleavage in which the caspase-8 activation occurred the first. Bromovulone III did not modify the protein levels of death receptors and ligands. Of note, the Fas clustering in PC-3 cells responsive to bromovulone III was observed by confocal immunofluorescence microscopy suggesting the involvement of Fas-mediated pathway. Bromovulone III also induced the cleavage of Mcl-1 in this study. The cleavage fragments (24, 19 and 17 kDa) may partly share the apoptotic insult. Although it has been suggested that Fas-mediated signaling may contribute to the caspase-8 activation induced by DNA-damaging agents; however, bromovulone III did not induce any DNA breakage, suggesting that bromovulone III-induced Fas/caspase-8-dependent signaling is not through the direct target on DNA damage. In summary, the data suggest that bromovulone III causes a rapid redistribution and clustering of Fas in PC-3 cells. Subsequently, the Fas event causes the activation and interaction of caspase-8/Bid/caspase-9 signaling cascades, and the activation of executor caspase-3.

RNA degradation via the ribonuclease H (RNase H) activity of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) is a critical component of the reverse transcription process. In this connection, mutations of RT that inactivate RNase H activity result in noninfectious virus particles. Thus, interfering with the RNase H activity of RT represents a potential vehicle for the inhibition of HIV-1 replication. Here, we demonstrate an approach for inhibiting the RNase H activity of HIV-1 RT by targeting its RNA.DNA hybrid substrates. Specifically, we show that the binding of the 4,5-disubstituted 2-deoxystreptamine aminoglycosides, neomycin, paromomycin, and ribostamycin, to two different chimeric RNA-DNA duplexes, which mimic two distinct intermediates in the reverse transcription process, inhibits specific RT-mediated RNase H cleavage, with this inhibition being competitive in nature. UV melting and isothermal titration calorimetry studies reveal a correlation between the relative binding affinities of the three drugs for each of the chimeric RNA-DNA host duplexes and the relative extents to which the drugs inhibit RT-mediated RNase H cleavage of the duplexes. Significantly, this correlation also extends to the relative efficacies with which the drugs inhibit HIV-1 replication. In the aggregate, our results highlight a potential strategy for AIDS chemotherapy that should not be compromised by the unusual genetic diversity of HIV-1.

Substituted benzo[i]phenanthridines that have incorporated within their structure an 8,9-methylenedioxy group can exhibit topoisomerase I-targeting activity. Structure-activity studies were performed to examine the influence of saturation at the 11,12-positions of several substituted 8,9-methylenedioxybenzo[i]phenanthridines. The activities of these dihydro analogues were compared to those of their unsaturated analogues. In addition, the influence of varying substituents at the 2- and 3-positions within the A-ring of these 8,9-methylenedioxybenzo[i]phenanthridines on their relative potency as topoisomerase I-targeting agents and cell proliferation as determined using the MTT assay was investigated. 2,3-Dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine and its 11,12-dihydro derivative were among the more potent analogues evaluated with regard to topoisomerase I-targeting activity and cytotoxicity.

Spectroscopic and calorimetric techniques were employed to characterize and contrast the binding of the aminoglycoside paromomycin to three octamer nucleic acid duplexes of identical sequence but different strand composition (a DNA.RNA hybrid duplex and the corresponding DNA.DNA and RNA.RNA duplexes). In addition, the impact of paromomycin binding on both RNase H- and RNase A-mediated cleavage of the RNA strand in the DNA.RNA duplex was also determined. Our results reveal the following significant features: (i) Paromomycin binding enhances the thermal stabilities of the RNA.RNA and DNA.RNA duplexes to similar extents, with this thermal enhancement being substantially greater in magnitude than that of the DNA.DNA duplex. (ii) Paromomycin binding to the DNA.RNA hybrid duplex induces CD changes consistent with a shift from an A-like to a more canonical A-conformation. (iii) Paromomycin binding to all three octamer duplexes is linked to the uptake of a similar number of protons, with the magnitude of this number being dependent on pH. (iv) The affinity of paromomycin for the three host duplexes follows the hierarchy, RNA.RNA > DNA.RNA >> DNA.DNA. (v) The observed affinity of paromomycin for the RNA.RNA and DNA.RNA duplexes decreases with increasing pH. (vi) The binding of paromomycin to the DNA.RNA hybrid duplex inhibits both RNase H- and RNase A-mediated cleavage of the RNA strand. We discuss the implications of our combined results with regard to the specific targeting of DNA.RNA hybrid duplex domains and potential antiretroviral applications.

5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-one exhibits potent TOP1-targeting activity and pronounced antitumor activity. It was hypothesized that replacement of the two methoxyl groups with a nitro substituent would allow for retention of similar activity. In this study 8-, 9-, and 10-nitro-5H-2,3-methylenedioxy-5-(2-N,N-dimethylaminoethyl)dibenzo[c,h][1,6]naphthyridin-6-one and their amino derivatives were synthesized and assessed for their relative TOP1-targeting activity and cytotoxicity. In the case of both the 8- and 9-nitro analogues, their TOP1-targeting activity and cytotoxicity are greater than that of camptothecin and comparable to that of 5H-8,9-dimethoxy-5-(2-N,N-dimethylaminoethyl)-2,3-methylenedioxydibenzo[c,h][1,6]naphthyridin-6-one.

Single-stranded DNA has been speculated to be the initial signal in the DNA damage signaling pathway. We showed that introduction of single-stranded DNA with diverse sequences into mammalian cells induced DNA damage as well as apoptosis signals. Like DNA damaging agents, single-stranded DNA up-regulated p53 and activated the nuclear kinase ataxia telangiectasia mutant (ATM) as evidenced by phosphorylation of histone 2AX, an endogenous ATM substrate. Single-stranded DNA also triggered apoptosis as evidenced by the formation of caspase-dependent chromosomal DNA strand breaks, cytochrome c release, and increase in reactive oxygen species production. Moreover, single-stranded DNA-induced apoptosis was reduced significantly in p53 null cells and in cells treated with ATM small interfering RNA. These results suggest that single-stranded DNA may act upstream of ATM/p53 in DNA damage signaling.

Several substituted dibenzo[ c,h ]cinnolines were synthesized and evaluated for their potential to target topoisomerase I and for their relative cytotoxic activity. Select benzo[ i ]phenanthridines are capable of stabilizing the cleavable complex formed with topoisomerase I and DNA. This study was initiated to examine whether dibenzo[ c , h ]cinnolines, which are in essence aza analogues of benzo[ i ]phenanthridines, possess similar pharmacological properties. 2,3-Dimethoxy-8,9-methylenedioxybenzo[ i ]phenanthridine is one of the more potent benzo[ i ]phenanthridine derivatives in regard to topoisomerase I-targeting activity and cytotoxicity. The structure–activity relationship observed with these substituted dibenzo[ c , h ]cinnolines parallels that observed for benzo[ i ]phenanthridine derivatives. Compared to similarly substituted benzo[ i ]phenanthridines, the dibenzo[ c , h ]cinnoline analogues exhibit more potent topoisomerase I-targeting activity and cytotoxicity. The relative IC 50 values obtained in assessing the cytotoxicity of 2,3-dimethoxy-8,9-methylenedioxydibenzo[ c , h ]cinnoline and 2,3-dimethoxy-8,9-methylenedioxybenzo[ i ]phenanthridine in the human lymphoblastma cell line, RPMI8402, are 70 and 400 nM, respectively. In tumor cell lines selected for resistance to camptothecin and known to express mutant topoisomerase I, benzo[ i ]phenanthridine derivatives were not cross-resistant. In contrast, similarly substituted dibenzo[ c , h ]cinnolines with significant topoisomerase I-targeting activity did exhibit cross-resistance in these camptothecin-resistant cell lines. The cytotoxicity of these dibenzo[ c , h ]cinnolines was not diminished in cells overexpressing the efflux transporter, MDR1. These data indicate that substituted dibenzo[ c,h ]cinnolines can exhibit potent topoisomerase I-targeting activity and are capable of overcoming the multi-drug resistance associated with this efflux transporter.

Several benzo[c]phenanthridine and protoberberine alkaloids, such as nitidine and berberrubine, are known to induce DNA cleavage in the presence of either topoisomerase I or II. Structure-activity studies performed on various analogues related to benzo[c]phenanthridine and protoberberine alkaloids have provided insights into structural features that influence this topoisomerase-targeting activity. Modifications within the A-ring of benzo[c]phenanthridine and protoberberine alkaloids can significantly alter their ability to enhance the cleavable complex formation that occurs between DNA and topoisomerases. Select benzo[i]phenanthridines were synthesized as potential bioisosteres of nitidine and its analogues. In the present study, 2,3-methylenedioxy-8,9-dimethoxybenzo[i]phenanthridine, 2,3-methylenedioxy-8,9-dimethoxy-5-methylbenzo[i]phenanthridine, 2,3,8,9-tetramethoxybenzo[i]phenanthridine and 5-methyl-2,3,8,9-tetramethoxybenzo[i]phenanthridine were synthesized. These benzo[i]phenanthridine derivatives were evaluated for their ability to enhance cleavable complex formation in the presence of topoisomerases and DNA as well as for their cytotoxicity against the human lymphoblastoma cell line, RPMI8402. 2,3-Methylenedioxy-8,9-dimethoxybenzo[i]phenanthridine (4a) and its 5-methyl derivative (4b) are active as topoisomerase I-targeting agents. In contrast to nitidine, the presence of the 5-methyl substituent in the case of 4b is not associated with enhanced activity. Consistent with previous structure-activity studies on nitidine and protoberberine alkaloids, 2,3,8,9-teramethoxybenzo[i]phenanthridine, 5a, and its 5-methyl derivative, 5b, are inactive as topoisomerase I-targeting agents. These studies were extended to an evaluation of the relative pharmacological activities of 2,8,9-trimethoxybenzo[i]phenanthridine, 3,8,9-trimethoxybenzo[i]phenanthridine, and 2,3-methylenedioxy-8,9-methylenedioxybenzo[i]phenanthridine.

Appropriately substituted benzo[i]phenanthridines structurally related to nitidine, a benzo[c]phenanthridine alkaloid with antitumor activity, are active as topoisomerase I-targeting agents. Studies on benzo[i]phenanthridines have indicated analogues that possess a 2,3-methylenedioxy moiety and at least one and preferably two methoxyl groups at the 8- and 9-positions, such as 8,9-dimethoxy-2,3-methylenedioxybenzo[i]phenanthridine, 2, are active as topoisomerase I-targeting agents. Tetramethoxylated benzo[i]phenanthridines, wherein the 2,3-methylenedioxy moiety is replaced with methoxyl groups at the 2- and 3-position, are inactive as a topoisomerase I-targeting agent. These results initially suggested that the 2,3-methylenedioxy moiety was critical to the retention of potent activity. Further studies revealed that 2,3-dimethoxy-8,9-methylenedioxybenzo[i]phenanthridine, 7a, is more potent than 2 as a topoisomerase I-targeting agent. The observation that 2,3-dimethoxylated benzo[i]phenanthridines can actually exhibit enhanced activity prompted the present study in which several 8-substituted 2,3-dimethoxybenzo[i]phenanthridines were prepared and their pharmacological activities evaluated. The influence of NH(2), CN, CH(2)OH, OBn, OCH(3), OH, and NHCOCH(3 )substituents at the 8-position on the relative activity of these 2,3-dimethoxybenzo[i]phenanthridines was examined. Relative to these derivatives, 7a was the most potent topoisomerase I-targeting agent, possessing similar cytotoxicity to that of nitidine in the human lymphoblast tumor cell line, RPMI8402.

Hydrogen peroxide (H2O2), a reactive oxygen species (ROS), is known to induce oxidative stress and apoptosis. U937 cells treated with H2O2 were shown to produce high molecular weight (HMW) DNA fragments approximately 50 to 100 kb in size in

Extracellular ATP is an important signaling molecule mediating quite divergent specific biological effects. Even though recent studies suggest a potential role of ATP in cancer progress, its real impact in chemotherapeutic efficacy remains unclear. In the present study, we investigated the effect of ATP on the cytotoxicity of doxorubicin in various cancer cell types and found that ATP had no effect on doxorubicin cytotoxicity in colon, prostate, breast, and cervical cancers or in osteosarcoma. In contrast, ATP has divergent effects on lung cancer cells: it can protect against doxorubicininduced cell death in non-metastatic lung cancer CL1.0 cells, but not in highly metastatic CL1.5 cells. Both apoptotic (characterized by sub-G1 peak, caspase 3 activation, poly(ADP-ribose) polymerase-1 cleavage) and necrotic (characterized by propidium iodide uptake and ROS production) features induced by doxorubicin in CL1.0 cells were reduced by ATP. In addition, ATP attenuated p53 accumulation, DNA damage (assessed by poly(ADP-ribose) formation and the comet assay) and topoisomerase II inhibition after doxorubicin treatment, and doxorubicin cytotoxicity was diminished by the p53 inhibitor pifithrin-a. Moreover, UTP, UDP, ADP, and pyrophosphate sodium pyrophosphate tetrabasic decahydrate diminished the antitumor effect of doxorubicin in CL1.0 cells, whereas purinergic P2 receptors antagonists did not abrogate the action of ATP. In summary, ATP fails to alter the antitumor efficacy of doxorubicin in most cancer cell types, except in CL1.0 cells, in which pyrophosphate mediates the cell protection afforded by ATP via attenuation of reactive oxygen species production, DNA damage, p53 accumulation, and caspase activation. (Cancer Sci 2012; 103: 1811‐1819)

Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid, has previously been shown to ameliorate obesity-associated metabolic syndrome. To decipher the mechanism responsible for the beneficial effects of DHA on energy/glucose homeostasis and the metabolic syndrome, 30 weaned cross-bred pigs were randomly assigned to three groups and fed ad libitum with a standard diet supplemented with 2% of beef tallow, soybean oil or DHA oil for 30 days, and the gene expression profile of various tissues was evaluated by quantitative real-time polymerase chain reaction. The DHA-supplemented diets reduced the expression of forkhead box O transcription factor (FoxO) 1 and FoxO3 in the liver and adipose tissue. DHA treatments also decreased the expression of FoxO1 and FoxO3 in human hepatoma cells, SK-HEP-1 and human and porcine primary adipocytes. In addition, DHA also down-regulated FoxO target genes, such as microsomal triacylglycerol transfer protein (MTP), glucose-6-phosphatase, apolipoprotein C-III (apoC-III) and insulin-like growth factor binding-protein 1 in the liver, as well as reduced total plasma levels of cholesterol and triacylglycerol in the pig. Transcriptional suppression of FoxO1, FoxO3, apoC-III and MTP by DHA was further confirmed by reporter assays with each promoter construct. Taken together, our study indicates that DHA modulates lipid and glucose homeostasis in part by down-regulating FoxO function. The down-regulation of genes associated with triacylglycerol metabolism and very low density lipoprotein assembly is likely to contribute to the beneficial effects of DHA on the metabolic syndrome.

Co-amplification and co-overexpression of ErbB2 and Grb7 are frequently found in various cancers, including breast cancer. Biochemical and functional correlations of the two molecules have identified Grb7 to be a pivotal mediator downstream of ErbB2-mediated oncogenesis. However, it remains largely unknown how Grb7 is involve in the ErbB2-mediated tumorigenesis. In this study, we show that Grb7-mediated cell proliferation and growth are essential for the tumorigenesis that occurs in ErbB2-Grb7-overexpressing breast cancer cells. Intrinsically, EGF-induced de novo Grb7 tyrosine phosphorylation/activation recruits and activates Ras-GTPases and subsequently promotes the phosphorylation of ERK1/2, thereby stimulating tumor growth. Furthermore, we also found the anti-tumor effect could be synergized by co-treatment with Herceptin plus Grb7 knockdown in Sk-Br3 breast cancer cells. Our findings illustrate an underlying mechanism by which Grb7 promotes tumorigenesis through the formation of a novel EGFR-Grb7-Ras signaling complex, thereby highlighting the potential strategy of targeting Grb7 as an anti-breast cancer therapy.

DNA topoisomerases are double-edged swords. They are essential for many vital functions of DNA during normal cell growth. However, they are also highly vulnerable under various physiological and nonphysiological stresses because of their delicate act on breaking and rejoining DNA. These stresses (e.g. exposure to topoisomerase poisons, acidic pH, and oxidative stresses) can convert DNA topoisomerases into DNA-breaking nucleases, resulting in cell death and/or genomic instability. The importance of topoisomerase-mediated DNA cleavage in tumor cell death and carcinogenesis has been recognized. This review focuses on recent findings concerning the molecular mechanisms of the stress responses to topoisomerase-mediated DNA damage. The involvement of ubiquitin/26S proteasome and SUMO/UBC9 in these processes, as well as the role of topoisomerase cleavable complexes in apoptotic cell death are discussed.

Camptothecin (CPT) class of compounds has been demonstrated to be effective against a broad spectrum of tumors. Their molecular target has been firmly established to be human DNA topoisomerase I (topo I). CPT inhibits topo I by blocking the rejoining step of the cleavage/religation reaction of topo-I, resulting in accumulation of a covalent reaction intermediate, the cleavable complex. The primary mechanism of cell killing by CPT is S-phase-specific killing through potentially lethal collisions between advancing replication forks and topo-I cleavable complexes. Collisions with the transcription machinery have also been shown to trigger the formation of long-lived covalent topo-I DNA complexes, which contribute to CPT cytotoxicity. Two novel repair responses to topo-I-mediated DNA damage involving covalent modifications of topo-I have been discovered. The first involves activation of the ubiquitin/26S proteasome pathway, leading to degradation of topo-I (CPT-induced topo-I downregulation). The second involves SUMO conjugation to topo-I. The potentials roles of these new mechanisms for repair of topo-I-mediated DNA damage in determining CPT sensitivity/resistance in tumor cells are discussed.

Protoberberines are a new class of organic cations that are dual poisons of topoisomerases I and II. Certain protoberberines exhibit greater in vitro cytotoxicity against cell lines derived from solid tumors than from leukemias. Using a group of seventeen different protoberberine analogs, the structural basis for selective cytotoxicity toward sensitive SF-268 glioblastoma cells as compared with resistant RPMI 8402 lymphoblast cells was explored. The selective cytotoxicity is associated with the presence of an imminium ion and other structural features of protoberberines, and is not shared by drugs such as camptothecin, doxorubicin, vinblastine, and etoposide, which are either equally or more cytotoxic against RPMI 8402 cells than SF-268 cells. The selective cytotoxicity of protoberberines against SF-268 over RPMI 8402 cells is not due to differences in topoisomerase levels or known drug efflux systems such as multidrug resistance (MDR1) and multidrug-resistance protein (MRP). Comparative in vitro studies of the accumulation of coralyne, a fluorescent protoberberine, into sensitive and resistant cells demonstrated a correlation between drug accumulation and selective cytotoxicity. Inhibitors of coralyne uptake included several protoberberine-related compounds. Of these, palmatine, a minimally cytotoxic protoberberine, both inhibited coralyne accumulation and reduced cytotoxicity against SF-268 cells, but not against RPMI 8402 cells. Despite the structural resemblance of protoberberines to catecholamines, our experiments using inhibitors and cells expressing biogenic amine uptake systems have ruled out the involvement of biogenic amine uptake1, uptake2, and vesicular monoamine transport systems. Uptake systems remaining as candidates, supported by preliminary data, include transport via vesicles derived from specialized membrane invaginations and selected carrier-mediated organic amine transport systems.

An uncoupler of oxidative phosphorylation, 2,4-dinitrophenol, and an aconitase inhibitor, fluoroacetic acid, both of which are known to lower the cellular ATP pool, protected Escherichia coli cells from the bactericidal actions of gyrase poisons including quinolone antibiotics, nalidixic acid and ciprofloxacin, and the epipodophyllotoxins VP-16 and VM-26. Using purified E. coli DNA gyrase, we examined the effect of ATP on gyrase-mediated DNA cleavage in the presence of these gyrase poisons. ATP was shown to stimulate gyrase-mediated DNA cleavage from 10- to more than 100-fold in the presence of these gyrase poisons. ADP antagonized the stimulatory effect of ATP. Consequently, gyrase-mediated DNA cleavage induced by gyrase poisons is modulated by the ATP concentration/ADP concentration ([ATP]/[ADP]) ratio. Coumermycin A1, an inhibitor of the ATPase subunit of DNA gyrase, like ADP, also effectively antagonized the stimulatory effect of ATP on gyrase-mediated DNA cleavage induced by gyrase poisons. Furthermore, coumermycin A1, like DNP and fluoroacetic acid, also protected cells from the bactericidal action of gyrase poisons. In the aggregate, our results are consistent with the notion that the [ATP]/[ADP] ratio, through its modulatory effect on the gyrase-mediated DNA cleavage, is an important determinant of cellular susceptibility to gyrase poisons.

It is now known that several antimicrobial and antitumor drugs destroy cells by interacting with cellular DNA topoisomerases. These enzymes are ubiquitous in nature and are required for the regulation of DNA structure (topology) in the cell. Specifically, DNA topoisomerases regulate the coiling of the DNA double helix, a parameter critical for processes such as replication and transcription. Topoisomerases alter DNA coiling by transiently cleaving DNA. They have been defined mechanistically as type I if they nick a single strand of the DNA double helix and type II if they cleave both strands. The discovery of antitumor drugs that specifically target topoisomerases has not only led to an improved understanding of tumor cell biology but has also fostered the rapid development of new anticancer drugs. This chapter reviews the anticancer drugs that target topoisomerases and focuses on cellular mechanisms of resistance to these drugs.

Activated inflammatory leukocytes generate a variety of reactive nitrogen and species (RNOS) which have been proposed to contribute to genome instability and cancer development. We previously reported that oxidative stress induces topoisomerase II (TOP2)-mediated DNA breaks via TOP2 cleavable complex (TOP2cc) formation. Moreover, drug-induced TOP2αcc and TOP2βcc are suggested to be anti-cancer and carcinogenic, respectively. Here we show that either inflammation-produced or chemical-released nitric oxide (NO), like TOP2-targeting etoposide (VP-16), induce TOP2cc-meidated DNA breakage, mutagenesis and carcinogenesis: (i) NO-releaser S-nitrosoglutathione (GSNO) induces formation of skin melanomas in 9,10-dimethyl-1,2-benzanthracene (DMBA)-initiated mice; (ii) the co-culture experiments revealed that inflamed macrophages causes activation of DNA damage response and chromosome DNA breakage in target cells (iii) NO production and two TOP2 isozymes are responsible for the generation of chromosome DNA breaks; (iv) consistently, treatment of a NO releaser GSNO induces hTOP2αcc and hTOP2βcc formation; (v) GSNO also increases mutation rate and kill cells in TOP2-dependent manner; and (vi) the incidences of GSNO- and VP-16-induced melanomas are found to be lower in skin-specific top2β-knockout mice. Thus, our results provide the first demonstration for the functional role of TOP2 in the NO-caused DNA damage, mutagenesis and carcinogenesis. The TOP2 involvement in pathological settings such as chronic inflammation-associated mutagenesis and subsequent cancer development were discussed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-394. doi:1538-7445.AM2012-LB-394

Growth factor receptor bound protein-7 (Grb7) regulates diverse cellular functions as a result of its multi-domain functionality in protein-protein interactions. A number of reports have indicated that Grb7 is essential for EGFR-mediated tumorigenesis and cancer progression. However, it remains unclear how Grb7 potentiates tumor progression. To understand the underlying mechanisms by which Grb7 participates in EGFR-mediated tumorigenesis, we utilized microarray analyses and yeast two-hybrid screening to explore Grb7-mediated signaling. Of which, a migration related protein, EphA4, was markedly up-regulated in associated with Grb7 overexpression but down-regulated while knocking down Grb7. Eventually, in response to EGF stimulation, we found that Grb7 was trans-located into the nucleus from the cytoplasm, which in turn promoted STAT3 transcriptional activity on EphA4 gene expression. The significance of this finding is further highlighted by the co-upregulation of Grb7 and EphA4 is concurrent with the highly invasive capability of lung adenocarcinoma. Moreover, we found that EphA4 could also potentiate EGF-induced reorganization of cytoskeleton, ERK1/2 phosphorylation, and cancer cell functions such as proliferation, migration and anchorage-independent growth. Based on these findings, we hypothesize that EphA4 can act as an amplifier involved EGF-mediated cancer progression in an EGF/Grb7/Stat3-mediated transcriptional manner. This study reveals a novel mechanism by which Grb7 regulates tumor progression through the EGF- by triggering EphA4 expression and then prolonging EGFR signaling in regulation of diverse cellular functions involved in cancer progression, thereby highlighting the potential strategy to target Grb7 for anti-cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-28. doi:1538-7445.AM2012-LB-28

The Escherichia coli rho-15 mutant (deficient in transcription termination) is known to be incompatible with pBR322 and other plasmids (J. S. Fassler, G. F. Arnold, and I. Tessman, Mol. Gen. Genet. 204:424-429, 1986). We show that failure of pBR322 to transform rho-15 is mediated by transcription from the tet promoter and readthrough from the tet gene into the rom region. Using an isopropyl-beta-D-thiogalactopyranoside-inducible promoter to replace the tet promoter, we have demonstrated that plasmid-specific transcription inhibits growth of the rho-15 host, possibly due to the expression of the Rom protein. The involvement of Rom protein in pBR322-rho-15 incompatibility is further indicated by the following two experiments. (i) Functional inactivation of the rom gene in pBR322 enabled plasmids to transform E. coli rho-15. (ii) Specific overexpression of the rom gene abolished plasmid transformation into E. coli rho-15. An rpoB8(Ts) mutant RNA polymerase which compensated for the termination defect in E. coli rho-15 also restored plasmid-host compatibility, suggesting that Rom-mediated plasmid-host incompatibility is linked to a defect in transcription termination.