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Metastatic dissemination from the primary tumor is a complex process that requires crosstalk between tumor cells and the surrounding milieu and involves the interplay between numerous cellular-signaling programs. Epithelial-mesenchymal transition (EMT) remains at the forefront of orchestrating a shift in numerous cellular programs, such as stemness, drug resistance, and apoptosis that allow for successful metastasis. Till date, there is limited success in therapeutically targeting EMT. Utilizing a high throughput screen of FDA-approved compounds, we uncovered a novel role of the topoisomerase inhibitor, Teniposide, in reversing EMT. Here, we demonstrate Teniposide as a potent modulator of the EMT program, specifically through an IRF7-NMI mediated response. Furthermore, Teniposide significantly reduces the expression of the key EMT transcriptional regulator, Zinc Finger E-Box Binding Homeobox 2 (ZEB2). ZEB2 downregulation by Teniposide inhibited RNA polymerase I (Pol I) activity and rRNA biogenesis. Importantly, Teniposide treatment markedly reduced pulmonary colonization of breast cancer cells. We have uncovered a novel role of Teniposide, which when used at a very low concentration, mitigates mesenchymal-like invasive phenotype. Overall, its ability to target EMT and rRNA biogenesis makes Teniposide a viable candidate to be repurposed as a therapeutic option to restrict breast cancer metastases., (© 2024. The Author(s).)

As an FDA-approved drug, teniposide, was utilized in cancer treatment but was accompanied by a strong side effect in long-term clinical trials. This work discovered potential candidate drugs with low toxicity by modifying the molecule structure of teniposide through a structure-guided drug design approach. The IC 50 value of novel 4,6- O -thenylidene-β-d-glucopyranoside-(2″-acetamido, 3″-acetyl-di- S -5-fluorobenzothizole/5-fluorobenzoxazole)-4'-demethylepipodophyllotoxin (compounds 15 and 16 ) was 120.4-125.1 μM, which was significantly improved by around 10 times more than teniposide (11.5-22.3 μM) against healthy human cells (i.e., HL-7702, H8, MRC-5, and HMEC). In vivo studies demonstrated compounds 15 and 16 significantly suppressed the tumor growth in the HepG2 cell xenograft model without exhibiting obvious toxicity (LD 50 values of 208.45 and 167.52 mg/kg), which was lower than that of teniposide (LD 50 = 46.12 mg/kg). Compounds 15 and 16 caused mild γH2AX phosphorylation for low DNA toxicity and less inhibition of PI3K/Akt. Compounds 15 and 16 might be potential antitumor drugs with low toxicity.

Checkpoint blockade antibodies have been approved as immunotherapy for multiple types of cancer, but the response rate and efficacy are still limited. There are few immunogenic cell death (ICD)-inducing drugs available that can kill cancer cells, enhance tumor immunogenicity, increase the in vivo immune infiltration, and thereby boosting a tumor response to immunotherapy. So far, the ICD markers have been identified as the few immuno-stimulating characteristics of dead cells, but whether the presence of such ICD markers on tumor cells translates into enhanced antitumor immunity in vivo is still investigational. To identify anticancer drugs that could induce tumor cell death and boost T cell response, we performed drug screenings based on both an ICD reporter assay and T cell activation assay. We identified that teniposide, a DNA topoisomerase II inhibitor, could induce high mobility group box 1 (HMGB1) release and type I interferon signaling in tumor cells, and teniposide-treated tumor cells could activate antitumor T cell response both in vitro and in vivo. Mechanistically, teniposide induced tumor cell DNA damage and innate immune signaling including NF-κB activation and STING-dependent type I interferon signaling, both of which contribute to the activation of dendritic cells and subsequent T cells. Furthermore, teniposide potentiated the antitumor efficacy of anti-PD1 on multiple types of mouse tumor models. Our findings showed that teniposide could trigger tumor immunogenicity, and enabled a potential chemo-immunotherapeutic approach to potentiate the therapeutic efficacy of anti-PD1 immunotherapy.

The switch of vascular smooth muscle cells (SMCs) from the contractile phenotype to proliferative one can make contributions to atherosclerosis and neointima formation. MiR-21 can prevent the rupture of advanced lesion plaques. We previously reported the protection of DNA topoisomerase II (Topo II) inhibitors against atherosclerosis and vascular calcification. However, it remains unknown if Topo II inhibitors can change SMC phenotypes. Herein, we show that teniposide protected SMC phenotype switching during atherosclerosis by enhancing expression of smooth muscle α-actin (SMA) while reducing osteopontin (OPN) expression in aortic lesion plaques. In vitro, teniposide induced expression of smooth muscle protein 22-α and calponin 1, but inhibited expression of OPN and epiregulin in human aortic SMCs (HASMCs). Moreover, teniposide attenuated platelet derived growth factor-BB-induced HASMC proliferation and migration. Mechanistically, the effect of teniposide on SMC phenotypes was completed, at least in part, by activating miR-21 expression. In addition, teniposide ameliorated ligation-induced carotid artery remodeling in C57BL/6J mice by regulating SMA and OPN expression. Taken together, our study demonstrates that teniposide regulates SMC phenotype switching by upregulating expression of contractile genes in a miR-21-dependent manner, and this function is an important anti-atherogenic mechanism of teniposide., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Objective- Vascular calcification is a major risk factor for rupture of atherosclerotic plaques. High expression of BMP2 (bone morphogenetic protein 2) in lesions suggests its importance in vascular calcification during atherosclerosis. Teniposide is a Topo II (DNA topoisomerase II) inhibitor and is used for cancer treatment. Previously, we reported that teniposide activated macrophage ABCA1 (ATP-binding cassette transporter A1) expression and free cholesterol efflux indicating Topo II inhibitors may demonstrate antiatherogenic properties. Herein, we investigated the effects of teniposide on the development of atherosclerosis and vascular calcification in apoE -/- (apoE deficient) mice. Approach and Results- apoE -/- mice were fed high-fat diet containing teniposide for 16 weeks, or prefed high-fat diet for 12 weeks followed by high-fat diet containing teniposide for 4 weeks. Atherosclerosis and vascular calcification were determined. Human aortic smooth muscle cells were used to determine the mechanisms for teniposide-inhibited vascular calcification. Teniposide reduced atherosclerotic lesions. It also substantially reduced vascular calcification without affecting bone structure. Mechanistically, teniposide reduced vascular calcification by inactivating BMP2/(pi-Smad1/5/8 [mothers against decapentaplegic homolog 1, 5, and 8])/RUNX2 (runt-related transcription factor 2) axis in a p53-dependent manner. Furthermore, activated miR-203-3p by teniposide functioned as a link between activated p53 expression and inhibited BMP2 expression in inhibition of calcification. Conclusions- Our study demonstrates that teniposide reduces vascular calcification by regulating p53-(miR-203-3p)-BMP2 signaling pathway, which contributes to the antiatherogenic properties of Topo II inhibitors.

The transcription factor MYB plays key roles in hematopoietic cells and has been implicated the development of leukemia. MYB has therefore emerged as an attractive target for drug development. Recent work has suggested that targeting MYB by small-molecule inhibitors is feasible and that inhibition of MYB has potential as a therapeutic approach against acute myeloid leukemia. To facilitate the identification of small-molecule MYB inhibitors we have re-designed and improved a previously established cell-based screening assay and have employed it to screen a natural product library for potential inhibitors. Our work shows that teniposide and etoposide, chemotherapeutic agents causing DNA-damage by inhibiting topoisomerase II, potently inhibit MYB activity and induce degradation of MYB in AML cell lines. MYB inhibition is suppressed by caffeine, suggesting that MYB is inhibited indirectly via DNA-damage signalling. Importantly, ectopic expression of an activated version of MYB in pro-myelocytic NB4 cells diminished the anti-proliferative effects of teniposide, suggesting that podophyllotoxins disrupt the proliferation of leukemia cells not simply by inducing general DNA-damage but that their anti-proliferative effects are boosted by inhibition of MYB. Teniposide and etoposide therefore act like double-edged swords that might be particularly effective to inhibit tumor cells with deregulated MYB.

Bone pain associated with advanced tumor metastasis is the most severe threat to life quality of patients. Highly efficient and low-toxic therapeutics is of urgent need for this complication. Bone tumor metastasis was established by direct bone inoculation of Walker 256 mammary gland carcinoma cells. Bone nociception was measured by mechanical allodynia, thermal hyperalgesia and spontaneous flinches. P2X7R level was determined by immunoblotting. The inward current was recorded by a patch clamp. The related cytokines were determined by ELISA. Our results showed that teniposide (TN) treatment significantly ameliorated bone nociception associated with tumor inoculation to a comparable extent with P2X7-specific inhibitor, BBG, in rat model. The efficient blockade of inward current generation and pro-inflammatory cytokines secretion were observed upon administration with TN. Our data highlighted the therapeutic potency of TN in this complication associated with tumor metastasis and warrants further clinical investigations.

Self-assembled polymeric micelles have been widely applied in anticancer drug delivery systems. Teniposide is a broad spectrum and effective anticancer drug, but its poor water-solubility and adverse effects of commercial formulation (VM-26) restrict its clinical application. In this work, teniposide-loaded polymeric micelles were prepared based on monomethoxy-poly(ethylene glycol)-poly(ε-caprolactone-co-d,l- lactide) (MPEG-PCLA) copolymers through a thin-film hydration method to improve the hydrophilic and reduce the systemic toxicity. The prepared teniposide micelles were without any surfactants or additives and monodisperse with a mean particle size of 29.6±0.3nm. The drug loading and encapsulation efficiency were 18.53±0.41% and 92.63±2.05%, respectively. The encapsulation of teniposide in MPEG-PCLA micelles showed a slow and sustained release behavior of teniposide in vitro and improved the terminal half-life (t 1/2 ), the area under the plasma concentration-time curve (AUC) and retention time of teniposide in vivo compared with VM-26. In addition, teniposide micelles also enhanced the cellular uptake by MCF-7 breast cancer cells in vitro and increased the distribution in tumors in vivo. Teniposide micelles showed an excellent safety with a maximum tolerated dose (MTD) of approximately 50mg teniposide/kg body weight, which was 2.5-fold higher than that of VM-26 (about 20mg teniposide/kg body weight). Furthermore, the intravenous application of teniposide micelles effectively suppressed the growth of subcutaneous MCF-7 tumor in vivo and exhibited a stronger anticancer effect than that of VM-26. These results suggested that we have successfully prepared teniposide-loaded MPEG-PCLA micelles with improved safety, hydrophilic and therapeutic efficiency, which are efficient for teniposide delivery. The prepared teniposide micelles may be promising in breast cancer therapy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The study was designed to identify the safety and antitumor activity of teniposide self-microemulsified drug delivery system (TEN-SMEDDS) previously developed, and to provide evidence for the feasibility and effectiveness of TEN-SMEDDS for application in clinic. The TEN-SMEDDS could form fine emulsion with mean diameter of 279 ± 19 nm, Zeta potential of -6.9 ± 1.4 mV, drug loading of 0.04 ± 0.001% and entrapment efficiency of 98.7 ± 1.6% after dilution with 5% glucose, respectively. The safety, including hemolysis, hypersensitivity, vein irritation and toxicity in vivo, and antitumor activity were assessed, VUNON as a reference. Sulforhodamine B assays demonstrated that the IC50 of TEN-SMEDDS against C6 and U87MG cells were higher than that of VUMON. But the effect of TEN-SMEDDS on the cell cycle distribution and cell apoptotic rate was similar to that of VUMON as observed by flow cytometry. Likewise, the antitumor activity of TEN-SMEDDS was considerable to that of VUMON. Finally, the TEN-SMEDDS exhibited less body weight loss, lower hemolysis and lower myelosuppression as compared with VUMON. In conclusion, promising TEN-SMEDDS retained the antitumor activity of teniposide and was less likely to cause some side effects compared to VUMON. It may be favorable for the application in clinic., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Teniposide (VM-26) is a semisynthetic derivative of podophyllotoxin effective for the treatment of many types of tumors. However, the poor water solubility and adverse effects restrict its clinical use. Our study aimed to develop a novel phospholipid complex albumin nanoparticle (VM-E80-AN) to reduce the systemic toxicity and enhance antitumor activity of VM-26. Egg yolk lecithin E80 and human serum albumin (HSA) were used as the main excipients to replace Cremophor EL in the commercial formulation. The physicochemical properties of VM-E80-AN were characterized to optimize the formulation. Cell and animal studies were further carried out to estimate its tumor inhibition efficacy, biodistribution, and toxicity. Comparison between VM-26 solution and VM-E80-AN showed that VM-E80-AN significantly reduced the toxicity of VM-26 and enhanced the anticancer efficacy of the drug. Thus, VM-E80-AN represents a safe and promising formulation of teniposide for clinical application., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from high density lipoprotein to triglyceride-rich lipoproteins. CETP expression can be transcriptionally activated by liver X receptor (LXR). Etoposide and teniposide are DNA topoisomerase II (Topo II) inhibitors. Etoposide has been reported to inhibit atherosclerosis in rabbits with un-fully elucidated mechanisms. In this study we determined if Topo II activity can influence cholesterol metabolism by regulating hepatic CETP expression. Inhibition of Topo II by etoposide, teniposide, or Topo II siRNA increased CETP expression in human hepatic cell line, HepG2 cells, which was associated with increased CETP secretion and mRNA expression. Meanwhile, inhibition of LXR expression by LXR siRNA attenuated induction of CETP expression by etoposide and teniposide. Etoposide and teniposide induced LXRα expression and LXRα/β nuclear translocation while inhibiting expression of receptor interacting protein 140 (RIP140), an LXR co-repressor. In vivo, administration of teniposide moderately reduced serum lipid profiles, induced CETP expression in the liver, and activated reverse cholesterol transport in CETP transgenic mice. Our study demonstrates a novel function of Topo II inhibitors in cholesterol metabolism by activating hepatic CETP expression and reverse cholesterol transport., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Although the incidence of glioma is relatively low, it is the most malignant tumor of the central nervous system. The prognosis of high-grade glioma patient is very poor due to the difficulties in complete resection and resistance to radio-/chemotherapy. Therefore, it is worth investigating the molecular mechanisms involved in glioma drug resistance. MicroRNAs have been found to play important roles in tumor progression and drug resistance. Our previous work showed that miR-181b is involved in the regulation of temozolomide resistance. In the current study, we investigated whether miR-181b also plays a role in antagonizing the effect of teniposide., Methods: MiR-181b expression was measured in 90 glioma patient tissues and its relationship to prognosis of these patients was analyzed. Cell sensitivity to teniposide was tested in 48 primary cultured glioma samples. Then miR-181b stably overexpressed U87 cells were generated. The candidate genes of miR-181b from our previous study were reanalyzed, and the interaction between miR-181b and target gene MDM2 was confirmed by dual luciferase assay. Cell sensitivity to teniposide was detected on miR-181b over expressed and MDM2 down regulated cells., Results: Our data confirmed the low expression levels of miR-181b in high-grade glioma tissues, which is related to teniposide resistance in primary cultured glioma cells. Overexpression of miR-181b increased glioma cell sensitivity to teniposide. Through target gene prediction, we found that MDM2 is a candidate target of miR-181b. MDM2 knockdown mimicked the sensitization effect of miR-181b. Further study revealed that miR-181b binds to the 3'-UTR region of MDM2 leading to the decrease in MDM2 levels and subsequent increase in teniposide sensitivity. Partial restoration of MDM2 attenuated the sensitivity enhancement by miR-181b., Conclusions: MiR-181b is an important positive regulator on glioma cell sensitivity to teniposide. It confers glioma cell sensitivity to teniposide through binding to the 3'-UTR region of MDM2 leading to its reduced expression. Our findings not only reveal the novel mechanism involved in teniposide resistance, but also shed light on the optimization of glioma treatment in the future.

Topoisomerases are a family of vital enzymes capable of resolving topological problems in DNA during various genetic processes. Topoisomerase poisons, blocking reunion of cleaved DNA strands and stabilizing enzyme-mediated DNA cleavage complex, are clinically important antineoplastic and anti-microbial agents. However, the rapid rise of drug resistance that impedes the therapeutic efficacy of these life-saving drugs makes the discovering of new lead compounds ever more urgent. We report here a facile high throughput screening system for agents targeting human topoisomerase IIα (Top2α). The assay is based on the measurement of fluorescence anisotropy of a 29 bp fluorophore-labeled oligonucleotide duplex. Since drug-stabilized Top2α-bound DNA has a higher anisotropy compared with free DNA, this assay can work if one can use a dissociating agent to specifically disrupt the enzyme/DNA binary complexes but not the drug-stabilized ternary complexes. Here we demonstrate that NaClO4, a chaotropic agent, serves a critical role in our screening method to differentiate the drug-stabilized enzyme/DNA complexes from those that are not. With this strategy we screened a chemical library of 100,000 compounds and obtained 54 positive hits. We characterized three of them on this list and demonstrated their effects on the Top2α-mediated reactions. Our results suggest that this new screening strategy can be useful in discovering additional candidates of anti-cancer agents.

ATP-binding cassette transporter A1 (ABCA1) facilitates cholesterol efflux and thereby inhibits lipid-laden macrophage/foam cell formation and atherosclerosis. ABCA1 expression is transcriptionally regulated by activation of liver X receptor (LXR). Both etoposide and teniposide are DNA topoisomerase II (Topo II) inhibitors and are chemotherapeutic medications used in the treatment of various cancers. Interestingly, etoposide inhibits atherosclerosis in rabbits by unclear mechanisms. Herein, we report the effects of etoposide and teniposide on macrophage ABCA1 expression and cholesterol efflux. Both etoposide and teniposide increased macrophage free cholesterol efflux. This increase was associated with increased ABCA1 mRNA and protein expression. Etoposide and teniposide also increased ABCA1 promoter activity in an LXR-dependent manner and formation of the LXRE-LXR/RXR complex indicating that transcriptional induction had occurred. Expression of ABCG1 and fatty acid synthase (FAS), another two LXR-targeted genes, was also induced by etoposide and teniposide. In vivo, administration of mice with either etoposide or teniposide induced macrophage ABCA1 expression and enhanced reverse cholesterol transport from macrophages to feces. Taken together, our study indicates that etoposide and teniposide increase macrophage ABCA1 expression and cholesterol efflux that may be attributed to the anti-atherogenic properties of etoposide. Our study also describes a new function for Topo II inhibitors in addition to their role in anti-tumorigenesis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We attempted to improve the oral delivery of lipophilic teniposide to achieve higher drug concentration in tumor by self-assembled nanocarrier for further oral chemotherapy. The teniposide loaded self-assembled nanocarrier (TSN) was spherical nanometric particles with narrow size distribution. The intestinal absorption of teniposide from TSN was obviously improved 4.09- and 6.35-fold in duodenum and jejunum at 0.5h after oral administration, then significantly decreased with the prolongation of time. The cellular uptake of TSN in Caco-2 cell monolayer was significantly enhanced over 3 folds and increased with incubation time. Moreover, TSN could be internalized into Caco-2 cell monolayer through clathrin-mediated endocytosis pathway, and then mainly transported into the systemic circulation via portal vein and intestinal lymphatic pathway. The pharmacokinetic results indicated that the AUC(0-t) value of TSN in rats was significantly improved 5.41-fold than that of teniposide solution, moreover, the teniposide concentration in tumor from TSN was obviously improved over 7-fold in tumor bearing mice. The captured image indicated that the oral administered TSN could specifically accumulate in tumor in the xenograft model. Therefore, the self-assembled nanocarrier was promising to enhance the oral delivery of lipophilic teniposide and its concentration in tumor for oral chemotherapy., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Medulloblastoma (MB) is one of the most common malignant brain tumors of childhood and is associated with a poor prognosis. Gap-junctional intercellular communication (GJIC) is an important mode for cell-to-cell communication. Dysfunctional GJIC is exhibited in most cancer cells. There is significant evidence that GJIC is important in at least some prodrug/suicide gene systems by augmenting the bystander effect (BE). GJIC is made up of connexins (Cxs), among which Cx43 is present in most tissues. Bcl-2, an important apoptosis blocker, is closely associated with the sensitivity to anticancer drugs. Our study showed that dibutyryl cyclic adenosine monophosphate (db-cAMP) upregulated the Cx43 expression and GJIC function in Daoy medulloblastoma cells. It directly enhanced the BE using a herpes simplex virus thymidine kinase (HSV‑tk)/ganciclovir (GCV) system, which was blocked by a Cx43 inhibitor. In addition, db-cAMP increased the cytotoxicity of temozolomide and teniposide, possibly by downregulating the Bcl-2 expression and inducing apoptosis. Taken together, we demonstrated the beneficial effect of db-cAMP in treating medulloblastoma depending on the upregulation of BE and chemosensitivity through Cx43 and Bcl-2-mediated pathways.

The AKT2 kinase (protein kinas Bβ) is frequently overexpressed in malignant gliomas. In this study, the human glioblastoma cell line U87 was stably transfected with a lentivirus vector expressing a short hairpin RNA (shRNA) targeting AKT2. Knockdown of AKT2 by the shRNA inhibited U87 cell proliferation and increased the rate of apoptosis. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and Western blot analysis revealed that cells stably underexpressing AKT2 showed lower expression of the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) and enhanced expression of the apoptosis effector caspase-3 compared to U87 cells stably transfected with a control vector. Furthermore, expression levels of AKT2 were correlated with the IC50 of the antitumor drug VM-26 (teniposide); the VM-26 IC50 was reduced from 6.46±0.42μg/ml in control glioma cells to 1.15±0.22μg/ml in U87 cells underexpressing AKT2. Combined AKT2 knockdown and VM-26 treatment inhibited cell proliferation in vitro more effectively than either treatment alone. Knockdown of AKT2 expression was associated with decreased expression of the multidrug resistance-associated protein 1 (MRP1) without affecting MRP1 mRNA expression. However, the mRNA and protein levels of MDR1 (p-glycoprotein) were unaffected by AKT2 knockdown. These results indicate that inhibition of AKT2 expression may be an effective means for overcoming AKT2-associated chemoresistance in human malignant glioma cells and may represent a potential gene-targeting approach to treat glioma., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The ability of topoisomerase II inhibitor, teniposide, to induce aneuploidy and meiotic delay in somatic and germinal cells of male mice was investigated by fluorescence in situ hybridisation (FISH) assay using labelled DNA probes and 5-bromo-2'-deoxyuridine (BrdU) incorporation assay, respectively. Colchicine and mitomycin C were used as a positive control aneugen and clastogen, respectively, and these compounds produced the expected responses. Using FISH assay with centromeric and telomeric DNA probes for erythrocyte, micronuclei (MN) showed that teniposide is not only clastogenic but also aneugenic in somatic cells in vivo. The assay also showed that chromosomes can be enclosed in the MN before and after centromere separation. By using the BrdU incorporation assay, it could be shown that the meiotic delay caused by teniposide in germ cells was ∼48 h. Disomic and diploid sperms were shown in epididymal sperm hybridised with DNA probes specific for chromosomes 8, X and Y after teniposide treatment. The prevalence of autodiploid (XX88 and YY88) sperm and disomic XX8 or YY8 sperm indicates that the second meiotic division was more sensitive to teniposide than the first meiotic division. The results also suggest that earlier prophase stages contribute relatively less to teniposide-induced aneuploidy. Both the clastogenic and the aneugenic potential of teniposide can give rise to the development of secondary tumours and abnormal reproductive outcomes in cured cancer patients and medical personnel exposing to drug regimens that include teniposide. Thus, genetic counselling of these patients should take place before the start of chemotherapy and should take the present results into consideration.

Previous studies have demonstrated that apoptosis repressor with caspase recruitment domain (ARC) is up-regulated in many forms of malignant tumors and low levels of ARC protein were expressed in normal human brain tissue. Little is known expression of ARC in glioma. Here, we found that ARC protein was highly expressed in primary human glioma when compared with normal brain tissues. A decrease in cell viability and an increase in apoptosis were observed in U251MG cells after ARC was knocked down. Knockdown of ARC was confirmed by western blotting. Knockdown of ARC promoted caspase-8, caspase-3 activation and Bax accumulation. These results indicate that ARC has a anti-apoptosis function in glioma.

Podophyllotoxin, one of the well-known naturally occurring aryltetralin lignans, has been used as the lead-compound for the preparation of potent anticancer agents, such as etoposide, teniposide, and etopophos. In our previous review, we described the advances of podophyllotoxin derivatives from 2003 and 2007. In recent years, an increased number of interesting research work has been carried out on the podophyllotoxins. As a continuation, the present review summarizes and highlights the update advances of podophyllotoxin derivatives from 2008 and 2010 in regard to semisynthesis, biosynthesis, biological activities, mode of action and structure-biological activity relationship.

Basic fibroblast growth factor (bFGF) is an important growth factor for glioma cell proliferation and invasion. BFGF is overexpressed in malignant gliomas and its level is associated with malignant grades and clinical outcome of patients with gliomas. Small interfering RNAs (siRNA) are synthetic forms of microRNA made of short double stranded RNA, and they effectively catalyze the degradation of their target mRNA. The use of siRNA has become a key method in the suppression of gene expression and the development of therapeutic agents. In this study, we used an adenovirus(Ad)-mediated transfer of siRNA against bFGF mRNA (Ad-bFGF-siRNA) to study the effect of down-regulating bFGF expression on the sensitivity of glioma cells to chemotherapeutics. An optimal siRNA sequence specific for bFGF mRNA was cloned into an adenoviral vector and transfected into three glioma cell lines: U251, A172, and LN229. Methyl thiazolyl tetrazolium (MTT) assays were used to examine changes in cell proliferation, and changes in bFGF mRNA and protein levels in U251 cells were detected using quantitative RT-PCR and Western blot, respectively. Apoptosis of U251 cells was detected using Hoechst staining and flow cytometry, with expression of apoptosis-related proteins evaluated by Western blot. Following the transfection of a bFGF-specific siRNA, mRNA and protein levels of bFGF decreased significantly. Lower rates of proliferation and increased levels of apoptosis also were associated with the Ad-bFGF-siRNA-transfected group compared to control group. Decreased expression of Bcl-2, Bcl-xL, Jak-1, and STAT-3 and increased expression of Bax also were detected in the Ad-bFGF-siRNA-transfected group. For cells treated with both Ad-bFGF-siRNA and chemotherapeutics, a significant reduction in cell survival was observed compared to treatment with Ad-bFGF-siRNA or chemotherapeutics alone. Overall, we found that targeting bFGF mRNA with a siRNA resulted in lower rates of proliferation, increased apoptosis, and enhanced sensitivity of glioma cells to chemotherapy drugs. This suggests that specific targeting of bFGF mRNA may provide a novel approach for the treatment of glioblastoma multiforme (GBM).

Brain tumor stem-like cells (BTSLCs) have been implied to play an important role in genesis and development of glioma. However, their characteristics on proliferation and drug-resistance are uncertain thoroughly. In this experiment, some of the biological characteristics about BTSLCs were explored. Twenty cases of different grades of human glioma tissues were obtained from clinic. The primary glioma cells were collected and CD133(+) cells from them were purified by magnetic cell sorting assay. The BTSLCs were identified by testing the expression of CD133, Nestin, NSE, and GFAP, along with the culture process. WST-8 assay kit was used to evaluate the proliferating situation of CD133(+) cells in the different grade gliomas, and to compare the drug-resistance between the CD133(+) and CD133( - ) cells in the medium containing different concentrations of teniposide (VM-26). The results showed that the CD133(+) cells could regenerate by self-renewal, then generate and different into NSE(+) and GFAP(+) cells, respectively. CD133(+) cells in the high grade of gliomas showed the faster generation than the ones in the low grade. The number of survived CD133(+) cells in the medium containing VM-26 was much more than the CD133(-) ones in it. Therefore, it was implied that the CD133(+) BTSLCs existed in the glioma tissues possessed the more tolerant ability to the VM-26, and could proliferate much more easily in the high-grade glioma.

Background: The topoisomerases Top1, Top2alpha and Top2beta are important molecular targets for antitumor drugs, which specifically poison Top1 or Top2 isomers. While it was previously demonstrated that poisoned Top1 and Top2beta are subject to proteasomal degradation, this phenomena was not demonstrated for Top2alpha., Methodology/principal Findings: We show here that Top2alpha is subject to drug induced proteasomal degradation as well, although at a lower rate than Top2beta. Using an siRNA screen we identified Bmi1 and Ring1A as subunits of an E3 ubiquitin ligase involved in this process. We show that silencing of Bmi1 inhibits drug-induced Top2alpha degradation, increases the persistence of Top2alpha-DNA cleavage complex, and increases Top2 drug efficacy. The Bmi1/Ring1A ligase ubiquitinates Top2alpha in-vitro and cellular overexpression of Bmi1 increases drug induced Top2alpha ubiquitination. A small-molecular weight compound, identified in a screen for inhibitors of Bmi1/Ring1A ubiquitination activity, also prevents Top2alpha ubiquitination and drug-induced Top2alpha degradation. This ubiquitination inhibitor increases the efficacy of topoisomerase 2 poisons in a synergistic manner., Conclusions/significance: The discovery that poisoned Top2alpha is undergoing proteasomal degradation combined with the involvement of Bmi1/Ring1A, allowed us to identify a small molecule that inhibits the degradation process. The Bmi1/Ring1A inhibitor sensitizes cells to Top2 drugs, suggesting that this type of drug combination will have a beneficial therapeutic outcome. As Bmi1 is also a known oncogene, elevated in numerous types of cancer, the identified Bmi1/Ring1A ubiquitin ligase inhibitors can also be potentially used to directly target the oncogenic properties of Bmi1.

MicroRNAs control a wide array of biological processes including cell differentiation, proliferation, and apoptosis whose dysregulation is a hallmark of cancer. MicroRNA-21 (miR-21) is overexpressed in many cancers including glioblastoma and contributes to tumor resistance to chemotherapy. We investigated whether miR-21 mediated chemoresistance to the chemotherapeutic agent VM-26 in glioblastoma cells and sought to identify the candidate target genes for miR-21 by gene expression profiling. Here we report that miR-21 was involved in mediating chemoresistance to VM-26 in glioblastoma cells. Suppression of miR-21 by specific antisense oligonucleotides in glioblastoma cell U373 MG led to enhanced cytotoxicities of VM-26 against U373 MG cells. We further identified and validated LRRFIP1, whose product is an inhibitor of NF-kappaB signaling, as a direct target gene of miR-21. Our findings suggest that miR-21 represents a promising target for therapeutic manipulation to increase the efficacy of chemotherapeutic agents in treating glioblastoma, a highly lethal type of cancer.

Podophyllotoxin is an important and much sought after antimitotic natural lead compound, since it paved the way for three hemisynthetic derivatives of podophyllotoxin, e.g., etoposide, teniposide and etopophos, which are widely used as anticancer drugs and show good clinical effects against several types of neoplasms. Although the publication of the recent reviews by Gordaliza in 2004 and You in 2005, which covered the literatures concerning podophyllotoxin until the early part of 2003, there have been significant number of works carried out on podophyllotoxin recently. Therefore, this review presents up-to-date coverage of podophyllotoxin in regard to hemisynthesis, biosynthesis, biological activities, mode of action and structure-activity relationship.

Although centromere function has been conserved through evolution, apparently no interspecies consensus DNA sequence exists. Instead, centromere DNA may be interconnected through the formation of certain DNA structures creating topological binding sites for centromeric proteins. DNA topoisomerase II is a protein, which is located at centromeres, and enzymatic topoisomerase II activity correlates with centromere activity in human cells. It is therefore possible that topoisomerase II recognizes and interacts with the alpha satellite DNA of human centromeres through an interaction with potential DNA structures formed solely at active centromeres. In the present study, human topoisomerase IIalpha-mediated cleavage at centromeric DNA sequences was examined in vitro. The investigation has revealed that the enzyme recognizes and cleaves a specific hairpin structure formed by alpha satellite DNA. The topoisomerase introduces a single-stranded break at the hairpin loop in a reaction, where DNA ligation is partly uncoupled from the cleavage reaction. A mutational analysis has revealed, which features of the hairpin are required for topoisomerease IIalpha-mediated cleavage. Based on this a model is discussed, where topoisomerase II interacts with two hairpins as a mediator of centromere cohesion.

Several DNA- and microtubule-binding agents are used to manage hematologic malignancies in the clinic. However, drug resistance has been a challenge, perhaps due to a few surviving cancer stem cells. Toxicity is another major impediment to successful chemotherapy, leading to an impoverished quality of life. Here, we show that a semisynthetic nontoxic tubulin-binding agent, 9-bromonoscapine (EM011), effectively inhibits growth and regresses multidrug resistance-associated protein (MRP)-overexpressing teniposide-resistant T-cell lymphoma xenografts and prolongs longevity. As expected, teniposide treatment failed to regress teniposide-resistant xenografts, rather, treated mice suffered tremendous body weight loss. Mechanistically, EM011 displays significant antiproliferative activity, perturbs cell cycle progression by arresting mitosis, and induces apoptosis in teniposide-resistant lymphoblastoid T cells both in vitro and in vivo. EM011-induced apoptosis has a mitochondrially-mediated component, which was attenuated by pretreatment with cyclosporin A. We also observed alterations of apoptosis-regulatory molecules such as inactivation of Bcl2, translocation of BAX to the mitochondrial membrane, cytochrome c release, and activation of downstream apoptotic signaling. EM011 caused DNA degradation as evident by terminal deoxynucleotidyl transferase-mediated dUTP-biotin end labeling staining of the increased concentration of 3'-DNA ends. Furthermore, the apoptotic induction was caspase dependent as shown by cleavage of the caspase substrate, poly(ADP)ribose polymerase. In addition, EM011 treatment caused a suppression of natural survival pathways such as the phosphatidylinositol-3'-kinase/Akt signaling. These preclinical findings suggest that EM011 is an excellent candidate for clinical evaluation.

Background & Objective: Some studies have showed that topoisomerase (Topo)I and II inhibitors have synergistic effects in tumor therapy, but the combinations have seldom been used in gastric cancer. This study was to investigate the effects of Topo I inhibitor hydroxycamptothecin (HCPT) and Topo II inhibitor teniposide (VM-26) on the proliferation and apoptosis of gastric cancer cell line BGC-823., Methods: MTT assay was used to examine the inhibitory effects of VM-26 and HCPT, used alone or in combination, on the proliferation of BGC-823 cells. Cell apoptosis was examined by flow cytometry (FCM)., Results: The inhibition rates of BGC-823 cell proliferation were 15.99%-80.83% when treated with 1.963-31.413 micromol/L VM-26; the apoptosis rates were 3.90%, 4.42%, 7.36%, 17.07% when exposed to 1.963 micromol/L VM-26 for 0, 12, 24, 48 h, respectively. The inhibition rates of BGC-823 cell proliferation were 7.89%-70.32% when treated with 8.577-137.227 micromol/L HCPT; the apoptosis rates were 2.80%, 8.50%, 10.50%, 13.30% when exposed to 8.577 micromol/L HCPT for 0, 12, 24, 48 h, respectively. When treated with 1.963 micromol/L VM-26 and 3.125 microg/ml HCPT for 0, 12, 24, 48 h, the inhibition rates of BGC-823 cell proliferation were 21.32%-87.74%, and the apoptosis rates were 2.80%, 15.50%, 15.70%, 20.20%, respectively. The combination index (CI) was 1.293., Conclusion: HCPT and VM-26 used alone could inhibit the proliferation and induce the apoptosis of BGC-823 cells, and they have antagonistic effect on gastric cancer BGC-823 cells.

Naturally occurring polysaccharides isolated from the yeasts are the substances with versatile intriguing biomodulatory activities. One of the novel derivatives prepared from the (1 --> 3)-beta-D-glucan isolated from the cell walls of baker's yeast Saccharomyces cerevisiae is sulfoethyl glucan (SEG). Its DNA-protective, antimutagenic, anticlastogenic and cytotoxic/cytostatic enhancing effect was evaluated using five eukaryotic systems. SEG showed bioprotective effect in recombination- repair-deficient strain of alga Chlamydomonas reinhardtii against methyl methanesulfonate-induced genotoxicity, antimutagenic effect against ofloxacin-induced genetic changes in yeast Saccharomyces cerevisiae assay and anticlastogenic activity in plants Vicia sativa and Vicia faba assays against maleic hydrazide-induced clastogenicity. In the combined application with cytostatic drug vumon, SEG exerted enhancement of the drug's cytotoxic/cytostatic effect in the cell revitalization assay using mouse leukemia cells. The study sheds light on the possible mechanisms of actions and utilization of this microbial polysaccharide derivative in the cancer prevention and therapy.

Purpose: The present study is designed to identify quantitatively the in vitro effect of VM-26 against oral squamous cell carcinoma Tca8113 and to explore the underlying mechanism., Methods: Human tongue squamous cell carcinoma cell line Tca8113 was used as subject. Cells were incubated with VM-26 of various concentrations for a variety of time spans. Cisplatin (CDDP) was employed as a control agent. MTT assay was used to assess the inhibition of cell growth by VM-26. Flow cytometer (FCM), transmission electron microscope (TEM) and fluorescence staining were employed for determining the apoptotic rate of cells induced by VM-26. Cell cycle distribution of Tca8113 cells incubated with VM-26 was examined through flow cytometer assay. SAS 6.12 was used for one-way ANOVA., Results: The IC50 of VM-26 against Tca8113 cells was 0.35microg/ml and that of CDDP was 1.1microg/ml. The changes in morphology and ultrastructure of Tca8113 cells were observed using fluorescence microscope and TEM, and apoptotic morphological feature could be found in the nucleus. Apoptotic rate of Tca8113 cells incubated with VM-26 of 5.0microg/ml for 72 hours was 81.01% and cell cycle appeared was arrested at S phase. However, exposed to VM-26 of 0.15microg/ml for 72 hours, Tca8113 cells in the G2/M phase increased from 12.75% to 98.71%, while the apoptotic rate of these cells was 17.38% and far below than that of cells treated with VM-26 of 5.0microg/ml., Conclusion: VM-26 could significantly induce apoptosis of oral squamous cell carcinoma cells and inhibit cell growth. There may be another pathway to induce apoptosis of oral squamous cell carcinoma cells except for G2/M phase arrest. Supported by National Natural Science Foundation of China (30300388, 30171014, 30330580) and Research Grant (No.036505013, 034107002) of Science and Technology Committee of Shanghai Municipality.

Background: Hypofractionated stereotactic radiotherapy (HFSRT) is one salvage treatment option in previously irradiated patients with recurrent malignant glioma. We analyzed the results of HFSRT and prognostic factors in a single-institution series., Methods: Between 1997 and 2003, 19 patients with recurrent malignant glioma (14 glioblastoma on most recent histology, 5 anaplastic astrocytoma) were treated with HFSRT. The median interval from post-operative radiotherapy to HFSRT was 19 (range 3-116) months, the median daily single dose 5 (4-10) Gy, the median total dose 30 (20-30) Gy and the median planning target volume 15 (4-70) ml., Results: The median overall survival (OS) was 9.3 (1.9-77.6+) months from the time of HFSRT, 15.4 months for grade III and 7.9 months for grade IV tumors (p = 0.029, log-rank test). Two patients were alive at 34.6 and 77.6 months. OS was longer after a total dose of 30 Gy (11.1 months) than after total doses of <30 Gy (7.4 months; p = 0.051). Of five (26%) reoperations, none was performed for presumed or histologically predominant radiation necrosis. Median time to tumor progression after HFSRT on imaging was 4.9 months (1.3 to 37.3) months., Conclusion: HFSRT with conservative total doses of no more than 30 Gy is safe and leads to similar OS times as more aggressive treatment schemes. In individual patients, HFSRT in combination with other salvage treatment modalities, was associated with long-term survival.

Purpose: The purpose of this study is to define the mechanistic basis for recovery of proliferative capacity in breast tumor cells after chemotherapy. Here, we test the hypothesis that evasion of senescence confers resistance to chemotherapeutic drugs and ionizing radiation., Experimental Design: MCF-7 cells were treated with a single, clinically relevant dose (0.75-1.0 micromol/L) of Adriamycin. Two weeks following induction of senescence, clonal outgrowths were expanded and characterized in terms of senescence-associated beta-galactosidase activity, gene expression profiles (Affymetrix U95 probe sets, Affymetrix, Santa Clara, CA) with confirmatory Western analyses, and telomerase activity following a second drug treatment. Levels of intracellular Adriamycin, as well as cross-resistance to other therapeutic agents, were also determined to define the resistance phenotype., Results: A senescence-resistant (SR) clone (clone 2) was identified that was largely refractory to both Adriamycin-induced and gamma-irradiation-induced senescence. Clone 2 continued to proliferate and maintain high levels of telomerase activity following a second drug treatment, when treated parental cells expressed very low levels of telomerase and many positive cell cycle regulators. SR clone 2 also expressed substantially more cdc-2 than parental cells and undetectable levels of MDR1, showed an intact p53 checkpoint and only a modestly lower level of intracellular drug accumulation, while exhibiting cross-resistance to other topoisomerase inhibitors., Conclusions: SR clone 2 is intrinsically resistant to DNA damage-induced senescence perhaps through an ability to prevent down-regulation of cdc-2. Telomerase is a marker of proliferative recovery for breast cancer cells after chemotherapy exposure. Evasion or escape from a single-step, drug-induced senescence may represent a unique and previously unrecognized drug-resistance phenotype.

Fluoroquinolones are broad-spectrum antimicrobial agents that target type II topoisomerases. Many fluoroquinolones are highly specific for bacterial type II topoisomerases and act against both DNA gyrase and topoisomerase IV. In Escherichia coli, mutations causing quinolone resistance are often found in the gene that encodes the A subunit of DNA gyrase. One common site for resistance-conferring mutations alters Ser83, and mutations to Leu or Trp result in high levels of resistance to fluoroquinolones. In the present study we demonstrate that the mutation of Ser83 to Trp in DNA gyrase (Gyr(S83W)) also results in sensitivity to agents that are potent inhibitors of eukaryotic topoisomerase II but that are normally inactive against prokaryotic enzymes. Epipodophyllotoxins, such as etoposide, teniposide and amino-azatoxin, inhibited the DNA supercoiling activity of Gyr(S83W), and the enzyme caused elevated levels of DNA cleavage in the presence of these agents. The DNA sequence preference for Gyr(S83W)-induced cleavage sites in the presence of etoposide was similar to that seen with eukaryotic type II topoisomerases. Introduction of the Gyr(S83W) mutation in E. coli strain RFM443-242 by site-directed mutagenesis sensitized it to epipodophyllotoxins and amino-azatoxin. Our results demonstrate that sensitivity to agents that target topoisomerase II is conserved between prokaryotic and eukaryotic enzymes, suggesting that drug interaction domains are also well conserved and likely occur in domains important for the biochemical activities of the enzymes.

Several podophyllotoxin derivatives modified in the A, B, C, D and E rings were prepared from podophyllotoxin and methyl isoxazopodophyllic acid and evaluated for their cytotoxicity on several neoplastic cell lines. Chemical transformations performed on these compounds have yielded derivatives more potent and more selective that the parent compound. Most of the compounds maintained their cytotoxicity at the microM level. Distribution, biosynthesis, production, biotechnology, applications and synthesis have also been reviewed.

Ubc9 is an E2-conjugating enzyme required for sumoylation and has been implicated in regulating several critical cellular pathways. We have shown previously that Ubc9 is important for sumoylation and nucleolar delocalization of topoisomerase (topo) I in response to topo I inhibitors such as topotecan. However, the role for Ubc9 in tumor drug responsiveness is not clear. In this study, we found that although MCF7 cells expressing a Ubc9 dominant-negative mutant (Ubc9-DN) display decreased activity of topo I, these cells are more sensitive to the topo I inhibitor topotecan and other anticancer agents such as VM-26 and cisplatin. In addition, we found that alteration of Ubc9 expression correlates with drug responsiveness in tumor cell lines. To understand possible mechanisms of Ubc9-associated drug responsiveness, we examined several proteins that have been shown to interact with Ubc9 and that may be involved in drug responsiveness. One such protein is Daxx, which is a Fas-associated protein that plays a role in Fas-mediated apoptosis by participating in a caspase-independent pathway through activation of apoptosis signal-regulating kinase 1 and c-Jun NH(2)-terminal kinase. We found that cells expressing Ubc9-DN accumulate more cytoplasmic Daxx than the control cells. Because cytoplasmic Daxx is believed to participate in cellular apoptosis, we suggest that the interaction of Ubc9 with Daxx and subsequent alteration in the subcellular localization of Daxx may contribute to the increased sensitivity to anticancer drugs in the cells expressing Ubc9-DN. Finally, we found that overexpression of Daxx sensitizes cells to anticancer drugs possibly in part through alterations of the ratio of cytoplasmic and nuclear Daxx. Together, our results suggest a role for Ubc9 in tumor drug responsiveness.

Objective: To explore the effect of antisense oligodeoxynucleotide (ASODN) of survivin gene on apoptosis and chemotherapy sensitivity of lymphoma cell line Raji., Methods: Anti-survivin phosphorothioate ASODN was synthesized and transfected into Raji cells by lipofectin. MTT assay was used to detect cytotoxicity. Apoptosis was observed by fluorescence microscopy and flow cytometry. Survivin expression was determined by RT-PCR and Western-blotting., Results: (1) survivin ASODN inhibited the cells proliferation in a dose and time dependent manner. (2) A higher apoptosis rate (33.0%) could be induced in Raji cells by survivin ASODN as compared with that induced by the sense oligodeoxynucleotide (11.5%) (P < 0.05). (3) The expression of survivin mRNA and protein significantly decreased after treatment with survivin ASODN. (4) There was a significant increase of cell inhibition rate after exposure to the combination of survivin ASODN and Vm26 as compared to Vm26 or survivin ASODN alone (both P < 0.05)., Conclusion: Survivin ASODN is able to inhibit the proliferation of Raji cells, induce the apoptosis, and enhance the sensitivity of Raji cell to chemotherapy via specific down-regulation of survivin expression.

Objective: To investigate the antitumor effectiveness of teniposide in oral squamous cell carcinomas (OSCC) and to find evidence for using teniposide for treatment of patients with OSCC., Methods: Seventy-five patients with OSCC from the School of Stomatology, Shanghai Second medical University during 1999 to 2001 were evaluated. Tumors were diagnosed pathologically, and drug sensitivity tested. The antitumor drugs tested were cisplatin (CDDP) and teniposide (VM-26). Fresh drug was diluted in complete medium at fold of five times of peak plasma concentration (PPC x 5) achieved by intravenous administration of clinical doses. The concentrations were VM-26 60 mg/L, CDDP 15 mg/L., Results: The MTT assay was performed in 75 of 81 patients (success rate 92.6%). The clinical stages of the 75 patients according to the UICC TNM classification of malignant tumors were 28 with stage IV, 34 with stage III, 11 with stage II and 2 with stage I. The pathological grades of the 75 patients according to three step classification were 18 with Grade I, 37 with Grade I approximately II and 20 with Grade III. At a drug concentration of PPC x 5, the inhibition rates of tumor cells for VM-26 and CDDP were 63.34% and 24.08%, respectively. The inhibition rates of tumor cells for VM-26 were significantly higher than those for CDDP (P < 0.01)., Conclusions: The inhibition rates of OSCC cells for VM-26 are significantly higher than for CDDP. VM-26 may be the first selected drug for treating patients with OSCC.

The two known antineoplastic quinoxaline topoisomerase II poisons, XK469 (NSC 697887) and CQS (chloroquinoxaline sulfonamide, NSC 339004), were compared for DNA cleavage site specificity, using purified human topoisomerase IIalpha and human topoisomerase IIbeta. The DNA cleavage intensity pattern for topoisomerase IIalpha poisoning by CQS closely resembled that of VM-26, despite the lack of any apparent common pharmacophore. In contrast, the topoisomerase IIalpha DNA cleavage intensity patterns of XK469 and CQS were very different from one another despite the similar overall structures of the two drugs. This suggests that the differences in DNA site specificity of topoisomerase II poisoning by XK469 and CQS may be caused by differences in their geometry, side chains, or electronic structure. The topoisomerase IIbeta-mediated DNA cleavage sites of CQS and XK469 were also very different from one another, adding further support to this idea. Earlier work has demonstrated that a number of specific topoisomerase II poisons show very similar patterns of DNA cleavage with either topoisomerase IIalpha or topoisomerase IIbeta, suggesting that the topoisomerase II isozymes play only a minor role in choices of DNA cleavage sites. However, both of the quinoxaline topoisomerase II poisons in this study showed distinctly different and unique DNA cleavage intensity patterns with each topoisomerase II isozyme. This indicates that topoisomerase II isozymes can play a major role in DNA cleavage site selection for some classes of topoisomerase II poisons.

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.

It has been proposed that the topoisomerase II (TOP2)beta-DNA covalent complex arrests transcription and triggers 26S proteasome-mediated degradation of TOP2beta. It is unclear whether the initial trigger for proteasomal degradation is due to DNA damage or transcriptional arrest. In the current study we show that the TOP2 catalytic inhibitor 4,4-(2,3-butanediyl)-bis(2,6-piperazinedione) (ICRF-193), which traps TOP2 into a circular clamp rather than the TOP2-DNA covalent complex, can also arrest transcription. Arrest of transcription, which is TOP2beta-dependent, is accompanied by proteasomal degradation of TOP2beta. Different from TOP2 poisons and other DNA-damaging agents, ICRF-193 did not induce proteasomal degradation of the large subunit of RNA polymerase II. These results suggest that proteasomal degradation of TOP2beta induced by the TOP2-DNA covalent complex or the TOP2 circular clamp is due to transcriptional arrest but not DNA damage. By contrast, degradation of the large subunit of RNA polymerase II is due to a DNA-damage signal.

The condensin complex and topoisomerase II (topo II) have different biochemical activities in vitro, and both are required for mitotic chromosome condensation. We have used Xenopus egg extracts to investigate the functional interplay between condensin and topo II in chromosome condensation. When unreplicated chromatin is directly converted into chromosomes with single chromatids, the two proteins must function together, although they are independently targeted to chromosomes. In contrast, the requirement for topo II is temporarily separable from that of condensin when chromosome assembly is induced after DNA replication. This experimental setting allows us to find that, in the absence of condensin, topo II becomes enriched in an axial structure within uncondensed chromatin. Subsequent addition of condensin converts this structure into mitotic chromosomes in an ATP hydrolysis-dependent manner. Strikingly, preventing DNA replication by the addition of geminin or aphidicolin disturbs the formation of topo II-containing axes and alters the binding property of topo II with chromatin. Our results suggest that topo II plays an important role in an early stage of chromosome condensation, and that this function of topo II is tightly coupled with prior DNA replication.

Mutations in human multidrug resistance protein 6 (MRP6, ABCC6), a member of the MRP family of drug efflux pumps, are the genetic basis of Pseudoxanthoma elasticum, a disease that affects elastin fibers in the skin, retina, and blood vessels. However, little is known about the functional characteristics of the protein, including its potential activity as a resistance factor for anticancer agents. Here, we report the results of investigations of the in vitro transport properties and drug resistance activity of MRP6. Using membrane vesicles prepared from Chinese hamster ovary cells transfected with MRP6 expression vector, it is shown that expression of MRP6 is specifically associated with the MgATP-dependent transport of the glutathione S-conjugates leukotriene C(4) and S-(2, 4-dinitrophenyl)glutathione and the cyclopentapeptide BQ123 but not glucuronate conjugates such as 17beta-estradiol 17-(beta-D-glucuronide). Analysis of the drug sensitivity of MRP6-transfected cells revealed low levels of resistance to several natural product agents, including etoposide, teniposide, doxorubicin, and daunorubicin. These results indicate that MRP6 is a glutathione conjugate pump that is able to confer low levels of resistance to certain anticancer agents.

Objective: To determine the chemosensitivity in fresh biopsy specimen of human oral and maxillofacial cancer, and the differential chemosensitivity among those drugs used popularly in clinic., Methods: Human biopsy cancer cells were obtained from 150 oral and maxillofacial malignant tumors. The antitumor drugs tested using modified MTT assay were cisplatin (CDDP), 5-fluorouracil (5-Fu), Pinyangmycin (PYM), Paclitaxel (Taxol), Teniposide (Vm-26), Epi-adriamycin (E-ADM), Vindesin (VDS) and Methortrexatum (MTX)., Results: The success rate of the MTT assay was 93.33% (140 of the 150 cases). At a drug concentration of Cmax x 5, the inhibition rates of oral tumor cells were 63.76% for Vm-26, 25.93% for CDDP, 25.86% for E-ADM, 23.52% for Taxol, 22.97% for PYM, 22.08% for 5-Fu, 18.42% for VDS and 18.93% for MTX. The inhibition rate of VM26 was significantly higher than any of other seven chemotherapeutic drugs (P < 0.05). Over forty percent patients with squamous cell carcinoma showed moderate chemosensitivity to VM-26, CDDP and E-ADM, and over forty percent cases with adenoid carcinoma showed moderate chemosensitivity to Vm-26, Taxol and E-ADM., Conclusions: Most oral and maxillofacial cancers showed chemosensitivity to Vm-26, CDDP, E-ADM and Taxol. Vm-26, E-ADM and Taxol were more potent drugs than VDS, 5-Fu and MTX against oral and maxillofacial cancer cells. Chemosensitivity testing using modified MTT assay was useful in selecting antitumor drugs for patients with oral and maxillofacial cancers.

Caspases are essential components of the mammalian cell death machinery. Caspase 9 is positioned at the apex of the pro-apoptotic signaling cascade induced by cytochrome c release from mitochondria. The splicing variant caspase 9-short (caspase 9S) lacks the catalytic domain and may therefore act as an endogenous inhibitor of caspase 9. Here, we report that human astrocytoma and neuroblastoma cell lines strongly express caspase 9 and 9S mRNA, whereas non-neoplastic human astrocytes show little caspase 9 and 9S mRNA expression. Transient overexpression of caspase 9S protects LN-229 astrocytoma cells from CD95 ligand (CD95L)-mediated apoptosis. However, stable overexpression of either caspase 9 or caspase 9S does not alter the sensitivity of LN-18 and LN-229 astrocytoma lines to CD95L or cytotoxic drugs. We conclude that the expression levels of caspase 9 or caspase 9S do not play a major role in determining vulnerability to apoptosis in human astrocytoma cells.

This randomized study of previously untreated patients with extensive disease small cell lung cancer was designed (a) to compare the survival of patients treated with either effective standard chemotherapy or an investigational anti-cancer drug as initial therapy and (b) to evaluate response rates and toxic effects of such therapies. One hundred and thirty-five patients were randomly assigned to receive as initial therapy, either the standard CAV regimen--cyclophosphamide (1000 mg/m(2)), doxorubicin (50 mg/m(2)) and vincristine (1.4 mg/m(2)) every 3 weeks--or the phase II drugs ifosfamide (1.5 gm/m(2)/days 1-5) with mesna (300 mg/m(2)) dose at 0, 4 and 8 h after IV daily ifosfamide every 3 weeks or teniposide (60 mg/m(2)/days 1-5) every 3 weeks. Nonresponders received salvage chemotherapy-etoposide (120 mg/m(2) on days 1, 2 and 3) and cisplatin (60 mg/m(2) on day 1), repeated every 3 weeks. Among the 46 patients on CAV, there were two complete and 24 partial responses (56%). Among the 43 patients on ifosfamide, there were three complete and 18 partial responses (49%), while among the 46 patients on teniposide, there were two complete and 18 partial responses (43%). Eighty-three of the patients proceeded onto salvage regimen, of which 81 were analyzable for response and toxicity. Among the 81 patients who continued on salvage therapy and were evaluable for response, the overall best response rate was 61% for CAV+salvage, 54% for ifosfamide+salvage, and 53% for teniposide+salvage. These rates were not significantly different (P=0.962). Of the 135 analyzable patients, 130 (96%) have died. The estimated median survival time was 42 weeks for CAV patients, 43 weeks for ifosfamide, and 38 weeks for teniposide. Seven patients survived longer than 2 years (four on CAV, one on ifosfamide and two on teniposide). There were 29 life-threatening complications to the induction regimen (22 (48%) on CAV, four (9%) on ifosfamide and three (7%) on teniposide) and seven lethal complications (two on CAV, four on ifosfamide and one on teniposide). The treatments were significantly different with respect to the overall degree of toxicity (P < 0.0001) with CAV being more toxic. The data of this study, like the previous ECOG study suggests that the administration of a new agent followed by effective salvage chemotherapy in the treatment of extensive disease small cell lung cancer may have no adverse effect on survival., (Eastern Cooperative Oncology Group)

A sensitive assay for quantitating DNA damage within individual genes would be a valuable tool for identifying the molecular mechanisms of disease and the sites of action of various carcinogens and anticancer drugs. This report describes a competitive PCR assay that was used to quantitate DNA damage induced by anticancer drugs within a 683-bp region of the c-myc gene in human CEM leukemia cells. Absolute quantitation of gene-specific DNA damage (attomoles or molecules of damaged DNA sequences) was achieved by coamplification of a homologous internal standard that has the same primer binding sites and PCR amplification efficiency as c-myc. The variability (standard error) associated with four separate determinations of the amount of c-myc sequence in 300 ng of DNA from untreated cells (6.80 +/- 0.05 SE amol) was less than 1% of the mean. The assay was capable of quantitating direct DNA damage that was induced by therapeutic concentrations of VM-26 and cisplatin prior to the onset of cellular apoptosis or necrosis. Both VM-26 (1-10 microM) and cisplatin (25-100 microM) induced a dose-dependent decrease in the amount of intact c-myc sequence. This assay should be readily adaptable to current real-time PCR protocols.

Topoisomerase II (topo II) is an essential enzyme that alters DNA topology. This activity is important for a variety of chromosome functions including DNA replication, transcription, recombination, and chromosome condensation and segregation. Previously we localized topo II in mouse gametes and preimplantation embryos using isoform-specific antibodies demonstrating the presence of the enzyme in oocytes and embryos, but not sperm. To probe functions of topo II during preimplantation development, we treated mouse zygotes with 100 nM teniposide, and assessed embryo morphology and DNA replication. Teniposide blocked cleavage in 69% embryos; the remainder cleaved once but had abnormal nuclei. Teniposide-treated embryos were devoid of topo II immunofluorescence. Teniposide also prevented DNA replication, implicating topo II in this process. Embryos treated with a 2 hr pulse of teniposide recovered and developed to the blastocyst stage, indicating 100 nM teniposide did not induce apoptosis. To more specifically analyze topo IIalpha function, we treated zygotes with topo IIalpha-targeted antisense oligodeoxynucleotides. Most zygotes arrested at the 2-cell stage while controls developed into blastocysts indicating topo IIalpha is essential for preimplantation development. The absence of topo IIalpha, but not beta immunofluorescence in antisense-treated embryos confirms the specificity and impact of the treatment. In addition, topo IIalpha is newly synthesized at the 2-cell stage. These results establish an essential function for topo II in mouse preimplantation embryonic development., (Copyright 2002 Wiley-Liss, Inc.)

Eukaryotic centromeres are composed of centromere DNA and the multiple proteins directly or indirectly associated with it. One important DNA-binding protein in the centromere is DNA topoisomerase II (topo II). In the genome in general, topo II has two functions, one structural and one enzymatic, the latter catalyzing DNA strand-passage reactions. It has been demonstrated that topo II accumulates at centromeres during the first part of mitosis, and disappears again at anaphase, but it has not been clear whether it serves a structural or an enzymatic function at the centromere. To investigate this issue, we developed the topo II-induced self-primed in situ assay (Topo-SPRINS). In this assay, DNA breaks created by topo II are stabilized with the topo II inhibitor VM-26 in vivo, and used as 'primers' for localized DNA synthesis in vitro. The assay revealed that topo II has enzymatic activity at mitotic centromeres and that the activity is relatively constant across centromeres. Furthermore, the activity was observed at a neocentromere, and, in multicentric chromosomes, the activity was restricted to the active centromere. The topo II activity is thus selectively present at functioning centromeres, indicating that it plays a role in mitotic centromere function.

DNA topoisomerase II (TOP2) cleavable complexes represent an unusual type of DNA damage characterized by reversible TOP2-DNA cross-links and DNA double strand breaks. Many antitumor drugs and physiological stresses are known to induce TOP2 cleavable complexes leading to apoptotic cell death and genomic instability. However, the molecular mechanism(s) for repair of TOP2 cleavable complexes remains unclear. In the current studies, we show that TOP2 cleavable complexes induced by the prototypic TOP2 poison VM-26 are proteolytically degraded by the ubiquitin/26 S proteasome pathway. Surprisingly the TOP2beta isozyme is preferentially degraded over TOP2alpha isozyme. In addition, transcription inhibitors such as 5,6-dichlorobenzimidazole riboside and camptothecin can substantially block VM-26-induced TOP2beta degradation. These results are consistent with a model in which the repair of TOP2beta cleavable complexes may involve transcription-dependent proteolysis of TOP2beta to reveal the protein-concealed double strand breaks.