Demethylepipodophyllotoxin Research Articles

Discovery of Epipodophyllotoxin-Derived B2 as Promising XooFtsZ Inhibitor for Controlling Bacterial Cell Division: Structure-Based Virtual Screening, Synthesis, and SAR Study.

The emergence of phytopathogenic bacteria resistant to antibacterial agents has rendered previously manageable plant diseases intractable, highlighting the need for safe and environmentally responsible agrochemicals. Inhibition of bacterial cell division by targeting bacterial cell division protein FtsZ has been proposed as a promising strategy for developing novel antibacterial agents. We previously identified 4′-demethylepipodophyllotoxin (DMEP), a naturally occurring substance isolated from the barberry species Dysosma versipellis, as a novel chemical scaffold for the development of inhibitors of FtsZ from the rice blight pathogen Xanthomonas oryzae pv. oryzae (Xoo). Therefore, constructing structure−activity relationship (SAR) studies of DMEP is indispensable for new agrochemical discovery. In this study, we performed a structure−activity relationship (SAR) study of DMEP derivatives as potential XooFtsZ inhibitors through introducing the structure-based virtual screening (SBVS) approach and various biochemical methods. Notably, prepared compound B2, a 4′-acyloxy DMEP analog, had a 50% inhibitory concentration of 159.4 µM for inhibition of recombinant XooFtsZ GTPase, which was lower than that of the parent DMEP (278.0 µM). Compound B2 potently inhibited Xoo growth in vitro (minimum inhibitory concentration 153 mg L−1) and had 54.9% and 48.4% curative and protective control efficiencies against rice blight in vivo. Moreover, compound B2 also showed low toxicity for non-target organisms, including rice plant and mammalian cell. Given these interesting results, we provide a novel strategy to discover and optimize promising bactericidal compounds for the management of plant bacterial diseases.

The discovery of natural 4'-demethylepipodophyllotoxin from renewable Dysosma versipellis species as a novel bacterial cell division inhibitor for controlling intractable diseases in rice

Natural products (NPs) are inexhaustible wellsprings for new agrochemicals discovery. To excavate novel natural products-based antibacterial leads or candidates potentially targeting FtsZ (filamentous temperature-sensitive protein Z) which is widely regarded as a promising antibacterial target for novel drug discovery, herein, a rapid and efficient screening platform was initially established to disclose the potential FtsZ inhibitors from a variety of commercial natural products. Screening results revealed that 4'-demethylepipodophyllotoxin (DMEP) from renewable Dysosma versipellis (D. versipellis) species could distinctly decrease the XooFtsZ’ GTPase activity and afforded a ponderable bacteriostatic effect (EC50 =38.7 mg L-1) against Xanthomonas oryzae pv. oryzae (Xoo). Further explorations found that DMEP could disorganize the assembly dynamics of XooFtsZ, and thereby yielding long and filamentous Xoo cells from the transmission electron microscopic and fluorescence microscopic imaging. Molecular docking study showed that DMEP displayed strong interactions with Asp38 and Arg205 residues of XooFtsZ, indicating that a natural potential FtsZ inhibitor was discovered gradually. The following bioassay result against rice bacterial blight in vivo presented good curative activity (50.0% at 200 mg L−1) and protective activity (46.8% at 200 mg L−1). Given that natural DMEP has many sites for structural modification, therefore, it can concurrently serve as a novel lead compound for exploring highly bioactive FtsZ inhibitors based on the molecular skeleton of DMEP.

Increasing the distance between two monomers of topoisomerase II\u03b2 under the action of antitumor agent 4\u03b2-sulfur-(benzimidazole) 4\u2032-demethylepipodophyllotoxin

Topoisomerases II (Top2s) are a group of essential enzymes involved in replication, transcription, chromosome condensation, and segregation via altering DNA topology. The mechanism of the Top2s poisons such as etoposide (VP-16) was reported as stabilizing the Top2-DNA complex and engendering permanent DNA breakage. As the structurally similar compound of VP-16, a novel 4β-sulfur-substituted 4′-demethylepipodophyllotoxin (DMEP) derivative (compound C-Bi) with superior antitumor activity was developed in our previous study. To understand the structural basis of the compound action, the crystal structure (2.54 Å) of human Top2 β-isoform (hTop2β) cleavage complexes stabilized by compound C-Bi was determined. However, compound C-Bi was not visible in the crystal structure. Through the comparison of the structures of hTop2β-DNA-etoposide ternary complex and hTop2β-DNA binary complex, it could be observed that the distance between drug-binding sites Arg503 of the two monomers was 26.62 Å in hTop2β-DNA-etoposide ternary complex and 34.54 Å in hTop2β-DNA binary complex, respectively. Significant twist were observed in the DNA chains of binary complex. It suggested that compound C-Bi played antitumor roles through increasing spacing of hTop2β monomers. The changes in hTop2β structure further caused double changes in the torsional direction and migration distance of the DNA chains, resulting in impeding religation of DNA.

Discover the leading compound of 4β-S-(5-fluorobenzoxazole)-4-deoxy-4′-demethylepipodophyllotoxin with millimolar-potency toxicity by modifying the molecule structure of 4′-demethylepipodophyllotoxin

4′-Demethylepipodophyllotoxin (DMEP) derivatives are broad-spectrum and potent antitumor leading compound. Because of their unacceptable toxicity, DMEP derivatives often failed in the development of new drug. Until now, there was no report on the millimolar-potency toxicity of DMEP derivatives by modifying the molecule structure of DMEP. For the first time, this work discovered leading compounds with millimolar-potency toxicity by modifying the molecule structure of DMEP. The IC50 value of 4β-S-(5-fluorobenzoxazole-2-)-4-deoxy-4′-demethylepipodophyllotoxin (Compound 2) was around 323.4–2000.9 μM on human healthy cells (i.e., HL-7702, H8, MRC-5 and HMEC), which was significantly reduced by 171–1999 times than podophyllotoxin (1.0–2.6 μM) and 9–80 times than etoposide (21.5–75.4 μM). Compared with the treatment of etoposide, DNA repair proteins HMGB1 and PARK7 were specifically activated and the expression of anti-apoptotic proteins were up-regulated in HL-7702 cells after the treatment of Compound 2. These indicated the toxicity of Compound 2 was synergistically reduced by DNA repair and anti-apoptosis pathway.

Surface dual-targeting equipped nanogels intracellularly deliver cytotoxic agent to suppress highly metastatic malignancy

In this work, two tertiary amine-derived 4′-demethylepipodophyllotoxin (DMEP) conjugates (DC and DP) have been designed and synthesized using N,N,N′-trimethyl-N′-(4-carboxyl benzyl)-1,3-propanediamine (CPDM) and 4-(4-methylpiperazinomethyl)benzoic acid (PBA) as the targeting ligands. Both DC and DP exhibited strong in vitro cytotoxicity against small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines. Cellular uptake efficiencies of DC and DP in human alveolar type II epithelial cells were significantly enhanced compared to DMEP and etoposide (VP-16), which were demonstrated to be concentration-, time- and energy-dependent. The active transport process of DC and DP might be mediated by organic cation transporters (OCTs). After systemic administration in mice, both DC and DP selectively accumulated in the lung, displaying the highest Cmax and AUC0-t values of all tested tissues. Compared with DMEP and VP-16, DC and DP remarkably reduced the lung weight and the number of lung metastases of B16 melanoma in mice, and further prolonged the survival of tumor-bearing mice. Also, DC and DP exhibited comparable levels of cell cycle arrest and cell apoptosis. Furthermore, DC and DP demonstrated minimum toxicity towards vital organs and reduced gastrointestinal injury compared to DMEP and VP-16. Taken together, tertiary amine-derived moieties such as CPDM and PBA represent an efficient yet safe strategy to achieve lung-targeted drug delivery.

Enzymatic glycosylation of 4'-demethylepipodophyllotoxin

The glycosides of 4'-demethylepipodophyllotoxin (DMEP) possess various pharmacological activities; however, the chemical synthesis of these glycosides faces challenges in regioselectivity, stereoselectivity, and the protection and de-protection of functional groups. In this work, a novel glycosyltransferase (GT) gene AbGT5 from Aloe barbadensis was successfully cloned, heterogeneously expressed and purified. Recombinant AbGT5 was able to catalyze the glycosylation of DMEP and the glycosylated product, which was separated from the preparative scale reaction, was characterized as DMEP 4'-O-β-D-glucoside via MS, 1H-NMR, 13C-NMR, HSQC and HMBC. According to the investigations of enzyme properties, AbGT5 show the highest activity around 20 ℃ in the buffer of pH 9.0, and it was independent of divalent metal ions. Under the optimum conditions, the conversion rate of DMEP can reach 80%. Above all, in this work the enzymatic glycosylation of DMEP was achieved with high efficiency by the novel GT AbGT5.

Tertiary amine mediated targeted therapy against metastatic lung cancer

In this work, two tertiary amine-derived 4′-demethylepipodophyllotoxin (DMEP) conjugates (DC and DP) have been designed and synthesized using N,N,N′-trimethyl-N′-(4-carboxyl benzyl)-1,3-propanediamine (CPDM) and 4-(4-methylpiperazinomethyl)benzoic acid (PBA) as the targeting ligands. Both DC and DP exhibited strong in vitro cytotoxicity against small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cell lines. Cellular uptake efficiencies of DC and DP in human alveolar type II epithelial cells were significantly enhanced compared to DMEP and etoposide (VP-16), which were demonstrated to be concentration-, time- and energy-dependent. The active transport process of DC and DP might be mediated by organic cation transporters (OCTs). After systemic administration in mice, both DC and DP selectively accumulated in the lung, displaying the highest Cmax and AUC0-t values of all tested tissues. Compared with DMEP and VP-16, DC and DP remarkably reduced the lung weight and the number of lung metastases of B16 melanoma in mice, and further prolonged the survival of tumor-bearing mice. Also, DC and DP exhibited comparable levels of cell cycle arrest and cell apoptosis. Furthermore, DC and DP demonstrated minimum toxicity towards vital organs and reduced gastrointestinal injury compared to DMEP and VP-16. Taken together, tertiary amine-derived moieties such as CPDM and PBA represent an efficient yet safe strategy to achieve lung-targeted drug delivery.

A rational design strategy of the novel topoisomerase II inhibitors for the synthesis of the 4-O-(2-pyrazinecarboxylic)-4′-demethylepipodophyllotoxin with antitumor activity by diminishing the relaxation reaction of topoisomerase II-DNA decatenation

A rational design strategy of the novel podophyllum topoisomerase II (Topo II) inhibitors for the synthesis of the esterification and amidation substituted 4′-demethylepipodophyllotoxin (DMEP) derivates was developed in order to discover the potential antitumor prodrug. Firstly, according to the structure–activity relationship, drug combination principle and bioisosterism, the –COO– and the –NH– bond substituents at the 4 position of cycloparaffin would be a great modification direction to improve antitumor activity of 4′-demethylepipodophyllotoxin (DMEP). Secondly, from the prodrug principle view, the esterification and amidation at the C-4 position of DMEP would be two useful structure modifications for improve solubility. Thirdly, from the activity pocket in Topo II-DNA cleavage complex point of view, a series of heterocyclic with pharmacological activity were chosen as module for improving antitumor activity by binding with Topo II. Finally, nine novel esterification and amidation DMEP derivates were designed and synthesized for the potential Topo II inhibitors with the superior biological activity. All the novel compounds exhibited promising in vitro antitumor activity, especially 4-O-(2-pyrazinecarboxylic)-4′-demethylepipodophyllotoxin (compound 1). The antitumor activity of compound 1 against tumor cell line HeLa (i.e., the IC50 value of 0.60±0.20μM), A549 (i.e., the IC50 value of 3.83±0.08μM), HepG2 (i.e., the IC50 value of 1.21±0.05μM), and BGC-823 (i.e., the IC50 value of 4.15±1.13μM) was significantly improved by 66, 16, 12, and 6 times than that of the clinically important podophyllum anticancer drug etoposide (i.e., the IC50 values of 15.32±0.10, 59.38±0.77, 67.25±7.05, and 30.74±5.13μM), respectively. Compound 1 could arrest HeLa cell cycle G2/M and induce apoptosis by strongly diminishing the relaxation reaction of Topo II-DNA decatenation. The correctness of rational drug design was strictly demonstrated by the bioactivity test.

Novel Biotransformation Process of Podophyllotoxin to 4 -Sulfur-Substituted Podophyllum Derivates with Anti-Tumor Activity by Penicillium purpurogenum Y.J. Tang

According to the structure-function relationship of podophyllotoxin (PTOX) and its analogue of 4'- demethylepipodophyllotoxin (DMEP), the 4 β-substitution of sulfur-containing heterocyclic compounds with a carbon-sulfur bond at 4 position of PTOX or DMEP is an essential modification direction for improving the anti-tumor activity. So, four novel 4 β-sulfursubstituted podophyllum derivatives (i.e., 4β -(1,2,4-triazole-3-yl)sulfanyl-4-deoxy-podophyllotoxin (4-MT-PTOX), 4β-(1,3,4- thiadiazole-2-yl)sulfanyl-4-deoxy-podophyllotoxin (4-MTD-PTOX), 4β-(1,2,4-triazole-3-yl)sulfanyl-4-deoxy-4' -demethylepipodophyllotoxin (4-MT-DMEP), and 4β-(1,3,4-thiadiazole-2-yl)sulfanyl-4-deoxy-4'-demethylepipodophyllotoxin (4-MTD-DMEP)) were designed and then successfully biosynthesized in this work. In the novel sulfur-substituted biotransformation processes, PTOX and DMEP was linked with sulfur-containing compounds (i.e., 3-mercapto-1,2,4-triazole (MT) and 2-mercapto-1,3,4-thiadiazole (MTD)) at 4 position of cycloparaffin to produce 4-MT-PTOX (1), 4-MTD-PTOX (2), 4-MT-DMEP (3), and 4-MTD-DMEP (4) by Penicillium purpurogenum Y.J. Tang, respectively, which was screened out from Diphylleia sinensis Li (Hubei, China). All the novel compounds exhibited promising in vitro bioactivity, especially 4-MT-PTOX (1). Compared with etoposide (i.e., a 50 % effective concentration [EC(50)] of 25.72, 167.97, and 1.15 M), the EC(50) values of 4-MT-PTOX (1) against tumor cell line BGC-823, A549 and HepG2 (i.e., 0.28, 0.76, and 0.42 M) were significantly improved by 91, 221 and 2.73 times, respectively. Moreover, the EC(50) value of 4-MT-PTOX (1) against the normal human cell line HK-2 (i.e., 182.4 μM) was 19 times higher than that of etoposide (i.e., 9.17 μM). Based on the rational design, four novel 4 β-sulfur-substituted podophyllum derivatives with superior in vitro anti-tumor activity were obtained for the first time. The correctness of structure-function relationship and rational drug design was strictly demonstrated by the in vitro biological activity tests.

Reversal of mdr1-mediated multidrug resistance in human leukemia cells by a new spin-labeled derivative of podophyllotoxin.

GP7 (4-[4"-(2", 2", 6", 6"-tetramethyl-l"-piperidinyloxy) amino]-4'-demethyl epipodophyllotoxin) is a promising anticancer drug of the podophyllotoxin class. However, little is known about its anti-multidrug resistance effects. In the present study, we investigated the effects of GP7 on P-glycoprotein (P-gp) overexpression multidrug-resistant human leukemia K562/ADM cells with the comparison of VP-16 and K562 cells. GP7 inhibited the proliferation of K562/ADM cells in a concentration- or time-dependent manner, and the inhibitory effect of GP7 on K562/ADM cells was 1.50-fold higher than that of VP-16. GP7 caused G2/M phase accumulation but VP-16 caused S phase accumulation in K562/ADM and K562 cells. GP7 could induce apoptosis of both K562/ADM and K562 cell lines, but there was no significant difference between GP7- and VP-16-induced apoptotic ratios. GP7 could also induce typical apoptotic morphological changes and internucleosomal DNA fragmentation of K562/ADM and K562 cells, but DNA fragmentation induced by GP7 in K562/ADM cells was weaker than that in K562 cells. When treated with GP7 or VP-16 for 48 h, 128-256 microM GP7 induced more DNA fragmentation than VP-16 did, but 32-64 microM GP7 induced less DNA fragmentation than VP-16 did. GP7 could down-regulate the expression of P-gp in K562/ADM cells but VP-16 could not. Our findings suggest that GP7 may reverse multidrug resistance in human leukemia K562/ADM cells via down-regulation of P-gp expression.

A novel biotransformation process of 4′-demethylepipodophyllotoxin to 4′-demethylepipodophyllic acid by Bacillus fusiformis CICC 20463, Part II: process optimization

This work optimized the novel biotransformation process of 4'-demethylepipodophyllotoxin (DMEP) into 4'-demethylepipodophyllic acid (DMEPA) by Bacillus fusiformis CICC 20463. Firstly, the biotransformation process was significantly affected by medium composition. 5 g/L of yeast extract and 10 g/L of peptone were optimal for DMEPA production (i.e., 2.81 + or - 0.21 mg/L), while not beneficial for the cell growth of B. fusiformis. This indicated that the biosynthesis of DMEPA was not corresponded well to the cell growth of B. fusiformis. 40 g/L of sucrose was optimal for DMEPA production (i.e., 2.94 + or - 0.17 mg/L), and 3 g/L of NaCl was the best for DMEPA production (i.e., 4.10 + or - 0.18 mg/L). Secondly, the production of DMEPA was significantly enhanced by the control of substrate concentration and culture pH. 100 mg/L of substrate was optimal for DMEPA production (i.e., 6.47 + or - 0.35 mg/L), and DMEPA concentration was enhanced to 38.78 mg/L by controlling culture pH at 9.0 in the stirred-tank bioreactors. The fundamental information obtained in this study provides a simple and efficient way to produce DMEPA by biotransformation.

Substituents on Etoposide That Interact with Human Topoisomerase IIα in the Binary Enzyme−Drug Complex: Contributions to Etoposide Binding and Activity

Etoposide is a widely prescribed anticancer agent that stabilizes topoisomerase II-mediated DNA strand breaks. The drug contains a polycyclic ring system (rings A-D), a glycosidic moiety at C4, and a pendant ring (E-ring) at C1. A recent study that focused on yeast topoisomerase II demonstrated that the H15 geminal protons of the etoposide A-ring, the H5 and H8 protons of the B-ring, and the H2', H6', 3'-methoxyl, and 5'-methoxyl protons of the E-ring contact topoisomerase II in the binary enzyme-drug complex [ Wilstermann et al. (2007) Biochemistry 46, 8217-8225 ]. No interactions with the C4 sugar were observed. The present study used DNA cleavage assays, saturation transfer difference [ (1)H] NMR spectroscopy, and enzyme-drug binding studies to further define interactions between etoposide and human topoisomerase IIalpha. Etoposide and three derivatives that lacked the C4 sugar were analyzed. Except for the sugar, 4'-demethyl epipodophyllotoxin is identical to etoposide, epipodophyllotoxin contains a 4'-methoxyl group on the E-ring, and 6,7- O, O-demethylenepipodophyllotoxin replaces the A-ring with a diol. Results suggest that etoposide-topoisomerase IIalpha binding is driven by interactions with the A- and B-rings and potentially by stacking interactions with the E-ring. We propose that the E-ring pocket on the enzyme is confined, because the addition of bulk to this ring adversely affects drug function. The A- and E-rings do not appear to contact DNA in the enzyme-drug-DNA complex. Conversely, the sugar moiety subtly alters DNA interactions. The identification of etoposide substituents that contact topoisomerase IIalpha in the binary complex has predictive value for drug behavior in the enzyme-etoposide-DNA complex.

Design, synthesis, and biological evaluation of novel pyridine acid esters of podophyllotoxin and esters of 4′-demethylepipodophyllotoxin

Podophyllotoxin and related analogs present numerous challenges associated with optimal antitumor activity and severe unpredictable toxicity. In the course of our ongoing investigation of quantitative structure–activity relationships to find biorational antitumor drugs, two series of pyridine acid ester derivatives of podophyllotoxin and 4′-demethylepipodophyllotoxin have been prepared by reacting the corresponding pyridine acids with the hydroxyl group of podophyllotoxin and 4′-demethyl epipodophyllotoxin in the presence of dimethylaminopyridine (DMAP) and N,N-dicyclohexylcarbodiimde (DCC). The structures of the compounds were extensively characterized by using 1H-nuclear magnetic resonance, mass spectroscopy, infrared, and elemental analysis and evaluated for their in vitro cytotoxicity on two neoplastic cell lines (P-388 murine leukemia, A-549 human lung carcinoma) using a MTT-based assay, the results indicated significantly higher efficacy of these compounds in comparison with the prototypical inhibitor etoposide. On the basis of the preliminary biological testing results, it suggested that the cytotoxic activity of OCH3 at C-4′ is more potent than that of its demethylated analogs and the podophyllotoxin substitution at C-4 may be optimal for synthesizing more potent cytotoxic compounds.

GP7 induces internucleosomal DNA fragmentation independent of caspase activation and DNA fragmentation factor in NB4 cells

DNA fragmentation into internucleosomal fragments is the best recognized biochemical event of apoptosis. Two major caspase pathways have been identified in the signal transduction leading to DNA fragmentation: the receptor pathway and the mitochondrial pathway. DNA fragmentation factor (DFF) has been identified as a major apoptotic endonuclease in the internucleosomal DNA fragmentation process. However, the potential roles of caspases and DFF in internucleosomal DNA fragmentation induced by specific stimuli still need to be investigated since caspase-independent pathways and nuclease(s) other than DFF also play important roles during this process. In the present study, we investigated the activity of GP7 (4-[4"-(2",2",6",6"-tetramethyl-l"-piperidinyloxy) amino]-4'-demethyl epipodophyllotoxin), a new spin-labeled derivative of podophyllotoxin semi-synthesized by our university, to induce apoptosis of the human leukemia cell line NB4. GP7 induced the release of cytochrome-c from mitochondria, activations of caspase-3, -8, and -9, cleavage of DFF45/inhibitor of caspase-activated DNase, activation of DFF40/caspase-activated DNase, and apoptotic DNA fragmentation in NB4 cells. The broad-spectrum caspase inhibitor zVAD-fmk abrogated GP7-induced caspase-3, -8, and -9 activations but could not inhibit GP7-induced apoptotic DNA fragmentation in NB4 cells. Our findings suggest that GP7-induced apoptotic DNA fragmentation in NB4 cells is independent of caspase activation and DFF, although they are closely involved in this process.

Determination of Podophyllotoxin and 4′-Demethylepipodophyllotoxin by Capillary Electrophoresis

A new simple, rapid and sensitive capillary zone electrophoresis (CZE) method has been developed for the simultaneous separation and determination of the anticancer active compounds podophyllotoxin (PPT) and 4′-demethylepipodophyllotoxin (DMEP) in ethanol extracts of the roots of Sinopophyllum emodi (Wall) Ying, and the roots, stems, leaves and fibrils of Dysosma versipellis (Hance) M. cheng. PPT and DMEP could be completely separated and sensitively determined at 214 nm within 12.5 min using 25 mM borate with pH 10.80 as running buffer (applied voltage 20 kV, temperature 20 °C, injection time 5 s). The linear range for the determination of PPT and DMEP were 1.5–171.0 μg mL−1 and 5.0–136.0 μg mL−1 with RSD of the relative migration time for PPT and DMEP of 1.50 and 3.26, and of the relative peak area of 0.82 and 1.13, respectively. The results showed that the method had requisite selectivity, sensitivity, reproducibility and wide linear range for the application to the rapid separation and accurate determination of PPT and DMEP in podophyllum plants. Furthermore, this is the first report about the analysis of podophyllotoxin analogues by CE.

Synthesis and in vitro drug release behavior of amphiphilic triblock copolymer nanoparticles based on poly (ethylene glycol) and polycaprolactone

Novel BAB type amphiphilic triblock copolymers consisting of poly (ethylene glycol) (PEG) (B) as hydrophilic segment and poly ( ε-caprolactone) (PCL) (A) as hydrophobic block were prepared by coupling reaction using l-lysine methyl ester diisocyanate (LDI) as the chain extender. The triblock copolymers obtained were characterized by FT-IR, 1H NMR, GPC, and DSC. Core-shell type nanoparticles were prepared by nanoprecipitation method and below 100 nm nanoparticles were obtained due to their specific structure. Transmission electron microscopy image demonstrated that these nanoparticles were spherical in shape. Stability of the nanoparticles in biological media was evaluated. Poorly water-soluble anticancer drug 4′-demethyl-epipodophyllotoxin (DMEP) was chosen for controlled drug release because it was easily encapsulated into polymeric nanoparticles via hydrophobic interaction. In vitro release behavior of DMEP from polymeric nanoparticles was investigated, the results showed that the drug release rate can be modulated by the variation of the copolymer composition.

GP7 can induce apoptotic DNA fragmentation of human leukemia cells through caspase-3-dependent and -independent pathways

GP7 (4-[4"-(2",2",6",6"-tetramethyl-l"-piperidinyloxy)amino]-4'-demethyl epipodophyllotoxin), a new spin-labeled derivative of podophyllotoxin, is a promising anticancer drug of podophyllotoxin class. The primary effect of GP7 is the anticancer activity on transplanted mouse tumors and cultured tumor cells. However, its molecular mechanism of action is still obscure. In this study, we investigated the activity of GP7 to induce apoptosis in human leukemia HL-60 and Jurkat cells. Apoptosis was determined by detection of DNA fragmentation in agarose gel electrophoresis. GP7 induced apoptotic DNA fragmentation of HL-60 and Jurkat cells in time- and dose-dependent manner. We further investigated the activity of caspase-3 in GP7-induced apoptotic DNA fragmentation of HL-60 and Jurkat cells. GP7 also induced time- and dose-dependent caspase-3 activation in both cell lines, and the kinetics of caspase-3 activation induced by GP7 was well correlated with that of apoptotic DNA fragmentation. To determine the role of caspase-3 in GP7-induced apoptotic DNA fragmentation, we examined the effect of specific caspase-3 inhibitor, Ac-DEVD-CHO, on GP7-induced apoptotic DNA fragmentation. Ac-DEVD-CHO prevented GP7-induced caspase-3 activation in both HL-60 and Jurkat cells, however, it only inhibited GP7-induced apoptotic internucleosomal DNA fragmentation in HL-60 cells. We then employed L-carnitine to investigate the role of caspase-3 in GP7-induced apoptotic DNA fragmentation. L-carnitine treatment prevented GP7-induced caspase-3 activation in both cell lines in a dose-dependent manner. Similar to Ac-DEVD-CHO, L-carnitine only inhibited GP7-induced apoptotic internucleosomal DNA fragmentation in HL-60 cells. These findings suggest that GP7 exerts an anti-leukemic effect by both caspase-3-dependent and -independent apoptotic signaling pathways.

Pulse radiolysis studies of etoposide and 4′-demethylepipodophyllotoxin in aqueous solution

Abstract The reactions of etoposide (VP 16, 4′-demethyl-epipodophyllotoxin ethylidene- β - d -glucoside) and 4′-demethylepipodophyllotoxin (DMEP) with the primary radiolytic products of water, such as e − aq , H and OH/O − radicals, and the secondary radical (SO − 4 ) in aqueous solution were studied by use of the techniques of pulse radiolysis, respectively. The absorption spectra of reaction products with e − aq , H and OH/O − and SO − 4 radicals were observed, and the rate constants of them were determined by following the build-up kinetics of radicals produced or the decay of hydrated electron observed at 600 nm, respectively.

Chemopreventive effect of 4'-demethyl epipodophyllotoxin on DMBA/TPA-induced mouse skin carcinogenesis.

The chemopreventive effect of topical application of 4'-demethyl epipodophyllotoxin (DMEP), an antimitotic agent, on a two-stage skin carcinogenesis model in Swiss Albino mice induced by 9, 10-dimethylbenz[a]anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) was investigated. Two topical applications with 0.24% DMBA over 1 week, followed later by 5 nmol of TPA twice weekly produced 100% incidence of tumors in these animals by 18 weeks. Treatment of animals with DMEP (until the end of the experiment), 30 min before TPA treatment, significantly reduced the tumor incidence, tumor volume and the conversion efficiency of papillomas to squamous cell carcinomas. The tumor formation and growth was also delayed by DMEP pre-treatment. Application of DMEP protected against the losses provoked in levels of glutathione and activity of catalase and superoxide dismutase in skin and liver of animals by the application of DMBA/TPA. Thus, DMEP might possibly be exerting its chemopreventive activity by acting as an antioxidant.

Altered subcellular distribution of topoisomerase IIα in a drug-resistant human small cell lung cancer cell line : Feldhoff PW, Mirski SEL, Cole SPC, Sullivan DM. James Graham Brown Cancer Center, University of Louisville, 529 South Jackson Street, Louisville, KY 40292. Cancer Res 1994;54: 756–762

A drug-resistant human small cell lung cancer cell line, H209/V6, selected in the presence of increasing concentrations of 9-(4,6-O-ethylidene-beta-D-glucopyranosyl)-4'-demethylepipodophylloto xin (VP-16) from parental H209 cells, is 22-, 9-, and 4-fold resistant to VP-16, 4'-(9-acridinyl-amino)methanesulfon-m-anisidide, and doxorubicin, respectively, but not cross-resistant to 1,4-dihydroxy-5,8-bis((2-[(2-hydroxyethyl)amino] ethyl]-amino)-9,10-anthracenedione. These cells do not overexpress P-glycoprotein or the multidrug resistance-associated protein. Immunoblotting demonstrates that H209 cells contain the M(r) 170,000 isoform of topoisomerase II (topo II), while H209/V6 cells have a M(r) 160,000 enzyme but none of the M(r) 170,000 isoform. The cell lines have equal amounts of topo II beta. The H209/V6 cells have a 5-fold decrease in total immunoreactive topo II alpha. The catalytic and VP-16-induced DNA cleavage activities of the topo II present in 0.35 M NaCl nuclear extracts are decreased 2- to 3-fold in the drug-resistant cell line. This decrease in enzymatic activity is not consistent with either the 22-fold VP-16 resistance of the H209/V6 cell line or the approximately 5-fold decrease in immunoreactive topo II alpha in the cells. The M(r) 160,000 isoform from the H209/V6 cell line and the M(r) 170,000 enzyme from the parental cell line were purified so that the enzymatic activity of the 2 isoforms could be evaluated. The catalytic activities of the purified isoforms were found to be very similar. The drug-induced DNA cleavage activity of the M(r) 160,000 enzyme was reduced compared to the M(r) 170,000 enzyme. However, as with the nuclear extracts, the differences in enzymatic activity of the purified enzymes are considerably less than the level of drug resistance. Investigations of the subcellular localization of topo II by immunocytochemical techniques and cytoplasm/nuclear fractionation studies demonstrated that the M(r) 160,000 topo II alpha-related enzyme is primarily localized in the cytoplasm, while the M(r) 170,000 topo II alpha enzyme and topo II beta are located in the nucleus. These data imply that the deleted sequence in the M(r) 160,000 enzyme is not necessary for catalytic activity but is required to facilitate nuclear localization.