Anticancer Drug Etoposide Research Articles

Microfluidic System for Automated Cell-Based Assays

Microfluidic cell culture is a promising technology for applications in the drug screening industry. Key benefits include improved biological function, higher quality cell-based data, reduced reagent consumption, and lower cost. In this work, we demonstrate how a microfluidic cell culture design was adapted to be compatible with the standard 96-well plate format. Key design features include the elimination of tubing and connectors, the ability to maintain long term continuous perfusion cell culture using a passive gravity driven pump, and direct analysis on the outlet wells of the microfluidic plate. A single microfluidic culture plate contained 8 independent flow units, each with 10(4) cells at a flow rate of 50 μl/day (6 minute residence time). The cytotoxicity of the anti-cancer drug etoposide was measured on HeLa cells cultured in this format, using a commercial lactate dehydrogenase (LDH) plate reader assay. The integration of microfluidic cell culture methods with commercial automation capabilities offers an exciting opportunity for improved cell-based screening.

A New Cytotoxic Lanostane‐Type Triterpene Glycoside from the Sea Cucumber Holothuria impatiens.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 200 leading journals. To access a ChemInform Abstract, please click on HTML or PDF.

The role of cordycepin in cancer treatment via induction or inhibition of apoptosis: implication of polyadenylation in a cell type specific manner

Most anticancer drugs show their antiproliferative and cytotoxic activity via induction of apoptosis. In the present study we assessed the implication and role of cordycepin, a polyadenylation-specific inhibitor and a well-known chemotherapeutic drug, in apoptosis, induced by the anticancer drug etoposide. For this purpose, a variety of leukemia and lymphoma cell lines (U937, K562, HL-60, Daudi, Molt-4) were treated with the anticancer drugs etoposide and/or cordycepin and assessed for poly(A) polymerase (PAP) activity and isoforms by the highly sensitive PAP activity assay and western blotting, respectively. Induction of apoptosis was determined by endonucleosomal DNA cleavage, DAPI staining, caspase-6 activity assay and DeltaPsi m reduction, whereas cytotoxicity and cell cycle status were assessed by Trypan blue staining, MTT assay and flow cytometry. The results showed that PAP changes in all cell lines, in response to apoptosis induced by etoposide, in many cases even prior to hallmarks of apoptosis (endonucleosomal cleavage of DNA, DeltaPsi(m) reduction). A further elucidation to this apoptosis-polyadenylation correlation was added, by cell treatment with cordycepin, resulting in either suppression (U937, K562) or induction (HL-60) of the apoptotic process, according to the cell type. However, inhibition of polyadenylation did not influence the cell lines Daudi and Molt-4 used, where alternative apoptotic pathways are induced through cleavage of DNA into high molecular weight fragments.

A New Cytotoxic Lanostane‐Type Triterpene Glycoside from the Sea Cucumber Holothuria impatiens

A new lanostane-type triterpene glycoside, impatienside A (1), was isolated from the sea cucumber Holothuria impatiens, together with a structurally related, known compound, bivittoside D (2). Their structures were elucidated by in-depth spectroscopic and mass-spectrometric methods, including (1)H-, (13)C-, and 2D-NMR, ESI-MS, and HR-ESI-MS experiments, as well as by chemical evidence. Compounds 1 and 2 possess the same hexasaccharide moieties, but differ slightly in their holostane-type triterpene aglycone. The two glycosides were found to exhibit in vitro cytotoxicities similar to or better than those of the potent anticancer drug etoposide (V-16) against seven different human tumor cells, with IC50 values of 0.37-2.75 microg/ml.

Immediate early gene-X1 interferes with 26 S proteasome activity by attenuating expression of the 19 S proteasomal components S5a/Rpn10 and S1/Rpn2

The stress response gene IEX-1 (immediate early gene-X-1) is involved in the regulation of cell growth and cellular viability. To some extent, these effects include an interference with the proteasomal turnover of certain regulatory proteins. Here, we show that IEX-1 directly attenuates the activity and formation of the 26 S proteasome in HEK-293 cells (human embryonic kidney cells). We further demonstrate that IEX-1 reduces the overall expression levels of certain protein components of the 19 S proteasomal subunit such as S5a/Rpn10 and S1/Rpn2, whereas the expression of other proteasomal proteins was less or not affected. In contrast with direct apoptotic stimuli, such as the anti-cancer drug etoposide, leading to caspase-dependent degradation of S1 and S5a, the effect of IEX-1 is independent of proteolytic cleavage of these proteins. Furthermore, the decreasing effect of IEX-1 on S5a and S1 expression is still seen in the presence of cycloheximide, but not in the presence of actinomycin D, and quantitative real-time PCR revealed lower mRNA levels of S5a and S1 in IEX-1-overexpressing cells, suggesting an interference of IEX-1 with the gene transcription of S5a and S1. Additionally, luciferase assays confirmed an interference of IEX-1 with the activity of the S5a promoter. These findings indicate a role of IEX-1 in the maintenance and assembly of the 26 S proteasome, obviously involving an altered gene expression of certain proteasomal proteins. Thereby, IEX-1 may essentially modulate signalling pathways related to 26 S proteasome activity and involved in cellular growth control and apoptosis.

In vitro germination of Podophyllum hexandrum seeds.

Considerable interest has centred on the Podophyllum-based lignans as lead compounds for the development of new drugs. The successful introduction of the anticancer drugs etoposide® and teniposide® and the development of new derivatives such as etopophos® have created a demand for podophyllotoxin. Currently it is obtained from the rhizomes and roots of wild populations of Podophyllum hexandrum, and thus the availability of this natural product is limited. There is an urgent need for a maintainable supply of P. hexandrum plants, a rare and threatened species. In the present work, a protocol was developed for the sterilisation and germination of seeds of P. hexandrum. Ripe fruits were harvested from seed-derived plants cultivated at the University of Nottingham. Seeds were removed from fruits, left for 20 – 30 min in running water, and washed three times with sterile, reverseosmosis water. Seeds were surface sterilised in (5, 10, 15 and 20%; v:v) Domestos bleach solution with 0.2% (v:v) Tween 20 for 5, 10, 15 and 20 min, followed by three washes in sterile, reverse-osmosis water. The effect of storage as a pre-treatment for the germination of seeds was also investigated. Sterilised seeds were kept in Petri dishes and maintained in the dark at 22 ± 1°C for 30 d. Ten stored seeds were cultured per dish containing a moist sterile filter paper disk. Cultures were incubated in the dark (22 ± 1 °C). Seeds with emerged radicles were individually transferred onto full-strength MS medium containing IAA (0.00875 mg l-1) in combination with kinetin (0.03 mg l-1) and folic acid (0.01 mg l-1) designated BGS medium. Seeds were also transferred onto full-strength MS medium lacking growth regulators (MSO medium). Media were supplemented with 3.0% (v:v) sucrose and solidified with 0.8% (w:v) agar. Cultures were maintained under diffuse light (3.8 µmol m-2 s-1) at 22 ± 1°C for three weeks and then transferred to a 16 h photoperiod under fluorescent light (42 µmol m-2 s-1) at 22 ± 1°C. Seed sterilisation was best achieved with 20% (v:v) Domestos and 0.2% (v:v) Tween 20 for 20 min. The results from these studies confirm that a post-harvest ripening period of 30 d was required for in vitro seed germination. If seeds are stored in moist and dark conditions, spontaneous germination occurs within 35 to 40 d. Axenic cultures were successfully established either on full-strength BGS medium with growth regulators or fullstrength MS medium lacking growth regulators. However, the overall growth of plants showing a normal morphology was superior on the latter medium. The in vitro-grown seedlings can be used as an alternative source of plant material for tissue culture experiments.

Three-component synthesis and anticancer evaluation of polycyclic indenopyridines lead to the discovery of a novel indenoheterocycle with potent apoptosis inducing properties

A multicomponent reaction of indane-1,3-dione, an aldehyde and an amine-containing aromatic compound leading to the formation of indenopyridine-based heterocyclic medicinal scaffolds has been investigated. It was found that the yields significantly improve when oxygen gas is bubbled through the reaction mixture, facilitating the oxidation of the intermediate dihydropyridine-containing compounds to their aromatic counterparts. Investigation of the reaction scope revealed that formaldehyde, as well as various aliphatic, aromatic and heteroaromatic aldehydes, works well as the aldehyde component. In addition, substituted anilines and diverse aminoheterocycles can be utilized in this process as the amine-containing component. Preliminary biological evaluation of the synthesized library identified a pyrimidine-based polycycle, which rivals the anticancer drug etoposide in its toxicity and apoptosis inducing properties toward a human T-cell leukemia cell line.

Lovastatin protects human endothelial cells from the genotoxic and cytotoxic effects of the anticancer drugs doxorubicin and etoposide

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are frequently used lipid-lowering drugs. Moreover, they exert pleiotropic effects on cellular stress responses and death. Here, we analysed whether lovastatin affects the sensitivity of primary human endothelial cells (HUVEC) to the anticancer drug doxorubicin. We investigated whether pretreatment of HUVEC with low dose of lovastatin influences the cellular sensitivity to doxorubicin. To this end, cell viability, proliferation and apoptosis as well as DNA damage-triggered stress response were analysed. Lovastatin reduced the cytotoxic potency of doxorubicin in HUVEC. Lovastatin attenuated the doxorubicin-induced increase in p53 as well as activation of checkpoint kinase (Chk-1) and stress-activated protein kinase/c-Jun-N-terminal kinase (SAPK/JNK). Acquired doxorubicin resistance was independent of alterations in doxorubicin efflux and cell cycle progression. Also, doxorubicin-triggered production of reactive oxygen species (ROS) and formation of oxidative DNA lesions remained unaffected by lovastatin. However, lovastatin impaired DNA strand break formation induced by doxorubicin. Notably, lovastatin also conferred cross-resistance to the cytotoxic and genotoxic effects of etoposide, indicating that lovastatin shields topoisomerase II against poisons. Based on these data, we suggest that lovastatin-mediated resistance to topoisomerase II inhibitors is due to a reduction in DNA damage and, hence, it attenuates stress responses leading to cell death that are triggered by DNA damage. Therefore, lovastatin might be useful clinically for alleviating side-effects of anticancer therapies that include topoisomerase II inhibitors.

Generation of a Stable Antioxidant Response Element–Driven Reporter Gene Cell Line and Its Use to Show Redox-Dependent Activation of Nrf2 by Cancer Chemotherapeutic Agents

The NF-E2 p45-related factor 2 (Nrf2) regulates cytoprotective genes that contain an antioxidant response element (ARE) in their promoters. To investigate whether anticancer drugs can induce ARE-driven gene expression, we have developed a stable human mammary MCF7-derived reporter cell line called AREc32, which contains a luciferase gene construct controlled by eight copies of the cis-element. In these cells, luciferase activity was increased up to 50-fold following treatment with 50 mumol/L tert-butylhydroquinone (t-BHQ). Basal and inducible luciferase activities in AREc32 cells were increased by forced overexpression of Nrf2 and reduced by knockdown of endogenous Nrf2 expression with RNA interference. Depletion of cellular reduced glutathione (GSH) by treatment of AREc32 cells with l-buthionine-S,R-sulfoximine (BSO) did not influence basal levels of luciferase activity, but pretreatment with BSO augmented induction of luciferase activity by t-BHQ. Induction of reporter activity by t-BHQ in AREc32 cells was suppressed markedly by the antioxidants N-acetylcysteine and GSH but only modestly by vitamins C or E, suggesting that ARE-luciferase expression is induced primarily by thiol-active electrophiles rather than free radicals. The anticancer drugs cisplatin, etoposide, mitoxantrone, chlorambucil, melphalan, and carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)] weakly induced luciferase activity in AREc32 cells. Moreover, treatment of AREc32 cells with BSO immediately before exposure to anticancer drugs enhanced induction of ARE-driven luciferase activity by cisplatin, BCNU, chlorambucil, and melphalan and also induced endogenous AKR1C (AKR1C refers to AKR1C1 and AKR1C2), a target gene of Nrf2. Our findings show that Nrf2 can be activated by certain anticancer agents, and this will influence the effectiveness of chemotherapy.

Peroxiredoxin V is essential for protection against apoptosis in human lung carcinoma cells

Sensitivity of tumor cells to treatment with anticancer drugs depends on expression and function of antiapoptotic and antioxidant proteins. The goal of our study was to determine the functional role of the novel antioxidant protein Peroxiredoxin V (PrxV), in protection of human lung carcinoma cell lines against apoptosis. Analysis of expression of PrxV in multiple lung carcinoma cell lines revealed a positive correlation between the expression of PrxV and radioresistance in vitro. Clones of the lung carcinoma cells U1810 with down-regulated expression of PrxV, or with its impaired enzymatic function (expression of redox-negative PrxV), demonstrated increased sensitivity to treatment with anticancer drugs etoposide and adriamycin. Pre-treatment of these clones with antioxidant N-acetyl-cysteine did not change their sensitivity to adriamycin, suggesting the involvement of a non-redox activity of PrxV. Expression of the redox-negative PrxV mainly affected the mitochondrial pathway of apoptosis, as assessed by cytochrome c release assay. Impairment of the PrxV enzymatic function also affected transmembrane potential and calcium loading capacity of mitochondria, as well as mitochondrial morphology. Altogether, these findings suggest that PrxV is a multifunctional protein, which is essential for protection against apoptosis induced by anticancer drugs.

Relationships between DNA strand breakage and apoptotic progression upon treatment of HL-60 leukemia cells with tafluposide or etoposide

Tafluposide (F11782), an epipodophyllotoxin derivative currently undergoing phase I clinical trials, is structurally close to the established anti-cancer drug etoposide, but mechanistically distinct. It is a dual inhibitor of topoisomerases I and II which impairs the binding of the enzyme to DNA, but does not stabilize the cleavage complex. Nevertheless, both etoposide and tafluposide induce DNA strand breaks and are potent pro-apoptotic agents. In this study, we have compared the cellular response of HL-60 human promyelocytic leukemia cells treated with etoposide and tafluposide. We show that tafluposide induces delayed, but extensive, DNA strand breaks, whereas etoposide provokes rapid and massive DNA damage. The two drugs trigger similar types of alterations at the mitochondrial and cell cycle levels, and lead to the generation of comparable levels of reactive oxygen species, but with different kinetics. Our data suggest that modification of the mitochondrial mass plays an important role in apoptosis induced by DNA-damaging anti-cancer agents, at least in the epipodophyllotoxin series. We suggest that drug-induced mitochondrial alterations can be divided into three successive steps: (i) hyperpolarization, (ii) depolarization and (iii) increase of the mitochondrial mass.

Post-harvest and scale-up extraction of American mayapple leaves for podophyllotoxin production

Leaves of American mayapple ( Podophyllum peltatum L. ) are of interest to the pharmaceutical industry as an alternative source of podophyllotoxin, an aryltetralin lignan that is the precursor of widely used anticancer drugs etoposide, teniposide, and etopophos. In this study, the effects of post-harvest handling were evaluated by inflicting physical damage to leaves of mayapple to simulate rough handling. The effects of storage conditions before and after being dried were also evaluated. In addition, techniques for conducting large-scale extractions of podophyllotoxin from bulk samples were investigated. Crushing injury, damaging the leaves in more than 70% of its area, has improved podophyllotoxin content of leaves when dried at 40 °C within 24 h of harvest. In contrast, podophyllotoxin content was greatly reduced when the leaves were dried at room temperature at 15% relative humidity and 24 °C. Podophyllotoxin was stable with no significant changes over time when the leaves were dried, ground, and stored under different conditions for up to 60 days. Based on these findings, mayapple leaves do not require careful handling at harvest. In fact, leaves can be handled in a manner consistent with mechanical injury as long as leaves are dried at 40 °C within 24 h. Leaves can then be stored for up to 60 days, and probably much longer, when dry. If leaves cannot be dried in a timely manner, they can be stored at 4 °C for up to 4 weeks without significant loss of podophyllotoxin.

DNA Topoisomerase II Poisons and the Etiology of Acute Leukemia in Infants.

Background: Chromosomal translocations leading to MLL chimeric oncoproteins are the primary aberrations in infant acute leukemias, but how these translocations are created has not been established. A Children's Oncology Group population epidemiology study recently validated that maternal consumption of dietary items containing topoisomerase II interacting compounds during pregnancy increases the risk of infant AML with MLL translocations. An inactivating polymorphism in the gene encoding NQO1, which detoxifies the topoisomerase II poison p-benzoquinone, also is associated with an increased risk of MLL-rearranged acute leukemias in infants, with the greatest risk for cases with t(4;11). Although there is heterogeneity, it has been suggested that infant leukemias share the same biased MLL translocation breakpoint distribution 3′ in the breakpoint cluster region (bcr) as secondary leukemias arising following chemotherapeutic topoisomerase II poisons. These observations led us to investigate the relationship between topoisomerase II cleavage complexes induced by dietary substances or p-benzoquinone and translocation breakpoints 3′ in the bcr.Methods: Southern blot analysis was used to identify the MLL rearrangement in the AML of an infant male harboring the inactivating NQO1 polymorphism, and the MLL-AF9 genomic breakpoint junction was characterized by panhandle variant PCR. DNA substrates containing the normal homologues of the breakpoint sequences were utilized in topoisomerase II in vitro cleavage assays in the presence of p-benzoquinone or dietary substances to determine whether topoisomerase II cleavage sites were stimulated by these agents at the observed translocation breakpoints.Results: There was a single MLL bcr rearrangement in the infant AML, and the cloning demonstrated a breakpoint junction fusing MLL bcr position 6774, 6775, 6776, 6777, 6778, 6779, 6780 or 6781 in intron 8 to position 35940, 35941, 35942, 35943, 35944, 35945, 35946 or 35947 relative to the start of AF9 intron 5. Identical 5′-GTTCTTA-3′ sequences suggesting the DNA double strand break repair mechanism of NHEJ were present at both breakpoints. The normal homologues of the breakpoints in MLL and AF9 were reciprocally cleaved by topoisomerase II in the presence of p-benzoquinone, and the cleavage sites were resolved to form the breakpoint junction. The MLL substrate contained another topoisomerase II cleavage site at position 6760 that not only was the strongest cleavage site in this substrate with the enzyme alone, but also was enhanced by p-benzoquinone, genistein, genistin, quercitin, and catechin at levels comparable to or greater than the anticancer drug etoposide. Further analysis of 3′ MLL bcr substrates revealed cleavage stimulation by natural topoisomerase II poisons near additional translocation breakpoints.Conclusions: These results establish the relationship between topoisomerase II mediated damage from p-benzoquinone and dietary exposures and MLL translocation breakpoints. MLL translocation breakpoints in infant leukemias can be explained by topoisomerase II cleavage. The combined evidence from population epidemiology studies on maternal-fetal exposures, the NQO1-leukemia association implicating p-benzoquinone, genomic breakpoint junction cloning and topoisomerase II cleavage assays corroborate that topoisomerase II poisons damage MLL in acute leukemia in infants.

Orally administered FPRL1 receptor agonist peptide MMK-1 inhibits etoposide-induced alopecia by a mechanism different from intraperitoneally administered MMK-1

Oral administration for 6 days of 100 mg/kg MMK-1, an agonist peptide selective for the FPRL1 receptor, suppressed alopecia induced by the anticancer drug etoposide in neonatal rats. The anti-alopecia effect of orally administered MMK-1 was not inhibited by pyrilamine or cimetidine, antagonists for histamine H 1 and H 2 receptors, respectively, which blocked the anti-alopecia effect of intraperitoneally administered MMK-1 at a dose of 10 mg/kg for 4 days. However, the anti-alopecia effect of orally administered MMK-1 was inhibited by indomethacin, an inhibitor of cyclooxygenase (COX), or AH-23848B, an antagonist of the EP 4 receptor for prostaglandin (PG) E 2, suggesting involvement of PGE 2 release and the EP 4 receptor in the oral MMK-1 anti-alopecia mechanism. The anti-alopecia effect of orally administered MMK-1 was also blocked by an inhibitor of nuclear factor-κB (NF-κB), pyrrolidine dithiocarbamate, suggesting that the oral anti-alopecia effect of MMK-1 may be mediated by activation of NF-κB. These results suggest that MMK-1 bound to FPRL1 receptor might suppress etoposide-induced apoptosis of hair follicle cells and alopecia by way of PGE 2 release and NF-κB activation.

Regulation of the Expression of Caspase-9 by the Transcription Factor Activator Protein-4 in Glucocorticoid-induced Apoptosis

Caspase-9 (Casp-9) induces death signals by triggering other types of caspase activation, and its expression greatly influences the onset of apoptosis. During the isolation of apoptosis-related genes involved in glucocorticoid (GC)-induced cell death in murine thymic lymphomas, we found that the antisense gene of the transcription factor activator protein-4 (AP-4) inhibited dexamethasone-induced apoptosis. Western blot analysis revealed that the expression of Bcl-xL, Bax, and Apaf-1 was not affected in cells transfected with sense or antisense AP-4 genes. In contrast, both the expression and activation of Casp-9 were inhibited in the antisense AP-4 transfectants. We isolated the 2.4-kb 5'-flanking region of Casp-9, and the promoter activity was investigated. We found the AP-4-binding sites at -1.55 and -1.38 kb to be responsible for the promoter activity. Furthermore, a negative cis-element was expected to exist between bases -1140 and -944. When the cells were treated with dexamethasone, a rapid down-regulation of AP-4 and Casp-9 was observed whether the cells were GC-sensitive lymphomas or GC-insensitive L929 fibroblast cells. In addition, L929 cells pretreated with dexamethasone were found to be resistant to subsequent treatment with etoposide, an apoptosis-inducing reagent. GC has a two-sided effect on apoptosis, i.e. a pro-apoptotic effect on certain cell types and a prosurvival effect on other cell types. Our findings will explain, at least in part, this effect.

Cooperative Cytotoxicity of Proteasome Inhibitors and Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand in Chemoresistant Bcl-2-Overexpressing Cells

Bcl-2 overexpression is frequently detected in lymphoid malignancies, being associated with poor prognosis and reduced response to therapy. Here, we evaluated whether Bcl-2 overexpression affects the cytotoxic activity of proteasome inhibitors taken alone or in association with conventional anticancer drugs or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Jurkat cells engineered to overexpress Bcl-2 were treated with proteasome inhibitors (MG132, epoxomicin, and bortezomib), anticancer drugs (etoposide and doxorubicin), TRAIL, or combinations of these compounds. Cell death and loss of mitochondrial transmembrane potential were detected by flow cytometry. Cytosolic relocalization of cytochrome c and SMAC/Diablo, caspase cleavage, and Bcl-2 and Mcl-1 levels were determined by immunoblotting. Nuclear factor-kappaB inhibition was done by retroviral transduction with a dominant-negative mutant of IkappaBalpha. Bcl-2 overexpression results in significant inhibition of apoptosis in response to proteasome inhibitors, antiblastics, and TRAIL. Addition of TRAIL to proteasome inhibitors results in a synergistic cytotoxic effect in Bcl-2-overexpressing cells, whereas this result is not reproduced by the combination of proteasome inhibitors with antiblastic drugs. Importantly, proteasome inhibitors plus TRAIL induce mitochondrial dysfunction irrespective of up-regulated Bcl-2. Bcl-2 cleavage to a fragment with putative proapoptotic activity and elimination of antiapoptotic Mcl-1 may both play a role in proteasome inhibitors-TRAIL cooperation. Conversely, nuclear factor-kappaB inhibition by proteasome inhibitors is per se insufficient to explain the observed synergy. Combined proteasome inhibitors and TRAIL overcome the apoptotic threshold raised by Bcl-2 and may prove useful in the treatment of chemoresistant malignancies with up-regulated Bcl-2.

Polyadenylate polymerase modulations in human epithelioid cervix and breast cancer cell lines, treated with etoposide or cordycepin, follow cell cycle rather than apoptosis induction

Cancer results from an imbalance between cell cycle progression and apoptosis. Therefore, most anticancer drugs exert their antiproliferative and cytotoxic activity via cell cycle arrest and induction of apoptosis, a controlled form of cell death that is dysregulated in cancer. Many polyadenylation trans-acting factors, including polyadenylate polymerase (PAP), are increasingly found to be involved in cell cycle, apoptosis and cancer prognosis. The objective of the present study was to identify PAP modulations in the response of two epithelial cancer cell lines (HeLa and MCF-7) to apoptosis induction by the anticancer drugs etoposide and cordycepin. Cells were assessed for PAP activity and isoforms by the highly sensitive PAP activity assay and Western blotting, respectively. Induction of apoptosis was determined by endonucleosomal DNA cleavage, 4'6-diamidino-2-phenylindol (DAPI) staining and caspase-6 activity assay, whereas cytotoxicity and cell cycle status were assessed by trypan blue staining, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Our results indicate that PAP changes very early in response to either etoposide or cordycepin treatment, even prior to the hallmarks of apoptosis (chromatin condensation and cleavage), in both cell lines tested, but in a different mode. Our results suggest, for the first time, that in the epithelial cancer cell lines used, PAP modulations follow cell cycle progression rather than the course of apoptosis.

Stimulation of topoisomerase II-mediated DNA cleavage by benzene metabolites

Benzene is a human carcinogen that induces hematopoietic malignancies. It is believed that benzene does not initiate leukemias directly, but rather generates DNA damage through a series of phenolic and quinone-based metabolites, especially 1,4-benzoquinone. Since the DNA damage induced by 1,4-benzoquinone is consistent with that of topoisomerase II-targeted drugs, it has been proposed that the compound initiates specific types of leukemia by acting as a topoisomerase II poison. This hypothesis, however, was not supported by initial in vitro studies. While 1,4-benzoquinone inhibited topoisomerase II catalysis, increases in enzyme-mediated DNA cleavage were not observed. Because of the potential involvement of topoisomerase II in benzene-induced leukemias, we re-examined the effects of benzene metabolites (including 1,4-benzoquinone, 1,4-hydroquinone, catechol, 1,2,4-benzenetriol, 2,2′-biphenol, and 4,4′-biphenol) on DNA cleavage mediated by human topoisomerase IIα. In contrast to previous reports, we found that 1,4-benzoquinone was a strong topoisomerase II poison and was more potent in vitro than the anticancer drug etoposide. Other metabolites displayed considerably less activity. DNA cleavage enhancement by 1,4-benzoquinone was unseen in previous studies due to the presence of reducing agents and the incubation of 1,4-benzoquinone with the enzyme prior to the addition of DNA. Unlike anticancer drugs such as etoposide that interact with topoisomerase IIα in a noncovalent manner, the actions of 1,4-benzoquinone appear to involve a covalent attachment to the enzyme. Finally, 1,4-benzoquinone stimulated DNA cleavage by topoisomerase IIα in cultured human cells. These findings are consistent with the hypothesis that topoisomerase IIα plays a role in the initiation of some benzene-induced leukemias.

Anti-alopecia mechanisms of soymetide-4, an immunostimulating peptide derived from soy β-conglycinin

Previously, we found that orally administered soymetide-4 (MITL), an immunostimulating peptide derived from soybean β-conglycinin α′ subunit, suppressed alopecia induced by the anti-cancer drug etoposide in neonatal rats. Soymetide-4 has weak affinity for N-formyl-methionyl-leucyl-phenylalanine (fMLP) receptor. fMLP showed an anti-alopecia effect after intraperitoneal administration, though it was inactive after oral administration. Anti-alopecia effect of fMLP was blocked by pyrilamine or cimetidine, antagonists for histamine H 1 or H 2 receptor, respectively. However, the anti-alopecia effect of soymetide-4 was not inhibited by the histamine antagonists but by indomethacin, an inhibitor of cyclooxygenase (COX), or AH-23848B, an antagonist of the EP 4 receptor for PGE 2. Anti-alopecia effect of soymetide-4 was also blocked by pyrrolidine dithiocarbamate, an inhibitor of nuclear factor-κB (NF-κB). These results suggest that PGE 2, which is produced after activation of COX by soymetide-4, might suppress apoptosis of hair matrix cells and etoposide-induced alopecia by activating NF-κB.

Regulation of multidrug resistance by ribosomal protein L6 in gastric cancer cells

Ribosomal proteins (RP) L6 was previously identified as an up-regulated gene in multidrug-resistant gastric cancer cells SGC7901/ADR comparing to its parental cells SGC7901 by subtractive hybridization. The aim of this study was to explore the roles of RPL6 in multidrug resistance (MDR) in gastric cancer cells. Northern and Western blot analysis confirmed that RPL6 was overexpressed in SGC7901/ADR cells. By gene transfection, RPL6 was genetically up-regulated in SGC7901 or down-regulated in SGC7901/ADR cells. Up-regulation of RPL6 was associated with enhanced resistance to multiple anticancer drugs (adriamycin, vincristine, etoposide, 5-fludrouracil and cisplatin) and to adriamycin-induced apoptosis. Down-regulation of RPL6 reversed MDR and sensitized cells to adriamycin-induced apoptosis. Alteration of RPL6 showed no obvious influence on intracellular adriamycin accumulation, glutathione content and expression of glutathione S-transferase. RPL6 could up-regulate Bcl-2 and down-regulate Bax in cells. Together, this work demonstrates that RPL6 could regulate MDR in gastric cancer cells by suppressing drug-induced apoptosis.