Peroxidative free radical formation and O-demethylation of etoposide(VP-16) and teniposide(VM-26)

The peroxidative activation of the antitumor drugs, etoposide (VP-16) and teniposide (VM-26), has been studied in vitro. Both of these drugs, in the presence of horseradish peroxidase or prostaglandin synthetase, formed phenoxy radical intermediates. Furthermore, this activation also resulted in the formation of two metabolites from each of the drugs. Using HPLC and mass spectrometry, one of the metabolites was shown to be the reactive o-quinone derivative of the parent drug which resulted from the peroxidative O-demethylation. It appears that O-demethylation catalyzed by peroxidases may be an important mechanism for the formation of reactive intermediates and may play a role in the mechanism of action of VP-16 and VM-26.     

Combination vincristine and VP-16-213: evaluation of drug sequence

The combination of vincristine and VP-16-213 has been found to have synergistic antitumor activity in a murine system in vivo when the sequence of drug administration was vincristine followed by VP-16-213. To investigate the potential influence of drug scheduling on this synergistic combination, the reverse sequence of drug administration was evaluated. DBA/2 mice were inoculated with 10(6) P-388 murine leukemia cells, after which saline only, VP-16-213 only, vincristine only, or VP-16-213 followed at various time intervals by vincristine, were administered. Probable cure (survival greater than 60 days) was observed in 0/20, 0/20, 0/120, and 46/115 (40%), respectively (p less than 0.001). The proportion of animals attaining probable cure was greatest in the group receiving vincristine 4-72 hours after VP-16-213 (40-50%). Similar results had been obtained previously with the reverse drug sequence. In this animal model, the synergistic antitumor activity of vincristine and VP-16-213 does not appear to be schedule-dependent with respect to the sequence of drug administration.     

Topoisomerase-II-mediated lesions in nascent DNA: comparison of the effects of epipodophyllotoxin derivatives, VM-26 and VP-16, and 9-anilinoacridine derivatives, m-AMSA and o-AMSA

This study compares the effects of the epipodophyllotoxin derivatives, VM-26 and VP-16, and the 9-anilinoacridine derivatives, m-AMSA and o-AMSA, on nascent and mature DNA. Two types of lesion which are putatively mediated by topoisomerase II, DNA-protein crosslinks and DNA double-strand breaks, were analyzed in drug-treated nuclei from 3H/14C labelled L1210 cells. Potassium/dodecyl sulfate precipitation assay was used to assess DNA-protein crosslinks in mature and nascent (1 min old) DNA. Both epipodophyllotoxins and m-AMSA showed a strong preference for nascent DNA. DNA double-strand cleavage induced by VM-26 and m-AMSA also showed a preference for nascent DNA as indicated by neutral elution technique. Sedimentation on neutral sucrose gradients revealed that these drugs generated highly degraded fragments (under 30 S) in nascent DNA substantially faster than in mature DNA. Lesions in nascent DNA were diminished substantially by the omission of ATP or the addition of novobiocin. The ability to induce lesions in nascent DNA correlates with cytotoxic potency of the agents studied. The results suggest that replicating DNA may constitute a preferential target for antitopoisomerase II drugs.     

A quantitative microdensitometric and autoradiographic study of the effect of 4''-demethyl-epipodophyllotoxin-beta-D-thenylidene glucoside (VM-26) on the cell cycle of cultured fibroblasts

Synopsis The effect of 4-demethyl-epipodophyllotoxin--d-thenylidene glucoside (VM-26), a semi-synthetic derivative of podophyllotoxin, on the cell cycle was studied with chick embryo fibroblasts cultivatedin vitro. DNA, RNA and protein content, as well as NADH-diaphorase activity were determined by quantitative microdensitometry and cytofluorometry. The incorporation of [³H]thymidine and [³H]leucine into DNA and proteins were analysed by autoradiography. These metabolic data correlated with morphological observation showed that VM-26 blocks the cell cycle at different moments of its kinetics depending on both the dose and the time exposure. NADH-diaphorase activity is the first to be affected, then biochemical changes (involving the metabolism of RNA and proteins) and morphological alterations (especially of mitochondria) follow. This suggests that VM-26 may act primarily upon the mechanism of respiration of the cell.Dedicated to Professor G. Conti on the occasion of his 60th birthday.     

Effects of the lipophilic anticancer drug teniposide (VM-26) on membrane transport

The epipodophyllotoxin glucopyranosides have previously been shown to interact with membrane lipids and to alter the activity of several lipid-embedded membrane proteins. To determine if these agents are acting as general membrane perturbants, we have further examined their effects on membrane processes in Ehrlich ascites tumor cells. [3H]VM-26 and [3H]VP-16 were taken up rapidly and concentrated within the cells in proportion to their lipophilicity. Neither agent was found to have any significant effect on the influx of L-[3H]leucine or alpha-[3H]aminoisobutyric acid. Likewise, these drugs had no significant effects on the hexose transporter. The nucleoside transporter, which is structurally and functionally similar to the hexose transporter, was dramatically affected, however. VM-26 was a non-competitive inhibitor of equilibrium-exchange influx of cytosine arabinoside in Ehrlich cells with a Ki of 15 microM. Equilibrium-exchange influx increased with temperature in control cells (Q10 = 2) but not in VM-26-treated cells; thus, VM-26 was a more potent inhibitor at higher temperatures. VM-26 also significantly reduced zero-trans influx in Ehrlich, P388, L5178Y, and ML-1 cells, and these effects were immediate in onset. VM-26 inhibited high-affinity binding of the nucleoside transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR), but VM-26 enhanced non-specific NBMPR binding to Ehrlich cells. The apparent specificity of the epipodophyllotoxins for the nucleoside transporter is discussed.     

VP-16-induced nucleotide pool changes and poly(ADP-ribose) synthesis: the role of VP-16 in interphase death

Exposure of human promyelocytic leukemia cell line (HL-60) to VP-16 resulted in accumulation of DNA strand breaks. Concomitantly, intracellular NAD levels fell at 1 h, followed by declines in ATP at 2 h and in GTP, CTP, and UTP at 3 h. Furthermore, marked morphological changes, such as loss of microvilli or bleb formation, appeared at 4 h and cell death by 8-10 h. The addition of an inhibitor of poly(ADP-ribose) polymerase, 3-aminobenzamide (5 mM), theophylline (2 mM), or thymidine (1 mM), prevented these sequential reductions of nucleotide pools and cell death. In fact, the activation of poly(ADP-ribose) synthesis was detectable within a few hours after treatment with VP-16, although it was smaller than that induced by N-methyl-N'-nitro-N-nitrosoguanidine. These results may suggest the possible role of activation of poly(ADP-ribosyl)ation in VP-16-induced nucleotide pool changes and subsequent interphase death.     

Inhibition of the DNA catenation activity of type II topoisomerase by VP16-213 and VM26

Studies suggest that the anticancer drugs VP16-213 and VM26 produce cytotoxicity by inducing protein-associated DNA breakage in vivo through interaction with a yet unknown nuclear component. The effects of these drugs and their congeners on topoisomerase activities was investigated. VP16-213, VM26, and congeners active toward inducing DNA breaks also inhibited the catenation activity of eukaryote type II topoisomerase in vitro at very low drug concentrations. A structure-activity relationship was obtained for inhibition of catenation that parallels in vivo DNA breakage and cytotoxic activities. Type I topoisomerase activity was totally unaffected by these drugs.     

Reversible accumulation of plant suspension cell cultures in g(1) phase and subsequent synchronous traverse of the cell cycle

The induction of DNA synthesis in Datura innoxia Mill. cell cultures was determined by flow cytometry. A large fraction of the total population of cells traversed the cell cycle in synchrony when exposed to fresh medium. One hour after transfer to fresh medium, 37% of the cells were found in the process of DNA synthesis. After 24 hours of culture, 66% of the cells had accumulated in G₂ phase, and underwent cell division simultaneously. Only 10% of the cells remained in G₀ or G₁. Transfer of cells into a medium, 80% (v/v) of which was conditioned by a sister culture for 2 days, was adequate to inhibit this simultaneous traverse of the cell cycle. A large proportion of dividing cells could be arrested at the G₀ + G₁/S boundary by exposure to 10 millimolar hydroxyurea (HU) for 12 to 24 hours. Inhibition of DNA synthesis by HU was reversible, and when resuspended into fresh culture medium synchronized cells resumed the cell cycle. Consequently, a large fraction of the cell population could be obtained in the G₂ phase. However, reversal of G₁ arrested cells was not complete and a fraction of cells did not initiate DNA synthesis. Seventy-four percent of the cells simultaneously reached 4C DNA content whereas the frequency of cells which remained in G₀ + G₁ phase was approximately 17%. Incorporation of radioactive precursors into DNA and proteins identified a population of nondividing cells which represents the fraction of cells in G₀. The frequency of cells entering G₀ was 11% at each generation. Our results indicate that almost 100% of the population of dividing cells synchronously traversed the cell cycle following suspension in fresh medium.     

Cytofluorometric analysis of cell proliferation and differentiation of the human erythroblasts

Summary The method of cytofluorometric measurement of the contents of Hb and nuclear DNA on a single erythroid cell (Fukuda et al., 1975, 1977 a) was used for the quantitative analysis of the erythropoiesis in normal human bone marrow.The intracellular Hb in an erythroid cell was converted to fluorescent porphyrin after removing the Giemsa staining by irradiation with violet light in the presence of SH-donor (mercaptoethylamine hydrochloride, MEA) and its nuclear DNA was subsequently stained with pararosaniline Feulgen staining. With the two quantitative parameters, Hb content and DNA amount, the erythroid cells in normal human bone narrow were classified into 6 classes of different maturation stages (EI-EIV).The morphological characteristics of the most primitive erythroblast (EI cells) were described. The proerythroblasts which were identified on the bases of morphological criteria had in general aneuploid amounts of nuclear DNA with disproportional contents of Hb, thereby indicating that they are rather aberrations from the normal steps of cell maturation. The DNA amounts of orthochromatic erythroblasts (EV cells) showed continuous decrease from diploid range to almost zero suggesting that the removal of nuclear DNA from the erythroblast is not exclusively due to mechanical expulsion of a whole intact nucleus.Partly supported by Alexander von Humboldt-Stiftung and Deutsche Forschungsgemeinschaft DFG-Grant Bo 395/3     

Etoposide (VP-16) sensitizes p53-deficient human non-small cell lung cancer cells to caspase-7-mediated apoptosis

Human non-small-cell-lung-cancer (NSCLC) cells of _p53-null genotype were exposed to low-dosage topoisomearse II inhibitor etoposide (VP-16). The cellular proliferation rate could be effectively inhibited by VP-16 in dose-dependent manner. The effective drug concentration for growth inhibition could be as low as 0.5 M and the apoptotic phenotype became evident 48 h later. In H1299 cells, VP-16-induced cytotoxic effect was demonstrated associated with apoptosis that disappeared when restored with wild-type p53. Cell cycle analysis revealed that, upon VP-16 induction, cell death began with growth arrest by accumulating cells at the G₂-M phase. The cells at sub-G₁ phase increased at the expense of those at G₂-M transition state. To assess the regulation of cell cycle modulators, western blot analysis of H1299 cell lysates showed the release of apoptosis initiator, cytochrome c and apaf-1 hours following drug induction. The cleavage of downstream effectors, procaspase-9 and procaspase-7, but not procaspase-3, was accompanied with proteolysis of poly-(ADP-ribose) polymerase (PARP). VP-16-activated procaspase-7 cleavage was abrogated in cells with ectopically expressed p53.On the other hand, the inhibited procaspase-7 fragmentation by caspase-specific inhibitor reversed apoptotic phenotype caused by drug induction. Thus, VP-16-induced apoptotic cell death was contributed by caspase-7 activation in_p53-deficient human NSCLC cells.     

Analysis of the anticancer drugs VP 16-213 and VM 26 and their metabolites by high-performance liquid chromatography

A rapid and convenient high-performance liquid chromatographic procedure for the analysis of the clinically useful anticancer agents VP 16-213 and VM 26 is described. The drugs, which are semi-synthetic derivatives of the natural product podophyllotoxin, are extracted from plasma with chloroform. The extracts are evaporated to dryness, reconstituted in methanol, and chromatographed on a reversed-phase microparticle C₁₈ column using isocratic elution with a mixture of methanol—water (60:40). Each drug is used as the internal standard for the other. Quantitation to 500 ng/ml (0.85 nmole/ml) plasma is based on peak height ratios using UV detection at 254 nm. Patient plasma concentration vresus time data agree well with previously published data obtained using radiolabelled drug.Investigations into the nature of the hydroxy acid metabolite of VP 16-213, carried out using paired-ion chromatography with tetrabutylammonium bromide and fluorescence detection, are described. Also, a unique separation of VP 16-213 and a possible metabolite, the isomer, picro VP 16-213, is described.     

The effects of ouabain on the cell mitotic cycle of mouse lymphoblasts

The inhibition of cell proliferation by ouabain has been analyzed with respect to the cell cycle. Three lines of evidence indicate that growth rate is modified by altering to different degrees the rate of progress through stages of the cell cycle: (1) a three hour lag occurs between the time of ouabain addition and the inhibition of proliferation; (2) ouabain must be present at least two to four hours prior to the mitotic burst of synchronized cells for inhibition of mitosis to occur; (3) parasynchrony is observed when cells are resuspended in ouabain-free medium after 12 hours of exposure to ouabain. Analysis of the distribution of cells in each of the stages of the cell cycle at various times during ouabain treatment reveals a progressive increase in the fraction of cells in S with a concomitant decrease in the percent of cells in each of the other stages. These results indicate that the prolongation of the cell cycle time in the presence of ouabain is due primarily to an S stage block.     

Daunomycin, cytosin-arabinoside and VP-16 (DAV) for myeloid blast crisis of CML

Nine patients with myeloid blast crisis of Philadelphia chromosome-positive chronic myelocytic leukemia received 1-3 courses of intensive induction chemotherapy with DAT (daunomycin, cytosin-arabinoside and 6-thioguanin) or DAV (daunomycin, cytosin-arabinoside and VP-16). Eight patients responded with clearing of blasts from peripheral blood giving a response rate of 89%. However, bone marrow aplasia with less than 5% blasts was seen in only 2 patients. These 2 patients subsequently received an allogeneic bone marrow transplant and achieved complete remissions of 3 and 6 month duration. All patients died due to progression of blast crisis. Median survival of the group was 164 days. These results were compared to a historical control group of 31 patients with myeloid blast crisis treated with vincristine and prednisone. Despite a significantly better response rate with DAV or DAT (8 of 9 versus 9 of 31, p = 0.01) survival was not significantly different than that of the control group.     

Teniposide (VM-26) treatment enhances the radiation-induced micronuclei in the bone marrow of mouse

The effect of Teniposide (VM-26) pretreatment was studied on the micronuclei induction in the bone marrow of mice exposed to 0, 0.5, 1, 2 and 3 Gy of gamma radiation at 12, 24 and 36 h post-irradiation. Administration of 0.05 mg/kg body weight of VM-26 to mice before irradiation resulted in the significant enhancement of micronucleated polychromatic erythrocytes (MPCE) at 12, 24 and 36 h post-irradiation. Highest elevation in the frequency of MPCE was observed in VM-26+irradiation group after exposure to 0.5 Gy when compared to concurrent DDW+irradiation group. This increase was two fold higher in VM-26+irradiation group at 12 and 24 h, while it was 3 fold higher at 36 h post-irradiation compared to DDW+irradiation group. The peak frequency of MPCE was observed at 24 h post-irradiation in both groups, which declined thereafter. The frequency of micronucleated normochromatic erythrocytes (MNCE) increased in a dose dependent manner in both DDW+irradiation and VM-26+irradiation groups. However, the frequency of MNCE was significantly higher in the latter when compared to the former group. The frequency of MNCE exhibited a continuous elevation up to 36 h post-irradiation in both DDW+irradiation and VM-26+irradiation groups. Treatment of mice with teniposide before irradiation resulted in a significant decline in the PCE/NCE ratio compared to DDW+irradiation group. The PCE/NCE ratio continued to decline up to 36 h post-irradiation in both the groups. The dose response for MPCE and PCE/NCE ratio was linear quadratic, while it was linear for MNCE.     

Mutagenicity and clastogenicity of teniposide (VM-26) in L5178Y/TK +/- -3.7.2C mouse lymphoma cells

The antitumor drug teniposide (VM-26) is a potent inducer of DNA breaks (Long et al., Cancer Res., (1985) 45, 3106), but it is only weakly mutagenic at the hprt locus in CHO cells (Singh and Gupta, Cancer Res., (1983) 43, 577). In the present study, the mutagenic and clastogenic activities of teniposide were evaluated in L5178Y/TK +/- -3.7.2C mouse lymphoma cells. Although teniposide is a weak mutagen at the hprt locus, it is a potent mutagen at the tk locus, with as little as 0.5 ng/ml producing 220 TK mutants/10(6) survivors at 96% survival (background = 100/10(6) survivors). This same dose of teniposide induced 38 aberrations per 100 metaphases (background = 7/100 cells). At 7 ng/ml, teniposide induced approximately 2700 TK mutants/10(6) survivors at approximately 10% survival. At the highest dose sampled for aberration analysis (5 ng/ml), teniposide induced 44 aberrations/100 cells. Most of the aberrations were chromosomal rather than chromatid events. As expected for a compound acting primarily by a clastogenic mechanism, most of the TK mutants were small colonies. Thus, teniposide is a potent clastogen, and it is a potent mutagen at the tk locus but not at the hprt locus. These results support the hypothesis that the location of the target gene affects the ability of the assay to detect both intragenic events and events causing functional multilocus effects. Thus, a heterozygous locus (like tk) but not a functionally hemizygous locus (like hprt) may permit the detection of mutagens that act primarily by a clastogenic mechanism. Because teniposide induces topoisomerase II-associated DNA breaks, and because there is evidence that teniposide may not interact directly with DNA, we discuss the possibility that the potent clastogenic/mutagenic activity of teniposide may be mediated by topoisomerase II.     

The relation between protein accumulation and cell cycle traverse of human NHIK 3025 cells in unbalanced growth

Human NHIK 3025 cells, synchronized by mitotic selection, were given 2 mM thymidine, which inhibited DNA synthesis without reducing the rate of protein accumulation. After removal of the thymidine the cells proceeded towards mitosis and cell division, with an S duration 2 hours shorter than, but a G2 and M duration nearly identical to that of the control cells. If cycloheximide (1.25 m?M) was present together with thymidine, no net protein accumulation took place during the treatment, and the subsequent duration of S, G2, and M was similar to that of the untreated cells. The shortening of S seen after treatment with thymidine alone would therefore indicate that the rate of DNA synthesis depended on the amount of some preaccumulated protein. The postreplicative period in thymidine-treated cells was lengthened by cycloheximide treatment although the protein content had already been doubled. This suggests that proteins required for the traverse of this part of the cell cycle might have to be synthesized after completion of DNA replication. Shortly after removal of thymidine, the rate of protein accumulation declined markedly, indicating the existence of some mechanism for negative control of cell mass. In addition, the daughters of thymidine-treated cells had their cell cycle shortened by 2 hours. As a result, the cells had returned to balanced growth already in the first cell cycle following the induction of unbalanced growth. In conclusion, our experiments suggest that NHIK 3025 cells might require a minimum time in order to traverse the cell cycle, which is independent of cell mass.     

Apoptosis of unstimulated human lymphocytes and DNA strand breaks induced by the topoisomerase II inhibitor etoposide (VP-16)

Etoposide (VP-16)-induced DNA strand breaks and repair and apoptosis of unstimulated human lymphocytes have been studied using DNA comet assay, electrophoresis of low-molecular-weight DNA extracts, and fluorescence microscopy. Incubation of unstimulated human lymphocytes with VP-16 (50-200 microg/ml) for 3 or 24 h induced apoptosis. This conclusion is supported by results of morphological studies, evaluation of the proportion of hypodiploidy and internucleosomal degradation of DNA in lymphocytes. Etoposide-induced formation of DNA strand breaks preceded the appearance of these conventional apoptotic manifestations. The number of single-strand breaks depended on VP-16 concentration, and 2-3 h after its removal from the incubation medium they were repaired. The hydroxyl group at the C-4; position of the etoposide dimethoxyphenol ring may be responsible for the formation of single-strand breaks. Double-strand breaks were unrepaired 20 h after the change of the incubation medium. The number of double-strand breaks and a proportion of apoptotic cells did not exhibit any dependence on VP-16 concentration and/or duration of cell exposure to this agent. We suggest that the cytotoxic effect of VP-16 on unstimulated lymphocytes is mediated by a topoisomerase II isoform, topoisomerase II-beta, which is localized in the nucleolus and is not related to the cell cycle.