Tyrosine kinase inhibitors vandetanib, lenvatinib and cabozantinib modulate oxidation of an anticancer agent ellipticine catalyzed by cytochromes P450 in vitro.

OBJECTIVES: Vandetanib¸ lenvatinib, and cabozantinib are tyrosine kinase inhibitors (TKIs) targeting VEGFR subtypes 1 and 2, EGFR and the RET-tyrosine kinase, thus considered as multiple TKIs. These TKIs have already been approved for treating patients suffering from thyroid cancer and renal cell carcinoma. Ellipticine, a DNA-damaging drug, is another anticancer agent that is effective against certain tumors of the thyroid gland, ovarian carcinoma, breast cancer and osteolytic breast cancer metastasis. Its anticancer efficiency is dictated by its oxidation with cytochrome P450 (CYP) and peroxidase enzymes. A number of studies testing the effectiveness of individual anticancer drugs, the pharmacological efficiencies of which are affected by their metabolism, alone or in a combination with other cytostatics demonstrated that such combination can have both positive and negative effects on treatment regimen. The aim of this study was to study the effect of vandetanib, lenvatinib and cabozantinib on oxidation of ellipticine which dictates its pharmacological efficiency. METHODS: Ellipticine oxidation catalyzed by hepatic microsomes, recombinant CYP enzymes and peroxidases (horseradish peroxidase, lactoperoxidase and myeloperoxidase) and the effect of TKIs (vandetanib, lenvatinib and cabozantinib) on this oxidation were analyzed by HPLC used for separation of ellipticine metabolites and quantification of their amounts formed during oxidation. RESULTS: The CYP enzymatic system oxidizes ellipticine up to five metabolites (9-hydroxy-, 12-hydroxy-, 13-hydroxy-, 7-hydroxyellipticine, and ellipticine N2- oxide), while peroxidases form predominantly ellipticine dimer. Ellipticine oxidation catalyzed by rat and human hepatic microsomes was inhibited by vandetanib and cabozantinib, but essentially no inhibition was caused by lenvatinib. Of individual CYP enzymes catalyzing oxidation of ellipticine, TKIs inhibited oxidation of ellipticine catalyzed by CYP2D6 > 2D1 > 2C9 > 3A1 > 3A4, the CYP enzymes participating in ellipticine oxidation to metabolites increasing the ellipticine anticancer efficiency. On the contrary, they have essentially no inhibition effect on ellipticine oxidation catalyzed by CYP1A1 and 1A2, which are the enzymes that predominantly detoxify this drug. All tested TKIs had essentially no effect on oxidation of ellipticine by used peroxidases. CONCLUSION: The results found demonstrate that TKIs vandetanib, lenvatinib and cabozantinib cause a decrease in oxidative activation of DNA-damaging drug ellipticine by several CYP enzymes in vitro which might lead to a decrease in its pharmacological efficiency. In contrast, they practically do not influence its detoxification catalyzed by CYP1A1, 1A2 and peroxidases. The present study indicates that tested TKIs seem not to have a potency to increase ellipticine anticancer efficiency.

The impact of chemotherapeutic drugs on the CYP1A1-catalysed metabolism of the environmental carcinogen benzo[a]pyrene: Effects in human colorectal HCT116 TP53(+/+), TP53(+/-) and TP53(-/-) cells.

Polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP) can induce cytochrome P450 1A1 (CYP1A1) via a p53-dependent mechanism. The effect of different p53-activating chemotherapeutic drugs on CYP1A1 expression, and the resultant effect on BaP metabolism, was investigated in a panel of isogenic human colorectal HCT116 cells with differing TP53 status. Cells that were TP53(+/+), TP53(+/-) or TP53(-/-) were treated for up to 48 h with 60 µM cisplatin, 50 µM etoposide or 5 µM ellipticine, each of which caused high p53 induction at moderate cytotoxicity (60-80% cell viability). We found that etoposide and ellipticine induced CYP1A1 in TP53(+/+) cells but not in TP53(-/-) cells, demonstrating that the mechanism of CYP1A1 induction is p53-dependent; cisplatin had no such effect. Co-incubation experiments with the drugs and 2.5 µM BaP showed that: (i) etoposide increased CYP1A1 expression in TP53(+/+) cells, and to a lesser extent in TP53(-/-) cells, compared to cells treated with BaP alone; (ii) ellipticine decreased CYP1A1 expression in TP53(+/+) cells in BaP co-incubations; and (iii) cisplatin did not affect BaP-mediated CYP1A1 expression. Further, whereas cisplatin and etoposide had virtually no influence on CYP1A1-catalysed BaP metabolism, ellipticine treatment strongly inhibited BaP bioactivation. Our results indicate that the underlying mechanisms whereby etoposide and ellipticine regulate CYP1A1 expression must be different and may not be linked to p53 activation alone. These results could be relevant for smokers, who are exposed to increased levels of BaP, when prescribing chemotherapeutic drugs. Beside gene-environment interactions, more considerations should be given to potential drug-environment interactions during chemotherapy.

Effect of Cholesterol on Cellular Uptake of Cancer Drugs Pirarubicin and Ellipticine.

The cell membrane is a major barrier for drug transport. Given that many cancer drugs must passively cross the cell membrane, understanding drug-membrane interactions is crucial. We used fluorescence-activated cell sorting to investigate how cholesterol influences the transport of the cancer drugs ellipticine and pirarubicin across cell membranes. We showed that cholesterol depletion helped pirarubicin cross the membranes of nonsmall cell lung carcinoma and Chinese hamster ovary cells. In contrast, the uptake of ellipticine was not strongly influenced by cholesterol depletion. To study the microscopic origins of these observations, atomistic molecular dynamics simulations were performed. Doxorubicin (similar in structure to pirarubicin) and ellipticine were simulated in model membranes of POPC and POPC with 40 mol % cholesterol. Atomistic free energy calculations for the translocation of a single ellipticine and doxorubicin across the lipid bilayers qualitatively matched the experiment results. The free energy barrier for doxorubicin crossing the bilayer was strongly increased when cholesterol was present, while for ellipticine the barrier remained similar with and without cholesterol. Molecular dynamics simulations showed that the different hydrogen-bonding propensities of the two drugs are likely the major factor for the different behaviors. The qualitative agreement between cell experiments and atomistic computer simulations illustrates the potential to link observed biological phenomena and single molecule mechanisms of actions. Our results suggest that the traditional understanding of drug permeation and the influence of cholesterol on the small molecule transport is naïve and needs to be re-examined.

Vacuolar-ATPase-mediated intracellular sequestration of ellipticine contributes to drug resistance in neuroblastoma cells.

Neuroblastoma is the most common cancer in infants and the fourth most common cancer in children. Aggressive cell growth and chemoresistance are notorious obstacles in neuroblastoma therapy. Exposure to the anticancer drug ellipticine inhibits efficiently growth of neuroblastoma cells and induces apoptosis in these cells. However, ellipticine induced resistance in these cells. The upregulation of a vacuolar (V)-ATPase gene is one of the factors associated with resistance development. In accordance with this finding, we found that levels of V-ATPase protein expression are higher in the ellipticine-resistant UKF-NB-4ELLI line than in the parental ellipticine-sensitive UKF-NB-4 cell line. Treatment of ellipticine-sensitive UKF-NB-4 and ellipticine-resistant UKF-NB-4ELLI cells with ellipticine-induced cytoplasmic vacuolization and ellipticine is concentrated in these vacuoles. Confocal microscopy and staining of the cells with a lysosomal marker suggested these vacuoles as lysosomes. Transmission electron microscopy and no effect of an autophagy inhibitor wortmannin ruled out autophagy. Pretreatment with a V-ATPase inhibitor bafilomycin A and/or the lysosomotropic drug chloroquine prior to ellipticine enhanced the ellipticine­mediated apoptosis and decreased ellipticine-resistance in UKF-NB-4ELLI cells. Moreover, pretreatment with these inhibitors increased formation of ellipticine-derived DNA adducts, one of the most important DNA-damaging mechanisms responsible for ellipticine cytotoxicity. In conclusion, resistance to ellipticine in the tested neuroblastoma cells is associated with V-ATPase-mediated vacuolar trapping of this drug, which may be decreased by bafilomycin A and/or chloroquine.

Ranking the Binding Energies of p53 Mutant Activators and Their ADMET Properties.

The guardian of the genome, p53, is the most mutated protein found in all cancer cells. Restoration of wild-type activity to mutant p53 offers promise to eradicate cancer cells using novel pharmacological agents. Several molecules have already been found to activate mutant p53. While the exact mechanism of action of these compounds has not been fully understood, a transiently open pocket has been identified in some mutants. In our study, we docked twelve known activators to p53 into the open pocket to further understand their mechanism of action and rank the best binders. In addition, we predicted the absorption, distribution, metabolism, excretion and toxicity properties of these compounds to assess their pharmaceutical usefulness. Our studies showed that alkylating ligands do not all bind at the same position, probably due to their varying sizes. In addition, we found that non-alkylating ligands are capable of binding at the same pocket and directly interacting with Cys124. The comparison of the different ligands demonstrates that stictic acid has a great potential as a p53 activator in terms of less adverse effects although it has poorer pharmacokinetic properties.

Formation of DNA adducts by ellipticine and its micellar form in rats - a comparative study.

The requirements for early diagnostics as well as effective treatment of cancer diseases have increased the pressure on development of efficient methods for targeted drug delivery as well as imaging of the treatment success. One of the most recent approaches covering the drug delivery aspects is benefitting from the unique properties of nanomaterials. Ellipticine and its derivatives are efficient anticancer compounds that function through multiple mechanisms. Formation of covalent DNA adducts after ellipticine enzymatic activation is one of the most important mechanisms of its pharmacological action. In this study, we investigated whether ellipticine might be released from its micellar (encapsulated) form to generate covalent adducts analogous to those formed by free ellipticine. The (32)P-postlabeling technique was used as a useful imaging method to detect and quantify covalent ellipticine-derived DNA adducts. We compared the efficiencies of free ellipticine and its micellar form (the poly(ethylene oxide)-block-poly(allyl glycidyl ether) (PAGE-PEO) block copolymer, P 119 nanoparticles) to form ellipticine-DNA adducts in rats in vivo. Here, we demonstrate for the first time that treatment of rats with ellipticine in micelles resulted in formation of ellipticine-derived DNA adducts in vivo and suggest that a gradual release of ellipticine from its micellar form might produce the enhanced permeation and retention effect of this ellipticine-micellar delivery system.

The effect of benzo[a]pyrene on metabolic activation of anticancer drug ellipticine in mice.

OBJECTIVES: The aim of this study was to investigate a role of cytochrome P450 (CYP) and peroxidase in ellipticine oxidative activation in two mouse strains differing in expression of NADPH:CYP reductase (POR) [the HRN (Hepatic Cytochrome P450 Reductase Null) mice, in which POR is deleted in hepatocytes and its wild-type (WT) counterpart], and in levels of CYP1A1/2 and cytochrome b5 that were modulated by treatment of these mouse models with a CYP1A inducer, benzo[a]pyrene (BaP). METHODS: Ellipticine-DNA adducts were detected by 32P-postlabeling. HPLC was employed for the separation and characterization of ellipticine metabolites. RESULTS: Hepatic microsomes of HRN and WT mice activate ellipticine to form ellipticine-derived DNA adducts. A 2.2- and 10.4-fold increase in amounts of ellipticine-derived DNA adducts formed by liver microsomes was caused by exposure of HRN and WT mice to BaP, respectively. The results found and utilization of NADPH and arachidonic acid, cofactors of CYP- and cyclooxygenase (COX)-dependent enzyme systems, respectively, as well as inhibitors of CYP1A1/2 and 3A, demonstrate that the CYP1A and 3A enzymes play a major role in ellipticine activation in liver microsomes. In addition, the COX enzyme is important in ellipticine activation in liver of HRN mice. CONCLUSION: The CYP1A and 3A enzymes activate ellipticine mainly in liver of WT mice, whereas peroxidase COX plays this role in liver of HRN mice. Treatment of mice with BaP increases an impact of CYP1A on ellipticine activation. A pattern of expression levels of these enzymes plays a crucial role in their impact on this process.

Pharmacokinetics of peptide mediated delivery of anticancer drug ellipticine.

The amino acid pairing peptide EAK16-II (EAK) has shown the ability to stabilize the hydrophobic anticancer agent ellipticine (EPT) in aqueous solution. In this study, we investigate pharmacokinetics of the formulation of EAK-EPT complexes in vivo. The developed formulation can achieve a sufficiently high drug concentration required in vivo animal models. The nanostructure and surface properties of EAK-EPT complexes or nanoparticle were characterized by transmission electron microscopy (TEM) and zeta potential measurements, respectively. 12 healthy male SD rats were divided into EPT group and EAK-EPT group randomly. Rats in EPT group were tail intravenously injected with the EPT (20 mg/kg); rats in EAK-EPT group were injected with EAK-EPT complexes (EPT's concentration is 20 mg/kg). EPT was extracted from rat plasma with dexamethasone sodium phosphate as internal standards (IS). The pharmacokinetic parameters were obtained using high pressure liquid chromatography (HPLC). Significant differences in main pharmacokinetic parameters between EPT and EAK-EPT complexes were observed, demonstrating that the complexation with EAK prolongs the residence time of the drug and enlarges the area under the concentration-time curve (AUC). This means that EAK can serve as a suitable carrier to increase the bioavailability of EPT.

Self-assembling peptide-based nanoparticles enhance anticancer effect of ellipticine in vitro and in vivo.

BACKGROUND AND METHODS: Applications of the anticancer agent, ellipticine, have been limited by its hydrophobicity and toxicity. An efficient delivery system is required to exploit the enormous potential of this compound. Recently, EAK16-II, an ionic-complementary, self-assembling peptide, has been found to stabilize ellipticine in aqueous solution. Here, the anticancer activity of ellipticine encapsulated in EAK16-II (EAK-EPT) was evaluated in vitro and in vivo. RESULTS: Our cellular uptake, toxicity, and apoptosis results in an A549 human lung carcinoma cell line indicate that EAK-EPT complexes are significantly more effective than treatment with EAK16-II or ellipticine alone. This is due to the ability of EAK16-II to stabilize ellipticine in a protonated state in well formed nanostructures approximately 200 nm in size. In vivo observations in an A549 nude mouse tumor model show higher antitumor activity and lower cytotoxicity of EAK-EPT complexes than in the control group treated with ellipticine alone. Tumor growth in animals was significantly inhibited after treatment with EAK-EPT complexes, and without any apparent side effects. CONCLUSION: The anticancer activity observed in this study coupled with minimal side effects encourages further development of peptide-mediated delivery of anticancer drugs, ellipticine in the present case, for clinical application.

Cytochromes P450 reconstituted with NADPH: P450 reductase mimic the activating and detoxicating metabolism of the anticancer drug ellipticine in microsomes.

OBJECTIVES: Ellipticine is a potent antineoplastic agent exhibiting multiple action mechanisms. Recently, we found that after cytochrome P450 (CYP)-mediated oxidation ellipticine forms covalent DNA adducts. Ellipticine oxidation by isolated CYP and its binding to DNA is the target of this study. METHODS: High performance liquid chromatography (HPLC) was employed for separation and characterization of ellipticine metabolites generated by CYPs. The (32)P-postlabeling technique was utilized to determine ellipticine-DNA adducts. RESULTS: Purified CYP enzymes reconstituted with NADPH:CYP reductase oxidized ellipticine to up to five metabolites, 7-hydroxy-, 9-hydroxy-, 12-hydroxy-, 13-hydroxyellipticine and ellipticine N(2)-oxide. However, only CYP1A1 was capable to form all metabolites. Using the reconstituted enzymatic system, we demonstrated that the detoxication ellipticine metabolites, 7-hydroxyellipticine and 9-hydroxyellipticine, are mainly generated by CYP1A1 and 1A2, while those responsible for DNA binding, 13-hydroxy-, 12-hydroxyellipticine and ellipticine N(2)-oxide, by CYP3A1 and 2C3. Likewise, the most efficient CYPs forming DNA adducts from ellipticine were CYP3A1 and 2C3. CONCLUSIONS: The results showed that the system of purified CYPs reconstituted with NADPH: CYP reductase proved for ellipticine oxidation provide a true reflection of the situation in the microsomal membrane.

The development of VIP-ellipticine conjugates.

The mechanism by which vasoactive intestinal peptide (VIP)-ellipticine (E) conjugates are cytotoxic for human lung cancer cells was investigated. VIP-alanyl-leucyl-alanyl-leucyl-alanine (ALALA)-E and VIP-leucyl-alanyl-leucyl-alanine (LALA)-E inhibited (125)I-VIP binding to NCI-H1299 cells with an IC50 values of 0.5 and 0.1 microM, respectively. VIP-ALALA-E and VIP-LALA-E caused elevation of cAMP in NCI-H1299 cells with ED50 values of 0.7 and 0.1 microM. Radiolabeled VIP-LALA-E was internalized at 37 degrees C and delivered the cytotoxic E into NCI-H1299 cells. VIP-LALA-E inhibited the growth of NCI-H1299 cells in vitro. Three days after the addition of VIP-LALA-E to NCI-H1299 cells, cell viability decreased based on trypan blue exclusion and reduced 3H-thymidine uptake. These results suggest that VIP-E conjugates are internalized in lung cancer cells as a result of VPAC1 receptor-mediated endocytosis.

DNA adduct formation by the anticancer drug ellipticine in rats determined by 32P postlabeling.

Ellipticine is a potent antineoplastic agent whose mode of action is considered to be based mainly on DNA intercalation and/or inhibition of topoisomerase II. Recently, we found that ellipticine also forms covalent DNA adducts in vitro and that the formation of the major adduct is dependent on the activation of ellipticine by cytochrome P450 (CYP). Here, we investigated the capacity of ellipticine to form DNA adducts in vivo. Male Wistar rats were treated with ellipticine, and DNA from various organs was analyzed by (32)P postlabeling. Ellipticine-specific DNA adduct patterns, similar to those found in vitro, were detected in most test organs. Only DNA of testes was free of the ellipticine-DNA adducts. The highest level of DNA adducts was found in liver (19.7 adducts per 10(7) nucleotides), followed by spleen, lung, kidney, heart and brain. One major and one minor ellipticine-DNA adducts were found in DNA of all these organs of rats exposed to ellipticine. Besides these, 2 or 3 additional adducts were detected in DNA of liver, kidney, lung and heart. The predominant adduct formed in rat tissues in vivo was identical to the deoxyguanosine adduct generated in DNA by ellipticine in vitro as shown by cochromatography in 2 independent systems. Correlation studies showed that the formation of this major DNA adduct in vivo is mediated by CYP3A1- and CYP1A-dependent reactions. The results presented here are the first report showing the formation of CYP-mediated covalent DNA adducts by ellipticine in vivo and confirm the formation of covalent DNA adducts as a new mode of ellipticine action.

Pharmacological evaluation of a Br-76 analog of epibatidine: a potent ligand for studying brain nicotinic acetylcholine receptors.

[(76)Br]-Norchlorobromoepibatidine ([(76)Br]BrPH) is a specific and high affinity radioligand for the nicotinic acetylcholine receptors (nAChRs). In vitro, on rat thalamus membranes [(76)Br]BrPH bound to two sites with apparent affinities of 8 pM and 3 nM. The density of binding sites were 1.9 and 70 fmol/mg protein, respectively. In vivo, in biodistribution and autoradiographic studies in rats the regional distribution of [(76)Br]BrPH paralleled the neuroanatomical localization of nAChRs. Two hours postinjection, the highest concentration in the brain was found in thalamus and colliculi (4% ID/g). Competition experiments with specific nicotinic, muscarinic, dopaminergic, and serotoninergic drugs confirmed that the in vivo binding of [(76)Br]BrPH was consistent with neuronal nicotinic receptors. PET imaging of [(76)Br]BrPH in baboon demonstrated a rapid and high uptake in the brain. Peak uptake occurred at 30-40 min for the thalamus. Due to the constant washout in the cerebellum, the thalamus to cerebellum ratio was 5 at 2 h postinjection. Subcutaneous injection of cytisine (1 mg/kg), 3 h postinjection of [(76)Br]BrPH reduced the radioactivity concentration in thalamus and cortex by 58 and 50%, respectively, as observed 1 h later. Cytisine pretreatment (5 mg/kg s.c.) inhibited completely the radioligand accumulation in the thalamus. Chronic MPTP pretreatment resulted in reduction of [(76)Br]BrPH uptake in all brain regions except in cerebellum. These preliminary results suggest that [(76)Br]BrPH has the potential to be a useful radioligand for studying the pharmacology of nicotinic acetylcholine receptors in preclinical experiments.

N-(2-hydroxypropyl)methacrylamide copolymer-6-(3-aminopropyl)-ellipticine conjugates. Synthesis, in vitro, and preliminary in vivo evaluation.

Ellipticine derivatives have potential as anticancer drugs. Their clinical use has been limited, however, by poor solubility and host toxicity. As N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-anticancer conjugates are showing promise in early clinical trials, a series of novel HPMA copolymer conjugates have been prepared containing the 6-(3-aminopropyl)-ellipticine derivative (APE, NSC176328). Drug was linked to the polymer via GFLG or GG peptide side chains. To optimize biological behavior, HPMA copolymer-GFLG-APE conjugates with different drug loading (total APE: 2.3-7% w/w; free APE: <0.1% w/w) were synthesized. Conjugation of APE to HPMA copolymers considerably increased its aqueous solubility (>10-fold). HPMA copolymer-GG-APE did not liberate drug in the presence of isolated lysosomal enzymes (tritosomes), but HPMA copolymer-GFLG-APE released APE to a maximum of 60% after 5 h. The rate of drug release was influenced by drug loading; lower loading led to greater release. Whereas free APE (35 microg/mL) caused significant hemolysis (50% after 1 h), HPMA copolymer-APE conjugates were not hemolytic up to 300 microg/mL (APE-equiv). As would be expected from its cellular pharmacokinetics, HPMA copolymer-GFLG-APE was >75 times less cytotoxic than free drug (IC(50) approximately 0.4 microg/mL) against B16F10 melanoma in vitro. However, in vivo when tested in mice bearing s.c. B16F10 melanoma, HPMA copolymer-GFLG-APE (1-10 mg/kg single dose, APE-equiv) given i.p. was somewhat more active (highest T/C value of 143%) than free APE (1 mg/kg) (T/C =127%). HPMA copolymer-APE conjugates warrant further evaluation as potential anticancer agents.

Preclinical evaluation of 9-chloro-2-methylellipticinium acetate alone and in combination with conventional anticancer drugs for the treatment of human brain tumor xenografts.

Some ellipticine derivative salts, including 9-chloro-2-methylellipticinium (CME), have been found to have a marked selectivity against all eight brain tumor cell lines of the U.S. National Cancer Institute's disease-oriented in vitro screen. We initiated in vivo antitumor studies to explore the feasibility for further development of this class of compounds. We found that CME was extremely toxic to nude mice when given i.p. at a dose of 25 mg/kg for 3 consecutive days. Animals treated by this route experienced an increase in hepatic transaminases and histopathological changes in the liver, compatible with mitochondrial damage. In contrast, when the portal circulation was bypassed and the same dose of CME was given i.v., animals tolerated daily bolus injections for 5 consecutive days. This 5-day i.v. bolus schedule had consistent antitumor activity, with 28.1% growth delay on s.c. implanted human U251 gliomas. When the potentially high peaks of CME in the portal circulation were avoided by using a 3-day continuous infusion with osmotic minipumps implanted i.p. to release 3.4 mg kg(-1) h(-1) or 6.6 mg kg(-1) h(-1) CME, there were only modest increases in liver enzymes and leukopenia, but no meaningful antitumor activity was observed. In contrast, continuous infusion in the s.c. space was well tolerated and was accompanied by a demonstrable growth delay in s.c. U251 human gliomas of 37.8%. When CME was used in conjunction with carmustine, etoposide or cisplatin, no synergistic activities were observed, but additive effects were demonstrated. Our pharmacokinetic and disposition studies with CME argue against the notion that large and invasive tumors in the brain lack blood-brain barrier features. When CME was used in animals bearing orthotopically implanted U251 gliomas in the brain of nude mice, the survival of the treated animals was not better than vehicle controls, and the addition of CME to carmustine therapy did not improve the survival of those animals treated with carmustine alone. We conclude that, in spite of its marked cytotoxicity in vitro on a variety of human brain tumor cell lines, including U251 glioma cells, CME has a modest antitumor effect on extracranially implanted U251 glioma tumors, and no beneficial effect in animals bearing the same U251 tumor in the brain, owing to a poor penetration into the brain parenchyma.

Monitoring uptake of ellipticine and its fluorescence lifetime in relation to the cell cycle phase by flow cytometry.

Ellipticine (5,11-dimethyl-6H-pyrido[4,3-b]carbazole) is a green fluorescent plant alkaloid that inhibits DNA topoisomerase II activity and possesses pharmacologic activity toward both murine and human leukemias in vivo. In this flow cytometric study, the uptake of ellipticine was monitored as a function of cell volume and cell cycle phase in viable human promyelocytic (HL-60) cells costained with the DNA fluorochrome Hoechst 33342. Uptake of ellipticine was time and dose dependent; however, drug content was quantitatively similar in all phases of the cell cycle when normalized for DNA content or similar to cell size when correlated with cell volume. The fluorescence lifetime values of ellipticine in HL-60 cells, as analyzed by novel flow cytometric analysis, reached a plateau when the intra-cellular ellipticine intensity was still rising with increasing drug concentration. Since the free drug and the different subcellular ellipticine complexes, including DNA and RNA, had different lifetime values, the changes in the lifetime values appear to reflect differing proportions of unbound drug to that bound to different cellular constituents in the cells. Further development of phase-sensitive flow cytometry will provide for multiple lifetime determinations so that quantitation of drugs bound to the different cellular components can be performed along with the simultaneous determination of total drug uptake and cell cycle position. Such analyses should provide useful information for the design of drugs with greater affinity for cytotoxic targets.

Synthesis and antitumor activity of 9-acyloxyellipticines.

Various kinds of water-soluble 9-acyloxyellipticine derivatives were synthesized in a search for compounds with potent antitumor activity. Antitumor activities against several tumors in mice (P388 leukemia, colon 26, Lewis lung carcinoma and B16 melanoma) were evaluated by using intravenous administration. Many compounds exhibited good antitumor activities; in particular, the glutarate derivative (5o) showed potent antitumor activity. This compound (5o) may be converted to 9-hydroxyellipticine (2) by enzyme-catalyzed hydrolysis in the body.

Role of membrane potential in the accumulation of quaternized ellipticines by human tumor cell lines.

9-Methoxy-N2-methylellipticinium acetate (MMEA) is representative of a series of quaternized ellipticines that exhibited selective cytotoxicity for human brain tumor cell lines of glial origin in the in vitro primary screen of the U.S. National Cancer Institute. The present investigation was initiated to determine whether membrane potential contributes to the cellular accumulation of this lipophilic cation by selected brain tumor and non-brain tumor cell lines. The results indicate that accumulation of MMEA by drug-sensitive cell lines, but not drug-resistant cell lines, is reduced by experimental conditions that depolarize the plasma membrane, e.g., stepped increases in the extracellular potassium concentration. These experimental conditions result in increased cellular fluorescence of cells stained with the voltage-sensitive anionic dye bis(1,3-dibutylbarbituric acid)trimethine oxonol, suggesting that decreased accumulation of MMEA is the result of decreased membrane potential. Membrane potential measurements using the null point method indicated that the mean membrane potential of selected MMEA-sensitive cell lines (-39.4 +/- 6.8 mV) was significantly lower (P < .005) than MMEA-resistant cell lines (-17 +/- 3.8 mV). Ultrastructural studies with the MMEA-sensitive U-251 glioblastoma indicated that the first morphological effects of MMEA occurred in mitochondria, where dissolution of cristae was observed, followed by engulfment of mitochondria in multilamellar phagocytic vesicles. Electron microscopic autoradiographic studies with tritium-labeled MMEA revealed that the drug was localized in mitochondria and nuclei.

Uptake and cytotoxicity of 9-methoxy-N2-methylellipticinium acetate in human brain and non-brain tumor cell lines.

9-Methoxy-N2-methylellipticinium acetate (MMEA) was preferentially cytotoxic to human brain tumor cell lines in the in vitro primary screen of the U.S. National Cancer Institute. In the present study, the average intracellular accumulation of radioactivity derived from [14C]MMEA concentrations that were selectively cytotoxic to sensitive brain tumor cell lines was nearly 4-fold greater than in human tumor cell lines derived from the lung, kidney, ovary and colon. The extent of peak cellular accumulation of [14C]MMEA-derived radioactivity, achieved after 10-15 hr of drug exposure, was correlated positively with relative MMEA cytotoxicity in brain tumor cell lines (r2 = 0.963). A similar correlation (r2 = 0.967) was observed in selected non-brain tumor cell lines but required substantially higher (18-fold) concentrations of MMEA. [14C]MMEA radioactivity accumulation by a selected glioblastoma cell line occurred via an energy-requiring system that was predominantly sodium and pH independent.

Microspectrofluorometry of the protonation state of ellipticine, an antitumor alkaloid, in single cells.

The protonation state and intracellular distribution of ellipticine were investigated in single human mammary T47D cells by confocal laser microspectrofluorimetry. In the cell nucleus, only the protonated form of ellipticine was detected as a direct consequence of its apparent pK increase upon DNA binding. Both protonated and neutral forms were present in the aqueous cytoplasm, where the pH is close to the drug pK. When cells were incubated in high concentrations of K+, a condition that depolarizes the plasma membrane potential, ellipticine cellular accumulation was reduced. In the cytoplasm, ellipticine was mainly bound to mitochondria, and its protonation equilibrium was shifted toward the neutral form. The fluorescence spectrum of ellipticine bound to mitochondria was insensitive to valinomycin, whereas it was markedly shifted toward the protonated form after carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or nigericin addition. Similar studies with ellipticine bound to isolated mitochondria suggest that it behaves as a fluorescent probe of mitochondrial pH in both isolated mitochondria and single living cells.