A radioligand binding assay for antitubulin activity in tumor cells

The benzamide RH-5854 is shown to be highly potent toward tumor cells and to arrest nuclear division by a highly specific covalent binding to the beta-subunit of tubulin in the colchicine binding region. Binding of 3H-RH-5854 to beta-tubulin in HCT-116 colon cancer cells is saturable and has been exploited in the development of a cell-based competitive binding assay, which allows antitubulin effects to be detected in whole cells. 3H-RH-5854 binding is strongly inhibited by preincubating the cells with compounds that bind to the colchicine site and with paclitaxel. Binding of 3H-RH-5854 is enhanced by preincubating the cells with vinblastine but not by other agents that bind at or near the vinblastine site (ansamitocin P-3 and phomopsin A). Various cytotoxic agents that do not act on tubulin do not affect binding of 3H-RH-5854 in HCT-116 cells, demonstrating specificity of the assay for detection of antitubulin activity. As an alternative to traditional assays that employ isolated brain tubulin, the 3HRH-5854 binding assay enables screening for antitubulin effects directly in tumor cells, providing an assay that accounts for cell-specific criteria that influence sensitivity such as different tubulin isotypes, tubulin mutations, drug metabolism, and efflux mechanisms.     

Calmodulin, activated cyclic nucleotide phosphodiesterase, microtubules, and vinca alkaloids

Calmodulin 1.8 and 10.6 microM, inhibited the polymerization of bovine brain microtubules by 30 and 50%, respectively. Two 55,000- to 68,000-dalton calmodulin-binding protein as well as calmodulin-dependent and -independent phosphodiesterases (PDE) were found associated with microtubule proteins. Among the antimicrotubule drugs, such as colchicine, podophyllotoxin, griseofulvin, and vinca alkaloids (vinblastine, desacetylvinblastine amide, and vincristine), the vinca alkaloids were selective inhibitors of calmodulin-activated PDE activity. This action of vinca alkaloids resides in the catharanthine moiety of vinblastine molecule. An alpha 2 inhibitor, yohimbine, affects the microtubules, and a series of alpha-adrenoceptor blocking agents were examined for their effects on calmodulin-dependent PDE. The relative order of the potency is phenoxybenzamine = dibenamine greater than phentolamine greater than yohimbine greater than prazosin greater than tolazoline, and the first four drugs in this series were selective inhibitors of calmodulin action. Inasmuch as phenoxybenzamine and dibenamine are alkylating agents, the effects of antineoplastic alkylating agents on the calmodulin action were also examined. Busulphan, melphalan, 1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea, and streptozotocin (up to 4 mM) were not selective inhibitors of calmodulin action. Maytansine, a vinca alkaloid-type antimicrotubule agents as well as an alkylating agent, selectively inhibited the calmodulin with a potency similar to vincristine. In addition, phenoxybenzamine affected Ca2+-dependent fluorescence induced by the interaction between calmodulin and hydrophobic fluorescent probes, whereas vinblastine was ineffective. However, the binding of vinblastine to calmodulin is calcium dependent. Studies such as these suggest the importance of physical and structural considerations in drugs binding to calmodulin as well as at least two different binding sites for drugs on calmodulin.     

Geldanamycin derivatives and neuroprotective effect on cultured P19-derived neurons

Geldanamycin (1), an antifungal and anticancer ansamycin, was reported as a neurotrophic and neuroprotective substance against antineoplastic drugs, paclitaxel, vincristine, and cisplatin, on cultured dorsal root ganglion neurons from chick embryos. In this study, 1 in a large quantity, together with a known 17-O-demethylgeldanamycin (2), and a new 17-O-demethylgeldanamycin hydroquinone (3) were obtained from a mangrove Streptomyces sp. A series of O-alkyl and N-alkyl derivatives of 1 were prepared by modification of C-17 and/or C-19 on the quinone ring and were evaluated for in vitro activity against P19-derived neurons. Compound 1 and 19-O-methylgeldanamycin (7) at a very low dose (1nM) enhanced survival and neurite outgrowth of P19-derived neurons and prevented neurotoxicity of paclitaxel and vinblastine. Compound 7, possessing the lowest cytotoxicity and neurotoxicity, is serving as the most promising candidate in neurodegenerative therapy against neurotoxic anticancer drugs.     

Effects of antineoplastic agents on cytoplasmic and membrane-bound heat shock protein 70 (Hsp70) levels

Here we report on the study of the effects of different antineoplastic agents, including cytarabine, 4-hydroperoxyifosfamide, the activated form of ifosfamide, vincristine, and paclitaxel, with regard to their capacity to modulate the amount of cytoplasmic and membrane-bound heat shock protein 70 (Hsp70). Hsp70 levels were measured in the myelogenous leukemic cell line K562, in the human colon carcinoma cell line CX2, and in peripheral blood lymphocytes (PBL) under physiological conditions (37 degrees C), and following non-lethal heat shock at 41.8 degrees C. A concentration of 1 microM and an incubation period of 2 h were determined as non-lethal, since none of the different antineoplastic agents induced necrosis or apoptosis in untreated or heat-shocked cells under these conditions. Our results show that tubulin-interacting agents, including vincristine and paclitaxel, but not DNA-interacting agents, including cytarabine and ifosfamide, selectively increase the amount of cytoplasmic Hsp70 in tumor and normal cells, as measured by semi-quantitative Western blot analysis. Mechanistically, a vincristine- and paclitaxel-induced tubulin assembly, as demonstrated by immunofluorescence microscopy, might be responsible for the elevated cytoplasmic Hsp70 levels. Interestingly, an increased membrane expression of Hsp70 following treatment with vincristine or paclitaxel was selectively observed on tumor cells, but not on normal cells.     

Biological activities of Ginkgo extracts

The biological activity of methanolic the extracts of leaves, roots, leaf-derived callus, root-derived callus, ginkolide A, ginkgolide B, bilobalide and a commercial Ginkgo product (Tanakan) was assessed. Bioassays consisted of the Agrobacterium tumefaciens-induced potato tumor assay and a Kirby-Bauer microbial sensitivity assay with pure strains of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes. Methanolic extracts of leaves, leaf-derived callus, root-derived callus, bilobalide and Tanakan inhibited tumor formation significantly, but more weakly than the positive control, camptothecin. No activity against E. coli was detected, but extracts from both callus types inhibited the growth of K. pneumonia, P. aeruginosa, S. aureus, S. epidermidis and S. pyogenes. All extracts and reference compounds inhibited the growth of S. pyogenes. Leaf and root tissues contained the highest levels of ginkgolide A, as compared to the callus tissues; leaf tissue contained more of all three marker compounds than the callus tissues.     

Sensitive liquid chromatography-mass spectrometry assay for quantitation of docetaxel and paclitaxel in human plasma

We have developed a high-performance liquid chromatography-electrospray ionization mass spectrometry (LC-MS) method for quantifying docetaxel and paclitaxel in human plasma. The assay fulfills the need for defining the lower plasma concentrations of these antineoplastic agents that result from a number of changes in how these agents are used clinically. The assay uses paclitaxel as the internal standard for docetaxel, and vice versa; solid-phase extraction; a Phenomenex Hypersil ODS (5 micrometer, 100x2 mm) reversed-phase analytical column; an isocratic mobile phase of 0.1% formic acid in methanol-water (70:30, v/v); and mass spectrometric detection using electrospray positive mode electron ionization. The assay has a lower limit of quantitation (LLOQ) of 0.3 nM and is linear between 0.3 nM and 1 microM for docetaxel. For paclitaxel, the LLOQ was 1 nM, and the assay is linear between 1 nM and 1 microM. We demonstrated the suitability of this assay for docetaxel by using it to quantify the docetaxel concentrations in plasma of a patient given 40 mg/m(2) of docetaxel and comparing those results to results produced when the same samples were assayed with an HPLC assay using absorbance detection. In a similar manner, the suitability of the assay for paclitaxel was demonstrated by using it to quantify the concentrations of paclitaxel in the plasma of a patient given 15 mg/m(2) of paclitaxel and comparing those results to results produced when the same samples were assayed with an HPLC assay using absorbance detection. The LC-MS assay, which proved superior because of its greater sensitivity and relatively short (7 min) run time, should be an important tool for future pharmacokinetic analyses of docetaxel and paclitaxel.     

Inhibition of phytohemagglutinin-, periodate- and A23187-induced lymphocyte transformation by Vinca alkaloids

The effect of the Vinca alkaloids, vincristine and vinblastine, on mitogen-induced transformation of isolated human peripheral blood lymphocytes has been investigated. Cells were subjected to a variety of mitogens (PHA, ionophore A23187 and sodium periodate) whose mechanism and site of action differ. Addition of vincristine or vinblastine to lymphocyte cultures prior to mitogen produced a concentration-dependent inhibition of cell transformation as determined by measurement of DNA synthesis and blast formation. The inhibitory effects were not due to decreased cell viability, since the drugs had little or no effect on cell viability. Vincristine and vinblastine were also found to impair [³H]thymidine incorporation by prestimulated blast cells at the higher drug concentrations tested. The results presented in this communication show that the Vinca alkaloids block lymphocyte transformation induced by either lectin or non-lectin mitogens. This suggests that the inhibitory step(s) may occur after mitogen stimulation.     

Three tumor sensitivity tests evaluated with mouse tumors

The predictive value of three types of tumor sensitivity tests was evaluated using mouse tumors. Sensitivities of osteosarcoma C22LR, Lewis lung and M2661 carcinoma were determined for the following drugs: DNA interacting or alkylating agent (doxorubicin, cisplatin, 1,3-bis(2-chloroethyl)-1-nitrosourea, melphalan), antimetabolite (5-fluorouracil, methotrexate) and microtubule inhibitor (vinblastine, vincristine). Volume measurements of the subcutaneously growing tumors after treatment with the same drugs were considered to be the traditional reference system with which the results of the in vitro clonogenic assay, the labeled precursor incorporation assay and the subrenal capsule assay were compared. Results obtained with the in vitro clonogenic assay were highly reproducible. With the 1-h drug exposure technique the predictive accuracy was 71%. This result is in the same range as those found by others for human tumors. Predictive accuracy after continuous drug exposure was only 25%. Vinblastine, vincristine and cisplatin caused no inhibition of labeled precursor incorporation. However, the assay is too unreliable to use, due to the extreme variability when used with the other drugs. From 31 consecutively performed duplicate tests in the subrenal capsule assay, nine showed opposite results. This degree of disagreement between duplicate test results was considered too high to make reliable predictions of tumor sensitivity with this assay.     

A fluorometric assay for L-asparaginase activity and monitoring of L-asparaginase therapy

The antineoplastic enzyme L-asparaginase is commonly used for the induction of remission in acute lymphoblastic leukemia (ALL). There is no simple method available for measuring the activity of this highly toxic drug. We incubated L-asparaginase from Erwinia chrysanthemi with L-aspartic acid beta-(7-amido-4-methylcoumarin) and measured the release of 7-amino-4-methylcoumarin fluorometrically for 30-300 min. The rate of the hydrolysis of the substrate was linear over a 50-fold range of the concentration of the enzyme. With increasing substrate concentration, the enzyme showed a saturable kinetic pattern with V(max) of 0.547 (SD 0.059) microM/min/mg of enzyme (n = 3) and Km of 0.302 (SD 0.095) mM (n = 3). This assay enables rapid analysis of L-asparaginase activity in biological samples and it can be used, for example, for monitoring of L-asparaginase activity in serum of ALL patients during their L-asparaginase therapy.     

In vitro testing of chemotherapeutic combinations in a rapid thymidine incorporation assay

A total of 199 solid human tumors were tested with a rapid thymidine incorporation assay for sensitivity to one of several clinically used multi-drug combinations and to each agent in the combination separately. Melanomas, lung and breast cancers accounted for the majority of specimens. In 120 specimens, at least one single agent exhibited in vitro activity (80% or greater inhibition of thymidine incorporation), and in 116 of these (96.7%) the drug combination was active in vitro. In the 79 specimens where no single agent was active in vitro, the combination was also inactive in 62 (78.5%). Overall, there was concordance of in vitro activity of the most active single agent and the combination of agents in 89.5% of specimens tested. A lack of significant in vitro synergy was noted in all of the drug combinations and tumor types tested. Of note is the fact that in 141 of the 199 tests (70.9%) either no drug (n = 79) or only 1 drug (n = 62) demonstrated in vitro activity. We conclude that the rapid thymidine incorporation assay can be used to test for in vitro sensitivity to drug combinations, and that sensitivity to a drug combination can be inferred if a tumor is sensitive to any component drug of the combination.     

A rapid bioassay to determine stabilities of anticancer agents under conditions of the clonogenic assay

Summary We developed a rapid bioassay to determine in vitro drug stabilities in the clonogenic assay. The in vitro half-lives of 11 standard antitumor agents, actinomycin D, Adriamycin, bleomycin, cis-Platinum, dacarbazine, 5-fluorouracil, melphalan, mitomycin C, 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), vinblastine, and vincristine, and four investigational drugs, Aziridinylbenzoquinone (AZQ) (NSC 182986), diamminecyclobutane-dicarboxylatoplatinum (CBDCA) (NSC 241240), Dihydroxyanthracenedione dihydrochloride (DHAD) (NSC 301739), and 1-(2-chloroethyl)-3-(2,6-dioxo-3-piperidyl-1-nitrosourea) (PCNU) (NSC 95466), were determined under conditions of the clonogenic assay as well as under storage conditions at −40° and−196°C. BCNU and AZQ at−40°C had t1/2 of 4.7 d and 2.5 d, respectively. All other drugs were stable at −40° and −196°C with t1/2>6 wk. Under assay conditions at 37°C, actinomycin D, bleomycin, dacarbazine, 5-fluorouracil, mitomycin C, vinblastine, vincristine, and DHAD were stable, with t1/2>14 d. CBDCA, AZQ, Adriamycin, cis-Platinum, melphalan, BCNU, and PCNU had t1/2 of 94,72,29,18.5,1.8,1, and 0.5 h, respectively. This work was supported by Veterans Administration Medical Research Service and by Contract CM57710 of the National Cancer Institute.     

Stathmin Potentiates Vinflunine and Inhibits Paclitaxel Activity

Cell biology and crystallographic studies have suggested a functional link between stathmin and microtubule targeting agents (MTAs). In a previous study we showed that stathmin increases vinblastine (VLB) binding to tubulin, and that conversely VLB increases stathmin binding to tubulin. This constituted the first biochemical evidence of the direct relationship between stathmin and an antimitotic drug, and revealed a new mechanism of action for VLB. The question remained if the observed interaction was specific for this drug or represented a general phenomenon for all MTAs. In the present study we investigated the binding of recombinant stathmin to purified tubulin in the presence of paclitaxel or another Vinca alkaloid, vinflunine, using Isothermal Titration Calorimetry (ITC). These experiments revealed that stathmin binding to tubulin is increased in the presence of vinflunine, whereas no signal is observed in the presence of paclitaxel. Further investigation using turbidity and co-sedimentation showed that stathmin inhibited paclitaxel microtubule-stabilizing activity. Taken together with the previous study using vinblastine, our results suggest that stathmin can be seen as a modulator of MTA activity and binding to tubulin, providing molecular explanation for multiple previous cellular and in vivo studies showing that stathmin expression level affects MTAs efficiency.     

Exposure to paclitaxel or vinblastine down-regulates CD11a and CD54 expression by P815 mastocytoma cells and renders the tumor cells resistant to killing by nonspecific cytotoxic T lymphocytes induced with anti-CD3 antibody

Paclitaxel and vinblastine are drugs with anti-microtubule activity that are commonly used in the treatment of numerous types of cancer. In this study, we investigated the effect of prior exposure to submaximal cytotoxic concentrations (EC(25) and EC(50)) of paclitaxel or vinblastine on the subsequent susceptibility of surviving P815 murine mastocytoma cells to cytolysis by major histocompatibility complex (MHC)-unrestricted mouse cytotoxic T lymphocytes that had been induced with anti-CD3 antibody. P815 cells that had survived culture for 24 h in the presence of paclitaxel (5 or 50 micro g/ml) or vinblastine (1.5 or 15 micro g/ml) were rendered resistant to anti-CD3-activated killer-T (AK-T) cell-mediated cytolysis in a standard (51)Cr-release assay. Resistance to killing was associated with a reduced ability of AK-T cells to form conjugates with drug-treated P815 target cells, suggesting a possible effect on adhesion molecules. Flow cytometric analysis of paclitaxel- or vinblastine-treated P815 cells revealed reduced cell-surface expression of the adhesion molecules LFA-1 (CD11a /CD18) and ICAM-1 (CD54). Similar results were obtained following paclitaxel or vinblastine treatment of Yac-1 lymphoma cells. RT-PCR analysis revealed reduced levels of mRNAs coding for CD11a and CD54 in paclitaxel- or vinblastine-pretreated P815 cells. Collectively, these data lead us to conclude that paclitaxel and vinblastine render P815 mastocytoma cells resistant to T cell-mediated cytotoxicity by interfering with CD11a and CD54 expression by the tumor cells. A similar effect by these drugs on tumor cells and/or leukocytes in cancer patients might compromise tumor-specific cell-mediated immune responses.     

Possible mechanism of the selectivity of the cytostatic action of antineoplastic preparations

A model of the phenomenon of selectivity of antineoplastic phase-specific preparations in leukemia, based on the assumption that any proliferating cells are equally sensitive to the action of such preparations, is suggested. Therefore, selectivity, the concept and fundamental significance of which were formulated and substantiated by the authors earlier, can be expressed in terms of the proliferative pathways of the normal and leukemic populations, the ratio between which thus plays the deciding role in the selection of the optimum system of therapy. The concept of maximum selectivity, which is a convenient numerical characteristic of a preparation, was introduced. A comparative quantitative analysis was made of a number of antineoplastic preparations (vinblastine, vincristine, amethopterin, arabinosylcytosine, azaserine); it showed that the conclusions of the model do not contradict the experimental data.     

Vinflunine, a novel microtubule inhibitor, suppresses calmodulin interaction with the microtubule-associated protein STOP

Vinca alkaloids vinblastine and vincristine and some of their derivatives such as vinorelbine are widely used in therapy of leukemia and several solid tumors. Their action is associated with alterations of the mitotic spindle functions that prevent the cell cycle progression and lead to mitotic block. A number of studies show that some Vinca alkaloids inhibit CaM-target interaction. The newest microtubule inhibitor, vinflunine (Javlor), currently in clinical trials, is remarkably more active than vinblastine against a number of tumors. Moreover, vinflunine is significantly less toxic than other Vinca alkaloids. The high antitumor activity of this molecule is not well understood since it binds to tubulin with an overall affinity several-fold lower than that of vinblastine or vincristine. In this study, we examined the interaction of Ca2+-CaM with vinflunine, vinblastine, and stable tubule only polypeptide (STOP) by using a combination of thermodynamic and mass spectrometric approaches. We characterized the influence of Vinca alkaloids on Ca2+-CaM-STOP complex formation. Our results revealed different binding modes to Ca2+-CaM for vinflunine and vinblastine, highlighting that adding fluorine atoms on the cleavamine moiety of the Vinca alkaloid molecule is critical for the localization of the drug on calmodulin. We demonstrate that vinflunine is a better inhibitor for STOP binding to calmodulin than vinblastine. We suggest that vinflunine action on calmodulin can have an effect on microtubule dynamics. These data may contribute to a better understanding of the superior antitumor efficiency and lower toxicity of vinflunine.     

Imatinib and Nilotinib Reverse Multidrug Resistance in Cancer Cells by Inhibiting the Efflux Activity of the MRP7 (ABCC10)

Background

One of the major mechanisms that could produce resistance to antineoplastic drugs in cancer cells is the ATP binding cassette (ABC) transporters. The ABC transporters can significantly decrease the intracellular concentration of antineoplastic drugs by increasing their efflux, thereby lowering the cytotoxic activity of antineoplastic drugs. One of these transporters, the multiple resistant protein 7 (MRP7, ABCC10), has recently been shown to produce resistance to antineoplastic drugs by increasing the efflux of paclitaxel. In this study, we examined the effects of BCR-Abl tyrosine kinase inhibitors imatinib, nilotinib and dasatinib on the activity and expression of MRP7 in HEK293 cells transfected with MRP7, designated HEK-MRP7-2.

Methodology and/or Principal Findings

We report for the first time that imatinib and nilotinib reversed MRP7-mediated multidrug resistance. Our MTT assay results indicated that MRP7 expression in HEK-MRP7-2 cells was not significantly altered by incubation with 5 µM of imatinib or nilotinib for up to 72 hours. In addition, imatinib and nilotinib (1-5 µM) produced a significant concentration-dependent reversal of MRP7-mediated multidrug resistance by enhancing the sensitivity of HEK-MRP7-2 cells to paclitaxel and vincristine. Imatinib and nilotinib, at 5 µM, significantly increased the accumulation of [³H]-paclitaxel in HEK-MRP7-2 cells. The incubation of the HEK-MRP7-2 cells with imatinib or nilotinib (5 µM) also significantly inhibited the efflux of paclitaxel.

Conclusions

Imatinib and nilotinib reverse MRP7-mediated paclitaxel resistance, most likely due to their inhibition of the efflux of paclitaxel via MRP7. These findings suggest that imatinib or nilotinib, in combination with other antineoplastic drugs, may be useful in the treatment of certain resistant cancers.     

Development of a rodent lung macrophage chromosome aberration assay

Lung macrophages are the first line of defense against inhaled xenobiotics. They are able to accumulate airborne particulates as well as having metabolic capability. They may thus be sensitive indicator cells for detecting inhalation exposure to environmental mutagens. Their usefulness as a short-term in vivo genotoxic assay has not, however, been adequately explored. We have systematically investigated the feasibility of developing a lung macrophage chromosome-aberration assay. It was found that with different types of spindle-binding chemicals (vinblastine and vincristine), and with improved harvesting procedures, an adequate number of metaphase cells can be collected from mice and Chinese hamsters. The chromosome aberration frequencies in macrophages from control mice and Chinese hamsters were found to be 1.2 +/- 2.3 and 0.75 +/- 2.2 per 100 cells respectively. These frequencies are within normal ranges for other somatic cells. After inhalation exposure to an occupational-exposure level of benzene (0, 0.1 and 1 ppm), significant dose-dependent induction of aberrations (1.2 +/- 2.3, 5.7 +/- 6.3 and 6.8 +/- 6.2 chromatid deletions per 100 cells resp.) were observed in the macrophages. Thus, these cells can be used as one of a battery of in vivo assays for inhalation exposure studies.     

Selective immunomodulation by the antineoplastic agent mitoxantrone. II. Nonspecific adherent suppressor cells derived from mitoxantrone-treated mice

Mitoxantrone exerts a potent suppressive influence upon humoral immune responses. The B cell is a likely target for this inhibitory effect, and we have reported evidence supporting this possibility. The impact of mitoxantrone upon T lymphocyte reactivity was assessed as a second mode of action of this novel antineoplastic drug. TH and TS lymphocyte induction were tested in the in vitro anti-sheep erythrocyte response, and a surprising differential effect of mitoxantrone was observed. Helper activity was abrogated and suppressor function was enhanced. In apparent disagreement with this result, mitoxantrone inhibited the in vivo induction of TS cells using trinitrophenylated spleen cells. Macrophages were investigated as potential mediators of these effects upon immunoregulatory function. Replacement of macrophages in mitoxantrone-treated spleen cell preparations by normal adherent cells allowed the induction and complete expression of TH lymphocyte function. Conversely, replacement of mitoxantrone-treated macrophages with normal adherent cells before induction of TS cells failed to generate TS cell function. Thus, TH cells were resistant and TS cells were completely susceptible to mitoxantrone. Furthermore, supplementation of normal TH cell cultures with splenic macrophages from mitoxantrone-treated mice inhibited the induction of helper function. Production of the lymphokines IL 2 and TRF in mitoxantrone-treated mice was normal. This is consistent with the retention of functional TH cells in drug-treated spleens. Macrophages in the spleens of mitoxantrone-treated mice were responsible for the abrogated helper function and the enhanced suppressor activity. Although TS cell induction was directly inhibited by the drug, the effect upon TH cell function was secondary to the action of mitoxantrone-induced suppressor macrophages. Mitogen-stimulated lymphokine production was normal. Thus, mitoxantrone is a selective immunomodulator. The macrophage-mediated suppression of TH cell induction and humoral immunity investigated in spleens from mitoxantrone-treated mice is an intriguing finding that may have significant implications for immunotherapy.